Tomohiro Sawa

8-Mercapto-Cyclic GMP Mediates Hydrogen Sulfide-Induced Stomatal Closure in Arabidopsis.

Plants are exposed to hydrogen sulfide (H2S) both exogenously, as it exists as a pollutant gas in the environment, and endogenously, as it is synthesized in cells. H2S has recently been found to function as a gaseous signaling molecule, but its signaling cascade remains unknown. Here, we examined H2S-mediated guard cell signaling in Arabidopsis. The H2S donor GYY4137 (morpholin-4-ium-4-methoxyphenyl [morpholino] phosphinodithioate) induced stomatal closure, which peaked after 150 min at 1 µM or after 90 min at 10 and 100 µM. After reaching maximal closure, stomatal apertures gradually increased in size in response to further exposure to GYY4137. GYY4137 induced nitric oxide (NO) generation in guard cells, and GYY4137-induced stomatal closure was reduced by an NO scavenger and inhibitors of NO-producing enzymes. Mass spectrometry analyses showed that GYY4137 induces the synthesis of 8-nitro-cGMP and 8-mercapto-cGMP and that this synthesis is mediated by NO. In addition, 8-mercapto-cGMP triggered stomatal closure. Moreover, inhibitor and genetic studies showed that calcium, cADP ribose and slow anion channel 1 act downstream of 8-mercapto-cGMP. This study therefore demonstrates that 8-mercapto-cGMP mediates the H2S signaling cascade in guard cells.

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.

Reactive sulfur species regulate tRNA methylthiolation and contribute to insulin secretion

The 2-methylthio (ms2) modification at A37 of tRNAs is critical for accurate decoding, and contributes to metabolic homeostasis in mammals. However, the regulatory mechanism of ms2 modification remains largely unknown. Here, we report that cysteine hydropersulfide (CysSSH), a newly identified reactive sulfur species, is involved in ms2 modification in cells. The suppression of intracellular CysSSH production rapidly reduced ms2 modification, which was rescued by the application of an exogenous CysSSH donor. Using a unique and stable isotope-labeled CysSSH donor, we show that CysSSH was capable of specifically transferring its reactive sulfur atom to the cysteine residues of ms2-modifying enzymes as well as ms2 modification. Furthermore, the suppression of CysSSH production impaired insulin secretion and caused glucose intolerance in both a pancreatic β-cell line and mouse model. These results demonstrate that intracellular CysSSH is a novel sulfur source for ms2 modification, and that it contributes to insulin secretion.

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.

8-Nitro-cGMP Enhances SNARE Complex Formation through S-Guanylation of Cys90 in SNAP25

Nitrated guanine nucleotide 8-nitroguanosine 3,5-cyclic monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP formation and its protein targets in the normal brain have not been identified. Here, we investigated 8-nitro-cGMP generation and protein S-guanylation in the rodent brain. Immunohistochemistry indicated that 8-nitro-cGMP was produced by neurons, such as pyramidal cells and interneurons. Using liquid chromatography-tandem mass spectrometry, we determined endogenous 8-nitro-cGMP levels in the brain as 2.92 ± 0.10 pmol/mg protein. Based on S-guanylation proteomics, we identified several S-guanylated neuronal proteins, including SNAP25 which is a core member of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. SNAP25 post-translational modification including palmitoylation, phosphorylation, and oxidation, are known to regulate neurotransmission. Our results demonstrate that S-guanylation of SNAP25 enhanced the stability of the SNARE complex, which was further promoted by Ca(2+)-dependent activation of neuronal nitric oxide synthase. Using site-directed mutagenesis, we identified SNAP25 cysteine 90 as the main target of S-guanylation which enhanced the stability of the SNARE complex. The present study revealed a novel target of redox signaling via protein S-guanylation in the nervous system and provided the first substantial evidence of 8-nitro-cGMP function in the nervous system.

Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation

We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3,5-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.

Endogenous occurrence of protein S-guanylation in Escherichia coli: Target identification and genetic regulation

8-Nitroguanosine 3,5-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E.xa0coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E.xa0coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E.xa0coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E.xa0coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5-triphosphate. More importantly, E.xa0coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E.xa0coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections.

Metabolomic profiling of reactive persulfides and polysulfides in the aqueous and vitreous humors

We investigate the metabolomic profile of reactive persulfides and polysulfides in the aqueous and vitreous humors. Eighteen eyes of 18 consecutive patients with diabetes mellitus (DM) and diabetic retinopathy underwent microincision vitrectomy combined with cataract surgery. Samples of the aqueous and vitreous humors were collected and underwent mass spectrometry-based metabolomic profiling of reactive persulfides and polysulfides (polysulfidomics). The effect of reactive polysulfide species on the viability of immortalized retinal cells (the RGC-5 cell line) under oxidative stress (induced with H2O2) was also evaluated with an Alamar Blue assay. The experiments showed that cysteine persulfides (CysSSH), oxidized glutathione trisulfide (GSSSG) and cystine were elevated in the aqueous humor, and CysSSH, Cys, and cystine were elevated in the vitreous. Furthermore, GSSSG, cystine, and CysSSH levels were correlated in the aqueous and vitreous humors. A comparison, in DM and control subjects, of plasma levels of reactive persulfides and polysulfides showed that they did not differ. In vitro findings revealed that reactive polysulfide species increased cell viability under oxidative stress. Thus, various reactive persulfides and polysulfides appear to be present in the eye, and some reactive sulfide species, which have a protective effect against oxidative stress, are upregulated in the aqueous and vitreous humors of DM eyes.

Synthesis of L-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome

Abstract Cysteine persulfide is an L‐cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L‐cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L‐cysteine. We cloned three cysteine synthases—CysE, CysK, and CysM—from the Gram‐negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34S‐labeled L‐cysteine from O‐acetyl‐L‐serine and 34S‐labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L‐cysteine at both sulfur (34S) and nitrogen (15N) atoms was also achieved by performing enzyme reactions with 15N‐labeled L‐serine, acetyl‐CoA, and 34S‐labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L‐cysteine derivatives including L‐cystine, L‐cystine persulfide, S‐sulfo‐L‐cysteine, L‐cysteine sulfonate, and L‐selenocystine. We also prepared 34S‐labeled N‐acetyl‐L‐cysteine (NAC) by incubating 34S‐labeled L‐cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low‐molecular‐weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34S‐labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L‐cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology. Graphical abstract Figure. No Caption available. HighlightsA method for incorporation of sulfur isotopes into cysteinyl thiols was developed.N‐Acetyl‐L‐cysteine (NAC) was labeled with 34S by using this method.Isotope labeling together with MS was applied to the sulfur metabolome of NAC.Bioconversion of NAC to cysteine persulfides in mammalian cells was identified.

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 22u2009µM 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.