Induction of reversible cysteine-targeted protein oxidation by an endogenous electrophile 15-deoxy-delta12,14-prostaglandin J2.

We have previously shown that a prostaglandin D(2) metabolite, 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress. J. Biol. Chem. 276, 12076-12083.]. In the present study, to investigate the correlation between the redox regulation and the 15d-PGJ(2)-induced oxidative stress and to establish the cellular mechanism for protection against the endogenous electrophile, we analyzed S-oxidized proteins using biotinylated cysteine as a molecular probe. In addition, the reversible regulation of protein function by S-oxidation/thiolation was characterized in vitro. When human neuroblastoma SH-SY5Y cells were exposed to 15d-PGJ(2), followed by treatment with biotinylated cysteine, 26 proteins, including glycolytic enzymes, cytoskeletal proteins, redox enzymes, and stress proteins, were identified as substrates for reversible cysteine-targeted oxidation. To investigate the regulatory mechanism of protein function by S-oxidation/thiolation, the binding of a low molecular weight thiol (glutathione) to a glycolytic enzyme alpha-enolase was characterized. Treatment of alpha-enolase with the thiol oxidant diamide in the presence of glutathione in vitro resulted in the binding of glutathione to the protein and concomitant loss of the enzymatic activity, whereas the glutathiolation and inactivation of alpha-enolase were fully reversed by dithiothreitol. Mass spectrometric analysis of the tryptic fragments from native and oxidized alpha-enolase identified two cysteine residues, Cys-118 and Cys-388, as the S-oxidation sites, which may play a role in the regulation of the biological activities of the protein and may be regulated by a reversible S-oxidation/thiolation reaction. These results suggest that cysteine-targeted oxidation/thiolation plays a critical role in the regulation of protein function under conditions of electrophile-induced oxidative stress.