ScMC4 is a Metacaspase Active in Cleaving Peptides after Arg Residues

Apoptosis is a form of programmed cell death (PCD) that is carefully regulated in all types of cells. Caspases are well-studied proteases, proteins that cleave other proteins, that have a role in activating PCD in mammals. Metacaspases are related proteins that have not been as well characterized as caspases, but are believed to also have a role in activating PCD in plants, fungi and protozoa. Based on current evidence, both caspases and metacaspases require a cysteine-histidine dyad to cleave protein substrates. A key difference between caspases and metacaspases is their substrate specificity, namely that caspases cleave protein substrates after aspartate amino acids while metacaspases cleave after lysine and arginine. A subset of metacaspases include a prodomain at the N-terminus of the protein, and the removal of this prodomain may be a prerequisite for activity. Five metacaspase genes, ScMC1-5, have been identified in the complete genome of Schizophyllum commune, a fungus and reported human pathogen. This project focuses on the expression, purification, and characterization of active ScMC4. The genes coding for the protein ScMC4 with and without the prodomain (ScMC4nopro) have been successfully inserted into plasmids for expression. Active forms of ScMC4 and ScMC4nopro have been expressed, and kinetic constants have been determined for ScMC4nopro. The optimal concentration of calcium and optimal pH have also been determined for ScMC4nopro. To further characterize these metacaspases, a protocol for determining the concentration of enzyme has been developed by titrating the active sites with a suicide inhibitor. Through a combination of these methods, the metacaspases found in S. commune can be further characterized, and this information could be used to develop antifungal pharmaceuticals targeting metacaspase proteins.