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 high-temperature requirement serine protease HtrA1. It plays important roles in protein quality control as well as the regulation of numerous signaling cascades via substrate degradation. 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. For HTRA1 proteolytic activity, it must be found in its homomultimeric state as a trimer.
  
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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, this makes it a very suitable candidate to be used and target various diseases caused by certain proteins.  
  
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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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<!-- -->
 
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===<span class='h3bb'>Sequence and Features</span>===
 
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<partinfo>BBa_K4165004 SequenceAndFeatures</partinfo>
 
  
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===Features and codon optimized===
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<html>
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the length of HTRA1 protein is 480 amino acids which contains:
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<p><img src="https://static.igem.wiki/teams/4165/wiki/project/description/htra1-domains.png" style="margin-left:200px;" alt="" width="500" /></p>
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IGFBP N-terminal: 33-100 mediates interaction with TSC2 substrate.
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</html>
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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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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.
+
                            Figure 1.: A graphical illustration showing the domains of HtrA1.
 +
 
 +
A more recent study found that HTRA1 may cleave recombinant tau (BBa_K4165009) in vitro into numerous pieces of various sizes, as well as breakdown insoluble and fibrillarized tau, some other studies attempted to boost HTRA1 activity by attaching the PDZ domain to other protease domains such as calpain and short peptides (sequence of H1A), which redeemed successful at the end, in addition, A study proposed that Mass spectrometry identified a comparable sequence of cleavage sites in Aβ-40, Aβ-42, and Amyloid precursor protein intracellular domain (AICD), and in vitro proteolysis tests verified breakdown of the pathogenic Aβ-42 peptide.  
 +
 
 +
We used HtrA1 to assemble a universal switchable system that could be used to target any protein, it is mediated by the presence of a synthetic switch. It is composed of 3 parts connected by different linkers; an HtrA1 PDZ peptide, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.
 +
 
 +
This system could be used to target any type of misfolded protein by changing the targeting peptides in the clamps.
  
 
<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/project/description/htra1.png" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
  
  
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                            Figure 1.: this figure shows HTRA1 system and its domains from uniprot.
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                            Figure 1.: Structural composition of the switchable system.  
  
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===Source===
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<!-- -->
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Q92743 in Uniport.
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Gene ID: 5654 in NCBI.
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===<span class='h3bb'>Sequence and Features</span>===
 +
<partinfo>BBa_K4165004 SequenceAndFeatures</partinfo>
  
  
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===Dry Lab===
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===Dry Lab Characterization===
 
<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
 
<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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After a long time of searching, we couldn't find any model for the HTRA1 monomer which contains the 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 and Multimer docking option that supports dimer and trimer formation. The results showed successful trimerization of the protease when compared to other experimental models, along with successful binding to the inhibitor, and HtrA1 peptides, providing full assembly of the system.
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<html>
 
<html>
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</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 1.: Predicted 3D structure of truncated HtrA1 trimer visualized by Pymol.
 +
 
  
 
<p style=" font-weight: bold; font-size:14px;"> Docking </p>
 
<p style=" font-weight: bold; font-size:14px;"> Docking </p>
 +
We docked the HTRA1 with the peptide binding the PDZ and the inhibitors binding the Catalytic domain to choose from which the top-ranked Inhibitors and peptides were able to follow our criteria, following this we docked it with the top-ranked switches to check the right choice of the linkers, whether the parts maintained its docking energy after linkers addition without adding extra tension to the assembled switch or not. Thus, the results have proven our theory that the flexible linkers didn/t affect the binding of the separate parts to either the PDZ Domain or the Catalytic domain of the HTRA1 
  
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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 = -32.325
  
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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/htra1/h1a-htra1-removebg-preview.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</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 2.: Docked structure of HtrA1 with PDZ binding peptide 1 visualized by Pymol.
  
