Function and molecular interactions of the Fasciola hepatica cathepsin L5 protease
Creators: name: Yap, Huan Yong
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310106
subject: Enzymes
classificationCode: 310112
subject: Structural biology (incl. macromolecular modelling)
classificationCode: 310299
subject: Bioinformatics and computational biology not elsewhere classified

description: Fasciolosis is a parasitic disease that damages the liver of the infected host and is caused by the migration and feeding activity of a trematode – Fasciola hepatica in temperate regions, or Fasciola gigantica in tropical regions, which mainly affects domestic ruminants. Fasciolosis has inflicted a significant economic burden on the agriculture sector worldwide due to the reduction of meat and milk production. Humans are susceptible to fasciolosis from ingestion of metacercaria on contaminated vegetables. Triclabendazole is the drug of choice used to control fasciolosis and is the only drug recommended by the WHO to treat human fasciolosis. Triclabendazole resistance in Fasciola has emerged however, and has been reported in many countries including Australia. Hence, new drugs/inhibitors against fasciolosis are needed. Cathepsin L5 is one of the cathepsin L isoforms secreted by F. hepatica while residing in the liver tissue and bile ducts. The specific substrates of other isoforms that are co-expressed with cathepsin L5 are known to be involved in parasite migration and feeding, while the specific natural substrates of cathepsin L5 are not known.<br><br>The focus of this project was therefore to inspect the potential specific natural substrates of cathepsin L5 that are involved in host immunomodulation, and the determinants of its substrate preference. In Chapter 3, recombinant cathepsin L1, L2, and L5 were expressed in the yeast Saccharomyces cerevisiae while the helminth defence molecule (HDM) was expressed in bacteria Escherichia coli. Subsequently, the cleavage efficiency of the isoforms to activate HDM were assessed at pH 4.5 and pH 7.3. Cathepsin L variants with point mutations were created to assess the involvement of the residue 163. Furthermore, in Chapters 4 &amp; 5, comparative molecular dynamics simulations were performed on apo and substrate-bound cathepsin L1 and L5 to identify differences in their structures and enzyme-ligand interactions and to propose further potential determinants of the substrate preference of cathepsin L5. In Chapter 6, three microRNA-adapted short hairpin RNAs (sh42, sh313 and sh420) aiming to knock-down cathepsin L5 in vivo were developed as potential tools for future study on the function of cathepsin L5 in F. hepatica.<br><br>The findings of this study confirmed that cathepsin L5 is most likely the specific enzyme to activate HDM and the activation site is predicted to be within the gut lumen of the parasite (pH 4.5). Furthermore, the results of enzyme kinetic assays using Z-Asp-Arg-AMC fluorogenic substrate and HDM digestion analysis suggests that a glycine at position 163 is crucial to the P2 Asp preference of cathepsin L5 however it is not the only determinant. Based on simulation results, including the contributions of individual residues to the overall binding free energy, it is proposed that in addition to position 163, a cluster of residues in its vicinity might also contribute to the differential substrate preference of the two cathepsin isoforms studied. MicroRNA-adapted shRNAs carried in a lentivector (pGIPZ) to knock-down F. hepatica L5 were developed for the first time however the knock-down effects were not specific. In conclusion, the findings from this study provide crucial information that could lead to the development of new drug/inhibitor against fasciolosis.