Strict control of metacaspase activity in trypanosomes: a matter of life or death

VANINA E. ALVAREZ

Caxambu - Minas Gerais

Encuentro; XXXII Annual Meeting of the Brazilian Society of Protozoology / XLIII Annual Meeting on Basic Research in Chagas Disease in Caxambu; 2016

Brazilian Society of Protozoology

Apoptosis represents a form of programmedcell death that is critical for the development and homeostasis ofmulticellular organisms. In unicellular organisms, however, its existence aswell as the possible role are still controversial. Despite the presence ofmorphological characteristics compatible with apoptosis, caspase orthologs areabsent in the genomes of these organisms. About a decade ago, sequences with certaindegree of similarity to those of caspases (including the conservation of theCys-His catalytic dyad and a predicted common secondary structure) wereidentified in the genomes of plants, fungi and protozoans, and they weregrouped into a new subfamily of peptidases named metacaspases. The first years of research in the field ofmetacaspases were marked by vast contradictions, mainly due to differentopinions about the biochemical and functional relationship between metacaspasesand caspases. However, it is clear now that metacaspases possess a completelydifferent substrate specificity compared to that of caspases. While caspasescleave peptides and proteins after aspartic acid residues, all metacaspasesstudied so far have specificity towards basic (Arg/Lys) aminoacids. Anotherimportant difference among caspases and metacaspases is that the latter areabsolutely dependent on calcium for activity, a property that has never beendescribed for a caspase to date. In spite of this, they seem to share withcaspases some functions, including the regulation of cell cycle and deathalthough these observations could be related to the recently reported role inproteostasis through the removal of protein aggregates. However, since naturalproteolytic substrates have scarcely been identified to date, the underlyingsignaling mechanisms remain an enigma. Here, I will present our current work on Trypanosoma cruzi metacaspases. To startunveiling the molecular pathways modulated by these enzymes 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 evaluatedproteolytic processing when co-expressing the potential substrate with theactive peptidase and by this method we identified the proteasome adaptorprotein Ddi1. The cleavage site, determined by N-terminal Edman sequencing offragments 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. bruceiand budding yeast metacaspase orthologs on their respective substrates.Interestingly, in each case cleavage occurs at a linker region that connectsdifferent domains. The in vivoproteolytic event and its consequences are currently being studied.