Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

ABSTRACT

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved inoligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, andtwo are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalianimmunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterialrecombinant EP24.15 with oxidized glutathione concentration as low as 10 ìM. The in vitro S-glutathionylation of EP24.15 was responsible for itsoxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showedincreased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also showthat EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the proteinoligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization byinterprotein thiol–disulfide exchange.