Programmed cell death (PCD), otherwise known as apoptosis, is implicit across all kingdoms of life especially in the processes of development, aging, reproduction, responses to stress, and maintaining homeostasis. In humans and other animals, apoptosis is mediated by caspases, proteases that cleave other proteins after Asp residues. Plants, fungi, and protozoa do not have caspases, but rather produce homologous proteins known as metascapases. In these organisms, metacaspases are the proteins primarily responsible for programmed cell death. Studying their mechanisms can provide insight into opportunistic fungal infections. In our research, Schizophyllum commune is the model organism for studying metacaspase proteolytic function in fungi. There are two types of metacaspases in S. commune with varying structures and organization (Type I and Type II). Both types contain a larger p20 catalytic region with a Cys-His catalytic dyad similar to casapses, and a smaller p10 region with a conserved sequence. The Type I metacaspases cleave preferentially after Lys and Arg and are activated by calcium ions. The focus of this research project is the characterization and analysis of Type II metacaspases (ScMCA-II) in S. commune. Type II metacaspases have a large linker region between the p20 and p10 subunits, and are hypothesized to be the chief enzyme involved in apoptosis. Previous literature has focused primarily on Type I metacaspases, making the studies of Type II metacaspase especially crucial. There are six Type II metacaspases that have been identified in the fungus S. commune (labelled ScMCAIIa-f). Full-length constructs of ScMCAIIa and ScMCAIIf are expressed at low levels in E. coli and expression is not improved by decreasing the growth temperature. Preliminary results with small amounts of partially-purified protein suggest that S. commune Type II metacaspases are cleaved during expression and are active against the metacaspase substrate Z-GGR-AMC in both the presence and absence of calcium.
