Metacaspase of Saccharomyces cerevisiae (ScMCA-Ia) presents different catalytic cysteine in a processed and non-processed form

Metacaspases are cysteine proteases belonging to the CD clan of the C14 family. They possess important characteristics, such as specificity for cleavage after basic residues (Arg/Lys) and dependence on calcium ions to exert their catalytic activity. They are defined by the presence of a large subunit (p20) and a small subunit (p10) and are classified into types I, II, and III. Type I metacaspases have a characteristic pro-domain at the N-terminal of the enzyme, preceding a region rich in glutamine and asparagine. In the yeast Saccharomyces cerevisiae, a type I metacaspase is found. This organism encodes a single metacaspase that participates in the process of programmed cell death by apoptosis. The study focuses on cloning, expressing, and mutating Saccharomyces cerevisiae metacaspase (ScMCA-Ia). Mutations in Cys¹⁵⁵ and Cys²⁷⁶ were introduced to investigate autoprocessing mechanisms. Results revealed that Cys¹⁵⁵ plays a crucial role in autoprocessing, initiating a conformational change that activates ScMCA-Ia. Comparative analysis with TbMCA-IIa highlighted the significance of the N-terminal region in substrate access to the active site. The study proposes a two-step processing mechanism for type I metacaspases, where an initial processing step generates the active form, followed by a distinct intermolecular processing step. This provides new insights into ScMCA-Ia's activation and function. The findings hold potential implications for understanding cellular processes regulated by metacaspases. Overall, this research significantly advances knowledge in metacaspase biology.