Formation of protein S-nitrosylation by reactive oxygen species.

ABSTRACT

In the present study, the formation of whole cellular S-nitrosylated proteins (protein-SNOs) by the reactive oxygen species (ROS), hydrogen peroxide (H2O2), and superoxide (O2(•-)) is demonstrated. A spectrum of protein cysteine oxidative modifications was detected upon incubation of serum-starved mouse embryonic fibroblasts with increasing concentrations of exogenous H2O2, ranging from exclusive protein-SNOs at low concentrations to a mixture of protein-SNOs and other protein oxidation at higher concentrations to exclusively non-SNO protein oxidation at the highest concentrations of the oxidant used. Furthermore, formation of protein-SNOs was also detected upon inhibition of the antioxidant protein Cu/Zn superoxide dismutase that results in an increase in intracellular concentration of O2(•-). These results were further validated using the phosphatase and tensin homologue, PTEN, as a model of a protein sensitive to oxidative modifications. The formation of protein-SNOs by H2O2 and O2(•-) was prevented by the NO scavenger, c-PTIO, as well as the peroxinitrite decomposition catalyst, FETPPS, and correlated with the production or the consumption of nitric oxide (NO), respectively. These data suggest that the formation of protein-SNOs by H2O2 or O2(•-) requires the presence or the production of NO and involves the formation of the nitrosylating intermediate, peroxinitrite.