The Antioxidant Properties of Glucosinolates in Cardiac Cells Are Independent of H2S Signaling.

The organic sulfur-containing compounds glucosinolates (GSLs) and the novel gasotransmitter H2S are known to have cardioprotective effects. This study investigated the antioxidant effects and H2S-releasing potential of three GSLs ((3E)-4-(methylsulfanyl)but-3-enyl GSL or glucoraphasatin, 4-hydroxybenzyl GSL or glucosinalbin, and (RS)-6-(methylsulfinyl)hexyl GSL or glucohesperin) in rat cardiac cells. It was found that all three GSLs had no effect on cardiac cell viability but were able to protect against H2O2-induced oxidative stress and cell death. NaHS, a H2S donor, also protected the cells from H2O2-stimulated oxidative stress and cell death. The GSLs alone or mixed with cysteine, N-acetylcysteine, glutathione, H2O2, iron and pyridoxal-5'-phosphate, or mouse liver lysates did not induce H2S release. The addition of GSLs also did not alter endogenous H2S levels in cardiac cells. H2O2 significantly induced cysteine oxidation in the cystathionine gamma-lyase (CSE) protein and inhibited the H2S production rate. In conclusion, this study found that the three tested GSLs protect cardiomyocytes from oxidative stress and cell death but independently of H2S signaling.

Deficiency of cystathionine gamma-lyase promotes aortic elastolysis and medial degeneration in aged mice.

Enzymatic degradation of elastin by matrix metalloproteinases (MMPs) leads to the permanent dilation of aortic wall and constitutes the most prominent characters of aortic aneurysm and aging-related medial degeneration. Hydrogen sulfide (H2S) as a gasotransmitter exhibits a wide variety of cardio-protective functions through its anti-inflammatory and anti-oxidative actions. Cystathionine gamma-lyase (CSE) is a main H2S-generating enzyme in cardiovascular system. The regulatory roles of CSE/H2S system on elastin homeostasis and blood vessel degeneration have not yet been explored. Here we found that aged CSE knockout mice had severe aortic dilation and elastic degradation in abdominal aorta and were more sensitive to angiotensin II-induced aortic elastolysis and medial degeneration. Administration of NaHS would protect the mice from angiotensin II-induced inflammation, gelatinolytic activity, elastin fragmentation, and aortic dilation. In addition, human aortic aneurysm samples had higher inflammatory infiltration and lower expression of CSE. In cultured smooth muscle cells (SMCs), TNFα-induced MMP2/9 hyperactivity and elastolysis could be attenuated by exogenously applied NaHS or CSE overexpression while further deteriorated by complete knockout of CSE. It was further found that H2S inhibited MMP2 transcription by posttranslational modification of Sp1 via S-sulfhydration. H2S also directly suppressed MMP hyperactivity by S-sulfhydrating the cysteine switch motif. Taken together, this study revealed the involvement of CSE/H2S system in the pathogenesis of aortic elastolysis and medial degeneration by maintaining the inactive form of MMPs, suggesting that CSE/H2S system can be a target for the prevention of age-related medial degeneration and treatment of aortic aneurysm.

Effect of hydrogen sulfide on glycolysis-based energy production in mouse erythrocytes.

Hydrogen sulfide (H2 S) is a gasotransmitter that regulates both physiological and pathophysiological processes in mammalian cells. Recent studies have demonstrated that H2 S promotes aerobic energy production in the mitochondria in response to hypoxia, but its effect on anaerobic energy production has yet to be established. Glycolysis is the anaerobic process by which ATP is produced through the metabolism of glucose. Mammalian red blood cells (RBCs) extrude mitochondria and nucleus during erythropoiesis. These cells would serve as a unique model to observe the effect of H2 S on glycolysis-mediated energy production. The purpose of this study was to determine the effect of H2 S on glycolysis-mediated energy production in mitochondria-free mouse RBCs. Western blot analysis showed that the only H2 S-generating enzyme expressed in mouse RBCs is 3-mercaptopyruvate sulfurtransferase (MST). Supplement of the substrate for MST stimulated, but the inhibition of the same suppressed, the endogenous production of H2 S. Both exogenously administered H2 S salt and MST-derived endogenous H2 S stimulated glycolysis-mediated ATP production. The effect of NaHS on ATP levels was not affected by oxygenation status. On the contrary, hypoxia increased intracellular H2 S levels and MST activity in mouse RBCs. The mitochondria-targeted H2 S donor, AP39, did not affect ATP levels of mouse RBCs. NaHS at low concentrations (3-100 µM) increased ATP levels and decreased cell viability after 3 days of incubation in vitro. Higher NaHS concentrations (300-1000 µM) lowered ATP levels, but prolonged cell viability. H2 S may offer a cytoprotective effect in mammalian RBCs to maintain oxygen-independent energy production.

