Proteome Screens for Cys Residues Oxidation

Abstract

The oxidation of the cysteine (Cys) residue to sulfenic (–S–OH), disulfide (–S–S–), or S-nitroso (S–NO) forms are thought to be a posttranslational modifications that regulate protein function. However, despite a few solid examples of its occurrence, thiol-redox regulation of protein function is still debated and often seen as an exotic phenomenon. A systematic and exhaustive characterization of all oxidized Cys residues, an experimental approach called redox proteomics or redoxome analysis, should help establish the physiological scope of Cys residue oxidation and give clues to its mechanisms. Redox proteomics still remains a technical challenge, mainly because of the labile nature of thiol-redox reactions and the lack of tools to directly detect the modified residues. Here we consider recent technical advances in redox proteomics, focusing on a gel-based fluorescent method and on the shotgun OxICAT technique.

Introduction

Until recently, cysteine (Cys) residue oxidation was thought to be confined to the endoplasmic-reticulum (ER), in which catalyzed disulfide bond formation contributes to the folding of proteins in their way to secretion (Ito & Inaba, 2008, Sevier & Kaiser, 2008), and to a few cytoplasmic enzymes that carry an oxidation–reduction step in their catalytic cycle, such as ribonucleotide reductase or the thiol- and selenothiol-based peroxiredoxins and glutathione peroxidases (Fourquet et al., 2008, Toledano et al., 2007). The paradigm concept of the ER as an oxidizing environment and the cytoplasm, and remaining compartments, as reducing ones has shifted as a result of an increasing number of observations indicating the occurrence of Cys residue oxidation as a posttranslational modification regulating the function of cytoplasmic and nuclear proteins (D'Autreaux & Toledano, 2007, Janssen-Heininger et al., 2008, Linke & Jakob, 2003, Rhee et al., 2005, Toledano et al., 2004). Cys residue oxidation to the sulfenic (–S–OH), disulfide (–S–S–), or S-nitro (S-nitrosylation, S–NO) forms have been identified in several proteins, and are proposed to drive cell signaling by H₂O₂ and nitric oxide (NO) (Hess et al., 2005). Further, import into the mitochondrial intermembrane space (IMS) of a protein subclass was recently shown to involve catalyzed disulfide formation that mediates folding of the polypeptide, thereby preventing its back-translocation to the cytoplasm (Mesecke et al., 2005). Cys residue oxidation in the ER and IMS is mechanistically well understood, as being a catalyzed event for which the enzyme is identified. In contrast, the occurrence of Cys residue oxidation in the cytoplasm is not well understood, except for a few cases for which an oxidation mechanism has been described (D'Autreaux and Toledano, 2007). A systematic and exhaustive characterization of all oxidized Cys residues, an experimental approach called redox proteomics or redoxome analysis, should help establish the inventory of all thiol-redox-based phenomena and their physiological scope. In addition, inventory of the targets of the thiol reductases thioredoxins and glutaredoxins might be established through redoxome analyses of cells in which either of these activities has been shut down. Here we consider the main experimental methods that have been devised to characterize Cys residue oxidation at the proteome-wide level. We then focus on a two-dimensional electrophoresis gel (2DE)-based fluorescent method and on the novel shotgun proteomics OxICAT method developed by Jakob and colleagues (Leichert et al., 2008), two approaches having complementary attributes (Fu et al., 2008). These methods have already provided important advances in understanding thiol-redox metabolism. Nevertheless, redox proteomics still remains a technological challenge and needs further improvements.