Reversible Oxidative Modification Research Articles

Hydrogen Sulphide: A Key Player in Plant Development and Stress Resilience.

Based on the research conducted so far, hydrogen sulphide (H2S) plays a crucial role in the development and stress resilience of plants. H2S, which acts as a signalling molecule, responds to different stresses such as heavy metals, drought, and salinity, and it regulates various aspects of plant growth and development including seed germination, root development, stomatal movement, flowering, and fruit ripening. Additionally, H2S is involved in mediating legume-Rhizobium symbiosis signalling. It modulates plant responses to external environmental stimuli by interacting with other signalling molecules like phytohormones, nitric oxide, and reactive oxygen species. Furthermore, H2S exerts these regulations since it can modify protein functions through a reversible thiol-based oxidative posttranslational modification called persulfidation, particularly in stress response and developmental processes. As a result, H2S is recognised as an important emerging signalling molecule with multiple roles in plants. Research in this field holds promise for engineering stress tolerance in crops and may lead to potential biotechnological applications in agriculture and environmental management.

Cysteine sulfenylation contributes to liver fibrosis via the regulation of EphB2-mediated signaling

Sulfenylation is a reversible oxidative posttranslational modification (PTM) of proteins on cysteine residues. Despite the dissection of various biological functions of cysteine sulfenylation, its roles in hepatic fibrosis remain elusive. Here, we report that EphB2, a receptor tyrosine kinase previously implicated in liver fibrosis, is regulated by cysteine sulfenylation during the fibrotic progression of liver. Specifically, EphB2 is sulfenylated at the residues of Cys636 and Cys862 in activated hepatic stellate cells (HSCs), leading to the elevation of tyrosine kinase activity and protein stability of EphB2 and stronger interactions with focal adhesion kinase for the activation of downstream mitogen-activated protein kinase signaling. The inhibitions of both EphB2 kinase activity and cysteine sulfenylation by idebenone (IDE), a marketed drug with potent antioxidant activity, can markedly suppress the activation of HSCs and ameliorate hepatic injury in two well-recognized mouse models of liver fibrosis. Collectively, this study reveals cysteine sulfenylation as a new type of PTM for EphB2 and sheds a light on the therapeutic potential of IDE for the treatment of liver fibrosis.

The role of ubiquitin and heat shock proteins 27 and 70 in the oxidative modification of proteins and the implementation of dexamethasone-induced apoptosis of tumor cells

BACKGROUND: Experimental studies of molecular control of the tumor cell’s redox status, which influences the implementation of apoptosis, are relevant for studying the pathogenesis of tumor growth.
 AIM: Studying the molecular mechanisms of the participation of ubiquitin and heat shock proteins 27 and 70 in the oxidative modification of proteins, amino acids and the implementation of dexamethasone-induced apoptosis of Jurkat tumor cells in conditions of decreased antioxidant protection by blocking the synthesis of reduced glutathione.
 MATERIAL AND METHODS: The effect of the inhibitor of de novo glutathione synthesis buthionine sulfoximine at a final concentration of 1 mM and/or the apoptosis inducer dexamethasone at a final concentration of 10 μM on the content of hydroxyl radical, protein-bound glutathione, carbonyl derivatives of proteins, oxidized tryptophan and bityrosine, ubiquitin, heat shock proteins 27 and 70, number of annexin V-positive cells and caspase-3 activity in Jurkat tumor cells was studied. Using the Shapiro–Wilk test, the normality of the distribution of indicators was assessed. Statistical hypotheses about the differences between the study groups were tested using the nonparametric Mann–Whitney test with a Bonferroni correction; correlation analysis was performed using the Spearman method at a significance level of p 0.05.
 RESULTS: In tumor cells of the Jurkat line, exposure to buthionine sulfoximine and dexamethasone was accompanied by a statistically significant decrease in the content of ubiquitin by 24% (p=0.004), protein-bound glutathione by 93% (p=0.003), oxidized tryptophan by 57% (p=0.003), and heat protein shock 70 by 56% (p=0.004), as well as an increase in the concentration of carbonyl derivatives of proteins by 53% (p=0.004), heat shock protein 27 by 104% (p=0.004), associated with an increase in the number of annexin-positive cells by 1296% (p=0.006) and caspase-3 activity by 258% relative to the values in intact cells. The relationship between an increase in the number of annexin-positive cells and caspase-3 activity with changes in the content of protein-bound glutathione, carbonylated proteins, oxidized tryptophan, ubiquitin and heat shock proteins 27 and 70 in tumor cells with simultaneous exposure to both buthionine sulfoximine and dexamethasone has been proven.
 CONCLUSION: Blocking de novo glutathione synthesis and stimulating apoptosis causes activation of reversible and irreversible oxidative modification of proteins and amino acids against the background of increased oxidative stress in Jurkat tumor cells.

