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.

Keywords::

• nitrosylation
• nitric oxide
• superoxide
• hydrogen peroxide
• peroxinitrite
• redox signaling

Acknowledgments

The authors thank Professor S. Pervaiz, National University of Singapore, for proofreading the manuscript and providing valuable feedback and Dr Sharon Lim for generating the preliminary results that led to the present study during the course of her PhD.

Declaration of interest

The authors declare that no competing financial interests exist. The study was supported by a grant from the National Medical Research council of Singapore to MVC (NMRC/1196/2008). MVC is employed by the National University of Singapore, Yong Loo Lin School of Medicine, and KHH was a graduate student from NUS Graduate School for Integrative Science and Engineering (NGS) at the time this study was designed and performed.