Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-ß-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

Recent data show that colon cancer cells selectively overexpress cystathionine-ß-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1-1 mM, while 3 mM was inhibitory. Longer-term (72 h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects.

Restoring assembly and activity of cystathionine ß-synthase mutants by ligands and chemical chaperones.

Misfolding and aggregation of mutant enzymes have been proposed to play role in the pathogenesis of homocystinuria due to cystathionine ß-synthase (CBS) deficiency. Chemical chaperones have been recently shown to facilitate proper assembly of several CBS mutants. To asses the number of patients that may respond to chaperone therapy, we examined the effect of selected CBS ligands and osmolytes on assembly and activity of 27 CBS mutants that represent 70% of known CBS alleles. The mutant enzymes were expressed in a bacterial system, and their properties were assessed by native Western blotting and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, respectively. We studied the chaperoning activity of δ-aminolevulinic acid (δ-ALA)-a heme precursor-and of three osmolytes betaine, 2-aminoethanesulfonic acid (taurine), and glycerol. Fourteen mutants responded by at least 30% increase in the amount of correctly assembled tetramers and enzymatic activity to the coexpressional presence of either 0.5 mM δ-ALA, 100 mM betaine, and/or 750 mM glycerol. Eight of these mutants (p.R266K, p.P49L, p.R125Q, p.K102N, p.R369C, p.V180A, p.P78R, p.S466L) were rescuable by all of these three substances. Four mutants showed increased formation of tetramers that was not accompanied by changes in activity. Topology of mutations appeared to determine the chaperone responsiveness, as 11 of 14 solvent-exposed mutations were substantially more responsive than three of 13 buried mutations. This study identified chaperone-responsive mutants that represent 56 of 713 known patient-derived CBS alleles and may serve as a basis for exploring pharmacological approaches aimed at correcting misfolding in homocystinuria.

Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats.

Hydrogen sulfide (H2S) is a naturally occurring gas that may act as an endogenous signaling molecule. In the brain, H2S is mainly produced by cystathionine beta-synthase (CBS) and its cellular effects have been attributed to interactions with N-methyl-D-aspartate (NMDA) receptors and cyclic adenosine 3',5'-monophosphate (cAMP). In contrast, direct vasodilator actions of H2S are most probably mediated by opening smooth muscle ATP-sensitive K+ (K(ATP)) channels. In the hypothalamus, K(ATP) channel-dependent mechanisms are involved in CNS-mediated regulation of blood pressure. In this report, we investigated the hypothesis that H2S may act via K(ATP) channels in the hypothalamus to regulate blood pressure. Mean arterial blood pressure (MAP) and heart rate were monitored in freely moving rats via a pressure transducer placed in the femoral artery. Drugs were infused via a cannula placed in the posterior hypothalamus. Infusion of 200 microM sodium hydrogen sulfide (NaHS), an H2S donor, into the hypothalamus of freely moving rats reduced MAP and heart rate. Infusion of 300 nM to 3 microM gliclazide dose-dependently blocked the effect of 200 microM NaHS. Infusion of the CBS activator, s-adenosyl-L-methionine (0.1 mM and 1 mM), likewise decreased MAP. Infusion of the CBS inhibitors aminooxyacetic acid (10 mM) and hydroxylamine (20 mM) increased MAP but did not block the effects of infusion of 200 microM NaHS. These data indicate that actions of H2S in the hypothalamus decrease blood pressure and heart rate in freely moving rats. This effect appears to be mediated by a K(ATP) channel-dependent mechanism and mimicked by endogenous H2S.

Molecular effects of homocysteine on cbEGF domain structure: insights into the pathogenesis of homocystinuria.

