| Texto completo | |
| Autor(es): |
Duarte, Ane Caroline Moreira
;
Oliveira, Vinicius Henrique De
;
Costa, Joao Pedro Martins Silva
;
Trujilho, Mariana Nascimento Romero
;
Judice, Wagner Alves De Souza
;
Machado, Mauricio Ferreira Marcondes
Número total de Autores: 6
|
| Tipo de documento: | Artigo Científico |
| Fonte: | BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1874, n. 2, p. 8-pg., 2026-02-01. |
| Resumo | |
Metacaspases are cysteine proteases structurally related to caspases, widely distributed in plants, fungi, and protozoa, but absent in metazoans. In Trypanosoma cruzi, the etiological agent of Chagas disease, the functional and biochemical properties of metacaspases remain poorly understood. In this study, we performed a detailed characterization of the metacaspase TcMCA3, focusing on its expression, purification, calcium-dependent processing, and enzymatic activity. SDS-PAGE and western blotting revealed that TcMCA3 is primarily expressed in a processed form and undergoes further autoproteolytic cleavage in the presence of calcium. Kinetic analyses showed that calcium activates TcMCA3, enhancing catalytic efficiency (kcat/KM) and turnover rate (kcat) up to 1 mM CaCl2, beyond which activity decreases, likely due to autodegradation. Optimal activity was observed at pH 8.5 and 25 mM NaCl, suggesting a requirement for mildly alkaline and moderate ionic strength environments. Fluorescence spectroscopy confirmed that TcMCA3 undergoes conformational changes upon calcium binding, with a high-affinity site responsible for structural activation. We observed that calcium-dependent processing correlates with changes in catalytic activity, suggesting structural and functional differences between TcMCA3 isoforms. Overall, our findings reveal that TcMCA3 activation is tightly regulated by calcium, pH, and ionic strength, and that structural rearrangements induced by calcium binding are essential for its enzymatic function. These findings advance our understanding of the regulatory mechanisms of metacaspases in protozoan parasites and may help inform future evaluation of TcMCA3 as a potential target for therapeutic strategies against T. cruzi. (AU) | |
| Processo FAPESP: | 22/06394-0 - Obtenção e caracterização bioquímica de metacaspases recombinantes de protozoários de interesse clínico |
| Beneficiário: | Mauricio Ferreira Marcondes Machado |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |
| Processo FAPESP: | 21/01503-2 - Avaliação dos efeitos de glicosaminoglicanos na atividade de inibidores de cisteíno proteases catepsinas |
| Beneficiário: | Wagner Alves de Souza Júdice |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |