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

BOUVIER, LEÓN ALBERTO; NIEMIROWICZ, GABRIELA TERESA; SALAS SARDUY, EMIR; PÉREZ, BRIAN; CAZZULO, JUAN JOSÉ; ALVAREZ, VANINA EDER

Penang

Conferencia; The 9th general meeting of the International Proteolysis Society (IPS 2015 Proteolysis Conference); 2015

International Proteolisis Society

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 putative substrates in Trypanosoma cruzi we tested 14 enzyme interactors previously identified by co-purification and mass spectrometry. Each one was co-expressed with metacaspase 5 (TcMCA5) using a dualvector 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.