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.     

Fluorescent and affinity-based tools to detect cysteine sulfenic acid formation in proteins

Cysteine sulfenic acid formation in proteins results from the oxidative modification of susceptible cysteine residues by hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This species represents a biologically significant modification occurring during oxidant signaling or oxidative stress, and it can modulate protein function. Most methods to identify such oxidatively modified proteins rely on monitoring the loss of one or more thiol group(s) or on selective labeling of nascent thiol groups following reduction of oxidized proteins. Our previous work reported the direct labeling of these chemically distinct modifications with a dimedone analogue, 1,3-cyclohexadione, to which a linker and functional group (an alcohol) had been added; further addition of a fluorescent isatoic acid or methoxycoumarin reporter allowed detection of the incorporated tag by fluorescence techniques ( Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. ( 2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjugate Chem . 16, 1624-1628 ). We have now expanded our arsenal of tagging reagents to include two fluorescein-, two rhodamine-, and three biotin-conjugated probes based on the original approach. The new tools provide readily detectable fluorescent and affinity probes to identify sulfenic acid modifications in proteins and have been used in subsequent mass spectrometric analyses to confirm covalent attachment of the conjugates and directly determine the site of modification.     

Molecular mechanism of oxidative stress perception by the Orp1 protein

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H(2)O(2) Orp1(Cys36) forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys(36) of Orp1(Cys36) upon exposure to H(2)O(2). Under similar conditions, neither Cys(82) of Orp1(Cys82) nor Cys(598) of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln(70) and Trp(125) form a catalytic triad with Cys(36) in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe(38), Asn(126), and Phe(127), which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H(2)O(2). Both mutants were unable to form Cys-SOH and did not form a H(2)O(2)-inducible disulfide-bonded complex with Yap1-cCRD. The pK(a) of Cys(36) was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys(82) (the resolving cysteine) has a pK(a) value of 8.3. The pK(a) of Cys(36) in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys(36). Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H(2)O(2) than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pK(a) cysteines.     

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.     

In vivo detection of protein cysteine sulfenylation in plastids

Protein cysteine thiols are post‐translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H₂O₂) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation‐sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C‐trapping technology to the plastids of light‐grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H₂O₂ pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H₂O₂ treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle‐tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.     

Detection of protein oxidation in rat-1 fibroblasts by fluorescently labeled tyramine.

Oxidative damage to proteins has been postulated as a major cause of various degenerative diseases including the loss of functional capacity during aging. A prominent target for oxidation by reactive oxygen species (ROS) is the tyrosine residue. Here we present a highly sensitive method for the detection of tyrosyl radical formation in cells. The method is based on the fluorescein-labeled tyrosine analogue, tyramine, which upon oxidation may couple to proteins carrying a tyrosyl radical. Coupling of the probe (denoted TyrFluo) to standard proteins could be induced by generating ROS with horseradish peroxidase/hydrogen peroxide, SIN-1 or with peroxides (cumene or hydrogen peroxide) in combination with a transition metal. TyrFluo added to rat-1 fibroblasts remained outside the cell, whereas the acetylated form (acetylTyrFluo) was membrane-permeable and accumulated in the cell. Exposure of the cells to oxidative stress in the presence of either TyrFluo or acetylTyrFluo gave a cellular labeling characteristic for each probe. Western blot analysis confirmed that each probe labeled a specific set of proteins. This new method for the detection of ROS-induced oxidation of proteins may mimic the tendency of oxidized proteins to form dityrosine bonds.     

