Sensing Cytosolic DNA Lowers Blood Pressure by Direct cGAMP-Dependent PKGI Activation.

Background: The major cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) has emerged as a key mediator of inflammation that underlies cardiovascular disease. On interaction with double-stranded DNA, cGAS generates the second messenger 2',3'-cyclic GMP-AMP (cGAMP) that directly binds to and activates the stimulator of interferon genes, which in turn leads to enhanced expression of genes encoding interferons and proinflammatory cytokines. Here, we show that cGAMP generated by cGAS also directly activates PKGI (cGMP-dependent protein kinase 1), a mechanism that underlies crosstalk between inflammation and blood pressure regulation.

IDH3γ functions as a redox switch regulating mitochondrial energy metabolism and contractility in the heart.

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (HO) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins.

The Regulation and Function of Histone Crotonylation.

Histone crotonylation is a newly identified epigenetic modification that has a pronounced ability to regulate gene expression. It belongs to an expanding group of short chain lysine acylations that also includes the extensively studied mark histone acetylation. Emerging evidence suggests that histone crotonylation is functionally distinct from histone acetylation and that competition for sites of modification, which reflects the cellular metabolic status, could be an important epigenetic mechanism that regulates diverse processes.

Complex interrelationships between nitro-alkene-dependent inhibition of soluble epoxide hydrolase, inflammation and tumor growth.

Nitro-oleate (10-nitro-octadec-9-enoic acid), which inhibits soluble epoxide hydrolase (sEH) by covalently adducting to C521, increases the abundance of epoxyeicosatrienoic acids (EETs) that can be health promoting, for example by lowering blood pressure or their anti-inflammatory actions. However, perhaps consistent with their impact on angiogenesis, increases in EETs may exacerbate progression of some cancers. To assess this, Lewis lung carcinoma (LLc1) cells were exposed to oleate or nitro-oleate, with the latter inhibiting the hydrolase and increasing their proliferation and migration in vitro.

Blood Pressure-Lowering by the Antioxidant Resveratrol Is Counterintuitively Mediated by Oxidation of cGMP-Dependent Protein Kinase.

Background: The health-promoting and disease-limiting abilities of resveratrol, a natural polyphenol, has led to considerable interest in understanding the mechanisms of its therapeutic actions. The polyphenolic rings of resveratrol enable it to react with and detoxify otherwise injurious oxidants. Whilst the protective actions of resveratrol are commonly ascribed to its antioxidant activity, here we show that this is a misconception.

Oxidative stress impairs autophagy through oxidation of ATG3 and ATG7.

Dysfunctional macroautophagy/autophagy has been causatively linked to aging and the pathogenesis of many diseases, which are also broadly characterized by dysregulated cellular redox. As the autophagy-related (ATG) conjugation systems that mediate autophagosome maturation are cysteine dependent, their oxidation may account for loss in this catabolic process under conditions of oxidative stress. During active autophagy, LC3 is transferred from the catalytic thiol of ATG7 to the active site thiol of ATG3, where it is conjugated to phosphatidylethanolamine.

Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation.

S-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide.

Oxidation of Atg3 and Atg7 mediates inhibition of autophagy.

Macroautophagy (autophagy) is a crucial cellular stress response for degrading defective macromolecules and organelles, as well as providing bioenergetic intermediates during hypoxia and nutrient deprivation. Here we report a thiol-dependent process that may account for impaired autophagy during aging. This is through direct oxidation of key autophagy-related (Atg) proteins Atg3 and Atg7.

β-Adrenergic regulation of cardiac type 2A protein phosphatase through phosphorylation of regulatory subunit B56δ at S573.

Background: Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ.

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.

Proof of Principle for a Novel Class of Antihypertensives That Target the Oxidative Activation of PKG Iα (Protein Kinase G Iα).

Arterial hypertension continues to be a major health burden. Development of new antihypertensive drugs that engage vasodilatory mechanisms not harnessed by available therapies offer therapeutic potential. Oxidants induce an interprotein disulfide in PKG Iα (protein kinase G Iα) at C42, which is associated with its targeting and activation, resulting in vasodilation and blood pressure lowering.

Phosphodiesterase 5 Inhibition Limits Doxorubicin-induced Heart Failure by Attenuating Protein Kinase G Iα Oxidation.

Phosphodiesterase 5 (PDE5) inhibitors limit myocardial injury caused by stresses, including doxorubicin chemotherapy. cGMP binding to PKG Iα attenuates oxidant-induced disulfide formation. Because PDE5 inhibition elevates cGMP and protects from doxorubicin-induced injury, we reasoned that this may be because it limits PKG Iα disulfide formation.

Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein S-Guanylation.

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of guanosine 3',5'-cyclic monophosphate (cGMP) formed endogenously under conditions associated with production of both reactive oxygen species and nitric oxide. It acts as an electrophilic second messenger in the regulation of cellular signaling by inducing a post-translational modification of redox-sensitive protein thiols via covalent adduction of cGMP moieties to protein thiols (protein S-guanylation). Here, we demonstrate that 8-nitro-cGMP potentially S-guanylates thiol groups of cGMP-dependent protein kinase (PKG), the enzyme that serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses.

Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling.

The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known.

Ca(2+) signaling in the myocardium by (redox) regulation of PKA/CaMKII.

Homeostatic cardiac function is maintained by a complex network of interdependent signaling pathways which become compromised during disease progression. Excitation-contraction-coupling, the translation of an electrical signal to a contractile response is critically dependent on a tightly controlled sequence of events culminating in a rise in intracellular Ca(2+) and subsequent contraction of the myocardium. Dysregulation of this Ca(2+) handling system as well as increases in the production of reactive oxygen species (ROS) are two major contributing factors to myocardial disease progression.

Deficient angiogenesis in redox-dead Cys17Ser PKARIα knock-in mice.

Angiogenesis is essential for tissue development, wound healing and tissue perfusion, with its dysregulation linked to tumorigenesis, rheumatoid arthritis and heart disease. Here we show that pro-angiogenic stimuli couple to NADPH oxidase-dependent generation of oxidants that catalyse an activating intermolecular-disulphide between regulatory-RIα subunits of protein kinase A (PKA), which stimulates PKA-dependent ERK signalling. This is crucial to blood vessel growth as 'redox-dead' Cys17Ser RIα knock-in mice fully resistant to PKA disulphide-activation have deficient angiogenesis in models of hind limb ischaemia and tumour-implant growth.

Protein kinase G Iα oxidation paradoxically underlies blood pressure lowering by the reductant hydrogen sulfide.

Dysregulated blood pressure control leading to hypertension is prevalent and is a risk factor for several common diseases. Fully understanding blood pressure regulation offers the possibility of developing rationale therapies to alleviate hypertension and associated disease risks. Although hydrogen sulfide (H2S) is a well-established endogenous vasodilator, the molecular basis of its blood-pressure lowering action is incompletely understood.

The PEG-switch assay: a fast semi-quantitative method to determine protein reversible cysteine oxidation.

Introduction: Reversible protein cysteine oxidation is recognised as a pivotal post-transitional modification that transduces physiological as well as pathological signalling. Pharmacological interventions that target specific sources of oxidant formation are currently being trialled to ascertain their potential ability to prevent disease progression. To determine the selectivity of such pharmacological treatments and to indentify new drug targets, a suitable method is required to detect target cysteine oxidation.

Detecting disulfide-bound complexes and the oxidative regulation of cyclic nucleotide-dependent protein kinases by H2O2.

Hydrogen peroxide regulates intracellular signaling by oxidatively converting susceptible cysteine thiols to a modified state, which includes the formation of intermolecular disulfides. This type of oxidative modification can occur within the cAMP- and cGMP-dependent protein kinases often referred to as PKA and PKG, which have important roles in regulating cardiac contractility and systemic blood pressure. Both kinases are stimulated through conical pathways that elevate their respective cyclic nucleotides leading to direct kinase stimulation.

Protein kinase G oxidation is a major cause of injury during sepsis.

Sepsis is a common life-threatening clinical syndrome involving complications as a result of severe infection. A cardinal feature of sepsis is inflammation that results in oxidative stress. Sepsis in wild-type mice induced oxidative activation of cGMP-dependent protein kinase 1 alpha (PKG Iα), which increased blood vessel dilation and permeability, and also lowered cardiac output.

Approaches for monitoring PKG1α oxidative activation.

cGMP-dependent protein kinase, also known as protein kinase G (PKG), is activated independently of cGMP by a novel thiol-reactive mechanism involving the formation of an intermolecular disulfide. This oxidative modification within PKG is generally not detected by conventional Western immunoblot analysis due to the experimental conditions used. Here, we describe the proteomic approach that lead to PKG being identified as a kinase susceptible to oxidant-dependent disulfide dimer formation, these methods being applicable for the identification of other disulfide bound protein complexes.

Endomembrane H-Ras controls vascular endothelial growth factor-induced nitric-oxide synthase-mediated endothelial cell migration.

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras.