Difference between revisions of "Part:BBa K2271105"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2271105 short</partinfo>
 
<partinfo>BBa_K2271105 short</partinfo>
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===Brief introduction===
 +
  [[Image:Artico_p5wt_polar.png|thumb|right|350px|alt=TPR|'''Figure 1:''' TPR domains of the yeasts PEX5 protein.]]
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    <p align="justify">
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      Protein import into the peroxisome is mediated by two so called peroxins &minus; PEX5 and PEX7. PEX5 is the protein that is responsible for most of the protein import into the peroxisomal membrane. It detects the very twelve amino acids at the C-terminus and then mediates the import of the protein attached to it. The PEX5 of <i>Saccharomyces cerevisiae</i> is a 612 amino acid long proteins, that contains seven tetratricopeptide (TPR) regions which are interacting motifs of the receptor (see figure 1).
 +
      <br>
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      Figure 2 shows all steps of the import mechanisms. It starts with the binding of the PTS1, then the transport to the membrane, where PEX5 interacts with PEX13, PEX14 and PEX17 which leads to membrane integration and pore formation of PEX5. Then the interaction with PEX8, which is bound to PEX2, PEX10 and PEX12, causes cargo release into the matrix. Subsequently ubiquitination of PEX5 lead either to receptor recycling or degradation. This depends on the degree of ubiquitination &minus; while mono- or di-ubiquitination cause recycling, polyubiquitination causes degradation.
 +
    </p>
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    [[Image:Artico_p5shuttle.jpeg|thumb|center|500px|'''Figure 2:''' Import mechanism (<cite>Peroxisomal matrix protein import: the transient pore model, Erdmann et al. (2005)</cite>)]]
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===Targeted mutagenesis===
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  [[Image:Artico_mdworkflow.png|thumb|right|150px|'''Figure 3:''' Workflow of our molecular dynamics approach]]
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    <p align="justify">
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      The achievement of an orthogonal import into the peroxisomes was our teams most important objective &minus; we wanted to offer a PEX5 variant with the following features:
 +
    </p>
 +
      <ul>
 +
        <li>
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          Interaction with a new PTS1* signal and no interaction with the natural PTS1 &minus; by implication this means that the wildtype does not interact with the PTS1*
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        </li>
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        <li>
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          Full functionality &minus; cargo release and receptor recycling should still work
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        </li>
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        <li>
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          Protein missfolding should not happen
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        </li>
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      </ul>
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    <p align="justify">
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      We wanted to achieve our aim with the help of molecular dynamics simulations. Therefore, we checked which amino acids of the receptor are interacting with the targeting signal and tried different mutations in this model.
 +
    </p>
 +
    <p align="justify">
 +
      This PEX5 variant was one of the promising candiates for experiments in the laboratory. We ordered the synthesis at IDT and started cloning once we got this part. Our plan for the validation of correct functionality was to tag a fluorescent protein &minus; in this case mTurqouise &minus; with the PTS1* and see if we have right localization.
 +
    </p>
  
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<h1>PEX5</h1>
 
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<h2>Brief introduction</h2>
 
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<p>
 
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PEX5 is one of two proteins that mediate most of the protein import into the peroxisomal matrix. It recognizes the very c-terminal peroxisomal targeting signal 1 (<b>PTS1</b>), then folds itself to a more compact version and interacts with a variety of enzymes.
 
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<br>
 
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The whole process is divided into five steps: binding, transport, docking, translocation and receptor recycling.
 
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</p>
 
  
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<h2>Variant</h2>
 
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<p>
 
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We followed a targeted mutagenesis approach to achieve an orthogonal import mechanism &minus; our PEX5 variant should recognize a non-native PTS1 variant which is not recognized with the wildtype PEX5.
 
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</p>
 
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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<p align="justify">
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  This part should be used in combination with the corresponding PTS1* in a PEX5 knock out strain to obtain an orthogonal import of any protein of interest. With that, one is able to e.g. relocate metabolic pathways into the empty peroxisome. Thus, it prevents interferences, ensures a higher metabolite concentration due to the small volume and increases resistance to toxic substances because of the spatial isolation.
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</p>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2271105 parameters</partinfo>
 
<partinfo>BBa_K2271105 parameters</partinfo>
 
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Latest revision as of 09:48, 29 October 2017


PEX5 variant R15

Brief introduction

TPR
Figure 1: TPR domains of the yeasts PEX5 protein.

Protein import into the peroxisome is mediated by two so called peroxins − PEX5 and PEX7. PEX5 is the protein that is responsible for most of the protein import into the peroxisomal membrane. It detects the very twelve amino acids at the C-terminus and then mediates the import of the protein attached to it. The PEX5 of Saccharomyces cerevisiae is a 612 amino acid long proteins, that contains seven tetratricopeptide (TPR) regions which are interacting motifs of the receptor (see figure 1).
Figure 2 shows all steps of the import mechanisms. It starts with the binding of the PTS1, then the transport to the membrane, where PEX5 interacts with PEX13, PEX14 and PEX17 which leads to membrane integration and pore formation of PEX5. Then the interaction with PEX8, which is bound to PEX2, PEX10 and PEX12, causes cargo release into the matrix. Subsequently ubiquitination of PEX5 lead either to receptor recycling or degradation. This depends on the degree of ubiquitination − while mono- or di-ubiquitination cause recycling, polyubiquitination causes degradation.

Figure 2: Import mechanism (Peroxisomal matrix protein import: the transient pore model, Erdmann et al. (2005))

Targeted mutagenesis

Figure 3: Workflow of our molecular dynamics approach

The achievement of an orthogonal import into the peroxisomes was our teams most important objective − we wanted to offer a PEX5 variant with the following features:

  • Interaction with a new PTS1* signal and no interaction with the natural PTS1 − by implication this means that the wildtype does not interact with the PTS1*
  • Full functionality − cargo release and receptor recycling should still work
  • Protein missfolding should not happen

We wanted to achieve our aim with the help of molecular dynamics simulations. Therefore, we checked which amino acids of the receptor are interacting with the targeting signal and tried different mutations in this model.

This PEX5 variant was one of the promising candiates for experiments in the laboratory. We ordered the synthesis at IDT and started cloning once we got this part. Our plan for the validation of correct functionality was to tag a fluorescent protein − in this case mTurqouise − with the PTS1* and see if we have right localization.


Usage and Biology

This part should be used in combination with the corresponding PTS1* in a PEX5 knock out strain to obtain an orthogonal import of any protein of interest. With that, one is able to e.g. relocate metabolic pathways into the empty peroxisome. Thus, it prevents interferences, ensures a higher metabolite concentration due to the small volume and increases resistance to toxic substances because of the spatial isolation.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 409
  • 1000
    COMPATIBLE WITH RFC[1000]