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| The project |
| Synthetic readout of cellular stress |
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| Project Description |
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The conceptual idea is to use the conformational change in the SNF1 protein complex to establish a FRET (Förster Resonance Energy Transfer) system for cellular stress detection. There are two chromophores tagged at appropriate locations and upon undergoing the conformational change, they come in close proximity such that the emission energy from one can excite the other, thus resulting in different emission energy. There are two possibilities to be tested:
1. To fuse the chromophores to the subunits of the Snf1 complex to build an active readout of the conformational change
2. To fuse the chromophores to a short peptide (SAMS peptide) that can be phosphorylated by Snf1 and is commonly used as a read-out of Snf1 activity.
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• Working package 1: Find positions for fluorescent markers in SNF1
For WP1 all present information about the protein complex is utilized, so that the fluorescent tags do not interfere with active sites or evolutionary conserved regions. Two alternatives should be investigated and presented with regard to present information and distance between fluorophores. The working package is ended with a 3D modeling of the tagging suggestions to further investigate functionality.
• Working Package 2: Decide what fluorescent protein to use
To apply FRET a compatible fluorphore pair is necessary. Also, efficiency and duration in respect to e.g. photonic bleaching should be taken into consideration.
• Working package 3: Find DNA sequence for SAMS-peptide
Beside the two tagging alternative from WP1 a SAMS-peptide will be used. Both ends will be tagged with fluorophores and conformational change should be indicated with FRET-signal during conformational change at phophorylation by active SNF1.
• Working package 4: Decide on plasmid backbone
The plasmid containing fusion protein should be able to amplify in E.coli and also contain a strong promoter for protein expression in yeast. Use VectorNTI to find good alternatives and locate RE sites.
• Working package 5: Design primers for fusion PCR
When WP 1-4 is finished the primer design can be initiated. Primers should be designed so that they have a high melting temperature, preferably around 60°C, with at least 20 bp homology with each side that should be fused. Primers at utter end of the fusion proteins should include restriction enzyme sites with GGCC repetition at the end. After rigorous proofreading the primers should be ordered together with a primer representing the SAMS peptide.
• Working package 6: Acquire plasmid backbone for transfection with fusion protein
Look up how the plasmid backbone from WP4 can be acquired. If it is a common backbone it should be avaible in the plasmid bank at the laboratory, otherwise it should be ordered.
• Working package 7: Perform fusion PCR
When the primers from WP5 has arrived the fusion PCR can be initiated. As a template for the protein subunits in SNF1 genomic DNA from yeast should be used, this should be avaible at the laboratory, and as template for fluorescent proteins dried DNA provided by iGEM will be used. Create a PCR mastermix that includes all ingredients except the DNA template. Each fusion is run in five steps according to the manual and each step is analyzed on agar gel so that the correct fragments are produced.
• Working package 8: Ligate insert into plasmids
After WP7 is successful the fusion protein should be inserted into the plasmid backbone. Use restriction enzymes as decided in WP6 and follow ligation protocol. Afterwards the plasmids should be purified with miniprep, they should be cut again and put on a gel to verify that the insert was successful.
• Working package 9: Amplify transfection plasmids
After WP8 the plasmid need to be amplified before being tranfected into yeast. Tranfect E.coli and amplify over night. Purify the amplified plasmids with a miniprep kit.
• Working package 10: Sporulate diploid yeast strains to acquire Snf1 deletion mutants
Grow diploid yeast strains with Snf1 deletions on sporulation plats to acquire haploid deletion mutants. Use protocol to clear the population from diploids and unwanted haploids.
• Working package 11: Transfect yeast with tagged fusion protein
After WP9-10 the hapoloid yeast deletion strain should be tranfected with the fusion protein plasmid. When the yeast has recovered from transfection the strain should be grown on sucrose overnight to show transfection result.