 +
 +
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ΔG = -25.0
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<html>
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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>
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</html>
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                            Figure 3.: Docked structure of HtrA1 with SPINK8 inhibitor visualized by Pymol.
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 +
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ΔG = -38.18
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 +
<html>
 +
<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 visualized by Pymol.
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 +
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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 Visualized by Pymol.
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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 Visualized by Pymol.
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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>
 +
</html>
 +
 +
                            Figure 7.: Docked structure of HtrA1 with Switch 15 Visualized by Pymol.
 +
 +
 +
 +
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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                              Figure 8.: Docked structure of HtrA1 with Switch 18 Visualized by Pymol.
 +
 +
<html>
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<p style=" font-weight: bold; font-size:14px;"> Mathematical modeling </p>
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<p style=" font-weight: bold; font-size:14px;">Transcription rate and translation rate under T7 promoter </p>
 +
the mathematical modeling was based on our code for the calculation of transcription and translation For more information: <a href="https://2022.igem.wiki/cu-egypt/ProgrammingClub.html">Programming club page code.</a>.</p> beside with the estimated results from the wet lab.
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 +
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/dry-lab/mathematical-modeling/mathematical-modeling/htra12.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
 +
 +
                Figure 9. this figure shows the results from the transcription and translation code, showing
 +
                mRNA and protein concentrations vary with time compared with the wet lab results.
 +
 +
 +
<p style=" font-weight: bold; font-size:14px;"> Enzyme Activity </p>
 +
<html>
 +
The Enzyme activity model was based on the Michaelis-Menten automation in which a MATLAB code was constructed by us to simulate according to this theory For more information: <a href="https://2022.igem.wiki/cu-egypt/ProgrammingClub.html">Programming club page code.</a>.</p>.
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 +
<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-inhibitors/dry-lab/htra1-with-tau-enzyme-activity-non-linear.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
 +
                Figure 10.: Enzyme activity with tau protein showing a high affinity of HtrA1 for tau at very low
 +
                                      concentrations of substrate for Tau (Non-linear curve).
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 +
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-inhibitors/dry-lab/htra1-with-tau-enzyme-activity-line-weaver-burk-plot.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
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 +
                Figure 11.: Enzyme activity with tau protein showing a high affinity of HtrA1 for tau at very low
 +
                                      concentrations of substrate for Tau (Line-Weaver Burk Plot).
 +
 +
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-inhibitors/dry-lab/htra1-with-amyloid-beta-enzyme-activity-non-linear.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                Figure 12.: Enzyme activity with tau protein showing a high affinity of HtrA1 for Amyloid beta at very low
 +
                                      concentrations of substrate for amyloid beta (Non-linear Curve).
 +
 +
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-inhibitors/dry-lab/htra1-with-tau-enzyme-activity-line-weaver-burk-plot.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
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                Figure 13.: Enzyme activity with tau protein showing a high affinity of HtrA1 for amyloid beta at very low
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                                      concentrations of substrate for amyloid beta (Line-Weaver Burk Plot).
  
 
===References===
 
===References===
Line 61: Line 177:
  
 
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.
 
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.
 +
 +
5- Clausen, T., Kaiser, M., Huber, R., & Ehrmann, M. (2011). HTRA proteases: Regulated proteolysis in protein quality control. Nature Reviews Molecular Cell Biology, 12(3), 152–162. https://doi.org/10.1038/nrm3065
 +
 +
6- Rey, J., Breiden, M., Lux, V., Bluemke, A., Steindel, M., Ripkens, K., Möllers, B., Bravo Rodriguez, K., Boisguerin, P., Volkmer, R., Mieres-Perez, J., Clausen, T., Sanchez-Garcia, E., & Ehrmann, M. (2022). An allosteric HTRA1-calpain 2 complex with restricted activation profile. Proceedings of the National Academy of Sciences, 119(14). https://doi.org/10.1073/pnas.2113520119
 +
 +
7- Proteomic analysis of amyloid corneal aggregates from TGFBI-H626R lattice corneal dystrophy patient implicates serine-protease HTRA1 in mutation-specific pathogenesis of tgfbip. (n.d.). https://doi.org/10.1021/acs.jproteome.7b00188.s001
  
  

Latest revision as of 04:06, 14 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 high-temperature requirement serine protease HtrA1. It plays important roles in protein quality control as well as the regulation of numerous signaling cascades via substrate degradation. 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. For HTRA1 proteolytic activity, it must be found in its homomultimeric state as a trimer.

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, this makes it a very suitable candidate to be used and target various diseases caused by certain proteins. 



                           Figure 1.: A graphical illustration showing the domains of HtrA1.

A more recent study found that HTRA1 may cleave recombinant tau (BBa_K4165009) in vitro into numerous pieces of various sizes, as well as breakdown insoluble and fibrillarized tau, some other studies attempted to boost HTRA1 activity by attaching the PDZ domain to other protease domains such as calpain and short peptides (sequence of H1A), which redeemed successful at the end, in addition, A study proposed that Mass spectrometry identified a comparable sequence of cleavage sites in Aβ-40, Aβ-42, and Amyloid precursor protein intracellular domain (AICD), and in vitro proteolysis tests verified breakdown of the pathogenic Aβ-42 peptide.

