Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation

Kathryn Wolhuter,Harry J Whitwell,Christopher H Switzer,Joseph R Burgoyne,John F Timms,Philip Eaton

doi:10.1016/j.molcel.2017.12.019

Kathryn Wolhuter, Harry J Whitwell + Show 4 more

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https://doi.org/10.1016/j.molcel.2017.12.019

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Abstract

SummaryS-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide. Proteins detected using the ascorbate-dependent biotin switch method are typically interpreted to be directly regulated by S-nitrosation. However, these S-nitrosated proteins are shown to predominantly comprise transient intermediates leading to disulfide bond formation. These disulfides are likely to be the dominant end effectors resulting from elevations in nitrosating cellular nitric oxide species. We propose that S-nitrosation primarily serves as a transient intermediate leading to disulfide formation. Overall, we conclude that the current widely held perception that stable S-nitrosation directly regulates the function of many proteins is significantly incorrect.

Highlights

Protein S-nitrosation, often referred to as S-nitrosylation, is a post-translational modification involving the covalent addition of a nitrosonium (NO+) equivalent to the sulfur atom of cysteine
It was especially notable that a significant increase in PKAR1 and PKG1a disulfide levels preceded the increase in detectable, stable S-nitrosothiols (Figure 1D; p < 0.05)
S-nitrosothiol and PKG1a disulfide formation in smooth muscle cells (SMCs) was measured over a time course of 30 min after the addition of 6 or 50 mM CysNO (Figures 1E and 1F)

Summary

Introduction

Protein S-nitrosation, often referred to as S-nitrosylation, is a post-translational modification involving the covalent addition of a nitrosonium (NO+) equivalent to the sulfur atom of cysteine. The mechanisms by which endogenous S-nitrosothiols are formed are unclear. Nitrosothiol formation may occur via oxidation of NO to N2O3 in the presences of an electron acceptor (Broniowska and Hogg, 2012) or radical recombination between a thiyl radical and NO (Madej et al, 2008). Dinitrosyl-iron or heme-NO pathways may be involved (Bosworth et al, 2009; Jia et al, 1996), the physiological occurrence of such reactions remains unclear (Gladwin et al, 2003). Protein S-nitrosation is widely held as a principal mechanism by which changes in cellular NO are transduced to exert its biological effects. The biological significance of S-nitrosation is supported by studies showing that nitrosothiol formation at the active site of proteins inhibits their activity (Mannick et al, 2001)

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