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Protein S-glutathionylation: From current basics to targeted modifications
Abstract
Abstract The interaction between antioxidant glutathione and the free thiol in susceptible cysteine residues of proteins leads to reversible protein S-glutathionylation. This reaction ensures cellular homeostasis control (as a common redox-dependent post-translational modification associated with signal transduction) and intervenes in oxidative stress-related cardiovascular pathology (as initiated by redox imbalance). The purpose of this review is to evaluate the recent knowledge on protein S-glutathionylation in terms of chemistry, broad cellular intervention, specific quantification, and potential for therapeutic exploitation. The data bases searched were Medline and PubMed, from 2009 to 2014 (term: glutathionylation). Protein S-glutathionylation ensures protection of protein thiols against irreversible over-oxidation, operates as a biological redox switch in both cell survival (influencing kinases and protein phosphatases pathways) and cell death (by potentiation of apoptosis), and cross-talks with phosphorylation and with S-nitrosylation. Collectively, protein S-glutathionylation appears as a valuable biomarker for oxidative stress, with potential for translation into novel therapeutic strategies.
... The multifaceted function of glutathione in these cellular processes also include a role as substrate for leukotriene biosynthesis [16], storage and transport of Cys [17], neutralization and transmembrane transport of drugs and lipophilic xenobiotics [18], and as substrate of the redox cycling of Cyscontaining enzymes, such as peroxiredoxin 6 (Prxd-6) [19], or protein S-glutathionylation [20]. Many other fundamental metabolic pathways like DNA and protein synthesis, enzyme activation or cell transport are affected by its fundamental protecting role [2,15]. ...
... The formation of protein mixed disulfides often occurs under oxidative and nitrosative stress [71] (further discussed in Section 1.4), and it is believed to represent a prevention mechanism for the irreversible oxidation of protein thiols. However, it also operates as a biological redox switch in the regulation of signal transduction and metabolic pathways [20,72]. Indeed, S-glutathionylation occurs on protein kinases, checkpoints of cell cycle regulation and death pathways, as well as on proteins involved in glycolysis/energy metabolism regulation, antioxidant enzymes, and on cytoskeletal and chaperone proteins [20,67,[73][74][75][76][77][78]. ...
... However, it also operates as a biological redox switch in the regulation of signal transduction and metabolic pathways [20,72]. Indeed, S-glutathionylation occurs on protein kinases, checkpoints of cell cycle regulation and death pathways, as well as on proteins involved in glycolysis/energy metabolism regulation, antioxidant enzymes, and on cytoskeletal and chaperone proteins [20,67,[73][74][75][76][77][78]. ...
Glutathione is considered the major non‐protein low molecular weight modulator of redox processes and the most important thiol reducing agent of the cell. The biosynthesis of glutathione occurs in the cytosol from its constituent amino acids, but this tripeptide is also present in the most important cellular districts, such as mitochondria, nucleus, and endoplasmic reticulum, thus playing a central role in several metabolic pathways and cytoprotection mechanisms. Indeed, glutathione is involved in the modulation of various cellular processes and, not by chance, it is a ubiquitous determinant for redox signaling, xenobiotic detoxification, and regulation of cell cycle and death programs. The balance between its concentration and redox state is due to a complex series of interactions between biosynthesis, utilization, degradation, and transport. All these factors are of great importance to understand the significance of cellular redox balance and its relationship with physiological responses and pathological conditions. The purpose of this review is to give an overview on glutathione cellular compartmentalization. Information on its subcellular distribution provides a deeper understanding of glutathione‐dependent processes and reflects the importance of compartmentalization in the regulation of specific cellular pathways.
... 14 Therefore the GSH:GSSG ratio provides an indication of redox metabolism within a particular cellular compartment. [14][15][16] Reduced GSH exists at high (2-3 mM) concentrations in brain tissues and contributes to the reducing status of the cell during normal homeostasis. 1,11,17 As a result of this reducing intracellular environment, many cytoplasmic proteins are rich in free cysteine thiols, which are available to undergo oxidative modification. ...
... 1,18,23,25,29,30 When combined, the S-glutathionylation and deglutathionylation of proteins compose the S-glutathionylation cycle (Fig. 1). 16 The low molecular weight of GSH, which increases its capacity to interact with cysteine residues, combined with its abundance makes it a critical regulator of redox homeostasis. 1 Although S-glutathionylation of proteins may be constitutive, that is, occurring under basal conditions, they are increased under conditions of increased ROS or RNS, which suggests that they are important in mediating the cellular response to oxidative and nitrosative stress. 1,21 ...
... 21,32,33 In addition, S-glutathionylation imparts a negative charge to the protein and as a result may produce structural and functional changes. 16 Importantly, modification of cysteine residues via S-glutathionylation is a reversible process and any loss-or gainof-function will be recovered following normalization of cellular redox status. 1 Therefore S-glutathionylation represents a mechanism by which changes in cellular redox status can be transduced into a functional response via post-translational protein modification, that is, S-glutathionylation is a means of cellular redox siganling. 1,20,34,35 The mammalian genome encodes 214,000 cysteine residues of which at least 10-20% are redox sensitive under biologic conditions. ...
Drug addiction is a chronic relapsing disorder that comes at a high cost to individuals and society. Therefore understanding the mechanisms by which drugs exert their effects is of prime importance. Drugs of abuse increase the production of reactive oxygen and nitrogen species resulting in oxidative stress. This change in redox homeostasis increases the conjugation of glutathione to protein cysteine residues; a process called S-glutathionylation. Although traditionally regarded as a protective mechanism against irreversible protein oxidation, accumulated evidence suggests a more nuanced role for S-glutathionylation, namely as a mediator in redox-sensitive protein signaling. The reversible modification of protein thiols leading to alteration in function under different physiologic/pathologic conditions provides a mechanism whereby change in redox status can be translated into a functional response. As such, S-glutathionylation represents an understudied means of post-translational protein modification that may be important in the mechanisms underlying drug addiction. This review will discuss the evidence for S-glutathionylation as a redox-sensing mechanism and how this may be involved in the response to drug-induced oxidative stress. The function of S-glutathionylated proteins involved in neurotransmission, dendritic spine structure, and drug-induced behavioral outputs will be reviewed with specific reference to alcohol, cocaine, and heroin.
... In the presence of highly oxidizing conditions, redox-sensitive proteins involved in many, if not all, cell functions may undergo posttranslational modifications, including protein S-glutathionylation (P-SSG) (28). This reversible modification consists of the interaction between molecules of GSH and/or GSSG with susceptible cysteine residues of proteins (55). P-SSG has been found in various structural and functional proteins, including actin (28), modifying their characteristics and functions (27). ...
... from 30:1 to 100:1(35). During high ROS emission conditions, cells put up reversible Sglutathionylation to guard highly reactive cysteine residues from permanent oxidative modifications(55). Here, for the first time, we performed a direct GSH/GSSG ratio assessment in human endothelial cells isolated from aortic valves at different stages of the disease, employing a state-of-the-art approach, such as LC-MS/MS, previously mentioned developed in our lab (68). ...
Aims:
During calcific aortic valve stenosis (CAVS) progression, oxidative stress and endothelial dysfunction mark the initial pathogenic steps with a parallel dysregulation of the antioxidant systems. Here, we tested whether oxidation-induced protein S-glutathionylation (P-SSG) accounts for a phenotypic switch in human aortic valvular tissue, eventually leading to calcium deposition. Next, we tested whether countering this reactive oxygen species (ROS) surge would prevent these perturbations.
Results:
We employed state-of-the-art technologies, such as electron paramagnetic resonance (EPR), liquid chromatography-tandem mass spectrometry, imaging flow-cytometry, and live-cell imaging on human excised aortic valves and primary valve endothelial cells. We observed that a net rise in EPR-detected ROS emission marked the transition from fibrotic to calcific in human CAVS specimens, coupled to a progressive increment in P-SSG deposition. In human VECs, treatment with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid triggered highly oxidizing conditions prompting P-SSG accumulation, damaging mitochondria, and inducing endothelial nitric oxide synthase uncoupling. All the events conjured up in morphing these cells from their native endothelial phenotype into a damaged calcification-inducing one. As proof of principle, the use of the antioxidant N-acetyl-L-cysteine prevented these alterations.
Innovation:
Borne as a compensatory system to face excessive oxidative burden, with time, P-SSG contributes to the morphing of human valve endothelial cells from their innate phenotype into a damaged one, paving the way to calcium deposition.
Conclusion:
Our data suggest that, in the human aortic valve, unremitted ROS emission along with a P-SSG build-up occurs and accounts, at least in part, for the morphological/functional changes leading to calcific aortic valve stenosis.
... In contrast, deglutathionylation reaction is reported to be catalyzed by GRXs and SRXs [35,36]. To monitor the deglutathionylation of PRX-IIE, PRX-IIE-SG was incubated with GSH in the presence or absence of GRX-S12, GRX-C5, and SRX Protein S-glutathionylation ensures the protection of critical protein thiols against irreversible hyperoxidation in vivo and is therefore considered a biomarker for oxidative stress [37,38]. To test for this redox-dependent posttranslational modification of PRX-IIE in vivo, A. thaliana plants were sprayed with a single high dose of 300 µM methylviologen, harvested after 3 h, and analyzed using non-reducing two-dimensional gel electrophoresis following Western blot with the specific anti-PRX-IIE antibody. ...
... Protein S-glutathionylation ensures the protection of critical protein thiols against irreversible hyperoxidation in vivo and is therefore considered a biomarker for oxidative stress [37,38]. To test for this redox-dependent posttranslational modification of PRX-IIE in vivo, A. thaliana plants were sprayed with a single high dose of 300 µM methylviologen, harvested after 3 h, and analyzed using non-reducing two-dimensional gel electrophoresis following Western blot with the specific anti-PRX-IIE antibody. ...
Peroxiredoxins (PRX) are thiol peroxidases that are highly conserved throughout all biological kingdoms. Increasing evidence suggests that their high reactivity toward peroxides has a function not only in antioxidant defense but in particular in redox regulation of the cell. Peroxiredoxin IIE (PRX-IIE) is one of three PRX types found in plastids and has previously been linked to pathogen defense and protection from protein nitration. However, its posttranslational regulation and its function in the chloroplast protein network remained to be explored. Using recombinant protein, it was shown that the peroxidatic Cys121 is subjected to multiple posttranslational modifications, namely disulfide formation, S-nitrosation, S-glutathionylation, and hyperoxidation. Slightly oxidized glutathione fostered S-glutathionylation and inhibited activity in vitro. Immobilized recombinant PRX-IIE allowed trapping and subsequent identification of interaction partners by mass spectrometry. Interaction with the 14-3-3 υ protein was confirmed in vitro and was shown to be stimulated under oxidizing conditions. Interactions did not depend on phosphorylation as revealed by testing phospho-mimicry variants of PRX-IIE. Based on these data it is proposed that 14-3-3υ guides PRX‑IIE to certain target proteins, possibly for redox regulation. These findings together with the other identified potential interaction partners of type II PRXs localized to plastids, mitochondria, and cytosol provide a new perspective on the redox regulatory network of the cell.
... Cysteine oxidation has recently emerged to be of significant importance in intracellular signal transduction, with diverse protein functions that are responsive to specific cysteine modifications, including disulfide, sulfenic acid, S-nitrosyl, and/or S-glutathionylated oxidized forms of cysteine. [4][5][6][7][8][9][10][11][12][13][14][15][16] These post-translational modifications provide the capability of specific cellular responses to defined redox stresses. 17 Proteomics approaches have applied a variety of techniques to identify specific oxidative modifications of cysteine in proteins, including broad observation of S-glutathionylation of proteins. ...
... Changes in glutathione oxidation state are associated with different cell types, subcellular compartments (increased oxidized glutathione in mitochondria and the ER), and oxidative stress response, with resultant S-glutathionylation at a subset of cysteine residues in proteins ( Figure 1). 11,12,14,33 For example, increased protein glutathionylation is observed in ischemia, with depletion of reduced glutathione associated with oxidative damage on cardiac reperfusion. 5,9 Increased glutathione oxidation and protein glutathionylation are also observed in some neurodegenerative diseases, which is associated with the increased susceptibility of neuronal proteins to oxidative damage. ...
Cysteine S-glutathionylation is a protein post-translational modification that promotes cellular responses to changes in oxidative conditions. The design of protein motifs that directly depend on defined changes to protein side chains provides new methods to probe diverse protein post-translational modifications. A canonical, 12-residue EF Hand motif was redesigned to be responsive to cysteine glutathionylation. The key design principle was the replacement of the metal-binding Glu12 carboxylate of an EF Hand with a motif capable of metal binding via a free carboxylate in the glutathione-conjugated peptide. In the optimized peptide (DKDADGWCG), metal binding and terbium luminescence were dependent on glutathionylation, with weaker metal binding in the presence of reduced cysteine, but increased metal affinity and a 3.5-fold increase in terbium luminescence at 544 nm when cysteine was glutathionylated. NMR spectroscopy indicated that the structure at all residues of the glutathionylated peptide changed in the presence of metal, with chemical shift changes consistent with the adoption of an EF-Hand-like structure in the metal-bound glutathionylated peptide. This small protein motif consists of canonical amino acids, and is thus genetically encodable, for its potential use as a localized tag to probe protein glutathionylation.
... S-glutathionylation has been shown to crosstalk with other PTMs to modify proteins that control cellular signaling events [27]. The interplay between S-glutathionylation and phosphorylation is involved in modulating functions of the cardiovascular system. ...
