Metacaspase-mediated cleavage of the proteasome adaptor protein Ddi1: possible consequences on cell proteostasis.

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

Renaissance Tuscany Il Ciocco Lucca (Barga)

Congreso; Gordon Research Conference-Proteolytic Enzymes & Their Inhibitors. Exploring and Exploiting Proteases in Development and Disease.; 2016

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. Here, we assessed if metacaspase interactors (identifi¬ed by mass spectrometry as pulled-down proteins from transgenic parasites expressing flag-tagged metacaspases using anti-Flag agarose resin) could also be substrates of these enzymes. Using a dual-vector E. coli system we evaluated proteolytic processing when co-expressing the potential substrate with the active peptidase and by this method we identified the proteasome adaptor protein Ddi1. The cleavage site, determined by N-terminal Edman sequencing of fragments produced in vitro with both recombinant purifi¬ed 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 orthologues on their respective substrates. Interestingly, in each case cleavage occurs at a linker region that connects different domains. The in vivo proteolytic event and its consequences are currently being studied in T. brucei, and wild type or metacaspase null mutant yeasts.