Difference between revisions of "Part:BBa K4165004"
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<partinfo>BBa_K4165004 short</partinfo>
 
<partinfo>BBa_K4165004 short</partinfo>
  
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This basic part encodes for Truncated human high temperature requirement A1 which is a serine protease which can degrade a variety of targets including extracellular matrix proteins.
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This basic part encodes for truncated human high-temperature requirement A1 serine protease (HtrA1) which can degrade a variety of targets including extracellular matrix proteins.
  
 
===Usage and Biology===
 
===Usage and Biology===
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The N-terminal domain of homotrimeric HtrA1 has homology to the IGFBP and Kazal proteins, setting it apart from the bacterial HtrA proteases with which it shares a similarity in terms of its trypsin-like catalytic and PDZ domains. The human HtrA1 protein has several different domains, including an N-terminal IGFBP-like module and a Kazal-like module, a protease domain with a trypsin-like structure, and a C-terminal PDZ domain<sup>[1]</sup>.
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This part encodes the truncated monomer of serine protease HtrA1 present in the human brain. This enzyme is involved in many biological functions ranging from regulating the transforming growth factor (TGF) pathway to degrading fibronectin. It mainly consists of four domains (Kazal - IGFBP - PDZ - Catalytic) all of which have different functions. We used the truncated version as it only contains the PDZ and catalytic domain necessary for its proteolytic activity in our system.
  
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The protease domain obtains an allosteric activation signal upon peptide binding via the L3 sensor loop (nomenclature according to refs<sup>[2,3]</sup>. The catalytic triad, the oxyanion hole, and the substrate-specificity pockets <sup>[3,4]</sup> all is present in the active conformation of the enzyme, which is achieved by a rearrangement of L3, which in turn triggers a remodeling of the activation domain (loops L1, L2, and LD). The transition in activity is reversible, allowing for fine-tuned and quick regulation in response to specific stress signals, unlike that seen for traditional serine proteases<sup>[4]</sup>.
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This protease is proven to degrade Tau (BBa_K4165009) and amyloid beta (Aβ) (BBa_K4165005) which are the main two proteins responsible for the pathogenesis of Alzheimer’s Disease (AD). Its presence both intra and extracellularly along with its ATP-independent characteristics make it a very suitable candidate to be used and target various diseases caused by certain proteins.
  
 
Due to the structure and function of HTRA1, we benefited from this system and its features to make our plug sink system.  
 
Due to the structure and function of HTRA1, we benefited from this system and its features to make our plug sink system.  
  
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===Source===
 
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Q92743 in Uniport.
 
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Gene ID: 5654 in NCBI.
 
  
 
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<partinfo>BBa_K4165004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4165004 SequenceAndFeatures</partinfo>
  
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===Features and codon optimized===
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the length of HTRA1 protein is 480 amino acids which contains:
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===Dry Lab Characterization===
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IGFBP N-terminal: 33-100 mediates interaction with TSC2 substrate.
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<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
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Kazal domain: 98-157, Kazal domains are tight binding inhibitors of serine proteases with three conserved disulfide bonds and are exemplified by the pancreatic secretory trypsin inhibitor SPINK1.
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Serine protease: 204 – 364
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After a long time of searching, we couldn't find any model for the HTRA1 monomer which contains whole PDZ domain so we modeled the HTRA1 monomer through multiple modeling tools (Alphafold – TrRrosetta – Rosettafold – iTASSER) to get the best model that we then trimerized using Cluspro server.
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PDZ domain: 365- 467, our proteins tau and Amyloid beta will bind to it and activate the catalytic domain when binding peptide binds to it.
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<html>
 
<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1.png" style="margin-left:200px;" alt="" width="500" /></p>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/picture10.png" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
  
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                                Figure 1.: Predicted 3D structure of truncated HtrA1 trimer
  
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                            Figure 1.: this figure shows HTRA1 system and its domains from uniprot.
 
  
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<p style=" font-weight: bold; font-size:14px;"> Docking </p>
  
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===Dry Lab===
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ΔG = -32.325
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<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
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After long time of searching , we couldn't find any model for the HTRA1 monomer which contain the whole domains so we decided to model the HTRA1 monomer through multiple modeling tools (Alphafold – Trosetta – Rosettafold – itasser) to get the best model with which we continued the whole project, based on that there was no trimer structure for the HTRA1 on registry and the model which we found as a trimer on RCSB had a problem in the amino acid number specially PDZ domain so we decided to trimerize the HTRA1 on cluspro website to get the final model which we continue the project with it.
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1/h1a-htra1-removebg-preview.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                                Figure 2.: Docked structure of HtrA1 with PDZ binding peptide 1
 +
 
 +
 
 +
ΔG = -25
  
 
<html>
 
<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/picture10.png" style="margin-left:200px;" alt="" width="500" /></p>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1/htra1-p0c7l1.jpeg" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
  
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              Figure 2.: the Trimer model of HTRA1 as each color is an indicator for each chain visualized by pymol.
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                                Figure 3.: Docked structure of HtrA1 with SPINK8 inhibitor
  
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<p style=" font-weight: bold; font-size:14px;"> Docking </p>
 
  
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To make sure of each part of the HTRA1 and the whole system, we needed to go through docking, we made docking of the HTRA1 model with the inhibitors to rank the best models of the inhibitors to use (P0C7L1 (BBa_K4165010) and Q8IUB5 (BBa_K4165008)) and to make sure that the inhibitor binds to the Kazal domain.  
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ΔG = -38.18
  
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Then after that we docked the HTRA1 with tau, Amyloid beta, and their binding peptides (BBa_K4165023-BBa_K4165026-BBa_K4165029-BBa_K4165032-BBa_K4165036-BBa_K4165037-BBa_K4165038-BBa_K4165039-BBa_K4165043-BBa_K4165044-BBa_K4165045-BBa_K4165046-BBa_K4165050-BBa_K4165051-BBa_K4165052-BBa_K4165053-BBa_K4165057-BBa_K4165058-BBa_K4165059-BBa_K4165060 for Amyloid-beta and other switches part for tau) to study the structure and the binding affinity with PDZ domain with different linker lengths.
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/q8iub5-htra1.jpeg" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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 +
                                Figure 4.: Docked structure of HtrA1 with WAP inhibitor
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ΔG = -41.09
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1/pep10-htra1.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                                Figure 5.: Docked structure of HtrA1 with Switch 10
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 +
 
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ΔG = -43.15
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1/pep12-htra1.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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 +
                                Figure 6.: Docked structure of HtrA1 with Switch 12
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 +
 
 +
ΔG = -43.15
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 +
<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1/pep12-htra1.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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 +
                                Figure 7.: Docked structure of HtrA1 with Switch 12
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 +
 
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ΔG = -41.04
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1/pep15-htra1.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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 +
                                Figure 8.: Docked structure of HtrA1 with Switch 15
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ΔG = -42.38
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1/pep18-htra1.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
  
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Finally, we docked the Htra1 model with our whole system (from BBa_K4165021 to BBa_K4165050).
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                                Figure 9.: Docked structure of HtrA1 with Switch 18
  
  

Revision as of 20:14, 10 October 2022

Truncated Serine Protease HtrA1

This basic part encodes for truncated human high-temperature requirement A1 serine protease (HtrA1) which can degrade a variety of targets including extracellular matrix proteins.

Usage and Biology

This part encodes the truncated monomer of serine protease HtrA1 present in the human brain. This enzyme is involved in many biological functions ranging from regulating the transforming growth factor (TGF) pathway to degrading fibronectin. It mainly consists of four domains (Kazal - IGFBP - PDZ - Catalytic) all of which have different functions. We used the truncated version as it only contains the PDZ and catalytic domain necessary for its proteolytic activity in our system.

This protease is proven to degrade Tau (BBa_K4165009) and amyloid beta (Aβ) (BBa_K4165005) which are the main two proteins responsible for the pathogenesis of Alzheimer’s Disease (AD). Its presence both intra and extracellularly along with its ATP-independent characteristics make it a very suitable candidate to be used and target various diseases caused by certain proteins.

Due to the structure and function of HTRA1, we benefited from this system and its features to make our plug sink system.


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 NgoMIV site found at 72
  • 1000
    COMPATIBLE WITH RFC[1000]


Dry Lab Characterization

Modeling

After a long time of searching, we couldn't find any model for the HTRA1 monomer which contains whole PDZ domain so we modeled the HTRA1 monomer through multiple modeling tools (Alphafold – TrRrosetta – Rosettafold – iTASSER) to get the best model that we then trimerized using Cluspro server. 

                                Figure 1.: Predicted 3D structure of truncated HtrA1 trimer


Docking

ΔG = -32.325 

                                Figure 2.: Docked structure of HtrA1 with PDZ binding peptide 1


ΔG = -25 

                                Figure 3.: Docked structure of HtrA1 with SPINK8 inhibitor


ΔG = -38.18 

                                Figure 4.: Docked structure of HtrA1 with WAP inhibitor

ΔG = -41.09 

                                Figure 5.: Docked structure of HtrA1 with Switch 10


ΔG = -43.15 

                                Figure 6.: Docked structure of HtrA1 with Switch 12


ΔG = -43.15 

                                Figure 7.: Docked structure of HtrA1 with Switch 12


ΔG = -41.04 

                                Figure 8.: Docked structure of HtrA1 with Switch 15


ΔG = -42.38 

                                Figure 9.: Docked structure of HtrA1 with Switch 18


References

1- Eigenbrot, C., Ultsch, M., Lipari, M. T., Moran, P., Lin, S. J., Ganesan, R., ... & Kirchhofer, D. (2012). Structural and functional analysis of HtrA1 and its subdomains. Structure, 20(6), 1040-1050.

2- Clausen, T., Southan, C. & Ehrmann, M. Mol. Cell 10, 443–455 (2002)

3- Perona, J.J. & Craik, C.S. J. Biol. Chem. 272, 29987–29990 (1997).

4- Truebestein, L., Tennstaedt, A., Mönig, T., Krojer, T., Canellas, F., Kaiser, M., ... & Ehrmann, M. (2011). Substrate-induced remodeling of the active site regulates human HTRA1 activity. Nature structural & molecular biology, 18(3), 386-388.