Site-Specific Proteomic Mapping Identifies Selectively Modified Regulatory Cysteine Residues in Functionally Distinct Protein Networks

Abstract

Summary S-acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct, modifications that regulate protein function, redox-sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remain unknown. Novel MS-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics that showed a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications.

Thiol-Based Redox Modulation of Soluble Guanylyl Cyclase, the Nitric Oxide Receptor

“…In fact, the association between solvent exposed Cys and thiol modification is not absolute according to a study using a bioinformatics structural analysis of known SNO sites showing that *35% of the sulfur group in SNO-Cys was predicted to be buried (68). This was confirmed by a recent proteomics mapping of Cys modifications in the liver showing only a slightly higher solvent access for the modified Cys versus the unmodified Cys (44). The same study also showed that basic amino acids were in low proportions within a 10 Å of the modified Cys suggesting that the reactivity of buried thiol might not be a primary determinant of modification.…”

“…The reliability and selectivity of the method were verified using in parallel eNOS knockout mice, in which a drastic reduction of SNO was observed (29). In a subsequent study, Gould et al identified four different Cys modifications (S-NO, S-OH, SS-G, and S-Ac) in the proteome with minimal overlap for specific Cys (11% between SNO and SSG, for instance) (44). The different outcomes between these studies could be explained by the methods used, the system (cells vs. tissues) and the treatment with NO-generating agents (nitrosative stress) or lack thereof.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…CysTMT utilises a dithiopyridine reactive group to label thiols and successfully revealed 171 proteins which were both S-nitrosated as well as S-glutathiolated, although this only relates to a 10% overlap in pools of proteins that were either S-nitrosated or S-glutathiolated. Furthermore, endothelial NOS knockout mice showed decreased S-nitrosothiol site occupancy with no significant difference in S-glutathiolation [139]. These data seemingly indicate functionally distinct protein networks are modulated by the two modifications.…”

Molecular Mechanisms of Allosteric Inhibition of Brain Glycogen Phosphorylase by Neurotoxic Dithiocarbamate Chemicals

“…Proteomic studies revealed highly reactive cysteine residues in bGP (Cys 109 , Cys 326 , and Cys 496 ) (25)(26)(27), which can constitute potent targets of electrophilic compounds such as DTCs. Consequently, DTCs may directly modulate glycogenolysis through the modification of key cysteine residues of bGP, known to redox regulate the enzyme activity.…”

“…Rat models and recent proteomic studies suggested that brain glycogen phosphorylase is a putative target of DTC chemicals in the brain (7). In addition, this isoenzyme has been described as presenting highly reactive cysteine residues (23,(25)(26)(27). To further determine the impact of DTCs on bGP activity and glycogen metabolism in the brain, we treated mice brain extracts with thiram (Fig.…”