Glutathionylation Research Articles

P 140 - GSTP expression influences the metabolism and redox of cellular glutathione

Recently we described the role of glutathione-S-transferase P-1 (GSTP) expression in regulating Nrf2 transcriptional activity by protein-protein interaction. A functional interplay between Nrf2 and GSTP to control glutathione (GSH) biosynthesis, detoxification function and signal transduction through protein Cys glutathionylation can be hypothesized. To explore such interplay the effect of GSTP gene manipulation on the cellular levels and redox of main thiols in murine embryonic fibroblasts (MEFs) was investigated. GSTP knockout MEFs showed increased levels of all the identified forms of thiols that included GSH, GSSG, GSSP, Cys, Cyss, CyssP, HCys, HCyss, HCyssP, γ-GluCys and CysGly. The exposure to the Se-organic thiol-peroxidase compound Ebselen, further and massively up-regulated GSH, Cys and HCys and their corresponding disulfides in GSTP-KO, but not in wild-type (WT) cells. GSSP (protein glutathionylation), and to a lower extent HCyssP, increased after treatment with Se-organic compounds only in WT cells (confirmed by both HPLC and immunoblot analysis). These findings confirms the role of GSTP as a feedback component of Nrf2 pathway (control of GSH levels) and identify with an unbiased approach the role of this gene in mediating protein glutathionylation signalling during the exposure to Se-organic thiol-peroxidases.

Does oxidative stress play any role in diabetic cataract formation? ----Re-evaluation using a thioltransferase gene knockout mouse model

Oxidative stress is a known risk factor in senile cataract formation. In recent years, it has been suggested that oxidation may also be associated with cataract induced by hyperglycemia, but this concept has not been well examined or validated. Since thioltransferase (TTase) is one of the key enzymes that regulates redox homeostasis and protects against oxidative stress in the lens, we have used TTase gene knockout (KO) mice as a model to examine this new concept. Lenses from 4 months old TTase KO and wild-type (WT) mice were incubated in TC199 culture medium containing 30 mM glucose for 48 h. Each lens was assessed for opacity, graded by LOCSII system, and the wet weight was recorded after which it was homogenized in lysis buffer and analyzed for water-soluble protein and free glutathione (GSH). In vivo studies were carried out using 4 months old TTase KO and WT mouse groups. Each mouse received two consecutive days of intraperitoneal streptozotozin (STZ) injections to induce diabetes. The lenses were examined weekly for 4 weeks using a slit-lamp biomicroscope, and then extracted and analyzed for levels of GSH, water-soluble protein, ATP and protein-GSH mixed disulfide (PSSG). TTase KO lenses cultured in high glucose developed a mild cortical opacity but slightly more than that of the WT lenses. Both groups had similar contents of soluble proteins and GSH. Exposure to high glucose did not change the soluble protein level but did suppress GSH by 20% in lenses with or without TTase. STZ-induced diabetic KO mice also developed a higher degree of mild cortical lens opacity compared to that of the diabetic WT controls. Similar 15–20% losses in lens GSH and ATP were found after one-month induced diabetes in WT and KO mice. There was a 20% greater amount of PSSG in the lenses of TTase KO than the WT control. Under diabetic condition, both groups displayed more glutathionylated proteins in the beta-actin (42 kDa) and lens crystallin proteins (18–22 kDa) regions, and some additional modified proteins at 15–17 kDa and 60–70 kDa, with a total 20–30% PSSG increment in both groups. In conclusion, we have found that hyperglycemia induced some oxidative stress-associated biochemical changes with mild lens opacity in both WT and KO mice. However, these changes were only marginally higher in the TTase KO mouse than that of the WT control, suggesting that TTase deletion may only play a minor role in the early stage of hyperglycemia-induced cataract formation in the mice.

Dual-Specificity Phosphatase 4 Overexpression in Cells Prevents Hypoxia/Reoxygenation-Induced Apoptosis via the Upregulation of eNOS.