Homocystinuria is an inborn error of methionine metabolism that results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Homocystinurics often exhibit phenotypic abnormalities that are similar to those found in Marfan syndrome (MFS), a heritable connective tissue disorder that is caused by reduced levels of, or defects in, the cysteine-rich extracellular matrix (ECM) protein fibrillin-1. The phenotypic similarities between homocystinuria and MFS suggest that elevated homocysteine levels may result in an altered function of fibrillin-1. We have used recombinant calcium binding epidermal growth factor-like (cbEGF) domain fragments from fibrillin-1, and an unrelated protein Notch1, to analyse the effects of homocysteine on the native disulphide (cystine) bonds of these domains. We show using analytical reverse phase, high performance liquid chromatography (HPLC), electrospray ionisation mass spectrometry (ESI-MS) and limited proteolysis that homocysteine attacks intramolecular disulphide bonds causing reduction of cystine and domain misfolding, and that the effects of homocysteine are dependent on its concentration. We also identify the importance of calcium binding to cbEGF domains for their stabilisation and protection against homocysteine attack. Collectively, these data suggest that reduction of intramolecular cbEGF domain disulphide bonds by homocysteine and the resulting disruption of this domain fold may contribute to the change in connective tissue function seen in homocystinuria. Furthermore, since we show that the effects of homocysteine are not unique to fibrillin-1, other cbEGF-containing proteins may be implicated in the pathogenic mechanisms underlying homocystinuria.

Zofenopril Protects Against Myocardial Ischemia-Reperfusion Injury by Increasing Nitric Oxide and Hydrogen Sulfide Bioavailability.

BACKGROUND: Zofenopril, a sulfhydrylated angiotensin-converting enzyme inhibitor (ACEI), reduces mortality and morbidity in infarcted patients to a greater extent than do other ACEIs. Zofenopril is a unique ACEI that has been shown to increase hydrogen sulfide (H2S) bioavailability and nitric oxide (NO) levels via bradykinin-dependent signaling. Both H2S and NO exert cytoprotective and antioxidant effects. We examined zofenopril effects on H2S and NO bioavailability and cardiac damage in murine and swine models of myocardial ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: Zofenopril (10 mg/kg PO) was administered for 1, 8, and 24 hours to establish optimal dosing in mice. Myocardial and plasma H2S and NO levels were measured along with the levels of H2S and NO enzymes (cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and endothelial nitric oxide synthase). Mice received 8 hours of zofenopril or vehicle pretreatment followed by 45 minutes of ischemia and 24 hours of reperfusion. Pigs received placebo or zofenopril (30 mg/daily orally) 7 days before 75 minutes of ischemia and 48 hours of reperfusion. Zofenopril significantly augmented both plasma and myocardial H2S and NO levels in mice and plasma H2S (sulfane sulfur) in pigs. Cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and total endothelial nitric oxide synthase levels were unaltered, while phospho-endothelial nitric oxide synthase(1177) was significantly increased in mice. Pretreatment with zofenopril significantly reduced myocardial infarct size and cardiac troponin I levels after I/R injury in both mice and swine. Zofenopril also significantly preserved ischemic zone endocardial blood flow at reperfusion in pigs after I/R. CONCLUSIONS: Zofenopril-mediated cardioprotection during I/R is associated with an increase in H2S and NO signaling.

Effects of streptozotocin-induced diabetes and of insulin treatment on homocysteine metabolism in the rat.

An elevation in the concentration of total plasma homocysteine is known to be an independent risk factor for the development of vascular disease. Alterations in homocysteine metabolism have also been observed clinically in diabetic patients. Patients with either type 1 or type 2 diabetes who have signs of renal dysfunction tend to exhibit elevated total plasma homocysteine levels, whereas type 1 diabetic patients who have no clinical signs of renal dysfunction have lower than normal plasma homocysteine levels. The purpose of this study was to investigate homocysteine metabolism in a type 1 diabetic animal model and to examine whether insulin plays a role in its regulation. Diabetes was induced by intravenous administration of 100 mg/kg streptozotocin to Sprague-Dawley rats. We observed a 30% reduction in plasma homocysteine in the untreated diabetic rat. This decrease in homocysteine was prevented when diabetic rats received insulin. Transsulfuration and remethylation enzymes were measured in both the liver and the kidney. We observed an increase in the activities of the hepatic transsulfuration enzymes (cystathionine beta-synthase and cystathionine gamma-lyase) in the untreated diabetic rat. Insulin treatment normalized the activities of these enzymes. The renal activities of these enzymes were unchanged. These results suggest that insulin is involved in the regulation of plasma homocysteine concentrations by affecting the hepatic transsulfuration pathway, which is involved in the catabolism of homocysteine.

