Redox modulation of thimet oligopeptidase activity by hydrogen peroxide.

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a cytosolic mammalian zinc protease that can process a diversity of bioactive peptides. TOP has been pointed out as one of the main postproteasomal enzymes that process peptide antigens in the MHC class I presentation route. In the present study, we describe a fine regulation of TOP activity by hydrogen peroxide (HO). Cells from a human embryonic kidney cell line (HEK293) underwent an ischemia/reoxygenation-like condition known to increase HO production. Immediately after reoxygenation, HEK293 cells exhibited a 32% increase in TOP activity, but no TOP activity was observed 2 h after reoxygenation. In another model, recombinant rat TOP (rTOP) was challenged by HO produced by rat liver mitoplasts (RLMt) alone, and in combination with antimycin A, succinate, and antimycin A plus succinate. In these conditions, rTOP activity increased 17, 30, 32 and 38%, respectively. Determination of HO concentration generated in reoxygenated cells and mitoplasts suggested a possible modulation of rTOP activity dependent on the concentration of HO. The measure of pure rTOP activity as a function of HO concentration corroborated this hypothesis. The data fitted to an asymmetrical bell-shaped curve in which the optimal activating HO concentration was 1.2 nM, and the maximal inhibition (75% about the control) was 1 μm. Contrary to the oxidation produced by aging associated with enzyme oligomerization and inhibition, HO oxidation produced sulfenic acid and maintained rTOP in the monomeric form. Consistent with the involvement of rTOP in a signaling redox cascade, the HO-oxidized rTOP reacted with dimeric thioredoxin-1 (TRx-1) and remained covalently bound to one subunit of TRx-1.