Cystathionine gamma-lyase/H2 S signaling facilitates myogenesis under aging and injury condition.

Hydrogen sulfide (H2 S) can be endogenously produced and belongs to the class of signaling molecules known as gasotransmitters. Cystathionine gamma-lyase (CSE)-derived H2 S is implicated in the regulation of cell differentiation and the aging process, but the involvements of the CSE/H2 S system in myogenesis upon aging and injury have not been explored. In this study, we demonstrated that CSE acts as a major H2 S-generating enzyme in skeletal muscles and is significantly down-regulated in aged skeletal muscles in mice. CSE deficiency exacerbated the age-dependent sarcopenia and cardiotoxin-induced injury/regeneration in mouse skeletal muscle, possibly attributed to inefficient myogenesis. In contrast, supplement of NaHS (an H2 S donor) induced the expressions of myogenic genes and promoted muscle regeneration in mice. In vitro, incubation of myoblast cells (C2C12) with H2 S promoted myogenesis, as evidenced by the inhibition of cell cycle progression and migration, altered expressions of myogenic markers, elongation of myoblasts, and formation of multinucleated myotubes. Myogenesis was also found to upregulate CSE expression, while blockage of CSE/H2 S signaling resulted in a suppression of myogenesis. Mechanically, H2 S significantly induced the heterodimer formation between MEF2c and MRF4 and promoted the binding of MEF2c/MRF4 to myogenin promoter. MEF2c was S-sulfhydrated at both cysteine 361 and 420 in the C-terminal transactivation domain, and blockage of MEF2c S-sulfhydration abolished the stimulatory role of H2 S on MEF2c/MRF4 heterodimer formation. These findings support an essential role for H2 S in maintaining myogenesis, presenting it as a potential candidate for the prevention of age-related sarcopenia and treatment of muscle injury.

Hydrogen sulfide and metal interaction: the pathophysiological implications.

Hydrogen sulfide (H2S), previously recognized as a toxic gas, has emerged as an important gaseous signaling molecule along with nitric oxide, carbon monoxide and also hydrogen. H2S can be endogenously produced in the mammalian body at a very low level for various pathophysiological processes. Notably, H2S can interact with several essential metals in the body such as iron, copper, nickel, and zinc to carry out specific functions. The interactions of H2S with metal-binding proteins have been shown to aid in its signal transduction and cellular metabolism. In addition, H2S is capable of providing a cytoprotective role against metal toxicity. As the research in the field of H2S signaling in biology and medicine increases, much progresses have been developed for detecting H2S via interaction with metals. In this review, the interaction of H2S with metals, specifically in regard to metal-driven metabolism of H2S, the protection against metal toxicity by H2S and the detection of H2S using metals will be discussed. Discovering the interactions of this gasotransmitter with metals is important for determining the mechanisms underlying the cellular functions of H2S as well as developing novel therapeutic avenues.

H2S protects from oxidative stress-driven ACE2 expression and cardiac aging.

Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H2S) plays an essential role in preserving cardiac functions. Angiotensin-converting enzyme 2 (ACE2) acts as the negative regulator of the renin-angiotensin system, exerting anti-oxidative stress and anti-inflammatory properties within the body. The interplays of CSE/H2S signaling and ACE2 in cardiac aging are unclear. In this study, the regulatory roles of H2S on ACE2 expression in mouse heart tissue and rat cardiomyocytes under different stress conditions were investigated. It was found that ACE2 protein level was lower in heart tissues from old mice (56-week-old) than young mice (8-week-old), and the knockout of CSE (CSE KO) induced moderate oxidative stress and further inhibited ACE2 protein level in mouse hearts at both young and old age. Incubation of rat cardiac cells (H9C2) with a low dose of H2O2 (50 µM) suppressed ACE2 protein level and induced cellular senescence, which was completely reversed by co-incubation with 30 µM NaHS (a H2S donor). Prolonged nutrient excess is an increased risk of heart disorders by causing metabolic dysfunction and cardiac remodeling. We further found high-fat diet feeding stimulated ACE2 expression and induced severe oxidative stress in CSE KO heart in comparison with wild-type heart. Lipid overload in H9C2 cells to mimic a status of nutrient excess also enhanced the expression of ACE2 protein and induced severe oxidative stress and cell senescence, which were significantly attenuated by the supplementation of exogenous H2S. Furthermore, the manipulation of ACE2 expression partially abolished the protective role of H2S against cellular senescence. These results demonstrate the dynamic roles of H2S in the maintenance of ACE2 levels under different levels of oxidative stress, pointing to the potential implications in targeting the CSE/H2S system for the interruption of aging and diabetes-related heart disorders.