The effect of glucose and insulin on the activity of methylene tetrahydrofolate reductase and cystathionine-beta-synthase: studies in hepatocytes.

Hyperhomocysteinemia is a well established risk factor for cardiovascular disease, and multiple factors likely lead to abnormal regulation of plasma homocysteine in patients with diabetes. To examine a possible role for insulin and glucose in homocysteine metabolism, we examined the activity of two important enzymes of homocysteine metabolism in hepatocytes. In various tissues of six mice, methylene tetrahydrofolate reductase (MTHFR) activity was present in all tissues tested and the highest concentration (per gram) was in the brain. In contrast, cystathionine beta-synthase (CBS) activity appeared to be present only in the liver and to a small extent in the kidney. Using HEP G2 cells in culture, MTHFR activity was 3.3+/-0.8 nmol/h when the glucose concentration in the medium was 100 mg/dl and fell to 2.3+/-0.3 nmol/h when glucose was increased to 300 mg/dl. MTHFR activity was 3.4+/-0.3 nmol/h when cells were exposed to an insulin concentration of 5 mU/ml and fell to 2.8+/-0.3 nmol/h when insulin concentration was increased to 200 mU/ml (P<0.01). In contrast CBS activity increased from 0.017 to 0.13 U/ml by increasing the glucose concentration in the medium (P<0.01), but decreased from 0.04 to 0.02 (P<0.01) when the insulin concentration was increased from 5 to 200 mU/ml, respectively. We conclude that CBS and MTHFR have different tissue distributions, with CBS being present predominantly in liver and kidney, and MTHFR found in many tissues. In addition, both insulin and glucose affect the activity of the two enzymes when added to hepatocytes in vitro. If such effects occur in humans with hyperglycemia and hyperinsulinemia, then alterations in homocysteine metabolism may contribute to the accelerated macrovascular disease associated with insulin resistance or type 2 diabetes.

Effects of red wine polyphenolic compounds on paraoxonase-1 and lectin-like oxidized low-density lipoprotein receptor-1 in hyperhomocysteinemic mice.

Hyperhomocysteinemia, or abnormally high plasma homocysteine (Hcy) concentration, has often been associated with vascular thrombosis and the development of premature atherosclerosis. Many studies have shown that moderate wine consumption has potential beneficial effects related to the prevention of atherosclerosis, in part attributed to the biological properties of polyphenolic components, mainly flavonoids. The aim of the present study is to determine the effects of a red wine polyphenolic extract (PE) administration on hyperhomocysteinemia due to cystathionine beta-synthase (CBS) deficiency and on the associated biochemical markers of hepatic and endothelial dysfunctions in mice. Red wine PE was added for 4 weeks to the drinking water of heterozygous CBS-deficient mice fed a high-methionine diet, a murine model of hyperhomocysteinemia. Red wine PE supplementation at low dose significantly reduced plasma Hcy levels and restored the hepatic and plasma-decreased paraoxonase-1 activity induced by chronic hyperhomocysteinemia. Moreover, aortic expression of proinflammatory cytokines and adhesion molecules and levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 were reduced in hyperhomocysteinemic mice fed the red wine PE supplementation. These findings suggest that red wine PE administration in low quantities has beneficial effects on biochemical markers of endothelial dysfunction due to hyperhomocysteinemia.

Suppression of methionine-induced hyperhomocysteinemia by dietary eritadenine in rats.

