Oxidative Cysteine Modifications Research Articles

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxICAT, Biotin Switch, and SP3-Rox, these methods typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. Here we establish the local cysteine capture (Cys-LoC) and local cysteine oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole-cell proteomic analysis. Application of the Cys-LOx method to LPS-stimulated immortalized murine bone marrow-derived macrophages (iBMDM), revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation, including those associated with oxidative mitochondrial metabolism.

Aurora Kinase A Regulation by Cysteine Oxidative Modification

Aurora kinase A (AURKA), which is a member of serine/threonine kinase family, plays a critical role in regulating mitosis. AURKA has drawn much attention as its dysregulation is critically associated with various cancers, leading to the development of AURKA inhibitors, a new class of anticancer drugs. As the spatiotemporal activity of AURKA critically depends on diverse intra- and inter-molecular factors, including its interaction with various protein cofactors and post-translational modifications, each of these pathways should be exploited for the development of a novel class of AURKA inhibitors other than ATP-competitive inhibitors. Several lines of evidence have recently shown that redox-active molecules can modify the cysteine residues located on the kinase domain of AURKA, thereby regulating its activity. In this review, we present the current understanding of how oxidative modifications of cysteine residues of AURKA, induced by redox-active molecules, structurally and functionally regulate AURKA and discuss their implications in the discovery of novel AURKA inhibitors.

Hsp70 in Redox Homeostasis.

Cellular redox homeostasis is precisely balanced by generation and elimination of reactive oxygen species (ROS). ROS are not only capable of causing oxidation of proteins, lipids and DNA to damage cells but can also act as signaling molecules to modulate transcription factors and epigenetic pathways that determine cell survival and death. Hsp70 proteins are central hubs for proteostasis and are important factors to ameliorate damage from different kinds of stress including oxidative stress. Hsp70 members often participate in different cellular signaling pathways via their clients and cochaperones. ROS can directly cause oxidative cysteine modifications of Hsp70 members to alter their structure and chaperone activity, resulting in changes in the interactions between Hsp70 and their clients or cochaperones, which can then transfer redox signals to Hsp70-related signaling pathways. On the other hand, ROS also activate some redox-related signaling pathways to indirectly modulate Hsp70 activity and expression. Post-translational modifications including phosphorylation together with elevated Hsp70 expression can expand the capacity of Hsp70 to deal with ROS-damaged proteins and support antioxidant enzymes. Knowledge about the response and role of Hsp70 in redox homeostasis will facilitate our understanding of the cellular knock-on effects of inhibitors targeting Hsp70 and the mechanisms of redox-related diseases and aging.

Oxidative Cysteine Modification of Thiol Isomerases in Thrombotic Disease: A Hypothesis.

Significance: Oxidative stress is a characteristic of many systemic diseases associated with thrombosis. Thiol isomerases are a family of oxidoreductases important in protein folding and are exquisitely sensitive to the redox environment. They are essential for thrombus formation and represent a previously unrecognized layer of control of the thrombotic process. Yet, the mechanisms by which thiol isomerases function in thrombus formation are unknown. Recent Advances: The oxidoreductase activity of thiol isomerases in thrombus formation is controlled by the redox environment via oxidative changes to active site cysteines. Specific alterations can now be detected owing to advances in the chemical biology of oxidative cysteine modifications. Critical Issues: Understanding of the role of thiol isomerases in thrombus formation has focused largely on identifying single disulfide bond modifications in isolated proteins (e.g., αIIbβ3, tissue factor, vitronectin, or glycoprotein Ibα [GPIbα]). An alternative approach is to conceptualize thiol isomerases as effectors in redox signaling pathways that control thrombotic potential by modifying substrate networks. Future Directions: Cysteine-based chemical biology will be employed to study thiol-dependent dynamics mediated by the redox state of thiol isomerases at the systems level. This approach could identify thiol isomerase-dependent modifications of the disulfide landscape that are prothrombotic.

