The role of conserved residues of chagasin in the inhibition of cysteine peptidases

dos Reis, Flavia C.G.; Smith, Brian O.; Santos, Camila C.; Costa, Tatiana, F.R.; Scharfstein, Julio; Coombs, Graham H.; Mottram, Jeremy C.; Lima, Ana Paula C.A.

doi:10.1016/j.febslet.2008.01.008

« Previous Next »FEBS Letters
Volume 582, Issue 4 , Pages 485-490, 20 February 2008

The role of conserved residues of chagasin in the inhibition of cysteine peptidases

Edited by Stuart Ferguson

Flavia C.G. dos Reis
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Bloco G, C.C.S., Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil
Brian O. Smith
- Divisions of Biochemistry and Molecular Biology, Institute of Biomedical Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
Camila C. Santos
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Bloco G, C.C.S., Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil
- Institute of Biomedical Life Sciences, Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, UK
Tatiana, F.R. Costa
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Bloco G, C.C.S., Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil
Julio Scharfstein
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Bloco G, C.C.S., Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil
Graham H. Coombs
- Institute of Biomedical Life Sciences, Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, UK
Jeremy C. Mottram
- Institute of Biomedical Life Sciences, Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, UK
Ana Paula C.A. Lima
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Bloco G, C.C.S., Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil
- Corresponding author. Fax: +55 21 2280 8193.

Received 22 November 2007; received in revised form 21 December 2007; accepted 7 January 2008. published online 15 January 2008.

Abstract

We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10–100-fold increases in the K_i for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

Abbreviations: Z-Phe-Arg-MCA, carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin, PBS, phosphate buffered saline, cruzain, recombinant cruzipain truncated at the C-terminal extension, DTT, dithiothreitol, EDTA, ethylenidiaminetetracetic acid disodium salt 2-hydrate, E-64, l-trans-epoxysuccinylleucylamido-(4-guanidino) butane, IPTG, isopropyl-β-d-thiogalactopyranoside