The interaction of disulfiram and H2S metabolism in inhibition of aldehyde dehydrogenase activity and liver cancer cell growth.

Disulfiram (DSF), a sulfur-containing compound, has been used to treat chronic alcoholism and cancer for decades by inactivating aldehyde dehydrogenase (ALDH). Hydrogen sulfide (H2S) is a new gasotransmitter and regulates various cellular functions by S-sulfhydrating cysteine in the target proteins. H2S exhibits similar properties to DSF in the sensitization of cancer cells. The interaction of DSF and H2S on ALDH activity and liver cancer cell survival are not clear. Here it was demonstrated that DSF facilitated H2S release from thiol-containing compounds, and DSF and H2S were both capable of regulating ALDH through inhibition of gene expression and enzymatic activity. The supplement of H2S sensitized human liver cancer cells (HepG2) to DSF-inhibited cell viability. The expression of cystathionine gamma-lyase (a major H2S-generating enzyme) was lower but ALDH was higher in mouse liver cancer stem cells (Dt81Hepa1-6) in comparison with their parental cells (Hepa1-6), and H2S was able to inhibit liver cancer stem cell adhesion. In conclusion, these data point to the potential of combining DSF and H2S for inhibition of cancer cell growth and tumor development by targeting ALDH.

An evolutionary perspective on the interplays between hydrogen sulfide and oxygen in cellular functions.

The physiological effects of the endogenously generated hydrogen sulfide (H2S) have been extensively studied in recent years. This review summarized the role of H2S in the origin of life and H2S metabolism in organisms from bacteria to vertebrates, examined the relationship between H2S and oxygen from an evolutionary perspective and emphasized the oxygen-dependent manner of H2S signaling in various physiological and pathological processes. H2S and oxygen are inextricably linked in various cellular functions. H2S is involved in aerobic respiration and stimulates oxidative phosphorylation and ATP production within the cell. Besides, H2S has protective effects on ischemia and reperfusion injury in several organs by acting as an oxygen sensor. Also, emerging evidence suggests the role of H2S is in an oxygen-dependent manner. All these findings indicate the subtle relationship between H2S and oxygen and further explain why H2S, a toxic molecule thriving in an anoxia environment several billion years ago, still affects homeostasis today despite the very low content in the body.

H2S signaling and extracellular matrix remodeling in cardiovascular diseases: A tale of tense relationship.

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network that not only provides mechanical support but also transduces essential molecular signals in organ functions. ECM is constantly remodeled to control tissue homeostasis, responsible for cell adhesion, cell migration, cell-to-cell communication, and cell differentiation, etc. The dysregulation of ECM components contributes to various diseases, including cardiovascular diseases, fibrosis, cancer, and neurodegenerative diseases, etc. Aberrant ECM remodeling is initiated by various stress, such as oxidative stress, inflammation, ischemia, and mechanical stress, etc. Hydrogen sulfide (H2S) is a gasotransmitter that exhibits a wide variety of cytoprotective and physiological functions through its anti-oxidative and anti-inflammatory actions. Amounting research shows that H2S can attenuate aberrant ECM remodeling. In this review, we discussed the implications and mechanisms of H2S in the regulation of ECM remodeling in cardiovascular diseases, and highlighted the potential of H2S in the prevention and treatment of cardiovascular diseases through attenuating adverse ECM remodeling.

Spermine Protects Cardiomyocytes from High Glucose-Induced Energy Disturbance by Targeting the CaSR-gp78-Ubiquitin Proteasome System.