Human sirtuins are differentially sensitive to inhibition by nitrosating agents and other cysteine oxidants

Sirtuins (e.g. human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD+-dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Although multiple reports suggest that sirtuin activity is regulated by oxidative post-translational modifications of cysteines during inflammation and aging, no systematic comparative study of potential direct sirtuin cysteine oxidative modifications has been performed. Here, using IC50 and kinact/KI analyses, we quantified the ability of nitrosothiols (S-nitrosoglutathione and S-nitroso-N-acetyl-d,l-penicillamine), nitric oxide, oxidized GSH, and hydrogen peroxide to post-translationally modify and inhibit the deacylase activity of Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6. The inhibition was correlated with cysteine modification and assessed with chemical-probe and blot-based assays for cysteine S-nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine S-nitrosation in response to exposure to both free nitric oxide and nitrosothiols (kinact/KI ≥ 5 m-1 s-1), which is the first report of Sirt2 and Sirt6 inhibition by S-nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development.

Upregulation of Thioredoxin-Interacting Protein in Brain of Amyloid-β Protein Precursor/Presenilin 1 Transgenic Mice and Amyloid-β Treated Neuronal Cells.

Oxidative stress has been hypothesized to play a role in the pathophysiology of Alzheimer's disease (AD). Previously, we found that total nitrosylated protein levels were increased in the brain of amyloid-β protein precursor (AβPP) and presenilin 1 (PS1) double transgenic mice, an animal model for AD, suggesting that cysteine oxidative protein modification may contribute to this disease. Thioredoxin (Trx) is a major oxidoreductase that can reverse cysteine oxidative modifications such as sulfenylation and nitrosylation, and inhibit oxidative stress. Thioredoxin-interacting protein (Txnip) is an endogenous Trx inhibitor. To understand the involvement of Trx and Txnip in AD development, we investigated Trx and Txnip in the brain of AβPP/PS1 mice. Using immunoblotting analysis, we found that although Trx protein levels were not changed, Txnip protein levels were significantly increased in hippocampus and frontal cortex of 9- and 12-month-old AβPP/PS1 mice when compared to wild-type mice. Txnip protein levels were also increased by amyloid-β treatment in primary cultured mouse cerebral cortical neurons and HT22 mouse hippocampal cells. Using biotin switch and dimedone conjugation methods, we found that amyloid-β treatment increased protein nitrosylation and sulfenylation in HT22 cells. We also found that downregulation of Txnip, using CRISPR/Cas9 method in HT22 cells, attenuated amyloid-β-induced protein nitrosylation and sulfenylation. Our findings suggest that amyloid-β may increase Txnip levels, subsequently inhibiting Trx reducing capability and enhancing protein cysteine oxidative modification. Our findings also indicate that Txnip may be a potential target for the treatment of AD.

Proteomic Methods to Evaluate NOX-Mediated Redox Signaling.

The NADPH oxidase (NOX) family of proteins is involved in regulating many diverse cellular processes, which is largely mediated by NOX-mediated reversible oxidation of target proteins in a process known as redox signaling. Protein cysteine residues are the most prominent targets in redox signaling, and to understand the mechanisms by which NOX affect cellular pathways, specific methodology is required to detect specific oxidative cysteine modifications and to identify targeted proteins. Among the many potential redox modifications involving cysteine residues, reversible modifications most relevant to NOX are sulfenylation (P-SOH) and S-glutathionylation (P-SSG), as both can induce structural or functional alterations. Various experimental approaches have been developed to detect these specific modifications, and this chapter will detail state-of-the-art methodology to selectively evaluate these modifications in specific target proteins in relation to NOX activation. We also discuss some of the limitations of these procedures and potential complementary approaches.

327 - Thioredoxin-interacting protein, a potential pharmacological target for the treatment of depression