• Working package 12: Apply FRET and analyze results
By using a microscopy with FRET set up the success of previous WP can be concluded from the presence or absence of a FRET signal. Analyze results and make conclusions together with Marcus Wilhelmsson.
• Working package 13:Write report
WP 13 is running alongside with the other WPs during the whole project. Initially aims and theory is the major work load and as the project progress results and analysis claims the major part of this WP.
• Working package 14: Apply for funding
Also running alongside the other WPs. Funding is initially applied at science funding committees and later, as there are results to be presented, at major companies interested in the possibilities presented by this project. Funding should cover laboratory expenses, travel to Boston and team t-shirts.
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| Theoretical background |
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Cellular stress is sensed by a key protein called AMP-activated protein kinase (AMPK). The AMPK protein complex is conserved among all eukaryotes, including yeast, plants and humans. In humans this is the target of most anti-diabetic drugs in the market today and is also implicated in many other metabolic disorders such as obesity and atherosclerosis and also in developmental processes such as cell cycle and ageing, etc. In yeast, this protein is called SNF1.
This protein functions as a heterotrimer consisting of α, β and γ subunits. The α-subunit contains the catalytic domain (actual kinase) while the β-subunit is the regulatory domain. It response to high levels of AMP and undergoes a conformational change, which exposes the phosphorylation site on the catalytic α-subunit. AMPK is activated by phosphorylation on this conserved site on the α-subunit (shown in the figure below).
Figure 1: (1)Schematic representation of the AMPK heterotrimer. The figure on the left shows the conformation in an inactive state. On the right, the conformational change in the γ-subunit triggers a structural change in the α-subunit, which is phosphorylated. (2) Schematic representation (stereo view) of the heterotrimer core of SNF1. The regulatory sequence of the a-subunit (Snf1) is shown in red and the rest is in yellow; theGBDof the b-subunit (Sip2) is shown in cyan and the rest is in magenta; and the c-subunit (Snf4) is shown in green. The positions of AMP (stick model in black), as observed from our studies and in the S. pombe enzyme9, as well as that of b-cyclodextrin (in grey) as bound in the rat GBD10, are shown for reference.
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| techniques |
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Fusion PCR:
Fusion PCR is a method used to ligate strands of DNA without using restriction enzymes or ligase (Horton 1989). For a three fragment fusion six primers is necessary: Two ordinary primers and four fusion primers. The fusion primers should optimally have at least 20 bp homology with the next fragment (figure.2) and fusion efficiency is highly dependent of GC-content within these regions (Kamochai et al. 2008). In other aspects than the fusion primer design and the number of steps to produce a final product fusion PCR follow ordinary PCR protocol.
Figure 2: Essential steps for fusing three DNA fragments using fusion PCR
1-3: Each fragment is run with primers that results in regions of homology corresponding to the next piece.
4: Two fragments with homology are run together, resulting in fusion and amplification.
5: The product from step 4 is run with the last homology fragment, resulting in fusion and amplification of the final product.
References:
- Kamonchai, Cha-aim., Tomoaki, Fukunaga., Hisashi, Hoshida & Rinji Akada (2009) Reliable fusion PCR mediated by GC-rich overlap sequences. Gene, 434: 43-49.
- Horton, Robert., Hunt, Henry., Ho, Stefan., Pullen, Jeffrey & Pease, Larry (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 77:61-68.
FRET:
FRET (Förster resonance energy transfer) is an electrodynamic phenomenon that occurs between two chromophores, where one is a donor and the other one is an acceptor. The donor molecule, in its excited state, transfers energy to the acceptor molecule through non-radiative dipole-dipole interactions and does not involve the appearance of a photon. The rate of energy transfer between the donor and acceptor depends on the spectral overlap, the quantum yield of the donor, the relative orientation of the transition dipoles and the distance between the molecules. Owing to the distance dependence, it is possible to measure distances between donor molecules and acceptor molecules. The most common application of FRET is to measure the distance between two sites on a macromolecule, typically a protein is covalently labeled with two chromophores. In the case of multi-domain protein, FRET can be used to measure conformational changes that move the domains closer or further apart.