PURPOSE: To determine the mediation of spermine on energy metabolism disorder and diabetic cardiomyopathy (DCM) development as well as the underlying mechanisms. METHODS: An in vitro model of DCM was established by incubating primary cultured neonatal rat cardiomyocytes with high glucose (HG). Spermine content was assessed by RP-HPLC. The protein levels were detected by western blot. Mitochondrial functions were analyzed using the respiratory chain complex assay kit and immunofluorescence staining. RESULTS: The endogenous content of spermine was decreased in the HG group, and the protein levels of ornithine decarboxylase, respiratory chain complex (I-V), mitochondrial fusion-related protein (Mfn1, Mfn2), Cx43, N-cadherin, CaSR, and ß-catenin (in cytomembrane) were also down-regulated by HG. In contrast, the protein levels of spermine-N1-acetyltransferase, gp78, Fis1, Drp1, and ß-catenin were up-regulated by HG. Meanwhile, we observed that HG increased ubiquitination levels of Mfn1, Mfn2, and Cx43, decreased membrane potential (ΔΨm), and the opening of mitochondrial permeability transport pore (mPTP) followed by intracellular ATP leakage. The supplement of spermine or siRNA-mediated knockdown of gp78 significantly alleviated the detrimental effects of HG, while downregulation of CaSR aggravated the development of DCM. We further confirmed that the lower level of spermine by HG activates the gp78-ubiquitin-proteasome pathway via downregulation of CaSR protein level, which in turn damages mitochondrial gap junction intercellular communication and leads to reduced ATP level. CONCLUSION: The protective role of spermine on energy metabolism disorder is based on higher CaSR protein level and lower gp78 activation, pointing to the possibility that spermine can be a target for the prevention and treatment of DCM.

H2S as a potential defense against COVID-19?

The outbreak of COVID-19 pneumonia caused by a new coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is posing a global health emergency and has led to more than 380,000 deaths worldwide. The cell entry of SARS-CoV-2 depends on two host proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). There is currently no vaccine available and also no effective drug for the treatment of COVID-19. Hydrogen sulfide (H2S) as a novel gasotransmitter has been shown to protect against lung damage via its anti-inflammation, antioxidative stress, antiviral, prosurvival, and antiaging effects. In light of the research advances on H2S signaling in biology and medicine, this review proposed H2S as a potential defense against COVID-19. It is suggested that H2S may block SARS-CoV-2 entry into host cells by interfering with ACE2 and TMPRSS2, inhibit SARS-CoV-2 replication by attenuating virus assembly/release, and protect SARS-CoV-2-induced lung damage by suppressing immune response and inflammation development. Preclinical studies and clinical trials with slow-releasing H2S donor(s) or the activators of endogenous H2S-generating enzymes should be considered as a preventative treatment or therapy for COVID-19.

Hydrogen sulfide signaling in regulation of cell behaviors.

Recent advances in the biomedical importance of H2S have help us understand various cellular functions and pathophysiological processes from a new aspect. Specially, H2S has been demonstrated to play multiple roles in regulating cell behaviors, including cell survival, cell differentiation, cell senescence, cell hypertrophy, cell atrophy, cell metaplasia, and cell death, etc. H2S contributes to cell behavior changes via various mechanisms, such as histone modification, DNA methylation, non-coding RNA changes, DNA damage repair, transcription factor activity, and post-translational modification of proteins by S-sulfhydration, etc. In this review, we summarized the recent research progress on H2S signaling in control of cell behaviors and discussed the ways of H2S regulation of gene expressions. Given the key roles of H2S in both health and diseases, a better understanding of the regulation of H2S on cell behavior change and the underlying molecular mechanisms will help us to develop novel and more effective strategies for clinical therapy.

Induction of cystathionine gamma-lyase expression and metallothionein-1 S-sulfhydration alleviate cadmium-induced cell death in myoblast cells.

Hydrogen sulfide (H2S), a multifunctional gasotransmitter, participates in a wide range of cellular signal transduction and pathophysiological processes. Cystathionine gamma-lyase (CSE) acts as a major H2S-generating enzyme in peripheral organs and tissues. As a cysteine-rich and heavy metal-binding protein, metallothionein-1 (MT-1) is known to protect cells from various environmental stresses. Here we demonstrated that exposure of cadmium (Cd) induced oxidative stress, depleted intracellular thiols, and stimulated apoptotic cell death in mouse myoblast cells. CSE expression and H2S production were significantly enhanced by Cd treatment. NaHS, a well-known H2S donor, at physiologically relevant concentration significantly alleviated Cd-induced damage in both myoblasts and mouse skeletal muscles. In contrast, down-regulation of CSE/H2S system deteriorated Cd-stimulated oxidative stress and cell death. Exposure of the cells to Cd lead to increased expressions of metal regulatory transcription factor 1 and MT-1, while siRNA-mediated MT-1 knockdown alleviated Cd-induced CSE expression and caused more oxidative stress and cell death. In addition, H2S post-translationally modified MT-1 by S-sulfhydration and stabilized zinc-protein complex. Taken together, these data suggest that CSE/H2S system would protect myoblasts and skeletal muscles from Cd-induced damage by S-sufhydrating MT-1.

