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Quantitative Proteomic Analysis of S-glutathionylated Proteins After Cocaine-induced Reinstatement

Joachim Uys, Departments of Cell and Molecular Pharmacology, Medical University of South Carolina
Cocaine administration leads to oxidative stress in the nucleus accumbens (NAc) and alters redox homeostasis. However, the role of redox-mediated signaling events in cocaine addiction is unknown. The glutathione (GSH) precursor, N-acetylcysteine, has been shown to restore glutamate homeostasis and reduce relapse to cocaine seeking. In addition to changes in GSH levels, chronic cocaine can also induce changes in S-glutathionylation, which is a post-translational modification (PTM) on cysteine residues following oxidative and/or nitrosative stress. S-glutathionylation of proteins can prevent further modification of cysteine residues to sulfinic and sulfonic acids and subsequent degradation. Target proteins for S-glutathionylation are considered redox switches and contribute to redox- mediated signaling events. Furthermore, the consequences of S-glutathionylation may be redox-mediated modifications of the structure/function of a target protein. Previously we have shown that after withdrawal from daily cocaine using a sensitization paradigm, an acute cocaine challenge elicited a marked increase in both NAc global protein S-glutathionylation and in the enzyme responsible for catalyzing the forward reaction of S-glutathionylation, glutathione-S-transferase Pi (GSTP), in rats. In addition, a single cocaine injection in saline withdrawn rats does not lead to a change in S-glutathionylation, suggesting that the observed changes can only be attributed to chronic cocaine exposure. Furthermore, genetic deletion or inhibition of GSTP increases cocaine sensitivity and cocaine self-administration in mice, implicating S-glutathionylation as a protective mechanism keeping neuronal plasticity in check. Taken together these data suggest an important role for protein S-glutathionylation in models of cocaine sensitization and self-administration. Known proteins that contain redox sensitive cysteines and have a regulatory role in cocaine addiction include cofilin, actin, and N-methyl-D-aspartate (NMDA) receptors. However, this small list of proteins will definitely be expanded through the application of novel PTM proteomic technologies. Given that S-glutathionylation of target proteins can result in altered protein signaling and is dysregulated after withdrawal from chronic cocaine, the proposed project has a direct relationship to the theme of the Center. This project will focus on the proteomic analysis of the NAc “glutathionome” after cocaine self-administration, withdrawal and cocaine-induced reinstatement in rats.

We will use powerful and unbiased iodoacetyl-activated Tandem Mass Tag (iodoTMT) approaches for de novo identification of candidate proteins and their S-glutathionylated modifications in the NAc. IodoTMT labeling will be used to quantitate both cysteine containing proteins and the cysteinyl modification, S-glutathionylation. For S-glutathionylation analysis we will use a sequential tagging method by which we first label all exposed cysteines with one of the irreversible iodoTMT labels. This step will be followed by reduction with the deglutathionylating enzyme, glutaredoxin, to remove GSH from modified cysteines (S-glutathionylated cysteines) and the newly exposed cysteines will be labeled with a second irreversible iodoTMT label (S-glutathionylation label). This will be followed by Lys-C/trypsin digestion and fractionation by Strong Cation Exchange prior to LC-MS/MS analysis on a Thermo Orbitrap Fusion Tribrid or Q Exactive Plus platform. The sequential labeling approach will allow us to generate quantitative data on both S-glutathionylated and non-modified cysteine-containing proteins as well as quantification of S-glutathionylated/total protein ratios.