Chronic stress is a key contributor to depression. Studies show that oxidative stress and inflammation are increased in rodents exposed to chronic stress and in patients with depression. Thioredoxin as a redox protein can reduce protein cysteine oxidative modification and inhibit apoptosis signal-regulating kinase 1 (ASK1) stress and inflammatory pathway. Therefore thioredoxin plays an important role in defense against oxidative stress and inflammation. Thioredoxin-interacting protein (Txnip) can inhibit thioredoxin. The purpose of this study was to investigate thioredoxin and Txnip in brain of mice exposed to chronic unpredictable stress (CUS) and in postmortem brain of patient with depression. We found that mice exposed to CUS displayed depressive-like behavior including decreased exploration, increased anhedonia and increased despair. Although CUS had no effect on thioredoxin levels, it increased Txnip levels in mouse hippocampus and frontal cortex. Further Txnip levels were upregulated by chronic treatment with stress hormone corticosterone (CORT) in cultured neuronal cells and in postmortem anterior cingulate of patient with depression. Txnip not only inhibits Trx, enhancing cysteine oxidative modification and ASK1 activation; but also binds to nod-like receptor protein 3 (NLRP3), facilitating NLRP3 inflammasome forming. We found that CUS increased protein sulfenylation and nitrosylation, and ASK1 phosphorylation in mouse brain. Knocking out Txnip using CRISPR/Cas9 technology blocked chronic CORT treatment-increased sulfenylation and nitrosylation in cultured neuronal cells. We also found that CUS and CORT treatment increased Txnip-NLRP3 binding in mouse brain and cultured microglia. Our results suggest that chronic stress may upregulate Txnip, enhancing Txnip binding to Trx, which inhibit Trx reducing activity, subsequently promoting cysteine oxidative modification; and inhibit Trx binding to ASK1, facilitating ASK1 phosphorylation. CUS-upregulated Txnip may also enhance NLRP3-mediated inflammatory signaling. Because chronic stress is a major risk for depression, Txnip may be a novel potential target for the treatment of depression.

Proteomic Analysis of Hippocampus in a Mouse Model of Depression Reveals Neuroprotective Function of Ubiquitin C-terminal Hydrolase L1 (UCH-L1) via Stress-induced Cysteine Oxidative Modifications

Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.

Integrated Dissection of Cysteine Oxidative Post-translational Modification Proteome During Cardiac Hypertrophy.

Cysteine oxidative modification of cellular proteins is crucial for many aspects of cardiac hypertrophy development. However, integrated dissection of multiple types of cysteine oxidative post-translational modifications (O-PTM) of proteomes in cardiac hypertrophy is currently missing. Here we developed a novel discovery platform that encompasses a customized biotin switch-based quantitative proteomics pipeline and an advanced analytic workflow to comprehensively profile the landscape of cysteine O-PTM in an ISO-induced cardiac hypertrophy mouse model. Specifically, we identified a total of 1655 proteins containing 3324 oxidized cysteine sites by at least one of the following three modifications: reversible cysteine O-PTM, cysteine sulfinylation (CysSO2H), and cysteine sulfonylation (CysSO3H). Analyzing the hypertrophy signatures that are reproducibly discovered from this computational workflow unveiled four biological processes with increased cysteine O-PTM. Among them, protein phosphorylation, creatine metabolism, and response to elevated Ca2+ pathways exhibited an elevation of cysteine O-PTM in early stages, whereas glucose metabolism enzymes were increasingly modified in later stages, illustrating a temporal regulatory map in cardiac hypertrophy. Our cysteine O-PTM platform depicts a dynamic and integrated landscape of the cysteine oxidative proteome, through the extracted molecular signatures, and provides critical mechanistic insights in cardiac hypertrophy. Data are available via ProteomeXchange with identifier PXD010336.

Increased thioredoxin-interacting protein in brain of mice exposed to chronic stress

Chronic stress is a key contributor to depression. Previous studies have shown that oxidative stress and inflammation are increased by chronic stress and in subjects with depression. Thioredoxin is a small redox protein that regulates cellular redox balance and signaling. This protein can reverse protein cysteine oxidative modifications such as sulfenylation and nitrosylation, and inhibit stress-regulated apoptosis signal-regulating kinase 1 pathway. Therefore thioredoxin plays an important role in cellular defense against oxidative stress. Thioredoxin-interacting protein is an endogenous thioredoxin inhibitor. In the present study, to understand the role of thioredoxin in chronic stress and depression, we have investigated thioredoxin, thioredoxin-interacting protein, sulfenylation, nitrosylation and apoptosis signal-regulating kinase 1 phosphorylation in brain of mice exposed to chronic unpredictable stress (CUS). We found that mice exposed to CUS displayed decreased exploratory, increased anhedonic and increased despair depressive-like behaviours. We also found that although CUS had no effect on thioredoxin protein levels, it significantly increased levels of thioredoxin-interacting protein in mouse hippocampus and frontal cortex. CUS also increased protein cysteine sulfenylation, protein cysteine nitrosylation and apoptosis signal-regulating kinase 1 phosphorylation in mouse hippocampus and frontal cortex. These findings suggest that chronic stress may upregulate thioredoxin-interacting protein, subsequently inhibiting thioredoxin activity and enhancing oxidative protein cysteine modification and apoptosis signal-regulating kinase 1 pathway. These results also indicate that thioredoxin-interacting protein may have potential for depression treatment.

