http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Maven2010.igem.org - User contributions [en]2024-03-28T09:48:48ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:UCSF/Project/PrecisionTeam:UCSF/Project/Precision2010-10-28T03:00:54Z<p>Maven: </p>
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<h3 style="font-weight:bold;">Greater Precision</h3><br />
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<p><b>Goal</b>: Engineer killer cells to increase their precision in detecting cancer cells.</p><br><br />
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<p><b>Approach</b>: After discussing our goals for the iGEM project, we have come up with an approach to increase precision by using:<br><br />
1. CARs that recognize many types of different cancer ligands.<br><br />
2. Logic gating to set higher restrictions on killer cell activation.</p><br><br />
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<p><b>Devices</b>:<br><br />
1. <b>ANDN gate</b> - we have successfully developed devices for this type of logic gate, and we have confirmed through testing data their ability to increase precision.<br><br />
2. <b>AND gate</b> - we have constructed potential devices for this type of logic gate, and we are now working on optimizing the assay to measure their effects on precision.</p><br><br />
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<h3 style="font-weight:bold;">Background information</h3><br />
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<p>In the same way that we recognize people based on their appearance, killer cells (cytotoxic T cells and NK cells) recognize target cells based on their different types of surface proteins <a href="#references">[1]</a>. This important ability to recognize the many different types of cells allows killers cells to eliminate unhealthy cells but avoid harming healthy cells. Unfortunately, killer cells can have trouble recognizing cancer cells among healthy cells due to complex profiles of surface markers on cancer cells. When it comes to cancer, killer cells are disadvantaged because they target foreign and dangerous organisms, but cancer cells originate from formerly healthy cells. Therefore, killer cells face difficulty in labeling cancer cells as dangerous entities because cancer cells express self antigens <a href="#references">[2]</a>. This fact is unsettling in that this method of differentiation is currently the only means by which killer cells can recognize cancerous cells.</p><br><br />
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<p>We hope to introduce a logic gating system as an engineering platform to make killer cell recognition more specific and precise. We took advantage of the fact that many of the killer cells’ receptors bind to specific target cell surface proteins, much like antibodies bind to specific antigens. So why not replace the receptors’ recognition domains with different antibodies to create new receptors to recognize the many surface proteins on cells? That’s exactly what we did. These chimeric antigen receptors (CARs) are the products of an immune receptor intracellular signaling chain and an antigen binding domain, which, when put together as a single unit, can bind specifically to target antigens and trigger signaling responses <a href="#references">[3]</a>. Killer cells engineered with such modularly constructed synthetic receptors can overcome the restriction imposed by the presence of self-antigens on cancer cells.</p><br><br />
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<img src="https://static.igem.org/mediawiki/2010/2/23/UCSF_CAR_structure.png" /><br><br />
<b>Domain structure and function of a chimeric antigen receptor <a href="#references">[4]</a>.</b><br />
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<p>We also took advantage of the fact that killer cells’ receptors relay extracellular information intracellular compartments using modular signaling motifs such as ITAM, ITAM-like activation motifs, and ITIM. When ITAM and ITAM-like activation motifs become activated, they recruit kinases in the cytoplasm that initiate cell killing. ITIM motifs recruit phosphatases that cancel out the effects of ITAM activation <a href="#references">[5]</a>.</p><br><br />
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<p>The modularity of CARs and killer cells’ receptors makes it feasible to create a multitude of recognition systems that function as logic gates in the killer cell. The logic gates should enable engineered killer cells to recognize specific combinations of surface proteins on target cells. Each specific combination of surface proteins acts as the prerequisite to activate a logic gate in order to trigger a killer cell action, which makes recognition a highly precise process. This in turn allows killer cells to distinguish cancer cells from normal cells more effectively because the specific combinations of antigens found only on cancerous cells can be set as logic gate prerequisites to trigger cell activation, whereas the normal cells do not fulfill the prerequisites and are left unharmed.</p><br><br><br />
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<h3 style="font-weight:bold;">Experimental Design and Results</h3><br />
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<p>For our project we have designed two main gates: ANDN and AND gate.</p><br />
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<h4 id="andn" style="color:black; font-weight:bold;">i. ANDN gate</h4><br />
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<p>In order to understand the ANDN gate, let us set up a hypothetical situation in which antigen A and antigen B are expressed on the membrane surface of healthy cells. Since cancer cells typically discard many surface proteins as a result of genetic mutation, we represent this discarded protein as antigen B in our scenario. Our ANDN gate is designed to address this issue by triggering cytotoxicity in the presence of antigen A and absence of antigen B. Therefore, cancerous cells that express antigen A and “hide” antigen B will be targeted. Healthy cells expressing both antigen A and B will not set off the activation of the ANDN gate. This concept is valuable for ensuring a level of specificity that prevents the overly indiscriminate activation of killer cells.</p><br><br />
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<p>We tested two different ANDN gate designs to determine their effects on target recognition. To achieve the level of specificity as described by our hypothetical situation, we have set two different antigen binding domains to recognize antigen A and antigen B, respectively. Attached to the domain that recognized antigen A was an ITAM-bearing intracellular chain, from either the CD3 zeta or Fc receptor gamma, that signaled for killer cell activation. The domain that recognized antigen B, the antigen found in healthy cells, was fused to the intracellular portion of the ITIM-based receptor KIR3DL1, which inhibits killer cell activation. As a result of this combination, target cells expressing only antigen A would trigger killer cell activation, and target cells that do not express antigen A would not. Target cells that express both antigens A and B would be unharmed due to ITIM inhibitory signals, meaning that the presence of antigen B overwrites the input of antigen A. In conclusion, only a specific combination of surface antigens can set off the chain of activation, resulting in increased precision of detection and cancer killing.</p><br><br />
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<img src="https://static.igem.org/mediawiki/2010/8/87/UCSF_ANDN_gate.png" /><br><br />
<b>Function of ANDN Gate</b><br />
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<p>To evaluate our ANDN gate designs, we presented the dually transfected killer cells with target cells expressing antigen A, antigen B, both antigens A and B, or none of those antigens. The killer cells had been engineered to express the GFP reporter from the promoter of NFAT, a gene induced during killer cell activation. This reporter cell line enabled us to quantify the percentage of activated killer cells that express using FACS (fluorescence activated cell sorting). As shown in the figure below, killer cells presented with target cells expressing neither antigen or only antigen B showed basal levels of activation. Target cells expressing only antigen A, which represent cancer cells in this experiment, increased the percentage of activated killer cells. Notably, killer cells presented with target cells expressing both antigens had basal levels of activation. Living up to expectations, the ANDN gates proved to increase specificity because more killer cells became activated only in the presence of antigen A and the absence of antigen B.</p><br><br />
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<img src="https://static.igem.org/mediawiki/2010/8/80/UCSF_ANDNgate_results.png" /><br><br />
<b>Experimental Data of ANDN Gate on T-Cell Activation</b><br />
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<h4 style="color:black; font-weight:bold;">ii. AND gate</h4><br />
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<p>Cancer cells are prone to overexpressing proteins on their cell surfaces. This fact allows us to detect the presence of cancer cells using AND gates. In our new hypothetical situation, normal cells express either antigen C or antigen D. In contrast, the overproduction characteristic of cancerous cells allows for the expression of both of these proteins <a href="#references">[6]</a>. AND gates are useful in this situation because they become activated only in the presence of two defined antigens. In the case of our new situation, the AND gate will trigger cytotoxicity only in the presence of antigen C and D, a condition that only applies to cancerous cells.</p><br><br />
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<p>Applying the AND gate scenario and concept to the lab, we have explored using the activation adaptor DAP10. In normal killer cells, DAP10 recruits two different proteins to two different motifs along its main body when activated. This recruitment will trigger the killing response only when both proteins are present. In order to ensure that killer cells will only kill in the presence of two specific antigens, we used two mutant versions of DAP10, each of which has a different motif that does not allow its complementary protein to bind. Each mutant version is fused to a different extracellular part that recognizes specific antigens on cell surfaces. As a result, when such CARs only recognize one antigen on healthy cells, they will not be able to trigger activation because only one motif is activated. When both CARs bind to both antigens found on cancerous cells, both motifs are able to become activated to induce cell cytotoxicity.</p><br><br />
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<img src="https://static.igem.org/mediawiki/2010/3/30/UCSF_AND_gate.png" /><br><br />
<b>AND gate design</b><br />
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<p>Due to time constraints, we have not been able to evaluate our AND gate design. We could not measure the AND gate’s effect in killer cells using the NFAT promoter reporter assay. We attempted to test our AND gate using assays directly measuring the level of target cell killing. However, we faced a major technical obstacle that prevented us from obtaining informative results before the summer ended. The technical challenge was that only a small percentage of killer cells expressed the logic gate CARs after transfection. Due to this low transfection efficiency, the vast majority of killer cells used in target cell killing assays did not express our constructs. This was problematic because untransfected killer cells have an innate ability to kill, which produces a high killing background that makes it difficult to pinpoint the killing ability of transfected cells.</p><br><br><br />
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<h3 style="font-weight:bold;">Future Directions</h3><br />
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<p>Our future goal would be to increase the efficiency for transfecting the killer cells (use viral vectors) and hopefully to obtain cells stably expressing our logic gate parts. This would allow us to test both the ANDN and AND gate designs directly based on cell killing efficiency with a significantly reduced basal killing level.</p><br><br><br />
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<h3 style="font-weight:bold;">References</h3><br />
<p><a name="references"></a></p><br />
<p><br />
1. <b>Formation and function of the lytic NK-cell immunological synapse.</b></p><br />
<p>Orange JS.</p><br />
<p>Nat Rev Immunol. 2008 Sep;8(9):713-25.</p><br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19172692">http://www.ncbi.nlm.nih.gov/pubmed/19172692</a></p><br />
<br><br />
<p><br />
2. <b>Learning how to discriminate between friends and enemies, a lesson from Natural Killer cells.</b><br />
<p>Bottino C, Moretta L, Pende D, Vitale M, Moretta A.<br />
<p>Mol Immunol. 2004 Jul;41(6-7):569-75.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/15219995">http://www.ncbi.nlm.nih.gov/pubmed/15219995</a></p><br />
<br><br />
<p><br />
3. <b>Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor.</b><br />
<p>Jena B, Dotti G, Cooper LJ.<br />
<p>Blood. 2010 Aug 19;116(7):1035-44. Epub 2010 May 3.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20439624">http://www.ncbi.nlm.nih.gov/pubmed/20439624</a></p><br />
<br><br />
<p><br />
4. <b>Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.</b><br />
<p>Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, Temme A, Schmitz M.<br />
<p>J Biomed Biotechnol. 2010;2010:956304. Epub 2010 May 5.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20467460">http://www.ncbi.nlm.nih.gov/pubmed/20467460</a></p><br />
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<p><br />
5. <b>Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse.</b><br />
<p>Tassi I, Klesney-Tait J, Colonna M.<br />
<p>Immunol Rev. 2006 Dec;214:92-105.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/17100878">http://www.ncbi.nlm.nih.gov/pubmed/17100878</a></p><br />
<br><br />
<p><br />
6. <b>Oncogenic stress sensed by the immune system: role of natural killer cell receptors.</b><br />
<p>Raulet DH, Guerra N.<br />
<p>Nat Rev Immunol. 2009 Aug;9(8):568-80.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19629084">http://www.ncbi.nlm.nih.gov/pubmed/19629084</a></p><br />
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__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/SandBox1Team:UCSF/SandBox12010-10-28T03:00:11Z<p>Maven: </p>
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===Project Description===<br />
<br />
Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
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Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
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<h4 id="andn">i. ANDN gate</h4><br />
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__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/SandBox1Team:UCSF/SandBox12010-10-28T02:58:39Z<p>Maven: </p>
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===Project Description===<br />
<br />
Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
<html><br />
<h4><span class="mw-headline">i. ANDN gate</span></h4><br />
</html><br />
{{Template:UCSF/LeftEnd}}<br />
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<div id="right"><br />
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<br />
__TOC__<br />
{{Template:UCSF/RightEnd}}<br />
<br />
__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/SandBox1Team:UCSF/SandBox12010-10-28T02:57:18Z<p>Maven: </p>
<hr />
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===Project Description===<br />
<br />
Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
<html><br />
<h4>i. ANDN gate</h4><br />
</html><br />
{{Template:UCSF/LeftEnd}}<br />
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<style><br />
#right{<br />
margin-top:-10px;<br />
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<div id="right"><br />
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<br />
__TOC__<br />
{{Template:UCSF/RightEnd}}<br />
<br />
__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/SandBox1Team:UCSF/SandBox12010-10-28T02:55:15Z<p>Maven: </p>
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===Project Description===<br />
<br />
Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
====i. ANDN gate====<br />
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__TOC__<br />
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<br />
__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/SandBox1Team:UCSF/SandBox12010-10-28T02:52:54Z<p>Maven: </p>
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===Project Description===<br />
<br />
Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
===i. ANDN gate===<br />
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<br />
__TOC__<br />
{{Template:UCSF/RightEnd}}<br />
<br />
__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-28T02:09:02Z<p>Maven: </p>
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
[[Image:UCSFnewgranuleagents-06.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br /><br />
[[Image:UCSFMailaddress-05.jpg|400px]]<br />
<br /><br /><br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br /><br />
[[Image:UCSFGranuleloadingaddress-04.jpg|400px]]<br />
<br /><br /><br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells and started to select for cells expressing our construct for our assay by selecting with the selection agent G418. The cells used for the assay were mixed populations as the selection was not complete.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
SUMMARY OF RESULTS<br />
(See below for more details)<br />
colocalization stats images ready for wiki<br />
{|cellspacing="0"<br />
! Construct<br />
! GFP looks granular<br />
! Granule stain worked<br />
! GFP and granule stain appear colocalized<br />
! Location of granules<br />
! Colocalization statistics<br />
|-<br />
|GZMB-eGFP-CDMPR (56)<br />
|yes<br />
|yes<br />
|yes<br />
|Center<br />
|Pearson's Correlation: 0.831 Mander's Overlap: 0.967<br />
|-<br />
|GZMB-eGFP-LIMP (59)<br />
|yes<br />
|Kind of<br />
|Not really<br />
|Edges<br />
|Pearson's Correlation: 0.577 Mander's Overlap: 0.993<br />
|-<br />
|GZMB-eGFP-Ymotif (60)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|throughout cell, mostly central<br />
|Pearson's Correlation: 0.829 Mander's Overlap: 0.936<br />
|-<br />
|GZMB-eGFP-STP (61)<br />
|Yes<br />
|Yes (with adjustment to visualization settings)<br />
|Yes<br />
|Edges<br />
|Pearson's Correlation: 0.774 Mander's Overlap: 0.976<br />
|-<br />
|pMAX-GFP<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|-<br />
|GZMM-eGFP-GILT (63)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|GZMM-eGFP-STP (67)<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-<br />
|NKL-untransfected<br />
|No<br />
|No<br />
|No<br />
|N/A<br />
|N/A<br />
|}<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
===Future application===<br />
<br /><br />
[[Image:UCSFnewgranuleagents-07.jpg|center|600px]]<br />
<br /><br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
http://www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/ProtocolsTeam:UCSF/Protocols2010-10-28T02:05:19Z<p>Maven: </p>
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<br />
===AarI Digest===<br />
1. Label PCR tubes.<br><br />
2. Add the following reagents into the PCR tubes:<br><br />
'''Aar1 Digest Reagents:'''<br><br />
5 ug DNA<br><br />
2.5ul Aar1 Enzyme<br><br />
0.9ul Aarl oligo<br><br />
6 ul 10x Aar1 Buffer<br><br />
x ul dH2O<br><br />
60 ul total reaction<br><br />
''Note: x ul dH2O may change in volume for different reactions in order to bring total volume up to 60 ul.''<br />
<br />