Mitogen-activated protein kinases (MAPKs) signaling cascades regulate several cellular functions, including differentiation, proliferation, survival, and apoptosis. The duration and magnitude of phosphorylation of these MAPKs are decisive determinants of their physiological functions. Dual-specificity phosphatases exert kinetic control over these signaling cascades. Previously, we demonstrated that DUSP4−/− hearts sustain a larger infarct and have poor functional recovery, when isolated hearts were subjected to ischemia/reperfusion. Uncontrolled p38 activation and upregulation of Nox4 expression are the main effectors for this functional alteration. Here, dual-specificity phosphatase 4 (DUSP4) overexpression in endothelial cells was used to investigate the role of DUSP4 on the modulation of reactive oxygen species (ROS) generation and vascular function, when cells were subjected to hypoxia/reoxygenation (H/R) insult. Immunostaining with cleaved caspase-3 revealed that DUSP4 overexpression prevents caspase-3 activation and apoptosis after H/R. The beneficial effects occur via modulating p38 activity, increased NO bioavailability, and reduced oxidative stress. More importantly, DUSP4 overexpression upregulates eNOS protein expression (1.62 ± 0.33 versus 0.65 ± 0.16) during H/R-induced stress. NO is a critical small molecule involved in regulating vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. The level of NO generation determined using DAF-2 fluorescence demonstrated that DUSP4 overexpression augments NO production and thus improves vascular function. The level of superoxide generated from cells after being subjected to H/R was determined using dihydroethidium-HPLC method. The results suggested that DUSP4 overexpression in cells decreases H/R-induced superoxide generation (1.56 ± 0.14 versus 1.19 ± 0.05) and thus reduces oxidant stress. This also correlates with the reduction in the total protein S-glutathionylation, an indicator of protein oxidation. These results further support our hypothesis that DUSP4 is an antioxidant gene and a key phosphatase in modulating MAPKs, especially p38, during oxidative stress, which regulates ROS generation and eNOS expression and thus protects against oxidant-induced injury or apoptosis. Overall, DUSP4 may serve as an excellent molecular target for the treatment of ischemic heart disease.

Modulation of the specific glutathionylation of mitochondrial proteins in the yeast Saccharomyces cerevisiae under basal and stress conditions.

The potential biological consequences of oxidative stress and changes in glutathione levels include the oxidation of susceptible protein thiols and reversible covalent binding of glutathione to the -SH groups of proteins by S-glutathionylation. Mitochondria are central to the response to oxidative stress and redox signaling. It is therefore crucial to explore the adaptive response to changes in thiol-dependent redox status in these organelles. We optimized the purification protocol of glutathionylated proteins in the yeast Saccharomyces cerevisiae and present a detailed proteomic analysis of the targets of protein glutathionylation in cells undergoing constitutive metabolism and after exposure to various stress conditions. This work establishes the physiological importance of the glutathionylation process in S. cerevisiae under basal conditions and provides evidence for an atypical and unexpected cellular distribution of the process between the cytosol and mitochondria. In addition, our data indicate that each oxidative condition (diamide, GSSG, H2O2, or the presence of iron) elicits an adaptive metabolic response affecting specific mitochondrial metabolic pathways, mainly involved in the energetic maintenance of the cells. The correlation of protein modifications with intracellular glutathione levels suggests that protein deglutathionylation may play a role in protecting mitochondria from oxidative stress. This work provides further insights into the diversity of proteins undergoing glutathionylation and the role of this post-translational modification as a regulatory process in the adaptive response of the cell.

Upregulated glutathione transferase omega-1 correlates with progression of urinary bladder carcinoma

ABSTRACTObjectives: Newly discovered glutathione transferase omega 1 (GSTO1-1) plays an important role in the glutathionylation cycle, a significant mechanism of protein function regulation. GSTO1-1 expression pattern has not been studied in transitional cell carcinoma (TCC), as yet.Methods: A total of 56 TCC tumor and corresponding non-tumor specimens were investigated. Glutathione content and thioltransferase activity were measured spectrophotometrically. Protein-glutathione mixed disulfides were measured fluorimetrically. GSTO1-1 expression was determined by immunoblot and qPCR. Immunoprecipitation with GSTO1-1 antibody was followed by immunoblot using anti-GSTO1, GSTP1, c-Jun, JNK, Akt, phospho-Akt, and ASK1 antibody, while for the total S-glutathionylation levels non-reducing electrophoresis was performed.Results: The contents of reduced glutathione and thioltransferase activity were significantly increased in tumor compared to non-tumor tissue. The increased GSTO1 expression in tumor tissue showed clear correlation with grade and stage. However, decreased total protein glutathionylation level in tumor compared to non-tumor samples was found. Immunoprecipitation has shown an association of GSTO1-1 with GSTP1, Akt, phospho-Akt, and ASK1 proteins.Conclusions: GSTO1 deglutathionylase activity suggests its potential important role in redox perturbations present in TCC. Increased GSTO1-1 expression might contribute to TCC development and/or progression supporting the notion that GSTO1-1 may be a promising novel cancer target.

Protein S-glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex

Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O2•-/ H2O2 emission from pyruvate dehydrogenase (Pdh) is altered by S-glutathionylation. Glutathione disulfide (GSSG) amplified O2•-/ H2O2 production by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-glutathionylation. S-glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NAD+ lowering O2•-/ H2O2 production. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O2•-/ H2O2 production in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-glutathionylation.

Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances.

Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation.

Protein Glutathionylation in the Pathogenesis of Neurodegenerative Diseases.

Protein glutathionylation is a redox-mediated posttranslational modification that regulates the function of target proteins by conjugating glutathione with a cysteine thiol group on the target proteins. Protein glutathionylation has several biological functions such as regulation of metabolic pathways, calcium homeostasis, signal transduction, remodeling of cytoskeleton, inflammation, and protein folding. However, the exact role and mechanism of glutathionylation during irreversible oxidative stress has not been completely defined. Irreversible oxidative damage is implicated in a number of neurological disorders. Here, we discuss and highlight the most recent findings and several evidences for the association of glutathionylation with neurodegenerative diseases and the role of glutathionylation of specific proteins in the pathogenesis of neurodegenerative diseases. Understanding the important role of glutathionylation in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions.

Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1).

Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

160 - Improved Redox State Increases Aged Skeletal Muscle Performance

Muscle redox status regulates diverse aspects of skeletal muscle function, including mitochondrial energetics and contractile performance. In aging skeletal muscle mitochondrial dysfunction contributes to altered redox homeostasis creating a feedforward cycle. Despite this, the role of redox status and its dynamic nature has not been adequately explored as it relates to the decline in skeletal muscle performance with age. To identify mechanisms linking redox status to aged skeletal muscle function we used two long-term treatment strategies to reduce oxidative stress and restore redox homeostasis in aged mice. The first treatment strategy used a short mitochondrial-targeted peptide, elamipretide (SS-31), that our lab has previously shown to acutely improve mitochondrial bioenergetics and muscle fatiguability while also enhancing glutathione redox potential in aged mice. Treating with SS-31 (3 mg/kg) for 8 weeks recapitulates all of the acute improvements while also increasing resting ATP levels and improving treadmill performance compared to both pre-treatment baseline performance and age matched controls. Increased performance was associated with improved redox status as indicated by lower 4-hydroxynonenal-protein adducts and reversal of age-related changes in protein S-glutathionylation. Our second treatment strategy used the powerful antioxidant astaxanthin delivered through the chow of mice at a target dose of 300 mg/kg/day. Astaxanthin treatment paired with exercise training for 8 weeks in aged animals showed a significant improvement in skeletal muscle specific force compared to untreated controls and a trend toward improved treadmill performance. Taken together these results suggest that altering skeletal muscle oxidative stress to restore redox homeostasis is a viable strategy for improving muscle function with age.

378 - Overexpression of Glutaredoxin Averts F-Actin Loss in Spines of Primary Cortical Neurons Derived from Mouse Model of Alzheimer's Disease

Oxidative stress is known to be one of the major contributors towards the pathogenesis of Alzheimer’s disease (AD) potentially leading to amyloid plaques, neurofibrillary tangles and loss of dendritic spine morphology. As a marker for ongoing oxidative stress, we observed increased levels of reactive oxygen species (ROS) in primary cortical neurons from APPSwe/PS1ΔE9 (APP/PS1) transgenic mouse model of AD, compared to wild type littermates. Thiol oxidation and protein glutathionylation are important targets during oxidative stress, resulting in perturbation of cellular thiol homeostasis. Glutaredoxins and other thiol disuflide oxidoreductases are important enzymes involved in reversing protein thiol oxidation in the brain. We hypothesized that overexpressing Grx1 (glutaredoxin) may partially reverse the adverse effects of oxidative stress in AD neurons. We overexpressed human glutaredoxin using AAV6 virus and confirmed overexpression of glutaredoxin using biochemical and immunostaining methods. Actin, an important neuronal cytoskeletal filament, is important for maintenance of spine morphology and function. It is also a target of cellular oxidation undergoing gluathionylation, leading to its conversion from the functional fibrillar form (F-actin) to the globular (G-actin) form. Other studies from our laboratory have shown increased actin oxidation in early stages of AD-related pathology leading to synaptic dysfunction using a combination of cellular, in vivo and post-mortem human brain samples. We also observed loss of F-actin in APP/PS1 neurons compared to their wild-type littermates, as indicated by reduced phalloidin staining. Importantly, overexpression of Grx1 reversed this F-actin loss, and preserved the F-actin microarchitecture in spines. Our results suggest that thiol oxidation events, such as protein glutathionylation including that of actin, play an important role in the synaptic structural and functional deficits seen in AD.

