Post-translational Modification Of Cysteine Research Articles

Old poisons, new signaling molecules: the case of hydrogen cyanide.

The high phenotypic plasticity developed by plants includes rapid responses and adaptations to aggressive or changing environments. To achieve this, they evolved extremely efficient mechanisms of signaling mediated by a wide range of molecules, including small signal molecules. Among them, hydrogen cyanide (HCN) has been largely ignored due to its toxic characteristics. However, not only is it present in living organisms, but it has been shown that it serves several functions in all kingdoms of life. Research using model plants has changed the traditional point of view, and it has been demonstrated that HCN plays a positive role in the plant response to pathogens independently of its toxicity. Indeed, HCN induces a response aimed at protecting the plant from pathogen attack, and the HCN is provided either exogenously (in vitro or by some cyanogenic bacteria species present in the rhizosphere) or endogenously (in reactions involving ethylene, camalexin, or other cyanide-containing compounds). The contribution of different mechanisms to HCN function, including a new post-translational modification of cysteines in proteins, namely S-cyanylation, is discussed here. This work opens up an expanding 'HCN field' of research related to plants and other organisms.

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

GSNOR deficiency promotes tumor growth via FAK1 S-nitrosylation

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer.

Nitrosative stress induced by homocysteine thiolactone drives vascular cognitive impairments via GTP cyclohydrolase 1 S-nitrosylation in vivo

Backgrounds: Hyperhomocysteinemia (HHcy) is one of risk factors for vascular cognitive impairment (VCI). GTP cyclohydrolase 1 (GCH1) deficiency is critical to oxidative stress in vascular dysfunction. The aim of this study was designed to examine whether HHcy induces VCI through GCH1 S-nitrosylation, a redox-related post-translational modification of cysteine. MethodsThe VCI model was induced by feeding mice homocysteine thiolactone (HTL) for 16 consecutive weeks. The cognitive functions were evaluated by step-down avoidance test, passive avoidance step-through task test, and Morris water maze (MWM) test. Protein S-nitrosylation was assayed using a biotin-switch method. ResultsIn cell-free system, nitric oxide (NO) donor induced GCH1 protein S-nitrosylation and decreased GCH1 activity. In endothelial cells, HTL increased GCH1 S-nitrosylation, reduced tetrahydrobiopterin, and induced oxidative stress, which were attenuated by N-acetyl-cysteine, L-N6-1-Iminoethyl-lysine, mutant of GCH1 cysteine 141 to alanine (MT-GCH1) or gene deletion of inducible NO synthase (iNOS). Further, HTL incubation or iNOS overexpression promoted endothelial cellular senescence, but abolished by exogenous expression of MT-GCH1 or pharmacological approaches including N-acetyl-cysteine, L-sepiapterin, and tempol. In wildtype mice, long-term administration of HTL induced GCH1 S-nitrosylation and vascular stiffness, decreased cerebral blood flow, and damaged the cognitive functions. However, these abnormalities induced by HTL administration were rescued by enforced expression of MT-GCH1 or gene knockout of iNOS. In human subjects, GCH1 S-nitrosylation was increased and cognitive functions were impaired in patients with HHcy. ConclusionThe iNOS-mediated nitrosative stress induced by HTL drives GCH1 S-nitrosylation to induce cerebral vascular stiffness and cognitive impairments.

Quantification of Redox-Sensitive GFP Cysteine Redox State via Gel-Based Read-Out.

To date, fluorescent protein biosensors are widely used in research. In vivo, they can be applied to dynamically monitor several physiological parameters in various subcellular compartments. Redox-sensitive green fluorescent protein2 (roGFP2) senses the glutathione redox potential via a disulfide bridge formed between neighboring beta-strands of its beta-barrel structure. As changes in redox state affect both excitation maxima of roGFP2 oppositely, sensor responses are ratiometric. The reaction mechanism of roGFP2 is well characterized and involves an intermediate S-glutathionylation step. Thus, roGFP2 is also used in enzymatic in vitro assays, e.g., assessing glutaredoxin kinetics. In addition to the fluorescent read-out, theroGFP2 redox state can also be determined by differential migration on a non-reducing SDS-PAGE. This read-out mode may be beneficial in some applications, e.g., if mass-spectrometric analysis of posttranslational cysteine modifications is desired. Here, we describe a protocol for gel-based fluorescent read-out of theroGFP2 redox state, as well as modification of free cysteines by maleimide-based reagents.

