Identification of a Metacaspase protein substrate conserved among early divergent eukaryotic cells

BOUVIER, LEÓN ALBERTO; NIEMIROWICZ, GABRIELA TERESA; D'ALESSIO, CECILIA; CAZZULO, JUAN JOSÉ; ALVAREZ, VANINA EDER

Rosario

Encuentro; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Metacaspases, distant relatives of metazoan caspases, have been shown to participate in programmed cell death, progression of the cell cycle and removal of protein aggregates in unicellular eukaryotes. However, since natural proteolytic substrates have scarcely been identified to date, their roles in these processes remain unclear. To find metacaspase?s putative substrates in Trypanosoma cruzi we tested 14 enzyme interactors previously identified by copurification and mass spectrometry. Each one was co-expressed with metacaspase 5 (TcMCA5) using a dual-vector E. coli system. By this method we identified for the first time a TcMCA5 protein substrate. The cleavage site, determined by N-terminal Edman sequencing of fragments produced in vitro with both recombinant purified proteins, presented an Arg residue upstream the hydrolyzed peptide bond matching perfectly the known metacaspase specificity. Moreover, replacement of this residue by Ala completely prevented cleavage. Similar results were obtained for T. brucei and budding yeast metacaspase orthologs on their respective substrates. Interestingly, in each case cleavage occurs at a linker region that connects different domains. The in vivo proteolytic event is currently being studied in T. brucei, and wild type or metacaspase null mutant yeasts.