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ScMC4 and ScMC5 are Metacaspases Active in Cleaving Peptides after Arg Residues
Author(s) -
McDonald Molly,
Fox Kristin
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.918.1
Subject(s) - proteases , caspase , deubiquitinating enzyme , cleave , chemistry , biochemistry , plasmid , cysteine , histidine , enzyme , gene , protease , microbiology and biotechnology , programmed cell death , biology , ubiquitin , apoptosis
Apoptosis is a highly organized form of programmed cell death (PCD) that can be initiated as part of development in higher eukaryotes, or in response to environmental stresses, toxic metabolites, and nutrient starvation. Caspases are well studied proteases that play a crucial role in activating PCD, and are found in all kingdoms of life. Metacaspases are caspase‐related proteases that are found in eukaryotes that do not have any caspases, such as plants and fungi. Though both metacaspases and caspases contain a catalytic cysteine‐histidine dyad, metacaspases cleave proteins after a lysine or arginine residue while caspases cleave after an aspartate residue. A subset of metacaspases contain an N‐terminal prodomain that may undergo autocatalysis, and is thought to be required for activity. Five metacaspase genes (ScMC1‐5) have been identified in the complete genome of Schizophyllum commune , a split gilled fungus that is a reported human pathogen. ScMC1 and ScMC3 have been previously characterized, and the genes for ScMC5 both with and without the prodomain (ScMC5Δpro) have been inserted into plasmids for expression. This project focuses on the expression, purification, and characterization of ScMC4 and ScMC5, as well as the development of protocols for determining enzyme concentration. The genes for ScMC4 and ScMC4Δpro were isolated using PCR and were successfully inserted into a plasmid fused with the small ubiquitin modifier (SUMO) gene using Gibson Assembly. Both ScMC4 and ScMC5 with and without the prodomain were expressed and showed activity and kinetic constants comparable to other metacaspases. The activity assay used to measure protein activity utilizes the substrate Z‐GRR‐AMC. To quantify the amount of cleavage, a standard curve of AMC fluorescence relative to concentration was created. A protocol for quantifying the amount of active metacaspase was developed by titrating the active site with the suicide inhibitor valine‐arginine‐proline‐arginine‐fluoromethylketone (VRPR‐FMK). Through these studies, S. commune metacaspases can be further characterized and a more in depth understanding of metacaspase activity can be utilized for the development of antifungal medications.