Auto-Proteolysis of Type I Metacaspases in Schizophyllum commune

Brianna Cummings, Union College - Schenectady, NY

Abstract

Metacaspases are cysteine proteases found in plants, protozoa and fungi, and are involved in cellular apoptotic pathways. These enzymes contain a catalytic histidine/cystine dyad which is responsible for the cleavage of peptide bonds after lysine and arginine residues, and many require calcium for activation. Type I metacaspases contain a proline-rich N-terminal prodomain that is typically cleaved to achieve the fully active form, and in some cases, continued auto-proteolysis is observed after the prodomain is removed. Within the fungus Schizophyllum commune genome five type I metacaspases (ScMC1-5) were characterized and expressed with an N-terminal His tag. Optimal assay conditions have been determined, and all five metacaspases require calcium ions for optimal activity. Time course studies revealed ScMC1, in the presence of calcium, auto-processes to form a 40 and 37kD fragment. Assays using small peptide substrates show a decrease in overall activity of ScMC1 over the same time course. Western Blot analysis indicates the cleaved fragments contain the N-terminal His-tag. Mutation of R389 and R447 confirmed MC1 auto-processes at both sites, with preference for the latter. While the unprocessed enzyme can digest the protein gelatin, the two fragments cannot, suggesting that they are inactive. Mutation of the active site residues H264Q and C320A produced an enzyme with decreased activity, and upon incubation with calcium, no auto-proteolysis was observed. Incubation of catalytic amounts of wild-type ScMC2-5 with the double mutant ScMC1 showed no proteolysis, indicating cleavage of ScMC1 by other Type I metacaspases does not occur. Time course studies of ScMC2, 3 and 5 indicate that the protein is fully processed when purified, and the proteins do not auto-process further when incubated with calcium. Interestingly, ScMC2 and its fragments showed activity towards the digestion of gelatin, whereas ScMC5 showed no activity. These results have furthered our understanding of metacaspase activation and function, and ultimately their role in fungal apoptosis.