3. Briefly vortex and spin down the reaction.<br><br />
4. Incubate reaction at 37C for 3 hours.<br />
<br />
''Note: PCR program can be set to 37C for 3 hours and cooled down to 4C indefinitely.''<br />
<br />
<br><br />
<br />
<br />
===PCR with Phusion enzyme===<br />
1. Label PCR tubes.<br><br />
2. Add the following reagents into the PCR tubes (in order):<br><br />
'''PCR Reagents'''<br><br />
23.7 ul dH2O<br><br />
10 ul 5x HF Buffer<br><br />
5 ul forward primer<br><br />
5 ul reverse primer<br><br />
5 ul dNTP<br><br />
0.3 ul template<br><br />
1 ul Phusion<br><br />
50 ul total<br> <br />
3. Vortex and spin down the reaction.<br><br />
4. Set up PCR program.<br><br />
<br />
{|cellspacing="0"<br />
|'''Cycle Step'''||'''Temperature'''||'''Time'''||'''# Cycles'''<br />
|+<br />
|Initial Denature||98C||3min||1<br />
|-<br />
|Denature<br>Annealing<br>Extension||98C<br>55C<br>72C||10s<br>30s<br>30s||30<br />
|-<br />
|Final Extension||72C||5min||1<br />
|-<br />
|Hold||4C|| || <br />
|}<br />
<br />
''Note: Times may differ for different reactions based on the size of the desired PCR product. Annealing temperature may also differ depending on primer properties.''<br />
<br />
<br />
<br><br />
===Ligation===<br />
''' Ligation Reagents:'''<br><br />
50 ng vector<br><br />
DNA insert(s)<br><br />
Buffer<br><br />
1 ul Ligase<br><br />
<br />
'''Ligation Reaction:'''<br><br />
1. Calculate amount of DNA insert and vector needed for reaction.<br><br />
150 ng / ( # bp in backbone/ # bp in insert) = ng needed<br><br />
Concentration of insert or vector divided by ng needed = volume of DNA needed<br />
<br />
2. Calculate amount of buffer needed depending on concentration for volume needed. A typical ligation reaction volume can be 20 ul.<br />
<br />
3. Add reagents together, from smallest volumes to largest. Distilled water can be used to bring the volume up so that the reaction has the proper buffer concentration for the reaction. Ligase or enzymes in general should be added at the end.<br />
<br />
4. Vortex or pipet up and down to mix. Spin down afterwards if vortexing.<br />
<br />
5. Let reaction sit at room temperature for 5 to 20 minutes depending on ligase used. The ligase may start acting up if left too long.<br />
<br />
<br><br />
===Agarose Gel Electrophoresis===<br />
'''Gel Electrophoresis Reagents/Materials:'''<br><br />
Gel caster<br><br />
Gel tray<br><br />
Agar powder<br><br />
TAE buffer<br><br />
10, 000X Sybr Safe<br><br />
50ml conical tube<br><br />
Gel comb<br><br />
Microwave<br><br />
Large flask (microwave safe)<br><br />
<br />
'''Preparing the agarose solution:'''<br />
<br />
1. Pour in flask the amount of TAE buffer desired. This will be the volume of agarose solution made.<br />
<br />
2. Measure out the amount of agar needed for desired concentration. For example to get a 1% agarose gel, you would add 1 gram of agarose powder to 100 ml of TAE buffer. Most concentrations commonly fall between 0.7- 2%. Higher concentrations of agarose are better suited for small fragment separation while lower concentrations are suitable for large fragments.<br />
<br />
3. Microwave three minutes, may vary depending on volume in flask. Make sure cap is on loosely.<br />
<br />
4. Using hot gloves, swirl bottle gently to ensure complete dissolving of agar. The solution should be clear when agar is completely dissolved. If it is not, microwave for another minute.<br />
<br />
5. Place flask of agarose solution in 80C bath to store as to prevent solidifying of agarose solution.<br />
<br />
<br />
'''Casting gel:'''<br />
<br />
1. Secure gel tray in caster<br />
<br />
2. Pour desired volume of premade agarose solution into 50 ml conical tube. Volume desired will vary depending on gel tray size and desired thickness of gel. Thin gels solidify quicker and can show faint bands clearer, but the wells of thicker gels can hold more DNA.<br />
<br />
3. Add Sybr safe to agarose solution. For 10 ml of agarose solution, add 1ul Sybr Safe. The Sybr safe is used to stain and visualize the DNA when viewed under a blue light.<br />
<br />
4. Invert tube several times until Sybr safe is equally distributed. Invert gently to avoid creating air bubbles.<br />
<br />
5. Pour contents of conical tube into secured gel tray.<br />
<br />
6. Place gel comb into notches of gel tray. Check that comb is evenly submerged in agarose. The comb will form wells in the gel to load DNA.<br />
<br />
7. Dry at room temperature. The gel will appear opaque when done.<br />
<br />
===Colony PCR===<br />
'''Colony PCR reagents:'''<br><br />
12ul dH20<br><br />
4 ul 5x gotaq green buffer<br><br />
2 ul 10mM dntp<br><br />
1 ul forward primer<br><br />
1 ul reverse primer<br><br />
0.1 ul GoTaq Polymerase<br><br />
<br />
1. After adding reagents to PCR tube, pick a colony and touch the bottom of the PCR tube with the pipette.<br />
<br />
2. Place PCR tubes into the PCR machine and set the cycling parameters to be optimal for the piece of DNA being copied.<br />
<br />
<br><br />
===Transformation of E. coli===<br />
'''Transformation Reagents'''<br><br />
LB Plates<br><br />
Ice box<br><br />
Water bath<br><br />
37C Incubator<br><br />
Competent cells & Plasmid<br><br />
Spreading tool (beads)<br><br />
<br />
1. Prewarm LB plates in the 37C incubator<br />
<br />
2. Remove desired cells from -80C freezer (or other storage location) and melt over ice for at least 10 minutes. (Make sure to aliquot the correct amount of cells for transformation)<br />
<br />
3. Pipette up 5ul of your plasmid DNA and gently pipette into the suspended cells. Do not mix by triterating the solution (pipetting up and down).<br />
<br />
4. Let the cells sit on ice for 15-30 minutes.<br />
<br />
5. Heat shock your cells for 75 seconds in a 42C water bath.<br />
<br />
6. Quickly return your tubes to the ice bucket for 2-3 minutes.<br />
<br />
7. When you are ready to plate, remove your prewarmed plate from the incubator. Using sterile technique, carefully pipette your entire tube of cells onto the LB plate.<br />
<br />
8. Again using sterile technique, spread the cells across your plate using either sterile beads or another spreading tool.<br />
<br />
9. Put the plate in the 37C incubator for 5-15 hours.<br />
<br />
===Gel Extraction/Purification of DNA===<br />
<br />
'''Gel Extraction/Purification Materials & Reagents:'''<br><br />
Sterile Blade<br><br />
2ml microcentrifuge tube<br><br />
1.5ml microcentrifuge tube<br><br />
QIAGEN Gel Extraction Kit<br><br />
Centrifuge<br><br />
Heat Block/Bath<br><br />
<br />
1. Using a sterile blade, carefully excise the desired band from your gel.<br />
<br />
2. Transfer the agarose chunk into a new 2ml microcentrifuge tube.<br />
<br />
3. Weigh the excised agarose chunk and add 3 times its weight in microliters of QG buffer.<br />
<br />
4. Move the tube to a melting block for 10 minutes at 50C or until the agarose is completely melted.<br />
<br />
5. Add isopropanol to the tube at a 1:3 ratio with QG, then vortex the solution briefly to help precipitate the DNA.<br />
<br />
6. Transfer the solution to a Qiagen gel extraction/PCR purification spin column.<br />
<br />
7. Spin for 1 minute at 13,000rpm and dump the supernatant.<br />
<br />
8. Add 500μl of QG buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum the flowthrough.<br />
<br />
9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum flowthrough.<br><br />
''NOTE: Wash the rim with the buffer.''<br />
<br />
10. Repeat Step 9<br />
<br />
11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum)<br />
<br />
12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column.<br />
<br />
13. Place tubes in a -20oC freezer box.<br />
<br />
<br />
<br><br />
===Miniprep===<br />
'''Miniprep Materials'''<br><br />
Qiagen Miniprep Kit<br><br />
Centrifuge<br><br />
<br />
1. Spin down bacterial culture at 3500 rpm for 5 minutes<br />
<br />
2. Aspirate supernatant. Change tips for every sample.<br />
<br />
3. Resuspend in 250μl of P1 Buffer. Vortex until cell pellet is not visible. Transfer into a 1.5 ml tube.<br />
<br />
4. Add 250μl of P2 Buffer. Invert 5-6 times. Wait 3-5 minutes.<br><br />
''NOTE: When you open the cap, you should see a string of DNA.''<br />
<br />
5. Add 350μl of N3 Buffer. Invert 5-6 times or until colorless<br />
<br />
6. Centrifuge at 13,000 rpm for 10 minutes. Label blue spin columns. Transfer supernatant into blur spin columns.<br />
<br />
7. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant.<br />
<br />
8. Add 500μl of PB buffer. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant.<br />
<br />
9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum supernatant.<br><br />
''NOTE: Wash the rim with the buffer.''<br />
<br />
10. Repeat Step 9<br />
<br />
11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum)<br />
<br />
12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column.<br />
<br />
13. Place tubes in a -20oC freezer box.<br />
<br />
<br><br />
===Maxiprep===<br />
<br />
'''Maxiprep Materials & Reagents'''<br><br />
250ml centrifuge tubes<br><br />
Isopropanol<br><br />
QIAGEN Maxiprep Stand<br><br />
QIAGEN Maxiprep Kit<br><br />
Large Centrifuge<br><br />
50ml conical tubes<br><br />
100% Ethanol<br><br />
<br />
1. Pick a single colony and inoculate in 2-5 ml LB medium containing the appropriate antibiotic, Incubate for around 8 hrs at 37C with vigorous shaking.<br />
<br />
2. Dilute 1/500 to 1/1000 into selective LB medium. For high copy plasmids, inoculate 100 ml medium with 100-200μl of starter culture. For low copy plasmids, inoculate 250 ml medium with 250-500μl of starter culture. Grow at 37C for 12-16 hrs with vigorous shaking.<br />
<br />
3. Spin at 6000 x g for 15 minutes at 4C<br />
<br />
4. Resuspend pellet in 10 ml P1 Buffer<br />
<br />
5. Add 10 ml P2 Buffer, mix thoroughly, Incubate at room temperature for 5 mins.<br />
<br />
6. Add 10 ml chilled P3 Buffer, mix immediately and thoroughly<br />
<br />
7. Pour into the barrel of the QIAfilter cartridge. Incubate at room temperature for 10 mins.<br />
<br />
8. Remove the cap from the QIAfilter cartridge outlet nozzle, insert the plunger into the QIAfilter Maxi cartridge and filter the cell lysate into a 50 ml tube.<br />
<br />
9. Add 2.5 ml ER Buffer, mix by inverting 10 times, Incubate on ice for 30 mins.<br />
<br />
10. Equilibrate a QIAgen-Tip 500 by applying 10 ml QBT Buffer and allow the column to empty by gravity flow<br />
<br />
11. Apply the filtered lysate from step 9 to the QIAGEN-Tip and allow it to enter the resin by gravity flow.<br />
<br />
12. Wash the QIAGEN-Tip with 30 ml QC Buffer, Repeat QC wash<br />
<br />
13. Elute DNA with 15 ml QN Buffer <br />
<br />
14. Precipitate DNA by adding 10.5 ml room temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥ 15,000 x g for 10 mins. Carefully decant supernatant without disturbing the pellet<br />
<br />
15. Air-dry the pellet for 5-10 min, redissolve the DNA in a suitable volume of endotoxin-free TE Buffer.<br />
<br />
<br><br />
<br />
<br />
===Antibody Staining===<br />
<br />
'''Antibody Staining Reagents:'''<br><br />
Transfected and Untransfected cells of the same cell type.<br><br />
Cells with empty vector or put through electroporation without vector<br><br />
Dylight 488-conjugated AffiniPure Goat anti-mouse IgG, F(ab’)2, <br>Jackson Immuno Research cat# 115-485-072, Lot# 83241 <br>[should be protected from light].<br><br />
Pierce Human IgG, whole molecule, ThermoScientific, Cat# 31154, <br>Lot# LE1311903. (concentration=11mg/ml).<br><br />
D-PBS CMF (Calcium, Magnesium free PBS).<br><br />
Wash Buffer: 0.5% BSA, 0.1% Sodium Azide in D-PBS CMF.<br><br />
FC Block: 1ul Human IgG in 1mL Wash Buffer, prepare fresh for each use.<br><br />
Propidium Iodide(2mg/ml).<br><br />
PI solution: 1ul Propidium Iodide in 1mL D-PBS CMF.<br><br />
<br />
Work in an ice bucket (ethanol the bucket)<br />
<br><br />
<br />
1. Pipette 1ml wash buffer into 1.5ml microcentrifuge tubes. <br />
<br />
2. Add 1x10^5 cells into wash buffer in each tube.<br />
<br />
3. Spin for 1 min for NKL and 2 minutes for CD8+ then aspirate. (Don’t aspirate the pellet!)<br />
<br />
4. Go to step 3, repeat one more time. (Meanwhile, prepare FC Block)<br />
<br />
5. Resuspend in 100ul of FC Block. Icubate in ice for 15 min.<br />
<br />
6. Add 1ul of Goat anti-F(ab’)2 to each tube. Wrap the tube with foil. Incubate in dark for 30 minutes at 4 degrees. For the samples not being labeled, don’t add the Goat antib-F(ab’)2.<br />
<br />
7. Add 1ml wash buffer to the Ab-labeled samples. Repeat step 3, two more times.<br />
<br />
8. Resuspend in 250ul of wash buffer.<br />
<br />
9. Take samples to the FACS machine.<br />
<br />
10. Add 250ul PI solution to the samples the moment before you run the samples.<br />
<br />
===TOPO cloning===<br />
'''Zero Blunt TOPO'''<br />
- Follow manufacturer's protocol<br />
<br />
===Transfection===<br />
'''Jurkat Cell Transfection Protocol'''<br><br />
1. Cultivate the required number of cells for samples.<br><br />
2. Prepare DNA for each sample.<br><br />
3. Pre-warm the supplemented Cell Line Nucleofector® Solution V to room temperature. Either pre-warm an aliquot of culture medium at 37°C in a 50 ml tube (500 μl per sample) or add an additional 500 μl in the step below.<br><br />
4. Prepare 12-well plates by filling appropriate number of wells with 1 ml of culture medium containing supplements and serum. Pre-incubate plates in a humidified37°C/5%CO2 incubator.<br><br />
5. Take an aliquot of cell suspension and count the cells to determine the cell density.<br><br />
6. Centrifuge the required number of cells (1 x 106 cells per nucleofection® sample) at 500xg at room temperature for 5 min. Discard supernatant completely so that noresidual medium covers the cell pellet.<br><br />
7. Resuspend the pellet in room temperature Cell Line Nucleofector® Solution V to a final concentration of 1 x 106 cells/100 μl. Avoid storing the cell suspension longer than 15 min in Nucleofector® Solution as this reduces cell viability and gene transfer efficiency.<br><br />
<br />
'''Important: Steps 8 - 13 should be performed for each sample separately.'''<br><br />
8. Transfer the nucleofection® sample into an amaxa certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the blue cap.<br><br />
9. Select the appropriate Nucleofector® program, X-01/X-001 or X-05/X-005 Insert the cuvette into the cuvette holder and press the “X” button to start the program.<br><br />
10. After the program has finished (display showing "OK") take the cuvette out of the holder and incubate the sample in the cuvette for 10 min at room temperature.<br><br />
11. If in step 3 you pre-warmed culture medium in a 50 ml tube, then after the 10 min incubation step, add 500 μl of this pre-warmed culture medium to the cuvette and transfer the sample into the prepared 12-well plates. If you added an additional 500μl to each well in step 4, then take 500 μl from the well, add to the cuvette, and transfer the sample back into the prepared 12-well plates.To transfer the cells from the cuvettes, we strongly recommend using the plastic pipettes provided in the kit to prevent damage and loss of cells.<br><br />
12. Press the “X” button to reset the Nucleofector®.<br><br />
13. Repeat steps 8 - 13 for the remaining samples. Cultivation after 15.Incubate cells in a humidified 37°C/5% CO2 incubator. Following nucleofection®,nucleofection® gene expression should be analyzed at different times. Depending on the gene, expression is often detectable after 4 - 8 hours. If this is not the case, the incubation period may be prolonged up to 24 hours.<br><br />
<br />
<br />
===FACS===<br />
'''T-Cell Activation (NFAT-GFP readout by FACS)'''<br><br />
Resuspend Jurkat and K562 cells at 1 million live cell/mL in RPMI supplemented with glutamine and 10% FBS (10G RPMI)<br><br />
Add 100ul of each cell into a 96 well plate<br><br />
Incubate overnight<br><br />
Jurkat induction with anti-TCR (anti-CD3 ( clone C305))<br><br />
Resuspend Jurkat at 1 million live cell/mL in FBS supplemented with glutamine and 10% FBS<br><br />
Add 100ul into a well in 96 well plate<br><br />
Add 100ul of 2X induction media (2X C305 = 1:1000 dilution of C305 in 10G RPMI)<br><br />
Incubate overnight<br><br />
FACS<br><br />
<br />
===Imaging===<br />
'''IMAGING PROTOCOL'''<br> <br />
Open up granules and stain with anti-GFP (fixed cells)<br><br />
Pre-warm all solutions/buffers that need to be warm (RPMI-1640, Myelocult, PBS)<br><br />
DiI-labeling<br><br />
1. Count NKLs and spin down 2e6 cells at 400g for 5 minutes <br><br />
2. Resuspend in 2mL of pre-warmed RPMI-1640 (at 1e6/mL).<br><br />
3. Add 10ul DiI stain and mix by swirling tube, etc<br><br />
4. Incubate cells and dye for 10 minutes at 37 C <br><br />
5. Centrifuge cells for 5 minutes at 400g<br><br />
6. Aspirate off supernatant<br><br />
7. Resuspend in 2ml Myelocult and incubate for ~5 minutes at 37C in TC incubator (“recovery”)<br><br><br />
<br />
Lysotracker Blue labeling<br><br />
LB1. Spin down cells at 400g for 5 minutes.<br><br />
LB2. Resuspend at ~1e6/mL in 5uM LysoTracker Blue (diluted into RPMI-1640; note: 1:200 of 1mM stock). Incubate ~30 minutes at 37C in TC incubator.<br><br />
LB3. Centrifuge cells for 5 minutes at 400g<br><br />
LB4. Resuspend in (0.5 ml) Myelocult and incubate for ~5 minutes at 37C in TC incubator<br><br />
Plating cells down on Fibronectin-coated Chambered Coverglass (8-well)<br> <br />
P1. Aspirate myelocult out of each well.<br><br />
P2. Transfer 0.2mL cells to a fibronectin-coated well incubate at 37C for 15-30 minutes <br><br />
P3. Check to make sure cells appear to be stuck (on scope)<br><br />
P4. If not enough cells stuck, add more cells (repeat steps P2-P3).<br><br><br />
<br />
Fix the cells / permeabilize / stain with anti-GFP Alexa647<br><br />
F1. Fix in fixative solution (4% formaldehyde in PBS) for 15 minutes at room temperature with gentle agitation in the dark. Remove the solution.<br><br />
F2. Wash cells twice in PBS for 1 minute each with gentle agitation. Remove PBS. <br><br />
F3. Permeabilize the specimen with Permeabilization solution (0.25% Triton® X-100 in PBS) for 5 minutes at room temperature with gentle agitation in the dark. Remove the solution.<br><br />
F4. Wash cells twice in PBS for 1 minute each with gentle agitation. Remove PBS.<br> <br />
F5. Add Blocking solution (5% FBS in PBS pH 7.4). Incubate for 15 min at room temperature with gentle agitation. <br><br />
F6. ANTIBODY STAINING<br><br />
-Add anti-GFP Alexa 647. (final conc: 10ug/mL, 1:20 overall dilution)<br><br />
-Incubate for 0.5 hour at room temperature with gentle agitation.<br><br />
F7. Decant antibody solution.<br><br />
F8. Wash cells twice in PBS for 2 minutes each with gentle agitation. After the final wash, add PBS+BSA to the sample. <br><br><br />
<br />
Prepare for microscopy<br><br />
10/13 - We’ve tried 1:20 dilution and will stick with it.<br><br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T18:28:46Z<p>Maven: /* References */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here] '''Sam Do you have a table here'''<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
<br />
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===Graphic showing the main assays used to show T cell Activation===<br />
<br />
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<br />
===AarI Digest===<br />
1. Label PCR tubes.<br><br />
2. Add the following reagents into the PCR tubes:<br><br />
'''Aar1 Digest Reagents:'''<br><br />
5 ug DNA<br><br />
2.5ul Aar1 Enzyme<br><br />
0.9ul Aarl oligo<br><br />
6 ul 10x Aar1 Buffer<br><br />
x ul dH2O<br><br />
60 ul total reaction<br><br />
''Note: x ul dH2O may change in volume for different reactions in order to bring total volume up to 60 ul.''<br />
<br />
3. Briefly vortex and spin down the reaction.<br><br />
4. Incubate reaction at 37C for 3 hours.<br />
<br />
''Note: PCR program can be set to 37C for 3 hours and cooled down to 4C indefinitely.''<br />
<br />
<br><br />
<br />
<br />
===PCR with Phusion enzyme===<br />