Cystathionine gamma-lyase/hydrogen sulfide system is essential for adipogenesis and fat mass accumulation in mice.

Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter analogous to nitric oxide and carbon monoxide. Cystathionine gamma-lyase (CSE)-derived H2S is implicated in the regulation of insulin resistance and glucose metabolism, but the involvement of CSE/H2S system in energy homeostasis and fat mass has not been extensively explored. In this study, a potential functional role of the CSE/H2S system in in vitro adipocyte differentiation and in vivo adipogenesis and the underlying mechanism was investigated. CSE expression and H2S production were increased during adipocyte differentiation, and that the pattern of CSE mRNA expression was similar to that of CCAAT/enhancer-binding protein (C/EBP) ß and δ, two key regulators for adipogenesis. C/EBPß and γ bind to the CCAAT box in CSE promoter and stimulate CSE gene transcription. H2S induced PPARγ transactivation activity by S-sulfhydrating all the cysteine residues in the DNA binding domain and stimulated adipogenesis. High fat diet-induced fat mass was lost in CSE deficient mice, and exogenously applied H2S promoted fat mass accumulation in fruit flies. In conclusion, CSE/H2S system is essential for adipogenesis and fat mass accumulation through enhancement of PPARγ function in adipocytes. This study suggests that the CSE/H2S system is involved in the pathogenesis of obesity in mice.

Reversal of Sp1 transactivation and TGFß1/SMAD1 signaling by H2S prevent nickel-induced fibroblast activation.

Nickel as a heavy metal is known to bring threat to human health, and nickel exposure is associated with changes in fibroblast activation which may contribute to its fibrotic properties. H2S has recently emerged as an important gasotransmitter involved in numerous cellular signal transduction and pathophysiological responses. Interaction of nickel and H2S on fibroblast cell activation has not been studied so far. Here, we showed that a lower dose of nickel (200 µM) induced the activation of human fibroblast cells, as evidenced by increased cell growth, migration and higher expressions of α-smooth muscle actin (αSMA) and fibronectin, while high dose of nickel (1 mM) inhibited cell viability. Nickel reduced intracellular thiol contents and stimulated oxidative stress. Nickel also repressed the mRNA and protein expression of cystathionine gamma-lyase (CSE, a H2S-generating gene) and blocked the endogenous production of H2S. Exogenously applied NaHS (a H2S donor) had no effect on nickel-induced cell viability but significantly attenuated nickel-stimulated cell migration and the expression of αSMA and fibronectin. In contrast, CSE deficiency worsened nickel-induced αSMA expression. Moreover, H2S incubation reversed nickel-stimulated TGFß1/SMAD1 signal and blocked TGFß1-initiated expressions of αSMA and fibronectin. Nickel inhibited the interaction of Sp1 with CSE promoter but strengthened the binding of Sp1 with TGFß1 promoter, which was reversed by exogenously applied NaHS. These data reveal that H2S protects from nickel-stimulated fibroblast activation and CSE/H2S system can be a potential target for the treatment of tissue fibrosis induced by nickel.

H2S protects lipopolysaccharide-induced inflammation by blocking NFκB transactivation in endothelial cells.

Hydrogen sulfide (H2S) is a novel gasotransmitter and acts as a multifunctional regulator in various cellular functions. Past studies have demonstrated a significant role of H2S and its generating enzyme cystathionine gamma-lyase (CSE) in the cardiovascular system. Lipopolysaccharide (LPS), a major pathogenic factor, is known to initiate the inflammatory immune response. The cross talk between LPS-induced inflammation and the CSE/H2S system in vascular cells has not yet been elucidated in detail. Here we showed that LPS decreased CSE mRNA and protein expression in human endothelial cells and blocked H2S production in mouse aorta tissues. Transfection of the cells with TLR4-specific siRNA knockdown TLR4 mRNA expression and abolished the inhibitory role of LPS on CSE expression. Higher dose of LPS (100µg/ml) decreased cell viability, which was reversed by exogenously applied H2S at physiologically relevant concentration (30µM). Lower dose of LPS (10µg/ml) had no effect on cell viability, but significantly induced inflammation gene expressions and cytokines secretion and stimulated cell hyper-permeability. H2S treatment prevented LPS-induced inflammation and hyper-permeability. Lower VE-cadherin expression in LPS-incubated cells would contribute to cell hyper-permeability, which was reversed by H2S co-incubation. In addition, H2S treatment blocked LPS-induced NFκB transactivation. We further validated that LPS-induced hyper-permeability was reversed by CSE overexpression but further deteriorated by CRISPR/Cas9-mediated knockout of CSE. In vivo, deficiency of CSE sensitized the mice to LPS-induced inflammation in vascular tissues. Take together, these data suggest that CSE/H2S system protects LPS-induced inflammation and cell hyper-permeability by blocking NFκB transactivation.

