Redox modulation of thimet oligopeptidase activity by hydrogen peroxide

Abstract

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 (H₂O₂). Cells from a human embryonic kidney cell line (HEK293) underwent an ischemia/reoxygenation-like condition known to increase H₂O₂ 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 H₂O₂ 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 H₂O₂ concentration generated in reoxygenated cells and mitoplasts suggested a possible modulation of rTOP activity dependent on the concentration of H₂O₂. The measure of pure rTOP activity as a function of H₂O₂ concentration corroborated this hypothesis. The data fitted to an asymmetrical bell-shaped curve in which the optimal activating H₂O₂ 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, H₂O₂ oxidation produced sulfenic acid and maintained rTOP in the monomeric form. Consistent with the involvement of rTOP in a signaling redox cascade, the H₂O₂-oxidized rTOP reacted with dimeric thioredoxin-1 (TRx-1) and remained covalently bound to one subunit of TRx-1.