1. Label PCR tubes.<br><br />
2. Add the following reagents into the PCR tubes (in order):<br><br />
'''PCR Reagents'''<br><br />
23.7 ul dH2O<br><br />
10 ul 5x HF Buffer<br><br />
5 ul forward primer<br><br />
5 ul reverse primer<br><br />
5 ul dNTP<br><br />
0.3 ul template<br><br />
1 ul Phusion<br><br />
50 ul total<br> <br />
3. Vortex and spin down the reaction.<br><br />
4. Set up PCR program.<br><br />
<br />
{|cellspacing="0"<br />
|'''Cycle Step'''||'''Temperature'''||'''Time'''||'''# Cycles'''<br />
|+<br />
|Initial Denature||98C||3min||1<br />
|-<br />
|Denature<br>Annealing<br>Extension||98C<br>55C<br>72C||10s<br>30s<br>30s||30<br />
|-<br />
|Final Extension||72C||5min||1<br />
|-<br />
|Hold||4C|| || <br />
|}<br />
<br />
''Note: Times may differ for different reactions based on the size of the desired PCR product. Annealing temperature may also differ depending on primer properties.''<br />
<br />
<br />
<br><br />
===Ligation===<br />
''' Ligation Reagents:'''<br><br />
50 ng vector<br><br />
DNA insert(s)<br><br />
Buffer<br><br />
1 ul Ligase<br><br />
<br />
'''Ligation Reaction:'''<br><br />
1. Calculate amount of DNA insert and vector needed for reaction.<br><br />
150 ng / ( # bp in backbone/ # bp in insert) = ng needed<br><br />
Concentration of insert or vector divided by ng needed = volume of DNA needed<br />
<br />
2. Calculate amount of buffer needed depending on concentration for volume needed. A typical ligation reaction volume can be 20 ul.<br />
<br />
3. Add reagents together, from smallest volumes to largest. Distilled water can be used to bring the volume up so that the reaction has the proper buffer concentration for the reaction. Ligase or enzymes in general should be added at the end.<br />
<br />
4. Vortex or pipet up and down to mix. Spin down afterwards if vortexing.<br />
<br />
5. Let reaction sit at room temperature for 5 to 20 minutes depending on ligase used. The ligase may start acting up if left too long.<br />
<br />
<br><br />
===Agarose Gel Electrophoresis===<br />
'''Gel Electrophoresis Reagents/Materials:'''<br><br />
Gel caster<br><br />
Gel tray<br><br />
Agar powder<br><br />
TAE buffer<br><br />
10, 000X Sybr Safe<br><br />
50ml conical tube<br><br />
Gel comb<br><br />
Microwave<br><br />
Large flask (microwave safe)<br><br />
<br />
'''Preparing the agarose solution:'''<br />
<br />
1. Pour in flask the amount of TAE buffer desired. This will be the volume of agarose solution made.<br />
<br />
2. Measure out the amount of agar needed for desired concentration. For example to get a 1% agarose gel, you would add 1 gram of agarose powder to 100 ml of TAE buffer. Most concentrations commonly fall between 0.7- 2%. Higher concentrations of agarose are better suited for small fragment separation while lower concentrations are suitable for large fragments.<br />
<br />
3. Microwave three minutes, may vary depending on volume in flask. Make sure cap is on loosely.<br />
<br />
4. Using hot gloves, swirl bottle gently to ensure complete dissolving of agar. The solution should be clear when agar is completely dissolved. If it is not, microwave for another minute.<br />
<br />
5. Place flask of agarose solution in 80C bath to store as to prevent solidifying of agarose solution.<br />
<br />
<br />
'''Casting gel:'''<br />
<br />
1. Secure gel tray in caster<br />
<br />
2. Pour desired volume of premade agarose solution into 50 ml conical tube. Volume desired will vary depending on gel tray size and desired thickness of gel. Thin gels solidify quicker and can show faint bands clearer, but the wells of thicker gels can hold more DNA.<br />
<br />
3. Add Sybr safe to agarose solution. For 10 ml of agarose solution, add 1ul Sybr Safe. The Sybr safe is used to stain and visualize the DNA when viewed under a blue light.<br />
<br />
4. Invert tube several times until Sybr safe is equally distributed. Invert gently to avoid creating air bubbles.<br />
<br />
5. Pour contents of conical tube into secured gel tray.<br />
<br />
6. Place gel comb into notches of gel tray. Check that comb is evenly submerged in agarose. The comb will form wells in the gel to load DNA.<br />
<br />
7. Dry at room temperature. The gel will appear opaque when done.<br />
<br />
===Colony PCR===<br />
'''Colony PCR reagents:'''<br><br />
12ul dH20<br><br />
4 ul 5x gotaq green buffer<br><br />
2 ul 10mM dntp<br><br />
1 ul forward primer<br><br />
1 ul reverse primer<br><br />
0.1 ul GoTaq Polymerase<br><br />
<br />
1. After adding reagents to PCR tube, pick a colony and touch the bottom of the PCR tube with the pipette.<br />
<br />
2. Place PCR tubes into the PCR machine and set the cycling parameters to be optimal for the piece of DNA being copied.<br />
<br />
<br><br />
===Transformation of E. coli===<br />
'''Transformation Reagents'''<br><br />
LB Plates<br><br />
Ice box<br><br />
Water bath<br><br />
37C Incubator<br><br />
Competent cells & Plasmid<br><br />
Spreading tool (beads)<br><br />
<br />
1. Prewarm LB plates in the 37C incubator<br />
<br />
2. Remove desired cells from -80C freezer (or other storage location) and melt over ice for at least 10 minutes. (Make sure to aliquot the correct amount of cells for transformation)<br />
<br />
3. Pipette up 5ul of your plasmid DNA and gently pipette into the suspended cells. Do not mix by triterating the solution (pipetting up and down).<br />
<br />
4. Let the cells sit on ice for 15-30 minutes.<br />
<br />
5. Heat shock your cells for 75 seconds in a 42C water bath.<br />
<br />
6. Quickly return your tubes to the ice bucket for 2-3 minutes.<br />
<br />
7. When you are ready to plate, remove your prewarmed plate from the incubator. Using sterile technique, carefully pipette your entire tube of cells onto the LB plate.<br />
<br />
8. Again using sterile technique, spread the cells across your plate using either sterile beads or another spreading tool.<br />
<br />
9. Put the plate in the 37C incubator for 5-15 hours.<br />
<br />
===Gel Extraction/Purification of DNA===<br />
<br />
'''Gel Extraction/Purification Materials & Reagents:'''<br><br />
Sterile Blade<br><br />
2ml microcentrifuge tube<br><br />
1.5ml microcentrifuge tube<br><br />
QIAGEN Gel Extraction Kit<br><br />
Centrifuge<br><br />
Heat Block/Bath<br><br />
<br />
1. Using a sterile blade, carefully excise the desired band from your gel.<br />
<br />
2. Transfer the agarose chunk into a new 2ml microcentrifuge tube.<br />
<br />
3. Weigh the excised agarose chunk and add 3 times its weight in microliters of QG buffer.<br />
<br />
4. Move the tube to a melting block for 10 minutes at 50C or until the agarose is completely melted.<br />
<br />
5. Add isopropanol to the tube at a 1:3 ratio with QG, then vortex the solution briefly to help precipitate the DNA.<br />
<br />
6. Transfer the solution to a Qiagen gel extraction/PCR purification spin column.<br />
<br />
7. Spin for 1 minute at 13,000rpm and dump the supernatant.<br />
<br />
8. Add 500μl of QG buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum the flowthrough.<br />
<br />
9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum flowthrough.<br><br />
''NOTE: Wash the rim with the buffer.''<br />
<br />
10. Repeat Step 9<br />
<br />
11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum)<br />
<br />
12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column.<br />
<br />
13. Place tubes in a -20oC freezer box.<br />
<br />
<br />
<br><br />
===Miniprep===<br />
'''Miniprep Materials'''<br><br />
Qiagen Miniprep Kit<br><br />
Centrifuge<br><br />
<br />
1. Spin down bacterial culture at 3500 rpm for 5 minutes<br />
<br />
2. Aspirate supernatant. Change tips for every sample.<br />
<br />
3. Resuspend in 250μl of P1 Buffer. Vortex until cell pellet is not visible. Transfer into a 1.5 ml tube.<br />
<br />
4. Add 250μl of P2 Buffer. Invert 5-6 times. Wait 3-5 minutes.<br><br />
''NOTE: When you open the cap, you should see a string of DNA.''<br />
<br />
5. Add 350μl of N3 Buffer. Invert 5-6 times or until colorless<br />
<br />
6. Centrifuge at 13,000 rpm for 10 minutes. Label blue spin columns. Transfer supernatant into blur spin columns.<br />
<br />
7. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant.<br />
<br />
8. Add 500μl of PB buffer. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant.<br />
<br />
9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum supernatant.<br><br />
''NOTE: Wash the rim with the buffer.''<br />
<br />
10. Repeat Step 9<br />
<br />
11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum)<br />
<br />
12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column.<br />
<br />
13. Place tubes in a -20oC freezer box.<br />
<br />
<br><br />
===Maxiprep===<br />
<br />
'''Maxiprep Materials & Reagents'''<br><br />
250ml centrifuge tubes<br><br />
Isopropanol<br><br />
QIAGEN Maxiprep Stand<br><br />
QIAGEN Maxiprep Kit<br><br />
Large Centrifuge<br><br />
50ml conical tubes<br><br />
100% Ethanol<br><br />
<br />
1. Pick a single colony and inoculate in 2-5 ml LB medium containing the appropriate antibiotic, Incubate for around 8 hrs at 37C with vigorous shaking.<br />
<br />
2. Dilute 1/500 to 1/1000 into selective LB medium. For high copy plasmids, inoculate 100 ml medium with 100-200μl of starter culture. For low copy plasmids, inoculate 250 ml medium with 250-500μl of starter culture. Grow at 37C for 12-16 hrs with vigorous shaking.<br />
<br />
3. Spin at 6000 x g for 15 minutes at 4C<br />
<br />
4. Resuspend pellet in 10 ml P1 Buffer<br />
<br />
5. Add 10 ml P2 Buffer, mix thoroughly, Incubate at room temperature for 5 mins.<br />
<br />
6. Add 10 ml chilled P3 Buffer, mix immediately and thoroughly<br />
<br />
7. Pour into the barrel of the QIAfilter cartridge. Incubate at room temperature for 10 mins.<br />
<br />
8. Remove the cap from the QIAfilter cartridge outlet nozzle, insert the plunger into the QIAfilter Maxi cartridge and filter the cell lysate into a 50 ml tube.<br />
<br />
9. Add 2.5 ml ER Buffer, mix by inverting 10 times, Incubate on ice for 30 mins.<br />
<br />
10. Equilibrate a QIAgen-Tip 500 by applying 10 ml QBT Buffer and allow the column to empty by gravity flow<br />
<br />
11. Apply the filtered lysate from step 9 to the QIAGEN-Tip and allow it to enter the resin by gravity flow.<br />
<br />
12. Wash the QIAGEN-Tip with 30 ml QC Buffer, Repeat QC wash<br />
<br />
13. Elute DNA with 15 ml QN Buffer <br />
<br />
14. Precipitate DNA by adding 10.5 ml room temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥ 15,000 x g for 10 mins. Carefully decant supernatant without disturbing the pellet<br />
<br />
15. Air-dry the pellet for 5-10 min, redissolve the DNA in a suitable volume of endotoxin-free TE Buffer.<br />
<br />
<br><br />
<br />
<br />
===Antibody Staining===<br />
<br />
'''Antibody Staining Reagents:'''<br><br />
Transfected and Untransfected cells of the same cell type.<br><br />
Cells with empty vector or put through electroporation without vector<br><br />
Dylight 488-conjugated AffiniPure Goat anti-mouse IgG, F(ab’)2, <br>Jackson Immuno Research cat# 115-485-072, Lot# 83241 <br>[should be protected from light].<br><br />
Pierce Human IgG, whole molecule, ThermoScientific, Cat# 31154, <br>Lot# LE1311903. (concentration=11mg/ml).<br><br />
D-PBS CMF (Calcium, Magnesium free PBS).<br><br />
Wash Buffer: 0.5% BSA, 0.1% Sodium Azide in D-PBS CMF.<br><br />
FC Block: 1ul Human IgG in 1mL Wash Buffer, prepare fresh for each use.<br><br />
Propidium Iodide(2mg/ml).<br><br />
PI solution: 1ul Propidium Iodide in 1mL D-PBS CMF.<br><br />
<br />
Work in an ice bucket (ethanol the bucket)<br />
<br><br />
<br />
1. Pipette 1ml wash buffer into 1.5ml microcentrifuge tubes. <br />
<br />
2. Add 1x10^5 cells into wash buffer in each tube.<br />
<br />
3. Spin for 1 min for NKL and 2 minutes for CD8+ then aspirate. (Don’t aspirate the pellet!)<br />
<br />
4. Go to step 3, repeat one more time. (Meanwhile, prepare FC Block)<br />
<br />
5. Resuspend in 100ul of FC Block. Icubate in ice for 15 min.<br />
<br />
6. Add 1ul of Goat anti-F(ab’)2 to each tube. Wrap the tube with foil. Incubate in dark for 30 minutes at 4 degrees. For the samples not being labeled, don’t add the Goat antib-F(ab’)2.<br />
<br />
7. Add 1ml wash buffer to the Ab-labeled samples. Repeat step 3, two more times.<br />
<br />
8. Resuspend in 250ul of wash buffer.<br />
<br />
9. Take samples to the FACS machine.<br />
<br />
10. Add 250ul PI solution to the samples the moment before you run the samples.<br />
<br />
===TOPO cloning===<br />
'''Zero Blunt TOPO'''<br />
- Follow manufacturer's protocol<br />
<br />
===Transfection===<br />
'''Jurkat Cell Transfection Protocol'''<br><br />
1. Cultivate the required number of cells for samples.<br><br />
2. Prepare DNA for each sample.<br><br />
3. Pre-warm the supplemented Cell Line Nucleofector® Solution V to room temperature. Either pre-warm an aliquot of culture medium at 37°C in a 50 ml tube (500 μl per sample) or add an additional 500 μl in the step below.<br><br />
4. Prepare 12-well plates by filling appropriate number of wells with 1 ml of culture medium containing supplements and serum. Pre-incubate plates in a humidified37°C/5%CO2 incubator.<br><br />
5. Take an aliquot of cell suspension and count the cells to determine the cell density.<br><br />
6. Centrifuge the required number of cells (1 x 106 cells per nucleofection® sample) at 500xg at room temperature for 5 min. Discard supernatant completely so that noresidual medium covers the cell pellet.<br><br />
7. Resuspend the pellet in room temperature Cell Line Nucleofector® Solution V to a final concentration of 1 x 106 cells/100 μl. Avoid storing the cell suspension longer than 15 min in Nucleofector® Solution as this reduces cell viability and gene transfer efficiency.<br><br />
<br />
'''Important: Steps 8 - 13 should be performed for each sample separately.'''<br><br />
8. Transfer the nucleofection® sample into an amaxa certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the blue cap.<br><br />
9. Select the appropriate Nucleofector® program, X-01/X-001 or X-05/X-005 Insert the cuvette into the cuvette holder and press the “X” button to start the program.<br><br />
10. After the program has finished (display showing "OK") take the cuvette out of the holder and incubate the sample in the cuvette for 10 min at room temperature.<br><br />
11. If in step 3 you pre-warmed culture medium in a 50 ml tube, then after the 10 min incubation step, add 500 μl of this pre-warmed culture medium to the cuvette and transfer the sample into the prepared 12-well plates. If you added an additional 500μl to each well in step 4, then take 500 μl from the well, add to the cuvette, and transfer the sample back into the prepared 12-well plates.To transfer the cells from the cuvettes, we strongly recommend using the plastic pipettes provided in the kit to prevent damage and loss of cells.<br><br />
12. Press the “X” button to reset the Nucleofector®.<br><br />
13. Repeat steps 8 - 13 for the remaining samples. Cultivation after 15.Incubate cells in a humidified 37°C/5% CO2 incubator. Following nucleofection®,nucleofection® gene expression should be analyzed at different times. Depending on the gene, expression is often detectable after 4 - 8 hours. If this is not the case, the incubation period may be prolonged up to 24 hours.<br><br />
<br />
<br />
===FACS===<br />
'''T-Cell Activation (NFAT-GFP readout by FACS)'''<br><br />
Resuspend Jurkat and K562 cells at 1 million live cell/mL in RPMI supplemented with glutamine and 10% FBS (10G RPMI)<br><br />
Add 100ul of each cell into a 96 well plate<br><br />
Incubate overnight<br><br />
Jurkat induction with anti-TCR (anti-CD3 ( clone C305))<br><br />
Resuspend Jurkat at 1 million live cell/mL in FBS supplemented with glutamine and 10% FBS<br><br />
Add 100ul into a well in 96 well plate<br><br />
Add 100ul of 2X induction media (2X C305 = 1:1000 dilution of C305 in 10G RPMI)<br><br />
Incubate overnight<br><br />
FACS<br><br />
<br />
===Imaging===<br />
'''IMAGING PROTOCOL'''<br> <br />
Open up granules and stain with anti-GFP (fixed cells)<br><br />
Pre-warm all solutions/buffers that need to be warm (RPMI-1640, Myelocult, PBS)<br><br />
DiI-labeling<br><br />
1. Count NKLs and spin down 2e6 cells at 400g for 5 minutes <br><br />
2. Resuspend in 2mL of pre-warmed RPMI-1640 (at 1e6/mL).<br><br />
3. Add 10ul DiI stain and mix by swirling tube, etc<br><br />
4. Incubate cells and dye for 10 minutes at 37 C <br><br />
5. Centrifuge cells for 5 minutes at 400g<br><br />
6. Aspirate off supernatant<br><br />
7. Resuspend in 2ml Myelocult and incubate for ~5 minutes at 37C in TC incubator (“recovery”)<br><br><br />
<br />
Lysotracker Blue labeling<br><br />
LB1. Spin down cells at 400g for 5 minutes.<br><br />
LB2. Resuspend at ~1e6/mL in 5uM LysoTracker Blue (diluted into RPMI-1640; note: 1:200 of 1mM stock). Incubate ~30 minutes at 37C in TC incubator.<br><br />
LB3. Centrifuge cells for 5 minutes at 400g<br><br />
LB4. Resuspend in (0.5 ml) Myelocult and incubate for ~5 minutes at 37C in TC incubator<br><br />
Plating cells down on Fibronectin-coated Chambered Coverglass (8-well)<br> <br />
P1. Aspirate myelocult out of each well.<br><br />
P2. Transfer 0.2mL cells to a fibronectin-coated well incubate at 37C for 15-30 minutes <br><br />
P3. Check to make sure cells appear to be stuck (on scope)<br><br />
P4. If not enough cells stuck, add more cells (repeat steps P2-P3).<br><br><br />
<br />
Fix the cells / permeabilize / stain with anti-GFP Alexa647<br><br />
F1. Fix in fixative solution (4% formaldehyde in PBS) for 15 minutes at room temperature with gentle agitation in the dark. Remove the solution.<br><br />
F2. Wash cells twice in PBS for 1 minute each with gentle agitation. Remove PBS. <br><br />
F3. Permeabilize the specimen with Permeabilization solution (0.25% Triton® X-100 in PBS) for 5 minutes at room temperature with gentle agitation in the dark. Remove the solution.<br><br />
F4. Wash cells twice in PBS for 1 minute each with gentle agitation. Remove PBS.<br> <br />
F5. Add Blocking solution (5% FBS in PBS pH 7.4). Incubate for 15 min at room temperature with gentle agitation. <br><br />
F6. ANTIBODY STAINING<br><br />
-Add anti-GFP Alexa 647. (final conc: 10ug/mL, 1:20 overall dilution)<br><br />
-Incubate for 0.5 hour at room temperature with gentle agitation.<br><br />
F7. Decant antibody solution.<br><br />
F8. Wash cells twice in PBS for 2 minutes each with gentle agitation. After the final wash, add PBS+BSA to the sample. <br><br><br />
<br />
Prepare for microscopy<br><br />
10/13 - We’ve tried 1:20 dilution and will stick with it.<br><br />
<br />
<br />
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<br />
__TOC__<br />
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here] '''Sam Do you have a table here'''<br />
<br />
Here are the visual results for each sample:<br />
<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T18:13:22Z<p>Maven: </p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg]]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T18:11:40Z<p>Maven: </p>
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<html><br />
<p><br />
Here are the visual results for each sample:<br />
<br />
[[Image:UCSF_56003.jpg|left|200px]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg|left|200px]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[[Image:UCSF_61001.jpg|left|200px]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[[Image:UCSF_61002.jpg|left|200px]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg|left|200px]]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
</p><br />
</html><br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T18:10:47Z<p>Maven: </p>
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cellspacing:0px;<br />
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<p><br />
[[Image:UCSF_56003.jpg|left|200px]]<br />
