Chaperone-directed ribosome repair after oxidative damage.

Because of the central role ribosomes play for protein translation and ribosome-mediated mRNA and protein quality control (RQC), the ribosome pool is surveyed and dysfunctional ribosomes degraded both during assembly, as well as the functional cycle. Oxidative stress downregulates translation and damages mRNAs and ribosomal proteins (RPs). Although damaged mRNAs are detected and degraded via RQC, how cells mitigate damage to RPs is not known. Here, we show that cysteines in Rps26 and Rpl10 are readily oxidized, rendering the proteins non-functional. Oxidized Rps26 and Rpl10 are released from ribosomes by their chaperones, Tsr2 and Sqt1, and the damaged ribosomes are subsequently repaired with newly made proteins. Ablation of this pathway impairs growth, which is exacerbated under oxidative stress. These findings reveal an unanticipated mechanism for chaperone-mediated ribosome repair, augment our understanding of ribosome quality control, and explain previous observations of protein exchange in ribosomes from dendrites, with broad implications for aging and health.

Chemical approaches to discovery and study of sources and targets of hydrogen peroxide redox signaling through NADPH oxidase proteins.

Hydrogen peroxide (H2O2) is a prime member of the reactive oxygen species (ROS) family of molecules produced during normal cell function and in response to various stimuli, but if left unchecked, it can inflict oxidative damage on all types of biological macromolecules and lead to cell death. In this context, a major source of H2O2 for redox signaling purposes is the NADPH oxidase (Nox) family of enzymes, which were classically studied for their roles in phagocytic immune response but have now been found to exist in virtually all mammalian cell types in various isoforms with distinct tissue and subcellular localizations. Downstream of this tightly regulated ROS generation, site-specific, reversible covalent modification of proteins, particularly oxidation of cysteine thiols to sulfenic acids, represents a prominent posttranslational modification akin to phosphorylation as an emerging molecular mechanism for transforming an oxidant signal into a dynamic biological response. We review two complementary types of chemical tools that enable (a) specific detection of H2O2 generated at its sources and (b) mapping of sulfenic acid posttranslational modification targets that mediate its signaling functions, which can be used to study this important chemical signal in biological systems.

Nucleophilic covalent ligand discovery for the cysteine redoxome.

With an eye toward expanding chemistries used for covalent ligand discovery, we elaborated an umpolung strategy that exploits the 'polarity reversal' of sulfur when cysteine is oxidized to sulfenic acid, a widespread post-translational modification, for selective bioconjugation with C-nucleophiles. Here we present a global map of a human sulfenome that is susceptible to covalent modification by members of a nucleophilic fragment library. More than 500 liganded sulfenic acids were identified on proteins across diverse functional classes, and, of these, more than 80% were not targeted by electrophilic fragment analogs. We further show that members of our nucleophilic fragment library can impair functional protein-protein interactions involved in nuclear oncoprotein transport and DNA damage repair. Our findings reveal a vast expanse of ligandable sulfenic acids in the human proteome and highlight the utility of nucleophilic small molecules in the fragment-based covalent ligand discovery pipeline, presaging further opportunities using non-traditional chemistries for targeting proteins.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein.

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites.

Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

Activity-Based Sensing for Site-Specific Proteomic Analysis of Cysteine Oxidation.

Oxidative post-translational modifications (OxiPTMs) of cysteine residues are the molecular foundation of thiol-based redox regulation that modulates physiological events such as cell proliferation, differentiation, and migration and, when dysregulated, can lead to biomolecule damage and cell death. Common OxiPTMs of cysteine thiols (-SH) include reversible modifications such as S-sulfenylation (-SOH), S-glutathionylation (-SSG), disulfide formation (-SSR), S-nitrosylation (-SNO), and S-sulfhydration (-SSH) as well as more biologically stable modifications like S-sulfinylation (-SO2H) and S-sulfonylation (-SO3H). In the past decade, our laboratory has developed first-in-class chemistry-based tools and proteomic methods to advance the field of thiol-based redox biology and oxidative stress. In this Account, we take the reader through the historical aspects of probe development and application in our laboratory, highlighting key advances in our understanding of sulfur chemistry, in the test tube and in living systems. Offering superior resolution, throughput, accuracy, and reproducibility, mass spectrometry (MS)-based proteomics coupled to chemoselective "activity-based" small-molecule probes is the most rigorous technique for global mapping of cysteine OxiPTMs. Herein, we describe the evolution of this field from indirect detection to state-of-the-art site-centric quantitative chemoproteomic approaches that enable mapping of physiological and pathological changes in cysteine oxidation. These methods enable protein and site-level identification, mechanistic studies, mapping fold-changes, and modification stoichiometry. In particular, this Account focuses on activity-based methods for profiling S-sulfenylation, S-sulfinylation, and S-sulfhydration with an eye toward new reactions and methodologies developed in our group as well as their applications that have shed new light on fundamental processes of redox biology. Among several classes of sulfenic acid probes, dimedone-based C-nucleophiles possess superior chemical selectivity and compatibility with tandem MS. Cell-permeable dimedone derivatives with a bioconjugation handle are capable of detecting of S-sulfenylation in living cells. In-depth screening of a C-nucleophile library has yielded several entities with significantly enhanced reactivity over dimedone while maintaining selectivity, and reversible linear C-nucleophiles that enable controlled target release. C-Nucleophiles have also been implemented in tag-switch methods to detect S-sulfhydration. Most recently, activity-based detection of protein S-sulfinylation with electrophilic nitrogen species (ENS), such as C-nitroso compounds and electron deficient diazines, offers significant advantages in simplicity-of-use and target specificity compared to label-free methods. When feasible, the rich information provided by site-centric quantitative proteomics should not be tainted by oxidation artifacts from cell lysis. Therefore, chemoselective probes that function in a native environment with low cytotoxicity, good cell-permeability, and competitive kinetics are desired in modern redox chemoproteomics approaches. As our understanding of sulfur chemistry and redox signaling evolves, newly discovered cysteine OxiPTMs in microorganisms, plants, cells, tissues, and disease models should innovatively promote mechanistic and therapeutic research.

Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.

Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1-4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species-associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.

The Expanding Landscape of the Thiol Redox Proteome.

Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO(2)H), S-sulfonylation (-SO(3)H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes. Here, we review chemical proteomic strategies used to expand the landscape of thiol redox modifications.

A universal entropy-driven mechanism for thioredoxin-target recognition.

Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx-protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx-target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein-protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx-target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition.

Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles.

Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compounds for biological inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-molecule fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidation in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive production of reactive oxygen species. Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid formed in proteins via oxidation or hydrolysis reactions. Here, we report analysis of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 S-sulfenylated cysteines present in "druggable" proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand-protein interactions identified was that of a pyrrolidinedione nucleophile that reacted preferentially with protein tyrosine phosphatases. Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable "redoxome" with significant implications for covalent inhibitor pharmacology and also affords new chemical tools to investigate redox-regulation of protein function.

Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution.

In human pathogens, the sulfate assimilation pathway provides reduced sulfur for biosynthesis of essential metabolites, including cysteine and low-molecular-weight thiol compounds. Sulfonucleotide reductases (SRs) catalyze the first committed step of sulfate reduction. In this reaction, activated sulfate in the form of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is reduced to sulfite. Gene knockout, transcriptomic and proteomic data have established the importance of SRs in oxidative stress-inducible antimicrobial resistance mechanisms. In previous work, we focused on rational and high-throughput design of small-molecule inhibitors that target the active site of SRs. However, another critical goal is to discover functionally important regions in SRs beyond the traditional active site. As an alternative to conservation analysis, we used directed evolution to rapidly identify functional sites in PAPS reductase (PAPR). Four new regions were discovered that are essential to PAPR function and lie outside the substrate binding pocket. Our results highlight the use of directed evolution as a tool to rapidly discover functionally important sites in proteins.

Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.

Mycobacterium tuberculosis (Mtb) survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, Mtb produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (EMSH ) within Mtb cells during infection. We demonstrate that coupling of Mtb MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial EMSH with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of EMSH in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in EMSH of the Mtb population. Further application of this new biosensor demonstrates that treatment of Mtb infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in EMSH , suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient Mtb killing. Lastly, we analyze the membrane integrity of Mtb cells with varied EMSH during infection and show that subpopulation with higher EMSH are susceptible to clinically relevant antibiotics, whereas lower EMSH promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of Mtb and will lead to novel strategies to target redox metabolism for controlling Mtb persistence.

Reactivity, Selectivity, and Stability in Sulfenic Acid Detection: A Comparative Study of Nucleophilic and Electrophilic Probes.

The comparative reaction efficiencies of currently used nucleophilic and electrophilic probes toward cysteine sulfenic acid have been thoroughly evaluated in two different settings-(i) a small molecule dipeptide based model and (ii) a recombinant protein model. We further evaluated the stability of corresponding thioether and sulfoxide adducts under reducing conditions which are commonly encountered during proteomic protocols and in cell analysis. Powered by the development of new cyclic and linear C-nucleophiles, the unsurpassed efficiency in the capture of sulfenic acid under competitive conditions is achieved and thus holds great promise as highly potent tools for activity-based sulfenome profiling.

Redox regulation of protein kinases.

Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous hydrogen peroxide (H2O2) by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiological and pathological H2O2 responses.

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.

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.

Chemical biology approaches to study protein cysteine sulfenylation.

The oxidation of cysteine thiol side chains by hydrogen peroxide to afford protein sulfenyl modifications is an important mechanism in signal transduction. In addition, aberrant protein sulfenylation contributes to a range of human pathologies, including cancer. Efforts to elucidate the roles of protein sulfenylation in physiology and disease have been hampered by the lack of techniques to probe these modifications in native environments with molecular specificity. In this review, we trace the history of chemical and biological methods that have been developed to detect protein sulfenylation and illustrate how a recent cell-permeable chemical reporter, DYn-2, has been used to detect and identify intracellular targets of endogenous H2 O2 during growth factor signaling, including the epidermal growth factor receptor. The array of new tools and methods discussed herein enables the discovery of new biological roles for cysteine sulfenylation in human health and disease.

Redox-sensitive sulfenic acid modification regulates surface expression of the cardiovascular voltage-gated potassium channel Kv1.5.

RATIONALE: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress. OBJECTIVE: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5. METHODS AND RESULTS: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress. CONCLUSIONS: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.