Exploiting S-nitrosylation for cancer therapy: facts and perspectives.

S-nitrosylation, the post-translational modification of cysteines by nitric oxide, has been implicated in several cellular processes and tissue homeostasis. As a result, alterations in the mechanisms controlling the levels of S-nitrosylated proteins have been found in pathological states. In the last few years, a role in cancer has been proposed, supported by the evidence that various oncoproteins undergo gain- or loss-of-function modifications upon S-nitrosylation. Here, we aim at providing insight into the current knowledge about the role of S-nitrosylation in different aspects of cancer biology and report the main anticancer strategies based on: (i) reducing S-nitrosylation-mediated oncogenic effects, (ii) boosting S-nitrosylation to stimulate cell death, (iii) exploiting S-nitrosylation through synthetic lethality.

Palmitoylation: A Fatty Regulator of Myocardial Electrophysiology.

Regulation of cardiac physiology is well known to occur through the action of kinases that reversibly phosphorylate ion channels, calcium handling machinery, and signaling effectors. However, it is becoming increasingly apparent that palmitoylation or S-acylation, the post-translational modification of cysteines with saturated fatty acids, plays instrumental roles in regulating the localization, activity, stability, sorting, and function of numerous proteins, including proteins known to have essential functions in cardiomyocytes. However, the impact of this modification on cardiac physiology requires further investigation. S-acylation is catalyzed by the zDHHC family of S-acyl transferases that localize to intracellular organelle membranes or the sarcolemma. Recent work has begun to uncover functions of S-acylation in the heart, particularly in the regulation of cardiac electrophysiology, including modification of the sodium-calcium exchanger, phospholemman and the cardiac sodium pump, as well as the voltage-gated sodium channel. Elucidating the regulatory functions of zDHHC enzymes in cardiomyocytes and determination of how S-acylation is altered in the diseased heart will shed light on how these modifications participate in cardiac pathogenesis and potentially identify novel targets for the treatment of cardiovascular disease. Indeed, proteins with critical signaling roles in the heart are also S-acylated, including receptors and G-proteins, yet the dynamics and functions of these modifications in myocardial physiology have not been interrogated. Here, we will review what is known about zDHHC enzymes and substrate S-acylation in myocardial physiology and highlight future areas of investigation that will uncover novel functions of S-acylation in cardiac homeostasis and pathophysiology.

Functionalization and Post-Translational Modification of Cysteines by Tsuji–Trost Reaction

Functionalization and Post-Translational Modification of Cysteines by Tsuji–Trost Reaction

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies

BackgroundSince malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites.MethodsPlasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium.ResultsHere the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity.ConclusionThe characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites’ redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.

S-Glutathionylation Influences the Property of Fibronectin

Fibronectin is a key extracellular matrix (ECM) protein that plays a role in cell growth, migration and differentiation through its engagement of cell surface receptors, known as integrins, in a spatiotemporal manner. Fibronectin is a mechanically sensitive protein displaying force-induced conformational changes that impact its multiple growth factor, integrin, and ECM protein binding domains. Fibronectin is a unique ECM protein in that it contains 4 free cysteine residues that are protected in cryptic sites, but that can be exposed under force. S-glutathionylation is a post-translational modification of cysteines that occurs in oxidative environments like the extracellular space during pathologies like cancer and fibrosis. Here, we show for the first time that fibronectin's cysteines can be glutathionylated when exposed either through chemical unfolding in its soluble form or mechanical unfolding in its fibrillar form (both on the single fiber and matrix level). Moreover, we proved that S-glutathionylation impacts the mechanical properties of fibronectin fibers. Specifically, our results reveal that glutathionylated fibronectin fibers are more extensible and less stiff compared to unmodified fibronectin. This is true for both single fibronectin fibers and fibronectin matrices. Thus S-glutathionylation of mechanically exposed cysteines on fibronectin may act as a mechanism of mechanical memory within fibronectin-rich ECMs, regulating subsequent mechanotransduction. One possible realism of this mechanism could be the enhancement fibronectin's integrin binding mechanoswitch due to fibronectin fibers being more easily stretched under lower force regimes.

