86 - Are S-Nitrosothiols Predominantly Stable End-Effectors of Protein Function or Transient Intermediates Leading to Disulfide Formation?

Abstract

S-nitrosation is considered to be a ubiquitous, stable post-translational modification that directly targets and modulates a vast number of proteins. This widespread role appears incompatible with the inherent lability of the nitroso bond and its propensity to rapidly react with thiols to generate disulfide bonds. We present evidence that S-nitrosothiols primarily exist as transient intermediates leading to disulfides. Exposure of primary rat aortic smooth muscle cells (SMCs) to the nitrosating reagent S-nitrosocysteine (CysNO) generated robust and widespread protein S-nitrosation. However, CysNO also induced disulfides in multiple candidate proteins which preceded a significant increase in widespread protein S-nitrosation. Quantitative analysis of SMCs exposed to CysNO demonstrated that disulfides, not S-nitrosothiols, were the dominant modification that accumulated. S-glutathiolation and S-cysteinylation were significantly increased after exposure of SMCs to biotinylated forms of CysNO or S-nitrosoglutathione respectively. 10 μM CysNO depleted non-protein thiols, predominantly reduced glutathione, 7-fold as measured by the Ellman's assay. Furthermore, this intervention also reduced the activity of NAPDH-dependent enzymes (MTS assay) 2-fold, which together indicates CysNO compromises the cells reducing system (p