SNO-DCA: A model for predicting S-nitrosylation sites based on densely connected convolutional networks and attention mechanism

Protein S-nitrosylation is a reversible oxidative reduction post-translational modification that is widely present in the biological community. S-nitrosylation can regulate protein function and is closely associated with a variety of diseases, thus identifying S-nitrosylation sites are crucial for revealing the function of proteins and related drug discovery. Traditional experimental methods are time-consuming and expensive; therefore, it is necessary to explore more efficient computational methods. Deep learning algorithms perform well in the field of bioinformatics sites prediction, and many studies show that they outperform existing machine learning algorithms. In this work, we proposed a deep learning algorithm-based predictor SNO-DCA for distinguishing between S-nitrosylated and non-S-nitrosylated sequences. First, one-hot encoding of protein sequences was performed. Second, the dense convolutional blocks were used to capture feature information, and an attention module was added to weigh different features to improve the prediction ability of the model. The 10-fold cross-validation and independent testing experimental results show that our SNO-DCA model outperforms existing S-nitrosylation sites prediction models under imbalanced data. In this paper, a web server prediction website: https://sno.cangmang.xyz/SNO-DCA/was established to provide an online prediction service for users. SNO-DCA can be available at https://github.com/peanono/SNO-DCA.

Abstract 12273: Crispr-Mutation of the Na + -k + Pump β1-Subunit Protects Against Cardiac Fibrosis and Heart Failure in Mice

Introduction: The Na+-K+ pump controls translocation of Na+ and K+ across the cell membrane which indirectly affects heart muscle contraction. Glutathionylation of the Na+-K+ pump’s β1 subunit (NKβ1) is a reversible post-translational oxidative modification, where a glutathione adduct forms a disulphide bond with the cysteine residue at site 45, leading to a reduction in pump activity. Using CRISPR/Cas9 technology, we created a transgenic mouse line (CRISPR-β1) that has a mutation in cysteine to serine at site 45 on NKβ1, causing insensitivity to β1-GSS. Hypothesis: We hypothesize that a mutation of the β1-GSS can inhibit transverse aortic constriction (TAC)-induced cardiac fibrosis. Methods and Results: TAC was induced in both the wild-type (WT) and CRISPR-NKβ1 mutated mice (CRISPR-β1) over a period of four weeks. Gene sequencing showed that cysteine 45 was replaced by serine, and immunoprecipitation of the free cysteine demonstrated that cysteine 45 was mutated in the β1 subunit of the CRISPR-β1 mice hearts. TAC significantly reduced cardiac function in the WT but not the CRISPR-β1 mice (ejection fraction: WT TAC: 36.24% ± 3.49%; CRISPR-β1 TAC: 57.5% ± 3.41%). Similarly, we found that the CRISPR-β1 mice were protected from cardiac hypertrophy, as evidenced by a significant reduction in cardiomyocytes size (WT TAC: 452.1 μm2 ± 30.74 μm2 ; CRISPR-β1 TAC: 271.1 μm 2 ± 11.0 μm 2 ). TAC significantly increased interstitial and perivascular fibrosis in the left ventricles of WT but not the CRISPR-β1 mice (interstitial: WT TAC: 18.4% ± 1.96%; CRISPR-β1 TAC: 5.2% ± 1.62%, perivascular: WT TAC: 42.6% ± 4.5%; CRISPR-β1 TAC: 22.01% ± 1.6%). Cardioprotective effects observed in the CRISPR-β1 mice were at least in part attributed to a significant reduction in the expression of ANP, BNP, Tgfb1, Fn1 and Ctgf genes. Conclusions: Our data indicate that CRISPR mutation of the Na+-K+ pump’s β1 subunit prevents the development of cardiac fibrosis and heart failure in a TAC-induced mouse model.

Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis

AimsS-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. ResultsExposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10–15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. ConclusionsAbundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.

Alcohol Binge Drinking Selectively Stimulates Protein S-Glutathionylation in Aorta and Liver of ApoE -/- Mice.

Background: Binge drinking has become the most common and deadly pattern of excessive alcohol use in the United States, especially among younger adults. It is closely related to the increased risk of cardiovascular disease. Oxidative stress as a result of ethanol metabolism is the primary pathogenic factor for alcohol-induced end organ injury, but the role of protein S-glutathionylation—a reversible oxidative modification of protein cysteine thiol groups that mediates cellular actions by oxidants—in binge drinking-associated cardiovascular disease has not been explored. The present study defines the effect of alcohol binge drinking on the formation of protein S-glutathionylation in a mouse model of atherosclerosis.Methods and Results: To mimic the weekend binge drinking pattern in humans, ApoE deficient (ApoE−/−) mice on the Lieber-DeCarli liquid diet received ethanol or isocaloric maltose (as a control) gavages (5 g/kg/day, 2 consecutive days/week) for 6 weeks. The primary alcohol-targeted organs (liver, brain), and cardiovascular system (heart, aorta, lung) of these two groups of the mice were determined by measuring the protein S-glutathionylation levels and its regulatory enzymes including [Glutaredoxin1(Grx1), glutathione reductase (GR), glutathione-S-transferase Pi (GST-π)], as well as by assessing aortic endothelial function and liver lipid levels. Our results showed that binge drinking selectively stimulated protein S-glutathionylation in aorta, liver, and brain, which coincided with altered glutathionylation regulatory enzyme expression that is downregulated Grx1 and upregulated GST-π in aorta, massive upregulation of GST-π in liver, and no changes in Grx1 and GST-π in brain. Functionally, binge drinking induced aortic endothelial cell function, as reflected by increased aortic permeability and reduced flow-mediated vasodilation.Conclusions: This study is the first to provide in vivo evidence for differential effects of binge drinking on formation of protein S-glutathionylation and its enzymatic regulation system in major alcohol-target organs and cardiovascular system. The selective induction of protein S-glutathionylation in aorta and liver is associated with aortic endothelial dysfunction and fatty liver, which may be a potential redox mechanism for the increased risk of vascular disease in human binge-drinkers.

S-glutathionylation of the Na+-K+ Pump: A Novel Redox Mechanism in Preeclampsia.

Reduced Na+-K+ pump activity is widely reported in preeclampsia and may be caused by a reversible oxidative modification that is a novel pathological feature of preeclampsia. This work aims to determine whether β 1 subunit (GSS-β 1) protein glutathionylation of the Na+-K + pump occurs in preeclampsia. The GSS-β1 of the Na+-K+ pump and its subunit expression in human placentas were compared between women with healthy pregnancies and women with preeclampsia. Human placental samples of pregnant women with preeclampsia (n = 11, mean gestational age 36.5 weeks) were used to examine the GSS-β 1 of the Na+-K+ pump, compared to healthy pregnancies (n = 11, mean gestational age 39 weeks).The potential pathogenetic role of GSS-β 1-mediated Na+-K+ pump dysfunction in preeclampsia was investigated. Protein expression of the β 1 subunit was unchanged in placentas from women with preeclampsia vs those with normotensive pregnancies. Preeclamptic placentas had a significantly increased GSS-β 1 of the Na+-K+ pump compared to those from healthy pregnancies, and this was linked to a decrease in α 1/β 1 subunit coimmunoprecipitation. The cytosolic p47phox nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase subunit and its coimmunoprecipitation with the α 1 Na+-K+ pump subunit was increased in preeclamptic placentas, thus implicating NADPH oxidase-dependent pump inhibition. The high level of β 1 pump subunit glutathionylation provides new insights into the mechanism of Na+-K+ pump dysfunction in preeclampsia.