The rate of energy transfer from a donor to an acceptor, kT(r) is given by the expression:
 eq 1
In equation 1, τD is the lifetime of the donor in the absence of acceptor and r is the donor to acceptor distance. The distance at which the resonance energy transfer efficiency is 50 % is called the Förster distance, R0. At this distance, half of the donor molecules are decaying by energy transfer and half are decaying by the radiative and non-radiative rates. This distance is typically in the rage of 20 to 60 Å. The energy transfer efficiency (E) is the fraction of energy absorbed by the donor which is transferred to the acceptor. This fraction is given by equation 2.
 eq 2
Combining these two equations gives an expression for how the energy transfer efficiency varies as the inverse sixth power of the distance between the donor and acceptor molecules, equation 3 (Lakowicz, 2006).
 eq 3
The expression is graphically demonstrated in figure 3. Because of the inverse sixth power dependence on the distance between the donor and acceptor molecule, the curve has a very sharp decline. When designing a biochemical experiment the distance between the donor and acceptor molecule is of great importance. The useful range for measuring FRET is between 0.5 times R0 and 1.5 times R0. For most applications in cell biology, FRET experiments have a relatively binary readout. Measurements will often only be able to distinguish between high-FRET and low-FRET, or simply between the presence and absence of FRET (Kremers, Piston and Davidson, n.d.).
Figure 3: Dependence of energy transfer efficiency (E) on donor to acceptor distance (r).
References:
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| snf1 & ampk |
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SNF1:
SNF1 belongs to a family of protein kinases which is highly conserved in eukaryotes (Hedbacker 2009). The SNF1 homologue in mammalian cells is called AMP- activated protein kinase (AMPK) and it regulates energy balance in mammals. It is therefore an attractive target for drug discovery against for example diabetes and obesity (Amodeo 2007). SNF1 was identified in Saccaromyces cerevisiae in 1981 when the snf1 mutation was found (Carlson 1981).
The main function of SNF1 is to help yeast cells adapt to glucose limitation and it is required for growth on other carbon sources than glucose such as sucrose (Hedbacker 2009). This is how SNF got its name, since SNF is an abbreviation for sucrose-nonfermenting (Hong 2007). SNF1 is consequently important in response to metabolic stress and it also regulates protective mechanisms against different stresses in response to glucose depletion (Hong 2007). Besides the primary role in nutrient stress, it is also important in environmental stresses including sodium ion stress, heat shock, alkaline pH, oxidative stress and genotoxic stress (Hedbacker 2009). SNF1 regulates transcription of many genes and controls the activity of metabolic enzymes involved in fatty acid metabolism and carbohydrate storage (Hong 2007). SNF1 also controls nutrient-responsive cellular developmental processes such as meiosis, sporulation and aging (Hedbacker 2009).
SNF1 is activated when phosphorylated by the upstream kinases Sak 1, Tos 3 and Elm 1 (Hedbacker 2009). Each of all three kinases is enough to activate SNF1 in response to stress but Sak1 has the major role (Hong 2007). SNF1 is activated by glucose limitation but the glucose signal regulating activation of SNF1 is not known (Hong 2007). Hong et al. studied mutants lacking all three kinases and their result was that SNF1 still was activated in response to sodium ion and alkaline stress which according to Hong et al. suggests that stress signals might activate SNF1 by a mechanism that is independent of the upstream kinases. SNF1 is inactivated by the Ref1-GLc7 protein phosphatase 1 (Hedbacker 2009). The SNF1 protein kinase pathway is robust and low levels of SNF1 activity is mostly enough for growth under many different stress conditions (Hong 2007). Apart from activation and deactivation, regulation of SNF1 is also achieved through autoinhibition and through control of the subcellular localization (Hedbacker 2009).