Heat shock protein 90 (Hsp90) is a ubiquitous chaperone to interact with numerous proteins to regulate multiple cellular processes, especially during cell proliferation and cell cycle progression. Hsp90 exists in a high level in tumor cells and tissues, and thus serves as a prognostic biomarker or therapeutic target in cancers. We herein report that Hsp90 is subjected to S-glutathionylation, a redox-dependent modification to form a disulfide bond between the tripeptide glutathione and cysteine residues of proteins, primarily at C366 and C412 in the presence of reactive oxygen species. The modification led to the loss of the ATPase activity. The level of Hsp90 was obviously reduced by S-glutathionylation, owing to C-terminus of Hsc70-interacting protein (CHIP)-mediated ubiquitin proteasome system. S-glutathionylation of Hsp90 was found to crosstalk with its C-terminal phosphorylation of Hsp90 that impedes the binding of Hsp90 with CHIP, demonstrating the importance of chaperone code in modulating Hsp90 function. Further biophysical analyses indicated that S-glutathionylation caused structural change of Hsp90, underlying the aforementioned functional regulation. Moreover, in accordance with the analysis of 64 samples collected from patients of breast cancer, the expression level of Hsp90 inversely correlated with the glutathionylated status of Hsp90. The ratio of total expression to glutathionylated status of Hsp90 was coherent to expression of biomarkers in breast cancer sample, potentiating the prognostic value in the cancer treatment.
... In the case of PKA and PKC, glutathionylation of cysteine 199 in the activation loop of the catalytic subunit blocks the phosphotransfer reaction and inhibits the creation of an intramolecular disulfide bond between cysteines 199 and 343 [64]. As a result of PKA and PKC negative regulation, the PI3K and protein kinase B (Akt) signaling pathways are activated [65], which in some cases can even result in cancer development [66]. Moreover, through PI3K/Akt activation, TBE virus replication and transmission are increased [67], regardless of bacterial co-infection. ...
Despite the increasing number of patients suffering from tick-borne encephalitis (TBE), Lyme disease, and their co-infection, the mechanisms of the development of these diseases and their effects on the human body are still unknown. Therefore, the aim of this study was to evaluate the changes in the proteomic profile of human plasma induced by the development of TBE and to compare it with changes in TBE patients co-infected with other tick-borne pathogens. The results obtained by proteomic analysis using a nanoLC-Q Exactive HF mass spectrometer showed that the most highly elevated groups of proteins in the plasma of TBE patients with co-infection were involved in the pro-inflammatory response and protein degradation, while the antioxidant proteins and factors responsible for protein biosynthesis were mainly downregulated. These results were accompanied by enhanced GSH- and 4-HNE-protein adducts formation, observed in TBE and co-infected patients at a higher level than in the case of patients with only TBE. In conclusion, the differences in the proteomic profiles between patients with TBE and co-infected patients indicate that these diseases are significantly diverse and, consequently, require different treatment, which is particularly important for further research, including the development of novel diagnostics tools.
... These include its interaction with glutathione (GSH), and the oxidation of cysteine residues leading to allosteric changes in a variety of proteins such as phosphatases, transcription factors and ion channels (Winterbourn and Hampton, 2008). Typically, the oxidation of redox-sensitive cysteines is a reversible process catalysed by enzymes that use GSH or nicotinamide adenine dinucleotide phosphatase (NADPH) (Xiong et al., 2011;Pastore and Piemonte, 2012;Sabens Liedhegner et al., 2012;Popov, 2014) as redox cofactors. Most studies of H 2 O 2 and T. gondii tachyzoites have focused on the ability of host cells to use the damaging oxidative properties of ROS as a component of innate defence, and strategies employed by tachyzoites to overcome this defence (Murray and Cohn, 1979;Ding et al., 2004;Akerman and Muller, 2005;Luder and Gross, 2005;Luder et al., 2009). ...
The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in apicomplexan parasites such as Toxoplasma gondii. The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 58 genes containing domains related to canonical antioxidant function, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H2O2 to human fibroblasts infected with T. gondii triggers a Ca²⁺ flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H2O2 is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression a glutaredoxin-roGFP2 redox sensor fusion protein in the parasitophorous vacuole severely impacts parasite replication. These data highlight the rich redox network that exists in T. gondii, evidencing a link between extracellular redox and intracellular Ca²⁺ signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasite growth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.
... GSH can also bind specific protein by disulfide linkages with Cys residues [98]. This process, defined as protein S-glutathionylation can be either spontaneous or enzymatically driven, depending on the redox state of intracellular glutathione pools [99,100]. ...
Under physio-pathological conditions, cells release membrane-surrounded structures named Extracellular Vesicles (EVs), which convey their molecular cargo to neighboring or distant cells influencing their metabolism. Besides their involvement in the intercellular communication, EVs might represent a tool used by cells to eliminate unnecessary/toxic material. Here, we revised the literature exploring the link between EVs and redox biology. The first proof of this link derives from evidence demonstrating that EVs from healthy cells protect target cells from oxidative insults through the transfer of antioxidants. Oxidative stress conditions influence the release and the molecular cargo of EVs that, in turn, modulate the redox status of target cells. Oxidative stress-related EVs exert both beneficial or harmful effects, as they can carry antioxidants or ROS-generating enzymes and oxidized molecules. As mediators of cell-to-cell communication, EVs are also implicated in the pathophysiology of oxidative stress-related diseases. The review found evidence that numerous studies speculated on the role of EVs in redox signaling and oxidative stress-related pathologies, but few of them unraveled molecular mechanisms behind this complex link. Thus, the purpose of this review is to report and discuss this evidence, highlighting that the analysis of the molecular content of oxidative stress-released EVs (reminiscent of the redox status of originating cells), is a starting point for the use of EVs as diagnostic and therapeutic tools in oxidative stress-related diseases.
... Cysteine residues of proteins can form disulfides with low-molecular-weight thiols [81]. Such post-translational modifications generate different forms of proteins with various physicochemical properties and may also affect their functions and, in particular, modify their enzymatic activity [82,83]. ...
The importance of coenzyme A (CoA) as a carrier of acyl residues in cell metabolism is well understood. Coenzyme A participates in more than 100 different catabolic and anabolic reactions, including those involved in the metabolism of lipids, carbohydrates, proteins, ethanol, bile acids, and xenobiotics. However, much less is known about the importance of the concentration of this cofactor in various cell compartments and the role of altered CoA concentration in various pathologies. Despite continuous research on these issues, the molecular mechanisms in the regulation of the intracellular level of CoA under pathological conditions are still not well understood. This review summarizes the current knowledge of (a) CoA subcellular concentrations; (b) the roles of CoA synthesis and degradation processes; and (c) protein modification by reversible CoA binding to proteins (CoAlation). Particular attention is paid to (a) the roles of changes in the level of CoA under pathological conditions, such as in neurodegenerative diseases, cancer, myopathies, and infectious diseases; and (b) the beneficial effect of CoA and pantethine (which like CoA is finally converted to Pan and cysteamine), used at pharmacological doses for the treatment of hyperlipidemia.
... Oxidative Cys PTMs that can be reduced are considered ''reversible'' and include S-nitrosylation (SNO), S-glutathionylation (SSG), S-acylation, S-cysteinylation, S-sulfhydration, and S-sulfenylation (Cys-SOH), as well as intra-and intermolecular disulfides (8,25,26,41,45,46,62); while those with no known means of reduction (Cys-sulfinic and -sulfonic acid; Cys-SO 2 H/SO 3 H) are regarded as ''irreversible,'' are associated with oxidative ''damage,'' and are considered markers of protein dysfunction and degradation (41,57). A single exception exists; enzymatic reduction of sulfinylated peroxiredoxins (Prx I-IV) to their active form by sulfiredoxin (4,86). ...
Aims:
Cysteine (Cys) is a major target for redox post-translational modifications (PTM) that occur in response to changes in the cellular redox environment. We describe multiplexed, peptide-based enrichment and quantitative mass spectrometry (MS) applied to globally profile reversible redox Cys PTM in rat hearts during ischemia / reperfusion (I/R) in the presence or absence of an aminothiol antioxidant, N -2-mercaptopropionylglycine (MPG). Parallel fractionation allowed identification of irreversibly oxidised Cys peptides (Cys-SO<sub>2</sub>H/SO<sub>3</sub>H).
Results:
We identified 4505 reversibly oxidised Cys peptides of which 1372 were significantly regulated by ischemia and/or I/R. An additional 219 peptides (247 sites) contained Cys-SO<sub>2</sub>H/Cys-SO<sub>3</sub>H modifications, and these were predominantly identified from hearts subjected to I/R ( n =168 peptides). Parallel reaction monitoring MS (PRM-MS) enabled relative quantitation of 34 irreversibly oxidised Cys peptides. MPG attenuated a large cluster of I/R-associated reversibly oxidised Cys peptides and irreversible Cys oxidation to less than non-ischemic controls ( n =24 and 34 peptides, respectively). PRM-MS showed that Cys sites oxidised during ischemia and/or I/R and 'protected' by MPG were largely mitochondrial and were associated with antioxidant functions (peroxiredoxins 5 and 6) and metabolic processes, including glycolysis. Metabolomics revealed I/R induced changes in glycolytic intermediates that were reversed in the presence of MPG, and which were consistent with irreversible PTM of triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), altered GAPDH activity, and reduced I/R glycolytic pay-off as evidenced by ATP and NADH levels.
Conclusions:
Cys sites identified here are targets of reactive oxygen species (ROS) that can contribute to protein dysfunction and the pathogenesis of I/R.
... While glutathionylation of the β1 subunit inhibits Na + /K + -ATPase [37], glutathionylation can also protect protein cysteine residues against irreversible oxidation, preserving proteins in a reversible functionally silent state. GSTπ promotes such protection [38]. Binding of GSH to GSTπ lowers GSH's cysteine thiol pKa, forming a nucleophilic thiolate anion that GSTπ can deliver to cysteine residues in hydrophobic domains. ...
Background
FXYD proteins associate closely with- and protect plasmalemmal Na⁺/K⁺-ATPase against oxidative inhibition. One of them, FXYD3, is often overexpressed in cancers, including those of breast and pancreas. Down-regulation of overexpression in MCF-7 breast cancer cells with siRNA augments doxorubicin-induced cytotoxicity. Because down-regulation with siRNA is not readily translated therapeutically, we developed a peptide as an alternative for suppression of FXYD3.
Methods
A shortened peptide derivative of the wild-type (WT) FXYD3 protein, FXYD3-pep has the four cysteine residues in the WT protein replaced by serine, which eliminates the WT protein’s protection against oxidative Na⁺/K⁺-ATPase inhibition. We exposed human cancer cells to FXYD3-pep and measured cytotoxicity and caspase 3/7 activity with co-exposure to doxorubicin. We also measured effects of the peptide on expression glutathione-S-transferase π (GST-π), implicated in treatment resistance, and on expression of tumor suppressor p53. Selected experiments were performed with parallel FXYD3 suppression with siRNA or FXYD3-pep.
Results
Exposure of cells to FXYD3-pep displaced WT FXYD3 from Na⁺/K⁺-ATPase. Exposure of MCF-7 breast or pancreatic BxPC-3 cancer cells that highly express FXYD3 to the peptide had little effect alone but combined with doxorubicin it significantly (P < 0.05) increased cytotoxicity. A peptide not mutated to eliminate FXYD3’s protective effect of Na⁺/K⁺-ATPase had no effect. FXYD3-pep did not augment doxorubicin’s cytotoxicity against MDA-MB-468 breast and Panc-1 pancreatic cancer cells that have low- or no FXYD3 expression. Cellular FXYD3 expressions was reflected by expression of the α1 Na⁺/K⁺-ATPase subunits but not by plasmalemmal Na⁺/K⁺-ATPase function. Signals from fluorescently labeled FXYD3-pep were detected in a perinuclear distribution in BxPC-3 cells as reported for overexpressed FXYD3, α- and β Na⁺/K⁺-ATPase subunits in cancer. Exposure to FXYD3-pep or to FXYD3 siRNA almost eliminated expression of GST-π. FXYD3-pep alone had no effect on p53 levels but significantly augmented a doxorubicin-induced increase, and, while the peptide alone had no effect on caspase 3/7 activity, it significantly augmented a doxorubicin-induced increase.
Conclusions
Overexpressed FXYD3 has intracellular roles beyond its accepted modulation of plasmalemmal Na⁺/K⁺-ATPase. These roles can be countered with a membrane-permeable peptide derivative of FXYD3 that suppresses GST-π and enhances chemosensitivity of cancer cells overexpressing FXYD3.
... Grxs are glutathionedependent, that may modulate protein activity by reversible glutathionylation in the presence of NADPH and glutathione reductase (GR), as a response against oxidative stress (Meyer et al., 2009). Reversible post-translational modification of thiol groups by the addition of GSH (S-glutathionylation) protects the cysteine residues of proteins from irreversible oxidation (Popov, 2014). Glutaredoxin forms a complex system, having glutaredoxin (Grx), glutathione (GSH), nicotine adenine dinucleotide phosphate (NADPH), and glutathione reductase (GR). ...
Glutaredoxins (Grxs) are small (10-15 kDa) glutathione (GSH) - dependent redox proteins. The role of Grxs are well documented in tolerance to heavy metal stress in prokaryotic and mammalian systems and a few plant genera but poorly understood in plants against drought. In the present study, two rice glutaredoxin (Osgrx) genes (LOC_Os02g40500 and LOC_Os01g27140) responsible for tolerance against heavy metal stress have been studied for investigating their role against drought. Each glutaredoxin gene was over-expressed in Arabidopsis thaliana to reveal their role in drought stress. The relative expression of both Osgrx genes was higher in the transgenic lines. Transgenic lines of both Osgrxs showed longer roots, higher seed germination, and survival efficiency during drought stress. The physiological parameters (PN, gs, E, WUE, qP, NPQ and ETR), antioxidant enzymes (GRX, GR, GPX, GST, APX, POD, SOD, CAT, DHAR, and MDHAR), antioxidant molecules (ascorbate and GSH) and stress-responsive amino acids (cysteine and proline) levels were additionally increased in transgenic lines of both Osgrxs to provide drought tolerance. The outcomes from this study strongly determined that each Osgrx gene participated in the moderation of drought and might be utilized in biological engineering strategies to overcome drought conditions in different crops.
... Thioredoxin (Trx), glutaredoxin (Grx) and peroxiredoxin (Prx) have been implicated in a large number of CVD, including ischaemic heart disease, cardiac hypertrophy, diabetes, obesity, atherosclerosis and hypertension (reviewed in Ref. [319]). Accordingly, these enzymes were proposed as biomarkers as well as for gene therapy in CVD [307,319,320]. The half-life for recombinant human Trx-1 in the plasma of rodents or monkeys is one to 8 h but can be prolonged by polyethyleneglycol modification [321]. ...
According to the latest Global Burden of Disease Study data, non-communicable diseases in general and cardiovascular disease (CVD) in particular are the leading cause of premature death and reduced quality of life. Demographic shifts, unhealthy lifestyles and a higher burden of adverse environmental factors provide an explanation for these findings. The expected growing prevalence of CVD requires enhanced research efforts for identification and characterisation of novel therapeutic targets and strategies. Cardiovascular risk factors including classical (e.g. hypertension, diabetes, hypercholesterolaemia) and non-classical (e.g. environmental stress) factors induce the development of endothelial dysfunction, which is closely associated with oxidant stress and vascular inflammation and results in CVD, particularly in older adults. Most classically successful therapies for CVD display vasoprotective, antioxidant and anti-inflammatory effects, but were originally designed with other therapeutic aims. So far, only a few ‘redox drugs’ are in clinical use and many antioxidant strategies have not met expectations. With the present review, we summarise the actual knowledge on CVD pathomechanisms, with special emphasis on endothelial dysfunction, adverse redox signalling and oxidative stress, highlighting the preclinical and clinical evidence. In addition, we provide a brief overview of established CVD therapies and their relation to endothelial dysfunction and oxidative stress. Finally, we discuss novel strategies for redox-based CVD therapies trying to explain why, despite a clear link between endothelial dysfunction and adverse redox signalling and oxidative stress, redox- and oxidative stress–based therapies have not yet provided a breakthrough in the treatment of endothelial dysfunction and CVD.
... Indeed, GSH stores most of the intracellular cysteine and modulates the activity of proteins via reversible protein glutathionylation, influencing cell cycle progression, cell death, transcription factor activity and signalling [1,2,4]. Reversible protein glutathionylation occurs non-enzymatically via thiol-disulphide exchange reactions between GSSG and a cysteinyl residue in a protein or via reaction of GSH with an activated thiol derivative such as sulfenic acid (-SOH), thiyl radical (-S•) and S-nitrosyl (-SNO) [4,7]. Due to the high intracellular GSH/GSSG ratio, thiol-disulphide exchange is an unlikely mechanism for protein glutathionylation even under severe oxidative stress. ...
Glutathione (GSH) is the predominant low-molecular-weight antioxidant with a ubiquitous distribution inside the cell. The steady-state level of cellular GSH is dependent on the balance between synthesis, hydrolysis, recycling of glutathione disulphide (GSSG) as well as cellular extrusion of reduced, oxidized, or conjugated-forms. The augmented oxidative stress typical of cancer cells is accompanied by an increase of glutathione levels that confers them growth advantage and resistance to a number of chemotherapeutic agents. Targeting glutathione metabolism has been widely investigated for cancer treatment although GSH depletion as single therapeutic strategy has resulted largely ineffective if compared with combinatorial approaches. In this review, we circumstantiate the role of glutathione in tumour development and progression focusing on how interfering with different steps of glutathione metabolism can be exploited for therapeutic purposes. A dedicated section on synthetic lethal interactions with GSH modulators will highlight the promising option of harnessing glutathione metabolism for patient-directed therapy in cancer.
... Наиболее чувствительными к усилению процессов свободно-радикального окисления и основными акцепторами активных форм кислорода являются белки [8]. В результате взаимодействия активных форм кислорода с протеинами возникает как обратимая, так и необратимая окислительная модификация этих молекул [9][10][11]. Конформационные изменения белков являются основополагающими в функционировании рецепторов, мембранных транспортных систем, изменении скорости биохимических реакций, регуляции фаз клеточного цикла, транскрипции, репликации, гибели клеток и других процессах. ...
В статье представлены данные, характеризующие уровень активных форм кисло-рода, необратимой и обратимой окислительной модификации белков, состояние компонентов системы глутатиона и активность каталазы, реализацию апоптоза в интактных лимфоцитах крови. Методом моделирования окислительного стресса in vitro исследовано влияние пероксида водорода в конечной концентрации 0.5 мМ на реализацию апоптоза; проведена оценка вклада компонентов системы глута-тиона, необратимой и обратимой окислительной модификации белков в молеку-лярные механизмы клеточной гибели лимфоцитов крови. Показано, что апопто-тический тип гибели лимфоцитов крови при экспериментальном окислительном стрессе характеризовался активированием глутатионилирования и карбонили-рования протеинов. Представленные данные доказывают, что молекулы белков являются потенциальными редокс-зависимыми мишенями для регуляции кле-точного метаболизма лимфоцитов крови.
Ключевые слова: лимфоциты крови, редокс-статус клетки, окислительный стресс, антиоксидантная система, окислительная модификация белков, апоптоз.
... When glutathione homeostasis is altered, endothelial and/or smooth muscle cells are subjected to the deleterious effects of ROS, which react with exposed cysteine residues of proteins. These susceptible cysteines can interact, in turn, with GSH and/or GSSG giving rise to reversible protein S-glutathionylation (Pr-SSG) [15,16]. ...
Aortic valve sclerosis (AVSc) is characterized by non-uniform thickening of the leaflets without hemodynamic changes. Endothelial dysfunction, also caused by dysregulation of glutathione homeostasis expressed as ratio between its reduced (GSH) and its oxidised form (GSSG), could represent one of the pathogenic triggers of AVSc. We prospectively enrolled 58 patients with overt atherosclerosis and requiring coronary artery bypass grafting (CABG). The incidence of AVSc in the studied population was 50%. The two groups (No-AVSc and AVSc) had similar clinical characteristics. Pre-operatively, AVSc group showed significantly lower GSH/GSSG ratio than No-AVSc group (p = 0.02). Asymmetric dimethylarginine (ADMA) concentration was significantly higher in AVSc patients compared to No-AVSc patients (p < 0.0001). Explanted sclerotic aortic valves presented a significantly increased protein glutathionylation (Pr-SSG) than No-AVSc ones (p = 0.01). In vitro, inhibition of glutathione reductase caused β-actin glutathionylation, activation of histone 2AX, upregulation of α2 smooth muscle actin (ACTA2), downregulation of platelet and endothelial cell adhesion molecule 1 (PECAM1) and cadherin 5 (CDH5). In this study, we showed for the first time that the dysregulation of glutathione homeostasis is associated with AVSc. We found that Pr-SSG is increased in AVSc leaflets and it could lead to EndMT via DNA damage. Further studies are warranted to elucidate the causal role of Pr-SSG in aortic valve degeneration.
... The cysteine oxidation/protein S-glutathionylation cycle has recently received a lot of attention for its ability to act as a redox switch in cells. There are a number of excellent recent reviews that discuss redox regulation via thiol switches and S-glutathionylation in depth [57,[59][60][61][62][63][64][65][66][67] among others. The purpose of this review is not to extensively review this field. ...
Superoxide dismutases play an important role in human health and disease. Three decades of effort have gone into synthesizing SOD mimics for clinical use. The result is the Mn porphyrins which have SOD-like activity. Several clinical trials are underway to test the efficacy of these compounds in patients, particularly as radioprotectors of normal tissue during cancer treatment. However, aqueous chemistry data indicate that the Mn porphyrins react equally well with multiple redox active species in cells including H 2 O 2 , O 2•- , ONOO ⁻ , thiols, and ascorbate among others. The redox potential of the Mn porphyrins is midway between the potentials for the oxidation and reduction of O 2•- . This positions them to react equally well as oxidants and reductants in cells. The result of this unique chemistry is that: 1) the species the Mn porphyrins react with in vivo will depend on the relative concentrations of the reactive species and Mn porphyrins in the cell of interest, and 2) the Mn porphyrins will act as catalytic (redox cycling) agents in vivo. The ability of the Mn porphyrins to catalyze protein S-glutathionylation means that Mn porphyrins have the potential to globally modulate cellular redox regulatory signaling networks. The purpose of this review is to summarize the data that indicate the Mn porphyrins have diverse reactions in vivo that are the basis of the observed biological effects. The ability to catalyze multiple reactions in vivo expands the potential therapeutic use of the Mn porphyrins to disease models that are not SOD based.
... Considering this fact, along with decreased level of total serum glutathione, it might be suggested that GSH is probably recruited during burn injury to protect cellular and extracellular proteins from irreversible oxidations such as the formation of sulfinic and sulfonic acid (32) through glutathionylation of these proteins ( figure 3) (33, 34). Since this posttranslational modification is reversible, proteins can retain their activities after controlling the acute phase of injury (35)(36)(37)(38). On the other hand, since the administration of ascorbic acid as a part of the routine therapeutic protocol in our hospital was started from the second day of hospitalization, the extent of oxidative damage as a consequence of burn injury could be much more significant if there were not any antioxidants in the drug regimen. ...
Introduction
Several studies have shown the role of oxidative stress in pathophysiology of burn injuries. This study aimed to evaluate the changes of oxidant-antioxidant levels during the week following burn injuries and its correlation with grade of burn.
Methods
In this prospective cross-sectional study, changes of total glutathione, reduced glutathione (GSH), oxidized GSH (GSSG), GSH/GSSG ratio, as well as Pro-oxidant-antioxidant balance (PAB) were investigated on the 1st, 2nd and 7th days of admission in patients with > 15 % burns.
Results
40 patients with the mean age of 21.1 ± 14.5 were studied (47.5% male). More than 50% of patients were in the 18 – 55 years age range and over 70% had 20% – 60% grade of burn. Total serum glutathione level and GSH had significant decreasing trends (P < 0.001) and GSSG and GSH/GSSG ratio had increasing trends (p < 0.001). No significant correlation was observed between serum GSH level and the total body surface area (TBSA) of burn injury (r = 0.047; p = 0.779). The evaluation of PAB and its correlation with TBSA showed a significant and direct association between them on the 1st (coefficient = 0.516; p = 0.001), 2nd (coefficient = 0.62; p <0.001), and 3rd (coefficient = 0.471; p = 0.002) day of follow up.
Conclusion
According to this study, the redox perturbation occurred in burn injury which was measured and proved by decreased GSH/GSSG ratio as well as the shift of PAB in favour of oxidants. Besides, since PAB positively correlated with the severity of dermal damage, it might suggest the application of antioxidants as a part of therapeutic protocol for which the dosage should be proportionate to the surface area of the damaged skin.
... Most likely the modulations occur through the role of hGSTO1 in the glutathionylation cycle for which hGSTO1 has been reported to possess both deglutathionylation and glutathionylation activity Menon et al. 2014;Board and Menon 2016). The hGSTO1 is consequently involved in the selective glutathionylation/ deglutathionylation of specific proteins that contribute to cell signaling and regulation, for example PKC, NFκB and several MAPK pathways (Pastore and Piemonte 2012;Popov 2014). Therefore further studies are still needed to elucidate the proteins regulated by hGSTO1 that may serve as molecular targets for the treatment of Parkinson's disease. ...
Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. The disease is associated with dopaminergic neuron losses in the substantia nigra area of the brain and the formation of cytoplasmic inclusion bodies. Human glutathione transferase omega 1 (hGSTO1) appears to have a role in modulating stress response. The study was aimed to elucidate differentially expressed proteins caused by oxidative stress induced by 6-hydroxydopamine (6-OHDA). Human neuronal cells SH-SY5Y overexpressing hGSTO1 were used to investigate protein glutathionylation and the modulation of cellular protein expression. Therefore SH-SY5Y/hGSTO1 and SH-SY5Y/control lysate proteins were separated by 2D-gel electrophoresis compared with untreated conditions in both standard and non-reducing conditions. In standard conditions, the analysis of protein profiles demonstrated 25 differentially expressed spots and 10 spots were chosen for further protein identification by LC-MS analysis. Several proteins were later identified as vimentin, galectin-1, high mobility group protein B2, clathrin, tropomyosin, heterogenous nuclear ribonucleoprotein and peroxiredoxin-2. Search Tool for Interactions of Chemicals (STITCH) analysis suggested that oxidative stress induced by 6-OHDA involved carbohydrate metabolism in SH-SY5Y via a lactose metabolic pathway. Our results raise the possibility that hGSTO1 modulates the functions of many proteins that play a role in the degenerative cell response of a Parkinson's model.
... The redox state of thiol groups is related to glutathionylation of proteins which occurs in unstressed cells under physiological conditions as well as during cellular redox defense [56]. The glutathionylation is either a spontaneous or enzymatically driven finely controlled reversible process, which can involve both the GSH and GSSG [57]. To investigate the modifications in protein glutathionylated residues (PSSG), whole proteins were separated under nonreducing conditions. ...
In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin -mutant early onset Parkinson’s disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson’s disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin -mutant fibroblasts.
... Protein S-glutathionylation consists in glutathione reaction with the free thiol (SH) in certain cysteine (Cys) residues of proteins and it has been proposed as a reversible process of storing GSH during oxidative stress, and has been regarded as a protective mechanism against irreversible protein thiol-oxidation [62,63]. Studies have shown that many proteins can be S-glutathionylated or deglutathionylated during oxidative stress [64][65][66] and in this study we demonstrate that the S-glutathionylation of proteins also present during 2-AAPA treatment. ...