We used HtrA1 to assemble a universal switchable system that could be used to target any protein, it is mediated by the presence of a synthetic switch. It is composed of 3 parts connected by different linkers; an HtrA1 PDZ peptide, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.

This system could be used to target any type of misfolded protein by changing the targeting peptides in the clamps. 


                           Figure 1.: Structural composition of the switchable 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 the 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 and Multimer docking option that supports dimer and trimer formation. The results showed successful trimerization of the protease when compared to other experimental models, along with successful binding to the inhibitor, and HtrA1 peptides, providing full assembly of the system. 


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


Docking

We docked the HTRA1 with the peptide binding the PDZ and the inhibitors binding the Catalytic domain to choose from which the top-ranked Inhibitors and peptides were able to follow our criteria, following this we docked it with the top-ranked switches to check the right choice of the linkers, whether the parts maintained its docking energy after linkers addition without adding extra tension to the assembled switch or not. Thus, the results have proven our theory that the flexible linkers didn/t affect the binding of the separate parts to either the PDZ Domain or the Catalytic domain of the HTRA1

ΔG = -32.325 

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


ΔG = -25.0 

                           Figure 3.: Docked structure of HtrA1 with SPINK8 inhibitor visualized by Pymol.


ΔG = -38.18 

                           Figure 4.: Docked structure of HtrA1 with WAP inhibitor visualized by Pymol.


ΔG = -41.09 

                            Figure 5.: Docked structure of HtrA1 with Switch 10 Visualized by Pymol.


ΔG = -43.15 

                            Figure 6.: Docked structure of HtrA1 with Switch 12 Visualized by Pymol.


ΔG = -41.04 

                            Figure 7.: Docked structure of HtrA1 with Switch 15 Visualized by Pymol.


ΔG = -42.38 

                             Figure 8.: Docked structure of HtrA1 with Switch 18 Visualized by Pymol.

Mathematical modeling

Transcription rate and translation rate under T7 promoter

the mathematical modeling was based on our code for the calculation of transcription and translation For more information: Programming club page code..

beside with the estimated results from the wet lab. 


               Figure 9. this figure shows the results from the transcription and translation code, showing 
               mRNA and protein concentrations vary with time compared with the wet lab results.


Enzyme Activity

The Enzyme activity model was based on the Michaelis-Menten automation in which a MATLAB code was constructed by us to simulate according to this theory For more information: Programming club page code..

. 

               Figure 10.: Enzyme activity with tau protein showing a high affinity of HtrA1 for tau at very low 
                                      concentrations of substrate for Tau (Non-linear curve).



               Figure 11.: Enzyme activity with tau protein showing a high affinity of HtrA1 for tau at very low 
                                      concentrations of substrate for Tau (Line-Weaver Burk Plot).



               Figure 12.: Enzyme activity with tau protein showing a high affinity of HtrA1 for Amyloid beta at very low 
                                      concentrations of substrate for amyloid beta (Non-linear Curve).



               Figure 13.: Enzyme activity with tau protein showing a high affinity of HtrA1 for amyloid beta at very low 
                                      concentrations of substrate for amyloid beta (Line-Weaver Burk Plot).

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.

5- Clausen, T., Kaiser, M., Huber, R., & Ehrmann, M. (2011). HTRA proteases: Regulated proteolysis in protein quality control. Nature Reviews Molecular Cell Biology, 12(3), 152–162. https://doi.org/10.1038/nrm3065

6- Rey, J., Breiden, M., Lux, V., Bluemke, A., Steindel, M., Ripkens, K., Möllers, B., Bravo Rodriguez, K., Boisguerin, P., Volkmer, R., Mieres-Perez, J., Clausen, T., Sanchez-Garcia, E., & Ehrmann, M. (2022). An allosteric HTRA1-calpain 2 complex with restricted activation profile. Proceedings of the National Academy of Sciences, 119(14). https://doi.org/10.1073/pnas.2113520119

7- Proteomic analysis of amyloid corneal aggregates from TGFBI-H626R lattice corneal dystrophy patient implicates serine-protease HTRA1 in mutation-specific pathogenesis of tgfbip. (n.d.). https://doi.org/10.1021/acs.jproteome.7b00188.s001