Subunit structure and function:
Like its ortholog AMPK, SNF1 is heterotrimeric, which means that it consists of three subunits. The yeast genome encodes the catalytic alpha subunit Snf1, three different beta subunits, Sip1, Sip2 and Gal83, and the gamma subunit Snf4 (Hedbacker 2009). The heterotrimeric interface is located at residue 531-586 on Snf1, residues 375-412 of Sip2 and residues 38-45 of Snf4 (Amodeo, Rudolph, & Tong, 2007).
Snf1- alpha subunit:
This catalytic subunit spans 633 amino acids and contains 3 major features, namely the kinase domain, the activation site and a regulatory domain (Hedbacker 2009). The regulatory domain consists of the AIS-domain (Auto Inhibitory Sequence) which functions by binding to the kinase domain and thereby silencing the catalytic activity. The kinase domain also contains the activation site of the full protein complex, Thr210, which is phosphorylated by upstream kinases when the cells are exposed to external stress (McCartney & Schmidt 2001).
Beta- subunits:
There are three known types of the beta subunit, Sip1, Sip2 and Gal83 (Hedbacker 2009). All three beta subunits in yeast share a conserved GBD (glucogen binding domain) with their common mammalian ortholog AMPK (Wiatrowski et al 2004). The three different subunits differ in their cellular localization when glucose is limited. Sip1 localizes to the vacuolar membrane, Sip2 remains cytoplasmic while Gal83 localizes to the nucleus (Vincent et al 2001). Due to this the subunits contain a few differing elements, for example the Gal83-subunit contains a nuclear localization signal on the c-terminus (Hedbacker and Carlson 2006)
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Snf4 – gamma subunit:
The Snf4-subunit consits of 2 bateman domains made up of CBS (cystathionine-beta synthase) repeats that enable them to bind to adenosine derivatives (Hedbacker 2009). This subunit is also a part of the SNF1 activity regulating machinery through binding to the AIS -region of the Snf1 subunit when the glucose supply is low. This binding inhibits the autoinhibition of the kinase activity performed by the AIS sequence (Celenza and Carlson 1989).
References:
- Carlson, M., Osmond, B. C., Botstein, D. (1981) Mutants of yeast defective in sucrose utilization. Genetics, 98:25-40.
- Celenza, J. L., Carson, M. (1989) Mutational Analysis of the Saccharomyces cerevisiae SNF1 Protein
Kinase and Evidence for Functional Interaction with the SNF4 Protein. Molecular and cellular biology 9: 5034-5044
- Hedbacker, K., Carlson, M. (2009) SNF1/AMPK pathways in yeast. Frontiers in Bioscience, 13:2408‑2420.
- Hedbacker, K., Carlson, M. (2006) Regulation of the Nucleocytoplasmic Distribution of Snf1-Gal83 Protein Kinase. Eukaryotic Cell, 5: 1950-1956.
- Amodeo, G. A., Rudolph, M. J., Tong, L. (2007) Crystal structure of the heterotrimer core of Saccaromyces cerevisiae AMPK homologue SNF1. Nature, 449:492-495.
- Hong, S-P., Carlson, M. (2007) Regulation of Snf1 Protein Kinase in Response to Environmental Stress. The Journal of Biological Chemistry, 282:16838-16845.
- McCartney, R. R , Schmidt, C. S. (2001) Regulation of Snf1 Kinase Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit. The Journal of Biological Chemistry, 276:36460-36466
- Vincent, O., Townley, R., Kuchin, S. and Carlson, M. (2001) Subcellular localization of the Snf1 kinase is regulated by specific β subunits and a novel glucose signaling mechanism. Genes and Development 15; 1104-1114
- Wiatrowski, H. A., van Denderen, B. J. W, Berkey, C. D., Kemp, B. E., Stapleton, D. and Carlson, M. (2004) Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83 Kinase Pathway by a Glycogen-Independent Mechanism. Molecular and Cellular Biology 24; 352-361
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| safety questions |
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to be continued
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