The effect of dietary eritadenine on the plasma homocysteine concentration was investigated in methionine-induced hyperhomocysteinemic rats. The rats were fed on the control or eritadenine-supplemented (50 mg/kg) diet for 10 d. The animals were then injected with saline or methionine at a level of 100 or 300 mg/kg of body weight, and sacrificed 2 h or a more appropriate time after injection. The methionine injection increased the post-2 h concentration of plasma homocysteine in a dose-dependent manner in the control rats, this increase being significantly suppressed in the eritadenine-fed rats. This effect persisted up to 8 h after the methionine injection. The hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine were increased by eritadenine, whereas the hepatic homocysteine concentration was inversely decreased. The cystathionine beta-synthase activity in the liver was increased by eritadenine. It is suggested from these results that eritadenine might suppress the methionine-induced increase in plasma homocysteine concentration by dual mechanisms: slowing the homocysteine production from S-adenosylhomocysteine and increasing the removal of homocysteine due to the enhanced activity of cystathionine beta-synthase.

Deletion of the regulatory domain in the pyridoxal phosphate-dependent heme protein cystathionine beta-synthase alleviates the defect observed in a catalytic site mutant.

The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine beta-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B(6)-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The approximately 13-fold diminution in activity because of the mutation corresponds to an approximately 7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine beta-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B(6)-responsive phenotype associated with a cystathionine beta-synthase-impaired homocystinuric genotype.

Trypsin cleavage of human cystathionine beta-synthase into an evolutionarily conserved active core: structural and functional consequences.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine and serine to cystathionine-an irreversible step in the eukaryotic transsulfuration pathway. The native enzyme is a homotetramer or multimer of 63-kDa (551 amino acids) subunits and is activated by S-adenosyl-l-methionine (AdoMet) or by partial cleavage with trypsin. Amino-terminal analysis of the early products of trypsinolysis demonstrated that the first cleavages occur at Lys 30, 36, and 39. The enzyme still retains the subunit organization as a tetramer or multimer composed of 58-kDa subunits. Analysis by electrospray ionization mass spectrometry showed that further trypsin treatment cleaves CBS in its COOH-terminal region at Arg 413 to yield 45-kDa subunits. This 45-kDa active core is the portion of CBS most conserved with the evolutionarily related enzymes isolated from plants, yeast, and bacteria. The active core of CBS forms a dimer of approximately 85 kDa. The dimer is about twice as active as the tetramer. It binds both pyridoxal 5'-phosphate and heme cofactors but is no longer activated by AdoMet. Further analysis suggests that the dissociation of CBS to dimers causes a decrease in enzyme thermostability and a threefold increase in affinity toward the sulfhydryl-containing substrate-homocysteine. We found that the COOH-terminal region, residues 414-551, is essential for maintaining the tetrameric structure and AdoMet activation of the enzyme. The inability of the active core to form multimeric aggregates has facilitated its crystallization and X-ray diffraction studies.

Delta-aminolevulinate increases heme saturation and yield of human cystathionine beta-synthase expressed in Escherichia coli.

We recently expressed human cystathionine beta-synthase (CBS) in Escherichia coli and purified it to homogeneity. We showed that CBS requires heme in addition to pyridoxal 5'-phosphate for its function. Previously, CBS, only about 20% saturated with heme, was purified from transformed bacteria. In the present study, we supplemented the bacteria with 0.3 mM delta-aminolevulinate (delta ALA), a precursor of heme. While growth of the bacteria did not change, a 50-fold elevation of the heme content per milligram of total protein was observed in the cell extracts of delta ALA-supplemented cells. The increase in heme biosynthesis depended on the overexpression of a heme acceptor--CBS. Our data suggest that bacterial heme synthesis is regulated beyond delta ALA synthase. The delta ALA treatment resulted in 8 times more total CBS activity with a 3.5-fold higher yield of the purified recombinant enzyme, more than 68% saturated with heme. Increased yield, higher specific activity, and improved heme saturation of CBS will facilitate large-scale preparation of the enzyme. This method should be applicable to the overexpression of other recombinant heme proteins in bacteria.