New aspects of redox signaling mediated by supersulfides in health and disease.

Oxygen molecules accept electrons from the respiratory chain in the mitochondria and are responsible for energy production in aerobic organisms. The reactive oxygen species formed via these oxygen reduction processes undergo complicated electron transfer reactions with other biological substances, which leads to alterations in their physiological functions and cause diverse biological and pathophysiological consequences (e.g., oxidative stress). Oxygen accounts for only a small proportion of the redox reactions in organisms, especially under aerobic or hypoxic conditions but not under anaerobic and hypoxic conditions. This article discusses a completely new concept of redox biology, which is governed by redox-active supersulfides, i.e., sulfur-catenated molecular species. These species are present in abundance in all organisms but remain largely unexplored in terms of redox biology and life science research. In fact, accumulating evidence shows that supersulfides have extensive redox chemical properties and that they can be readily ionized or radicalized to participate in energy metabolism, redox signaling, and oxidative stress responses in cells and in vivo. Thus, pharmacological intervention and medicinal modulation of supersulfide activities have been shown to benefit the regulation of disease pathogenesis as well as disease control.

Isocitrate dehydrogenase 1 upregulation in urinary extracellular vesicles from proximal tubules of type 2 diabetic rats.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. Microalbuminuria is currently the most common non-invasive biomarker for the early diagnosis of DN. However, renal structural damage may have advanced when albuminuria is detected. In this study, we sought biomarkers for early DN diagnosis through proteomic analysis of urinary extracellular vesicles (uEVs) from type 2 diabetic model rats and normal controls. Isocitrate dehydrogenase 1 (IDH1) was significantly increased in uEVs from diabetic model rats at the early stage despite minimal differences in albuminuria between the groups. Calorie restriction significantly suppressed the increase in IDH1 in uEVs and 24-hour urinary albumin excretion, suggesting that the increase in IDH1 in uEVs was associated with the progression of DN. Additionally, we investigated the origin of IDH1-containing uEVs based on their surface sugar chains. Lectin affinity enrichment and immunohistochemical staining showed that IDH1-containing uEVs were derived from proximal tubules. These findings suggest that the increase in IDH1 in uEVs reflects pathophysiological alterations in the proximal tubules and that IDH1 in uEVs may serve as a potential biomarker of DN in the proximal tubules.

2H-Thiopyran-2-thione sulfine, a compound for converting H2S to HSOH/H2S2 and increasing intracellular sulfane sulfur levels.

Reactive sulfane sulfur species such as persulfides (RSSH) and H2S2 are important redox regulators and closely linked to H2S signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert H2S to HSOH, and then to H2S2 in the presence of excess H2S. Meanwhile, the reaction product 2H-thiopyran-2-thione (TT) can be oxidized to reform TTS by biological oxidants. The reaction mechanism of TTS is studied experimentally and computationally. TTS can be conjugated to proteins to achieve specific delivery, and the combination of TTS and H2S leads to highly efficient protein persulfidation. When TTS is applied in conjunction with established H2S donors, the corresponding donors of H2S2 (or its equivalents) are obtained. Cell-based studies reveal that TTS can effectively increase intracellular sulfane sulfur levels and compensate for certain aspects of sulfide:quinone oxidoreductase (SQR) deficiency. These properties make TTS a conceptually new strategy for the design of donors of reactive sulfane sulfur species.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.

Rapid Isolation of Extracellular Vesicles Using a Hydrophilic Porous Silica Gel-Based Size-Exclusion Chromatography Column.

Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by almost all cell types and can be found in biological fluids, such as blood and urine. EVs play an important role in various physiological and pathological processes via cell-cell communication, highlighting their potential applications as diagnostic markers for diseases and therapeutic drug delivery carriers. Although various methods have been developed for the isolation of EVs from biological fluids, most of them exhibit major limitations, including low purity, long processing times, and high cost. In this study, we developed a size-exclusion chromatography (SEC) column device using hydrophilic porous silica gel (PSG). Owing to the resistance to pressure of the device, a rapid system for EV isolation was developed by connecting it to a flash liquid chromatography system furnished with a UV detector and a fraction collector. This system can be used for the real-time monitoring of eluted EVs by UV absorption without further analysis and separation of high-purity EVs from urine samples with high durability, reusability, and reproducibility. In addition, there were no significant differences between the PSG column- and conventional SEC column-isolated EVs in the proteome profiles and cellular uptake activities, suggesting the good quality of the EVs isolated by the PSG column. These findings suggest that the PSG column device offers an effective and rapid method for the isolation of intact EVs from biological fluids.

Histone functions as a cell-surface receptor for AGEs.