An isomerase completes the circuit for a redox switch

The activity of human transglutaminase 2 (TG2), which forms protein cross-links between glutamine and lysine residues, is controlled by an allosteric disulfide bond. However, the mechanism by which this bond is formed, like many systems regulated by oxidative cysteine modifications, was not clear. A new study from Khosla and colleagues shows that TG2 is oxidatively inactivated by the protein disulfide isomerase ERp57, providing the first example of a defined and reversible protein-controlled redox switch and pointing to new strategies to inhibit undesirable TG2 activity in pathological states.

Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics.

63 - Redox-Regulation of Ca2+-Independent Phospholipase A2γ: From Computational Predictions to Experimental Procedures

Redox reactions of cysteine residues lead to a range of reversible post-translational modifications of proteins, regulating their functions. Ca2+-independent phospholipase A2γ (iPLA2γ) belongs to a family of enzymes which catalyze the hydrolysis of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids, contributing to cellular signaling, membrane homeostasis and energy metabolism. The activity of iPLA2γ is regulated by a redox-sensitive process, suggesting a thiol-based modification. Using recent computational methods, we searched the primary sequence of human iPLA2γ for potential cysteine residues that could be involved in a thiol-based redox regulation of the protein. Two cysteines, Cys233 and Cys329, were pointed out as highly probable S-sulfenylation target sites. In addition, we used protein structure homology modeling to create a three-dimensional structure of iPLA2γ to visualize the topology of the predicted cysteine residues. The primary sequence was aligned to search for suitable templates and the model was optimized by energy minimization using the GROMOS96 parameter set and checked for errors in the 3D protein structure. The final 3D structure with visualized cysteine residues was designed using molecular graphic system PyMOL. Using isolated recombinant human iPLA2γ, both Cys233Ser and Cys329Ser targeted mutations revealed altered regulation of the catalytic activity by H2O2. These results are consistent with the participation of iPLA2γ in cellular redox homeostasis and serve as preliminary tools leading to a direct identification of cysteine oxidative modifications of the enzyme. Supported by grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.

Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line.

Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca2+ homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca2+ entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca2+ entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype-targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca2+ influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5′-dithiobis (2-nitrobenzoic acid)-2biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1, TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP-induced Ca2+ entry and subsequent hepatocellular death are regulated by multiple redox-activated cation channels, among which TRPV1 and TRPC1 play a prominent role.

Does the Transcription Factor NemR Use a Regulatory Sulfenamide Bond to Sense Bleach?

Reactive chlorine species (RCS), such as hypochlorous acid (i.e., bleach), are antimicrobial oxidants produced by the innate immune system. Like many redox-regulated transcription factors, the Escherichia coli repressor NemR responds to RCS by using the reversible oxidation of highly conserved cysteines to alter its DNA-binding affinity. However, earlier work showed that RCS response in NemR does not depend on any commonly known oxidative cysteine modifications. We have now determined the crystal structure of NemR, showing that the regulatory cysteine, Cys106, is in close proximity to a highly conserved lysine (Lys175). We used crystallographic, biochemical, and mass spectrometric analyses to analyze the role of this lysine residue in RCS sensing. Based on our results, we hypothesize that RCS treatment of NemR results in the formation of a reversible Cys106-Lys175 sulfenamide bond. This is, to our knowledge, the first description of a protein whose function is regulated by a cysteine-lysine sulfenamide thiol switch, constituting a novel addition to the biological repertoire of functional redox switches.

