Rebecca L. Charles

Cysteine Redox Sensor in PKGIa Enables Oxidant-Induced Activation

Changes in the concentration of oxidants in cells can regulate biochemical signaling mechanisms that control cell function. We have found that guanosine 3′,5′-monophosphate (cGMP)–dependent protein kinase (PKG) functions directly as a redox sensor. The Iα isoform, PKGIα, formed an interprotein disulfide linking its two subunits in cells exposed to exogenous hydrogen peroxide. This oxidation directly activated the kinase in vitro, and in rat cells and tissues. The affinity of the kinase for substrates it phosphorylates was enhanced by disulfide formation. This oxidation-induced activation represents an alternate mechanism for regulation along with the classical activation involving nitric oxide and cGMP. This mechanism underlies cGMP-independent vasorelaxation in response to oxidants in the cardiovascular system and provides a molecular explantion for how hydrogen peroxide can operate as an endothelium-derived hyperpolarizing factor.

Endothelial Nox4 NADPH Oxidase Enhances Vasodilatation and Reduces Blood Pressure In Vivo

Objective—Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H2O2), but its role in vivo remains unclear. Methods and Results—We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H2O2 production and H2O2-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. Conclusion—Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H2O2 production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.

Protein Sulfenation as a Redox Sensor Proteomics Studies Using a Novel Biotinylated Dimedone Analogue

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively “tagging” them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H2O2 (0.1–10,000 μm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H2O2 and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H2O2-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.

Protection from hypertension in mice by the Mediterranean diet is mediated by nitro fatty acid inhibition of soluble epoxide hydrolase

Significance The Mediterranean diet is characterized by consumption of unsaturated fats with vegetables rich in nitrite and nitrate, resulting in endogenous formation of nitro fatty acids. These reactive lipids adduct to soluble epoxide hydrolase, inhibiting it to lower blood pressure. Mice genetically engineered to be resistant to this adductive inhibition had high blood pressure basally and their hydrolase activity was fully resistant to inhibition by nitro fatty acid supplied directly or generated via the Mediterranean diet. Similarly nitro fatty acid lowered blood pressure and abrogated cardiac hypertrophy in a hypertension model in wild-type mice, but was ineffective in mutant mice. Thus, protection from hypertension afforded by the Mediterranean diet is mediated by nitro-fatty acid-dependent inhibition of soluble epoxide hydrolase. Soluble epoxide hydrolase (sEH) is inhibited by electrophilic lipids by their adduction to Cys521 proximal to its catalytic center. This inhibition prevents hydrolysis of the enzymes’ epoxyeicosatrienoic acid (EET) substrates, so they accumulate inducing vasodilation to lower blood pressure (BP). We generated a Cys521Ser sEH redox-dead knockin (KI) mouse model that was resistant to this mode of inhibition. The electrophilic lipid 10-nitro-oleic acid (NO2-OA) inhibited hydrolase activity and also lowered BP in an angiotensin II-induced hypertension model in wild-type (WT) but not KI mice. Furthermore, EET/dihydroxy-epoxyeicosatrienoic acid isomer ratios were elevated in plasma from WT but not KI mice following NO2-OA treatment, consistent with the redox-dead mutant being resistant to inhibition by lipid electrophiles. sEH was inhibited in WT mice fed linoleic acid and nitrite, key constituents of the Mediterranean diet that elevates electrophilic nitro fatty acid levels, whereas KIs were unaffected. These observations reveal that lipid electrophiles such as NO2-OA mediate antihypertensive signaling actions by inhibiting sEH and suggest a mechanism accounting for protection from hypertension afforded by the Mediterranean diet.

Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury

The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na+/K+-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 μM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na+/K+-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na+/K+-ATPase activation during reperfusion would attenuate Na+ overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na+/K+-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

Redox Regulation of Soluble Epoxide Hydrolase by 15-Deoxy-Δ-Prostaglandin J2 Controls Coronary Hypoxic Vasodilation

Rationale: 15-Deoxy-&Dgr;-prostaglandin (15d-PG)J2 is an electrophilic oxidant that dilates the coronary vasculature. This lipid can adduct to redox active protein thiols to induce oxidative posttranslational modifications that modulate protein and tissue function. Objective: To investigate the role of oxidative protein modifications in 15d-PGJ2–mediated coronary vasodilation and define the distal signaling pathways leading to enhanced perfusion. Methods and Results: Proteomic screening with biotinylated 15d-PGJ2 identified novel vascular targets to which it adducts, most notably soluble epoxide hydrolase (sEH). 15d-PGJ2 inhibited sEH by specifically adducting to a highly conserved thiol (Cys521) adjacent to the catalytic center of the hydrolase. Indeed a Cys521Ser sEH “redox-dead” mutant was resistant to 15d-PGJ2–induced hydrolase inhibition. 15d-PGJ2 dilated coronary vessels and a role for hydrolase inhibition was supported by 2 structurally different sEH antagonists each independently inducing vasorelaxation. Furthermore, 15d-PGJ2 and sEH antagonists also increased coronary effluent epoxyeicosatrienoic acids consistent with their vasodilatory actions. Indeed 14,15-EET alone induced relaxation and 15d-PGJ2–mediated vasodilation was blocked by the EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Additionally, the coronary vasculature of sEH-null mice was basally dilated compared to wild-type controls and failed to vasodilate in response to 15d-PGJ2. Coronary vasodilation to hypoxia in wild-types was accompanied by 15d-PGJ2 adduction to and inhibition of sEH. Consistent with the importance of hydrolase inhibition, sEH-null mice failed to vasodilate during hypoxia. Conclusion: This represents a new paradigm for the regulation of sEH by an endogenous lipid, which is integral to the fundamental physiological response of coronary hypoxic vasodilation.