Reducing sugars can covalently react with proteins to generate a heterogeneous and complex group of compounds called advanced glycation end products (AGEs). AGEs are generally considered as pathogenic molecules, mediating a pro-inflammatory response and contributing to the development of a number of human diseases. However, the intrinsic function of AGEs remains to be elucidated. We now provide multiple lines of evidence showing that AGEs can specifically bind histone localized on the cell surface as an AGE-binding protein, regulate the function of histone as a plasminogen receptor, and result in the regulation of monocytes/macrophage recruitment to the site of inflammation. Our finding of histone as a cell-surface receptor for AGEs suggests that, beside our common concept of AGEs as danger-associated molecular patterns mediating a pro-inflammatory response, they may also be involved in the homeostatic response via binding to histone.

Metabolomics of small extracellular vesicles derived from isocitrate dehydrogenase 1-mutant HCT116 cells collected by semi-automated size exclusion chromatography.

Cancer-derived small extracellular vesicles (sEVs) are multifunctional particles with a lipid bilayer structure that are involved in cancer progression, such as malignant proliferation, distant metastasis, and cancer immunity evasion. The separation protocol used to isolate sEVs is an important process and thus, several have been developed, including ultracentrifugation (UC), size exclusion chromatography (SEC), and affinity purification using antibodies against sEV surface antigens. However, the effects of different separation methods on sEV components have not been adequately examined. Here, we developed a semi-automated system for collecting sEVs by combining SEC and preparative high-performance liquid chromatography and applied it to metabolome analysis. The developed SEC system could recover sEVs more efficiently and non-destructively than UC, suggesting that it is an appropriate recovery method for metabolic analysis and reflects biological conditions. Furthermore, using the developed SEC system, we performed metabolome analysis of sEVs from isocitrate dehydrogenase 1 (IDH)-mutated human colon HCT116 cells, which produce the oncogenic metabolite, 2-hydroxyglutaric acid (2-HG). IDH1-mutated HCT116 cells released significantly more sEVs than wild-type (WT) cells. The metabolomic profiles of IDH1 mutant and WT cells showed distinct differences between the cells and their sEVs. Notably, in IDH mutant cells, large amounts of 2-HG were detected not only in cells, but also in sEVs. These results indicate that the SEC system we developed has wide potential applications in sEVs research.

Autoxidation of ascorbate mediates lysine N-pyrrolation.

Protein N-pyrrolation, which converts lysine residues to Nε-pyrrole-l-lysine (pyrK), is a naturally occurring covalent modification. The pyrrolated proteins have a unique property of binding to DNA-staining agents, such as SYBR Green I (SG), and anti-DNA antibodies, suggesting a physiologically relevant modification that gives rise to DNA mimic protein. These properties of pyrrolated protein are suggested to be associated with innate and autoimmune responses. Short-chain aldehydes derived from lipid peroxidation are thought to be involved in the formation of pyrK. We now report that similar lysine N-pyrrolation also occurs during the metal-catalyzed oxidation of proteins with ascorbate. When human serum albumin (HSA) was incubated with Fe2+/ascorbate in the presence and absence of docosahexaenoic acid, the protein was converted to SG-binding proteins even without the polyunsaturated fatty acid. The formation of SG-binding proteins by Fe2+/ascorbate was accompanied by the formation of pyrK, which was also detected in ascorbate-treated hemoglobin. Moreover, the metal-catalyzed oxidation of ascorbate produced the pyrrolation factors, glycolaldehyde and glyoxal. These results and the observations that sera from autoimmune-prone MRL-lpr mice recognized modified proteins with Fe2+/ascorbate and with glycolaldehyde/glyoxal suggest that the autoxidation of ascorbate, as well as lipid peroxidation, can be a source of autoantigenic N-pyrrolated proteins. Our findings revealed a possible function of ascorbate as an endogenous source of pyrrolated proteins and suggested that the pyrK residues generated in proteins may play a role in the innate and autoimmune responses associated with the oxidative metabolism of ascorbate.

Low temperature plasma irradiation products of sodium lactate solution that induce cell death on U251SP glioblastoma cells were identified.

