Essential Role of Lysine 375 in a fungal Metacaspase

Cell death occurs in all domains of life. Like other eukaryotes species, fungi undergo programmed cell death (PCD) to control cellular growth. Caspases are proteases functioning as a trigger that stimulates the PCD process, and the metacaspases are known to be an ancestor of the caspase family. Metacaspases are cysteine-dependent proteases in bacteria, algae, fungi, and plants. Most metacaspases are calcium-dependent, undergo autoproteolysis and are cleaved into smaller fragments. Determination of the autoproteolytic cleavage sites is important for two reasons. It is important to recognize how cleavage affects the activity of the enzyme as this may be a form of regulation of metacaspase activity. Practically, it is important to be able to prevent cleavage if we want a homogeneous sample of protein for study. Prior research has been done on cleavage site identification, and mutants have been made at the proposed cleavage sites of the Type I metacaspase from the fungus S. commune (ScMCA-Ia). This specific project will focus mainly on the study of the potential proteolysis site at Lys375 in ScMCA-Ia. Three primary mutations were made, including mutating lysine at 375 to alanine (K375A), a triple mutant that includes mutations at both active site residues in addition to K375A, and a deletion mutant that removed the p10 domain at the C-terminus of the protein. These mutants were used to confirm that cleavage occurs at K375 and that the mutation of K375 to Ala also affects the activity of the metacaspase. Further investigation is still needed for the complete identification of all cleavage sites.