The protease activity of human ATG4B is regulated by reversible oxidative modification

ABSTRACT Macroautophagy/autophagy plays a pivotal role in cytoplasmic material recycling and metabolic turnover, in which ATG4B functions as a “scissor” for processing pro-LC3 and lipidated LC3 to drive the autophagy progress. Mounting evidence has demonstrated the tight connection between ROS and autophagy during various pathological situations. Coincidentally, several studies have shown that ATG4B is potentially regulated by redox modification, but the underlying molecular mechanism and its relationship with autophagy is ambiguous. In this study, we verified that ATG4B activity was definitely regulated in a reversible redox manner. We also determined that Cys292 and Cys361 are essential sites of ATG4B to form reversible intramolecular disulfide bonds that respond to oxidative stress. Interestingly, we unraveled a new phenomenon that ATG4B concurrently formed disulfide-linked oligomers at Cys292 and Cys361, and that both sites underwent redox modifications thereby modulating ATG4B activity. Finally, increased autophagic flux and decreased oxidation sensitivity were observed in Cys292 and Cys361 double site-mutated cells under normal growth conditions. In conclusion, our research reveals a novel molecular mechanism that oxidative modification at Cys292 and Cys361 sites regulates ATG4B function, which modulates autophagy.Abbreviations: Air-ox: air-oxidation; ATG4B: autophagy related 4B cysteine peptidase; BCNU: 1,3-bis(2-chloroethyl)-1-nitrosourea; CBB: Coomassie Brilliant Blue; CM: complete medium; CQ: chloroquine; DTT: dithiothreitol; GSH: reduced glutathione; GSNO: S-nitrosoglutathione; GSSG: oxidized glutathione; HMW: high molecular weight; H2O2: hydrogen peroxide; NAC: N-acetyl-L-cysteine; NEM: N-ethylmaleimide; PE: phosphatidylethanolamine; PTM: post-translational modification; ROS, reactive oxygen species; WT: wild type

Diurnal oscillations of endogenous H2O2 sustained by p66Shc regulate circadian clocks.

Redox balance, an essential feature of healthy physiological steady states, is regulated by circadian clocks, but whether or how endogenous redox signalling conversely regulates clockworks in mammals remains unknown. Here, we report circadian rhythms in the levels of endogenous H2O2 in mammalian cells and mouse livers. Using an unbiased method to screen for H2O2-sensitive transcription factors, we discovered that rhythmic redox control of CLOCK directly by endogenous H2O2 oscillations is required for proper intracellular clock function. Importantly, perturbations in the rhythm of H2O2 levels induced by the loss of p66Shc, which oscillates rhythmically in the liver and suprachiasmatic nucleus (SCN) of mice, disturb the rhythmic redox control of CLOCK function, reprogram hepatic transcriptome oscillations, lengthen the circadian period in mice and modulate light-induced clock resetting. Our findings suggest that redox signalling rhythms are intrinsically coupled to the circadian system through reversible oxidative modification of CLOCK and constitute essential mechanistic timekeeping components in mammals.

Gender differences in albumin and ascorbic acid in the vitreous antioxidant system

Ascorbic acid is present at high concentrations in the vitreous and plays a central role in vitreous redox chemistry. Albumin is the main protein in the vitreous with antioxidant properties and occurs in different oxidation states, which can be used as redox indicators, but have not been studied in the vitreous. This study, therefore, addressed the vitreous redox state of cysteine-34 of albumin in relation to the ascorbic acid content, which has been suggested to exert a main function in detoxifying reactive oxygen in the vitreous.A total of 58 vitreous samples obtained from patients undergoing vitrectomy were analyzed for (i) human mercaptalbumin (HMA), the reduced thiol form; (ii) human non-mercaptalbumin1 (HNA1), a reversible oxidative modification with a disulfide at cysteine-34; and (iii) human non-mercaptalbumin2 (HNA2), a non-reversibly (highly) oxidized form of albumin; as well as (iv) ascorbic acid concentrations, to study possible relations. In addition, blood samples were taken to compare albumin redox state between plasma and the vitreous.Vitreous albumin showed greater variability in the redox state of cysteine-34 and a shift to the oxidized fractions compared to plasma albumin (P < 0.001). A strong positive relation was observed between the vitreous ascorbic acid concentrations and the reversibly oxidized form, HNA1 (P < 0.001), and a negative relation with the reduced form, HMA. Positive relations between ascorbic acid and HNA1 in the vitreous were stronger in men than in women. In contrast to HMA and HNA1, there was a distinct gender difference noted for the irreversibly oxidized form, HNA2. While males showed a positive relation between the vitreous ascorbic acid concentrations and HNA2, there was no correlation found with HNA2 in females.Our results support the view that ascorbic acid, by decreasing either directly or indirectly the concentrations of molecular oxygen, generates hydrogen peroxide, and that thiols, including HMA, are acting as antioxidants. This study for the first time provides evidence that vitreous albumin can be used as a marker molecule for the appearance of reactive oxygen species in the vitreous of patients undergoing vitrectomy. Moreover, it can be shown that there are gender differences in vitreous ascorbic acid and albumin concentrations as well as in oxidation state of vitreous albumin.