</p><br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg|left|200px]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[[Image:UCSF_61001.jpg|left|200px]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[[Image:UCSF_61002.jpg|left|200px]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg|left|200px]]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T18:09:31Z<p>Maven: </p>
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==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[[Image:UCSF_56003.jpg|left|200px]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg|left|200px]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[[Image:UCSF_61001.jpg|left|200px]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[[Image:UCSF_61002.jpg|left|200px]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg|left|200px]]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T18:08:39Z<p>Maven: </p>
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[[Image:UCSF_56003.jpg|left]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg|left|60px]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[[Image:UCSF_61001.jpg|left]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[[Image:UCSF_61002.jpg|left]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg|left]]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
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<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T18:07:51Z<p>Maven: </p>
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[[Image:UCSF_56003.jpg|left]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg|left]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[[Image:UCSF_61001.jpg|left]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[[Image:UCSF_61002.jpg|left]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[[Image:UCSF_pMax_GFP.jpg|left]]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[[Image:UCSF_56003.jpg]]<br />
<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[[Image:UCSF_59002.jpg|left]]<br />
<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[[Image:UCSF_61001.jpg]]<br />
<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[[Image:UCSF_61002.jpg]]<br />
<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[Insert pMAX GFP]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
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{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/File:UCSF_61001.jpgFile:UCSF 61001.jpg2010-10-27T18:05:11Z<p>Maven: </p>
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<div></div>Mavenhttp://2010.igem.org/File:UCSF_61002.jpgFile:UCSF 61002.jpg2010-10-27T18:03:14Z<p>Maven: </p>
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<div></div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:57:53Z<p>Maven: /* References */</p>
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<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[insert 56003]<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[insert 59002]<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[Insert 60001]<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[Insert 61002]<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[Insert pMAX GFP]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''References for sorting signals:'''<br />
<br />
<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:57:02Z<p>Maven: /* References */</p>
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==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[insert 56003]<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[insert 59002]<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[Insert 60001]<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[Insert 61002]<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[Insert pMAX GFP]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
'''1. L is for lytic granules: lysosomes that kill'''<br />
<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
<br />
'''2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.'''<br />
<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
<br />
'''3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.'''<br />
<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
<br />
'''4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).'''<br />
<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
<br />
'''5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.'''<br />
<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
<br />
'''6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
'''7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.'''<br />
<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
<br />
<br />
'''references for sorting signals:'''<br />
<br />
'''Sorting of lysosomal proteins'''<br />
<br />
Thomas Braulke and Juan S. Bonifacino<br />
<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
<br />
'''Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.'''<br />
<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:54:33Z<p>Maven: </p>
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells' granules by fusing "address" tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal "address tag"sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
===Results===<br />
<br />
<br />
We were able to test six of our devices in the assay: pCPL_056, pCPL_059, pCPL_060, pCPL_061, pCPL_063, and pCPL_067.<br />
<br />
We also had two negative controls for comparison:<br />
<br />
<br />
'''NKL (untransfected control):'''<br />
<br />
We analyzed this condition because we wanted to have a negative control for auto-fluorescence in the cell line. However, we had some problems because the background staining intensity of the fluorescent anti-GFP antibody was different from the experimental samples. We hypothesized that this was because of the different health of these cells, which had not been growing under G418 selection.<br />
<br />
<br />
'''pMAX GFP:'''<br />
<br />
This control is the same cell line (NKL) transfected with the pMAX-GFP plasmid. pMAX-GFP is a powerful expression vector for GFP available from Lonza. Because it only expresses GFP, and doesn’t contain the N-terminal granzyme signals or the C-terminal sorting tags, the GFP will not be sent to the granules. The purpose of this control is to illustrate the difference between GFP left in the cytosol and GFP localized into the granules.<br />
<br />
<br />
'''Complications / troubleshooting in the assay'''<br />
<br />
- At times, staining with the anti-GFP antibody did not appear to be uniform between all of the different samples. This was likely a technical error and results probably would be improved with more practice.<br />
- Staining the granules with the lysosomal stain Lysotracker Blue (Invitrogen Molecular Probes) was often problematic. Most cells appeared to have high levels of non-specific background staining in the cytoplasm, especially after the cells were fixed and permeabilized. In addition, we were told that fluorescent dyes in the blue part of the light spectrum are often more challenging to visualize (more noise). However, we were able to observe reasonable Lysotracker Blue staining in some of our images.<br />
<br />
In some of our images, we had to independently adjust the LUTs settings for the Lysotracker to visualize the staining in some of our images (noted in the results tables below). We performed the image processing function “Smoothing” (NIS-Elements AR software) on all of our images (identical settings for all images).<br />
<br />
We were able to observe evidence of granular anti-GFP staining and workable granule staining in samples pCPL_056, pCPL_059, pCPL_060, and pCPL_061. (See summary table below.) In some samples, the granular anti-GFP staining and granule staining appear to be well-colocalized, suggesting that EGFP was loaded into the killer cell granules.<br />
<br />
[insert table here]<br />
<br />
Here are the visual results for each sample:<br />
<br />
[insert 56003]<br />
This is our best result. The anti-GFP stain (green) is well colocalized with the Granule stain (red). It also appears to be well centralized in the cell which seems to show that it is stably located in the correct granules, since it is not being sent out of the cell.<br />
<br />
<br />
[insert 59002]<br />
In this image we can see granular looking anti-GFP staining (green). However, it is located near the cell membrane and does not seem to overlap with the granule stain (red).<br />
<br />
[Insert 60001]<br />
This image also shows well colocalized anti-GFP (green) and granules (red) (the yellow part represnts the heavily overlapped area) the results here are less clear than in image 56003, but nevertheless show centralized, colocalized anti-GFP and Granule stain<br />
<br />
[Insert 61002]<br />
As you can see in this image, the granular anti-GFP is mostly at the edge of the membrane. The granule stain seems to slightly overlap the anti-GFP in some places, but is largely not colocalized with it. Since this construct only has the granzyme signal sequence we hypothesized that it would serve as a control for localization.<br />
<br />
<br />
[Insert pMAX GFP]<br />
In this image we have stained the pMAX-GFP with anti-GFP. As you can see, since the GFP is expressed throughout the cell, the stain is also everywhere. Unlike the EGFP constructs, this image has no granular GFP, but rather an entirely lite up cell.<br />
<br />
<br />
===Future application===<br />
<br />
We are encouraged by our preliminary results suggesting that we can successfully target the model protein EGFP into killer cell granules. With a few more tests we can fully optimize the process and figure out what combination of N- and C- terminal address tags work the best. Then, we can use the best combinations of address tags to route novel cytotoxic cargo into the granules for cancer killing. Some of the various ideas we have explored for cargo that we could load into granules include: proteins important for normal regulation such as p53, and the cytochrome and BL family proteins; Stronger killing agents, such as TNF and Par-4.<br />
We can also use it as a new means for delivering existing cancer therapies, such as growth inhibitors, with more specificity. Once this process has been further investigated and fine tuned, it will offer a new revolutionary means of combating cancer and creating more powerful killer cells.<br />
<br />
<br />
===References===<br />
<br />
<br />
<br />
1. L is for lytic granules: lysosomes that kill<br />
Page LJ, Darmon AJ, Uellner R, Griffiths GM.<br />
Biochim Biophys Acta. 1998 Feb 4;1401(2):146-56.<br />
http://www.ncbi.nlm.nih.gov/pubmed/9531970<br />
<br />
2. The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.<br />
Lefrancois S, Zeng J, Hassan AJ, Canuel M, Morales CR.<br />
EMBO J. 2003 Dec 15;22(24):6430-7.<br />
http://www.ncbi.nlm.nih.gov/pubmed/14657016<br />
<br />
3. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.<br />
Reczek D, Schwake M, Schr?der J, Hughes H, Blanz J, Jin X, Brondyk W, Van Patten S, Edmunds T, Saftig P.<br />
Cell. 2007 Nov 16;131(4):770-83.<br />
http://www.cell.com/abstract/S0092-8674%2807%2901290-1<br />
<br />
4. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).<br />
Hiesberger T, Hüttler S, Rohlmann A, Schneider W, Sandhoff K, Herz J.<br />
EMBO J. 1998 Aug 17;17(16):4617-25.<br />
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170791/<br />
<br />
5. Granzymes A and B Are Targeted to the Lytic Granules of Lymphocytes by the Mannose-6-Phosphate Receptor<br />
Griffiths GM, Isaaz S.<br />
J Cell Biol. 1993 Feb;120(4):885-96.<br />
http://www.ncbi.nlm.nih.gov/pubmed/8432729<br />
<br />
reference for how granzymes work: http://genomebiology.com/2001/2/12/reviews/3014<br />
reference for how signal peptides work:<br />
http://www.signalpeptide.de/<br />
<br />
6. Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
<br />
7. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.<br />
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS.<br />
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3083-8. Epub 2004 Feb 19.<br />
http://www.ncbi.nlm.nih.gov/pubmed/14976248<br />
<br />
references for sorting signals:<br />
<br />
Sorting of lysosomal proteins<br />
Thomas Braulke and Juan S. Bonifacino<br />
doi:10.1016/j.bbamcr.2008.10.016<br />
www.cimr.cam.ac.uk/investigators/robinson/docs/Bonifacino%2009.pdf<br />
<br />
Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.<br />
Hansson M, Gao Y, Rosén H, Tapper H, Olsson I.<br />
Eur Cytokine Netw. 2004 Jul-Sep;15(3):167-76.<br />
http://www.ncbi.nlm.nih.gov/pubmed/15542440<br />
<br />
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{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:50:20Z<p>Maven: /* Cloning Strategy */</p>
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the <html><a href="http://dspace.mit.edu/bitstream/handle/1721.1/46721/BBFRFC28.pdf?sequence=1">BBF RFC 28</a></html> strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:48:22Z<p>Maven: </p>
<hr />
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#left table, #left td, #left tr{<br />
cellspacing:0px;<br />
cellpadding:0px;<br />
border:1px black solid;<br />
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text-align:center;<br />
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{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br><br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
<br />
<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:46:58Z<p>Maven: </p>
<hr />
<div><html><br />
<br />
<style><br />
#left table, #left td, #left tr{<br />
cellspacing:0px;<br />
cellpadding:0px;<br />
border:1px black solid;<br />
margin-left:35px;<br />
text-align:center;<br />
}<br />
</style><br />
<script><br />
var Numbering="N3 N3-4";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:46:12Z<p>Maven: </p>
<hr />
<div><html><br />
<br />
<style><br />
#left table, #left td, #left tr{<br />
cellspacing:0px;<br />
cellpadding:0px;<br />
border:1px black solid;<br />
margin-left:35px;<br />
text-align:center;<br />
}<br />
</style><br />
<script><br />
var Numbering="N3 N3-4";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:44:22Z<p>Maven: </p>
<hr />
<div><html><br />
<br />
<style><br />
#left table, #left td, #left tr{<br />
cellspacing:0px;<br />
cellpadding:0px;<br />
border:1px black solid;<br />
margin-left:35px;<br />
text-align:center;<br />
}<br />
</style><br />
<script><br />
var Numbering="N3 N3-4";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
<br />
'''1. N-terminal signal peptides'''<br />
<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
<br />
'''Strategy 1'''<br />
<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
<br />
<br />
'''Y-motif:'''<br />
<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
<br />
<br />
'''Strategy 2'''<br />
<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
<br />
'''GILT:'''<br />
<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
<br />
Signal sequences are made into AB parts<br />
<br />
the cargo (GFP) is made into BC part<br />
<br />
and all the granule localization adress tags are made into CD parts.<br />
<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:41:02Z<p>Maven: </p>
<hr />
<div><html><br />
<br />
<style><br />
#left table, #left td, #left tr{<br />
cellspacing:0px;<br />
cellpadding:0px;<br />
border:1px black solid;<br />
margin-left:35px;<br />
text-align:center;<br />
}<br />
</style><br />
<script><br />
var Numbering="N3 N3-4";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted <html><a href="#References">[1]</a></html>. Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;<html><a href="#References">[2]</a></html><html><a href="#References">[3]</a></html><html><a href="#References">[4]</a></html>the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway<html><a href="#References">[5]</a></html>.The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
'''1. N-terminal signal peptides'''<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
'''Strategy 1'''<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
'''Y-motif:'''<br />
In a previous work<html><a href="#References">[6]</a></html>, scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXXØ motif (X represents any Amino Acid, Ø stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
'''Strategy 2'''<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR<html><a href="#References">[7]</a></html>.<br />
<br />
'''GILT:'''<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
Signal sequences are made into AB parts<br />
the cargo (GFP) is made into BC part<br />
and all the granule localization adress tags are made into CD parts.<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:34:12Z<p>Maven: </p>
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{{Template:UCSF/BannerAndNav}}<br />
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<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
<br /><br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted [1]. <br />
<br />
Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;[2][3][4]the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway[5].The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
'''1. N-terminal signal peptides'''<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
'''Strategy 1'''<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
'''Y-motif:'''<br />
In a previous work[6], scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXX? motif (X represents any Amino Acid, ? stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
'''Strategy 2'''<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR. [7].<br />
<br />
'''GILT:'''<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
Signal sequences are made into AB parts<br />
the cargo (GFP) is made into BC part<br />
and all the granule localization adress tags are made into CD parts.<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:33:34Z<p>Maven: </p>
<hr />
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}<br />
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{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
<br /><br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted [1]. <br />
<br />
Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;[2][3][4]the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway[5].The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
'''1. N-terminal signal peptides'''<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
'''Strategy 1'''<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
'''Y-motif:'''<br />
In a previous work[6], scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXX? motif (X represents any Amino Acid, ? stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
'''Strategy 2'''<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR. [7].<br />
<br />
'''GILT:'''<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
Signal sequences are made into AB parts<br />
the cargo (GFP) is made into BC part<br />
and all the granule localization adress tags are made into CD parts.<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:32:26Z<p>Maven: </p>
<hr />
<div><html><br />
<script><br />
var Numbering="N3 N3-4";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
<br /><br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted [1]. <br />
<br />
Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;[2][3][4]the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway[5].The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
'''1. N-terminal signal peptides'''<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
'''Strategy 1'''<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
'''Y-motif:'''<br />
In a previous work[6], scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXX? motif (X represents any Amino Acid, ? stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
'''Strategy 2'''<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR. [7].<br />
<br />
'''GILT:'''<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
Signal sequences are made into AB parts<br />
the cargo (GFP) is made into BC part<br />
