Auto-proteolysis of Type 1 Metacaspases in Schizophyllum commune

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 they may 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. To further understand the auto-proteolysis of these metacaspases, time course studies were performed on ScMC1-5 with the exclusion of ScMC4. ScMC1, in the presence of calcium, auto-processes to form a 40kDa fragment within a 24 hour period. Assays using small peptide substrates show a decrease in overall activity of ScMC1 over the same time course. Western Blot analysis indicates the cleaved 40kDa fragment does not contain the N-terminal His-tag. While the unprocessed enzyme can digest the protein gelatin, the 40kDa fragment cannot suggesting that the fragment is inactive. Mutation of the active site residues H211Q and C267A 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 already fully processed when purified, and the proteins do not undergo further auto-processing during a 24 hour incubation period with calcium. This is supported by Western Blot analysis in which the intensity of the bands does not change over the time course. 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.