Hydrogen peroxide (H2O2) acts as a second messenger that can mediate intracellular signal transduction via chemoselective oxidation of cysteine residues in signaling proteins. This Review presents current mechanistic insights into signal-mediated H2O2 production and highlights recent advances in methods to detect reactive oxygen species (ROS) and cysteine oxidation both in vitro and in cells. Selected examples from the recent literature are used to illustrate the diverse mechanisms by which H2O2 can regulate protein function. The continued development of methods to detect and quantify discrete cysteine oxoforms should further our mechanistic understanding of redox regulation of protein function and may lead to the development of new therapeutic strategies.Keywords: Chemoselective/chemospecific probes: Small molecules that react specifically with one chemical moiety to either remove a protecting group or to generate a covalent adduct. These probes are an attractive means by which to chemically trap reversible/transient PTMs, such as sulfenic acids.; NADPH oxidases (NOX): A family of heme/flavin-containing protein complexes of which there are seven human isoforms that generate superoxide and hydrogen peroxide by translocating electrons from NADPH to molecular oxygen. These enzymes are an inducible source of ROS production for cellular signaling events.; Oxidative stress: A condition wherein the production of ROS exceeds the biological system’s ability to readily detoxify these intermediates with its peroxiredoxin, peroxidase, Trx/TrxR, and GSH/GSR systems. This condition can result in oxidative damage of proteins, lipids, and DNA.; Oxoform: A general term referring to an oxidized form of the thiol side chain of a protein cysteine residue such as a disulfide or sulfenic acid.; Posttranslational modification (PTM): The chemical modification of a protein after its translation. Examples of PTMs include O-phosphorylation, acetylation, SUMOylation, and cysteine oxidation.Keywords: Ratiometric labeling: The use of isotopically labeled small molecules to derivatize unmodified (e.g., thiol) versus modified (e.g., disulfide) proteins to obtain quantitative information about the fraction of modified protein in terms of total protein available in a given sample. This method facilitates the direct comparison of the percentage of modified protein between different samples (e.g., ± stimulus) since fluctuations in protein expression are compensated for in the ratio.; Reactive oxygen species (ROS): Reduced forms of oxygen that are ions, radicals, or peroxides. These species are reactive as a result of the presence of unpaired valance shell electrons or a labile peroxide bond.; Redox signaling: The regulation of protein activity and the transduction of signals to downstream proteins through oxidative modification of reactive cysteine residues by ROS.; Second messenger: A diffusible molecule produced in cellular signal transduction pathways that modulates the activity of effector proteins thereby propagating the signaling event. Examples of second messengers are cAMP, phosphoinositols, and more recently hydrogen peroxide.; Trx/TrxR and GSH/GSR: The buffering systems of the cell that use electrons from NADPH to reduce protein disulfides and thereby act to maintain cysteine residues in their reduced thiol form.

Saccharomyces cerevisiae responds to elevated levels of hydrogen peroxide in its environment via a redox relay system comprising the thiol peroxidase Gpx3 and transcription factor Yap1. In this signaling pathway, a central unresolved question is whether cysteine sulfenic acid modification of Gpx3 is required for Yap1 activation in cells. Here we report that cell-permeable chemical probes, which are selective for sulfenic acid, inhibit peroxide-dependent nuclear accumulation of Yap1, trap the Gpx3 sulfenic acid intermediate, and block formation of the Yap1-Gpx3 intermolecular disulfide directly in cells. In addition, we present electrostatic calculations that show cysteine oxidation is accompanied by significant changes in charge distribution, which might facilitate essential conformational rearrangements in Gpx3 during catalysis and intermolecular disulfide formation with Yap1.

Mycobacterium tuberculosis adenosine-5′-phosphosulfate (APS) reductase is an iron−sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.

Oxidation of cysteine to sulfenic acid has emerged as a biologically relevant post-translational modification with particular importance in redox-mediated signal transduction; however, the identity of modified proteins remains largely unknown. We recently reported DAz-1, a cell-permeable chemical probe capable of detecting sulfenic acid modified proteins directly in living cells. Here we describe DAz-2, an analogue of DAz-1 that exhibits significantly improved potency in vitro and in cells. Application of this new probe for global analysis of the sulfenome in a tumor cell line identifies most known sulfenic acid modified proteins: 14 in total, plus more than 175 new candidates, with further testing confirming oxidation in several candidates. The newly identified proteins have roles in signal transduction, DNA repair, metabolism, protein synthesis, redox homeostasis, nuclear transport, vesicle trafficking, and ER quality control. Cross-comparison of these results with those from disulfide, S-glutathionylation, and S-nitrosylation proteomes reveals moderate overlap, suggesting fundamental differences in the chemical and biological basis for target specificity. The combination of selective chemical enrichment and live-cell compatibility makes DAz-2 a powerful new tool with the potential to reveal new regulatory mechanisms in signaling pathways and identify new therapeutic targets.

Hydrogen peroxide (H2O2) functions as a second messenger that can activate cell proliferation through chemoselective oxidation of cysteine residues in signaling proteins. The connection between H2O2 signaling, thiol oxidation, and activation of growth pathways has emerged as fertile ground for the development of strategies for cancer treatment. Central to achieving this goal is the development of tools and assays that facilitate characterization of the molecular events associated with tumorigenesis and evaluation of patient response to therapy. Here we report on the development of an immunochemical method for detecting sulfenic acid, the initial oxidation product that results when a thiolate reacts with H2O2. For this approach, the sulfenic acid is derivatized with a chemical tag to generate a unique epitope for recognition. The elicited antibody is exquisitely specific, context-independent, and capable of visualizing sulfenic acid formation in cells. Applying this approach to several systems, including cancer cell lines, shows it can be used to monitor differences in thiol redox status and reveals a diverse pattern of sulfenic acid modifications across different subtypes of breast tumors. These studies demonstrate a general strategy for producing antibodies against a specific oxidation state of cysteine and show the utility of these reagents for profiling thiol oxidation associated with pathological conditions such as breast cancer.

The polarizable sulfur atom in cysteine is subject to numerous post-translational oxidative modifications in the cellular milieu, which regulates a wide variety of biological phenomena such as catalysis, metal binding, protein turnover, and signal transduction. The application of chemical rationale to describe the features of different cysteine oxoforms affords a unique perspective on this rapidly expanding field. Moreover, a chemical framework broadens our understanding of the functional roles that specific cysteine oxidation states can play and facilitates the development of mechanistic proposals, which can be tested in both biochemical and cellular studies.

Oxidation of the thiol functional group in cysteine (Cys–SH) to sulfenic (Cys–SOH), sulfinic (Cys–SO2H) and sulfonic acids (Cys–SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acid-modified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting proteinsulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteineresidues.