Cystathionine Beta-synthase Enzyme Research Articles

Abstract 952: Determining the contribution of cystathionine beta synthase to lung cancer lineage fate

Abstract The two major pathologies of non-small cell lung cancer, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are historically treated as separate diseases, even though mounting evidence shows that epigenetic reprogramming from ADC to a more SCC fate allows lung cancers to evade therapies. Our team recently developed an isogenic lung cancer model in which lineage switching from ADC to SCC was epigenetically controlled through loss of Polycomb Repressive Complex 2 (PRC2) gene repression of squamous genes. Levels of S-adenosyl methionine (SAM), the methyl donor for the complex, influence PRC2 stability and activity. Steady state metabolism showed that SAM levels were decreased significantly and cystathionine levels were increased significantly in SCC cells relative to ADC cells. Our hypothesis is that increased activity of the enzyme cystathionine beta synthase (CBS) drives epigenetic reprogramming of lung ADC to an aggressive and therapy-resistant state through reduction of SAM pools and destabilization of PRC2, leading to expression of squamous genes. First, we stained a series of human lung cancer tissues for CBS and H3K27me3 and queried TCGA data to validate that CBS is up-regulated in squamous tumors. In addition, we leveraged 3-dimensional organoid cultures and traditional 2-dimensional human non-small cell lung cancer cell lines to explore the contribution of CBS to lung cancer lineage fate. With both of these systems, we used lentiviral small hairpins targeting Cbs/CBS and a lentivirus encoding a full length Cbs/CBS cDNA to modulate gene and protein levels. Lastly, we tested differential sensitivities to the common chemotherapeutic carboplatin on matched isogenic cultures that differ only in lineage state. In human tumors, SCCs have significantly higher levels of CBS and significantly lower levels of H3K27me3 than ADCs. Knock-down of CBS decreased cystathionine and increased both homocysteine and S-adenosyl methionine levels in 2-dimensional human NSCLC cultures. We also observed a global increase in H3K27me3 mark in shCBS cultures relative to shGFP controls. In contrast, CBS over-expression decreased global levels of H3K27me3. Importantly, we observed that squamous organoids are significantly less sensitive to the common chemotherapeutic carboplatin than are ADC organoids, suggesting that lineage switching driven by increased CBS activity will drive chemotherapy resistance. We are now characterizing changes in lineage fate and carboplatin sensitivity that are caused by these genetic manipulations of CBS. Our data suggest that lung cancer lineage fate may be governed in part through expression and activity of the enzyme CBS. Because CBS is a redox-sensitive enzyme, this could be the missing link between increased oxidative stress and ultimate epigenetic reprogramming to a therapy-resistant squamous state in lung cancer. Funded by ACS IRG-85-001-25, K22 CA201036 and KY LCRP to CFB Citation Format: Mojtaba Bakhtiari, Christine Fillmore Brainson. Determining the contribution of cystathionine beta synthase to lung cancer lineage fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 952.

Age‐dependence of Methylation, Transsulfuration and Glutathione Synthesis Pathways in Human Frontal Cortex

Neuronal synthesis of the antioxidant glutathione (GSH) depends upon availability of cysteine, provided either by cellular uptake or by transsulfuration of homocysteine generated by the methionine cycle of methylation (Fig. 1). To evaluate age‐dependent changes in the relative contribution of these two cysteine sources, we measured redox and methylation metabolite levels in postmortem human frontal cortex from subjects across the lifespan. Levels of the transsulfuration intermediate cystathionine were significantly decreased in 60–80 yr‐old subjects, indicating that the balance between these two cysteine sources shifts to transsulfuration in older age. This shift was accompanied by decreased methionine and S‐adenosylmethionine, and an increase in homocysteine, indicating lower activity of the folate and B12‐dependent enzyme methionine synthase (MS), consistent with earlier finding of decrease MS expression with age. Cystathionine levels in 10 yr‐old autistic subjects were decreased, similar to those of 60 yr‐old subjects. In vitro studies with human neuronal cells showed that the pro‐inflammatory cytokine TNF‐alpha increased transsulfuration and inhibited MS activity in association with decreased cysteine uptake. Transsulfuration pathway enzymes cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL) both produce hydrogen sulfide, which inhibits oxidative respiration and increases glycolysis. This action decreases production of reactive oxygen species (ROS), limiting the demand for antioxidant. Together these observations illustrate how an age‐dependent increase in transsulfuration links antioxidant synthesis to methylation status in human brain. TNF‐alpha, whose levels increase with age, may be a regulator of transsulfuration, with important implications for epigenetic regulation during early brain development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolomic studies identify changes in transmethylation and polyamine metabolism in a brain-specific mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and the quintessential disorder of mechanistic Target of Rapamycin Complex 1 (mTORC1) dysregulation. Loss of either causative gene, TSC1 or TSC2, leads to constitutive mTORC1 kinase activation and a pathologically anabolic state of macromolecular biosynthesis. Little is known about the organ-specific metabolic reprogramming that occurs in TSC-affected organs. Using a mouse model of TSC in which Tsc2 is disrupted in radial glial precursors and their neuronal and glial descendants, we performed an unbiased metabolomic analysis of hippocampi to identify Tsc2-dependent metabolic changes. Significant metabolic reprogramming was found in well-established pathways associated with mTORC1 activation, including redox homeostasis, glutamine/tricarboxylic acid cycle, pentose and nucleotide metabolism. Changes in two novel pathways were identified: transmethylation and polyamine metabolism. Changes in transmethylation included reduced methionine, cystathionine, S-adenosylmethionine (SAM-the major methyl donor), reduced SAM/S-adenosylhomocysteine ratio (cellular methylation potential), and elevated betaine, an alternative methyl donor. These changes were associated with alterations in SAM-dependent methylation pathways and expression of the enzymes methionine adenosyltransferase 2A and cystathionine beta synthase. We also found increased levels of the polyamine putrescine due to increased activity of ornithine decarboxylase, the rate-determining enzyme in polyamine synthesis. Treatment of Tsc2+/- mice with the ornithine decarboxylase inhibitor α-difluoromethylornithine, to reduce putrescine synthesis dose-dependently reduced hippocampal astrogliosis. These data establish roles for SAM-dependent methylation reactions and polyamine metabolism in TSC neuropathology. Importantly, both pathways are amenable to nutritional or pharmacologic therapy.

Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase.

The transsulfuration pathway enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (H2S). In this study, we assessed the role of CBS in H2S biogenesis. We show that despite discouraging enzyme kinetics of alternative H2S-producing reactions utilizing cysteine compared with the canonical condensation of serine and homocysteine, our simulations of substrate competitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine on CBS, thus leading to generation of appreciable amounts of H2S. The leading H2S-producing reaction is condensation of cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine is more abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the main determinant of CBS H2S productivity. Abundance of cysteine over serine, for example, in plasma, allowed for up to 43% of CBS activity being responsible for H2S production, while excess of serine typical for intracellular levels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine and cysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H2S pool. Our study characterizes the H2S-producing potential of CBS under biologically relevant conditions and highlights the serine-to-cysteine ratio as the main determinant of H2S production by CBS in vivo. Our data clarify the function of CBS in H2S biogenesis and the role of thioethers as surrogate H2S markers. Antioxid. Redox Signal. 28, 311-323.

The c.797 G>A (p.R266K) cystathionine β-synthase mutation causes homocystinuria by affecting protein stability.

Mutations in the cystathionine beta-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The c.797 G>A (p.R266K) mutation in CBS was originally described in several Norwegian pyridoxine responsive CBS deficient patients, and heterologous gene expression studies have shown that the protein has near wild-type levels of enzyme activity. Here, we characterize a transgenic mouse lacking endogenous Cbs and expressing p.R266K human CBS protein from a zinc inducible metallothionein promoter (Tg-R266K Cbs-/- ). Unlike mice expressing other mutant CBS alleles, the Tg-R266K transgene is unable to efficiently rescue neonatal lethality of Cbs-/- on a C57BL/6J background. On a C3H/HeJ background, zinc-induced Tg-R266K Cbs-/- mice express CBS mRNA, but have very low levels of CBS protein and enzyme activity, resulting in extreme elevations in serum total homocysteine (tHcy). Treatment with pyridoxine did not have any appreciable effect on tHcy, indicating this allele is not pyridoxine responsive in mice. However, treatment with the proteasome inhibitor bortezomib resulted in an 97% reduction in tHcy and a 2381% increase in liver CBS activity. These studies show that the p.R266K mutation causes increased proteasomal degradation in vivo, and that treatments that stabilize the protein can be used to reverse its effect.

Thioethers as markers of hydrogen sulfide production in homocystinurias.

Two enzymes in the transsulfuration pathway of homocysteine -cystathionine beta-synthase (CBS) and gamma-cystathionase (CTH)-use cysteine and/or homocysteine to produce the important signaling molecule hydrogen sulfide (H2S) and simultaneously the thioethers lanthionine, cystathionine or homolanthionine. In this study we explored whether impaired flux of substrates for H2S synthesis and/or deficient enzyme activities alter production of hydrogen sulfide in patients with homocystinurias. As an indirect measure of H2S synthesis we determined by LC-MS/MS concentrations of thioethers in plasma samples from 33 patients with different types of homocystinurias, in 8 patient derived fibroblast cell lines, and as reaction products of seven purified mutant CBS enzymes. Since chaperoned recombinant mutant CBS enzymes retained capacity of H2S synthesis invitro it can be stipulated that deficient CBS activity invivo may impair H2S production. Indeed, in patients with classical homocystinuria we observed significantly decreased cystathionine and lanthionine concentrations in plasma (46% and 74% of median control levels, respectively) and significantly lower cystathionine in fibroblasts (8% of median control concentrations) indicating that H2S production from cysteine and homocysteine may be also impaired. In contrast, the grossly elevated plasma levels of homolanthionine in CBS deficient patients (32-times elevation compared to median of controls) clearly demonstrates a simultaneous overproduction of H2S from homocysteine by CTH. In the remethylation defects the accumulation of homocysteine and the increased flux of metabolites through the transsulfuration pathway resulted in elevation of cystathionine and homolanthionine (857% and 400% of median control values, respectively) indicating a possibility of an increased biosynthesis of H2S by both CBS and CTH. This study shows clearly disturbed thioether concentrations in homocystinurias, and modeling using these data indicates that H2S synthesis may be increased in these conditions. Further studies are needed to confirm our findings and to explore the possible implications for pathophysiology of these disorders.

Fitting homocysteine to disease models, as well as adjusting the models to the disease.

Homocysteine is a nonprotein α-amino acid and differs from cysteine by having an additional methylene bridge. Homocysteine is not present in the diet, and all homocysteine in the body is biosynthesized from the essential amino acid methionine. Homocysteine is a product of methionine demethylation and is recycled into methionine or alternatively converted into cysteine with the assistance of B vitamins. Homocysteine is metabolized through two pathways, namely remethylation and transsulfuration. Much information has been generated from mouse models. A summary of this model information, as presented by Dayal and Lentz [1], is shown (Fig. 1). The remethylation pathway requires vitamin B12, folate, and the enzyme 5,10-methylene-tetrahydrofolate reductase (MTHFR). In the kidney and liver, homocysteine is also remethylated by the enzyme betaine homocysteine methyltransferase (BHMT), which transfers a methyl group to homocysteine via the demethylation of betaine to dimethylglycine (DMG). The transsulfuration pathway requires the enzyme cystathionine beta-synthase (CBS) and vitamin B6 (pyridoxal-5′-phosphate). Once formed from cystathionine, cysteine can be utilized in protein synthesis and glutathione (GSH) production. Genetics has contributed greatly to our understanding of homocysteine, its metabolism, and cardiovascular risk. Classical homocystinuria, an autosomal-recessive disease, is caused by CBS deficiency. The defect leads to a multisystem disorder involving connective tissue, muscle, the central nervous system, and the cardiovascular system [2]. Genetic variation in the MTHFR gene influences the susceptibility to occlusive vascular disease, neural tube defects, Alzheimer’s disease and other dementias, colon cancer, and acute leukemia [2]. Thus, homocysteine, its metabolism, and the enzymes involved are of great interest in biomedical research. Homocysteine contributes to endothelial injury and subsequent atherosclerosis and is a specific risk factor for cardiovascular disease. As a result, much effort has been expended in clinical trials for various vascular diseases in the hopes that by reducing homocysteine levels, cardiovascular endpoints could be ameliorated. For instance, an updated Cochrane review found no evidence to suggest that homocysteinelowering interventions in the form of supplements of vitamins B6, B9, or B12 given alone or in combination should be used for preventing cardiovascular events [3, 4]. Neither has the supplementation of dietary folic acid to lower homocysteine levels led to more favorable outcomes [5, 6]. Nevertheless, folic acid supplementation has a major impact on reducing neural tube defects [7]. J Mol Med this month features a homocysteine-based hypothesis in a complicated animal model. Muthuramu et al. point out that a Bcausal role of homocysteine in myocardial biology and function cannot be proven since residual confounding in multivariable models may occur^ [8]. How very true! They studied Cbs heterozygous gene-deleted (Cbs +/−) mice. These mice were crossed with low-density lipoprotein receptor (Ldlr −/−) gene-deleted mice to generate Cbs +/−/ Ldlr −/− mice. The mice were divided into three groups and fed regular chow, folic acid-depleted chow, or methionineenriched chow. Three weeks after diet initiation, the mice were intravenously injected with a hepatocyte-specific adenoviral vector expressing Cbs (AdCBS), with the same dose of control vector, or with saline buffer. Two weeks after that, the mice were subjected to thoracic aortic constriction (TAC), an experimental aortic coarctation, or sham operation to induce * Friedrich C. Luft luft@charite.de

Domain organization, catalysis and regulation of eukaryotic cystathionine beta-synthases.

Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.

P24 Renal protection through CBS/H2S pathway in mammalian hibernation: A natural model of hypothermic organ preservation during cold ischemia and reperfusion

Hydrogen sulfide (H2S) can induce a hypometabolic, hibernation-like state in small mammals when given in sublethal concentrations. Hibernating animals undergo a repetitive cycle of cooling (torpor) and rewarming without reperfusion injury or other ill effects. Recently, we observed in the ductus deferens cells of the Syrian hamster (hibernating animal) that one of the protective mechanisms against hypothermia-rewarming injury consists of endogenous production of H2S through the enzyme cystathionine-beta-synthase (CBS). We also showed that endogenous H2S is highly produced during torpor in hibernating hamsters and less produced when metabolism is normalized. Therefore in this study, we investigated the role of CBS and H2S in the induction of torpor and renal protection by blocking the CBS enzyme during torpor in hamsters. Our results show that hamsters during hibernation are able to preserve their kidney integrity through activation of CBS enzyme leading to endogenous H2S production under the extreme physiological conditions of torpor. The results demonstrate profound protective effects of CBS/H2S pathway on survival, renal function, and inflammation during hibernation. These findings might have therapeutic potential to protect kidneys that suffer from hypoxia during transplantation, ischemia–reperfusion and other related clinical conditions.

S7-6 Role of H2S and NO in Bacillus anthracis spore formation and virulence

Many prokaryotic species generate hydrogen sulfide (H2S) and nitric oxide (NO) enzymatically, from cysteine and arginine, respectively, in their natural environments. Both gases are small freely diffusible signaling molecules that are known to be involved in numerous physiological and pathological processes in mammals. However the biochemistry and physiological role of these gases in bacteria remains largely unknown. We have shown that inactivation of H2S producing enzymes (cystathionine beta-synthase, cystathionine gamma lyase, or 3-mercaptopyruvate sulfurtransferase) and NO-synthase in several Gram (+) and Gram (−) bacteria render them highly sensitive to different classes of antibiotics (Gusarov et al., Science 325 (2009) 1380–1384; Shatalin et al. Science 334 (2011) 986–990). We also presented evidence that Bacillus anthracis-derived NO is critical at the early stage of infection (Shatalin et al. PNAS 105 (2008) 1009–1013). Here we show that: (1) cbs/cse and nos mutations change Bacilli global gene transcription profile; (2) apore formation process in cbs/cse and nos mutants ofB. anthracis is affected; (3) virulence of cbs/cse and nos mutants of B. anthracis is diminished. These results demonstrate that bacterial H2S and NO are an important virulence factors, and that enzymes generated these gases may serve as an attractive target for antimicrobial therapy.

P58: Cystathionine-gamma-lyase is essential in maintaining retinal redox balance

Retina is one of the tissues that exposed to highest oxidative stress due to its high metabolic rate, abundant photosensitizing activity and visible light exposure. With these high chances of oxidative damage, antioxidative capacity of retinal tissues is essential for maintaining the proper functions of retina. Cytoprotective roles of hydrogen sulfide (H 2 S) have been documented and were largely attributed to its antioxidative activity. However, whether are these three H 2 S-producing enzymes (cystathionine-gamma-lyase (CSE), cystathionine-beta-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) plays antioxidative roles in retinal tissue is unclear. In this study, we examined the role of CSE, CBS and MPST enzymes in human retinal pigmented epithelial cell line ARPE19. ARPE19 expressed comparatively similar protein levels of the three enzymes. Surprisingly, gene silencing of CSE sensitized the cells to hydrogen peroxide (H 2 O 2 ) challenge to a much greater extent as compared to gene silencing in CBS or MPST. We also observed that loss of CSE in ARPE19 resulted in decreased level of glutathione, higher oxidative stress and accelerated senescence. Taken together, our data suggest that CSE is essential for retinal functions by maintaining its redox balance.

Correction of Cystathionine β-Synthase Deficiency in Mice by Treatment with Proteasome Inhibitors

Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism characterized by extremely elevated levels of plasma total homocysteine. The vast majority of CBS-deficient patients have missense mutations located in the CBS gene that result in the production of either misfolded or unstable protein. Here, we examine the effect of proteasome inhibitors on mutant CBS function using two different mouse models of CBS deficiency. These mice lack mouse CBS and express a missense mutant human CBS enzyme (either p.I278T or p.S466L) that has less than 5% of normal liver CBS activity, resulting in a 10-30-fold elevation in plasma homocysteine levels. We show that treatment of these mice with two different proteasome inhibitors can induce liver Hsp70, Hsp40, and Hsp27, increase levels of active CBS, and lower plasma homocysteine levels to within the normal range. However, response rates varied, with 100% (8/8) of the p.S466L animals showing correction, but only 38% (10/26) of the p.I278T animals. In total, our data show that treatment with proteostasis modulators can restore significant enzymatic activity to mutant misfolded CBS enzymes and suggests that they may be useful in treating certain types of genetic diseases caused by missense mutations.

Frequent Epigenetic Silencing of the Folate-Metabolising Gene Cystathionine-Beta-Synthase in Gastrointestinal Cancer

BackgroundBoth gastric and colorectal cancers (CRC) are the most frequently occurring malignancies worldwide with the overall survival of these patients remains unsatisfied. Identification of tumor suppressor genes (TSG) silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection and prognosis assessment. Cystathionine-beta-synthase (CBS) functions in the folate metabolism pathway, which is intricately linked to methylation of genomic DNA. Dysregulation of DNA methylation contributes substantially to cancer development.Methodology/Principal FindingsTo identify potential TSGs silenced by aberrant promoter methylation in CRC, we analyzed tumor and adjacent tissues from CRC cases using the Illumina Human Methylation45 BeadChip. We identified hypermethylation of the CBS gene in CRC samples, compared to adjacent tissues. Methylation and decreased mRNA expression of CBS were detected in most CRC cell lines by methylation-specific PCR and semiquantitative RT-PCR, as well as in gastric cancer. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A reversed methylation and restored CBS mRNA expression indicating a direct effect. Aberrant methylation was further detected in 31% of primary CRCs (29 of 96) and 55% of gastric tumors (11 of 20). In contrast, methylation was seldom found in normal tissues adjacent to the tumor. CBS methylation was associated with KRAS mutations in primary CRCs (P = 0.04, by χ2-test). However, no association was found between CBS methylation or KRAS mutations with cancer relapse/metastasis in Stage II CRC patients.ConclusionA novel finding from this study is that the folate metabolism enzyme CBS mRNA levels are frequently downregulated through CpG methylation of the CBS gene in gastric cancer and CRC, suggesting that CBS functions as a tumor suppressor gene. These findings warrant further study of CBS as an epigenetic biomarker for molecular diagnosis of gastrointestinal cancers.

A novel 110-bp insertion in a patient with homocysteinuria.

Homocysteinuria is an inherited recessive disorder caused by cystathionine beta-synthase (CBS) deficiency that has a wide spectrum of clinical manifestations, including ocular lens dislocation, skeletal disproportion, osteoporosis, vascular thrombosis, and central nervous system dysfunction [1]. The CBS enzyme catalyzes the synthesis of cystathionine from homocysteine and serine in the methionine pathway. This results in accumulation of homocysteine and methionine in plasma, leading to excretion of excessive urinary homocysteine. CBS deficiency has a worldwide incidence of 1/344,000 live births (range: 1/58,000-1/1000 live births) [2]. Human CBS is located in the cytoplasm of cells and is composed of 4 identical 63-kDa subunits. The CBS gene is located on the long (q) arm of chromosome 21 at position 22.3, encodes a protein consisting of 551 amino acids, and has 16 exons [3]. Herein we report a patient with homocysteinuria that was a carrier of compound heterozygote mutations in the CBS gene, 1 of which is novel, and a discussion of the clinical and molecular findings. A 14-year-old boy was referred to our hospital for evaluation of ocular symptoms that began approximately 1 year earlier. He was the first born of non-consanguineous parents that had lost 1 fetus. Family history of neurologic and thromboembolic disease was negative. Pregnancy and delivery were uneventful. The proband had neuromotor developmental delay. At the age of 10 years he had a clonic seizure of the left side. He had a marfanoid face and neurological examination showed left-sided central facial palsy, and mild left hemiparesis. Complete blood count results were normal. Cranial CT showed an area of low attenuation in the left frontoparietal region consistent with infarction. Prothrombotic work-up results were as follows: serum methionine level: 4.7 mg / dL (normal: 0.09-0.6 mg/dL); free homocysteine concentration: 50 mmol/L (normal: <12.5 mmol/L); urine homocysteine concentration: 360 mmol/L (normal: 5-15 mmol/L). The patient was diagnosed as homocysteinuria, and hydroxy cobalamin injections, vitamin B6, acetyl-salicylic acid, and a low-methionine diet were started and maintained.

The 844ins68 cystathionine beta-synthase and C677T MTHFR gene polymorphism and the vaso-occlusive event risk in sickle cell disease

IntroductionSickle cell disease (SCD) is an inflammatory condition with an increase in the adhesion of sickled erythrocytes, and it is a potential cause of vaso-occlusive episodes, an event related to clinical manifestations, morbidity and mortality. The cystathionine beta-synthase enzyme gene (CBS) and the methylenetetrahydrofolate reductase enzyme gene (MTHFR) are risk factors for thromboembolic disorders. This study evaluated the frequency of the 844ins68 CBS and C677T MTHFR gene polymorphisms and their possibility to be risk factors for vaso-occlusive crises.Material and methodsIn total 91 blood samples from SCD patients were studied by PCR-RFLP and PCR-allele-specific, for the SCD genotype confirmation and polymorphism identification.ResultsThe presence of clinical manifestations related to vaso-occlusive crises were more frequent among patients with the Hb SS genotype (p = 0.007). The CBS enzyme gene was three times more frequent (p = 0.011) among patients with vaso-occlusive complications. The MTHFR gene mutation frequency showed no increased risk for vaso-occlusive crises in SCD patients (p = 0.193). The interaction between the two polymorphisms was evaluated in 12.08% of the SCD patients and doubled the vaso-occlusive disease risk (relative risk: 2.16).ConclusionsWe conclude that the presence of 844ins68 CBS and C677T MTHFR gene polymorphism was a risk factor for vaso-occlusive episodes in the SCD patients evaluated.

Cystathionine γ-Lyase-deficient Mice Require Dietary Cysteine to Protect against Acute Lethal Myopathy and Oxidative Injury

Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine gamma-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth(-/-)) mice as an animal model of cystathioninemia/cystathioninuria. Cth(-/-) mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth(-/-) mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth(-/-) hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth(-/-) mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine beta-synthase (Cbs(-/-)). Because Cbs(-/-) mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie's method, which detects hypermethioninemia.

The Production of hydrogen Sulphide and the Effects of Nitric Oxide (NO) and Low Oxygen Conditions in Intrauterine Tissues in Rats

Hydrogen sulphide (H2S) is a gas signaling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). H2S may mediate hypoxic responses in vascular smooth muscle. H2S also appears to be a signaling molecule in mammalian non-vascular smooth muscle. Hypoxia is associated with pre-eclampsia where poor placental function can reduce the supply of oxygen and nutrients to the fetus resulting in intrauterine growth restriction (IUGR) and other placental dysfunctions. Hypoxia can also bring about other pre-eclamptic features such as the release of pro-inflammatory cytokines and oxidative stress.Hypoxic conditions can also reduce the uteroplacental perfusion, which may lead to inflammatory conditions i.e. oxidative stress. However, there are no reports to date on the production of H2S in reproductive tissues and the possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. We investigated the endogenous production of H2S in rat intrauterine tissues and the effectof NO and low oxygen condition on H2S production in intrauterine tissues. The production of H2S in rat intrauterine tissues was measured in vitro using a standard technique. The expression of CBS and CSE was also investigated in rat intrauterine tissues via western blotting. Furthermore, the effects of nitric oxide (NO) and low oxygen conditions on the production rates of hydrogen sulphide were investigated. The order of H2S production rates for rat tissues were: liver (488±28.9 nM/min/g) > uterus (310±36.7 nM/min/g) > fetal membranes (88.2±3.8 nM/min/g)> placenta (42.7± 6.8 nM/min/g). Under the effect of NO donor, NO significantly increased H2S production in rat fetal membranes only (from 88.2±3.8 nM/min/g to 103.2±7.4 nM/min/g). Under low oxygen conditions, production of H2S was significantly increased compared to room air oxygen conditions for rat liver (from 422±31.6 nM/min/g to 583±38.7 nM/min/g), uterus (from 328±11.8 nM/min/g to 5913±21.8 nM/min/g) and fetal membranes (from 78.2±9.1 to 189±17.1) , but not rat placenta. Western blotting detected the expression of CBS and CSE in all rat intrauterine tissues. Rat intrauterine tissues produce H2S in vitro possibly via CBS and CSE enzymes.

Taurine-deficient diet up-regulated cystathionine β-synthase monoallele in hemizygous cystathionine β-synthase knockout mice

Impaired cystathionine beta-synthase (CBS) causes hyperhomocystinuria and hyperhomocysteinemia, both risk factors for cardiovascular diseases. Reduced CBS activity could decrease cysteine and taurine biosyntheses (metabolites of homocysteine degradation) and lead to less taurocholic acid production with a resultant increased cholesterol content. We hypothesized that a deficiency in CBS genetic material and enzyme activity would reduce taurine synthesis, which would lead to an elevated cholesterol concentration. Both sexes of hemizygous C57BL/6J-Cbs(tm1Unc) [CBS (+/-)] and wild-type C57BL/6J mice [CBS (+/+)] were divided into 2 groups. One group of CBS (+/-) and CBS (+/+) mice was fed a cysteine- and taurine-deficient diet for 8 weeks, and the other group was fed a cysteine, taurine, and vitamin B6-deficient diet for 8 weeks. Significantly higher plasma total homocysteine concentrations occurred in the CBS (+/-) mice than their CBS (+/+) cohorts. Female mice of both genotypes had significantly higher plasma total homocysteine concentrations and significantly lower relative CBS mRNA levels than did male mice. During vitamin B(6) deficiency, plasma total homocysteine concentrations were significantly elevated. Three important findings were a differential sex response of CBS mRNA to feeding the vitamin B(6) diet; CBS (+/-) mice had a significantly lower plasma cholesterol concentration, contrary to what was anticipated; and during feeding, the taurine- and cysteine-deficient diet, CBS mRNA levels in CBS (+/-) mice were reduced only 13% rather than the expected 50%. We conclude that the remaining CBS monoallele is up-regulated in mice when fed a taurine-deficient diet to produce additional CBS mRNA.

The endogenous production of hydrogen sulphide in intrauterine tissues

BackgroundHydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro.MethodsThe production of hydrogen sulphide in rat and human intrauterine tissues was measured in vitro using a standard technique. The expression of CBS and CSE was also investigated in rat and human intrauterine tissues via Western blotting. Furthermore, the effects of nitric oxide (NO) and low oxygen conditions on the production rates of hydrogen sulphide were investigated.ResultsThe order of hydrogen sulphide production rates (mean +/- SD, n = 4) for rat tissues were: liver (777 +/- 163 nM/min/g) > uterus (168 +/- 100 nM/min/g) > fetal membranes (22.3 +/- 15.0 nM/min/g) > placenta (11.1 +/- 4.7 nM/min/g), compared to human placenta (200 +/- 102 nM/min/g). NO significantly increased hydrogen sulphide production in rat fetal membranes (P < 0.05). Under low oxygen conditions the production of hydrogen sulphide was significantly elevated in human placenta, rat liver, uterus and fetal membranes (P < 0.05). Western blotting (n = 4) detected the expression of CBS and CSE in all rat intrauterine tissues, and in human placenta, myometrium, amnion and chorion.ConclusionRat and human intrauterine tissues produce hydrogen sulphide in vitro possibly via CBS and CSE enzymes. NO increased the production of hydrogen sulphide in rat fetal membranes. The augmentation of hydrogen sulphide production in human intrauterine tissues in a low oxygen environment could have a role in pathophysiology of pregnancy.

Active Cystathionine β-Synthase Can Be Expressed in Heme-free Systems in the Presence of Metal-substituted Porphyrins or a Chemical Chaperone

Cystathionine beta-synthase (CBS), a key enzyme in the metabolism of homocysteine, has previously been shown to require a heme co-factor for maximal activity. However, the biochemical function of the CBS heme is not well defined. Here, we show that expression of human CBS in heme-deficient strains of Saccharomyces cerevisiae and Escherichia coli results in production of an enzyme that is misfolded and degraded. Addition of exogenous heme, porphyrins with non-iron metal, or porphyrin lacking metal entirely produced stable and active CBS enzyme. Purification of recombinant CBS enzyme expressed in the presence of various metalloporphyrins confirmed that Mn(III) and Co(III) had 30-60% of the specific activity of Fe(III)-CBS, and still responded to allosteric activation by S-adenosyl-L-methionine. Treatment of S. cerevisiae with the chemical chaperone trimethylamine-N-oxide resulted in near complete restoration of function to human CBS produced in a heme-deficient strain. Taken together, these results suggest that porphyrin moiety of the heme plays a critical role in proper CBS folding and assembly, but that the metal ion is not essential for this function or for allosteric regulation by S-adenosyl-L-methionine.