title: Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity.
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 Lys55and Lys268increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55and Lys268with 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.
authors.affiliation: Departmento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 7° andar, São Paulo, Brazil.
authors.forename: Joyce M
authors.initials: JM
authors.lastname: Gilio
authors.affiliation: Departmento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 7° andar, São Paulo, Brazil.
authors.forename: Marcelo F
authors.initials: MF
authors.lastname: Marcondes
authors.affiliation: Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes, Av. Cândido Xavier de Almeida e Souza, 200, Mogi das Cruzes, SP, Brazil.
authors.forename: Débora
authors.initials: D
authors.lastname: Ferrari
authors.affiliation: Departmento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 7° andar, São Paulo, Brazil.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.affiliation: Departmento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 7° andar, São Paulo, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Departmento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 7° andar, São Paulo, Brazil.
authors.forename: Vitor
authors.initials: V
authors.lastname: Oliveira
authors.affiliation: Departmento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 7° andar, São Paulo, Brazil; Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes, Av. Cândido Xavier de Almeida e Souza, 200, Mogi das Cruzes, SP, Brazil. Electronic address: mauriciomachado@umc.br.
authors.forename: Maurício F M
authors.initials: MFM
authors.lastname: Machado
mesh_terms: D019943:Amino Acid Substitution; D020169:Caspases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D020125:Mutation, Missense; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D015800:Protozoan Proteins; D020169:Caspases
keywords: Calcium binding; Kinetic parameters; Metacaspase; Mutations; Structural modification; Substrate specificity