Esophageal squamous cell carcinoma (ESCC) is a highly malignant cancer with poor response to both of chemotherapy and radiotherapy. 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino) phenyl carbamoylsulfanyl] propionic acid (2-AAPA), an irreversible inhibitor of glutathione reductase (GR), is able to induce intracellular oxidative stress, and has shown anticancer activity in many cancer cell lines. In this study, we investigated the effects of 2-AAPA on the cell proliferation, cell cycle and apoptosis and aimed to explore its mechanism of action in human esophageal cancer TE-13 cells. It was found that 2-AAPA inhibited growth of ESCC cells in a dose-dependent manner and it did not deplete reduced glutathione (GSH), but significantly increased the oxidized form glutathione (GSSG), resulting in decreased GSH/GSSG ratio. In consequence, significant reactive oxygen species (ROS) production was observed. The flow cytometric analysis revealed that 2-AAPA inhibited growth of esophageal cancer cells through arresting cell cycle in G2/M phase, but apoptosis-independent mechanism. The G2/M arrest was partially contributed by down-regulation of protein expression of Cdc-25c and up-regulation of phosphorylated Cdc-2 (Tyr15), Cyclin B1 (Ser147) and p53. Meanwhile, 2-AAPA-induced thiol oxidative stress led to increased protein S-glutathionylation, which resulted in α-tubulin S-glutathionylation-dependent depolymerization of microtubule in the TE-13 cells. In conclusion, we identified that 2-AAPA as an effective thiol oxidative stress inducer and proliferation of TE-13 cells were suppressed by G2/M phase cell cycle arrest, mainly, through α-tubulin S-glutathionylation-mediated microtubule depolymerization. Our results may introduce new target and approach for esophageal cancer therapy through generation of GR-mediated thiol oxidative stress.
... Indeed, hydrogen peroxide is the most abundant ROS released by mitochondria (79). Hydrogen peroxide is a signaling molecule, which interacts with cysteine residues in reduction-oxidation (redox)-sensitive proteins to induce the rapid formation of disulfide bonds between cysteines or with glutathione (GSH) to form S-glutathionylated proteins (43,208,215,235,293). These disulfide bonds are easily broken by reducing agents or enzymes that depend on GSH or nicotinamide adenine dinucleotide phosphate (NADPH). ...
Significance:
Oxidative stress increases in the brain with aging and neurodegenerative diseases. Previous work emphasized irreversible oxidative damage in relation to cognitive impairment. This research has evolved to consider a continuum of alterations, from redox signaling to oxidative damage, which provides a basis for understanding the onset and progression of cognitive impairment. This review provides an update on research linking redox signaling to altered function of neural circuits involved in information processing and memory.
Recent advances:
Starting in middle-age, redox signaling triggers changes in nervous system physiology described as senescent physiology. Recent studies indicates N-methyl-D-aspartate and ryanodine receptors, and Ca2+ signaling molecules as molecular substrates of redox-mediated senescent physiology.
Critical issues:
We review redox homeostasis mechanisms and consider the chemical character of reactive oxygen and nitrogen species, and their role in regulating different transmitter systems. In this regard, senescent physiology may represent the co-opting of pathways normally responsible for feedback regulation of synaptic transmission.
Future directions:
It will be important to identify the intrinsic mechanisms for the shift in oxidative/reductive processes. Intrinsic mechanism will depend on the transmitter system, the oxidative stressors, and the expression/activity of antioxidant enzymes. In addition, it will be important to identify how intrinsic processes interact with other aging factors including changes in inflammatory or hormonal signals.
Innovation:
Results suggest that physiological rather than pathological mechanisms underlie for the initial diagnosis of cognitive impairment. Because redox signaling is reversible, it provides hope for early identification and treatment of cognitive decline.
... One of the mechanisms for regulation of protein activity is glutathionylation of cysteines, shown to be involved in the regulation of P-type ATPases such as Na,K-ATPase and sarcoendoplasmic reticulum Ca-ATPase (SERCA) (64). ATP7A and ATP7B are susceptible to glutathionylation and interact with glutaredoxin 1, thiol oxidoreductase, which catalyzes reduction of disulfides into sulfhydryls (65). ...
Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2017 IUBMB Life, 2017.
... S-glutathiolated proteins can then be reduced by another GSH molecule, resulting in a free protein thiol and GSSG [53]. This process highlights how, when viewed from an alternate perspective, S-nitrosation is fundamentally regulated by non-enzymatic chemistry. ...
Over the last 25 years protein S-nitrosylation, also known more correctly as S-nitrosation, has been progressively implicated in virtually every nitric oxide-regulated process within the cardiovascular system. The current, widely-held paradigm is that S-nitrosylation plays an equivalent role as phosphorylation, providing a stable and controllable post-translational modification that directly regulates end-effector target proteins to elicit biological responses. However, this concept largely ignores the intrinsic instability of the nitrosothiol bond, which rapidly reacts with typically abundant thiol-containing molecules to generate more stable disulfide bonds. These protein disulfides, formed via a nitrosothiol intermediate redox state, are rationally anticipated to be the predominant end-effector modification that mediates functional alterations when cells encounter nitrosative stimuli. In this review we present evidence and explain our reasoning for arriving at this conclusion that may be controversial to some researchers in the field.
... Nevertheless, due to the pro-oxidant nature of MND, the increase in GSH-hTTR might also be a consequence of increased GSH formation and it remains to be verified in future research. This hypothesis is corroborated by studies reporting that protein glutathionylation could be observed in response to oxidative stress which can further affect both the stability and activity of target proteins 42,43 . ...
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
... Protein S-glutathionylation is generally associated with increased oxidative or nitrosative stress during such pathological conditions as ischemia-reperfusion and hypertension 42,43 , regulating cardiovascular responses to stress and injury. Sustained overproduction of ROS has emerged as a common element in a variety of mechanisms controlling vascular complications of metabolic disorders 44 , but how protein S-glutathionylation in endothelial cells is involved in these pathogenic processes has not been investigated. ...
Background: Oxidative stress is implicated in increased vascular permeability associated with metabolic disorders, but the underlying redox mechanism is poorly defined. S-glutathionylation, a stable adduct of glutathione with protein sulfhydryl, is a reversible oxidative modification of protein and is emerging as an important redox signaling paradigm in cardiovascular physiopathology. The present study determines the role of protein S-glutathionylation in metabolic stress-induced endothelial cell permeability.
Methods and results: In endothelial cells isolated from patients with type-2 diabetes mellitus, protein S-glutathionylation level was increased. This change was also observed in aortic endothelium in ApoE deficient (ApoE-/-) mice fed on Western diet. Metabolic stress-induced protein S-glutathionylation in human aortic endothelial cells (HAEC) was positively correlated with elevated endothelial cell permeability, as reflected by disassembly of cell-cell adherens junctions and cortical actin structures. These impairments were reversed by adenoviral overexpression of a specific de-glutathionylation enzyme, glutaredoxin-1 in cultured HAECs. Consistently, transgenic overexpression of human Glrx-1 in ApoE-/- mice fed the Western diet attenuated endothelial protein S-glutathionylation, actin cytoskeletal disorganization, and vascular permeability in the aorta. Mechanistically, glutathionylation and inactivation of Rac1, a small RhoGPase, were associated with endothelial hyperpermeability caused by metabolic stress. Glutathionylation of Rac1 on cysteine 81 and 157 located adjacent to guanine nucleotide binding site was required for the metabolic stress to inhibit Rac1 activity and promote endothelial hyperpermeability.
Conclusions: Glutathionylation and inactivation of Rac1 in endothelial cells represent a novel redox mechanism of vascular barrier dysfunction associated with metabolic disorders.
... Protein S-nitrosylation, a covalent reaction of NO moiety to a protein reactive cysteine thiol, has emerged as a ubiquitous protein posttranslational modification [13]. S-nitrosylation can mediate either protective or neurotoxic signaling depending on the action of target proteins, regulating protein function by directly inhibiting catalytically active cysteines, possibly via impacting protein-protein interaction or through reacting with allosteric sites [14]. Mounting evidences have demonstrated that Snitrosylation plays neuropathogenic role in neuroal loss and a number of S-nitrosylated proteins contribute to the pathogenesis of neurodegenerative diseases [15]. ...
Galectin-1 (Gal-1) shows neuroprotective activity in brain ischemia, spinal cord injury, and autoimmune neuroinflammation. To evaluate the Gal-1 situation in the brains of prion disease, the brain levels of Gal-1 in several scrapie-infected experimental rodent models were tested by Western blot, including agents 263K-infected hamsters, 139A-, ME7-, and S15-infected mice. Remarkable increases of brain Gal-1 were observed in all tested scrapie-infected rodents at the terminal stage. The brain levels of Gal-1 showed time-dependent increases along with the prolonging of incubation times. Immunohistochemical assays illustrated much stronger stainings in the brain sections of scrapie-infected rodents. Quantitative RT-PCR of Gal-1 gene demonstrated increased transcription in the brains of scrapie-infected mice. Gal-1 was colocalized with GFAP- and NeuN-positive cells, but not with Iba-1-positive cells in immunofluorescent test. Increases of Gal-1 were also detected in the several postmortem cortex regions of human prion diseases. Moreover, the S-nitrosylated forms of Gal-1 in the brains of scrapie-infected rodents were significantly higher than those of normal ones. Our finding here demonstrates markedly increased brain Gal-1 and S-nitrosylated Gal-1 both in scrapie-infected rodents and human prion diseases.
... Protein S-glutathionylation ensures protection of protein thiols against irreversible overoxidation, operates as a biological redox switch in both cell survival (influencing kinases and J U S T A C C E P T E D protein phosphatases pathways) and cell death (by potentiation of apoptosis), and cross-talks with phosphorylation and with S-nitrosylation (70). Protein S-glutathionylation occurs under increase of free radical species but also under physiological ROS generation, proceeds spontaneously through thiol-disulfide exchange, and for most proteins occurs only under nonphysiological (decreased) GSH/GSSG status (16). ...
Metastatic spread, not primary tumors, is the leading cause of cancer death. Glutathione (γ-glutamyl-cysteinyl-glycine, GSH) is particularly relevant in cancer cells as it is involved in regulating carcinogenic mechanisms, growth and dissemination, and multidrug and radiation resistance. Upon interaction of metastatic cells with the vascular endothelium, a high percentage of metastatic cells with high GSH levels survive the combined nitrosative and oxidative stresses elicited by the vascular endothelium. GSH release from different organs, mainly the liver, and its interorgan transport through the blood circulation to metastatic foci, promote their growth. The present review focuses on the relationship among GSH and different key mechanisms that facilitate metastatic cell survival and growth, i.e. adaptive responses to stress, cell death evasion, and utilization of physiological neuroendocrine mechanisms. Different strategies that are aimed at sensitizing metastases to cancer therapy by depleting metastatic cell GSH are analyzed.
... Protein oxidation has also been described in the BS of Ndufs4 KO mice (28), and reduced glutathione levels are lower in lymphocytes and whole blood (39,40), as well as in muscle of mitochondrial disease patients (41). We also studied protein glutathionylation in our samples, as this is a mechanism that protects sensitive sulfhydryl groups from irreversible oxidation (42). We did not detect any changes in the glutathionylation status of proteins in the BS of Ndufs4 KO mice versus age-matched controls (Fig. 1D, GSH panel). ...
Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus (VN). Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to VN pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels (VDAC) 1 and 2 as specific targets of succination in the Ndufs4 KO. Using targeted mass spectrometry, Cys77 and Cys48 were identified as endogenous sites of succination in VDAC2. Given the important role of VDAC isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.
... Protein S-glutathionylation is generally associated with increased oxidative or nitrosative stress during such pathological conditions as ischemia-reperfusion and hypertension [42,43], regulating cardiovascular responses to stress and injury. Sustained overproduction of ROS has emerged as a common element in a variety of mechanisms controlling vascular complications of metabolic disorders [44], but how protein S-glutathionylation in endothelial cells is involved in these pathogenic processes has not been investigated. ...
Cellulose nanocrystals (CNCs) display remarkable strength and physicochemical properties with significant potential applications. To better understand the potential adjuvanticity of a nanomaterial, it is important to investigate the extent of the immunological response, the mechanisms by which they elicit this response, and how this response is associated with their physicochemical characteristics. In this study, we investigated the potential mechanisms of immunomodulation and redox activity of two chemically related cationic CNC derivatives (CNC-METAC-1B and CNC-METAC-2B), using human peripheral blood mononuclear cells and mouse macrophage cells (J774A.1). Our data demonstrated that the biological effects caused by these nanomaterials occurred mainly with short term exposure. We observed opposite immunomodulatory activity between the tested nanomaterials. CNC-METAC-2B, induced IL-1β secretion at 2 h while CNC-METAC-1B decreased it at 24 h of treatment. In addition, both nanomaterials caused more noticeable increases in mitochondrial reactive oxygen species (ROS) at early time. The differences in apparent sizes of the two cationic nanomaterials could explain, at least in part, the discrepancies in biological effects, despite their closely related surface charges. This work provides initial insights about the complexity of the in vitro mechanism of action of these nanomaterials as well as foundation knowledge for the development of cationic CNCs as potential immunomodulators.
Background:
Glutathionylation is a protein post-translational modification triggered by oxidative stress. The susceptible proteins are modified by the addition of glutathione to specific cysteine residues. Virus infection also induces oxidative stress in the cell, which affects cellular homeostasis. It is not just the cellular proteins but the viral proteins that can also be modified by glutathionylation events, thereby impacting the function of the viral proteins.
Objectives:
This study was conducted to identify the effects of modification by glutathionylation on the guanylyltransferase activity of NS5 and identify the cysteine residues modified for the three flavivirus NS5 proteins.
Methods:
The capping domain of NS5 proteins from 3 flaviviruses was cloned and expressed as recombinant proteins. A gel-based assay for guanylyltransferase activity was performed using a GTP analog labeled with the fluorescent dye Cy5 as substrate. The protein modification by glutathionylation was induced by GSSG and evaluated by western blot. The reactive cysteine residues were identified by mass spectrometry.
Results:
It was found that the three flavivirus proteins behaved in a similar fashion with increasing glutathionylation yielding decreased guanylyltransferase activity. The three proteins also possessed conserved cysteines and they appeared to be modified for all three proteins.
Conclusion:
The glutathionylation appeared to induce conformational changes that affect enzyme activity. The conformational changes might also create binding sites for host cell protein interactions at later stages of viral propagation with the glutathionylation event, thereby serving as a switch for function change.
Three arboviruses, dengue virus, Zika virus and Japanese encephalitis virus, have wide distribution putting millions of people at risk of infection. These three flaviviruses show evolutionarily conserved features for the viral proteins, which consist of seven non-structural and three structural proteins. Non-structural protein 5 (NS5) is important for viral replication as it possesses multiple functions including both enzyme and non-enzyme roles. Oxidative stress induced by virus infection triggers glutathionylation of cell proteins. This study was to identify the effects of modification by glutathionylation on the guanylyltransferase activity of NS5 and identify the cysteine residues modified for the three flavivirus NS5 proteins. We found the three flavivirus proteins behaved in a similar fashion with increasing glutathionylation yielding decreasing guanylyltransferase activity. The three proteins also possessed conserved cysteines and these appeared to be modified for all three proteins. The glutathionylation appears to induce conformational changes that affect enzyme activity but possibly also create binding sites for host cell protein interactions that occur at later stages of viral propagation.
Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described as a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.
The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in the apicomplexan parasite Toxoplasma gondii . The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 49 genes containing domains related to canonical antioxidant groups, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H 2 O 2 to human fibroblasts infected with T. gondii triggers a Ca ²⁺ flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H 2 O 2 is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression of the active catalytic domain of glutaredoxin in the parasitophorous vacuole severely impacts parasite replication. These data shed light on the rich redox network that exists in T. gondii , evidencing a link between extracellular redox and intracellular Ca ²⁺ signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasite growth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.
AIMS: This study aimed to investigate the effects of crude extract of Carica papaya leaves on oxidative stress in mice induced by cyclophosphamide, as well as phytochemical profile characterization of this extract.METHODS: The male Swiss mice received 15 days of treatment with the extract (500 mg kg-1, via gavage) and intraperitoneal injection of cyclophosphamide (75 mg kg-1) or saline (0.9%) on the 15th day. After 24 h the last treatment, the animals were anesthetized for blood withdrawal, sacrificed and removal of the organs for analyses (liver, kidney and heart). In the biochemical tests were determined: hematological parameters in blood, aminotransferases, alkaline phosphatase, glucose and total cholesterol dosages in plasma, enzymatic and non-enzymatic antioxidants and lipid damage marker were evaluated in different tissues, besides genotoxic and histopathological analyzes.RESULTS: In the extract of Carica papaya leaves, the flavonoids quercetin-3β-D-glucoside and rutin were identified, besides present positive results for alkaloids, saponins and tannins. This extract increased the activity of glutathione-S-transferase and catalase enzymes in the liver and reduced the levels of reduced glutathione in the kidneys and hematocrit levels, red cell count, and hemoglobin. It promoted the decrease of the reactive species of thiobarbituric acid (TBARS) in the kidneys and the activity of enzyme aspartate aminotransferase in the plasma and was antimutagenic in the micronucleus test.CONCLUSIONS: The study showed that extract of Carica papaya was beneficial against oxidative events and prevented DNA damage. The extract also showed hepatotoxicity, therefore prolonged infusion of papaya leaves is not advisable.
AIMS: The objective of this study was to identify the phytochemical profile and to evaluate the biological effects of the crude ethanolic extract (EE) and the ethanolic fraction (EF) of leaves of the species Cissus spinosa Cambess, after oxidative stress induced by cyclophosphamide (CP) in mice.METHODS: Phytochemical profile was performed detecting functional groups and, analysis of total flavonoids and phenols concentration, as well as the antiradical activity in EE and EF. The phytochemical characterization was done for the identification of flavonoids present in the leaves of the plant. In the biochemical tests, hematological parameters, glucose and total cholesterol dosages in plasma, enzymatic and non-enzymatic antioxidants and lipid damage marker were evaluated in different tissues (liver, kidney and heart), besides genotoxic and immunological analyzes. The animals received 15 days of treatment, via gavage, with EE (50 mg kg-1) or EF (50 mg kg-1) and on the 15th day, an intraperitoneal injection of CP (100 mg kg-1) or saline (0.9%). After 24 h the last treatment, the animals were anesthetized for blood withdrawal, sacrificed and removal of the organs.RESULTS: In the phytochemical analyzes, the presence of alkaloids, flavonoids and phenols was identified, the latter presented a higher concentration for EF. Eight flavonoids were identified - Rutin, Quercetin-3-β-D-glucoside, Quercitrin, Taxifolin, Quercetin, Canferol, Luteolin and Apigenin. In the biochemical analyzes, in general, EE showed a better antioxidant action against oxidative damages, hypoglycemic and antitilipemic action when comparing with EF, probably due to the synergism caused by flavonoids. It was observed the reduction and an increase of micronucleated polychromatic erythrocytes, due to the action of antioxidant compounds and alkaloids present in the plant, also considering the question of the seasonal period that directly interferes in the production of these compounds. In the immunological analysis, the extracts did not stimulate the spontaneous production of oxygen peroxide (H2O2) and nitric oxide (NO•). CONCLUSIONS: Other studies, such as the variation of the chemical composition of the plant by local seasonality, hypoglycemic and antilipemic action, should be carried out to better delineate the biological action present in this plant.
α4 integrin plays a crucial role in retention and release of neutrophils from bone marrow. Although α4 integrin is known to be a potential target of reactive oxygen species (ROS)-induced cysteine glutathionylation, the physiological significance and underlying regulatory mechanism of this event remain elusive. Here, using in vitro and in vivo biochemical and cell biology approaches, we show that physiological ROS-induced glutathionylation of α4 integrin in neutrophils increases the binding of neutrophil-associated α4 integrin to vascular cell adhesion molecule 1 (VCAM-1) on human endothelial cells. This enhanced binding was reversed by extracellular glutaredoxin 1 (Grx1), a thiol disulfide oxidoreductase promoting protein deglutathionylation. Furthermore, in a murine inflammation model, Grx1 disruption dramatically elevated α4 glutathionylation and subsequently enhanced neutrophil egress from the bone marrow. Corroborating this observation, intravenous injection of recombinant Grx1 into mice inhibited α4 glutathionylation and thereby suppressed inflammation-induced neutrophil mobilization from the bone marrow. Taken together, our results establish ROS-elicited glutathionylation and its modulation by Grx1 as pivotal regulatory mechanisms controlling α4 integrin affinity and neutrophil mobilization from the bone marrow under physiological conditions.
Heart failure is the most common cause of morbidity and hospitalization in the western civilization. Protein phosphatases play a key role in the basal cardiac contractility and in the responses to β-adrenergic stimulation with type-1 phosphatase (PP-1) being major contributor. We propose here that formation of transient disulfide bridges in PP-1α might play a leading role in oxidative stress response. First, we established an optimized workflow, the so-called “cross-over-read” search method, for the identification of disulfide-linked species using permutated databases. By applying this method, we demonstrate the formation of unexpected transient disulfides in PP-1α to shelter against over-oxidation. This protection mechanism strongly depends on the fast response in the presence of reduced glutathione. Our work points out that the dimerization of PP-1α involving Cys39 and Cys127 is presumably important for the protection of PP-1α active surface in the absence of a substrate. We finally give insight into the electron transport from the PP-1α catalytic core to the surface. Our data suggest that the formation of transient disulfides might be a general mechanism of proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation.
The glutathione (GSH) S-transferase family of detoxification and signalling proteins represents a major hub for the metabolism of Selenium-derived compounds. At the same time, these compounds can be used to modulate the expression and multiple activities of GSTs and other glutathione-dependent genes, that are important aspects in both the chemoprevention and therapy of drug-resistant cancers.
In this context, the isoform GSTP-1 (GSTP) appears to play a fundamental role. Besides promoting GSH-dependent detoxification of cellular electrophiles, GSTP physically interacts with a number of small molecules and cellular proteins producing regulatory effects across the main signal transduction and transcription pathways (identified as the “regulatory interactome of GSTP”). An emerging molecular mechanism behind such regulatory function is the activity of GSTP as a redox chaperonine responsible for the selective glutathionylation of protein Cys residues in the different subcellular compartments.
The redox-sensitive transcription factor Nrf2 was recently identified as one of the regulatory nodes of this interactome at the interface between inflammation, adaptive stress response, and cell death pathways.
The influence of Nrf2 in the stress response to cellular electrophiles and its regulatory interaction with GSTP are discussed in this review suggesting the hypothesis that this interaction may represent the actual pharmacological target of Se compounds with thiol peroxidase activity. These points are critically evaluated with a view to further development of these compounds in cancer prevention and the chemotherapy of drug-resistant tumours.
Large-scale characterisation of cysteine modification is enabling study of the physicochemical determinants of reactivity. We find that location of cysteine at the amino terminus of an α-helix, associated with activity in thioredoxins, is under-represented in human protein structures, perhaps indicative of selection against background reactivity. An amino-terminal helix location underpins the covalent linkage for one class of kinase inhibitors. Cysteine targets for S-palmitoylation, S-glutathionylation, and S-nitrosylation show little correlation with pKa values predicted from structures, although flanking sequences of S-palmitoylated sites are enriched in positively-charged amino acids, which could facilitate palmitoyl group transfer to substrate cysteine. A surprisingly large fraction of modified sites, across the three modifications, would be buried in native protein structure. Furthermore, modified cysteines are (on average) closer to lysine ubiquitinations than are unmodified cysteines, indicating that cysteine redox biology could be associated with protein degradation and degron recognition.
We previously reported that oral L-citrulline (L-Cit) administration antagonizes neuronal cell death in hippocampus following transient brain ischemia and that oral glutathione (GSH) administration prevents neuronal death through antioxidant activity. Here, we tested potential synergy of combined L-Cit and GSH administration in protection against neuronal death following cerebral ischemia. One day after a 20-min bilateral common carotid artery occlusion (BCCAO), mice were orally administered L-Cit or GSH alone (at 40 or 100 mg/kg p.o.) or both (at 40 mg/kg p.o. each) daily for 10 days. The combination, but not L-Cit or GSH alone at 40 mg/kg p.o., significantly prevented neuronal death in the hippocampal CA1 region in BCCAO mice. Consistently, combined L-Cit and GSH administration improved memory-related behavioral deficits observed in BCCAO mice. Combination treatment also significantly rescued reduced endothelial nitric oxide synthase (eNOS) protein levels and antagonized eNOS S-glutathionylation seen following BCCAO ischemia. Recovery of eNOS activity was confirmed by in vivo NO production in hippocampus of BCCAO mice. Taken together, combined administration of L-Cit with GSH rescues eNOS function, thereby inhibiting delayed neuronal death in hippocampus.
Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein–protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.
The vascular endothelium is a regulatory interface between blood and tissues. It plays multifaceted and vitally important functions in the body. Pathological alterations in endothelial cells represent an important component of many human health maladies and targets for therapeutic interventions. Oxidative stress caused by surplus reactive oxygen species (ROS) and inflammation represent mutually reinforcing pathways underlying mechanisms of many human pathological conditions. Antioxidants may have some utility for the alleviation of chronic oxidative stress, but provide no tangible benefits in the treatment of acute, dangerous conditions that lack pharmacotherapy including ischemia-reperfusion, adult respiratory distress syndrome, and sepsis. Diverse carriers and drug delivery systems are being pursued to improve problematic delivery of antioxidants in the vasculature. Since ROS act on nanometer distances, targeted delivery of antioxidants to desirable compartments, in particular, to endothelial cells, is necessary. Here, we briefly review nanocarriers providing endothelial targeting of antioxidants with particular focus on antioxidant enzymes and discuss the current challenges and perspectives of this area of drug delivery and nanomedicine.
The oxidation of biothiols participates not only in the defense against oxidative damage but also in enzymatic catalytic mechanisms and signal transduction processes. Thiols are versatile reductants that react with oxidizing species by one- and two-electron mechanisms, leading to thiyl radicals and sulfenic acids, respectively. These intermediates, depending on the conditions, participate in further reactions that converge on different stable products. Through this review we will describe the biologically relevant species that are able to perform these oxidations and we will analyze the mechanisms and kinetics of the one- and two-electron reactions.The processes undergone by typical low molecular weight thiols as well as the particularities of specific thiol proteins will be described, including the molecular determinants proposed to account for the extraordinary reactivities of peroxidatic thiols. Finally, the main fates of the thiyl radical and sulfenic acid intermediates will be summarized.
The recent discoveries on organelles autoregulation and their molecular mechanisms motivate a novel perspective on mitochondria involvement in cardiovascular dysfunctions related to diabetes mellitus and obesity. We present here an up-dated view on the latter topic along with a condensed perception on morphological details resultant from diabetes mellitus experimental models. This study is organized into sections covering the following topics: (I) mitochondrial stress/dysfunction, (II) the "quality controller" role of mitochondria exerted by fusion, fission, and mitophagy events, (111) the connection between mitochondria and metabolic diseases, and (IV) the perspectives of potential application of mitochondrial-targeted compounds in metabolic diseases. Critical analysis of the knowledge available so far on mitochondria-metabolic diseases relationship allows a two sides conclusion: a doubtful view, as the correlation between impaired mitochondrial function and insulin resistance is still unclear, even after 40 years since its first publication, and a hopeful view based on the novel traits of this organelle uncovered recently, such as plasticity, the "quality controller" role, the "retrograde signalling", and the coordinate interaction with the nucleus, endoplasmic reticulum, Golgi apparatus and lysosomes. At the horizon, the essential issue of targeting mitochondria for the alleviation of diabetes/obesity complications remains to be resolved.