and all the granule localization adress tags are made into CD parts.<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:30:08Z<p>Maven: </p>
<hr />
<div><html><br />
<script><br />
var Numbering="N3 N3-4";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
<br /><br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br /><br />
<br />
'''Goal:'''<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br /><br />
<br />
'''Approach:'''<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted [1]. <br />
<br />
Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;[2][3][4]the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway[5].The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags''===<br />
<br />
''Concept and experimental design''<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
''1. N-terminal signal peptides''<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
''2. C-terminal “address tag” sequences''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
''Strategy 1''<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
''Y-motif:''<br />
In a previous work[6], scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXX? motif (X represents any Amino Acid, ? stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
''MPR:''<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
''LIMP-II & LAMP1:''<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
''Sortilin:''<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
''Strategy 2''<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR. [7].<br />
<br />
''GILT:''<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
Signal sequences are made into AB parts<br />
the cargo (GFP) is made into BC part<br />
and all the granule localization adress tags are made into CD parts.<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
<br />
<br />
<br />
==='''Better Arsenal'''===<br />
<br />
'''Goal:''' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
'''Approach:''' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted [1]. <br />
<br />
Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;[2][3][4]the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway[5].The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags===<br />
<br />
'''Concept and experimental design'''<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
'''1. N-terminal signal peptides'''<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
'''2. C-terminal “address tag” sequences'''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
'''Strategy 1'''<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
'''Y-motif:'''<br />
In a previous work[6], scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXX? motif (X represents any Amino Acid, ? stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
'''MPR:'''<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
'''LIMP-II & LAMP1:'''<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
'''Sortilin:'''<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
'''Strategy 2'''<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR. [7].<br />
<br />
'''GILT:'''<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
Signal sequences are made into AB parts<br />
the cargo (GFP) is made into BC part<br />
and all the granule localization adress tags are made into CD parts.<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|''''#''''||''''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]''''||''''Plasmid Name''''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/ArsenalTeam:UCSF/Project/Arsenal2010-10-27T17:27:37Z<p>Maven: </p>
<hr />
<div><html><br />
<script><br />
var Numbering="N3 N3-4";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
<br />
<br />
==='''BETTER ARSENAL'''===<br />
<br />
<br /><br />
<br /><br />
[[Image:Immune_synapse_killing-01.jpg|center|500px|Immune synapse]]<br />
<br /><br />
<br />
<br />
''Goal:'' <br />
<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to the protein.<br />
<br />
''Approach:'' <br />
<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells’ granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells’ granules by identifying and fusing granule localization “address” tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the “address” tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Brief Introduction===<br />
<br />
Current opinion on secretory granules is that the secreted granules are closely linked to lysosomes, they serve the function of lysosome when not secreted [1]. <br />
<br />
Proteins without signal peptides remain in the cytoplasm, while those with a signal peptide can be transported into the Endpplasmic reticulum, proteins that go into the ER are sorted according to its sorting signal, which can be regarded as an address tag. For Example, proteins with KEDL motif is maintained in the Endo reticulum. So “KEDL” motif is the Endo reticulum tag. Proteins with this tag finally stay in the endo reticulum. There are also tags that mark protein for granule, proteins with such tag are finally transported into the granule.<br />
<br />
The address tags of the proteins utilize the cellular transportation system to go to different compartments of the cell. It is like mails, although there are addresses on them, they themselves will not go to the address; it depends on the post system to pick them up and deliver them to the right place.<br />
<br />
So what’s the transportation system of the cell like? They are made of transmembrane proteins. The rumen sides of the transporter proteins can recognize and bind to the address tags on cargo proteins. The cytoplasmic side of the transporter proteins contains specific motifs. When the golgi bubbles out, different bubbles contains different sets of transporter proteins whose cytoplasmic motifs will decide where the bubble should go (The mechanism will not be discussed here).<br />
<br />
Now let’s take a look at how granule resident proteins are sorted to the granule. By transporter, the sorting pathways can be divided into Mannose Phosphate Receptor (MPR)-dependent pathway and MPR-independent pathways.<br />
<br />
MPR-dependent pathways use MPR as the transporter. And the address tag is mannose-6-phosphate. Proteins transported in this pathway are glycosylated with mannose-6-phosphate groups, these groups serve as the address tag. The rumen side of the MPR can bind to the mannose-6-phosphate motif (We can see the from the naming of MPR). The cytoplasmic side of MPR contains “DXXLL” motif, which decides that MPR will be sorted to lysosome/granule.<br />
<br />
MPR-independent pathways, from the name, use transporters other than MPR. It may or may not depend on the mannose-6-phosphate tag on the cargo. This includes sortilin, LIMPII, LRP;[2][3][4]the addressing tags involved are not very clear.<br />
<br />
Granzymes are transported through the MPR-dependent pathway[5].The signal peptide of the granzymes get cleaved upon entering the Endo reticulum. However, they are inactive in the Endo reticulum in the Endo reticulum and the golgi. Only after they reached granule, the N-terminal dipeptide can be cleaved off by DPPI aka cathepsin C, this leaves behind the active protein which begins with the conserved IIGG sequence. For Example, the N-terminal of Granzyme B is QPILLLLAFLLLPRADAGEIIGG underlined Amino acids are the signal sequence. The GE in italic is the dipeptide which inhibits the activity of granzyme B. In the granule, GE will get cleaved off by cathepsin C, and then granzyme B is activated.<br />
<br />
===Directing GFP to killer cells’ granules by fusing “address” tags''===<br />
<br />
''Concept and experimental design''<br />
We hypothesized that fusing address tags from proteins that are naturally sent to the granule may work to send GFP to the granule as well.<br />
<br />
''1. N-terminal signal peptides''<br />
Proteins should be translocated into the endoplasmic reticulum (ER) first in order to be further sorted to granule. Since it is not sure whether the signal sequence would play some role in the granule sorting, we decided to use the signal sequences from the granzymes. Each Granzyme has an individual signal peptide sequence that delivers it to the Endo Reticulum. Every signal sequence is distinct in size and amino acid content.<br />
<br />
''2. C-terminal “address tag” sequences''<br />
<br />
We have two strategies to send our cargo from the endoplasmic reticulum (ER) to the granule:<br />
1. Directly fuse our cargo to the granule specific transporters.<br />
2. Fuse our cargo to some address tag which can bind to the transporter.<br />
<br />
''Strategy 1''<br />
The proteins listed below all have granule localization motif on their cytoplasmic tails.<br />
<br />
''Y-motif:''<br />
In a previous work[6], scientists delivered TNFR1 to the granule by fusing it to the CD63 trans-membrane and cytoplasmic fragment. The mechanism is that the CD63 protein is a lysosome resident protein, it has signal sequence “SIRSGYEVM” on its cytoplasmic end, which contains the YXX? motif (X represents any Amino Acid, ? stands for hydrophobic amino acid). Similarly, other protein cargos can also be fused to the CD63 protein to be transported to the granule.<br />
<br />
''MPR:''<br />
Many of the normal granule resident proteins in the cell are glycosylated and form a mannose-6-phosphate in the Golgi. MPR(mannose phosphate receptor) are membrane receptors for the mannose-6-phosphate motif. The cytoplasmic motif “DXXLL” of the MPR decided that it will be sorted to the granule together with the cargos bound. We make it another candidate to fuse our cargo to.<br />
<br />
''LIMP-II & LAMP1:''<br />
Lysosomal membrane proteins have been proposed to reach lysosomes by two pathways referred to as “direct” and “indirect”. In the direct pathway, lysosomal membrane proteins are transported intracellularly from the TGN to either early or late endosomes and then to lysosomes without ever appearing at the cell surface. In contrast, the indirect pathway involves constitutive transport from the TGN to the plasma membrane, followed by internalization into early endosomes and eventual delivery to late endosomes and lysosomes. Members of the LAMP/LIMP class are the preeminent example of proteins thought to traffic via the direct pathway, that’s why we picked LIMP-II and LAMP1. We hope that by employing the direct pathway we can reduce the leakiness of our cargo to extracellular compartment.<br />
<br />
''Sortilin:''<br />
Sortilin is identified to be an alternative receptor than MPR in transporting lysosomal proteins. Sortilin has been reported to mediate lysosomal trafficking of prosaposin and acid sphingomyelinase. The cytoplasmic tail “GYHDDSDEDLL” contains “DXXLL” motif.<br />
<br />
''Strategy 2''<br />
<br />
Most of the soluble proteins in the granule are transported through the mannose-6-phosphate route, labeling the cargo protein with mannose-6-phosphate is the most direct and obvious method to use. However, mannose-6-phosphate is not directly genetically coded in the DNA sequence, instead it is the post-translational modification in the Golgi that delivers the glycosyl group to the protein. It is clear that enzymes only selectively create mannose-6-phosphate on those lysosome resident proteins, leaving the other proteins unmodified. So we turn to find the determining motif for glycosylation. Unfortunately, it turns out that this motif depends on the structure of the protein. No such domains that decides a protein should be decorated by mannose-6-phosphate, in other words, it is not modular.<br />
<br />
However, by looking at the works on the treatment of lysosomal storage diseases, we found a glycosylation independent motif that binds to MPR. [7].<br />
<br />
''GILT:''<br />
GILT stands for “glycosylation-independent lysosomal targeting”, it actually uses a truncation of insulin-like grow factor II (IGF II). The Cation Independent mannose phosphate receptor has a IGF II binding motif. Since CI-MPR can also go to the cell membrane, its natural function is to internalize the circulating IGF II proteins and send them to the lysosome for degradation, thus regulating IGF II concentration. Therapy of lysosome storage disease takes advantage of the IGF II, and fuse exogenous lysosomal enzymes to the GILT tag. These genetically modified enzymes can be taken up by patient cells to fulfill some deficient function. We use the GILT tag to deliver endogenous proteins to lysosome, which also looks feasible.<br />
<br />
<br />
===Cloning Strategy===<br />
<br />
We used the BBF RFC 28 strategy to make fusion protein.<br />
Signal sequences are made into AB parts<br />
the cargo (GFP) is made into BC part<br />
and all the granule localization adress tags are made into CD parts.<br />
After assembling the AB-BC and CD part, we can get the constructs we want and express them in CTL cells.<br />
<br />
Since the signal sequences and the granule localization sequences are all short in length, we decided to make the basic AB and BC parts fused together through overlap extension PCR-based methods used in gene synthesis. These methods are robust for assembling oligos of 40-50nt into sequences up to and greater than 3kb. (Protocols from personal communication, Jason Park). We used the web-based software Helix System DNAworks (http://helixweb.nih.gov/dnaworks/) for human codon-optimization and oligonucleotide parsing.<br />
<br />
After we get all the AB, BC and CD parts, we were able the assemble them in different combination and make devices for testing.<br />
<br />
===Device List===<br />
<br />
{|cellspacing="0"<br />
|'''#'''||'''[N-terminal Granzyme sequence]-[cargo]-[C-terminal “address” tag]'''||'''Plasmid Name'''<br />
|-<br />
|1||GRZM B-EGFP-CDMPR||pCPL_056<br />
|-<br />
|2||GRZM B-EGFP-GILT||pCPL_057<br />
|-<br />
|3||GRZM B-EGFP-LAMP||pCPL_058<br />
|-<br />
|4||GRZM B-EGFP-LIMP||pCPL_059<br />
|-<br />
|5||GRZM B-EGFP-Y TAIL||pCPL_060<br />
|-<br />
|6||GRZM B-EGFP-STOP||pCPL_061)<br />
|-<br />
|7||GRZM M-EGFP-CDMPR||pCPL_062<br />
|-<br />
|8||GRZM M-EGFP-GILT||pCPL_063<br />
|-<br />
|9||GRZM M-EGFP-LAMP||pCPL_064<br />
|-<br />
|10||GRZM M-EGFP-LIMP||pCPL_065<br />
|-<br />
|11||GRZM M-EGFP-Y TAIL||pCPL_066<br />
|-<br />
|12||GRZM M-EGFP-STOP||pCPL_067<br />
|}<br />
<br />
===Testing the Constructs===<br />
<br />
In this part of our project we used Amaxa electroporation to transfect NKL cells (an NK cell line) with plasmids containing our devices. Then we grew cells stably expressing our construct for our assay by selecting with the selection agent G418.<br />
<br />
Our assay consisted of using fluorescence microscopy to determine how well we were able to target EGFP to the granules. Before microscopy, we first stained our NKL cell membranes with the membrane Vybrant DiI stain and then with the Lysotracker Blue lysosomal stain (killer cell granules are specialized lysosomes). After our initial assay we saw that the EGFP wasn’t fluorescing well, which we attributed to quenching due to the low pH levels of the granules, or degradation due to other lysosomal proteins. To address this issue we decided to fixed and permeabilized our cells, and stained the EGFP inside with a fluorescent anti-GFP antibody. This showed much clearer results.<br />
<br />
<br />
<br />
<br />
<br />
<br />
==='''Better Arsenal'''===<br />
<br />
'''Goal:'''<br />
Direct a model protein, green fluorescent protein (GFP), to killer cells' granules by identifying and fusing granule localization "address" tags to the protein.<br />
<br />
<br /><br />
'''Approach:'''<br />
One way that cancer cells can evade the immune system is by evolving resistance to the arsenal of cytotoxic proteins found in killer cells' granules. Our goal for this summer was to direct a model protein, green fluorescent protein (GFP), to killer cells' granules by identifying and fusing granule localization "address" tags to GFP. This would be an important first step toward loading granules with novel cytotoxic proteins against which cancer cells would likely be defenseless!<br />
<br />
While it is not completely understood how killer cells send proteins to cytotoxic granules, we were able to learn a great deal about the "address" tags of various proteins that are sent to granules by searching and reading the literature. We designed a number of parts using these tags and made devices by fusing them in various combinations to GFP.<br />
<br />
===Devices: ===<br />
<br />
===Brief Introduction:===<br />
<br />
===Concept and experimental design:===<br />
<br />
===Results:===<br />
<br />
===Future applications:===<br />
<br />
{{Template:UCSF/LeftEnd}}<br />
<br />
{{Template:UCSF/RightStart}}<br />
{{Template:UCSF/RightEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSFTeam:UCSF2010-10-27T17:03:49Z<p>Maven: </p>
<hr />
<div><html><br />
<script><br />
var Numbering="N1";<br />
</script><br />
</html><br />
{{Template:UCSF/BannerAndNav}}<br />
{{Template:UCSF/LeftStart}}<br />
===Project Description===<br />
<br />
Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
<br />
<br />
<html><br />
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===Project Description===<br />
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Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
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===Project Description===<br />
<br />
Natural killer (NK) cells of the immune system identify cancer and virally-infected cells and kill them. These potent killers travel throughout the body, recognizing proteins and other molecules on the surface of cells. In order to differentiate between healthy and diseased cells, NK cells use a variety of receptors, which bind to specific ligands at the target cells’ surface. The balance between activating and inhibitory signals will tell the NK cell if the target cell is diseased or healthy, respectively. If the target cell is deemed potentially dangerous, the NK cell grips the target cell tightly and creates an immunological synapse at the site of adhesion. Within this immunological synapse, the NK cell releases cytotoxic granules to kill the target cell without harming any nearby cells allowing for a direct, apoptotic death.<br />
<br />
Our team will focus on improving NK cells’ specificity and killing efficiency towards certain cancer types. By using synthetic biology tools and logic gates’ design, we hope to create powerful killing biomachines for the fight against cancer. Our newly engineered synthetic devices would have the potential to enhance current adoptive cell-based immunotherapy for cancer patients.<br />
<br />
<br />
<html><br />
<a href="https://2010.igem.org/Team:UCSF/Project/Precision"><img src="https://static.igem.org/mediawiki/2010/2/28/UCSF_precision_home_icon.png" width="208" border="0" alt="Greater Precision" /><br />
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__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/PrecisionTeam:UCSF/Project/Precision2010-10-27T15:28:58Z<p>Maven: </p>
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<h3 style="font-weight:bold;">Greater Precision</h3><br />
<br />
<p><b>Goal</b>: Engineer killer cells to increase their precision in detecting cancer cells.</p><br><br />
<br />
<br />
<p><b>Approach</b>: After discussing our goals for the iGEM project, we have come up with an approach to increase precision by using:<br><br />
1. CARs that recognize many types of different cancer ligands.<br><br />
2. Logic gating to set higher restrictions on killer cell activation.</p><br><br />
<br />
<p><b>Devices</b>:<br><br />
1. <b>ANDN gate</b> - we have successfully developed devices for this type of logic gate, and we have confirmed through testing data their ability to increase precision.<br><br />
2. <b>AND gate</b> - we have successfully built devices for this type of logic gate, and we are now working on optimizing the assay to measure their effects on precision.</p><br><br />
<br />
<br><br />
<br />
<h3>Background information</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/7/79/UCSF_immune_signaling.png" /><br><br><br />
</div><br />
<br />