DYn-2 Based Identification of Arabidopsis Sulfenomes

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H₂O₂) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H₂O₂ dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H₂O₂ treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.Among the different amino acids, the sulfur containing amino acids like cysteine are particularly susceptible to oxidation by reactive oxygen species (ROS)¹ (1, 2). Recent studies suggest that the sulfenome, the initial oxidation products of cysteine residues, functions as an intermediate state of redox signaling (3 –5). Thus, identifying the sulfenome under oxidative stress is a way to detect potential redox sensors (6, 7).This central role of the sulfenome in redox signaling provoked chemical biologists to develop strategies for sensitive detection and identification of sulfenylated proteins. The in situ trapping of the sulfenome is challenging because of two major factors: (1) the highly reactive, transient nature of sulfenic acids, which might be over-oxidized in excess of ROS, unless immediately protected by disulfide formation (7); (2) the intracellular compartmentalization of the redox state that might be disrupted during extraction procedures, resulting in artificial non-native protein oxidations (8, 9). Having a sulfur oxidation state of zero, sulfenic acids can react as both electrophile and nucleophile, however, direct detection methods are based on the electrophilic character of sulfenic acid (10). In 1974, Allison and coworkers reported a condensation reaction between the electrophilic sulfenic acid and the nucleophile dimedone (5,5-dimethyl-1,3-cyclohexanedione), producing a corresponding thioether derivative (11). This chemistry is highly selective and, since then, has been exploited to detect dimedone modified sulfenic acids using mass spectrometry (12). However, dimedone has limited applications for cellular sulfenome identification because of the lack of a functional group to enrich the dimedone tagged sulfenic acids. Later, dimedone-biotin/fluorophores conjugates have been developed, which allowed sensitive detection and enrichment of sulfenic acid modified proteins (13 –15). This approach, however, was not always compatible with in vivo cellular sulfenome analysis, because the biotin/fluorophores-conjugated dimedone is membrane impermeable (9) and endogenous biotinylated proteins might appear as false positives.More recently, the Carroll lab has developed DYn-2, a sulfenic acid specific chemical probe. This chemical probe consists of two functional units: a dimedone scaffold for sulfenic acid recognition and an alkyne chemical handle for enrichment of labeled proteins (9). Once the sulfenic acids are tagged with the DYn-2 probe, they can be biotinylated through click chemistry (16). The click reaction used here is a copper (I)-catalyzed azide-alkyne cycloaddition reaction (17), also known as azide-alkyne Huisgen cycloaddition (16). With this chemistry, a complex is formed between the alkyne functionalized DYn-2 and the azide functionalized biotin. This biotin functional group facilitates downstream detection, enrichment, and mass spectrometry based identification (Fig. 1). In an evaluation experiment, DYn-2 was found to efficiently detect H₂O₂-dependent sulfenic acid modifications in recombinant glutathione peroxidase 3 (Gpx3) of budding yeast (18). Moreover, it was reported that DYn-2 is membrane permeable, non-toxic, and a non-influencer of the intracellular redox balance (17, 18). Therefore, DYn-2 has been suggested as a global sulfenome reader in living cells (17, 18), and has been applied to investigate epidermal growth factor (EGF) mediated protein sulfenylation in a human epidermoid carcinoma A431 cell line and to identify intracellular protein targets of H₂O₂ during cell signaling (17).Open in a separate windowFig. 1.Schematic views of the molecular mechanism of the DYn-2 probe and the strategy to identify DYn-2 trapped sulfenylated proteins. A, DYn-2 specifically detects sulfenic acid modifications, but no other thiol modifications. B, Biotinylation of the DYn-2 tagged proteins by click reaction. C, Once DYn-2 tagged proteins are biotinylated, a streptavidin-HRP (Strep-HRP) blot visualizes sulfenylation, or alternatively, after enrichment on avidin beads, proteins are identified by mass spectrometry analysis.Here, we selected the DYn-2 probe to identify the sulfenome in plant cells under oxidative stress. Through a combination of biochemical, immunoblot and mass spectrometry techniques, and TAIR10 database and SUBA3-software predictions, we can claim that DYn-2 is able to detect sulfenic acids on proteins located in different subcellular compartments of plant cells. We identified 226 sulfenylated proteins in response to an H₂O₂ treatment of Arabidopsis cell suspensions, of which 123 proteins are new candidates for cysteine oxidative post-translational modification (PTM) events.     

Redox-regulatory mechanisms induced by oxidative stress in Brassica juncea roots monitored by 2-DE proteomics

ROS, including hydrogen peroxide (H(2)O(2)), can serve as cellular signaling molecules following oxidative stress. Analysis of the redox state of proteins in Brassica juncea roots by 2-DE proteomics following treatment with either exogenous H(2)O(2) or buthionine sulfoximine, which depletes glutathione to cause accumulation of endogenous H(2)O(2), led to the identification of different sets of proteins. These data suggest that exogenous and endogenous oxidative stresses trigger specialized responses.     

The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide

Neurodegenerative diseases, a subset of age-driven diseases, have been known to exhibit increased oxidative stress. The resultant increase in reactive oxygen species (ROS) has long been viewed as a detrimental byproduct of many cellular processes. Despite this, therapeutic approaches using antioxidants were deemed unsuccessful in circumventing neurodegenerative diseases. In recent times, it is widely accepted that these toxic by-products could act as secondary messengers, such as hydrogen peroxide (H₂O₂), to drive important signaling pathways. Notably, mitochondria are considered one of the major producers of ROS, especially in the production of mitochondrial H₂O₂. As a secondary messenger, cellular H₂O₂ can initiate redox signaling through oxidative post-translational modifications (oxPTMs) on the thiol group of the amino acid cysteine. With the current consensus that cellular ROS could drive important biological signaling pathways through redox signaling, researchers have started to investigate the role of cellular ROS in the pathogenesis of neurodegenerative diseases. Moreover, mitochondrial dysfunction has been linked to various neurodegenerative diseases, and recent studies have started to focus on the implications of mitochondrial ROS from dysfunctional mitochondria on the dysregulation of redox signaling. Henceforth, in this review, we will focus our attention on the redox signaling of mitochondrial ROS, particularly on mitochondrial H₂O₂, and its potential implications with neurodegenerative diseases.Subject terms: Post-translational modifications, Neurodegenerative diseases     