191 - Redox-Active Mn Porphyrins, MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+ But Not Redox-Inert MnTBAP3-Suppress Tumor Growth in an Environment Where H2O2 Is Produced

Cationic Mn(III) N-substituted pyridylporphyrins (MnPs), such as MnTnBuOE-2-PyP5+ and MnTE-2-PyP5+, initially optimized as powerful mimics of superoxide dismutase, also impact cellular redox-based signaling pathways, most so redox sensitive thiol bearing proteins such as NF-κB, whereby affecting cellular apoptotic and proliferative pathways. Such actions lead to S-glutathionylation of proteins and their subsequent inactivation. In addition to MnP catalyst, both H2O2 and glutathione are required for such process, which includes initial oxidation of Mn porphyrin by H2O2, followed by oxidation of protein thiols by high-valent MnP in a GPx-like fashion. While tumors already have higher H2O2 levels, they are sensitive to any further increase in H2O2 which strategy is used to induce tumor killing. Mn porphyrins do not differentiate tumor from normal tissue and thereby interact at identical rate constants with small reactive species and proteins in both types of tissues. Yet, the yield of reactions depends upon the MnPs levels and levels of reactive species. In the case of NF-kB, the higher are the levels of reactants (MnPs and reactive species) the larger would be the yield of its oxidative damage. Consequently, the larger would be the inhibition of NF-κB activation and in turn the suppression of anti-apoptotic pathways (for involvement of NF-κB pathways in MnP/Asc anticancer effects see Tovmasyan et al abstract on Redox Proteomics). Using LC-MS/MS we showed the preferential accumulation of 3 different MnPs in tumor over normal tissue in a 4T1 flank mouse tumor model. In such model, the already high tumor H2O2 levels were increased with either radiation (RT) or ascorbate or both. The higher tumor accumulation of MnTE-2-PyP5+ relative to MnTnBuOE-2-PyP5+ resulted in larger tumor suppression. Regardless of much higher tumor vs normal tissue accumulation, the redox-inert MnTBAP3- sensitized tumor neither to ascorbate nor to radiation. As anticipated the tumor GSSG levels were increased only when redox-active MnTE-2-PyP5+, but not MnTBAP3-, was combined with RT/Ascorbate treatment. Grant support NIH 1R03-NS082704-01, 5-P30-CA14236-29, CNPq and CAPES from Brazil.

318 - FXYD1 Protects Against Redox-Dependent Endothelial Dysfunction and Angiotensin II-Induced Hypertension

Background Protein S-glutathionylation (GSS) can increase the generation of reactive oxygen species and cause eNOS uncoupling, thereby reducing nitric oxide (NO) bioavailability. Thus, mechanisms that protect against eNOS-GSS could improve vascular function and cardiovascular health. We hypothesised that caveolar co-localisation of FXYD1 with eNOS may result in functional interaction and ‘protective’ redox signalling in the vasculature. Methods and results: Blood pressure (MABP), measured by arterial cannulation was not different between anaesthetised FXYD1 wildtype (WT) and knockout (KO) mice (MABP (mmHg): WT 88.7±1.3 vs. KO 91.1 ± 2.2, n=7); confirmed by telemetry in conscious mice. Endothelium-dependent vasodilation, determined by MABP changes after increasing bolus doses of bradykinin acetate, was impaired in KO mice (max ΔmmHg at 10 µg/kg: KO -9.6±1.4 vs. WT -16.6±1.7, P Conclusions We have identified a functional partnership of FXYD1 with eNOS, protecting this vital enzyme from GSS-mediated eNOS uncoupling. This could lead to novel treatment options with recombinant FXYD1 for redox-dependent vascular disease.

240 - Decreased Intracellular Glutathione in Peripheral Neutrophils Decreases Chemotactic Behaviour towards Interleukin 8 and FMLP

The innate immune system is the first line of defence against invading microorganisms, and neutrophils are the most abundant leukocyte and the first to arrive at a site of infection. Periodontitis is a highly prevalent chronic non communicable disease and in its most severe form is the sixth most prevalent human disease, affecting 11.2% of the world’s population. It has been associated various systemic diseases and conditions, such as diabetes. In periodontitis it is known that neutrophils have aberrant chemotactic behaviour (Roberts et al PMID: 25360483), as well as increased reactive oxygen species production whilst resting (hyper activity) and when stimulated (hyper-reactivity) (Matthews et al PMID: 17223966). This combination may lead to enhanced tissue damage in the periodontal lesion. We have also shown that levels of glutathione in the periodontal lesion (Grant et al PMID: 19968740) and within circulating peripheral neutrophils (Dias et al PMID: 23826097) are decreased, demonstrating a loss in redox buffering and capacity. Previous studies have shown the importance of glutathionylation of actin in chemotactic movement of neutrophils (Sakai et al 2012 PMID: 23159440). With the long term aim to examine how diminished glutathione levels in periodontitis effect chemotaxis, we examined the effect of glutathione modulation by buthioninesulfoximine (BSO), N-acetylcysteine (NAC), bis-chloroethylnitrosourea (BCNU) and 1-chloro-2,4-dinitrobenzene (CDNB) on neutrophil intracellular glutathione, viability and chemotaxis towards interleukin 8 and N-formylmethionyl-leucyl-phenylalanine (fMLP). BSO, BCNU and CDNB decreased the chemotaxis and glutathione levels in peripheral neutrophils isolated from healthy donors. Future studies will translate this work into a periodontal cohort and examine protein glutathionylation by mass spectrometry.