<p>In the same way that we recognize people based on their appearance, killer cells (cytotoxic T cells and NK cells) recognize target cells based on their different types of surface proteins <a href="#references">[1]</a>. This important ability to recognize the many different types of cells allows killers cells to eliminate unhealthy cells but avoid harming healthy cells. Unfortunately, killer cells can have trouble recognizing cancer cells among healthy cells due to complex profiles of surface markers on cancer cells. When it comes to cancer, killer cells are disadvantaged because they target foreign and dangerous organisms, but cancer cells originate from formerly healthy cells. Therefore, killer cells face difficulty in labeling cancer cells as dangerous entities because cancer cells express self antigens <a href="#references">[2]</a>. This fact is unsettling in that this method of differentiation is currently the only means by which killer cells can recognize cancerous cells.</p><br><br />
<br />
<p>We hope to introduce a logic gating system as an engineering platform to make killer cell recognition more specific and precise. We took advantage of the fact that many of the killer cells’ receptors bind to specific target cell surface proteins, much like antibodies bind to specific antigens. So why not replace the receptors’ recognition domains with different antibodies to create new receptors to recognize the many surface proteins on cells? That’s exactly what we did. These chimeric antigen receptors (CARs) are the products of an immune receptor intracellular signaling chain and an antigen binding domain, which, when put together as a single unit, can bind specifically to target antigens and trigger signaling responses <a href="#references">[3]</a>. Killer cells engineered with such modularly constructed synthetic receptors can overcome the restriction imposed by the presence of self-antigens on cancer cells.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/2/23/UCSF_CAR_structure.png" /><br><br />
<b>Domain structure and function of a chimeric antigen receptor <a href="#references">[4]</a>.</b><br />
</div><br />
<br />
<p>We also took advantage of the fact that killer cells’ receptors relay extracellular information intracellular compartments using modular signaling motifs such as ITAM, ITAM-like activation motifs, and ITIM. When ITAM and ITAM-like activation motifs become activated, they recruit kinases in the cytoplasm that initiate cell killing. ITIM motifs recruit phosphatases that cancel out the effects of ITAM activation <a href="#references">[5]</a>.</p><br><br />
<br />
<p>The modularity of CARs and killer cells’ receptors makes it feasible to create a multitude of recognition systems that function as logic gates in the killer cell. The logic gates should enable engineered killer cells to recognize specific combinations of surface proteins on target cells. Each specific combination of surface proteins acts as the prerequisite to activate a logic gate in order to trigger a killer cell action, which makes recognition a highly precise process. This in turn allows killer cells to distinguish cancer cells from normal cells more effectively because the specific combinations of antigens found only on cancerous cells can be set as logic gate prerequisites to trigger cell activation, whereas the normal cells do not fulfill the prerequisites and are left unharmed.</p><br><br><br />
<br />
<h3>Experimental Design and Results</h3><br />
<br />
<p>For our project we have designed two main gates: ANDN and AND gate.</p><br />
<br />
<h4 style="color:black; font-weight:bold;">i. ANDN gate</h4><br />
<br />
<p>In order to understand the ANDN gate, let us set up a hypothetical situation in which antigen A and antigen B are expressed on the membrane surface of healthy cells. Since cancer cells typically discard many surface proteins as a result of genetic mutation, we represent this discarded protein as antigen B in our scenario. Our ANDN gate is designed to address this issue by triggering cytotoxicity in the presence of antigen A and absence of antigen B. Therefore, cancerous cells that express antigen A and “hide” antigen B will be targeted. Healthy cells expressing both antigen A and B will not set off the activation of the ANDN gate. This concept is valuable for ensuring a level of specificity that prevents the overly indiscriminate activation of killer cells.</p><br><br />
<br />
<p>We tested two different ANDN gate designs to determine their effects on target recognition. To achieve the level of specificity as described by our hypothetical situation, we have set two different antigen binding domains to recognize antigen A and antigen B, respectively. Attached to the domain that recognized antigen A was an ITAM-bearing intracellular chain, from either the CD3 zeta or Fc receptor gamma, that signaled for killer cell activation. The domain that recognized antigen B, the antigen found in healthy cells, was fused to the intracellular portion of the ITIM-based receptor KIR3DL1, which inhibits killer cell activation. As a result of this combination, target cells expressing only antigen A would trigger killer cell activation, and target cells that do not express antigen A would not. Target cells that express both antigens A and B would be unharmed due to ITIM inhibitory signals, meaning that the presence of antigen B overwrites the input of antigen A. In conclusion, only a specific combination of surface antigens can set off the chain of activation, resulting in increased precision of detection and cancer killing.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/87/UCSF_ANDN_gate.png" /><br><br />
<b>Function of ANDN Gate</b><br />
</div><br><br><br />
<br />
<p>To evaluate our ANDN gate designs, we presented the dually transfected killer cells with target cells expressing antigen A, antigen B, both antigens A and B, or none of those antigens. The killer cells had been engineered to express the GFP reporter from the promoter of NFAT, a gene induced during killer cell activation. This reporter cell line enabled us to quantify the percentage of activated killer cells that express using FACS (fluorescence activated cell sorting). As shown in the figure below, killer cells presented with target cells expressing neither antigen or only antigen B showed basal levels of activation. Target cells expressing only antigen A, which represent cancer cells in this experiment, increased the percentage of activated killer cells. Notably, killer cells presented with target cells expressing both antigens had basal levels of activation. Living up to expectations, the ANDN gates proved to increase specificity because more killer cells became activated only in the presence of antigen A and the absence of antigen B.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/80/UCSF_ANDNgate_results.png" /><br><br />
<b>Experimental Data of ANDN Gate on T-Cell Activation</b><br />
</div><br><br><br />
<br />
<br><br />
<h4 style="color:black; font-weight:bold;">ii. AND gate</h4><br />
<br />
<p>Cancer cells are prone to overexpressing proteins on their cell surfaces. This fact allows us to detect the presence of cancer cells using AND gates. In our new hypothetical situation, normal cells express either antigen C or antigen D. In contrast, the overproduction characteristic of cancerous cells allows for the expression of both of these proteins <a href="#references">[6]</a>. AND gates are useful in this situation because they become activated only in the presence of two defined antigens. In the case of our new situation, the AND gate will trigger cytotoxicity only in the presence of antigen C and D, a condition that only applies to cancerous cells.</p><br><br />
<br />
<p>Applying the AND gate scenario and concept to the lab, we have manipulated the function of activation adaptor DAP10. In normal killer cells, DAP10 recruits two different proteins to two different motifs along its main body when activated. This recruitment will trigger the killing response only when both proteins are present. In order to ensure that killer cells will only kill in the presence of two specific antigens, we used two mutant versions of DAP10, each of which has a different motif that does not allow its complementary protein to bind. Each mutant version is fused to a different extracellular part that recognizes specific antigens on cell surfaces. As a result, when such CARs only recognize one antigen on healthy cells, they will not be able to trigger activation because only one motif is activated. When both CARs bind to both antigens found on cancerous cells, both motifs are able to become activated to induce cell cytotoxicity.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/3/30/UCSF_AND_gate.png" /><br><br />
<b>AND gate design</b><br />
</div><br />
<br><br><br />
<br />
<br />
<p>Due to time constraints, we have not been able to evaluate our AND gate design. We could not measure the AND gate’s effect in killer cells using the GFP reporter assay described above because DAP10 signaling does not induce NFAT expression. We attempted to test our AND gate using assays directly measuring the level of target cell killing. However, we faced a major technical obstacle that prevented us from obtaining informative results before the summer ended. The technical challenge was that only a small percentage of killer cells expressed the logic gate CARs after transfection. Due to this low transfection efficiency, the vast majority of killer cells used in target cell killing assays did not express our constructs. This was problematic because untransfected killer cells have an innate ability to kill, which produces a high killing background that makes it difficult to pinpoint the killing ability of transfected cells.</p><br><br><br />
<br />
<h3>Future Directions</h3><br />
<br />
<p>Our immediate goal is to optimize the efficiency for transfecting the killer cells and hopefully to obtain cells stably expressing our logic gate parts. This will allow us to test both the ANDN and AND gate designs directly based on cell killing efficiency with a significantly reduced basal killing level.</p><br><br><br />
<br />
<br />
<h3>References</h3><br />
<p><a name="references"></a></p><br />
<p><br />
1. <b>Formation and function of the lytic NK-cell immunological synapse.</b></p><br />
<p>Orange JS.</p><br />
<p>Nat Rev Immunol. 2008 Sep;8(9):713-25.</p><br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19172692">http://www.ncbi.nlm.nih.gov/pubmed/19172692</a></p><br />
<br><br />
<p><br />
2. <b>Learning how to discriminate between friends and enemies, a lesson from Natural Killer cells.</b><br />
<p>Bottino C, Moretta L, Pende D, Vitale M, Moretta A.<br />
<p>Mol Immunol. 2004 Jul;41(6-7):569-75.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/15219995">http://www.ncbi.nlm.nih.gov/pubmed/15219995</a></p><br />
<br><br />
<p><br />
3. <b>Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor.</b><br />
<p>Jena B, Dotti G, Cooper LJ.<br />
<p>Blood. 2010 Aug 19;116(7):1035-44. Epub 2010 May 3.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20439624">http://www.ncbi.nlm.nih.gov/pubmed/20439624</a></p><br />
<br><br />
<p><br />
4. <b>Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.</b><br />
<p>Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, Temme A, Schmitz M.<br />
<p>J Biomed Biotechnol. 2010;2010:956304. Epub 2010 May 5.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20467460">http://www.ncbi.nlm.nih.gov/pubmed/20467460</a></p><br />
<br><br />
<p><br />
5. <b>Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse.</b><br />
<p>Tassi I, Klesney-Tait J, Colonna M.<br />
<p>Immunol Rev. 2006 Dec;214:92-105.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/17100878">http://www.ncbi.nlm.nih.gov/pubmed/17100878</a></p><br />
<br><br />
<p><br />
6. <b>Oncogenic stress sensed by the immune system: role of natural killer cell receptors.</b><br />
<p>Raulet DH, Guerra N.<br />
<p>Nat Rev Immunol. 2009 Aug;9(8):568-80.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19629084">http://www.ncbi.nlm.nih.gov/pubmed/19629084</a></p><br />
<br><br />
</p><br />
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__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/PrecisionTeam:UCSF/Project/Precision2010-10-27T15:28:20Z<p>Maven: </p>
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<h2 style="font-weight:bold;">Greater Precision</h2><br />
<br />
<p><b>Goal</b>: Engineer killer cells to increase their precision in detecting cancer cells.</p><br><br />
<br />
<br />
<p><b>Approach</b>: After discussing our goals for the iGEM project, we have come up with an approach to increase precision by using:<br><br />
1. CARs that recognize many types of different cancer ligands.<br><br />
2. Logic gating to set higher restrictions on killer cell activation.</p><br><br />
<br />
<p><b>Devices</b>:<br><br />
1. <b>ANDN gate</b> - we have successfully developed devices for this type of logic gate, and we have confirmed through testing data their ability to increase precision.<br><br />
2. <b>AND gate</b> - we have successfully built devices for this type of logic gate, and we are now working on optimizing the assay to measure their effects on precision.</p><br><br />
<br />
<br><br />
<br />
<h3>Background information</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/7/79/UCSF_immune_signaling.png" /><br><br><br />
</div><br />
<br />
<p>In the same way that we recognize people based on their appearance, killer cells (cytotoxic T cells and NK cells) recognize target cells based on their different types of surface proteins <a href="#references">[1]</a>. This important ability to recognize the many different types of cells allows killers cells to eliminate unhealthy cells but avoid harming healthy cells. Unfortunately, killer cells can have trouble recognizing cancer cells among healthy cells due to complex profiles of surface markers on cancer cells. When it comes to cancer, killer cells are disadvantaged because they target foreign and dangerous organisms, but cancer cells originate from formerly healthy cells. Therefore, killer cells face difficulty in labeling cancer cells as dangerous entities because cancer cells express self antigens <a href="#references">[2]</a>. This fact is unsettling in that this method of differentiation is currently the only means by which killer cells can recognize cancerous cells.</p><br><br />
<br />
<p>We hope to introduce a logic gating system as an engineering platform to make killer cell recognition more specific and precise. We took advantage of the fact that many of the killer cells’ receptors bind to specific target cell surface proteins, much like antibodies bind to specific antigens. So why not replace the receptors’ recognition domains with different antibodies to create new receptors to recognize the many surface proteins on cells? That’s exactly what we did. These chimeric antigen receptors (CARs) are the products of an immune receptor intracellular signaling chain and an antigen binding domain, which, when put together as a single unit, can bind specifically to target antigens and trigger signaling responses <a href="#references">[3]</a>. Killer cells engineered with such modularly constructed synthetic receptors can overcome the restriction imposed by the presence of self-antigens on cancer cells.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/2/23/UCSF_CAR_structure.png" /><br><br />
<b>Domain structure and function of a chimeric antigen receptor <a href="#references">[4]</a>.</b><br />
</div><br />
<br />
<p>We also took advantage of the fact that killer cells’ receptors relay extracellular information intracellular compartments using modular signaling motifs such as ITAM, ITAM-like activation motifs, and ITIM. When ITAM and ITAM-like activation motifs become activated, they recruit kinases in the cytoplasm that initiate cell killing. ITIM motifs recruit phosphatases that cancel out the effects of ITAM activation <a href="#references">[5]</a>.</p><br><br />
<br />
<p>The modularity of CARs and killer cells’ receptors makes it feasible to create a multitude of recognition systems that function as logic gates in the killer cell. The logic gates should enable engineered killer cells to recognize specific combinations of surface proteins on target cells. Each specific combination of surface proteins acts as the prerequisite to activate a logic gate in order to trigger a killer cell action, which makes recognition a highly precise process. This in turn allows killer cells to distinguish cancer cells from normal cells more effectively because the specific combinations of antigens found only on cancerous cells can be set as logic gate prerequisites to trigger cell activation, whereas the normal cells do not fulfill the prerequisites and are left unharmed.</p><br><br><br />
<br />
<h3>Experimental Design and Results</h3><br />
<br />
<p>For our project we have designed two main gates: ANDN and AND gate.</p><br />
<br />
<h4 style="color:black; font-weight:bold;">i. ANDN gate</h4><br />
<br />
<p>In order to understand the ANDN gate, let us set up a hypothetical situation in which antigen A and antigen B are expressed on the membrane surface of healthy cells. Since cancer cells typically discard many surface proteins as a result of genetic mutation, we represent this discarded protein as antigen B in our scenario. Our ANDN gate is designed to address this issue by triggering cytotoxicity in the presence of antigen A and absence of antigen B. Therefore, cancerous cells that express antigen A and “hide” antigen B will be targeted. Healthy cells expressing both antigen A and B will not set off the activation of the ANDN gate. This concept is valuable for ensuring a level of specificity that prevents the overly indiscriminate activation of killer cells.</p><br><br />
<br />
<p>We tested two different ANDN gate designs to determine their effects on target recognition. To achieve the level of specificity as described by our hypothetical situation, we have set two different antigen binding domains to recognize antigen A and antigen B, respectively. Attached to the domain that recognized antigen A was an ITAM-bearing intracellular chain, from either the CD3 zeta or Fc receptor gamma, that signaled for killer cell activation. The domain that recognized antigen B, the antigen found in healthy cells, was fused to the intracellular portion of the ITIM-based receptor KIR3DL1, which inhibits killer cell activation. As a result of this combination, target cells expressing only antigen A would trigger killer cell activation, and target cells that do not express antigen A would not. Target cells that express both antigens A and B would be unharmed due to ITIM inhibitory signals, meaning that the presence of antigen B overwrites the input of antigen A. In conclusion, only a specific combination of surface antigens can set off the chain of activation, resulting in increased precision of detection and cancer killing.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/87/UCSF_ANDN_gate.png" /><br><br />
<b>Function of ANDN Gate</b><br />
</div><br><br><br />
<br />
<p>To evaluate our ANDN gate designs, we presented the dually transfected killer cells with target cells expressing antigen A, antigen B, both antigens A and B, or none of those antigens. The killer cells had been engineered to express the GFP reporter from the promoter of NFAT, a gene induced during killer cell activation. This reporter cell line enabled us to quantify the percentage of activated killer cells that express using FACS (fluorescence activated cell sorting). As shown in the figure below, killer cells presented with target cells expressing neither antigen or only antigen B showed basal levels of activation. Target cells expressing only antigen A, which represent cancer cells in this experiment, increased the percentage of activated killer cells. Notably, killer cells presented with target cells expressing both antigens had basal levels of activation. Living up to expectations, the ANDN gates proved to increase specificity because more killer cells became activated only in the presence of antigen A and the absence of antigen B.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/80/UCSF_ANDNgate_results.png" /><br><br />
