Unexpected Activity of a Novel Kunitz-type Inhibitor: INHIBITION OF CYSTEINE PROTEASES BUT NOT SERINE PROTEASES.

Autor:	Smith D; From the School of Biological Sciences and dsmith37@qub.ac.uk., Tikhonova IG; School of Pharmacy, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom., Jewhurst HL; From the School of Biological Sciences and., Drysdale OC; From the School of Biological Sciences and., Dvořák J; From the School of Biological Sciences and., Robinson MW; From the School of Biological Sciences and., Cwiklinski K; From the School of Biological Sciences and., Dalton JP; From the School of Biological Sciences and.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2016 Sep 09; Vol. 291 (37), pp. 19220-34. Date of Electronic Publication: 2016 Jul 15.
DOI:	10.1074/jbc.M116.724344
Abstrakt:	Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu(15) within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu(15)/Arg(15)) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu(15) in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host. (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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