Heme-dependent Metabolite Switching Regulates H2S Synthesis in Response to Endoplasmic Reticulum (ER) Stress.
ABSTRACT: Substrate ambiguity and relaxed reaction specificity underlie the diversity of reactions catalyzed by the transsulfuration pathway enzymes, cystathionine ?-synthase (CBS) and ?-cystathionase (CSE). These enzymes either commit sulfur metabolism to cysteine synthesis from homocysteine or utilize cysteine and/or homocysteine for synthesis of H2S, a signaling molecule. We demonstrate that a kinetically controlled heme-dependent metabolite switch in CBS regulates these competing reactions where by cystathionine, the product of CBS, inhibits H2S synthesis by the second enzyme, CSE. Under endoplasmic reticulum stress conditions, induction of CSE and up-regulation of the CBS inhibitor, CO, a product of heme oxygenase-1, flip the operating preference of CSE from cystathionine to cysteine, transiently stimulating H2S production. In contrast, genetic deficiency of CBS leads to chronic stimulation of H2S production. This metabolite switch from cystathionine to cysteine and/or homocysteine renders H2S synthesis by CSE responsive to the known modulators of CBS: S-adenosylmethionine, NO, and CO. Used acutely, it regulates H2S synthesis; used chronically, it might contribute to disease pathology.
Project description:The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine ?-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine ?-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine ? cystathionine ? cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 ?mol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812.
Project description:In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H2S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H2S generation. CBS from both organisms shows a marked preference for H2S generation by beta-replacement of cysteine by homocysteine. The alternative H2S-generating reactions, i.e. beta-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H2S production by CBS would account for approximately 25-70% of the total H2S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H2S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H2S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H2S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.
Project description:Hydrogen sulfide (H2S), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of H2S or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and H2S regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or Na2S/GYY4137, a H2S donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas Na2S/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the Na2S-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/-, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for H2S-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.
Project description:Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys-SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys-SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys-SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys-SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and ?-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys-SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys-SSH product is short and decomposition leads to a mixture of polysulfides (Cys-S-(S)n-S-Cys). These in vitro data, together with the intrinsic reactivity of Cys-SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.
Project description:Human cystathionine beta-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits homocysteine to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that interact with CBS had previously identified several components of the sumoylation pathway and resulted in the demonstration that CBS is a substrate for sumoylation. In this study, we demonstrate that sumoylation of CBS is enhanced in the presence of human polycomb group protein 2 (hPc2), an interacting partner that was identified in the initial yeast two-hybrid screen. When the substrates for CBS, homocysteine and serine for cystathionine generation and homocysteine and cysteine for H(2)S generation, are added to the sumoylation mixture, they inhibit the sumoylation reaction, but only in the absence of hPc2. Similarly, the product of the CBS reaction, cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn decreases CBS activity by approximately 28% in the absence of hPc2 and by 70% in its presence. Based on these results, we conclude that hPc2 serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We also demonstrate that gamma-cystathionase, the second enzyme in the transsulfuration pathway is a substrate for sumoylation under in vitro conditions. We speculate that the role of this modification may be for nuclear localization of the cysteine-generating pathway under conditions where nuclear glutathione demand is high.
Project description:Cystathionine ?-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. In addition to pyridoxal phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe(III)-CBS is inert to exogenous ligands, Fe(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O2 to yield superoxide radical. In this study, we have examined the kinetics of Fe(II)CO-CBS formation and reoxidation. Reduction of Fe(III)-CBS by dithionite showed a square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2(•-)) that exists in rapid equilibrium with S2O4(2-). Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred with a rate constant of (3.1 ± 0.4) × 10(-3) s(-1) (pH 7.4, 25 °C). The reaction of Fe(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe(II)CO-CBS. Fe(II)-CBS was formed as an intermediate with a rate constant of (9.3 ± 2.5) × 10(2) M(-1) s(-1). Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed a hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence of an effect of exogenous H2S on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for cross talk among the CO, H2S, and superoxide signaling pathways.
Project description:In recent years, the gasotransmitter hydrogen sulphide (H2S), produced by the transsulphuration pathway, has been recognized as a biological mediator playing an important role under normal conditions and in various pathologies in both eukaryotes and prokaryotes. The transsulphuration pathway (TSP) includes the conversion of homocysteine to cysteine following the breakdown of methionine. In Drosophila melanogaster and other eukaryotes, H2S is produced by cystathionine ?-synthase (CBS), cystathionine ?-lyase (CSE), and 3-mercaptopyruvate sulphurtransferase (MST). In the experiments performed in this study, we were able to explore the CRISPR/Cas9 technique to obtain single and double deletions in homozygotes of these three major genes responsible for H2S production in Drosophila melanogaster. In most cases, the deletion of one studied gene does not result in the compensatory induction of two other genes responsible for H2S production. Transcriptomic studies demonstrated that the deletions of the above CBS and CSE genes alter genome expression to different degrees, with a more pronounced effect being exerted by deletion of the CBS gene. Furthermore, the double deletion of both CBS and CSE resulted in a cumulative effect on transcription in the resulting strains. Overall, we found that the obtained deletions affect numerous genes involved in various biological pathways. Specifically, genes involved in the oxidative reduction process, stress-response genes, housekeeping genes, and genes participating in olfactory and reproduction are among the most strongly affected. Furthermore, characteristic differences in the response to the deletions of the studied genes are apparently organ-specific and have clear-cut sex-specific characteristics. Single and double deletions of the three genes responsible for the production of H2S helped to elucidate new aspects of the biological significance of this vital physiological mediator.