Trend in H2S Biology and Medicine Research-A Bibliometric Analysis.

The biological and medical importance of hydrogen sulfide (H2S) has been recognized for decades. The aim of this bibliometric study is to analyze the quantity and quality of publications in H2S biology and medicine (H2SBM) based on the databases of Web of Science and Google Scholar. A total of 5881 publications published between 1990 and 2016 were analyzed. The number of H2SBM papers published before 2004 was below 100 annually, but thereafter this number rapidly increased and peaked in 2015 with more than 7-fold increase. All publications related to H2SBM research achieved a total h-index of 136 and were cited 123,074 times. The most published disciplines in H2S biomedicine research were the cardiovascular system (8.5%), neuroscience (6.5%), and gastroenterology hepatology (4.7%). The country with the greatest number of publications in the H2SBM research field was the USA with 1765 (30.0%) publications, followed by China with 995 (16.9%) publications and Japan with 555 (9.4%) publications. The top 3 most published institutes were National University of Singapore, Peking University in China, and University of Groningen in Netherlands. Nitric Oxide Biology and Chemistry was the most exploited journal for H2SBM publications with 461 articles, followed by FASEB Journal with 200 publications and Antioxidants Redox Signaling with 116 publications. The most highly cited publications and researchers in H2SBM research were also unmasked from this bibliometric analysis. Collectively, H2SBM publications exhibit a continuous trend of increase, reflecting the increased H2SBM research intensity and diversity globally.

H2S-Mediated Protein S-Sulfhydration: A Prediction for Its Formation and Regulation.

Protein S-sulfhydration is a newly discovered post-translational modification of specific cysteine residue(s) in target proteins, which is involved in a broad range of cellular functions and metabolic pathways. By changing local conformation and the final activity of target proteins, S-sulfhydration is believed to mediate most cellular responses initiated by H2S, a novel gasotransmitter. In comparison to protein S-sulfhydration, nitric oxide-mediated protein S-nitrosylation has been extensively investigated, including its formation, regulation, transfer and metabolism. Although the investigation on the regulatory mechanisms associated with protein S-sulfhydration is still in its infancy, accumulated evidence suggested that protein S-sulfhydration may share similar chemical features with protein S-nitrosylation. Glutathione persulfide acts as a major donor for protein S-sulfhydration. Here, we review the present knowledge on protein S-sulfhydration, and also predict its formation and regulation mechanisms based on the knowledge from protein S-nitrosylation.

H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells.

BACKGROUND: Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H(2)S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H(2)S S-sulfhydration is now proposed as a major mechanism for H(2)S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H(2)S and its implication in gluconeogenesis in the liver have been unknown. METHODS: Gene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. RESULTS: Exogenously applied H(2)S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H(2)S-induced gluconeogenesis. H(2)S had no effect on the expressions of PC mRNA and protein, while H(2)S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H(2)S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H(2)S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. CONCLUSIONS: CSE/H(2)S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. GENERAL SIGNIFICANCE: Tissue-specific regulation of CSE/H(2)S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.

S-sulfhydration of MEK1 leads to PARP-1 activation and DNA damage repair.

The repair of DNA damage is fundamental to normal cell development and replication. Hydrogen sulfide (H2S) is a novel gasotransmitter that has been reported to protect cellular aging. Here, we show that H2S attenuates DNA damage in human endothelial cells and fibroblasts by S-sulfhydrating MEK1 at cysteine 341, which leads to PARP-1 activation. H2S-induced MEK1 S-sulfhydration facilitates the translocation of phosphorylated ERK1/2 into nucleus, where it activates PARP-1 through direct interaction. Mutation of MEK1 cysteine 341 inhibits ERK phosphorylation and PARP-1 activation. In the presence of H2S, activated PARP-1 recruits XRCC1 and DNA ligase III to DNA breaks to mediate DNA damage repair, and cells are protected from senescence.