Low-temperature plasma is being widely used in the various fields of life science, such as medicine and agriculture. Plasma-activated solutions have been proposed as potential cancer therapeutic reagents. We previously reported that plasma-activated Ringer's lactate solution exhibited selective cancer-killing effects, and that the plasma-treated L-sodium lactate in the solution was an anti-tumor factor; however, the components that are generated through the interactions between plasma and L-sodium lactate and the components responsible for the selective killing of cancer cells remain unidentified. In this study, we quantified several major chemical products, such as pyruvate, formate, and acetate, in plasma-activated L-sodium lactate solution by nuclear magnetic resonance analysis. We further identified novel chemical products, such as glyoxylate and 2,3-dimethyltartrate, in the solution by direct infusion-electrospray ionization with tandem mass spectrometry analysis. We found that 2,3-dimethyltartrate exhibited cytotoxic effects in glioblastoma cells, but not in normal astrocytes. These findings shed light on the identities of the components that are responsible for the selective cytotoxic effect of plasma-activated solutions on cancer cells, and provide useful data for the potential development of cancer treatments using plasma-activated L-sodium lactate solution.

Extracellular vesicles derived from inflamed murine colorectal tissue induce fibroblast proliferation via epidermal growth factor receptor.

Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract. Although IBDs increase the risk of colitis-associated colon cancer, the underlying mechanisms are not fully understood. Extracellular vesicles (EVs) are lipid-bound sacs that transport proteins, RNA, and lipids between cells and are key mediators of cellular communication in both physiological and pathological settings. EVs have been implicated in many cancer hallmarks, including uncontrolled tumor growth and metastasis. In this study, we investigated the effects of colon-derived EVs on the proliferation of fibroblasts. We used comparative proteomics to characterize protein profiles of colorectal EVs isolated from healthy mice (Con-EVs) and those with dextran sulfate sodium-induced colitis (IBD-EVs). The results showed that 109 proteins were upregulated in IBD-EVs. Notably, expression of epidermal growth factor receptor (EGFR), which plays important roles in cell proliferation and development, was increased in IBD-EVs. We then examined the effect of EVs on murine NIH3T3 fibroblasts and found that IBD-EVs significantly promoted cell proliferation in EGFR- and ERK-dependent manner. Our findings suggest that inflamed colon-derived EVs promote tumor development thorough activation of fibroblasts.

Glycolaldehyde is an endogenous source of lysine N-pyrrolation.

Lysine N-pyrrolation converts lysine residues to Nϵ-pyrrole-l-lysine (pyrK) in a covalent modification reaction that significantly affects the chemical properties of proteins, causing them to mimic DNA. pyrK in proteins has been detected in vivo, indicating that pyrrolation occurs as an endogenous reaction. However, the source of pyrK remains unknown. In this study, on the basis of our observation in vitro that pyrK is present in oxidized low-density lipoprotein and in modified proteins with oxidized polyunsaturated fatty acids, we used LC-electrospray ionization-MS/MS coupled with a stable isotope dilution method to perform activity-guided separation of active molecules in oxidized lipids and identified glycolaldehyde (GA) as a pyrK source. The results from mechanistic experiments to study GA-mediated lysine N-pyrrolation suggested that the reactions might include GA oxidation, generating the dialdehyde glyoxal, followed by condensation reactions of lysine amino groups with GA and glyoxal. We also studied the functional significance of GA-mediated lysine N-pyrrolation in proteins and found that GA-modified proteins are recognized by apolipoprotein E, a binding target of pyrrolated proteins. Moreover, GA-modified proteins triggered an immune response to pyrrolated proteins, and monoclonal antibodies generated from mice immunized with GA-modified proteins specifically recognized pyrrolated proteins. These findings reveal that GA is an endogenous source of DNA-mimicking pyrrolated proteins and may provide mechanistic insights relevant for innate and autoimmune responses associated with glucose metabolism and oxidative stress.

Apolipoprotein E binds to and reduces serum levels of DNA-mimicking, pyrrolated proteins.

Lysine N-pyrrolation, converting lysine residues to Nϵ-pyrrole-l-lysine, is a recently discovered post-translational modification. This naturally occurring reaction confers electrochemical properties onto proteins that potentially produce an electrical mimic to DNA and result in specificity toward DNA-binding molecules such as anti-DNA autoantibodies. The discovery of this unique covalent protein modification provides a rationale for establishing the molecular mechanism and broad functional significance of the formation and regulation of Nϵ-pyrrole-l-lysine-containing proteins. In this study, we used microbeads coupled to pyrrolated or nonpyrrolated protein to screen for binding activities of human serum-resident nonimmunoglobin proteins to the pyrrolated proteins. This screen identified apolipoprotein E (apoE) as a protein that innately binds the DNA-mimicking proteins in serum. Using an array of biochemical assays, we observed that the pyrrolated proteins bind to the N-terminal domain of apoE and that oligomeric apoE binds these proteins better than does monomeric apoE. Employing surface plasmon resonance and confocal microscopy, we further observed that apoE deficiency leads to significant accumulation of pyrrolated serum albumin and is associated with an enhanced immune response. These results, along with the observation that apoE facilitates the binding of pyrrolated proteins to cells, suggest that apoE may contribute to the clearance of pyrrolated serum proteins. Our findings uncover apoE as a binding target of pyrrolated proteins, providing a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.

