Redox regulation of protein tyrosine phosphatase activity by hydroxyl radical

Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H₂O₂ include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H₂O₂ abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H₂O₂ is poorly reactive in chemical terms and the second order rate constants for the H₂O₂-mediated PTP inactivation are ~ 10 M^− 1 s^− 1, which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H₂O₂. We find that hydroxyl radical is produced from H₂O₂ solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H₂O₂ for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2–3 orders of magnitude higher than those mediated by H₂O₂ under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.     

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

Site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin affects specific amino acid residues and the rate of cytochrome b5-mediated heme reduction

Myeloperoxidase catalyzes the reaction of chloride ions with H₂O₂ to yield hypochlorous acid (HOCl), which can damage proteins. Human myoglobin (HMb) differs from other Mbs by the presence of a cysteine residue at position 110 (Cys110). This study has (i) compared wild-type and a Cys110Ala variant of HMb to assess the influence of Cys110 on HOCl-induced amino acid modification and (ii) determined whether HOCl oxidation of HMb affects the rate of ferric heme reduction by cytochrome b₅. For wild-type HMb (HOCl:Mb ratio of 5:1 mol:mol), Cys110 was preferentially oxidized to a homodimeric or cysteic acid product—sulfenic/sulfinic acids were not detected. At a HOCl:Mb ratio 10:1 mol:mol, methionine (Met) oxidation was detected, and this was enhanced in the Cys110Ala variant. Tryptophan (Trp) oxidation was detected only in the Cys110Ala variant at the highest HOCl dose tested, with oxidation susceptibility following the order Cys > Met > Trp. Tyrosine chlorination was evident only in reactions between HOCl and the Cys110Ala variant and at a longer incubation time (24 h), consistent with the formation via chlorine-transfer reactions from preformed chloramines. HOCl-mediated oxidation of wild-type HMb resulted in a dose-dependent decrease in the observed rate constant for ferric heme reduction (approx two-fold at HOCl:Mb of 10:1 mol:mol). These data indicate that Cys110 influences the oxidation of HMb by HOCl and that oxidation of Cys, Met, and Trp residues is associated with a decrease in the one-electron reduction of ferric HMb by other proteins; such heme-Fe³⁺ reduction is critical to the maintenance of function as an oxygen storage protein in tissues.     

Structural and functional regulation of eukaryotic 2-Cys peroxiredoxins including the plant ones in cellular defense-signaling mechanisms against oxidative stress

The ubiquitously distributed peroxiredoxins (Prxs) have been shown to have diverse functions in cellular defense‐signaling pathways. They have been largely classified into three Prx classes, 2‐Cys Prx, atypical 2‐Cys Prx and 1‐Cys Prx, which can be distinguished by how many Cys residues they possess and by their catalytic mechanisms. Proteins belonging to the typical 2‐Cys Prx group containing the N‐terminal peroxidatic Cys residue undergo a cycle of peroxide‐dependent oxidation to sulfenic acid and thiol‐dependent reduction during H₂O₂ catalysis. However, in the presence of high concentrations of H₂O₂ and catalytic components, including thioredoxin (Trx), Trx reductase and NADPH, the sulfenic acid can be hyperoxidized to cysteine sulfinic acid. The overoxidized 2‐Cys Prxs are slowly reduced by the action of the adenosine 5′‐triphosphate‐dependent enzyme, sulfiredoxin. Upon exposure of cells to strong oxidative or heat‐shock stress conditions, 2‐Cys Prxs change their protein structures from low‐molecular weight to high‐molecular weight complexes, which trigger their functional switching from peroxidases to molecular chaperones. The C‐terminal region of 2‐Cys Prx also plays an essential role in this structural conversion. Thus, proteins with truncated C‐termini are resistant to overoxidation and cannot regulate their structures or functions. These reactions are primarily guided by the active site peroxidatic Cys residue, which serves as an ‘H₂O₂‐sensor’ in cells. The reversible structural and functional switching of 2‐Cys Prxs provides cells with a means to adapt to external stresses by presumably activating intracellular defense‐signaling systems. In particular, plant 2‐Cys Prxs localized in chloroplasts have dynamic protein structures that undergo major conformational changes during catalysis, forming super‐complexes and reversibly attaching to thylakoid membranes in a redox‐dependent manner.     

