Redox Modulation of Endothelial Nitric Oxide Synthase by Glutaredoxin-1 through Reversible Oxidative Post-Translational Modification

Chen, Chun-An; Pascali, Francesco De; Basye, Ariel; Hemann, Craig; Zweíer, Jay L.

doi:10.1021/bi400404s

Cited by 59 publications

(65 citation statements)

References 53 publications

(171 reference statements)

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“…The procedure for the immunoblotting was followed as previously described [28, 30, 34]. Samples were first separated on either an 8% or 12% Tris-glycine polyacrylamide gel, and then electrophoretically transferred to a nitrocellulose membrane.…”

Section: Methodsmentioning

confidence: 99%

Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

Barajas-Espinosa

Basye

Angelos

2015

Free Radical Biology and Medicine

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Over-activation of p38 is implicated in many cardiovascular diseases (CVDs), including myocardial infarction, hypertrophy, heart failure, and ischemic heart disease. Numerous therapeutic interventions for CVDs have been directed towards the inhibition of the p38 mitogen-activated protein kinase activation that contributes to the detrimental effect after ischemia/reperfusion (I/R) injuries. However, the efficacy of these treatments is far from ideal as they lack specificity and are associated with high toxicity. Previously, we demonstrated that N-acetyl cysteine (NAC) pre-treatment up-regulates DUSP4 expression in endothelial cells, regulating p38 and ERK1/2 activities, thus providing a protective effect against oxidative stress. Here, endothelial cells under hypoxia/reoxygenation (H/R) insult and isolated heart I/R injury were used to investigate the role of DUSP4 on the modulation of the p38 pathway. In rat endothelial cells, DUSP4 is time-dependently degraded with H/R (0.25 ± 0.07 fold change of control after 2 h H/R). Its degradation is closely associated with hyper-phosphorylation of p38 (2.1 ± 0.36 fold change) and cell apoptosis, as indicated by the increase in cells immunopositive for cleaved caspase-3 (12.59% ± 3.38%) or TUNEL labeling (29.46% ± 3.75%). The inhibition of p38 kinase activity with 20 µM SB203580 during H/R prevents H/R-induced apoptosis, assessed via TUNEL (12.99% ± 1.89%). Conversely, DUSP4 gene silencing in endothelial cells augments their sensitivity to H/R-induced apoptosis (45.81% ± 5.23%). This sensitivity is diminished via the inhibition of p38 activity (total apoptotic cells drop to 17.47% ± 1.45%). Interestingly, DUSP4 gene silencing contributes to the increase in superoxide generation from cells. Isolated Langendorff-perfused mouse hearts were subjected to global I/R injury. DUSP4−/− hearts had significantly larger infarct size than WT. The increase in I/R-induced infarct in DUSP4−/− mice significantly correlates with reduced functional recovery (assessed by: RPP%, LVDP%, HR%, and dP/dtmax) as well as lower CF% and a higher initial LVEDP. From immunoblotting analysis, it is evident that p38 is significantly over-activated in DUSP4−/− mice after I/R injury. The activation of cleaved caspase-3 is seen in both WT and DUSP4−/− I/R hearts. Infusion of a p38 inhibitor prior to ischemia and during the reperfusion improves both WT and DUSP4−/− cardiac function. Therefore, the identification of p38 kinase modulation by DUSP4 provides a novel therapeutic target for oxidant-induced diseases, especially myocardial infarction.

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Section: Methodsmentioning

confidence: 99%

Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

Barajas-Espinosa

Basye

Angelos

2015

Free Radical Biology and Medicine

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“…Grx1 is the major deglutathionylating enzyme with selective preference for PrS-SG (19). The catalytic efficiency of Grx1 is much higher for PrS-SG compared with Trx (41).…”

Section: Journal Of Biological Chemistry 23385mentioning

confidence: 99%

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction

Kundumani‐Sridharan

Hilgers

Owens

et al. 2016

Journal of Biological Chemistry

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Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress.

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“…As a sulfhydryl-disulfide bond oxidoreductase -by catalyzing the reduction of protein disulfide bonds, mixed disulfide bonds, and of glutathione protein (Pr-S-S-G) -the Grx1 enzyme regulates post-translational modifications of thiol-containing proteins including those in JNK/c-Jun signaling pathways (Allen and Mieyal, 2012;Bansal et al, 2013;Chen et al, 2013a;Lu and Holmgren, 2014). One group recently reported that Grx1-mediated uncoupling of glutathione from IKKb Cys-179 plays a key role in diabetic complications by regulating the autocrine and paracrine actions of proinflammatory cytokines (Shelton et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

“…It is also involved in the regulation of various cellular functions, maintains a stable cellular redox state, produces a reducing environment within the cell under varying environmental conditions, and is involved in the pathogenesis of many diseases including diabetes and heart disease (Lekli et al, 2010;Chen et al, 2013a;De Benedetto et al, 2014;Du et al, 2014). In a physiological state of the cytoplasm, NADPH, GSH, GR, and Grx1 constitute the Grx1redox system, which reduces the oxidation state of proteinglutathione mixed disulfides (PSSG) in vivo and regulates post-translational modifications of thiol-containing proteins.…”

Section: Introductionmentioning

confidence: 99%

Cardiac damage and dysfunction in diabetic cardiomyopathy are ameliorated by Grx1

Gao

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Glutaredoxin 1 (Grx1) has been found to be an important endogenous antioxidant enzyme closely related to the pathogenesis of diabetes and cardiovascular diseases caused by oxidative stress. In this study, the functional changes of the Grx1 redox system in blood of hyperglycemic patients were examined. Furthermore, using a rat model of streptozotocin (STZ)-and high-fat-diet-induced type 2 diabetes, we explored the correlation between functional changes of the Grx1 redox system in the left ventricular tissue and blood of the diabetic rats. Moreover, we studied the protective effect of Grx1 against cardiac toxicity caused by the high-glucose-induced expression of cardiac matrix metalloproteinases (MMPs) in primary cultured cardiac fibroblasts. Finally, we investigated the protective effects and signaling regulatory mechanism of Grx1 against diabetic cardiomyopathy (DCM) in terms of oxidative stress and NF-kB-mediated fibrosis-associated signaling pathways. In the serum of hyperglycemic patients, Grx1 levels were elevated, total/protein thiol or sulfhydryl (Total-SH/P-SH) levels were decreased, glutathione was downregulated, and oxidized glutathione was upregulated. In addition, in the left ventricular myocardium and blood of the diabetic rats, Grx1 levels were significantly increased and glutathione reductase and P-SH levels were decreased. Moreover, endogenous Grx1 was highly expressed in cardiac fibroblasts during high-glucose treatment, and exogenous Grx1 can prevent DCM by controlling oxidative damage and MMP expression. These findings are suggestive of changes in the Grx1 redox system, and Grx1-regulated protein oxidative modifications may serve as molecular markers for diabetes caused by high-glucose-induced oxidative stress.

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Redox Modulation of Endothelial Nitric Oxide Synthase by Glutaredoxin-1 through Reversible Oxidative Post-Translational Modification

Cited by 59 publications

References 53 publications

Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction

Cardiac damage and dysfunction in diabetic cardiomyopathy are ameliorated by Grx1

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