2-Alkenal modification of hemoglobin: Identification of a novel hemoglobin-specific alkanoic acid-histidine adduct.

α,ß-Unsaturated aldehydes generated during lipid peroxidation, such as 2-alkenals, give rise to protein degeneration in a variety of pathological states. 2-Alkenals are highly reactive toward nucleophilic amino acid residues, such as histidine and lysine, to form Schiff base adducts or Michael addition adducts. In this study, upon the reaction of hemoglobin with 2-octenal, we unexpectedly detected a product corresponding to the reduced form of the 2-octenal-histidine Michael adduct plus 14 mass unit. Based on the LC-ESI-MS/MS analysis of synthetic adduct candidates, the adduct was identified to be Nτ-(1-carboxyheptan-2-yl)-histidine (CHH), a novel alkanoic acid-type histidine adduct. The alkanoic acid-histidine adducts were detected in the 2-alkenal-treated hemoglobin and myoglobin, but not in the 2-alkenal-treated cytochrome c and transferrin. The addition of hemin to the reaction mixture, containing a non-heme protein and 2-alkenals, resulted in the formation of the alkanoic acid-histidine adducts, suggesting that a heme iron may play a role in the oxidation of covalently modified proteins. Moreover, using the stable isotope dilution method, we showed evidence for the endogenous formation of CHH in red blood cells exposed to hydrogen peroxide. Thus, this study establishes a novel mechanism for covalent modification of proteins by 2-alkenals, in which heme iron is involved in the formation of the alkanoic acid-histidine adducts. The potential implications of this novel adduct are discussed.

Structural and functional insights into S-thiolation of human serum albumins.

Human serum albumin (HSA) is the most abundant serum protein, contributing to the maintenance of redox balance in the extracellular fluids. One single free cysteine residue at position 34 is believed to be a target of oxidation. However, the molecular details and functions of oxidized HSAs remain obscure. Here we analyzed serum samples from normal subjects and hyperlipidemia patients and observed an enhanced S-thiolation of HSA in the hyperlipidemia patients as compared to the control individuals. Both cysteine and homocysteine were identified as the low molecular weight thiols bound to the HSAs. Intriguingly, S-thiolations were observed not only at Cys34, but also at multiple cysteine residues in the disulfide bonds of HSA. When the serum albumins from genetically modified mice that exhibit high levels of total homocysteine in serum were analyzed, we observed an enhanced S-homocysteinylation at multiple cysteine residues. In addition, the cysteine residues in the disulfide bonds were also thiolated in recombinant HSA that had been treated with the disulfide molecules. These findings and the result that S-homocysteinylation mediated increased surface hydrophobicity and ligand binding activity of HSA offer new insights into structural and functional alternation of serum albumins via S-thiolation.

Modification of Tau by 8-Nitroguanosine 3',5'-Cyclic Monophosphate (8-Nitro-cGMP): EFFECTS OF NITRIC OXIDE-LINKED CHEMICAL MODIFICATION ON TAU AGGREGATION.

Neurofibrillar tangles caused by intracellular hyperphosphorylated tau inclusion and extracellular amyloid ß peptide deposition are hallmarks of Alzheimer's disease. Tau contains one or two cysteine residues in three or four repeats of the microtubule binding region following alternative splicing of exon 10, and formation of intermolecular cysteine disulfide bonds accelerates tau aggregation. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) acts as a novel second messenger of nitric oxide (NO) by covalently binding cGMP to cysteine residues by electrophilic properties, a process termed protein S-guanylation. Here we studied S-guanylation of tau and its effects on tau aggregation. 8-Nitro-cGMP exposure induced S-guanylation of tau both in vitro and in tau-overexpressed HEK293T cells. S-guanylated tau inhibited heparin-induced tau aggregation in a thioflavin T assay. Atomic force microscopy observations indicated that S-guanylated tau could not form tau granules and fibrils. Further biochemical analyses showed that S-guanylated tau was inhibited at the step of tau oligomer formation. In P301L tau-expressing Neuro2A cells, 8-nitro-cGMP treatment significantly reduced the amount of sarcosyl-insoluble tau. NO-linked chemical modification on cysteine residues of tau could block tau aggregation, and therefore, increasing 8-nitro-cGMP levels in the brain could become a potential therapeutic strategy for Alzheimer's disease.

Degradation of bradykinin by a metalloendopeptidase from Streptococcus pyogenes.