Effects of S-glutathionylation on the passive force-length relationship in skeletal muscle fibres of rats and humans.

This study investigated the effect of S-glutathionylation on passive force in skeletal muscle fibres, to determine whether activity-related redox reactions could modulate the passive force properties of muscle. Mechanically-skinned fibres were freshly obtained from human and rat muscle, setting sarcomere length (SL) by laser diffraction. Larger stretches were required to produce passive force in human fibres compared to rat fibres, but there were no fibre-type differences in either species. When fibres were exposed to glutathione disulfide (GSSG; 20mM, 15min) whilst stretched (at a SL where passive force reached ~ 20% of maximal Ca2+-activated force, denoted as SL20% max), passive force was subsequently decreased at all SLs in both type I and type II fibres of rat and human (e.g., passive force at SL20% max decreased by 12 to 25%). This decrease was fully reversed by subsequent reducing treatment with dithiothreitol (DTT; 10mM for 10min). If freshly skinned fibres were initially treated with DTT, there was an increase in passive force in type II fibres (by 10 ± 3% and 9 ± 2% in rat and human fibres, respectively), but not in type I fibres. These results indicate that (i) S-glutathionylation, presumably in titin, causes a decrease in passive force in skeletal muscle fibres, but the reduction is relatively smaller than that reported in cardiac muscle, (ii) in rested muscle in vivo, there appears to be some level of reversible oxidative modification, probably involving S-glutathionylation of titin, in type II fibres, but not in type I fibres.

A unique mechanism for thiolation of serum albumins by disulphide molecules.

Protein S-thiolation is a reversible oxidative modification that serves as an oxidative regulatory mechanism for certain enzymes and binding proteins with reactive cysteine residues. It is generally believed that the thiolation occurs at free sulphydryl group of cysteine residues. Meanwhile, despite the fact that disulphide linkages, serving structural and energetic roles in proteins, are stable and inert to oxidative modification, a recent study shows that the thiolation could also occur at protein disulphide linkages when human serum albumin (HSA) was treated with disulphide molecules, such as cystine and homocystine. A chain reaction mechanism has been proposed for the thiolation at disulphide linkages, in which free cysteine (Cys34) is involved in the reaction with disulphide molecules to form free thiols (cysteine or homocysteine) that further react with protein disulphide linkages to form the thiolated cysteine residues in the protein. This review focuses on the recent finding of this unique chain reaction mechanism of protein thiolation.

SAT-552 Oxidative Stress Induces Type 3 Deiodinase on Multiple Tissues in Disease: Implications to Nonthyroidal Illness Syndrome Pathophysiology

Imbalances in the redox status are involved in type 3 deiodinase (D3) induction in the non-thyroidal illness syndrome (NTIS). The mechanisms responsible for D3 dysfunction under systemic redox alterations in disease are poorly understood. Recently, the resemblance of D3 with peroxiredoxin-like proteins has been suggested. Objective: Evaluate the paths of redox alterations leading to D3 induction in liver, muscle, and brain in an animal model of NTIS. Methods: Male Wistar rats submitted to left anterior coronary artery occlusion (MI) were treated or not with N-acetylcysteine (NAC), an antioxidant that replenishes cysteine levels. Ten days post-MI animals were sacrificed and tissues collected. D3 activity was measured in liver, muscle, and brain. Total carbonyl and sulphydryl contents, as well as enzymatic and nonenzymatic oxidative parameters, were determined. Results: Compared to sham, D3 activity was increased in liver (P=0.002), muscle (P=0.03) and brain (P=0.01) in MI-placebo animals. All tissues from placebo group showed increased carbonyls, marker of oxidative damage to protein, and diminished sulphydryl levels, indicating cysteine and thiol consumption (all P=0.001). GSH/GSSG ratio was altered in all tissues (P=0.001). GSH is a critical antioxidant that depends on sulphydryl groups as a ready source of reducing equivalents. Liver and muscle had augmented glutathione peroxidase (GPx), glutathione reductase (GR) and thioredoxin reductase (TRx) levels (P=0.001). NAC prevented all the alterations described above. Conclusion: D3 induction in tissues correlates to MI-induced redox changes. NAC preventing D3 dysfunction indicates a role for altered cysteine/thiol in this process. The enzymatic profile suggests a functional regenerating GSH system. The set of these results adds to the present knowledge of NTIS physiopathology since they indicate a reversible oxidative post-transcriptional modification of D3 probably through a putative formation of mixed disulfides with cysteine and glutathione (glutathionylation process) that could be reduced by TRx as one of the causes for D3 induction in disease. The possibility that the thiols are being consumed to form disulphide bonds from augmented GSSG could also be considered. Antioxidant treatment prevents D3 dysfunction in multiple tissues, which probably contribute to avoiding NTIS.