Cofactor binding protects flavodoxin against oxidative stress

In organisms, various protective mechanisms against oxidative damaging of proteins exist. Here, we show that cofactor binding is among these mechanisms, because flavin mononucleotide (FMN) protects Azotobacter vinelandii flavodoxin against hydrogen peroxide-induced oxidation. We identify an oxidation sensitive cysteine residue in a functionally important loop close to the cofactor, i.e., Cys69. Oxidative stress causes dimerization of apoflavodoxin (i.e., flavodoxin without cofactor), and leads to consecutive formation of sulfinate and sulfonate states of Cys69. Use of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reveals that Cys69 modification to a sulfenic acid is a transient intermediate during oxidation. Dithiothreitol converts sulfenic acid and disulfide into thiols, whereas the sulfinate and sulfonate forms of Cys69 are irreversible with respect to this reagent. A variable fraction of Cys69 in freshly isolated flavodoxin is in the sulfenic acid state, but neither oxidation to sulfinic and sulfonic acid nor formation of intermolecular disulfides is observed under oxidising conditions. Furthermore, flavodoxin does not react appreciably with NBD-Cl. Besides its primary role as redox-active moiety, binding of flavin leads to considerably improved stability against protein unfolding and to strong protection against irreversible oxidation and other covalent thiol modifications. Thus, cofactors can protect proteins against oxidation and modification.     

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

Reactive oxidative species (ROS) and S‐glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI‐Cys13 and At‐Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI‐Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI‐Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI‐Cys13, mutations in AtcTPI‐Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI‐Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent‐exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S‐glutathionylation protects AtcTPI from irreversible chemical modifications and re‐routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.     

RegB Kinase Activity Is Repressed by Oxidative Formation of Cysteine Sulfenic Acid

RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.     

Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1): SITES AND NATURE OF OXIDATIVE MODIFICATION*

In mammalian skeletal muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca²⁺-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO₂) through O₂-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O₂) versus low pO₂ (1% O₂), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO₂-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO₂ only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO₂-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO₂-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca²⁺ channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in “hot spot” regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO₂-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.     

Identification of dimedone-trapped sulfenylated proteins in plants under stress

In stressed plants, the reactive oxygen species (ROS) levels rise. Key to ROS signaling research are detection and identification of the protein cysteine sulfenylation (-SOH), the ROS-mediated oxidative product of a thiol (-SH). Arabidopsis thaliana seedlings were stressed with hydrogen peroxide (H₂O₂) and the sulfenylated proteins were tagged with dimedone. Dimedone-tagged sulfenic acid proteins were visualized on a two-dimensional electrophoresis (2DE) immunoblot with an anticysteine sulfenic acid antibody and were subsequently detected by mass spectrometry. We optimized the detection method for protein sulfenylation in Arabidopsis. We conclude that dimedone can penetrate the cell wall, does not stress plants, and can “read” the changes in the protein sulfenylation pattern under oxidative stress. We observed that the number of sulfenylated proteins in plants treated with 10 mM H₂O₂ was higher than that in untreated plants. A total of 39 sulfenylated protein spots were found on 2DE immunoblots. By means of mass spectrometry, 11 sulfenylated proteins were discovered involved in primary metabolism, redox regulation, translation and signaling pathways. Hence, by combining an immunochemical 2DE strategy with mass spectrometry, we were able to identify sulfenylated proteins in H₂O₂-stressed Arabidopsis seedlings. The sulfenylated proteins can be considered for further validation as redox regulators in plants.     

Proposed role of primary protein carbonylation in cell signaling

Abstract

Reactive oxygen species (ROS) mediate various cell signaling processes, but the mechanism for how ROS promote cell signaling is poorly understood. Protein carbonylation occurs because of the direct metal-catalyzed oxidation of amino acid side chains (primary protein carbonylation) or the addition of reactive aldehydes to amino acid side chains (secondary protein carbonylation). We hypothesize that primary protein carbonylation plays a role in the mechanism of ROS signaling. Specifically, we propose that (i) primary protein carbonylation mediates cell signaling and (ii) primary protein carbonylation is reversible.     