Formation of protein S-nitrosylation by reactive oxygen species

In the present study, the formation of whole cellular S-nitrosylated proteins (protein-SNOs) by the reactive oxygen species (ROS), hydrogen peroxide (H2O2), and superoxide (O2•−) is demonstrated. A spectrum of protein cysteine oxidative modifications was detected upon incubation of serum-starved mouse embryonic fibroblasts with increasing concentrations of exogenous H2O2, ranging from exclusive protein-SNOs at low concentrations to a mixture of protein-SNOs and other protein oxidation at higher concentrations to exclusively non-SNO protein oxidation at the highest concentrations of the oxidant used. Furthermore, formation of protein-SNOs was also detected upon inhibition of the antioxidant protein Cu/Zn superoxide dismutase that results in an increase in intracellular concentration of O2•−. These results were further validated using the phosphatase and tensin homologue, PTEN, as a model of a protein sensitive to oxidative modifications. The formation of protein-SNOs by H2O2 and O2•− was prevented by the NO scavenger, c-PTIO, as well as the peroxinitrite decomposition catalyst, FETPPS, and correlated with the production or the consumption of nitric oxide (NO), respectively. These data suggest that the formation of protein-SNOs by H2O2 or O2•− requires the presence or the production of NO and involves the formation of the nitrosylating intermediate, peroxinitrite.

Protein glutathionylation in cardiovascular diseases.

The perturbation of thiol-disulfide homeostasis is an important consequence of many diseases, with redox signals implicated in several physio-pathological processes. A prevalent form of cysteine modification is the reversible formation of protein mixed disulfides with glutathione (S-glutathionylation). The abundance of glutathione in cells and the ready conversion of sulfenic acids to S-glutathione mixed disulfides supports the reversible protein S-glutathionylation as a common feature of redox signal transduction, able to regulate the activities of several redox sensitive proteins. In particular, protein S-glutathionylation is emerging as a critical signaling mechanism in cardiovascular diseases, because it regulates numerous physiological processes involved in cardiovascular homeostasis, including myocyte contraction, oxidative phosphorylation, protein synthesis, vasodilation, glycolytic metabolism and response to insulin. Thus, perturbations in protein glutathionylation status may contribute to the etiology of many cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy and atherosclerosis. Various reports show the importance of oxidative cysteine modifications in modulating cardiovascular function. In this review, we illustrate tools and strategies to monitor protein S-glutathionylation and describe the proteins so far identified as glutathionylated in myocardial contraction, hypertrophy and inflammation.

Chasing cysteine oxidative modifications: proteomic tools for characterizing cysteine redox status.

Redox-proteomics involves the large scale analysis of oxidative protein post-translational modifications. In particular, cysteine residues have become the subject of intensifying research interest because of their redox-reactive thiol side chain. Certain reactive cysteine residues can function as redox-switches, which sense changes in the local redox-environment by flipping between the reduced and oxidized state. Depending on the reactive oxygen or nitrogen species, cysteine residues can receive one of several oxidative modifications, each with the potential to confer a functional effect. Modification of these redox-switches has been found to play an important role in oxidative-signaling in the cardiovascular system and elsewhere. Due to the labile and dynamic nature of these modifications, several targeted approaches have been developed to enrich, identify and characterize the status of these critical residues. Here, we review the various proteomic strategies and limitations for the large scale analysis of the different oxidative cysteine modifications.

Thiol redox proteomics seen with fluorescent eyes: The detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE

There is increasing evidence that several reversible oxidative post-translational modifications of protein cysteines participate in cell signalling. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes, that are collectively known as thiol redox proteomics. Recently, fluorescence derivatization methods have been developed that enable these post-translational modifications to be studied using proteomic workflows based on two-dimensional electrophoresis, which is a relatively accessible and affordable technique. As well as enabling a large number of samples to be processed, two-dimensional electrophoresis has the advantage that it does not rely on the intensive use of mass spectrometers. This methodology allows to "visualise" redox changes in a broad context and, although identification of the modified residues is not so straightforward, complementary derivatization can overcome this drawback. Here we review the different derivatization strategies that have been employed in these studies, comparing their advantages and potential limitations. We also review the applications and results obtained, with particular emphasis on those involving (patho)physiological stimuli, thereby showing the potential of these techniques to study the thiol redox proteome.