Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.

Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.

Protein polysulfidation-dependent persulfide dioxygenase activity of ethylmalonic encephalopathy protein 1.

Reactive persulfide species such as glutathione persulfide (GSSH) are highly abundant biomolecules. Persulfide dioxygenase (also called ethylmalonic encephalopathy protein 1, ETHE1) reportedly metabolizes GSSH to GSH with simultaneous oxygen consumption. How ETHE1 activity is regulated is still unclear, however. In this study, we describe the possible role of protein polysulfidation in the catalytic activity of ETHE1. We first found that ETHE1 catalyzed the persulfide dioxygenase reaction mostly for glutathione polysulfides, GS-(S)n-H, as well as for GSSH, but not for other endogenous persulfides such as cysteine and homocysteine persulfides/polysulfides. We then developed a novel method to detect protein polysulfidation and named it the polyethylene glycol-conjugated maleimide-labeling gel shift assay (PMSA). PMSA analysis indicated that most cysteine residues in ETHE1 were polysulfidated. Site-directed mutagenesis of cysteine residues in ETHE1 combined with liquid chromatography tandem mass spectrometry for polysulfidation determination surprisingly indicated that the Cys247 residue was important for polysulfidation of other Cys residues and that the C247S mutant possessed no persulfide dioxygenase activity. These results suggested that ETHE1 is a major enzyme regulating endogenous GSSH/GS-(S)n-H and that its activity is controlled by polysulfidation of the Cys247 residue.

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. S-Guanylation of PKG was found to occur in a site specific manner; Cys42 and Cys195 were the susceptible residues among 11 Cys residues. Importantly, S-guanylation at Cys195, which is located in the high-affinity cGMP binding domain of PKG, causes persistent enzyme activation as determined by in vitro kinase assay as well as by an organ bath assay. In vivo, S-guanylation of PKG was demonstrated to occur in mice without any specific treatment and was significantly enhanced by lipopolysaccharide administration. These findings warrant further investigation in terms of the physiological and pathophysiological roles of S-guanylation-dependent persistent PKG activation.

Endogenous nitrated nucleotide is a key mediator of autophagy and innate defense against bacteria.

Autophagy is a cellular self-catabolic process wherein organelles, macromolecules, and invading microbes are sequestered in autophagosomes that fuse with lysosomes. In this study, we uncover the role of nitric oxide (NO) as a signaling molecule for autophagy induction via its downstream mediator, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). We found that 8-nitro-cGMP-induced autophagy is mediated by Lys63-linked polyubiquitination and that endogenous 8-nitro-cGMP promotes autophagic exclusion of invading group A Streptococcus (GAS) from cells. 8-nitro-cGMP can modify Cys residues by S-guanylation of proteins. We showed that intracellular GAS is modified with S-guanylation extensively in autophagosomes-like vacuoles, suggesting the role of S-guanylation as a marker for selective autophagic degradation. This finding is supported by the fact that S-guanylated bacteria were selectively marked with polyubiquitin, a known molecular tag for selective transport to autophagosomes. These results collectively indicate that 8-nitro-cGMP plays a crucial role in cytoprotection during bacterial infections or inflammations via autophagy upregulation.

Bicarbonate plays a critical role in the generation of cytotoxicity during SIN-1 decomposition in culture medium.

3-Morpholinosydnonimine (SIN-1) is used as a donor of peroxynitrite (ONOO(-)) in various studies. We demonstrated, however, that, the cell-culture medium remains cytotoxic to PC12 cells even after almost complete SIN-1 decomposition, suggesting that reaction product(s) in the medium, rather than ONOO(-), exert cytotoxic effects. Here, we clarified that significant cytotoxicity persists after SIN-1 decomposes in bicarbonate, a component of the culture medium, but not in NaOH. Cytotoxic SIN-1-decomposed bicarbonate, which lacks both oxidizing and nitrosating activities, degrades to innocuous state over time. The extent of SIN-1 cytotoxicity, irrespective of its fresh or decomposed state, appears to depend on the total number of initial SIN-1 molecules per cell, rather than its concentration, and involves oxidative/nitrosative stress-related cell damage. These results suggest that, despite its low abundance, the bicarbonate-dependent cytotoxic substance that accumulates in the medium during SIN-1 breakdown is the cytotoxic entity of SIN-1.

Neoechinulin a imparts resistance to acute nitrosative stress in PC12 cells: a potential link of an elevated cellular reserve capacity for pyridine nucleotide redox turnover with cytoprotection.

Treatment of PC12 cells with fungus-derived alkaloid neoechinulin A for more than 12 h renders the cells resistant to subsequent superoxide (O2⁻)/nitric oxide (NO) insults derived from 3-morpholinosydnonimine (SIN-1). However, the underlying mechanism(s) remains largely unclear. To elucidate the mechanism(s), we assessed the specificity of the cytoprotection afforded by neoechinulin A treatment using other cytocidal stressors and also clarified the resulting cellular alterations, focusing on the antioxidant and metabolic enzymes systems. Neoechinulin A treatment for more than 12 h endowed PC12 cells with significant resistance to transient NO toxicity, but not persistent NO toxicity, bolus H2O2 toxicity, or oxidative insult from the redox cycling quinone menadione. Cellular antioxidant system profiling revealed no substantial potentiation of the activity of any antioxidant enzyme in lysate from the neoechinulin A-treated cells excluding glutathione (GSH) content, which was significantly decreased (>50%), resulting in a proportional compromise in the thiol-reducing activity of the intact cells. In addition, no differences were observed in the activity for any nicotinamide adenine dinucleotide (phosphate) reduced form (NAD(P)H)-generating enzyme, steady-state NAD(P)H/nicotinamide adenine dinucleotide (phosphate) oxidized form (NAD(P)⁺) ratios, or the levels of total NAD(P)H. Nevertheless, the neoechinulin A-treated intact cells exhibited increased NAD(P)H redox turnover when driven by extracellular tetrazolium. The structurally inactive analog preechinulin failed to protect cells against NO toxicity or induce these alterations, suggesting their link with the cytoprotective mechanism. These results suggest that neoechinulin A, despite disabling the GSH defense system, confers cytoprotection against nitrosative stresses by elevating the cellular reserve capacity for NAD(P)H generation, which could offset crippling of energy-supplying systems due to nitrosative stress.

Neoechinulin a impedes the progression of rotenone-induced cytotoxicity in PC12 cells.

Neoechinulin A, an indole alkaloid from marine fungi, can protect PC12 cells from the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)), a Parkinson disease-inducing neurotoxin, by ameliorating downstream events resulting from mitochondrial complex I inactivation. However, the cytoprotective mechanisms remained unclear. In this study, by using rotenone, another parkinsonian-inducing neurotoxin targeting mitochondrial complex I, we investigated the cytoprotective mechanism of neoechinulin A. Rotenone-induced cell death was associated with accelerated glucose consumption, and excess glucose supplementation in the culture medium almost completely suppressed cell death, suggesting that glucose deficiency in the medium is critical for triggering cell death in this model. Co-treatment with neoechinulin A, but not neoechinulin A pre-treatment before rotenone exposure, significantly impeded cell death by rotenone. Although the presence of neoechinulin A did not affect the accelerated glycolytic turnover in rotenone-treated cells, it paradoxically decreased ATP levels in the cells, suggesting increased ATP consumption. Although the link between the decreased ATP levels and cytoprotection is not clear at present, it suggests that neoechinulin A may ameliorate rotenone toxicity by activating a cytoprotective machinery that requires ATP.