A propeptide ‘clamp’ mechanism is required for inhibition of Fasciola hepatica Collagenolytic Cathepsin L3

Wed11  Apr10:30am(15 mins)
Where:
Stream 3 - Physics 0.15 Main

Authors

I Pritsch1; I Tikhonova2; H Jewhurst2; O C Drysdale2; K Cwiklinski2; M Molento1; J P Dalton2C M Verissimo2
1 Department of Basic Pathology, Federal University of Parana, Brazil;  2 Queen's University Belfast, UK

Discussion

Cysteine proteinases are important Fasciola hepatica virulence molecules. Among the various cathepsins that F. hepatica expresses, cathepsin L3 (FhCL3) secreted by the newly excysted juveniles (NEJs) is of special interest due to its central role in host invasion. Its unique collagenolytic activity facilitates the rapid passage of the NEJ through the host gut wall. To protect cells and tissues integrity cathepsins are initially produced as inactive zymogens whereby the enzyme-specific N-terminal propeptide (pp) is responsible for regulating the catalytic activity. Accordingly, it has been suggested that propeptides represent a structural template on which to develop specific cathepsin inhibitors. Here, differential immunolocalization of the FhCL3 zymogen and its pp in NEJ’s and immunoblotting of NEJ excretory-secretory products show that most of FhCL3 zymogen is releasing the pp in the parasite gut. We also investigated the inhibitory properties and mechanisms of FhCL3 propeptide (ppFhCL3), revealing that it is a highly potent and selective inhibitor of F. hepatica cathepsin L’s. Using 3-D structural data we made amino acids substitutions in the ppFhCL3 at residues that we found are involved in interaction within the propeptide bind loop (PBL) of the mature enzyme (Tyr46Lys47/Ala46Ala47) or within the active site (Leu66/Gly66). Our enzyme kinetics and inhibitory studies unveiled a ‘clamp’ mechanism that is required for the proper binding and inhibitory activity of the pp to FhCL3. In summary, our results give remarkable insights regarding the propeptide-cathepsin interaction and open up the possibility of exploring these features in order to design new and selective inhibitors for F. hepatica cathepsins.
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