Project description:The cystathionine ß-synthase (CBS) is a critical enzyme in the transsulfuration pathway and is responsible for the synthesis of cystathionine from serine and homocysteine. Cystathionine is a precursor to amino acid cysteine. CBS is also responsible for generation of hydrogen sulfide (H2S) from cysteine. Mutation in CBS enzyme causes homocysteine levels to rise, and gives rise to a condition called hyperhomocysteinuria. To date, numerous mouse knockout models for CBS enzyme has been generated, which show panoply of defects, reflecting the importance of this enzyme in development. In zebrafish, we and others have identified two orthologs of cbs, which we call cbsa and cbsb. Previous gene knockdown studies in zebrafish have reported a function for cbsb ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining H2S homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for cbsa and cbsb primarily along the embryonic axis of the developing embryo. Loss-of-function analysis using a combination of approaches which include splice morpholinos and CRISPR/Cas9 genomic engineering show evidence that cbsb ortholog is responsible for anterior-posterior axis development, and cbsa function is redundant. Cbsb loss of function fish embryos show shortened and bent axis, along with less H2S and more homocysteine, effects resulting from loss of Cbsb. Using a chemical biology approach, we rescued the axis defects with betaine, a compound known to reduce homocysteine levels in plasma, and GYY4137, a long term H2S donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and H2S levels, pathways that are controlled by Cbsb, and thus is essential for development.
Project description:Hydrogen sulfide (H2S) is substantially converted from cysteine by the enzymes cystathionine ?-synthase (CBS) and cystathionine ?-lyase (CSE). H2S can profoundly affect most organ systems in animals and humans by inducing a wide range of physiological functions. However, the roles of H2S in the progression of endometriosis remain unknown. The aim of the current study was to test the hypothesis that H2S might play a role in the pathogenesis of endometriosis via modulating the biological behavior of endometrial stromal cells (ESCs). First, we explored the expression level of CBS and CSE in ESCs via immunohistochemistry and immunocytochemistry. Second, cell Count Kit-8 (CCK-8) assays were utilized to investigate the cell viability of human ESCs (HESCs) in vitro. Third, we studied the potential effects of H2S in a rodent model of endometriosis. Both CBS and CSE were overexpressed in endometriotic lesions. Exogenous and endogenous H2S could promote HESC proliferation in vitro. Furthermore, this pro-proliferation effect could be reversed by treating with inhibitors of CBS, CSE, or the NF-?B pathway. In vivo, we uncovered that inhibitors of CBS and CSE could remarkably reduce the number and weight of mouse endometriotic lesions. These data suggested that H2S promotes ESC proliferation via activation of the NF-?B pathway, which provides a scientific basis for the clinical application of blocking H2S to treat endometriosis.
Project description:Endogenous hydrogen sulfide (H2S) synthesized via metabolizing L-cysteine by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) is a potent vasodilator and angiogenic factor. The objectives of this study were to determine if human uterine artery (UA) H2S production increases with augmented expression and/or activity of CBS and/or CSE during the menstrual cycle and pregnancy and whether exogenous H2S dilates UA. Uterine arteries from nonpregnant (NP) premenopausal proliferative (pPRM) and secretory (sPRM) phases of the menstrual cycle and pregnant (P) women were studied. H2S production was measured by the methylene blue assay. CBS and CSE mRNAs were assessed by quantitative real-time PCR, and proteins were assessed by immunoblotting and semiquantitative immunofluorescence microscopy. Effects of H2S on rat UA relaxation were determined by wire myography ex vivo. H2S production was greater in NP pPRM and P than NP sPRM UAs and inhibited by the specific CBS but not CSE inhibitor. CBS but not CSE mRNA and protein were greater in NP pPRM and P than NP sPRM UAs. CBS protein was localized to endothelium and smooth muscle and its levels were in a quantitative order of P >NP UAs of pPRM>sPRM. CSE protein was localized in UA endothelium and smooth muscle with no difference among groups. A H2S donor relaxed P > NP UAs but not mesentery artery. Thus, human UA H2S production is augmented with endothelium and smooth muscle CBS upregulation, contributing to UA vasodilation in the estrogen-dominant physiological states in the proliferative phase of the menstrual cycle and pregnancy.