Kate S. Carroll

Expanding the functional diversity of proteins through cysteine oxidation

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.

Cysteine-Mediated Redox Signaling: Chemistry, Biology, and Tools for Discovery

Reactive oxygen, nitrogen, and sulfur species, referred to as ROS, RNS, and RSS, respectively, are produced during normal cell function and in response to various stimuli. An imbalance in the metabolism of these reactive intermediates results in the phenomenon known as oxidative stress. If left unchecked, oxidative molecules can inflict damage on all classes of biological macromolecules and eventually lead to cell death. Indeed, sustained elevated levels of reactive species have been implicated in the etiology (e.g., atherosclerosis, hypertension, diabetes) or the progression (e.g., stroke, cancer, and neurodegenerative disorders) of a number of human diseases.1 Over the past several decades, however, a new paradigm has emerged in which the aforementioned species have also been shown to function as targeted, intracellular second messengers with regulatory roles in an array of physiological processes.2 Against this backdrop, it is not surprising that considerable ongoing efforts are aimed at elucidating the role that these reactive intermediates play in health and disease. Site-specific, covalent modification of proteins represents a prominent molecular mechanism for transforming an oxidant signal into a biological response. Amino acids that are candidates for reversible modification include cysteines whose thiol (i.e., sulfhydryl) side chain is deprotonated at physiological pH, which is an important attribute for enhancing reactivity. While reactive species can modify other amino acids (e.g., histidine, methionine, tryptophan, and tyrosine), this Review will focus exclusively on cysteine, whose identity as cellular target or “sensor” of reactive intermediates is most prevalent and established.3 Oxidation of thiols results in a range of sulfur-containing products, not just disulfide bridges, as typically presented in biochemistry textbooks. An overview of the most relevant forms of oxidized sulfur species found in vivo is presented in Chart 1. Chart 1 Biologically Relevant Cysteine Chemotypesa

Orchestrating redox signaling networks through regulatory cysteine switches.

Hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2) 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.

Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity

Protein sulfenylation is a post-translational modification of emerging importance in higher eukaryotes. However, investigation of its diverse roles remains challenging, particularly within a native cellular environment. Herein we report the development and application of DYn-2, a new chemoselective probe for detecting sulfenylated proteins in human cells. These studies show that epidermal growth factor receptor-mediated signaling results in H(2)O(2) production and oxidation of downstream proteins. In addition, we demonstrate that DYn-2 has the ability to detect differences in sulfenylation rates within the cell, which are associated with differences in target protein localization. We also show that the direct modification of epidermal growth factor receptor by H(2)O(2) at a critical active site cysteine (Cys797) enhances its tyrosine kinase activity. Collectively, our findings reveal sulfenylation as a global signaling mechanism that is akin to phosphorylation and has regulatory implications for other receptor tyrosine kinases and irreversible inhibitors that target oxidant-sensitive cysteines in proteins.

Mining the Thiol Proteome for Sulfenic Acid Modifications Reveals New Targets for Oxidation in Cells

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.

Sulfenic acid chemistry, detection and cellular lifetime

BACKGROUND Reactive oxygen species-mediated cysteine sulfenic acid modification has emerged as an important regulatory mechanism in cell signaling. The stability of sulfenic acid in proteins is dictated by the local microenvironment and ability of antioxidants to reduce this modification. Several techniques for detecting this cysteine modification have been developed, including direct and in situ methods. SCOPE OF REVIEW This review presents a historical discussion of sulfenic acid chemistry and highlights key examples of this modification in proteins. A comprehensive survey of available detection techniques with advantages and limitations is discussed. Finally, issues pertaining to rates of sulfenic acid formation, reduction, and chemical trapping methods are also covered. MAJOR CONCLUSIONS Early chemical models of sulfenic acid yielded important insights into the unique reactivity of this species. Subsequent pioneering studies led to the characterization of sulfenic acid formation in proteins. In parallel, the discovery of oxidant-mediated cell signaling pathways and pathological oxidative stress has led to significant interest in methods to detect these modifications. Advanced methods allow for direct chemical trapping of protein sulfenic acids directly in cells and tissues. At the same time, many sulfenic acids are short-lived and the reactivity of current probes must be improved to sample these species, while at the same time, preserving their chemical selectivity. Inhibitors with binding scaffolds can be rationally designed to target sulfenic acid modifications in specific proteins. GENERAL SIGNIFICANCE Ever increasing roles for protein sulfenic acids have been uncovered in physiology and pathology. A more complete understanding of sulfenic acid-mediated regulatory mechanisms will continue to require rigorous and new chemical insights. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies

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.

Chemical 'omics' approaches for understanding protein cysteine oxidation in biology.

Oxidative cysteine modifications have emerged as a central mechanism for dynamic post-translational regulation of all major protein classes and correlate with many disease states. Elucidating the precise roles of cysteine oxidation in physiology and pathology presents a major challenge. This article reviews the current, targeted proteomic strategies that are available to detect and quantify cysteine oxidation. A number of indirect methods have been developed to monitor changes in the redox state of cysteines, with the majority relying on the loss of reactivity with thiol-modifying reagents or restoration of labeling by reducing agents. Recent advances in chemical biology allow for the direct detection of specific cysteine oxoforms based on their distinct chemical attributes. In addition, new chemical reporters of cysteine oxidation have enabled in situ detection of labile modifications and improved proteomic analysis of redox-regulated proteins. Progress in the field of redox proteomics should advance our knowledge of regulatory mechanisms that involve oxidation of cysteine residues and lead to a better understanding of oxidative biochemistry in health and disease.

A periplasmic reducing system protects single cysteine residues from oxidation.

Periplasmic Redox Regulation The oxidation state of intracellular and extracellular proteins are carefully managed by cellular redox machineries. Depuydt et al. (p. 1109) discovered a reducing system that protects single cysteine residues from oxidation in the bacterial periplasm. DsbG, a thioredoxin-related protein, appears to be a key player in that system and is the first reductase identified in the periplasm of Escherichia coli. Together with DsbC, DsbG controls the global sulfenic acid content of this compartment. Sulfenic acid formation is a major posttranslational modification in the periplasm, and three homologous L,D-transpeptidases are substrates of DsbG. Sulfenic acid formation is not restricted to E. coli, but is ubiquitous. Because proteins from the thioredoxin superfamily are widespread, similar thioredoxin-related proteins may control cellular sulfenic acid more widely. A thioredoxin-like enzyme controls the oxidation state of the bacterial periplasm. The thiol group of the amino acid cysteine can be modified to regulate protein activity. The Escherichia coli periplasm is an oxidizing environment in which most cysteine residues are involved in disulfide bonds. However, many periplasmic proteins contain single cysteine residues, which are vulnerable to oxidation to sulfenic acids and then irreversibly modified to sulfinic and sulfonic acids. We discovered that DsbG and DsbC, two thioredoxin-related proteins, control the global sulfenic acid content of the periplasm and protect single cysteine residues from oxidation. DsbG interacts with the YbiS protein and, along with DsbC, regulates oxidation of its catalytic cysteine residue. Thus, a potentially widespread mechanism controls sulfenic acid modification in the cellular environment.

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

Controlled generation of reactive oxygen species orchestrates numerous physiological signaling events (Finkel, T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15). A major cellular target of reactive oxygen species is the thiol side chain (RSH) of Cys, which may assume a wide range of oxidation states (i.e. −2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO2H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and biological role of sulfenic/sulfinic acid modifications have been recently expanded with the introduction of new tools for monitoring cysteine oxidation in vitro and directly in cells. This minireview discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications in cell signaling.