Team:Calgary/Project/misfolding overview

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<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li>
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li>
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<span id="bodytitle"><h1>Misfolding detection circuit overview</h1></span>
<span id="bodytitle"><h1>Misfolding detection circuit overview</h1></span>
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<p> Protein misfolding
 
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<h2 style="color:#0066CC">How Does Protein Misfolding Occur?</h2>
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Protein misfolding can occur as a result of a variety of factors.  Overproduction of proteins in the cell is a good example.  When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein.  Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein.  This can cause it to misfold into a non-functional state.  Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media.  Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example,  it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.
Protein misfolding can occur as a result of a variety of factors.  Overproduction of proteins in the cell is a good example.  When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein.  Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein.  This can cause it to misfold into a non-functional state.  Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media.  Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example,  it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.
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Why do we care?
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<h2 style="color:#0066CC">Why do we care?</h2>
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Protein misfolding is an important topic in mnay regards.  Many diseases, particularly neurodegenerative disorders such as Alzheimer’s Disease and __ result from misfolding proteins.  The production recmonbaint proteins in prokaryoes such as E. Coli can also pose a problem.  Non-native proteins are more susceptible to misfolding.  This can compliacte many lab projectas such as the deisgn of protein drugs.
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Protein misfolding is an important topic in many regards.  Many diseases, particularly neurodegenerative disorders such as prion diseases and Alzhemier's disease result from misfolding proteins.  The production of recombinant proteins in prokaryotes such as E. Coli can also pose a problem.  Non-native proteins are more susceptible to misfolding than native proteins.  This can complicate many lab projects such as the design of peptide based drugs. With this in mind, a detection system for protein misfolding could be a very useful tool.
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How does our system detect protein misfolding?
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Current methods
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<h2 style="color:#0066CC">How does our system detect protein misfolding?</h2>
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<h3>Current methods</h3>
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GFP fusions are a method commonly used to detect protein misfolding.  Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase.  If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output.  If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either,  Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system.  A more recent system has been the use of a split GFP system.  Cabantous et al (2005) describe a system using two fractions of GFP.  The smaller part is fused to the target protein.  The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest.  Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein.   
GFP fusions are a method commonly used to detect protein misfolding.  Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase.  If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output.  If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either,  Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system.  A more recent system has been the use of a split GFP system.  Cabantous et al (2005) describe a system using two fractions of GFP.  The smaller part is fused to the target protein.  The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest.  Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein.   
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Our System
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<h3>Our System</h3>
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Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters.  By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels.  Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell.   
Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters.  By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels.  Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell.   
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Our stress promoters
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<h3>Our stress promoters</h3>
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We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli (click here) and one that monitirs stress in the cytoplasm of E. Coli (click here)
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We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli: <a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a>, and one that monitors stress in the cytoplasm of E. Coli: <a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a>
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Cytoplamsic Stress
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The Cytplasmic Stress Pathway
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In the cytoplasm, stress, in particular misfolded protein, is largely regulated through the sigma32 pathway.  Normally, sigma factor 32 is bound to heat shock proteins such as GroE and DnaK.  In the presence of misfolding protein however, these heat shock proteins bind to the misfolded proteins, levaing sigma 32 free to form a complex with RNA Polymerase. This allows for transcription from various sigma-32 dependent promoters, driving the expression of anything downstream,. Many studies have found sigma-32 dependent promoters to be very effective at measuring levels of cytoplasm protein misfolding in E. Coli. One such promoter is the ibpAB promoter, which controls a heat shock operon in E. Coli. 
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The ibpAB Promoter
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The ibpAB promoter contorls the trasncription of two small proteins: ibpA and ibpB.  These are small heat shock proteins called inclusion body binding proteins. In the presence of inclusion bodies within the cytoplasm, they are thought to form mixed complexes, ibpA allowing ibpB to bind to the inclusoon body at higher temperatures.  The binding of these proteins to the misfolded protein lowers its hydrofobicity, previngin firtyher binding of exposed peptide chains, thus stabilizing the protein and mediating its refolding by the DnaK/DnaJ/GrpE chaperone protein system (Matuszewska at al., 2005).  Transcription levels from this promoter have been found to increase the most upon heat shock as compared to other heat shock promoters (Chuang et al., 1993).
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The ibpAB fusion promoter
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We chose to use the ibpAB promoter in our system in order to monitor cytoplasm misfolding .  We specifically chose to use a fusion promoter, which fuses fxsa, another heat shock promter in E. Coli that is not well known, to the ibpAb promoter.  Kraft et al (1997) designed this fusion promoter and found it to be considerably more sensitive to misfoled protein in the cytoplasm than either of the promoters alone.  We coupled this promoter with GFP downstream as our reporter.  We then proceeded to measure GFP output in the presence of folded and msifolded proteins.  For more information please visit our characterization page.
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Latest revision as of 02:43, 28 October 2010

Project Descriptions

Misfolding detection circuit overview

How Does Protein Misfolding Occur?

Protein misfolding can occur as a result of a variety of factors. Overproduction of proteins in the cell is a good example. When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein. Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein. This can cause it to misfold into a non-functional state. Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media. Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example, it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.


Why do we care?

Protein misfolding is an important topic in many regards. Many diseases, particularly neurodegenerative disorders such as prion diseases and Alzhemier's disease result from misfolding proteins. The production of recombinant proteins in prokaryotes such as E. Coli can also pose a problem. Non-native proteins are more susceptible to misfolding than native proteins. This can complicate many lab projects such as the design of peptide based drugs. With this in mind, a detection system for protein misfolding could be a very useful tool.


How does our system detect protein misfolding?

Current methods

GFP fusions are a method commonly used to detect protein misfolding. Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase. If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output. If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either, Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system. A more recent system has been the use of a split GFP system. Cabantous et al (2005) describe a system using two fractions of GFP. The smaller part is fused to the target protein. The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest. Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein.

Our System

Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters. By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels. Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell.

Our stress promoters

We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli: Periplasmic Stress Detectors, and one that monitors stress in the cytoplasm of E. Coli: Cytoplasmic Stress Detectors

Retrieved from "http://2010.igem.org/Team:Calgary/Project/misfolding_overview"