Nucleotides function as endogenous chemical sensors for oxidative stress signaling

Oxidized and nitrated nucleotides including 8-oxogunanine and 8-nitroguanine derivatives such as 8-nitroguanosine 3',5'-cyclic monophosphate were generated by reactive nitrogen oxides and reactive oxygen species in cultured cells and in tissues. 8-oxoguanine and 8-nitroguanine in DNA and RNA are potentially mutagenic, and the former also induces cell death. Some derivative, 8-nitroguanosine 3',5'-cyclic monophosphate a major nitrated guanine nucleotide, was identified as a novel second messenger. Surprisingly, the amount of 8-nitroguanosine 3',5'-cyclic monophosphate generated was found to be higher than that of guanosine 3',5'-cyclic monophosphate in cells expressing inducible nitric oxide synthase. More important, 8-nitroguanosine 3',5'-cyclic monophosphate is electrophilic and reacted efficiently with sulfhydryls of proteins to produce a novel posttranslational modification (named S-guanylation) via guanosine 3',5'-cyclic monophosphate adduction. For example, 8-nitroguanosine 3',5'-cyclic monophosphate-induced S-guanylation of Kelch-like ECH-associated protein 1 led to NF-E2-related factor activation and induction of antioxidant enzymes. 8-nitroguanosine 3',5'-cyclic monophosphate may thus protect cells against oxidative stress-related cytotoxicity. Therefore, although chemically modified nucleotides produced via oxidative and nitrative stress are regarded simply as endogenous mutagens, the endogenous nucleotides stored in cells per se may serve functionally as a sensing mechanism for reactive nitrogen oxides and oxygen species to induce cellular adaptive responses to oxidative stress

Characterization of a novel monoclonal antibody that senses nitric oxide-dependent activation of soluble guanylate cyclase

AbstractTwo monoclonal antibodies (mAbs) against bovine lung soluble guanylate cyclase (sGC) were prepared and characterized. mAb 3221 recognized both the α- and β-subunits of sGC and had greater binding affinity to the enzyme in the presence of NO. mAb 28131 recognized only the β-subunit and its affinity did not change with NO. Neither mAb cross-reacted with particulate GC. Cultured Purkinje cells from rats were treated with S-nitroso-N-acetylpenicillamine, an NO donor, and examined by immunocytochemical methods. The immunoreactivity associated with mAb 3221 increased with the cGMP content in a crude extract of cerebellum and the NO2 generated in the culture medium increased

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke

peer reviewedIn addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke

Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction

Sulfide Catabolism Ameliorates Hypoxic Brain Injury

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain’s sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury

Protein S-guanylation by the biological signal 8-nitroguanosine 3\u27,5\u27-cyclic monophosphate

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3′,5′-cyclic monophosphate (cGMP, 1). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP, 2), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO

Quantitative Determination of 2-Oxo-Imidazole-Containing Dipeptides by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

2-Oxo-imidazole-containing dipeptides (2-oxo-IDPs), novel imidazole-containing dipeptide (IDP) derivatives, exhibit a much higher antioxidant capacity than that of IDPs. However, quantitative methods have only been developed for IDPs, and methods for the quantitative analysis of 2-oxo-IDPs are needed. In this study, we developed methods for the quantitative analysis of 2-oxo-IDPs by high-performance liquid chromatography with online electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) coupled with a stable isotope dilution method. First, we prepared stable isotope-labeled IDP and 2-oxo-IDP standards for MS analyses. Next, using these standards, we established highly sensitive, selective, and absolute quantitative analysis methods for five IDPs and five 2-oxo-IDPs by HPLC-ESI-MS/MS, achieving a limit of detection in the fmol range. Finally, we applied the method to various types of meat, such as beef, pork, chicken, and whale meat, demonstrating the detection of both IDPs and 2-oxo-IDPs. Furthermore, we provide the first evidence for the endogenous production of 2-oxo-balenine in meats. The methods developed in this study enable the detection of trace levels of 2-oxo-IDPs in biological samples and could be helpful for understanding the biological relevance of 2-oxo-IDPs

Cell signaling mediated by nitrated cyclic guanine nucleotide

We recently clarified the physiological formation of 8-nitroguanosine 3\u27,5\u27-cyclic monophosphate (8-nitro-cGMP) and its critical roles in nitric oxide (NO) signal transductions. This discovery of 8-nitro-cGMP is the first demonstration of a nitrated cyclic nucleotide functioning as a new second messenger in mammals since the identification of cGMP more than 40. years ago. By means of chemical analyses, e.g., liquid chromatography-tandem mass spectrometry, we unequivocally identified 8-nitro-cGMP formation, which depended on NO production, in several types of cultured cells, including macrophages and glial cells. Most important, we previously showed that 8-nitro-cGMP as an electrophile reacted with particular sulfhydryls of proteins to generate a unique post-translational modification that we called protein S-guanylation. In fact, certain specific intracellular proteins, such as the redox-sensor protein Keap1, readily underwent S-guanylation induced by 8-nitro-cGMP. 8-Nitro-cGMP activated the Nrf2 signaling pathway by triggering dissociation of Keap1, via S-guanylation of its highly nucleophilic cysteine sulfhydryls. We also determined that S-guanylation of Keap1 was involved in cytoprotective actions of NO and 8-nitro-cGMP by inducing oxidative stress response genes such as heme oxygenase-1. Such unique chemical properties of 8-nitro-cGMP shed light on new areas of NO and cGMP signal transduction. Protein S-guanylation induced by 8-nitro-cGMP may thus have important implications in NO-related physiology and pathology, pharmaceutical chemistry, and development of therapeutics for many diseases

Characterization of Fusarium oxysporum β-1,6-Galactanase, an Enzyme That Hydrolyzes Larch Wood Arabinogalactan▿

A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a β-1,6-galactanase that preferentially debranches β-1,6-galactobiose from the substrate