OBJECTIVES: Streptococcus pyogenes secretes streptococcal pyrogenic exotoxin B (SpeB), which cleaves kininogen to liberate bradykinin. In addition, this bacterium also has cell-associated bradykinin-degrading activity. Here, we characterized the bradykinin-degrading enzyme produced by S. pyogenes. METHODS: The effects of various peptidase inhibitors on bradykinin degradation by intact S. pyogenes and cell lysates were assessed. Cleavage of bradykinin and other peptides by a recombinant putative metalloendopeptidase (Sp-Pep) from S. pyogenes was analyzed by mass spectrometry. The enhancement of vascular permeability induced by bradykinin (before and after treatment with Sp-Pep) was evaluated in guinea pig skin. RESULTS: Various S. pyogenes strains expressed Sp-Pep. Immunoadsorption of S. pyogenes with an anti-Sp-Pep antibody showed that 80% of the bradykinin-degrading activity in S. pyogenes was due to Sp-Pep. Recombinant Sp-Pep cleaved bradykinin, and cleavage caused a loss of its extravasation-inducing potential. Sp-Pep-mediated degradation of bradykinin was 40 times more efficient than degradation of substance P and angiotensin II. While S. pyogenes secreted mature SpeB in stationary phase, this bacterium produced Sp-Pep during all tested growth phases. CONCLUSIONS: S. pyogenes produces a cell-associated metalloendopeptidase that degrades bradykinin.

Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.

Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80(+) foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.

Nitrated cyclic GMP modulates guard cell signaling in Arabidopsis.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-5'-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells.

Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR.

Helicobacter cinaedi is the most frequently reported enterohepatic Helicobacter species isolated from humans. Earlier research suggested that certain patients with H. cinaedi infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods. Here, we report a nested PCR assay that rapidly detects the cytolethal distending toxin gene (cdt) of H. cinaedi with high specificity and sensitivity. Specificity of the assay was validated by using different species of Helicobacter and Campylobacter, as well as known H. cinaedi-positive and -negative samples. The sensitivity of detection for the cdt gene in the assay was 10(2) CFU/ml urine or 10(2) CFU/10(5) infected RAW 264.7 cells. In an H. cinaedi-infected mouse model, the cdt gene of H. cinaedi was effectively detected via the assay with urine (6/7), stool (2/3), and blood (2/6) samples. Importantly, it detected H. cinaedi in blood, urine, and stool samples from one patient with a suspected H. cinaedi infection and three patients with known infections. The assay was further used clinically to follow up two H. cinaedi-infected patients after antibiotic treatment. Stool samples from these two patients evaluated by nested PCR after antibiotic therapy showed clearance of bacterial DNA. Finally, analysis of stool specimens from healthy volunteers showed occasional positive reactions (4/30) to H. cinaedi DNA, which suggests intestinal colonization by H. cinaedi in healthy subjects. In conclusion, this nested PCR assay may be useful for the rapid diagnosis, antimicrobial treatment evaluation, and epidemiological study of H. cinaedi infection.

Nitric oxide promotes recycling of 8-nitro-cGMP, a cytoprotective mediator, into intact cGMP in cells.

8-Nitro-cGMP is an endogenous nucleotide discovered under inflammation conditions as an important mediator of nitric oxide (NO) signaling. Besides cGMP-like behaviour, 8-nitro-cGMP exerts unique cytoprotective effects against oxidative stress. Although the formation of 8-nitro-cGMP from 8-nitro-GTP has previously been proposed, the mechanism by which excess or unused 8-nitro-cGMP is removed from cells remains unknown. In this study, we report a nitric oxide-dependent cellular conversion of 8-nitro-cGMP to intact cGMP in RAW 264.7 macrophage cells. In our experiments, we synthesized isotopically labeled 8-nitro-cGMP as a tool for metabolites analysis and identified 8-amino-cGMP as an initial metabolite of 8-nitro-cGMP using a LC-MS/MS technique. We also proved that endogenous 8-nitro-cGMP can be converted into 8-amino-cGMP by immunocytochemical staining with an antibody that specifically recognizes 8-amino-cGMP. Moreover, we showed that isotopically labeled 8-amino-cGMP is metabolized into cGMP under inflammation conditions. We propose that nitrosylation of 8-amino-cGMP occurs by NO formation under stress conditions and gives putative 8-diazonium-cGMP, which subsequently decomposes into cGMP. To the best of our knowledge, this study is the first to report reductive deamination of aminoguanine nucleotide at the C-8 position. The findings of this study collectively indicate that NO plays a crucial role not only in the production of 8-nitro-cGMP but also in its elimination under oxidative stress or inflammation.