The oxido-redox potential of albumin: Methodological approach and relevance to human diseases

In humans, an increased synthesis of reactive oxygen species (ROS) may be a relevant cause of amplification of physiologic processes resulting in inflammatory organ damage or neoplasia. Efficient anti-oxidative systems targeting oxidative stress are thus essential to prevent tissue damage. In plasma, proteins proved to be the first line of defence against ROS and albumin, the highest concentration plasma protein, has a key role in this antioxidant function. Recent studies have clearly documented that albumin oxido-redox potential changes upon oxidation by different oxidants thus becoming a deputy biomarker of this process.ROS react primarily with the free ³⁴Cysteine (³⁴Cys) residue of albumin to form two reversible intermediate derivatives, sulfenic-(SOH-alb) and sulfinic acid (SO₂H-alb), resulting in sulfonic acid (SO₃H-alb), the final stable product of the reaction.Upon stable oxidation (SO₃H-alb), albumin properties are altered: the protein becomes more susceptible to trypsin digestion and is degraded faster compared to the non-oxidized counterpart.The present review focuses on the characterization of albumin chemical changes induced by ROS, their relevance in human pathology and the most recent advances in the approach to oxidation adduct analysis.     

Escherichia coli alanyl-tRNA synthetase maintains proofreading activity and translational accuracy under oxidative stress

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNA^Ala formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNA^Ala aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species “sinks” to protect catalytically critical sites from oxidative damage. Combined, our results indicate that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.     

S-Oxygenation of N-substituted thioureas catalyzed by the pig liver microsomal FAD-containing monooxygenase

The microsomal FAD-containing monooxygenase (EC 1.14.13.8, dimethylaniline monooxygenase) purified to homogeneity from hog liver catalyzes NADPH- and oxygen-dependent S-oxygenation of phenylthiourea, ethylenethiourea, thiocarbanilide, N-methylthiourea, and thiourea to their corresponding formamidine sulfinic acids. The sulfinic acids are formed by sequential enzymic oxidation of the thioureas through intermediate sulfenic acids. The reaction sequence was established by separating intermediate and final oxygenated metabolites of phenylthiourea and ethylenethiourea. The sulfenic and sulfinic acids of these two thioureas, produced enzymically, were chromatographically and spectrally identical with chemically synthesized reference compounds. Phenylformamidine and ethyleneformamidine sulfinic acids are slowly converted to their sulfonic acids upon prolonged incubation. While N-substituted formamidine sulfinic acids oxidize spontaneously to formamidine sulfonic acids at 37 °C, the further oxidation of ethyleneformamidine sulfinic acid may be, at least in part, enzyme catalyzed. The purified monooxygenase also catalyzes rapid oxygenation of mercaptoimidazoles to the corresponding imidazole sulfinic acids. The instability of S-oxygenated mercaptoimidazoles prevented their isolation and positive identification, but analysis of kinetic data obtained with sulfenic acid trapping agents suggests that these compounds are oxygenated by the same reaction sequence established for N-substituted thioureas. The NADPH- and oxygen-dependent oxidation of thiocarbamates and of 2-mercaptoimidazoles catalyzed by hog or hamster liver microsomes correlates with dimethylaniline N-oxidase activity and appears completely independent from cytochrome P-450. The S-oxidation of thiourea and its derivatives is not inhibited by n-octylamine, a known inhibitor of cytochrome P-450 dependent oxygenations. Furthermore, differential thermal inactivation of the flavin-containing monooxygenase totally abolishes phenylthiourea S-oxidase activity of hamster liver microsomes.     

Parvalbumin as a metal-dependent antioxidant

Parvalbumin (PA) is a Ca²⁺-binding protein of vertebrates massively expressed in tissues with high oxygen uptake and respectively elevated level of reactive oxygen species (ROS). To characterize antioxidant properties of PA, antioxidant capacity (AOC) of intact rat α-PA has been explored. ORAC, TEAC and hydrogen peroxide AOC assays evidence conformation-dependent oxidation of the PA. AOC value for the apo-PA 4-11-fold exceeds that for the Ca²⁺-loaded protein. Despite folded conformation of apo-PA, it has AOC equivalent to that of the proteolized protein. The most populated under resting conditions PA form, Mg²⁺-bound PA, has AOC similar to that of apo-PA. ROS-induced changes in absorption spectrum of PA evidence an oxidation of PA's phenylalanines in the ORAC assay. Sensitivity of PA oxidation to its conformation enabled characterization of its metal affinity and pH-dependent behavior: a transition with pK_a of 7.6 has been revealed for the Ca²⁺-loaded PA. Since total AOC of PA under in vivo conditions may reach the level of reduced glutathione, we propose that PA might modulate intracellular redox equilibria and/or signaling in a calcium-dependent manner. We speculate that the oxidation-mediated damage of some of PA-GABAergic interneurons observed in schizophrenia is due to a decline in total AOC of the reduced glutathione–PA pair.     

Reactive oxygen species in vascular biology: implications in hypertension

Reactive oxygen species (ROS), including superoxide (·O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl anion (OH-), and reactive nitrogen species, such as nitric oxide (NO) and peroxynitrite (ONOO^–), are biologically important O₂ derivatives that are increasingly recognized to be important in vascular biology through their oxidation/reduction (redox) potential. All vascular cell types (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) produce ROS, primarily via cell membrane-associated NAD(P)H oxidase. Reactive oxygen species regulate vascular function by modulating cell growth, apoptosis/anoikis, migration, inflammation, secretion, and extracellular matrix protein production. An imbalance in redox state where pro-oxidants overwhelm anti-oxidant capacity results in oxidative stress. Oxidative stress and associated oxidative damage are mediators of vascular injury and inflammation in many cardiovascular diseases, including hypertension, hyperlipidemia, and diabetes. Increased generation of ROS has been demonstrated in experimental and human hypertension. Anti-oxidants and agents that interrupt NAD(P)H oxidase-driven ·O₂^– production regress vascular remodeling, improve endothelial function, reduce inflammation, and decrease blood pressure in hypertensive models. This experimental evidence has evoked considerable interest because of the possibilities that therapies targeted against reactive oxygen intermediates, by decreasing generation of ROS and/or by increasing availability of antioxidants, may be useful in minimizing vascular injury and hypertensive end organ damage. The present chapter focuses on the importance of ROS in vascular biology and discusses the role of oxidative stress in vascular damage in hypertension.     

Gel-free proteomic methodologies to study reversible cysteine oxidation and irreversible protein carbonyl formation

Abstract

Oxidative modifications in proteins have been traditionally considered as hallmarks of damage by oxidative stress and aging. However, oxidants can generate a huge variety of reversible and irreversible modifications in amino acid side chains as well as in the protein backbones, and these post-translational modifications can contribute to the activation of signal transduction pathways, and also mediate the toxicity of oxidants. Among the reversible modifications, the most relevant ones are those arising from cysteine oxidation. Thus, formation of sulfenic acid or disulfide bonds is known to occur in many enzymes as part of their catalytic cycles, and it also participates in the activation of signaling cascades. Furthermore, these reversible modifications have been usually attributed with a protective role, since they may prevent the formation of irreversible damage by scavenging reactive oxygen species. Among irreversible modifications, protein carbonyl formation has been linked to damage and death, since it cannot be repaired and can lead to protein loss-of-function and to the formation of protein aggregates. This review is aimed at researchers interested on the biological consequences of oxidative stress, both at the level of signaling and toxicity. Here we are providing a concise overview on current mass-spectrometry-based methodologies to detect reversible cysteine oxidation and irreversible protein carbonyl formation in proteomes. We do not pretend to impose any of the different methodologies, but rather to provide an objective catwalk on published gel-free approaches to detect those two types of modifications, from a biologist's point of view.     

Generator-specific targets of mitochondrial reactive oxygen species

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for “signaling” and “damaging” ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc₁ complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H₂O₂) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.     

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.