Gel-based methods in redox proteomics.

BACKGROUND The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification. SCOPE OF THE REVIEW This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated. MAJOR CONCLUSIONS A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein. GENERAL SIGNIFICANCE By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Redox signalling in cardiovascular disease

Oxidative stress has almost universally and unequivocally been implicated in the pathogenesis of all major diseases, including those of the cardiovascular system. Oxidative stress in cells and cardiovascular biology was once considered only in terms of injury, disease and dysfunction. However, it is now appreciated that oxidants are also produced in healthy tissues, and they function as signalling molecules transmitting information throughout the cell. Conversely, when cells move to a more reduced state, as can occur when oxygen is limiting, this can also result in alterations in the function of biomolecules and subsequently cells. At the centre of this ‘redox signalling’ are oxidoreductive chemical reactions involving oxidants or reductants post translationally modifying proteins. These structural alterations allow changes in cellular redox state to be coupled to alterations in cell function. In this review, we consider aspects of redox signalling in the cardiovascular system, focusing on the molecular basis of redox sensing by proteins and the array of post‐translational oxidative modifications that can occur. In addition, we discuss studies utilising proteomic methods to identify redox‐sensitive cardiac proteins, as well as those using this technology more broadly to assess redox signalling in cardiovascular disease.

Redox-dependent dimerization of p38α mitogen-activated protein kinase with mitogen-activated protein kinase kinase 3

The kinase p38α MAPK (p38α) plays a pivotal role in many biological processes. p38α is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38α. p38α was activated and formed a disulfide-bound heterodimer with MAP2K3 (MKK3) in rat cardiomyocytes and isolated hearts exposed to H2O2. This disulfide heterodimer was sensitive to reduction by mercaptoethanol and was enhanced by the thioredoxin-reductase inhibitor auranofin. We predicted that Cys-119 or Cys-162 of p38α, close to the known MKK3 docking domain, were relevant for these redox characteristics. The C119S mutation decreased whereas the C162S mutation increased the dimer formation, suggesting that these two Cys residues act as vicinal thiols, consistent with C119S/C162S being incapable of sensing H2O2. Similarly, disulfide heterodimer formation was abolished in H9C2 cells expressing both MKK3 and p38α C119S/C162S and subjected to simulated ischemia and reperfusion. However, the p38α C119S/C162S mutants did not exhibit appreciable alteration in activating dual phosphorylation. In contrast, the anti-inflammatory agent 10-nitro-oleic acid (NO2-OA), a component of the Mediterranean diet, reduced p38α activation and covalently modified Cys-119/Cys-162, probably obstructing MKK3 access. Moreover, NO2-OA reduced the dephosphorylation of p38α by hematopoietic tyrosine phosphatase (HePTP). Furthermore, steric obstruction of Cys-119/Cys-162 by NO2-OA pretreatment in Langendorff-perfused murine hearts prevented the p38-MKK3 disulfide dimer formation and attenuated H2O2-induced contractile dysfunction. Our findings suggest that cysteine residues within p38α act as redox sensors that can dynamically regulate the association between p38 and MKK3.

Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain

The human soluble Epoxide Hydrolase (hsEH) is an enzyme involved in the hydrolysis of endogenous anti-inflammatory and cardio-protective signalling mediators known as epoxyeicosatrienoic acids (EETs). EETs’ conversion into the corresponding diols by hsEH generates non-bioactive molecules, thereby the enzyme inhibition would be expected to enhance the EETs bioavailability, and their beneficial properties. Numerous inhibitors have been developed to target the enzyme, some of which are showing promising antihypertensive and anti-inflammatory properties in vivo. Thus far, the preparation of the recombinant enzyme for enzymatic and structural in vitro studies has been performed mainly using a baculovirus expression system. More recently, it was reported that the enzyme could be exogenously expressed and isolated from E. coli, although limited amounts of active protein were obtained. We herein describe two novel methods to yield pure recombinant enzyme. The first describes the expression and purification of the full-length enzyme from eukaryotic cells HEK293-F, whilst the second concerns the C-terminal domain of hsEH obtained from the cost-effective and rapid E. coli prokaryotic system. The two methods successfully generated satisfactory amounts of functional enzyme, with virtually identical enzymatic activity. Overall, the protocols described in this paper can be employed for the recombinant expression and purification of active hsEH, to be used in future biomedical investigations and for high-throughput screening of inhibitors for potential use in the treatment of cardiovascular disease.