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

LEÓN A. BOUVIER; GABRIELA T. NIEMIROWICZ; BRIAN PÉREZ; EMIR SALAS SARDUY; JUAN J. CAZZULO; VANINA E. ALVAREZ

Lucca (Barga)

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

Gordon Research Conference

Metacaspases, distant relatives of metazoancaspases, have been shown to participate in programmed cell death, progressionof the cell cycle and removal of protein aggregates in unicellular eukaryotes.However, since natural proteolytic substrates have scarcely been identified todate, their roles in these processes remain unclear. Here, we assessed ifmetacaspase interactors (identified by mass spectrometry as pulled-downproteins from transgenic parasites expressing flag-tagged metacaspases usinganti-Flag agarose resin) could also be substrates of these enzymes. Using adual-vector E. coli system we evaluated proteolytic processing when co-expressingthe potential substrate with the active peptidase and by this method weidentified the proteasome adaptor protein Ddi1. The cleavage site, determinedby N-terminal Edman sequencing of fragments produced in vitro with bothrecombinant purified proteins, presented an Arg residue upstream the hydrolyzedpeptide bond matching perfectly the known metacaspase specificity. Moreover,replacement of this residue by Ala completely prevented cleavage. Similarresults were obtained for T. brucei and budding yeast metacaspase orthologs ontheir respective substrates. Interestingly, in each case cleavage occurs at alinker region that connects different domains. The in vivo proteolytic event andits consequences are currently being studied in T. brucei, and wild type ormetacaspase null mutant yeasts.