Basal levels of oxidants are indispensible for redox signaling to produce adaptive cellular responses such as vitagenes linked to cell survival; however, at higher levels, they are detrimental to cells, contributing to aging and to the pathogenesis of numerous age-related diseases. Aging is a complex systemic process and the major gap in aging research reminds the insufficient knowledge about pathways shifting from normal "healthy" aging to disease-associated pathological aging. The major complication of normal "healthy" aging is in fact the increasing risk of age-related diseases such as cardiovascular diseases, diabetes mellitus, and neurodegenerative pathologies that can adversely affect the quality of life in general, with enhanced incidences of comorbidities and mortality. In this context, global "omics" approaches may help to dissect and fully study the cellular and molecular mechanisms of aging and age-associated processes. The proteome, being more close to the phenotype than the transcriptome and more stable than the metabolome, represents the most promising "omics" field in aging research. In the present study, we exploit recent advances in the redox biology of aging and discuss the potential of proteomics approaches as innovative tools for monitoring at the proteome level the extent of protein oxidative insult and related modifications with the identification of targeted proteins.
Glutathionylation of endothelial nitric oxide synthase (eNOS) "uncouples" the enzyme, switching its function from nitric oxide (NO) to O2(•-) generation. We examined whether this reversible redox modification plays a role in angiotensin II (Ang II)-induced endothelial dysfunction.
Ang II increased eNOS glutathionylation in cultured human umbilical vein endothelial cells (HUVECs), rabbit aorta, and human arteries in vitro. This was associated with decreased NO bioavailability and eNOS activity as well as increased O2(•-) generation. Ang II-induced decrease in eNOS activity was mediated by glutathionylation, as shown by restoration of function by glutaredoxin-1. Moreover, Ang II-induced increase in O2(•-) and decrease in NO were abolished in HUVECs transiently transfected, with mutant eNOS rendered resistant to glutathionylation. Ang II effects were nicotinamide adenine dinucleotide phosphate (NADPH) oxidase dependent because preincubation with gp 91ds-tat, an inhibitor of NADPH oxidase, abolished the increase in eNOS glutathionylation and loss of eNOS activity. Functional significance of glutathionylation in intact vessels was supported by Ang II-induced impairment of endothelium-dependent vasorelaxation that was abolished by the disulfide reducing agent, dithiothreitol. Furthermore, attenuation of Ang II signaling in vivo by administration of an angiotensin converting enzyme (ACE) inhibitor reduced eNOS glutathionylation, increased NO, diminished O2(•-), improved endothelium-dependent vasorelaxation and reduced blood pressure.
Uncoupling of eNOS by glutathionylation is a key mediator of Ang II-induced endothelial dysfunction, and its reversal is a mechanism for cardiovascular protection by ACE inhibition. We suggest that Ang II-induced O2(•-) generation in endothelial cells, although dependent on NADPH oxidase, is amplified by glutathionylation-dependent eNOS uncoupling.
Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation
reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report
that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation
of mitochondrial proteins. Deletion of Grx2 (Grx2−/−) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration
was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly
increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring
the deglutathionylated states of proteins. Grx2−/− hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from
Grx2 heterozygotes (Grx2+/−) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2+/− mice were far less hypertensive than Grx2−/− mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to
pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel
redox-related therapies in cardiac disease.
Key Points
The x-ray crystal structure of the N2 domain from HRG at 1.93 Å resolution is presented. The structure reveals an S-glutathionyl adduct at Cys185, which has implications for angiogenic regulation.
The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, various signaling processes, antigen presentation, and control of protein synthesis. The central catalytic complex of the proteasome is called the 20 S core particle. The majority of these are flanked on one or both sides by regulatory units. Most common among these units is the 19 S regulatory unit. When coupled to the 19 S unit, the complex is termed the asymmetric or symmetric 26 S proteasome depending on whether one or both sides are coupled to the 19 S unit, respectively. The 26 S proteasome recognizes poly-ubiquitinylated substrates targeted for proteolysis. Targeted proteins interact with the 19 S unit where they are deubiquitinylated, unfolded, and translocated to the 20 S catalytic chamber for degradation. The 26 S proteasome is responsible for the degradation of major proteins involved in the regulation of the cellular cycle, antigen presentation and control of protein synthesis. Alternatively, the proteasome is also active when dissociated from regulatory units. This free pool of 20 S proteasome is described in yeast to mammalian cells. The free 20 S proteasome degrades proteins by a process independent of poly-ubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20 S proteasome comprises two central heptamers (β-rings) where the catalytic sites are located and two external heptamers (α-rings) that are responsible for proteasomal gating. Because the 20 S proteasome lacks regulatory units, it is unclear what mechanisms regulate the gating of α-rings between open and closed forms. In the present review, we discuss 20 S proteasomal gating modulation through a redox mechanism, namely, S-glutathionylation of cysteine residues located in the α-rings, and the consequence of this post-translational modification on 20 S proteasomal function.
Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER) stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI) is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO-) containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI) in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future.
Significance
Embryonic development is one of the most amazing miracles in nature. The proteins and signaling events driving this highly complex process are far from being elucidated completely. For a long time, an important role of protein reduction and oxidation during development has been assumed. Here, we demonstrate the essential role of such a regulation during cardiovascular development: The modification of a single cysteine in the protein sirtuin 1 by the vertebrate-specific oxidoreductase glutaredoxin 2 is required for vessel formation and guidance. Our data indicate that this redox-signaling pathway based on glutaredoxin-dependent reversible S -glutathionylation may be also important for diseases of the cardiovascular system and pathological situations connected to angiogenesis, e.g., malignancies.
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.
Obesity-induced insulin resistance has been linked to adipose tissue lipid aldehyde production and protein carbonylation. Trans-4-hydroxy-2-nonenal (4-HNE) is the most abundant lipid aldehyde in murine adipose tissue and is metabolized by glutathione S-transferase A4 (GSTA4) producing glutathionyl-HNE (GS-HNE) and its metabolite glutathionyl-1,4-dihydroxynonene (GS-DHN). The objective of this study was to evaluate adipocyte production of GS-HNE and GS-DHN and their effect on macrophage inflammation. Compared to lean controls, GS-HNE and GS-DHN were more abundant in visceral adipose tissue of ob/ob mice and diet-induced obese, insulin resistant mice. High glucose and oxidative stress induced production of GS-HNE and GS-DHNE by 3T3-L1 adipocytes in a GSTA4-dependent manner and both glutathionylated metabolites induced secretion of TNFα from RAW264.7 and primary peritoneal macrophages. Targeted microarray analysis revealed GS-HNE and GS-DHN induced expression of inflammatory genes including C3, C4b, c-Fos, igtb2, Nfkb1, and Nos2. Transgenic overexpression of GSTA4 in mouse adipose tissue led to increased levels of GS-HNE associated with higher fasting glucose levels and moderately impaired glucose tolerance. These results indicated adipocyte oxidative stress results in GSTA4-dependent production of pro-inflammatory glutathione metabolites, GS-HNE and GS-DHN, which may represent a novel mechanism by which adipocyte dysfunction results in tissue inflammation and insulin resistance.
Abstract Glutathione is considered the main regulator of redox balance in the cellular milieu due to its capacity for detoxifying deleterious molecules. The oxidative stress induced as a result of a variety of stimuli promotes protein oxidation, usually at cysteine residues, leading to changes in their activity. Mild oxidative stress, which may take place in physiological conditions, induces the reversible oxidation of cysteines to sulfenic acid form, while pathological conditions are associated with higher rates of reactive oxygen species production, inducing the irreversible oxidation of cysteines. Among these, neurodegenerative disorders, cardiovascular diseases and diabetes have been proposed to be pathogenetically linked to this state. In diabetes-associated vascular complications, lower levels of glutathione and increased oxidative stress have been reported. S-glutathionylation has been proposed as a posttranslational modification able to protect proteins from over-oxidizing environments. S-glutathionylation has been identified in proteins involved in diabetic models both in vitro and in vivo. In all of them, S-glutathionylation represents a mechanism that regulates the response to diabetic conditions, and has been described to occur in erythrocytes and neutrophils from diabetic patients. However, additional studies are necessary to discern whether this modification represents a biomarker for the early onset of diabetic vascular complications.
The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1(-/-)) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-β receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-β receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.
The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch
regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant
role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast
cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine
residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1
in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry
and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells.
In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation
of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic
variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism
affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.
Atherosclerosis is a chronic inflammatory disease involving the accumulation of monocytes and macrophages in the vascular wall. Monocytes and macrophages play a central role in the initiation and progression of atherosclerotic lesion development. Oxidative stress, which occurs when reactive oxygen species (ROS) overwhelm cellular antioxidant systems, contributes to the pathophysiology of many chronic inflammatory diseases, including atherosclerosis. Major targets of ROS are reactive thiols on cysteine residues in proteins, which when oxidized can alter cellular processes, including signaling pathways, metabolic pathways, transcription, and translation. Protein-S-glutathionylation is the process of mixed disulfide formation between glutathione (GSH) and protein thiols. Until recently, protein-S-glutathionylation was associated with increased cellular oxidative stress, but S-glutathionylation of key protein targets has now emerged as a physiologically important redox signaling mechanism, which when dysregulated contributes to a variety of disease processes. In this review, we will explore the role of thiol oxidative stress and protein-S-glutathionylation in monocyte and macrophage dysfunction as a mechanistic link between oxidative stress associated with metabolic disorders and chronic inflammatory diseases, including atherosclerosis.
The dynamics of redox metabolism necessitate cellular strategies for sensing redox changes and for responding to them. A common
mechanism for receiving and transmitting redox changes is via reversible modifications of protein cysteine residues. A plethora
of cysteine modifications have been described, including sulfenylation, glutathionylation, and disulfide formation. These
post-translational modifications have the potential to alter protein structure and/or function and to modulate cellular processes
ranging from division to death and from circadian rhythms to secretion. The focus of this thematic minireview series is cysteine
modifications in response to reactive oxygen and nitrogen species.
Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines
in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation
from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional
clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione
S-transferase P) exposed to agents that cause oxidative or nitrosative stress.
Maintenance of the cellular redox balance is crucial for cell survival. An increase in reactive oxygen, nitrogen, or chlorine
species can lead to oxidative stress conditions, potentially damaging DNA, lipids, and proteins. Proteins are very sensitive
to oxidative modifications, particularly methionine and cysteine residues. The reversibility of some of these oxidative protein
modifications makes them ideally suited to take on regulatory roles in protein function. This is especially true for disulfide
bond formation, which has the potential to mediate extensive yet fully reversible structural and functional changes, rapidly
adjusting the protein's activity to the prevailing oxidant levels.
Mitochondria are strategically and dynamically positioned in the cell to spatially coordinate ATP production with energy needs and to allow the local exchange of material with other organelles. Interactions of mitochondria with the sarco-endoplasmic reticulum (SR/ER) have been receiving much attention owing to emerging evidence on the role these sites have in cell signaling, dynamics and biosynthetic pathways. One of the most important physiological and pathophysiological paradigms for SR/ER-mitochondria interactions is in cardiac and skeletal muscle. The contractile activity of these tissues has to be matched by mitochondrial ATP generation that is achieved, at least in part, by propagation of Ca(2+) signals from SR to mitochondria. However, the muscle has a highly ordered structure, providing only limited opportunity for mitochondrial dynamics and interorganellar interactions. This Commentary focuses on the latest advances in the structure, function and disease relevance of the communication between SR/ER and mitochondria in muscle. In particular, we discuss the recent demonstration of SR/ER-mitochondria tethers that are formed by multiple proteins, and local Ca(2+) transfer between SR/ER and mitochondria.
Protein oxidative modifications, also known as protein oxidation, are a major class of protein posttranslational modifications. They are caused by reactions between protein amino acid residues and reactive oxygen species (ROS) or reactive nitrogen species (RNS) and can be classified into two categories: irreversible modifications and reversible modifications. Protein oxidation has been often associated with functional decline of the target proteins, which are thought to contribute to normal aging and age-related pathogenesis. However, it has now been recognized that protein oxidative modifications can also play beneficial roles in disease and health. This review summarizes and highlights certain positive roles of protein oxidative modifications that have been documented in the literature. Covered oxidatively modified protein adducts include carbonylation, 3-nitrotyrosine, s-sulfenation, s-nitrosylation, s-glutathionylation, and disulfide formation. All of which have been widely analyzed in numerous experimental systems associated with redox stress conditions. The authors believe that selected protein targets, when modified in a reversible manner in prophylactic approaches such as preconditioning or ischemic tolerance, may provide potential promise in maintaining health and fighting disease.
Oxidative stress has been identified as a common mechanism for cellular damage and dysfunction in a wide variety of disease states. Current understanding of the metabolic changes associated with obesity and the development of insulin resistance has focused on the role of oxidative stress and its interaction with inflammatory processes at both the tissue and organismal level. Obesity-related oxidative stress is an important contributing factor in the development of insulin resistance in the adipocyte as well as the myocyte. Moreover, oxidative stress has been linked to mitochondrial dysfunction, and this is thought to play a role in the metabolic defects associated with oxidative stress. Of the various effects of oxidative stress, protein carbonylation has been identified as a potential mechanism underlying mitochondrial dysfunction. As such, this review focuses on the relationship between protein carbonylation and mitochondrial biology and addresses those features that point to either the causal or casual relationship of lipid peroxidation-induced protein carbonylation as a determining factor in mitochondrial dysfunction.
