Free ATP inhibits thimet oligopeptidase (EC 3.4.24.15) activity, induces autophosphorylation in vitro, and controls oligopeptide degradation in macrophage

The fate of the proteasome-generated peptides depends upon the cytosolic pe ptidases whose activities ought to be regulated. One of the most important oligopeptide-degrading and -binding proteins in the cytosol is the thimet o ligopeptidase (EC 3.4.24.15), ubiquitously found in mammallian tissues. To date, there is no indication whether thimet oligopeptidase activities are p hysiologically regulated. Here, we present evidences suggesting that the co ncentration of unbound ATP in the cytosol regulates the thimet oligopeptida se activities both, in vitro and ex vivo. To perform these studies two olig opeptides were used: a quenched fluorescent peptide, which is susceptible t o thimet oligopeptidase degradation, and the ovalbumin(257-264) (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopepti dase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect t he peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a sit e directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo-peptidase motif. Altogether, the results presented here suggest that Zn2+ at the active cent er of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP may help to explain the fate of the proteasomal-gene rated oligopeptides in the cytosol.