<b>Experimental Data of ANDN Gate on T-Cell Activation</b><br />
</div><br><br><br />
<br />
<br><br />
<h4 style="color:black; font-weight:bold;">ii. AND gate</h4><br />
<br />
<p>Cancer cells are prone to overexpressing proteins on their cell surfaces. This fact allows us to detect the presence of cancer cells using AND gates. In our new hypothetical situation, normal cells express either antigen C or antigen D. In contrast, the overproduction characteristic of cancerous cells allows for the expression of both of these proteins <a href="#references">[6]</a>. AND gates are useful in this situation because they become activated only in the presence of two defined antigens. In the case of our new situation, the AND gate will trigger cytotoxicity only in the presence of antigen C and D, a condition that only applies to cancerous cells.</p><br><br />
<br />
<p>Applying the AND gate scenario and concept to the lab, we have manipulated the function of activation adaptor DAP10. In normal killer cells, DAP10 recruits two different proteins to two different motifs along its main body when activated. This recruitment will trigger the killing response only when both proteins are present. In order to ensure that killer cells will only kill in the presence of two specific antigens, we used two mutant versions of DAP10, each of which has a different motif that does not allow its complementary protein to bind. Each mutant version is fused to a different extracellular part that recognizes specific antigens on cell surfaces. As a result, when such CARs only recognize one antigen on healthy cells, they will not be able to trigger activation because only one motif is activated. When both CARs bind to both antigens found on cancerous cells, both motifs are able to become activated to induce cell cytotoxicity.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/3/30/UCSF_AND_gate.png" /><br><br />
<b>AND gate design</b><br />
</div><br />
<br><br><br />
<br />
<br />
<p>Due to time constraints, we have not been able to evaluate our AND gate design. We could not measure the AND gate’s effect in killer cells using the GFP reporter assay described above because DAP10 signaling does not induce NFAT expression. We attempted to test our AND gate using assays directly measuring the level of target cell killing. However, we faced a major technical obstacle that prevented us from obtaining informative results before the summer ended. The technical challenge was that only a small percentage of killer cells expressed the logic gate CARs after transfection. Due to this low transfection efficiency, the vast majority of killer cells used in target cell killing assays did not express our constructs. This was problematic because untransfected killer cells have an innate ability to kill, which produces a high killing background that makes it difficult to pinpoint the killing ability of transfected cells.</p><br><br><br />
<br />
<h3>Future Directions</h3><br />
<br />
<p>Our immediate goal is to optimize the efficiency for transfecting the killer cells and hopefully to obtain cells stably expressing our logic gate parts. This will allow us to test both the ANDN and AND gate designs directly based on cell killing efficiency with a significantly reduced basal killing level.</p><br><br><br />
<br />
<br />
<h3>References</h3><br />
<p><a name="references"></a></p><br />
<p><br />
1. <b>Formation and function of the lytic NK-cell immunological synapse.</b></p><br />
<p>Orange JS.</p><br />
<p>Nat Rev Immunol. 2008 Sep;8(9):713-25.</p><br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19172692">http://www.ncbi.nlm.nih.gov/pubmed/19172692</a></p><br />
<br><br />
<p><br />
2. <b>Learning how to discriminate between friends and enemies, a lesson from Natural Killer cells.</b><br />
<p>Bottino C, Moretta L, Pende D, Vitale M, Moretta A.<br />
<p>Mol Immunol. 2004 Jul;41(6-7):569-75.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/15219995">http://www.ncbi.nlm.nih.gov/pubmed/15219995</a></p><br />
<br><br />
<p><br />
3. <b>Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor.</b><br />
<p>Jena B, Dotti G, Cooper LJ.<br />
<p>Blood. 2010 Aug 19;116(7):1035-44. Epub 2010 May 3.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20439624">http://www.ncbi.nlm.nih.gov/pubmed/20439624</a></p><br />
<br><br />
<p><br />
4. <b>Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.</b><br />
<p>Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, Temme A, Schmitz M.<br />
<p>J Biomed Biotechnol. 2010;2010:956304. Epub 2010 May 5.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20467460">http://www.ncbi.nlm.nih.gov/pubmed/20467460</a></p><br />
<br><br />
<p><br />
5. <b>Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse.</b><br />
<p>Tassi I, Klesney-Tait J, Colonna M.<br />
<p>Immunol Rev. 2006 Dec;214:92-105.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/17100878">http://www.ncbi.nlm.nih.gov/pubmed/17100878</a></p><br />
<br><br />
<p><br />
6. <b>Oncogenic stress sensed by the immune system: role of natural killer cell receptors.</b><br />
<p>Raulet DH, Guerra N.<br />
<p>Nat Rev Immunol. 2009 Aug;9(8):568-80.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19629084">http://www.ncbi.nlm.nih.gov/pubmed/19629084</a></p><br />
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__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/Project/PrecisionTeam:UCSF/Project/Precision2010-10-27T15:27:48Z<p>Maven: </p>
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<h2 style="font-weight:bold;">Greater Precision</h2><br />
<br />
<p><b>Goal</b>: Engineer killer cells to increase their precision in detecting cancer cells.</p><br><br />
<br />
<br />
<p><b>Approach</b>: After discussing our goals for the iGEM project, we have come up with an approach to increase precision by using:<br><br />
1. CARs that recognize many types of different cancer ligands.<br><br />
2. Logic gating to set higher restrictions on killer cell activation.</p><br><br />
<br />
<p><b>Devices</b>:<br><br />
1. <b>ANDN gate</b> - we have successfully developed devices for this type of logic gate, and we have confirmed through testing data their ability to increase precision.<br><br />
2. <b>AND gate</b> - we have successfully built devices for this type of logic gate, and we are now working on optimizing the assay to measure their effects on precision.</p><br><br />
<br />
<br><br />
<br />
<h3>Background information</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/7/79/UCSF_immune_signaling.png" /><br><br><br />
</div><br />
<br />
<p>In the same way that we recognize people based on their appearance, killer cells (cytotoxic T cells and NK cells) recognize target cells based on their different types of surface proteins <a href="#references">[1]</a>. This important ability to recognize the many different types of cells allows killers cells to eliminate unhealthy cells but avoid harming healthy cells. Unfortunately, killer cells can have trouble recognizing cancer cells among healthy cells due to complex profiles of surface markers on cancer cells. When it comes to cancer, killer cells are disadvantaged because they target foreign and dangerous organisms, but cancer cells originate from formerly healthy cells. Therefore, killer cells face difficulty in labeling cancer cells as dangerous entities because cancer cells express self antigens <a href="#references">[2]</a>. This fact is unsettling in that this method of differentiation is currently the only means by which killer cells can recognize cancerous cells.</p><br><br />
<br />
<p>We hope to introduce a logic gating system as an engineering platform to make killer cell recognition more specific and precise. We took advantage of the fact that many of the killer cells’ receptors bind to specific target cell surface proteins, much like antibodies bind to specific antigens. So why not replace the receptors’ recognition domains with different antibodies to create new receptors to recognize the many surface proteins on cells? That’s exactly what we did. These chimeric antigen receptors (CARs) are the products of an immune receptor intracellular signaling chain and an antigen binding domain, which, when put together as a single unit, can bind specifically to target antigens and trigger signaling responses <a href="#references">[3]</a>. Killer cells engineered with such modularly constructed synthetic receptors can overcome the restriction imposed by the presence of self-antigens on cancer cells.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/2/23/UCSF_CAR_structure.png" /><br><br />
<b>Domain structure and function of a chimeric antigen receptor <a href="#references">[4]</a>.</b><br />
</div><br />
<br />
<p>We also took advantage of the fact that killer cells’ receptors relay extracellular information intracellular compartments using modular signaling motifs such as ITAM, ITAM-like activation motifs, and ITIM. When ITAM and ITAM-like activation motifs become activated, they recruit kinases in the cytoplasm that initiate cell killing. ITIM motifs recruit phosphatases that cancel out the effects of ITAM activation <a href="#references">[5]</a>.</p><br><br />
<br />
<p>The modularity of CARs and killer cells’ receptors makes it feasible to create a multitude of recognition systems that function as logic gates in the killer cell. The logic gates should enable engineered killer cells to recognize specific combinations of surface proteins on target cells. Each specific combination of surface proteins acts as the prerequisite to activate a logic gate in order to trigger a killer cell action, which makes recognition a highly precise process. This in turn allows killer cells to distinguish cancer cells from normal cells more effectively because the specific combinations of antigens found only on cancerous cells can be set as logic gate prerequisites to trigger cell activation, whereas the normal cells do not fulfill the prerequisites and are left unharmed.</p><br><br><br />
<br />
<h3>Experimental Design and Results</h3><br />
<br />
<p>For our project we have designed two main gates: ANDN and AND gate.</p><br />
<br />
<h4 style="color:black; font-weight:bold;">i. ANDN gate</h4><br />
<br />
<p>In order to understand the ANDN gate, let us set up a hypothetical situation in which antigen A and antigen B are expressed on the membrane surface of healthy cells. Since cancer cells typically discard many surface proteins as a result of genetic mutation, we represent this discarded protein as antigen B in our scenario. Our ANDN gate is designed to address this issue by triggering cytotoxicity in the presence of antigen A and absence of antigen B. Therefore, cancerous cells that express antigen A and “hide” antigen B will be targeted. Healthy cells expressing both antigen A and B will not set off the activation of the ANDN gate. This concept is valuable for ensuring a level of specificity that prevents the overly indiscriminate activation of killer cells.</p><br><br />
<br />
<p>We tested two different ANDN gate designs to determine their effects on target recognition. To achieve the level of specificity as described by our hypothetical situation, we have set two different antigen binding domains to recognize antigen A and antigen B, respectively. Attached to the domain that recognized antigen A was an ITAM-bearing intracellular chain, from either the CD3 zeta or Fc receptor gamma, that signaled for killer cell activation. The domain that recognized antigen B, the antigen found in healthy cells, was fused to the intracellular portion of the ITIM-based receptor KIR3DL1, which inhibits killer cell activation. As a result of this combination, target cells expressing only antigen A would trigger killer cell activation, and target cells that do not express antigen A would not. Target cells that express both antigens A and B would be unharmed due to ITIM inhibitory signals, meaning that the presence of antigen B overwrites the input of antigen A. In conclusion, only a specific combination of surface antigens can set off the chain of activation, resulting in increased precision of detection and cancer killing.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/87/UCSF_ANDN_gate.png" /><br><br />
<b>Function of ANDN Gate</b><br />
</div><br><br><br />
<br />
<p>To evaluate our ANDN gate designs, we presented the dually transfected killer cells with target cells expressing antigen A, antigen B, both antigens A and B, or none of those antigens. The killer cells had been engineered to express the GFP reporter from the promoter of NFAT, a gene induced during killer cell activation. This reporter cell line enabled us to quantify the percentage of activated killer cells that express using FACS (fluorescence activated cell sorting). As shown in the figure below, killer cells presented with target cells expressing neither antigen or only antigen B showed basal levels of activation. Target cells expressing only antigen A, which represent cancer cells in this experiment, increased the percentage of activated killer cells. Notably, killer cells presented with target cells expressing both antigens had basal levels of activation. Living up to expectations, the ANDN gates proved to increase specificity because more killer cells became activated only in the presence of antigen A and the absence of antigen B.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/80/UCSF_ANDNgate_results.png" /><br><br />
<b>Experimental Data of ANDN Gate on T-Cell Activation</b><br />
</div><br><br><br />
<br />
<br><br />
<h4 style="color:black; font-weight:bold;">ii. AND gate</h4><br />
<br />
<p>Cancer cells are prone to overexpressing proteins on their cell surfaces. This fact allows us to detect the presence of cancer cells using AND gates. In our new hypothetical situation, normal cells express either antigen C or antigen D. In contrast, the overproduction characteristic of cancerous cells allows for the expression of both of these proteins <a href="#references">[6]</a>. AND gates are useful in this situation because they become activated only in the presence of two defined antigens. In the case of our new situation, the AND gate will trigger cytotoxicity only in the presence of antigen C and D, a condition that only applies to cancerous cells.</p><br><br />
<br />
<p>Applying the AND gate scenario and concept to the lab, we have manipulated the function of activation adaptor DAP10. In normal killer cells, DAP10 recruits two different proteins to two different motifs along its main body when activated. This recruitment will trigger the killing response only when both proteins are present. In order to ensure that killer cells will only kill in the presence of two specific antigens, we used two mutant versions of DAP10, each of which has a different motif that does not allow its complementary protein to bind. Each mutant version is fused to a different extracellular part that recognizes specific antigens on cell surfaces. As a result, when such CARs only recognize one antigen on healthy cells, they will not be able to trigger activation because only one motif is activated. When both CARs bind to both antigens found on cancerous cells, both motifs are able to become activated to induce cell cytotoxicity.</p><br><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/3/30/UCSF_AND_gate.png" /><br><br />
<b>AND gate design</b><br />
</div><br />
<br><br><br />
<br />
<br />
<p>Due to time constraints, we have not been able to evaluate our AND gate design. We could not measure the AND gate’s effect in killer cells using the GFP reporter assay described above because DAP10 signaling does not induce NFAT expression. We attempted to test our AND gate using assays directly measuring the level of target cell killing. However, we faced a major technical obstacle that prevented us from obtaining informative results before the summer ended. The technical challenge was that only a small percentage of killer cells expressed the logic gate CARs after transfection. Due to this low transfection efficiency, the vast majority of killer cells used in target cell killing assays did not express our constructs. This was problematic because untransfected killer cells have an innate ability to kill, which produces a high killing background that makes it difficult to pinpoint the killing ability of transfected cells.</p><br><br><br />
<br />
<h3>Future Directions</h3><br />
<br />
<p>Our immediate goal is to optimize the efficiency for transfecting the killer cells and hopefully to obtain cells stably expressing our logic gate parts. This will allow us to test both the ANDN and AND gate designs directly based on cell killing efficiency with a significantly reduced basal killing level.</p><br><br><br />
<br />
<br />
<h3>References</h3><br />
<p><a name="references"></a></p><br />
<p><br />
1. <b>Formation and function of the lytic NK-cell immunological synapse.</b></p><br />
<p>Orange JS.</p><br />
<p>Nat Rev Immunol. 2008 Sep;8(9):713-25.</p><br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19172692">http://www.ncbi.nlm.nih.gov/pubmed/19172692</a></p><br />
<br><br />
<p><br />
2. <b>Learning how to discriminate between friends and enemies, a lesson from Natural Killer cells.</b><br />
<p>Bottino C, Moretta L, Pende D, Vitale M, Moretta A.<br />
<p>Mol Immunol. 2004 Jul;41(6-7):569-75.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/15219995">http://www.ncbi.nlm.nih.gov/pubmed/15219995</a></p><br />
<br><br />
<p><br />
3. <b>Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor.</b><br />
<p>Jena B, Dotti G, Cooper LJ.<br />
<p>Blood. 2010 Aug 19;116(7):1035-44. Epub 2010 May 3.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20439624">http://www.ncbi.nlm.nih.gov/pubmed/20439624</a></p><br />
<br><br />
<p><br />
4. <b>Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.</b><br />
<p>Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, Temme A, Schmitz M.<br />
<p>J Biomed Biotechnol. 2010;2010:956304. Epub 2010 May 5.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/20467460">http://www.ncbi.nlm.nih.gov/pubmed/20467460</a></p><br />
<br><br />
<p><br />
5. <b>Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse.</b><br />
<p>Tassi I, Klesney-Tait J, Colonna M.<br />
<p>Immunol Rev. 2006 Dec;214:92-105.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/17100878">http://www.ncbi.nlm.nih.gov/pubmed/17100878</a></p><br />
<br><br />
<p><br />
6. <b>Oncogenic stress sensed by the immune system: role of natural killer cell receptors.</b><br />
<p>Raulet DH, Guerra N.<br />
<p>Nat Rev Immunol. 2009 Aug;9(8):568-80.<br />
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/19629084">http://www.ncbi.nlm.nih.gov/pubmed/19629084</a></p><br />
<br><br />
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__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/FunStuffTeam:UCSF/FunStuff2010-10-27T14:11:29Z<p>Maven: </p>
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<h3>Killer Cell Lovin' Flyer ($19.95)</h3><br />
<hr><br><br />
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<a href="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" border="0" alt="Get Some Killer Cell Loving'" width="400"/></a><br />
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<br><br />
Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
</div><br />
<br />
<br><br><br />
<h3>Team Photo Gallery</h3><hr><br><br />
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<a href="http://www.dropbox.com/gallery/12793331/1/WikiGalleryPicks?h=8fb6ea" target="_blank"><img src="https://static.igem.org/mediawiki/2010/4/44/UCSF_team_gallery_framed.png" border="0"/ alt="UCSF Team Gallery"></a><br />
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<h3>The Irresistible Frisbee (& its pouch!) </h3><hr><br><br />
<br />
<div align="center"><br />