Novel Regulatory Mechanism of STAT1 through Redox Post-translational Modification of Cysteines

STAT1 plays a central role in the immune system, in which it mediates signaling induced by different cytokines, including type I (α/β) and type II Interferon (IFN), and regulates expression of genes involved in many physiological processes, including antiviral and antibacterial defense, immune tolerance, suppression of cell proliferation, and induction of apoptosis. Dysregulation or mutation of STAT1 causes life threatening conditions resulting from inefficient defense against pathogens, autoimmunity or cancer development. Single nucleotide polymorphisms of STAT1 Cys residues are associated with chronic mucocutaneous candidiasis disease (CMCD), suggesting a role of Cys in the capacity of STAT1 to drive an appropriate anti-pathogen response. Regulation of STAT1 activity through different post-translational modifications is well-characterized. However, the role of Reactive Oxygen species (ROS) and Cys reversible oxidative post-translational modifications (ox-PTMs) on STAT1 function remained to be investigated. Using maleimide-derivative switch methods to label Cys ox-PTMs, we found that Diamide, a thiol-oxidizing agent, and the myeloperoxidase-generated HOCl induce ox-PTMs of STAT1 Cys in epithelial cells (A549), monocytes (THP-1) and ectopically expressed STAT1 in STAT1 deficient fibroblasts (U3A). Analysis of Cys/Ala (C/A) mutants of STAT1 in response to IFNγ and IFNβ revealed gain of function phenotype of mutations in two different functional domains of STAT1. Altogether, our data supports a model in which previously unrecognized ox-PTMs of STAT1 Cys residues are essential to dampen STAT1 activity. Current studies are aimed at further characterizing the specific Cys ox-PTMs and their consequences on STAT1-dependent transcriptional program induction and antiviral immune response.

First Report of Hb Kent [β37(C3)Trp→Cys (TGG>TGC) HBB: c.114G>C] in a Chinese Family

We report a novel HBB: c.114G>C mutation in a Chinese family. This mutation resulted in a β37(C3)Trp→Cys amino acid substitution and was synonymous with Hb Kent, a hemoglobin (Hb) variant that was reported exclusively in patients of European descent. Though Hb Kent has a normal oxygen affinity and molecular stability, it has a characteristic dual variant appearance on cellulose acetate electrophoresis (CAE) and high performance liquid chromatography (HPLC) caused by the posttranslational modification of cysteine. We also report the phenotypic expression of this variant when coinherited with the Southeast Asian (– –SEA) double α-globin gene deletion.

Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1.

Sulfatases are a family of enzymes (sulfuric ester hydrolases, EC 3.1.6.-) that catalyze the hydrolysis of a wide array of sulfate esters. To date, despite the discovery of many sulfatase genes and the accumulation of data on numerous sulfated molecules, the number of characterized enzymes that are key players in sulfur metabolism remains extremely limited. While mammalian sulfatases are well studied due to their involvement in a wide range of normal and pathological biological processes, lower eukaryotic sulfatases, especially fungal sulfatases, have not been thoroughly investigated at the biochemical and structural level. In this paper, we describe the molecular cloning of Fusarium proliferatum sulfatase (F.p.Sulf-6His), its recombinant expression in Pichia pastoris as a soluble and active cytosolic enzyme and its detailed characterization. Gel filtration and native electrophoretic experiments showed that this recombinant enzyme exists as a tetramer in solution. The enzyme is thermo-sensitive, with an optimal temperature of 25°C. The optimal pH value for the hydrolysis of sulfate esters and stability of the enzyme was 6.0. Despite the absence of the post-translational modification of cysteine into Cα-formylglycine, the recombinant F.p.Sulf-6His has remarkably stable catalytic activity against p-nitrophenol sulfate, with kcat = 0.28 s-1 and Km = 2.45 mM, which indicates potential use in the desulfating processes. The currently proposed enzymatic mechanisms of sulfate ester hydrolysis do not explain the appearance of catalytic activity for the unmodified enzyme. According to the available models, the unmodified enzyme is not able to perform multiple catalytic acts; therefore, the enzymatic mechanism of sulfate esters hydrolysis remains to be fully elucidated.

Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1.

Sulfatases are a family of enzymes (sulfuric ester hydrolases, EC 3.1.6.-) that catalyze the hydrolysis of a wide array of sulfate esters. To date, despite the discovery of many sulfatase genes and the accumulation of data on numerous sulfated molecules, the number of characterized enzymes that are key players in sulfur metabolism remains extremely limited. While mammalian sulfatases are well studied due to their involvement in a wide range of normal and pathological biological processes, lower eukaryotic sulfatases, especially fungal sulfatases, have not been thoroughly investigated at the biochemical and structural level. In this paper, we describe the molecular cloning of Fusarium proliferatum sulfatase (F.p.Sulf-6His), its recombinant expression in Pichia pastoris as a soluble and active cytosolic enzyme and its detailed characterization. Gel filtration and native electrophoretic experiments showed that this recombinant enzyme exists as a tetramer in solution. The enzyme is thermo-sensitive, with an optimal temperature of 25°C. The optimal pH value for the hydrolysis of sulfate esters and stability of the enzyme was 6.0. Despite the absence of the post-translational modification of cysteine into Cα-formylglycine, the recombinant F.p.Sulf-6His has remarkably stable catalytic activity against p-nitrophenol sulfate, with kcat = 0.28 s-1 and Km = 2.45 mM, which indicates potential use in the desulfating processes. The currently proposed enzymatic mechanisms of sulfate ester hydrolysis do not explain the appearance of catalytic activity for the unmodified enzyme. According to the available models, the unmodified enzyme is not able to perform multiple catalytic acts; therefore, the enzymatic mechanism of sulfate esters hydrolysis remains to be fully elucidated.

Persulfidation (S-sulfhydration) and H2S.

The past decade has witnessed the discovery of hydrogen sulfide (H2S) as a new signalling molecule. Its ability to act as a neurotransmitter, regulator of blood pressure, immunomodulator or anti-apoptotic agent, together with its great pharmacological potential, is now well established. Notwithstanding the growing body of evidence showing the biological roles of H2S, the gap between the macroscopic descriptions and the actual mechanism(s) behind these processes is getting larger. The reactivity towards reactive oxygen and nitrogen species and/or metal centres cannot explain this plethora of biological effects. Therefore, a mechanism involving modification of protein cysteine residues to form protein persulfides is proposed. It is alternatively called S-sulfhydration. Persulfides are not particularly stable and show increased reactivity when compared to free thiols. Detection of protein persulfides is still facing methodological limitations, and mechanisms by which H2S causes this modification are still largely scarce. Persulfidation of protein such as KATP could contribute to H2S-induced vasodilation, while S-sulfhydration of GAPDH and NF-κB inhibits apoptosis. H2S regulates endoplasmic reticulum stress by causing persulfidation of PTP-1B. Several other proteins have been found to be regulated by this posttranslational modification of cysteine. This review article provides a critical overview of the current state of the literature addressing protein S-sulfhydration, with particular emphasis on the challenges and future research directions in this particular field.

Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology.

The perception of reactive oxygen species has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g., cancer). New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically distinct alterations to the protein (e.g., sulfenic/sulfinic/sulfonic acid, disulfides). These post-translational modifications (PTM) are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology.

Profiling and inhibiting reversible palmitoylation

Protein palmitoylation describes the posttranslational modification of cysteines by a thioester-linked long-chain fatty acid. This modification is critical for membrane association, spatial organization, and the proper activity of hundreds of membrane-associated proteins. Palmitoylation is continuously remodeled, both by spontaneous hydrolysis and enzyme-mediated de-palmitoylation. Bioorthogonal pulse-chase labeling approaches have highlighted the role of protein thioesterases as key regulators of palmitoylation dynamics. Importantly, thioesterases are critical for regulating the spatial organization of key oncogenic proteins, such as Ras GTPases. New inhibitors, probes, and proteomics methods have put a spotlight on this emerging posttranslational modification. These tools promise to advance our understanding the enzymatic regulation of dynamic palmitoylation, and present new opportunities for drug development.

