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 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. For HTRA1 protyolitic 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 make it a very suitable candidate to be used and target various diseases caused by certain proteins.

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.

                          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 promotor

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.