S-glutathionylation, the reversible formation of mixed disulfides between glutathione(GSH) and cysteine residues in proteins, is a specific form of post-translational modification that plays important roles in various biological processes, including signal transduction, redox homeostasis, and metabolism inside cells. Experimentally identifying S-glutathionylation sites is labor-intensive and time consuming, whereas bioinformatics methods provide an alternative way to this problem by predicting S-glutathionylation sites in silico. The bioinformatics approaches give not only candidate sites for further experimental verification but also bio-chemical insights into the mechanism of S-glutathionylation. In this paper, we firstly collect experimentally determined S-glutathionylated proteins and their corresponding modification sites from the literature, and then propose a new method for predicting S-glutathionylation sites by employing machine learning methods based on protein sequence data. Promising results are obtained by our method with an AUC (area under ROC curve) score of 0.879 in 5-fold cross-validation, which demonstrates the predictive power of our proposed method. The datasets used in this work are available at http://csb.shu.edu.cn/SGDB.
The ionized cysteines present on the surfaces of many redox-sensitive proteins play functionally essential roles and are readily targeted by the reactive oxygen- and reactive nitrogen species. Using disulfiram (DSF) and nitroaspirin (NCX4016) as the model compounds that mediate thiol-conjugating and nitrosylating reactions respectively, we investigated the fate of p53, NF-κB, and other redox-responsive proteins following the exposure of human cancer cell lines to the drugs. Both drugs induced glutathionylation of bulk proteins in tumor cells and cell-free extracts. A prominent finding of this study was a time- and dose-dependent degradation of the redox-regulated proteins after brief treatments of tumor cells with DSF or nitroaspirin. DSF and copper-chelated DSF at concentrations of 50-200 CM induced the disappearance of wild type p53, mutant p53, NF-κB subunit p50, and the ubiquitin-activating enzyme E1 (UBE1) in tumor cell lines. DSF also induced the glutathionylation of p53. The recombinant p53 protein modified by DSF was preferentially degraded by rabbit reticulocyte lysates. The proteasome inhibitor PS341 curtailed the DSF-induced degradation of p53 in HCT116 cells. Further, the nitroaspirin induced a dose-dependent disappearance of the UBE1 and NF-κB p50 proteins in cell lines, besides a time-dependent degradation of aldehyde dehydrogenase in mouse liver after a single injection of 150 mg/kg. The loss of p53 and NF-kB proteins correlated with decreases in their specific binding to DNA. Our results demonstrate the hitherto unrecognized ability of the nontoxic thiolating and nitrosylating agents to degrade regulatory proteins and highlight the exploitable therapeutic benefits.
No abstract available.
The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.
Endothelial nitric-oxide synthase (eNOS) is a critical regulator of vascular homeostasis by generation of NO that is dependent
on the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, eNOS becomes “uncoupled,” resulting in superoxide
production in place of NO. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, although the functional relationships between BH4 and S-glutathionylation remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we expressed either WT or mutant eNOS rendered resistant to S-glutathionylation in cells with Tet-regulated expression of human GTP cyclohydrolase I to regulate intracellular BH4 availability.
We reveal that S-glutathionylation of eNOS, by exposure to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase-specific
siRNA, results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction
in BH4 levels and BH4:7,8-dihydrobiopterin ratio. In eNOS uncoupling induced by BH4 deficiency, BCNU exposure further exacerbates
superoxide production, BH4 oxidation, and eNOS activity. Following mutation of C908S, BCNU-induced eNOS uncoupling and BH4
oxidation are abolished, whereas uncoupling induced by BH4 deficiency was preserved. Furthermore, BH4 deficiency alone is
alone sufficient to reduce intracellular GSH:GSSG ratio and cause eNOS S-glutathionylation. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling, although mechanistically distinct, are functionally related. We
propose that uncoupling of eNOS by S-glutathionylation- or by BH4-dependent mechanisms exemplifies eNOS as an integrated redox “hub” linking upstream redox-sensitive
effects of BH4 and glutathione with redox-dependent targets and pathways that lie downstream of eNOS.
Monocytic adhesion and chemotaxis are regulated by MAPK pathways, which in turn are controlled by redox-sensitive MAPK phosphatases (MKPs). We recently reported that metabolic disorders prime monocytes for enhanced recruitment into vascular lesions by increasing monocytes' responsiveness to chemoattractants. However, the molecular details of this proatherogenic mechanism were not known. Here we show that monocyte priming results in the S-glutathionylation and subsequent inactivation and degradation of MKP-1. Chronic exposure of human THP-1 monocytes to diabetic conditions resulted in the loss of MKP-1 protein levels, the hyperactivation of ERK and p38 in response to monocyte chemoattractant protein-1 (MCP-1), and increased monocyte adhesion and chemotaxis. Knockdown of MKP-1 mimicked the priming effects of metabolic stress, whereas MKP-1 overexpression blunted both MAPK activation and monocyte adhesion and migration induced by MCP-1. Metabolic stress promoted the S-glutathionylation of MKP-1, targeting MKP-1 for proteasomal degradation. Preventing MKP-1 S-glutathionylation in metabolically stressed monocytes by overexpressing glutaredoxin 1 protected MKP-1 from degradation and normalized monocyte adhesion and chemotaxis in response to MCP-1. Blood monocytes isolated from diabetic mice showed a 55% reduction in MKP-1 activity compared with nondiabetic mice. Hematopoietic MKP-1 deficiency in atherosclerosis-prone mice mimicked monocyte priming and dysfunction associated with metabolic disorders, increased monocyte chemotaxis in vivo, and accelerated atherosclerotic lesion formation. In conclusion, we identified MKP-1 as a central redox-sensitive regulator of monocyte adhesion and migration and showed that the loss of MKP-1 activity is a critical step in monocyte priming and the metabolic stress-induced conversion of blood monocytes into a proatherogenic phenotype.
Oxygen-induced regulation of Na,K-ATPase was studied in rat myocardium. In rat heart Na,K-ATPase responded to hypoxia with a dose-dependent inhibition in hydrolytic activity. Inhibition of Na,K-ATPase in hypoxic rat heart was associated with decrease in NO production and progressive oxidative stress. Accumulation of oxidized glutathione (GSSG) and decrease in NO availability in hypoxic rat heart were followed by a decrease in S-nitrosylation and up-regulation of S-glutathionylation of the catalytic α subunit of the Na,K-ATPase. Induction of S-glutathionylation of the α subunit by treatment of tissue homogenate with GSSG resulted in complete inhibition of the enzyme in rat a myocardial tissue homogenate. Inhibitory effect of GSSG in rat sarcolemma could be significantly decreased upon activation of NO synthases. we have further tested if oxidative stress and suppression of the Na,K-ATPase activity are observed in hypoxic heart of two subterranean hypoxia-tolerant blind mole species (Spalax galili and Spalax judaei). In both hypoxia-tolerant Spalax species activity of the enzyme and tissue redox state were maintained under hypoxic conditions. However, localisation of cysteines within the catalytic subunit of the Na,K-ATPase was preserved and induction of S-glutathionylation by GSSG in tissue homogenate inhibited the Spalax ATPase as efficiently as in rat heart. The obtained data indicate that oxygen-induced regulation of the Na,K-ATPase in the heart is mediated by a switch between S-glutathionylation and S-nitrosylation of the regulatory thiol groups localized at the catalytic subunit of the enzyme.
Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10-50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.
Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.
4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P
S-Glutathionylation of cysteine residues within target proteins is a posttranslational modification that alters structure and function. We have shown that S-glutathionylation of protein disulfide isomerase (PDI) disrupts protein folding and leads to the activation of the unfolded protein response (UPR). PDI is a molecular chaperone for estrogen receptor alpha (ERα). Our present data show in breast cancer cells that S-glutathionylation of PDI interferes with its chaperone activity and abolishes its capacity to form a complex with ERα. Such drug treatment also reverses estradiol-induced upregulation of c-Myc, cyclinD1, and P21(Cip), gene products involved in cell proliferation. Expression of an S-glutathionylation refractory PDI mutant diminishes the toxic effects of PABA/NO. Thus, redox regulation of PDI causes its S-glutathionylation, thereby mediating cell death through activation of the UPR and abrogation of ERα stability and signaling.
Purpose:
Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR.
Methods:
Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 μM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19).
Results:
In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation.
Conclusions:
In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.
Mitochondrial reactive oxygen species (ROS) have emerged as an important mechanism of disease and redox signaling in the cardiovascular system. Under basal or pathological conditions, electron leakage for ROS production is primarily mediated by the electron transport chain and the proton motive force consisting of a membrane potential (ΔΨ) and a proton gradient (ΔpH). Several factors controlling ROS production in the mitochondria include flavin mononucleotide and flavin mononucleotide-binding domain of complex I, ubisemiquinone and quinone-binding domain of complex I, flavin adenine nucleotide-binding moiety and quinone-binding pocket of complex II, and unstable semiquinone mediated by the Q cycle of complex III. In mitochondrial complex I, specific cysteinyl redox domains modulate ROS production from the flavin mononucleotide moiety and iron-sulfur clusters. In the cardiovascular system, mitochondrial ROS have been linked to mediating the physiological effects of metabolic dilation and preconditioning-like mitochondrial ATP-sensitive potassium channel activation. Furthermore, oxidative post-translational modification by glutathione in complex I and complex II has been shown to affect enzymatic catalysis, protein-protein interactions, and enzyme-mediated ROS production. Conditions associated with oxidative or nitrosative stress, such as myocardial ischemia and reperfusion, increase mitochondrial ROS production via oxidative injury of complexes I and II and superoxide anion radical-induced hydroxyl radical production by aconitase. Further insight into cellular mechanisms by which specific redox post-translational modifications regulate ROS production in the mitochondria will enrich our understanding of redox signal transduction and identify new therapeutic targets for cardiovascular diseases in which oxidative stress perturbs normal redox signaling.
Recently, we demonstrated that gene ablation of mitochondrial manganese superoxide dismutase and aldehyde dehydrogenase-2 markedly contributed to age-related vascular dysfunction and mitochondrial oxidative stress. The present study has sought to investigate the extent of vascular dysfunction and oxidant formation in glutathione peroxidase-1-deficient (GPx-1(-/-)) mice during the aging process with special emphasis on dysregulation (uncoupling) of the endothelial NO synthase. GPx-1(-/-) mice on a C57 black 6 (C57BL/6) background at 2, 6, and 12 months of age were used. Vascular function was significantly impaired in 12-month-old GPx-1(-/-) -mice as compared with age-matched controls. Oxidant formation, detected by 3-nitrotyrosine staining and dihydroethidine-based fluorescence microtopography, was increased in the aged GPx-1(-/-) mice. Aging per se caused a substantial protein kinase C- and protein tyrosine kinase-dependent phosphorylation as well as S-glutathionylation of endothelial NO synthase associated with uncoupling, a phenomenon that was more pronounced in aged GPx-1(-/-) mice. GPx-1 ablation increased adhesion of leukocytes to cultured endothelial cells and CD68 and F4/80 staining in cardiac tissue. Aged GPx-1(-/-) mice displayed increased oxidant formation as compared with their wild-type littermates, triggering redox-signaling pathways associated with endothelial NO synthase dysfunction and uncoupling. Thus, our data demonstrate that aging leads to decreased NO bioavailability because of endothelial NO synthase dysfunction and uncoupling of the enzyme leading to endothelial dysfunction, vascular remodeling, and promotion of adhesion and infiltration of leukocytes into cardiovascular tissue, all of which was more prominent in aged GPx-1(-/-) mice.
Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress and results in the generation of various bioactive compounds, of which 4-hydroxyalkenals are prominent. During the progression of type 2 diabetes mellitus, the ensuing hyperglycemia promotes the generation of reactive oxygen species (ROS) that contribute to the development of diabetic complications. It has been suggested that ROS-induced lipid peroxidation and the resulting 4-hydroxyalkenals markedly contribute to the development and progression of these pathologies. Recent findings, however, also suggest that non-cytotoxic levels of 4-hydroxyalkenals play important signaling functions in the early phase of diabetes and act as hormetic factors to induce adaptive and protective responses in cells, enabling them to function in the hyperglycemic milieu. Our studies and others' have proposed such regulatory functions for 4-hydroxynonenal and 4-hydroxydodecadienal in insulin secreting β-cells and vascular endothelial cells, respectively. This review presents and discusses the mechanisms regulating the generation of 4-hydroxyalkenals under high glucose conditions and the molecular interactions underlying the reciprocal transition from hormetic to cytotoxic agents.
It is well-established that maintenance of the intracellular redox (i.e., reduction-oxidation) state is critical for cell survival and that prolonged or abnormal perturbations toward oxidation result in cell dysfunction. This is exemplified by the widespread observation of oxidative stress in many pathological conditions, as well as the positive effects of antioxidants in treating certain conditions or extending the life span itself. In addition to the effects of oxidation on the lipid bilayer and modification of DNA in the nucleus, proteins are also modulated by the redox state. One of the primary targets of oxidation within a protein is the AA cysteine, whose thiol side chain is highly sensitive to all types of oxidizing agents. Although this sensitivity is used to prevent oxidation within the cell by potent defense mechanisms, such as glutathione, the use of cysteine in the active site of enzymes leaves them open to oxidant-mediated damage. Whether the damage is due to a pathological condition or to postmortem mediated loss of redox homeostasis, cysteine-dependent enzymes are targets of all forms of reactive oxygen, nitrogen, and sulfur species. A greater understanding of the redox-mediated control of cysteine-dependent enzymes opens the door to the selective use of antioxidants to prevent or reverse the cellular damage their inhibition causes.
Glutathionylation of the Na(+)-K(+) pump's β1 subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating Angiotensin II (Ang II)-induced increase in vascular reactivity.
β1 subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs) and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by pre-incubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1 subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1 subunit glutathionylation, and enhanced K(+)-induced vasorelaxation.
Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1 subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an important protective role in the vasculature under conditions of oxidative stress.
Abstract Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous hydrogen peroxide (H2O2) by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiological and pathological H2O2 responses.
Background:
It is now recognized that protein cysteines exist not only as free thiols or intramolecular disulfides, that help maintain the 3D structure of proteins, but can also undergo different types of oxidation, one of which is glutathionylation, or the formation of mixed disulfides with glutathione (GSH).
