Philip Eaton

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.

Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin–maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin–horseradish peroxidase and also their purification by streptavidin–agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.

Redox Signaling in Cardiac Physiology and Pathology

Redox signaling refers to the specific and usually reversible oxidation/reduction modification of molecules involved in cellular signaling pathways. In the heart, redox signaling regulates several physiological processes (eg, excitation-contraction coupling) and is involved in a wide variety of pathophysiological and homoeostatic or stress response pathways. Reactive oxygen species involved in cardiac redox signaling may derive from many sources, but NADPH oxidases, as dedicated sources of signaling reactive oxygen species, seem to be especially important. An increasing number of specific posttranslational oxidative modifications involved in cardiac redox signaling are being defined, along with the reactive oxygen species sources that are involved. Here, we review current knowledge on the molecular targets of signaling reactive oxygen species in cardiac cells and their involvement in cardiac physiopathology. Advances in this field may allow the development of targeted therapeutic strategies for conditions such as heart failure as opposed to the general antioxidant approaches that have failed to date.

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.

Oxidant-induced Activation of Type I Protein Kinase A Is Mediated by RI Subunit Interprotein Disulfide Bond Formation

Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for α-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of β-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.

Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

We have developed methods that allow detection, quantitation, purification, and identification of cardiac proteinsS-thiolated during ischemia and reperfusion. Cysteine was biotinylated and loaded into isolated rat hearts. During oxidative stress, biotin-cysteine forms a disulfide bond with reactive protein cysteines, and these can be detected by probing Western blots with streptavidin-horseradish peroxidase. S-Thiolated proteins were purified using streptavidin-agarose. Thus, we demonstrated that reperfusion and diamide treatment increased S-thiolation of a number of cardiac proteins by 3- and 10-fold, respectively. Dithiothreitol treatment of homogenates fully abolished the signals detected. Fractionation studies indicated that the modified proteins are located within the cytosol, membrane, and myofilament/cytoskeletal compartments of the cardiac cells. This shows that biotin-cysteine gains rapid and efficient intracellular access and acts as a probe for reactive protein cysteines in all cellular locations. Using Western blotting of affinity-purified proteins we identified actin, glyceraldehyde-3-phosphate dehydrogenase, HSP27, protein-tyrosine phosphatase 1B, protein kinase Cα, and the small G-protein ras as substrates for S-thiolation during reperfusion of the ischemic rat heart. MALDI-TOF mass fingerprint analysis of tryptic peptides independently confirmed actin and glyceraldehyde-3-phosphate dehydrogenase S-thiolation during reperfusion. This approach has also shown that triosephosphate isomerase, aconitate hydratase, M-protein, nucleoside diphosphate kinase B, and myoglobin are S-thiolated during post-ischemic reperfusion.

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.

Hydrogen peroxide as an endogenous mediator and exogenous tool in cardiovascular research: issues and considerations.

Exogenous H(2)O(2) is widely applied to cardiovascular tissues in order to elicit oxidant-dependent responses relevant to signalling and disease. Lower levels of endogenous H(2)O(2) are essential for normal physiological functioning and signalling, whereas higher levels are associated with disease. Within diseased tissues, concentrations in excess of 100 microM have been measured, though 1-15 microM appears to be the upper limit of the healthy physiological range. Analysing the kinetic constants and abundance of peroxidases suggests that they may, on occasion, encounter tissue H(2)O(2) concentrations as high as 1 mM. Extracellular application of 0.01-1 mM peroxide appears to be directly relevant to biology and broadly mimics the release of H(2)O(2) endogenously by growth factors and other effectors. However, the intracellular H(2)O(2) may only ever reach 1-15% of the applied exogenous concentration.

Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart

4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion.

The Utility of N,N-Biotinyl Glutathione Disulfide in the Study of Protein S-Glutathiolation

Glutathione disulfide (GSSG) accumulates in cells under an increased oxidant load, which occurs during neurohormonal or metabolic stimulation as well as in many disease states. Elevated GSSG promotes protein S-glutathiolation, a reversible post-translational modification, which can directly alter or regulate protein function. We developed novel strategies for the study of protein S-glutathiolation that involved the simple synthesis of N,N-biotinyl glutathione disulfide (biotin-GSSG). Biotin-GSSG treatment of cells mimics a defined component of oxidative stress, namely a shift in the glutathione redox couple to the oxidized disulfide state. This induces widespread protein S-glutathiolation, which was detected on non-reducing Western blots probed with streptavidin-horseradish peroxidase and imaged using confocal fluorescence microscopy and ExtrAvidin-FITC. S-Glutathiolated proteins were purified using streptavidin-agarose and identified using proteomic methods. We conclude that biotin-GSSG is a useful tool in the investigation of protein S-glutathiolation and offers significant advantages over conventional methods or antibody-based strategies. These novel approaches may find widespread utility in the study of disease or redox signaling models where GSSG accumulation occurs.