Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity

Autor(es)

Gilio, Joyce M. ; Marcondes, Marcelo F. ; Ferrari, Debora ; Juliano, Maria A. ; Juliano, Luiz ; Oliveira, Vitor ; Machado, Mauricio F. M.

• Tipo de documento: Artigo Científico
• Fonte: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1865, n. 4, p. 388-394, APR 2017.
• Citações Web of Science: 6

Resumo

Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys(55) and Lys(268) increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys(55) and Lys(268) with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2. (C) 2017 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 15/11190-0 - Caracterização bioquímica de caspases-símile envolvidas no ciclo celular de eucariotos simples
• Beneficiário: Mauricio Ferreira Marcondes Machado
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/20847-0 - Incorporação sítio específica de aminoácidos não naturais em proteínas: desenvolvimento de novas tecnologias de incorporação e aplicação no estudo de proteínas
• Beneficiário: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 12/50191-4 - Síntese, estudo cinético e aplicações de substratos e inibidores de enzimas proteolíticas
• Beneficiário: Maria Aparecida Juliano
• Modalidade de apoio: Auxílio à Pesquisa - Temático