Difference between revisions of "Part:BBa K4165004"

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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

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

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 (you can find it in the code section) beside with the estimated results from the wet lab.

               Figure 9. this figure shows the results from the transcription and translation code showing 
               the variation of mRNA and protein concentrations 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 (you can find it in the code section of our project).

               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