Scope of the review:
We will discuss how proteins can undergo glutathionylation and how this can affect the protein characteristics/function. Glutathionylation is reversible and de-glutathionylation can be catalysed by protein thiol-disulfide oxidoreductases. Genetic modification of the expression of these enzymes, particularly glutaredoxin, using overexpression, knockout mice or siRNA, is becoming an important tool to study the role of protein glutathionylation. While in the past this post-translational modification was mainly known in the context of oxidative stress, measurement of glutathionylated proteins in patients is pointing out a potential importance if this modification in pathogenesis and could identify new biomarkers. We also wanted to point out the main findings in the role of glutathionylation in diseases and drug action.
Major conclusions:
We identify two major open problems in the field, namely the complexity of the mechanisms responsible for glutathionylation and de-glutathionylation, as well as what makes a protein susceptible to glutathionylation.
General significance:
This review underlines the peculiarities of this post-translational modification and their biological role. This article is part of a Special Issue entitled Cellular functions of glutathione.
A number of clinical and biochemical studies demonstrate that obesity and insulin resistance are associated with increases in oxidative stress and inflammation. Paradoxically, insulin sensitivity can be enhanced by oxidative inactivation of cysteine residues of phosphatases, and inflammation can be reduced by S-glutathionylation with formation of protein-glutathione mixed disulfides (PSSG). Although oxidation of protein-bound thiols (PSH) is increased in multiple diseases, it is not known whether there are changes in PSH oxidation species in obesity.
In this work, the hypothesis that obesity is associated with decreased levels of proteins containing oxidized protein thiols was tested.
The tissue levels of protein sulfenic acids (PSOH) and PSSG in liver, visceral adipose tissue, and skeletal muscle derived from glucose intolerant, obese-prone Sprague-Dawley rats were examined.
The data in this study indicate that decreases in PSSG content occurred in liver (44%) and adipose (26%) but not skeletal muscle in obese rats that were fed a 45% fat-calorie diet versus lean rats that were fed a 10% fat-calorie diet. PSOH content did not change in the tissue between the two groups. The activity of the enzyme glutaredoxin (GLRX) responsible for reversal of PSSG formation did not change in muscle and liver between the two groups. However, levels of GLRX1 were elevated 70% in the adipose tissue of the obese, 45% fat calorie-fed rats.
These are the first data to link changes in S-glutathionylation and GLRX1 to adipose tissue in the obese and demonstrate that redox changes in thiol status occur in adipose tissue as a result of obesity.
In the cardiovascular system, changes in oxidative balance can affect many aspects of cellular physiology through redox-signaling. Depending on the magnitude, fluctuations in the cell's production of reactive oxygen and nitrogen species can regulate normal metabolic processes, activate protective mechanisms, or be cytotoxic. Reactive oxygen and nitrogen species can have many effects including the posttranslational modification of proteins at critical cysteine thiols. A subset can act as redox-switches, which elicit functional effects in response to changes in oxidative state. Although the general concepts of redox-signaling have been established, the identity and function of many regulatory switches remains unclear. Characterizing the effects of individual modifications is the key to understand how the cell interprets oxidative signals under physiological and pathological conditions. Here, we review the various cysteine oxidative posttranslational modifications and their ability to function as redox-switches that regulate the cell's response to oxidative stimuli. In addition, we discuss how these modifications have the potential to influence other posttranslational modifications' signaling pathways though cross-talk. Finally, we review the increasing number of tools being developed to identify and quantify the various cysteine oxidative posttranslational modifications and how this will advance our understanding of redox-regulation.
AimsChronic depletion of myocardial glutathione (GSH) may play a role in cardiac remodelling and dysfunction. This study examined the relationship between chronic GSH depletion and cardiac failure induced by pressure overload in mice lacking the modifier subunit (GCLM) of glutamate-cysteine ligase, the rate-limiting enzyme for GSH synthesis. In addition, we examined the association between idiopathic dilated cardiomyopathy (DCM) in humans and-588C/T polymorphism of the GCLM gene, which reduces plasma levels of GSH.Methods and resultsPressure overload in mice was created by transverse aortic constriction (TAC). Myocardial GSH levels after TAC in GCLM-/- mice were 31% of those in GCLM+/+ mice. TAC resulted in greater heart and lung-weight-to-body-weight ratios, greater dilation and dysfunction of left ventricle, more extensive myocardial fibrosis, and worse survival in GCLM -/- than GCLM+/+ mice. Supplementation of GSH diethyl ester reversed the left-ventricular dilation and contractile dysfunction and the increased myocardial fibrosis after TAC in GCLM-/- mice. The prevalence of-588T polymorphism of the GCLM gene was significantly higher in DCM patients (n = 205) than in age-and sex-matched control subjects (n = 253) (36 vs. 19%, respectively, P < 0.001). The-588T polymorphism increased the risk of DCM that was independent of age, diabetes, and systolic blood pressure (OR 3.13, 95% CI: 2.28-4.44; P < 0.0001).Conclusion
Chronic depletion of GSH exacerbates remodelling and dysfunction in the pressure-overloaded heart. The clinical relevance of this mouse model is supported by a significant association between-588T polymorphism of the GCLM gene and patients with DCM.
Redox proteomics involves the large-scale analysis of oxidative protein posttranslational modifications. In particular, cysteine residues have become the subject of intensifying research 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. Because of 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.
Reactive oxygen and nitrogen species (RO/NS) have been found to play a dual role in the cardiovascular system, acting both as second messengers in physiological redox signaling and as agents of oxidative damage, leading to pathological conditions.1,2 This dual role is determined largely by the balance of oxidant production and the capacity of the cell’s antioxidant defense. RO/NS in cardiomyocytes can be produced by several sources, including, in the mitochondria, nicotinamide adenine dinucleotide phosphate oxidases and NO synthases.3–5 The levels of these species are held in check by antioxidant scavengers such as catalase, glutathione (GSH) peroxidase, superoxide dismutase, peroxiredoxin, and free GSH, which patrol the cell neutralizing them.6 Changes in redox balance occur when the levels of RO/NS production exceed the local antioxidant capacity. Small changes in the concentration of some species (eg, superoxide, hydrogen peroxide, or NO) are more likely to participate in redox-signaling …
Redox signaling refers to the specific and usually reversible oxidation/reduction modification of molecules involved in cellular signaling pathways. In the heart, redox signaling regulates several physiological processes (eg, excitation-contraction coupling) and is involved in a wide variety of pathophysiological and homoeostatic or stress response pathways. Reactive oxygen species involved in cardiac redox signaling may derive from many sources, but NADPH oxidases, as dedicated sources of signaling reactive oxygen species, seem to be especially important. An increasing number of specific posttranslational oxidative modifications involved in cardiac redox signaling are being defined, along with the reactive oxygen species sources that are involved. Here, we review current knowledge on the molecular targets of signaling reactive oxygen species in cardiac cells and their involvement in cardiac physiopathology. Advances in this field may allow the development of targeted therapeutic strategies for conditions such as heart failure as opposed to the general antioxidant approaches that have failed to date.
In non-excitable cells, thiol-oxidizing agents have been shown to evoke oscillations in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by increasing the sensitivity of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) to IP(3). Although thiol modification of the IP(3)R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein–glutathione (P-SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H(2)O(2)), on oscillations in [Ca(2+)](i) in fura-2-loaded bovine (BAECs) and human (HAECs) aortic endo-thelial cells using time-lapse fluorescence video microscopy. In the absence of extracellular Ca(2+), acute treatment with either diamide or H(2)O(2) increased the number of BAECs exhibiting asynchronous Ca(2+) oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine-stimulated Ca(2+) oscillations and BAECs to bradykinin-stimulated Ca(2+) oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin-induced Ca(2+) transient suggesting that Ca(2+)-induced Ca(2+) release (CICR) via the IP(3)R is enhanced by glutathionylation. Similar to diamide, H(2)O(2) increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP(3)R(1) is increased in cells challenged with H(2)O(2). Collectively our results reveal that thiol-oxidizing agents primarily increase the sensitivity of the IP(3)R to Ca(2+), i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP(3)R activity during physiological redox signalling and during pathologicalical oxidative stress.
Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H(2)O(2) include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H(2)O(2) abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H(2)O(2) is poorly reactive in chemical terms and the second order rate constants for the H(2)O(2)-mediated PTP inactivation are ~10M(-1)s(-1), which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H(2)O(2). We find that hydroxyl radical is produced from H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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.
Oxidative stress is linked to the production of reactive lipid aldehydes that non-enzymatically alkylate cysteine, histidine, or lysine residues in a reaction termed protein carbonylation. Reactive lipid aldehydes and their derivatives are detoxified via a variety of phase I and phase II systems, and when antioxidant defenses are compromised or oxidative conditions are increased, protein carbonylation is increased. The resulting modification has been implicated as causative in a variety of metabolic states including neurodegeneration, muscle wasting, insulin resistance, and aging. Although such modifications usually result in loss of protein function, protein carbonylation may be regulatory and activate signaling pathways involved in antioxidant biology and cellular homeostasis.
Oxidative stress has been recognized as a contributing factor in the toxicity of a large number of developmental toxicants. Traditional definitions of oxidative stress state that a shift in the balance between reduced and oxidized biomolecules within cells, in favor of the latter, result in changes that are deleterious to vital cell functions and can culminate in malformations and death. The glutathione (GSH)/glutathione disulfide (GSSG) redox couple has been the traditional marker of choice for characterization of oxidative stress because of its high concentrations and direct roles as antioxidant and cellular protectant. Steady state depletion of GSH through conjugation, oxidation, or export has often been reported as the sole criteria for invoking oxidative stress and a myriad of associated deleterious consequences. Numerous other, mostly qualitative, observations have also been reported to suggest oxidative stress has occurred but it is not always clear how well they reflect the state of a cell or its functions. Our emerging understanding of redox signaling and the roles of reactive oxygen species (ROS), thiols, oxidant molecules, and cellular antioxidants, all acting as second messengers, has prompted a redefinition of oxidative stress based on changes in the real posttranslational protein thiol modifications that are central to redox regulation and control. Thiol-based redox couples such as GSH/GSSG, cysteine/cystine (cys/cySS), thioredoxin-reduced/thioredoxin-oxidized (TRX(red)/TRX(ox)) form independent signaling nodes that selectively regulate developmental events and are closely linked to changes in intracellular redox potentials. Accurate assessment of the consequences of increased free radicals in developing conceptuses should best be made using a battery of measurements including the quantitative assessment of intracellular redox potential, ROS, redox status of biomolecules, and induced changes in specific redox signaling nodes. Methods are presented for a determination of ROS production, soluble thiol oxidation, redox potential, and a proteomic approach to evaluate the thiol oxidation state of specific proteins.
Key points
In non‐excitable cells, oxidative stress increases inositol 1,4,5‐trisphosphate (IP 3 ) receptor (IP 3 R) activity, which can cause Ca ²⁺ oscillations under basal conditions and enhance agonist‐stimulated changes in cytosolic free Ca ²⁺ concentration.
Protein S ‐glutathionylation, the reversible modification of cysteine thiols by glutathione, is elevated in response to oxidative stress, but the consequence of glutathionylation for IP 3 R function is not known.
In this study we provide evidence that Ca ²⁺ ‐induced Ca ²⁺ ‐release (CICR) via the IP 3 R is enhanced by oxidant‐induced glutathionylation in cultured aortic endothelial cells.
Our results suggest glutathionylation may represent a fundamental mechanism for regulating IP 3 R activity during physiological redox signalling and during pathological oxidative stress.
Abstract In non‐excitable cells, thiol‐oxidizing agents have been shown to evoke oscillations in cytosolic free Ca ²⁺ concentration ([Ca ²⁺ ] i ) by increasing the sensitivity of the inositol 1,4,5‐trisphosphate (IP 3 ) receptor (IP 3 R) to IP 3 . Although thiol modification of the IP 3 R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein–glutathione (P‐SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H 2 O 2 ), on oscillations in [Ca ²⁺ ] i in fura‐2‐loaded bovine (BAECs) and human (HAECs) aortic endo‐thelial cells using time‐lapse fluorescence video microscopy. In the absence of extracellular Ca ²⁺ , acute treatment with either diamide or H 2 O 2 increased the number of BAECs exhibiting asynchronous Ca ²⁺ oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine‐stimulated Ca ²⁺ oscillations and BAECs to bradykinin‐stimulated Ca ²⁺ oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin‐induced Ca ²⁺ transient suggesting that Ca ²⁺ ‐induced Ca ²⁺ release (CICR) via the IP 3 R is enhanced by glutathionylation. Similar to diamide, H 2 O 2 increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP 3 R 1 is increased in cells challenged with H 2 O 2 . Collectively our results reveal that thiol‐oxidizing agents primarily increase the sensitivity of the IP 3 R to Ca ²⁺ , i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP 3 R activity during physiological redox signalling and during pathologicalical oxidative stress.
Complex I is a critical site of O(2)(•-) production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, complex I protein thiols at the 51- and 75-kDa subunits are reversibly S-glutathionylated. The mechanism of complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (J. Chen et al., J. Biol. Chem. 285:3168-3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75-kDa subunit from S-glutathionylated complex I. Under the conditions of O(2)(•-) production in the presence of GSH, we detected complex I S-glutathionylation at Cys-226, Cys-367, and Cys-727 of the 75-kDa subunit. Addition of a radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), significantly decreased complex I S-glutathionylation and subsequently increased the protein radical adduct of complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO binding, confirming that formation of the complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O(2)(•-) generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O(2)(•-) generation, complex I protein radical and S-glutathionylation were increased. Thus mediation of complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.
Reactive oxygen species (ROS), particularly hydrogen peroxide (H(2) O(2) ), act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pK(a) . This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling.