Protein Characterization and Mutant Studies of ScMC1-5Δpro

Metacaspases belong to a class of cysteine peptidase enzymes and are known to be involved in several programmed cell death (PCD) mechanisms in plants and lower eukaryotes. Similar to caspases, metacaspases contain a cysteine-histidine catalytic dyad and can undergo autoprocessing for increased activity. However, these proteases cleave after arginine or lysine residues, while caspases are aspartate specific. Previous students in the Fox lab have identified five metacaspase genes from the split-gilled fungus Schizophyllum commune labeled Schizophyllum commune Metacaspase 1-5 (ScMC1-5). These enzymes belong to a subset of metacaspases containing a N-terminal prodomain. All genes for ScMC1-5 have been cloned with and without the prodomain (Δpro) into a vector construct, expressed, purified and tested for enzymatic activity. This project focuses on characterizing all ScMC1-5Δpro enzymes by identifying optimal pH and Ca²⁺ conditions, determining critical protein sites, and understanding enzyme-substrate specificity. Optimal pH and Ca²⁺ conditions and substrate specificity was determined using a metacaspase activity assay. Incubation of our enzyme with a fluorogenic peptide, Z-GlyGlyArg-AMC, results in cleavage after the arginine residue and measureable fluorescence correlated with enzymatic activity. ScMC1-5Δpro have optimal activity at similar pH valueswhen compared with metacaspases that have been characterized in other organisms. Only ScMC1Δpro is completely inactive at 0 mM calcium. ScMC1&5Δpro have a much higher optimal concentration (50 mM), compared to ScMC2-4Δpro (5 mM), suggesting that calcium may have different binding affinities and play different roles in each enzyme. All tested metacaspases showed arginine and lysine specificity, with higher efficiency for arginine. Mutations in the ScMC4Δpro gene were also introduced using site-directed mutagenesis to study protein active sites and cleavage patterns. Because metacaspases are not present in animals, understanding their roles in programmed cell death can contribute to the development of safer antibiotics and antifungals.