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<a href="https://static.igem.org/mediawiki/2010/e/e4/UCSF_fling_disk_lg.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e4/UCSF_fling_disk_lg.png" alt="UCSF frisbee design" width="400" border="0" /></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2010/a/a3/UCSF_fling_bag.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a3/UCSF_fling_bag.png" alt="UCSF frisbee pouch" width="230" border="0" /></a><br />
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<h3>Killer Animations</h3><hr><br><br />
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Click on image to watch the ninja move!<br />
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{{Template:UCSF/WholeBlockEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/FunStuffTeam:UCSF/FunStuff2010-10-27T14:10:27Z<p>Maven: </p>
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</head><br />
<br />
<body><br />
<br />
<br><br />
<h3>Killer Cell Lovin' Flyer ($19.95)</h3><br />
<hr><br><br />
<br />
<div align="center"><br />
<a href="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/c2/UCSF_HAHAHAH.jpg" border="0" alt="Get Some Killer Cell Loving'" width="400"/></a><br />
<br />
<br><br />
Not sure what this flyer means? Check out <a href="http://www.youtube.com/watch?v=qHQ9CtCv778&feature=related" target="_blank">this video</a>.<br />
</div><br />
<br />
<br><br><br />
<h3>Team Photo Gallery</h3><hr><br><br />
<br />
<div align="center"><br />
<a href="http://www.dropbox.com/gallery/12793331/1/WikiGalleryPicks?h=8fb6ea" target="_blank"><img src="https://static.igem.org/mediawiki/2010/4/44/UCSF_team_gallery_framed.png" border="0"/ alt="UCSF Team Gallery"></a><br />
</div><br />
<br />
<br><br><br />
<h3>The Irresistible Frisbee (& its pouch!) </h3><hr><br><br />
<br />
<div align="center"><br />
<br />
<a href="https://static.igem.org/mediawiki/2010/e/e4/UCSF_fling_disk_lg.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2010/e/e4/UCSF_fling_disk_lg.png" alt="UCSF frisbee design" width="400" border="0" /></a><br />
<br />
<a href="https://static.igem.org/mediawiki/2010/a/a3/UCSF_fling_bag.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a3/UCSF_fling_bag.png" alt="UCSF frisbee pouch" width="230" border="0" /></a><br />
<br />
</div><br />
<br />
<br />
<br />
<br><br><br />
<h3>Killer Animations</h3><hr><br><br />
<br />
<div align="center"><br />
<table border="0" style="margin:0px;padding:0px;margin-top:0px;"><br />
<tbody><br />
<tr><br />
<td width="446px" style="padding: 0 0 0 0; background-color:#d5651b;margin:0;"><br />
<br><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8f/UCSF_Igem_Ninjas_animated_web_transparent.gif"/><br />
</div><br />
<br><br />
</td><br />
<br />
</tr><br />
</tbody><br />
</table><br />
<br />
<br><br><br />
<br />
<img border="0" alt="UCSF banner" src="https://static.igem.org/mediawiki/2010/d/dc/UCSF-SF-GGB.jpg" id="b1" <br />
onmouseover="mouseOver()" <br />
onmouseout="mouseOut()" <br />
onclick="onClick()" <br />
height="250"><br />
<br><br />
Click on image to watch the ninja move!<br />
<br />
<br><br><br><br><br />
<br />
</div><br />
<br />
</body><br />
<br />
</html><br />
<br />
{{Template:UCSF/WholeBlockEnd}}</div>Mavenhttp://2010.igem.org/Team:UCSF/Notes/TimelineTeam:UCSF/Notes/Timeline2010-10-27T14:06:37Z<p>Maven: </p>
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<br><br />
<h3>Responsibilities of Team Members</h3><br />
<br />
<table id="customers"><br />
<tr><br />
<th>Member</th><br />
<th>Lab Work</th><br />
<th>Other Responsibilities</th><br />
</tr><br />
<br />
<tr><br />
<td>Lianna Fung</td><br />
<td>Cloning, Transfections</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Hannah Yan</td><br />
<td>Cloning</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>Ethan Chan</td><br />
<td>Cloning, Cell Culturing, Transfections,Killing Assay</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Ryan Liang</td><br />
<td>Cloning, Cell Culturing, Transfections, Antibody Staining, Killing Assay, Reporter Assay</td><br />
<td>Wiki Design</td><br />
</tr><br />
<br />
<tr><br />
<td>Crystal Liu</td><br />
<td>Cloning, Killing Assay</td><br />
<td>Presentation, Wiki Design</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Carmen Zhou</td><br />
<td>Cloning, Transfections, Killing Assay</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>John Elam</td><br />
<td>Cloning, Transfections, Killing Assay</td><br />
<td>Presentation</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Connor Grant</td><br />
<td>Cloning</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>Samuel Zorn</td><br />
<td>Cloning, Antibody Staining, Reporter Assay</td><br />
<td>Presentation</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Eric Wong</td><br />
<td>Cloning, Cell Culturing, Transfections, Antibody Staining, Killing Assay</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>Min Lin</td><br />
<td>Cloning, Transfections, Antibody Staining, Reporter Assay</td><br />
<td>Wiki Content, Wiki Design, EVERYTHING</td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
<br><br><br />
<h3>Project Timeline</h3><br />
<br />
</html><br />
<br />
'''FEBRUARY'''<br><br />
<br />
'''17th''' – Arrival of NK cell lines – NKL, YTS/eco, KHYG1<br><br />
'''18th''' – Ryan and Ethan begin NK cell cytotoxicity research on cell lines NKL, KHYG-1, NK92MI & YTS/eco<br><br />
<br><br />
<br />
'''MARCH'''<br><br />
<br />
'''16th''' – Ryan and Ethan starts optimization of NKL cell line<br><br />
'''18th''' – Ryan and Ethan starts optimization of KHYG-1 cell line<br><br />
<br><br />
<br />
'''APRIL'''<br><br />
<br />
'''9th''' – Troubleshooting of FACS readout due to Propidium Iodide staining<br><br />
'''16th''' – Reduction of Propidium Iodide, cell concentration, and event readout, FACS analysis continues.<br><br />
'''20th''' – Ryan and Ethan starts optimization of NK92MI cell line<br><br />
'''22nd''' – Ryan and Ethan starts optimization of YTS/eco cell line<br><br />
'''29th''' – Second round of optimization begins; NK92MI cell line is discontinued <br><br />
'''30th''' – Preparation of presentation for May 4th meeting with other iGEM students<br><br />
<br><br />
<br />
'''MAY'''<br><br />
<br />
'''4th''' – Ryan, Ethan and advisor meet with other iGEM students from Abraham Lincoln High School<br><br />
'''13th''' – Ethan and Ryan gain full responsibility of NKL, KHYG-1, YTS/eco & K562 cell lines<br><br />
'''18th''' – Arrival of Anti-Meso/CD19 plasmids from Mike Milone (University of Pennsylvania)<br><br />
<br><br />
<br />
'''JUNE'''<br><br />
<br />
'''11th''' – Arrival of K562 cell line from Mike Milone (UPenn)<br><br />
'''14th''' – Full local iGEM team arrive at UCSF and begin 2-week boot camp session.<br><br />
'''15th''' – Seminar by Raquel G. on immune response, signaling cascades, feedback loops, receptor adaptors, and general cancer detection<br><br />
'''16th''' – Seminar by Derek W. on cellular cytoskeleton, actin system, microtubules and other related proteins<br><br />
'''17th''' - Seminar by Daniel H. on cell death, killing process, cytotoxic agents and inducers of apoptosis<br><br />
'''18th''' – Seminar by Reid W. on logic gates, combinatorial qualities of proteins and behavioral changes<br><br />
'''21st''' – Seminar by David P. on modularity, synthetic biology, and Boolean gates<br><br />
'''22rd''' – iGEM Team challenge; brainstorming and consulting with advisors and grad students<br><br />
'''24th''' – General project in mind: granzyme linking, stronger signaling and greater arsenal<br><br />
'''25th''' – Lab safety training begins, first set of primers are designed, first set of source plasmids are ordered, and lab protocols are learned. <br><br />
'''28th''' – Lab work begins<br><br />
<br><br />
<br />
'''JULY'''<br><br />
<br />
'''1st''' – International student, Min L. arrives from China. Source plasmids arrive for transformation<br><br />
'''6th''' – Primers were incorrect so all labwork had to be redone and primers had to be reordered<br><br />
'''8th''' – Transformations of first bulk source plasmids begin<br><br />
'''9th''' – Tilden Park BBQ with Berkeley<br><br />
'''12th''' – Gel Extraction, Restriction Digests & Colony PCR begin<br><br />
'''13th''' – Minipreps begin<br><br />
'''14th''' – Sequencing of parts begin<br><br />
'''16th''' – Sequences show contamination in CD28, Grb2, and mDAP10 plasmids, team project description due<br><br />
'''19th''' – Ly49 mRNA Contamination, restriction digest failures, and mix ups become prevalent<br><br />
'''22nd''' – Second source plasmid bulk arrives<br><br />
'''23rd''' – Positively sequenced primers are preserved in glycerol.<br><br />
'''24th''' – Ryan, Ethan, and Eric starts first dry run transfection in NKL cell line with anti-Mesothelin CAR for killing assays<br><br />
'''26th''' – AB parts were incorrect and Sam and Connor redo these parts<br><br />
'''28th''' – iCLEM visit<br><br />
'''29th''' – Carmen and Ryan makes BD backbone and retrieve AB-start codon (UCSF iGEM 2009) for ligations<br><br />
'''30th''' – Ryan, Ethan, and Eric starts transfection in NKL cell line with anti-CD19 for killing assays<br><br />
<br><br />
<br />
'''AUGUST'''<br><br />
<br />
'''2nd''' – Ligations of AB, BC, CD parts<br><br />
'''3rd''' – Granule project theories with advisors<br><br />
'''6th''' – Ryan and Min start antibody staining and blocking for killing assay <br><br />
'''7th''' – Killing assay used the wrong plasmids, redo with correct plasmid<br><br />
'''9th''' – Transfection Assay Optimization continues<br><br />
'''16th''' – Ryan, Ethan, Sam and Eric leave for school<br><br />
'''17th''' – Sam and Min begins oligo synthesis<br><br />
'''19th''' – Ethan and Eric continue killing assay with correct plasmid<br><br />
'''22nd''' – Hannah leaves for school<br><br />
'''23rd''' – Granule oligos synthesized<br><br />
'''27th''' – Begin endo-free maxipreps of completed constructs<br><br />
'''30th''' – Start pH sensitive GFP, LIMP, LAMP, and eGFP for granule side project<br><br />
<br><br />
<br />
'''SEPTEMBER'''<br><br />
<br />
'''5th''' – Connor leaves for UCSD for soccer tryouts<br><br />
'''6th''' – pH sensitive GFP, LIMP, LAMP granule project discontinued<br><br />
'''9th''' – Killing assay discontinued, transfection efficiency too low<br><br />
'''20th''' – Connor, Crystal, John, Carmen, and Lianna leave for school<br><br />
'''21st''' – T-cell activation assays begin, presentation draft created<br><br />
'''26th''' – Connor returns from UCSD<br><br />
'''27th''' – Min starts first granule project assay on eGFP<br><br />
<br><br />
<br />
'''OCTOBER'''<br><br />
<br />
'''3rd''' – Min leaves for China<br><br />
'''4th''' – Sam takes over granule project and begins cell imaging<br><br />
'''7th''' – End of construct production, 59 constructs completed from endo-free maxipreps, 17 have been preserved in glycerol<br><br />
'''8th''' – Ryan starts imaging on live KHYG1 for images of killing; results for T-cell activation assays data is revealed, final T-cell activation assay<br><br />
'''12th''' – Preparation for presentation begins<br><br />
'''15th''' – Killing imaging completed<br><br />
'''20th''' – Granule project: eGFP imaging completed<br><br />
'''23th''' – NorCal Jamboree with Stanford, UC Davis, and UC Berkeley iGEM teams<br><br />
'''27th''' – iGEM Wiki freeze<br><br />
'''30th''' – Continue presentation preparation<br><br />
<br />
<br><br />
'''NOVEMBER'''<br><br />
<br />
'''5th''' – iGEM 2010 Jamboree!<br><br />
<br><br />
<br />
{{Template:UCSF/WholeBlockEnd}}<br />
__NOTOC__</div>Mavenhttp://2010.igem.org/Team:UCSF/Notes/TimelineTeam:UCSF/Notes/Timeline2010-10-27T14:06:15Z<p>Maven: </p>
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#customers<br />
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<br />
<br><br />
<h3>Responsibilities of Team Members</h3><br />
<br />
<table id="customers"><br />
<tr><br />
<th>Member</th><br />
<th>Lab Work</th><br />
<th>Other Responsibilities</th><br />
</tr><br />
<br />
<tr><br />
<td>Lianna Fung</td><br />
<td>Cloning, Transfections</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Hannah Yan</td><br />
<td>Cloning</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>Ethan Chan</td><br />
<td>Cloning, Cell Culturing, Transfections,Killing Assay</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Ryan Liang</td><br />
<td>Cloning, Cell Culturing, Transfections, Antibody Staining, Killing Assay, Reporter Assay</td><br />
<td>Wiki Design</td><br />
</tr><br />
<br />
<tr><br />
<td>Crystal Liu</td><br />
<td>Cloning, Killing Assay</td><br />
<td>Presentation, Wiki Design</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Carmen Zhou</td><br />
<td>Cloning, Transfections, Killing Assay</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>John Elam</td><br />
<td>Cloning, Transfections, Killing Assay</td><br />
<td>Presentation</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Connor Grant</td><br />
<td>Cloning</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>Samuel Zorn</td><br />
<td>Cloning, Antibody Staining, Reporter Assay</td><br />
<td>Presentation</td><br />
</tr><br />
<br />
<tr class="alt"><br />
<td>Eric Wong</td><br />
<td>Cloning, Cell Culturing, Transfections, Antibody Staining, Killing Assay</td><br />
<td>Wiki Content</td><br />
</tr><br />
<br />
<tr><br />
<td>Min Lin</td><br />
<td>Cloning, Transfections, Antibody Staining, Reporter Assay</td><br />
<td>Wiki Content, Wiki Design, EVERYTHING</td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
<br><br><br />
<h3>Project Timeline</h3><br />
<br />
</html><br />
<br />
'''FEBRUARY'''<br><br />
<br />
'''17th''' – Arrival of NK cell lines – NKL, YTS/eco, KHYG1<br><br />
'''18th''' – Ryan and Ethan begin NK cell cytotoxicity research on cell lines NKL, KHYG-1, NK92MI & YTS/eco<br><br />
<br><br />
<br />
'''MARCH'''<br><br />
<br />
'''16th''' – Ryan and Ethan starts optimization of NKL cell line<br><br />
'''18th''' – Ryan and Ethan starts optimization of KHYG-1 cell line<br><br />
<br><br />
<br />
'''APRIL'''<br><br />
<br />
'''9th''' – Troubleshooting of FACS readout due to Propidium Iodide staining<br><br />
'''16th''' – Reduction of Propidium Iodide, cell concentration, and event readout, FACS analysis continues.<br><br />
'''20th''' – Ryan and Ethan starts optimization of NK92MI cell line<br><br />
'''22nd''' – Ryan and Ethan starts optimization of YTS/eco cell line<br><br />
'''29th''' – Second round of optimization begins; NK92MI cell line is discontinued <br><br />
'''30th''' – Preparation of presentation for May 4th meeting with other iGEM students<br><br />
<br><br />
<br />
'''MAY'''<br><br />
<br />
'''4th''' – Ryan, Ethan and advisor meet with other iGEM students from Abraham Lincoln High School<br><br />
'''13th''' – Ethan and Ryan gain full responsibility of NKL, KHYG-1, YTS/eco & K562 cell lines<br><br />
'''18th''' – Arrival of Anti-Meso/CD19 plasmids from Mike Milone (University of Pennsylvania)<br><br />
<br><br />
<br />
'''JUNE'''<br><br />
<br />
'''11th''' – Arrival of K562 cell line from Mike Milone (UPenn)<br><br />
'''14th''' – Full local iGEM team arrive at UCSF and begin 2-week boot camp session.<br><br />
'''15th''' – Seminar by Raquel G. on immune response, signaling cascades, feedback loops, receptor adaptors, and general cancer detection<br><br />
'''16th''' – Seminar by Derek W. on cellular cytoskeleton, actin system, microtubules and other related proteins<br><br />
'''17th''' - Seminar by Daniel H. on cell death, killing process, cytotoxic agents and inducers of apoptosis<br><br />
'''18th''' – Seminar by Reid W. on logic gates, combinatorial qualities of proteins and behavioral changes<br><br />
'''21st''' – Seminar by David P. on modularity, synthetic biology, and Boolean gates<br><br />
'''22rd''' – iGEM Team challenge; brainstorming and consulting with advisors and grad students<br><br />
'''24th''' – General project in mind: granzyme linking, stronger signaling and greater arsenal<br><br />
'''25th''' – Lab safety training begins, first set of primers are designed, first set of source plasmids are ordered, and lab protocols are learned. <br><br />
'''28th''' – Lab work begins<br><br />
<br><br />
<br />
'''JULY'''<br><br />
<br />
'''1st''' – International student, Min L. arrives from China. Source plasmids arrive for transformation<br><br />
'''6th''' – Primers were incorrect so all labwork had to be redone and primers had to be reordered<br><br />
'''8th''' – Transformations of first bulk source plasmids begin<br><br />
'''9th''' – Tilden Park BBQ with Berkeley<br><br />
'''12th''' – Gel Extraction, Restriction Digests & Colony PCR begin<br><br />
'''13th''' – Minipreps begin<br><br />
'''14th''' – Sequencing of parts begin<br><br />
'''16th''' – Sequences show contamination in CD28, Grb2, and mDAP10 plasmids, team project description due<br><br />
'''19th''' – Ly49 mRNA Contamination, restriction digest failures, and mix ups become prevalent<br><br />
'''22nd''' – Second source plasmid bulk arrives<br><br />
'''23rd''' – Positively sequenced primers are preserved in glycerol.<br><br />
'''24th''' – Ryan, Ethan, and Eric starts first dry run transfection in NKL cell line with anti-Mesothelin CAR for killing assays<br><br />
'''26th''' – AB parts were incorrect and Sam and Connor redo these parts<br><br />
'''28th''' – iCLEM visit<br><br />
'''29th''' – Carmen and Ryan makes BD backbone and retrieve AB-start codon (UCSF iGEM 2009) for ligations<br><br />
'''30th''' – Ryan, Ethan, and Eric starts transfection in NKL cell line with anti-CD19 for killing assays<br><br />
<br><br />
<br />
'''AUGUST'''<br><br />
<br />
'''2nd''' – Ligations of AB, BC, CD parts<br><br />
'''3rd''' – Granule project theories with advisors<br><br />
'''6th''' – Ryan and Min start antibody staining and blocking for killing assay <br><br />
'''7th''' – Killing assay used the wrong plasmids, redo with correct plasmid<br><br />
'''9th''' – Transfection Assay Optimization continues<br><br />
'''16th''' – Ryan, Ethan, Sam and Eric leave for school<br><br />
'''17th''' – Sam and Min begins oligo synthesis<br><br />
'''19th''' – Ethan and Eric continue killing assay with correct plasmid<br><br />
'''22nd''' – Hannah leaves for school<br><br />
'''23rd''' – Granule oligos synthesized<br><br />
'''27th''' – Begin endo-free maxipreps of completed constructs<br><br />
'''30th''' – Start pH sensitive GFP, LIMP, LAMP, and eGFP for granule side project<br><br />
<br><br />
<br />
'''SEPTEMBER'''<br><br />
<br />
'''5th''' – Connor leaves for UCSD for soccer tryouts<br><br />
'''6th''' – pH sensitive GFP, LIMP, LAMP granule project discontinued<br><br />
'''9th''' – Killing assay discontinued, transfection efficiency too low<br><br />
'''20th''' – Connor, Crystal, John, Carmen, and Lianna leave for school<br><br />
'''21st''' – T-cell activation assays begin, presentation draft created<br><br />
'''26th''' – Connor returns from UCSD<br><br />
'''27th''' – Min starts first granule project assay on eGFP<br><br />
<br><br />
<br />
'''OCTOBER'''<br><br />
<br />
'''3rd''' – Min leaves for China<br><br />
'''4th''' – Sam takes over granule project and begins cell imaging<br><br />
'''7th''' – End of construct production, 59 constructs completed from endo-free maxipreps, 17 have been preserved in glycerol<br><br />
'''8th''' – Ryan starts imaging on live KHYG1 for images of killing; results for T-cell activation assays data is revealed, final T-cell activation assay<br><br />
'''12th''' – Preparation for presentation begins<br><br />
'''15th''' – Killing imaging completed<br><br />
'''20th''' – Granule project: eGFP imaging completed<br><br />
'''23th''' – NorCal Jamboree with Stanford, UC Davis, and UC Berkeley iGEM teams<br><br />
'''27th''' – iGEM Wiki freeze<br><br />
'''30th''' – Continue presentation preparation<br><br />
<br />
<br><br />
'''NOVEMBER'''<br><br />
<br />
'''5th''' – iGEM 2010 Jamboree!<br><br />
<br><br />
<br />
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__NOTOC__</div>Maven