Abstract B48: Integrin α6 S-nitrosylation induces prostate cancer cell migration by loss of adhesion to laminin-1

Abstract Prostate cancer is the second most common cancer in American males. Elevated nitric oxide and nitric oxide synthase levels are associated with the progression of prostate tumors. S-nitrosylation, a reversible nitric oxide post-translational modification of cysteines, was investigated here. This report showed that low dose nitric oxide and inducible nitric oxide synthase induced the migration of prostate cancer cells, which was blocked using selective integrin alpha 6 and inducible nitric oxide synthase inhibitors. Furthermore, both nitric oxide donor and inducible nitric oxide synthase targeted integrin alpha 6 for S-nitrosylation on cysteine 86 in prostate cancer cells. Nitric oxide reduced prostate cancer cell adhesion to laminin-1 and integrin alpha 6 binding to laminin beta 1 but theses effects were not seen after mutation of cysteine 86 in integrin alpha 6. S-nitrosylation of integrin alpha 6 cysteine 86 ultimately led to increased migration of prostate cancer cells, which is a potential mechanism of prostate cancer metastasis. Citation Format: Jared Isaac, Ying Wai Lam, Pheruza Tarapore, Shuk-mei Ho. Integrin α6 S-nitrosylation induces prostate cancer cell migration by loss of adhesion to laminin-1 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B48.

Thiol-Based Posttranslational Modifications in Parasites

Cysteine residues of proteins participate in the catalysis of biochemical reactions, are crucial for redox reactions, and influence protein structure by the formation of disulfide bonds. Covalent posttranslational modifications (PTMs) of cysteine residues are important mediators of redox regulation and signaling by coupling protein activity to the cellular redox state, and moreover influence stability, function, and localization of proteins. A diverse group of protozoan and metazoan parasites are a major cause of diseases in humans, such as malaria, African trypanosomiasis, leishmaniasis, toxoplasmosis, filariasis, and schistosomiasis. Human parasites undergo dramatic morphological and metabolic changes while they pass complex life cycles and adapt to changing environments in host and vector. These processes are in part regulated by PTMs of parasitic proteins. In human parasites, posttranslational cysteine modifications are involved in crucial cellular events such as signal transduction (S-glutathionylation and S-nitrosylation), redox regulation of proteins (S-glutathionylation and S-nitrosylation), protein trafficking and subcellular localization (palmitoylation and prenylation), as well as invasion into and egress from host cells (palmitoylation). This review focuses on the occurrence and mechanisms of these cysteine modifications in parasites. Studies on cysteine modifications in human parasites are so far largely based on in vitro experiments. The in vivo regulation of cysteine modifications and their role in parasite development will be of great interest in order to understand redox signaling in parasites.

Posttranslational Modification of Cysteine in Redox Signaling and Oxidative Stress: Focus on S-Glutathionylation

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have become recognized as second messengers for initiating and/or regulating vital cellular signaling pathways, and they are known also as deleterious mediators of cellular stress and cell death. ROS and RNS, and their cross products like peroxynitrite, react primarily with cysteine residues whose oxidative modification leads to functional alterations in the proteins. In this Forum, the collection of six review articles presents a perspective on the broad biological impact of cysteine modifications in health and disease from the molecular to the cellular and organismal levels, focusing in particular on reversible protein-S-glutathionylation and its central role in transducing redox signals as well as protecting proteins from irreversible cysteine oxidation. The Forum review articles consider the role of S-glutationylation in regulation of the peroxiredoxin enzymes, the special redox environment of the mitochondria, redox regulation pertinent to the function of the cardiovascular system, mechanisms of redox-activated apoptosis in the pulmonary system, and the role of glutathionylation in the initiation, propagation, and treatment of neurodegenerative diseases. Several common themes emerge from these reviews; notably, the probability of crosstalk between signaling/regulation mechanisms involving protein-S-nitrosylation and protein-S-glutathionylation, and the need for quantitative analysis of the relationship between specific cysteine modifications and corresponding functional changes in various cellular contexts.