Part:BBa_K4165004

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.