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.

Application of Mitochondria-Targeted Pharmaceuticals for the Treatment of Heart Disease.

Mitochondria fulfill the massive energy demands of the human heart through oxidative phosphorylation (OXPHOS) which couples nutrient oxidation and the reduction of molecular oxygen (O2) to the phosphorylation of ADP. Reactive oxygen species (ROS) are also generated during OXPHOS which can be damaging at high levels but serve as secondary messengers when produced in a controlled manner. Here, I review how disruption of control over mitochondrial ROS production can lead to the pathogenesis of a range of cardiovascular diseases (CVD) including decompensated left ventricular hypertrophy, alcoholic and diabetic hypertrophy, myocardial infarction (MI), ischemic-reperfusion injury (IR), and heart failure. In particular I focus on the function of protein S-glutathionylation (PGlu) reactions, a rapid and reversible redox signaling mechanism that involves the conjugation and removal of glutathione from cysteine switches, in the modulation of ROS production in myocardial mitochondria and how these reactions become deregulated in heart disease. I also discuss the use of mitochondria penetrating antioxidants in the treatment of heart disease. I propose that heart disease related to deregulated PGlu reactions can be treated with a novel and hypothetical class of mitochondria penetrating reduced glutathione (GSH) molecules called MitoGSH. This synthetic form of GSH can be tagged with either SS peptides or triphenylphosphonium ions to ensure accumulation in mitochondria which could restore glutathione levels and preserve redox buffering networks. Mitochondria penetrating antioxidants have been shown to be efficient at restoring mitochondrial antioxidant defense in CVD. However, CVD and various other disorders are associated with a depletion of GSH pools. Use of mitochondria-targeted GSH analogs could serve as a more efficient means of treating heart disease since it would allow for the direct restoration of GSH levels and preserve mitochondrial redox buffering and signaling capacity.

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.

Ablation of Glutaredoxin-1 Modulates House Dust Mite-Induced Allergic Airways Disease in Mice.

Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.

Prediction of glutathionylation sites in proteins using minimal sequence information and their experimental validation

S-glutathionylation of proteins plays an important role in various biological processes and is known to be protective modification during oxidative stress. Since, experimental detection of S-glutathionylation is labor intensive and time consuming, bioinformatics based approach is a viable alternative. Available methods require relatively longer sequence information, which may prevent prediction if sequence information is incomplete. Here, we present a model to predict glutathionylation sites from pentapeptide sequences. It is based upon differential association of amino acids with glutathionylated and non-glutathionylated cysteines from a database of experimentally verified sequences. This data was used to calculate position dependent F-scores, which measure how a particular amino acid at a particular position may affect the likelihood of glutathionylation event. Glutathionylation-score (G-score), indicating propensity of a sequence to undergo glutathionylation, was calculated using position-dependent F-scores for each amino-acid. Cut-off values were used for prediction. Our model returned an accuracy of 58% with Matthew’s correlation-coefficient (MCC) value of 0.165. On an independent dataset, our model outperformed the currently available model, in spite of needing much less sequence information. Pentapeptide motifs having high abundance among glutathionylated proteins were identified. A list of potential glutathionylation hotspot sequences were obtained by assigning G-scores and subsequent Protein-BLAST analysis revealed a total of 254 putative glutathionable proteins, a number of which were already known to be glutathionylated. Our model predicted glutathionylation sites in 93.93% of experimentally verified glutathionylated proteins. Outcome of this study may assist in discovering novel glutathionylation sites and finding candidate proteins for glutathionylation.

Regulation of DJ-1 by Glutaredoxin 1 in Vivo: Implications for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH, and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in Caenorhabditis elegans; however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx(-/-) mice we provide show that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents, and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homologue in a C. elegans in vivo model of PD. Therefore, our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.