Proteomics analysis of cellular response to oxidative stress. Evidence for in vivo overoxidation of peroxiredoxins at their active site

The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the destruction of peroxides. In doing so, a reactive cysteine in the peroxiredoxin active site is weakly oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being both important peroxide-destroying enzymes and peroxide targets. Results obtained in a primary culture of Leydig cells challenged with tumor necrosis factor alpha suggested that this oxidized/native balance of peroxiredoxin 2 may play an active role in resistance or susceptibility to tumor necrosis factor alpha-induced apoptosis.     

GRP78 Suppresses Lipid Peroxidation and Promotes Cellular Antioxidant Levels in Glial Cells following Hydrogen Peroxide Exposure

Oxidative stress, caused by the over production of reactive oxygen species (ROS), has been shown to contribute to cell damage associated with neurotrauma and neurodegenerative diseases. ROS mediates cell damage either through direct oxidation of lipids, proteins and DNA or by acting as signaling molecules to trigger cellular apoptotic pathways. The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that has been suggested to protect cells against ROS-induced damage. However, the protective mechanism of GRP78 remains unclear. In this study, we used C6 glioma cells transiently overexpressing GRP78 to investigate the protective effect of GRP78 against oxidative stress (hydrogen peroxide)-induced injury. Our results showed that the overexpression of GRP78 significantly protected cells from ROS-induced cell damage when compared to non-GRP78 overexpressing cells, which was most likely due to GRP78-overexpressing cells having higher levels of glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two antioxidants that protect cells against oxidative stress. Although hydrogen peroxide treatment increased lipid peroxidation in non-GRP78 overexpressing cells, this increase was significantly reduced in GRP78-overexpressing cells. Overall, these results indicate that GRP78 plays an important role in protecting glial cells against oxidative stress via regulating the expression of GSH and NQO1.     

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.     

Antibacterial activity of CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) generating reactive oxygen species

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) is an antifungal and chemosensitizing agent that induces oxidative stress in yeast and filamentous fungi and enhances the cytotoxic activity of 5-fluorocytosine and azole antimycotics. This study reports the effect of CTBT on bacterial cells. CTBT inhibited the growth of both Gram-positive and Gram-negative bacterial species. The action of CTBT was bactericidal. In Escherichia coli, CTBT induced an increased formation of reactive oxygen species (ROS), as determined with a ROS specific probe 2′,7′-dichlorodihydrofluorescein diacetate. In zone inhibition assays, bacterial cells were more sensitive to CTBT compared with paraquat, menadione and hydrogen peroxide. The deletion of oxidative stress related genes resulted in increased susceptibility of E. coli mutant strains to CTBT treatment. Exogenous antioxidants such as ascorbic acid, cysteine and glutathione exhibited a protective effect against the growth inhibition induced by CTBT. CTBT may be a useful tool in the studies of ROS generation, oxidant sensing and oxidative stress response in different bacterial species.     

Novel oxidative modifications in redox-active cysteine residues

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.     

A critical evaluation of probes for cysteine sulfenic acid

Cysteine oxidation is important in cellular redox regulation, signaling, and biocatalysis. To understand the biological relevance of cysteine oxidation, it is desirable to identify the proteins involved, the site of the oxidized cysteine, and the relevant oxidation states. Because the thiol of cysteine can be converted to a wide range of oxidation states, mapping these oxidative modifications is challenging. The dynamic and reversible nature of many cysteine oxidation states compounds the difficulty in such proteomic analyses. In this review, we examine methods to detect cysteine sulfenic acid — a particularly challenging functional group to analyze because of its reactive nature. We focus on the selectivity of recently reported probes and discuss some challenges and opportunities in this field.     

Activity of artichoke leaf extract on reactive oxygen species in human leukocytes

Artichoke leaf extract was studied in human leukocytes for activity against oxidative stress using flow cytometry and dichlorofluorescin diacetate as a fluorescence probe. It produces a concentration-dependent inhibition of oxidative stress when cells are stimulated with agents that generate reactive oxygen species (ROS): hydrogen peroxide, phorbol-12-myristate-13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Cynarin, caffeic acid, chlorogenic acid, and luteolin, constituents of artichoke leaf extract, also show a concentration-dependent inhibitory activity in the above models, contributing to the antioxidant activity of the extract in human neutrophils.