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

Author(s)

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

• Document type: Journal article
• Source: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1865, n. 4, p. 388-394, APR 2017.
• Web of Science Citations: 6

Abstract

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)

• FAPESP's process: 15/11190-0 - Biochemical characterization of caspase-like involved in a cell cycle of simple eukariotic.
• Grantee: Mauricio Ferreira Marcondes Machado
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/20847-0 - Site-specific incorporation of non-natural amino acids in proteins: development of new incorporation technologies and the use in the study of proteins
• Grantee: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Support Opportunities: Regular Research Grants
• FAPESP's process: 12/50191-4 - Synthesis, kinetic studies and applications of substrates and inhibitors for proteolytic enzymes
• Grantee: Maria Aparecida Juliano
• Support Opportunities: Research Projects - Thematic Grants