Роль редокс-статуса и окислительной модификации белков в реализации апоптоза лимфоцитов крови человека в норме и при экспериментальном окислительном стрессе

The article presents the data on the level of reactive oxygen species, irreversible and reversible oxidative modification of proteins, the state of glutathione system components, activity of catalase, and implementation of apoptosis in intact blood lymphocytes. By modulating oxidative stress in vitro, we studied the role of hydrogen peroxide in the final concentration of 0.5 mM in apoptosis implementation. We also evaluated the contribution of the glutathione system components and irreversible and reversible oxidative modification of proteins to molecular mechanisms of apoptosis in blood lymphocytes. We showed that apoptotic death in blood lymphocytes under experimental oxidative stress was characterized by activation of protein glutathionylation and carbonylation. The obtained findings prove that protein molecules are potential redox-dependent targets for regulating cell metabolism in blood lymphocytes.

Critical Role of Flavin and Glutathione in Complex I-Mediated Bioenergetic Failure in Brain Ischemia/Reperfusion Injury.

Ischemic brain injury is characterized by 2 temporally distinct but interrelated phases: ischemia (primary energy failure) and reperfusion (secondary energy failure). Loss of cerebral blood flow leads to decreased oxygen levels and energy crisis in the ischemic area, initiating a sequence of pathophysiological events that after reoxygenation lead to ischemia/reperfusion (I/R) brain damage. Mitochondrial impairment and oxidative stress are known to be early events in I/R injury. However, the biochemical mechanisms of mitochondria damage in I/R are not completely understood. We used a mouse model of transient focal cerebral ischemia to investigate acute I/R-induced changes of mitochondrial function, focusing on mechanisms of primary and secondary energy failure. Ischemia induced a reversible loss of flavin mononucleotide from mitochondrial complex I leading to a transient decrease in its enzymatic activity, which is rapidly reversed on reoxygenation. Reestablishing blood flow led to a reversible oxidative modification of mitochondrial complex I thiol residues and inhibition of the enzyme. Administration of glutathione-ethyl ester at the onset of reperfusion prevented the decline of complex I activity and was associated with smaller infarct size and improved neurological outcome, suggesting that decreased oxidation of complex I thiols during I/R-induced oxidative stress may contribute to the neuroprotective effect of glutathione ester. Our results unveil a key role of mitochondrial complex I in the development of I/R brain injury and provide the mechanistic basis for the well-established mitochondrial dysfunction caused by I/R. Targeting the functional integrity of complex I in the early phase of reperfusion may provide a novel therapeutic strategy to prevent tissue injury after stroke.

A conserved R type Methionine Sulfoxide Reductase reverses oxidized GrpEL1/Mge1 to regulate Hsp70 chaperone cycle

Cells across evolution employ reversible oxidative modification of methionine and cysteine amino acids within proteins to regulate responses to redox stress. Previously we have shown that mitochondrial localized methionine sulfoxide reductase (Mxr2) reversibly regulates oxidized yeast Mge1 (yMge1), a co-chaperone of Hsp70/Ssc1 to maintain protein homeostasis during oxidative stress. However, the specificity and the conservation of the reversible methionine oxidation mechanism in higher eukaryotes is debatable as human GrpEL1 (hGrpEL1) unlike its homolog yMge1 harbors two methionine residues and multiple cysteines besides the mammalian mitochondria hosting R and S types of Mxrs/Msrs. In this study, using yeast as a surrogate system, we show that hGRPEL1 and R type MSRs but not the S type MSRs complement the deletion of yeast MGE1 or MXR2 respectively. Our investigations show that R type Msrs interact selectively with oxidized hGrpEL1/yMge1 in an oxidative stress dependent manner, reduce the conserved hGrpEL1-Met146-SO and rescue the Hsp70 ATPase activity. In addition, a single point mutation in hGrpEL1-M146L rescues the slow growth phenotype of yeast MXR2 deletion under oxidative duress. Our study illustrates the evolutionarily conserved formation of specific Met-R-SO in hGrpEL1/yMge1 and the essential and canonical role of R type Msrs/Mxrs in mitochondrial redox mechanism.

Abstract 21314: Glutaredoxin-1: An Endothelial Protector in Hyperlipidemia

Background: Hypercholesterolemia (HC) dominates the risk factors for atherosclerosis. Sustained oxidative stress is a central mechanism driving endothelial dysfunction (ED) in HC. But the underlying redox mechanism remains unclear. Protein S-glutathionylation, a reversible oxidative modification of cysteine residues is tightly controlled by a specific de-glutathionylation enzyme: glutaredoxin-1 (Glrx-1), which thereby plays a critical role in redox regulation of cellular function. Methods and Results: The protein levels of Glrx1 in aorta and lung tissues are inversely correlated with circulating cholesterol in WT and ApoE -/- mice. Overexpression of Glrx1 in ApoE -/- mice attenuates HC-induced ED as measured by flow-mediated dilation of femoral artery using a new vascular imaging method with optical coherence tomography technique, and impaired eNOS function as evidenced by increased eNOS monomer to dimer ratio and suppressed phosphorylation of vasodilator-stimulated phosphoprotein in the aorta. In HAECs oxidized LDL promotes downregulation of Glrx1 without altering its mRNA level. The consequent elevated PrS-SG inactivates Akt/eNOS and stimulates degradation of Rac1, a key regulator of eNOS. All of these deteriorate effects of oxLDL are prevented by adenoviral overexpression of Glrx1. Conclusions: HC-induced downregulation of Glrx1 promotes ED by redox inactivation of eNOS and its upstream regulators: Akt1 and Rac1.

Protein glutathionylation protects wheat (Triticum aestivum Var. Sonalika) against Fusarium induced oxidative stress

Fusarium induced oxidative stress could be recovered by reversible protein oxidative modification through the process of glutathionylation in co-stressed (low-dose (50 μM) Cd2+ pre-treatment followed by Fusarium inoculation) wheat seedlings. Co-stressed seedlings showed low disease severity index as compared to Fusarium infected seedlings. A reduced level of hydrogen peroxide (H2O2) and carbonyl contents due to irreversible protein oxidation were observed in co-stressed seedlings as compared to Fusarium infected seedlings. Further, a comparative biochemical assay showed an enhanced glutathione content in co-stressed tissues as compared to Fusarium infected tissues. In an investigation, reduced glutathione pre-coated agarose gel beads were used to pull down proteins having affinity with GSH. Fructose-1, 6-bisphosphate aldolase and 3-Phosphoglycerate kinase were observed to be co-existed in co-stressed seedlings when analysed by LC-MS/MS after being processed through protein-pull assay. Co-stressed tissues showed an enhanced free protein thiol content as compared to Fusarium infected tissues. The ratio of free thiol to thiol disulfides was also observed to be increased in co-stressed tissues as compared to Fusarium infected tissues. In contrast, the quantitative assay by Ellman's reagent and qualitative analysis by diagonal gel electrophoresis showed enhanced protein thiol disulfides in Fusarium infected tissues as compared to co-stressed tissues. Further, glutaredoxin, responsible for the reverse reduction of proteins was observed to be enhanced in co-stressed tissues as compared to Fusarium infected tissues. Thus, a low dose Cd2+ triggered glutathionylation is suggestive of offering tolerance against Fusarium induced oxidative stress and protects target proteins from irreversible modification and permanent damage in wheat.

The redox mechanism for vascular barrier dysfunction associated with metabolic disorders: Glutathionylation of Rac1 in endothelial cells

BackgroundOxidative 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 resultsIn 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. ConclusionsGlutathionylation and inactivation of Rac1 in endothelial cells represent a novel redox mechanism of vascular barrier dysfunction associated with metabolic disorders.