Cystathionine Beta-synthase Protein Research Articles

Benserazide, a cystathionine beta-synthase (CBS) inhibitor, potentially enhances the anticancer effects of paclitaxel via inhibiting the S-sulfhydration of SIRT1 and the HIF1-α/VEGF pathway.

Cancer targeted therapy is essential to minimize damage to normal cells and improve treatment outcomes. The elevated activity of Cystathionine beta-synthase (CBS), an enzyme responsible for producing endogenous hydrogen sulfide (H2S), plays a significant role in promoting tumor growth, invasiveness, and metastatic potential. Consequently, the selective inhibition of CBS could represent a promising therapeutic strategy for cancer. Currently, there is much interest in combining paclitaxel with other drugs for cancer treatment. This study aimed to investigate the efficacy of combining benserazide, a CBS inhibitor, with paclitaxel in treating tumors. Firstly, we demonstrated CBS is indeed involved in the progression of multiple cancers. Then it was observed that the total binding free energy between the protein and the small molecule is -98.241kJ/mol. The release of H2S in the group treated with 100μM benserazide was reduced by approximately 90% compared to the negative control, and the thermal denaturation curve of the complex protein shifted to the right, suggesting that benserazide binds to and blocks the CBS protein. Next, it was found that compared to paclitaxel monotherapy, the combination of benserazide with paclitaxel demonstrated stronger antitumor activity in KYSE450, A549, and HCT8 cells, accompanied by reduced cell viability, cell migration and invasion, as well as diminished angiogenic and lymphangiogenic capabilities. In vivo studies showed that the combined administration of benserazide and paclitaxel significantly reduced the volume and weight of axillary lymph nodes in comparison to the control group and single administration group. Further mechanistic studies revealed that the combination of benserazide and paclitaxel significantly suppressed the S-sulfhydration of SIRT1 protein, thereby inhibiting the expression of SIRT1 protein and activating SIRT1 downstream Notch1/Hes1 signaling pathway in KYSE450, A549, and HCT8 cells. Meanwhile, we observed that benserazide combined with paclitaxel induced a more significant downregulation of HIF-1α, VEGF-A, VEGF-C, and VEGF-D proteins expression levels in KYSE450, A549, and HCT8 cells compared to paclitaxel alone. These findings indicated that benserazide enhances the anticancer effects of paclitaxel via inhibiting the S-sulfhydration of SIRT1 and down-regulating HIF-1α/VEGF signaling pathway. This study suggests that benserazide may have potential as a chemosensitizer in cancer treatment.

Pyridoxine non-responsive p.R336C mutation alters the molecular properties of cystathionine beta-synthase leading to severe homocystinuria phenotype

•The prevalence of homocystinuria in Qatar is 1:1800, mainly due to a founder missense mutation p.R336C.•The cystathionine beta-synthase (CBS) R336C mutant was bacterially expressed, purified and its molecular properties were compared to CBS wild type (WT) recombinant protein.•Our data revealed that p.R336C mutation results in a dramatic reduction (∼86%) of CBS enzymatic activity.•Circular Dichroism experiments suggested that the p.R336C mutation does not significantly alter the secondary structure of the CBS protein.•CD spectra also revealed distinct differences in the thermal unfolding mechanisms of CBS WT and R336C mutant protein species.•Chemical denaturation experiments indicated that the WT CBS protein is thermodynamically more stable than the R336C mutant, suggesting a destabilizing effect of the p.R336C mutation.•This study provides mechanistic insight into the pathogenicity of the p.R336C mutation that leads to a severe homocystinuria phenotype.

Analysis of differential neonatal lethality in cystathionine β-synthase deficient mouse models using metabolic profiling.

Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine β-synthase (Cbs-/- ) gene or an inactive human CBS protein (Tg-G307S Cbs-/- ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs-/- ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs+/- , Tg-G307S Cbs-/- , and Tg-I278T Cbs-/- mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs-/- and Tg-I278T Cbs-/- compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs-/- mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs-/- animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis.

An eQTL in the cystathionine beta synthase gene is linked to osteoporosis in laying hens

BackgroundSkeletal damage is a challenge for laying hens because the physiological adaptations required for egg laying make them susceptible to osteoporosis. Previously, we showed that genetic factors explain 40% of the variation in end of lay bone quality and we detected a quantitative trait locus (QTL) of large effect on chicken chromosome 1. The aim of this study was to combine data from the commercial founder White Leghorn population and the F2 mapping population to fine-map this QTL and understand its function in terms of gene expression and physiology.ResultsSeveral single nucleotide polymorphisms on chromosome 1 between 104 and 110 Mb (galGal6) had highly significant associations with tibial breaking strength. The alternative genotypes of markers of large effect that flanked the region had tibial breaking strengths of 200.4 vs. 218.1 Newton (P < 0.002) and, in a subsequent founder generation, the higher breaking strength genotype was again associated with higher breaking strength. In a subsequent generation, cortical bone density and volume were increased in individuals with the better bone genotype but with significantly reduced medullary bone quality. The effects on cortical bone density were confirmed in a further generation and was accompanied by increased mineral maturity of the cortical bone as measured by infrared spectrometry and there was evidence of better collagen cross-linking in the cortical bone. Comparing the transcriptome of the tibia from individuals with good or poor bone quality genotypes indicated four differentially-expressed genes at the locus, one gene, cystathionine beta synthase (CBS), having a nine-fold higher expression in the genotype for low bone quality. The mechanism was cis-acting and although there was an amino-acid difference in the CBS protein between the genotypes, there was no difference in the activity of the enzyme. Plasma homocysteine concentration, the substrate of CBS, was higher in the poor bone quality genotype.ConclusionsValidated markers that predict bone strength have been defined for selective breeding and a gene was identified that may suggest alternative ways to improve bone health in addition to genetic selection. The identification of how genetic variants affect different aspects of bone turnover shows potential for translational medicine.

Analysis of the Qatari R336C cystathionine β-synthase protein in mice.

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs -/- ). Zinc-treated Tg-R336C Cbs -/- mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs -/- mice are significantly reduced compared to that found in Tg-hCBS Cbs -/- mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs -/- mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs -/- wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.

Alterations in the Serotonin and Dopamine Pathways by Cystathionine Beta Synthase Overexpression in Murine Brain.

Cystathionine beta synthase (CBS) is one of the 225 genes on chromosome 21 (HSA 21) that are triplicated in persons with trisomy 21 (Down syndrome). Although most triplicate HSA21 genes have their orthologous genes on murine chromosome 16, the murine ortholog of hCBS is on murine chromosome 17 and thus is not present in the well-studied Ts65Dn mouse model of trisomy 21. Persons with trisomy 21 (T21) present deficits in neurotransmission and exhibit early brain aging that can partially be explained by monoamine neurotransmitter alterations. We used transgenic mice for the hCBS gene, which overexpress the CBS protein in various brain regions, to study if CBS overexpression induces modifications in the monoamine neurotransmitters in the hypothalamus, thalamus, hippocampus, and striatum from transgenic and control female and male mice aged 3-4months and 11-12months. Sex, age, and brain area each influenced neurotransmitter levels. Briefly, the serotonin pathway was modified by CBS overexpression in various brain areas in female mice but not in male mice. The dopamine pathway was modified in brain regions according to sex and age. These results may allow us to better understand the role of the transsulfuration pathway and especially CBS overexpression in the metabolism of biogenic amines and the catecholamine catabolism in persons with trisomy 21.

Abstract 2980: Cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H2S) supports proliferation, migration and bioenergetics in colon cancer-associated fibroblasts (CAFs)

Abstract Introduction: Our group has recently demonstrated that CBS, one of the H2S-producing enzymes, is abundantly overexpressed in human colorectal cancer (CRC) tissue specimens resulting in an increased H2S production. In order to extend our observations, we have now compared, using immunohistochemistry, CBS expression in carcinoma-associated fibroblasts (CAFs) compared to normal colonic fibroblasts (NCFs) in human colon adenocarcinoma samples. The overall goal of our studies is to characterize how the CBS/H2S axis contributes to the malignant phenotype of the CAFs in the tumor microenvironment. Methods: Human CAFs and NCFs were collected from CRC under an IRB approved protocol and cultured in vitro. Western blot analysis of both NCF and CAF was performed to determine CBS protein expression levels. SiRNA/shRNA-mediated silencing of CBS in CAFs was performed. A pharmacological inhibitor of CBS, aminooxyacetic acid (AOAA), in both CAF and NCF cultures was also utilized. Hydrogen sulfide (H2S) levels were measured using the fluorogenic probe 7-Azido-4-methylcoumarin. We also performed proliferation assays using a coulter counter in CAFs, NCFs, and CAFs subjected to siRNA/shRNA-mediated CBS silencing or AOAA. Control cells were transduced with shNT vectors. Cells were also treated with the slow-releasing H2S donor GYY4137. We also conducted cell migration assays in transwell chambers (migration of CAFs toward conditioned media (CM) collected either from HCT116 cells or normal colon mucosal cells (NCM356). Finally, the XF24 Extracellular Flux Analyzer (Seahorse) was used to measure bioenergetic function in CAFs subjected to siRNA/shRNA-mediated silencing of CBS or treated with AOAA. Results: CBS protein expression and H2S production in CAFs was increased compared to NCFs. The H2S donor, GYY 4137, increased the proliferation rate of CAFs but not of NCFs. CBS silencing or AOAA treatment reduced CAF migration toward conditioned media from HCT116 cells. Genetic or pharmacological inhibition of CBS in CAF cells suppressed key bioenergetic parameters including basal respiration, ATP production, maximal respiratory capacity, and spare respiratory capacity compared to NCF cells or CAF shNT cells. Conclusions: Thus, CBS/H2S axis serves as a mechanism to support CAFs’ function. In these cells, H2S contributes to maintaining their bioenergetic functions, which, in turn, promotes their proliferation and migration toward colon cancer cells. We hypothesize that these processes may contribute CRC progression. Citation Format: Katalin Modis, Manjit Maskey, Paul Johnson, Csaba Szabo, Mark R. Hellmich, Celia Chao. Cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H2S) supports proliferation, migration and bioenergetics in colon cancer-associated fibroblasts (CAFs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2017-2980

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.

Augmented H2S production via cystathionine-beta-synthase upregulation plays a role in pregnancy-associated uterine vasodilation.

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.

Abstract 1857: Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis

Abstract Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC. Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed. Results: Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p&amp;lt;0.001) and number (p&amp;lt;0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified. Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future. Citation Format: Paul Johnson, Ches’Que M. Phillips, Carl Grim, John R. Zatarain, Aakash H. Gajjar, Suimin Qiu, Rui Wang, Celia Chao, Iryna V. Pinchuk, Mark R. Hellmich. Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1857.

Exendin-4 regulates redox homeostasis in rats fed with high-fat diet.

Non-alcoholic fatty liver disease (NAFLD) is associated with increased plasma homocysteine level, which is caused by down-regulation of hepatic cystathionine beta-synthase (CBS) activity. CBS catalyzes the first step in the transsulfuration of homocysteine to cysteine, which contributes ∼50% of the cysteine required for hepatic biosynthesis of glutathione (GSH), the most abundant antioxidant in cells. As the glucagon-like peptide-1 (GLP-1) receptor agonists (e.g. exendin-4) effectively reverse hepatic steatosis, the effect of exendin-4 on both homocysteine and redox status was investigated in the livers of rats fed with high-fat diet (HFD). It was found that HFD down-regulated CBS protein expression, which was probably due to induction of rno-miR-376c expression in the liver. The level of GSH was markedly reduced, whereas the level of malonydialdehyde, an indicator of lipid peroxidation, was significantly increased in the livers of rats fed with HFD. Exendin-4 treatment increased hepatic CBS protein and GSH levels, and reduced malonydialdehyde level in hyperlipidemic rats. Our findings suggest that GLP-1 receptor agonists have beneficial effects on redox homeostasis in NAFLD.

P91: Cystathionine-beta-synthase expression in medulla oblongata can be up-regulated by in utero cigarette exposure in neonatal rats

H2S has been found to act as a neuroprotectant. Cystathionine-beta-synthase (CBS) is one of three enzymes for production of H2S in mammals. The present study was conducted to investigate whether CBS expression can be affected by in utero cigarette smoke exposure (CS) in the neonatal rats. SD pregnant rats were randomly divided into control and CS groups. The neonatal offspring rats were used in experimental observation. The methylene blue assay method was used to examine H2S production rate by medulla oblongata. CBS mRNA and protein expression in medulla oblongata were analysed by RT-PCR and Western blot, respectively, and the levels of CBS expression in neurons and glia of medullary nuclei were examined with immunohistochemical technique. In medulla oblongata of CS group, compared to control group, H2S production rate was increased (P

P96 Chronic intermittent hypoxia upregulates cystathionine-beta-synthase-hydrogen sulfide pathway in adult rat medulla oblongata

We previously revealed that cystathionine-beta-synthase (CBS) for production of H 2 S is expressed in neonatal rat medulla oblongata and CBS-H 2 S pathway contributes to protection of medullary respiratory centers against injury induced by acute hypoxia in both neonatal and adult rats. The present study was carried out to explore if expression of CBS/H 2 S in medulla oblongata is upregulated by chronic intermittent hypoxia (CIH) in adult rats. Adult SD rats were randomly divided into control and CIH groups. The levels of mRNA and protein of CBS in medulla oblongata were analyzed by RT-PCR and Western blot, respectively. The expression of CBS protein in medullary respiration-related nuclei was examined with immunohistochemistry. H 2 S production rate by medulla oblongata was measured by methylene blue assay. It was indicated that the levels of both mRNA and protein of CBS in medulla oblongata were higher in CIH group ( P P 2 S by medulla oblongata was elevated in CIH group ( P 2 S in medulla oblongata can be upregulated by CIH, suggesting that CBS-H 2 S pathway may be involved in protection of medullary respiratory centers against CIH injury in adult rats.

Increased protein aggregation in Zucker Diabetic Fatty rat brain: identification of key mechanistic targets and the therapeutic application of hydrogen sulfide

BackgroundDiabetes and particularly high blood glucose levels are implicated in neurodegeneration. One of the hallmarks of neurodegeneration is protein aggregation. We investigated the presence of protein aggregation in the frontal brain of Zucker diabetic fatty (ZDF) rats, an animal model for diabetes. Further, the effect of NaHS in suppressing protein aggregation in cultured brain slices from ZDF was assessed.ResultsThe levels of protein synthesis, protein/gene expression, autophagy and anti-oxidant defense were evaluated in ZDF and control (Lean) brains.Compared to Lean, ZDF brains displayed a significant increase in protein aggregates, p-tau, fibronectin expression and protein glycosylation. Increased phosphorylation of mTOR and S6 ribosomal protein in ZDF indicated higher protein synthesis, while the increase in ubiquitinated proteins and LC3-I in ZDF brains accompanied by lower LC3-II expression and LC3-II/LC3-I levels indicated the blockage of proteolytic pathways. CBS (cystathionine beta synthase) protein and mRNA expression and thiol group levels in ZDF brains were lower compared to Lean. ZDF brains show a higher level of reactive oxygen species. In vitro NaHS treatment normalized proteostasis while counteracting oxidative stress.ConclusionOur data demonstrate increased protein synthesis and aggregation in the diabetic ZDF rat brain, which was reversible by NaHS treatment.This is the first report on the potential use of NaHS as a novel strategy against protein aggregation in diabetic brain.

P15 The vascular effect of zofenopril, a sulfhydrylated ACE inhibitor, involves l-cys/H2S pathway

Background Hydrogen sulphide (H2S) is a novel gaseous mediator enzymatically produced by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). It is involved in physiological homeostatic processes and several pathological conditions; however, more importantly, H2S has a prominent role in cardiovascular system, where CSE deletion results in hypertension [1] .ACE inhibitors are widely used in controlling blood pressure in hypertensive patients and they represent first line treatment in different cardiovascular diseases, since they also show additional beneficial effects unrelated to ACE inhibition [2] , [3] .In particular, therapeutic use of zofenopril, a sulfhydrylated ACE inhibitor, has raised hypothesis over a potential role of thiol group in such beneficial effects, other than antioxidant activity.Here, we aimed to investigate on vascular effect of zofenoprilat, water-soluble metabolite of zofenopril, with respect to H2S pathway activation. Methods In order to pursue our goal, we performed isolated organ bath experiments by using aorta and carotid arteries harvested from Wistar Kyoto rats, where we tested vascular effect of zofenoprilat, compared to other ACE inhibitors, in presence of CSE inhibitor PAG or after endothelium removal.In addition, we also performed in vivo experiment, where we tested vascular response after two weeks treatment with zofenopril compared to enalapril in spontaneously hypertensive rats.We also determined vascular response to l -cys, precursor for H2S production, and quantified H2S levels in plasma and tissues and CSE and CBS protein levels upon different treatments. Results In vitro data showed that zofenoprilat, but not enalaprilat, was able to relax both aorta and carotid arteries in a concentration dependent fashion (100 nM–1 mM), reaching ∼60% vasodilation in both cases.Endothelium removal significantly reduced zofenoprilat induced vasorelaxation in aorta (EC50:8.5 μM vs 381 μM), but this effect was not observed in carotid.Next, CSE inhibition by PAG nearly abrogated zofenoprilat vasodilation in both aorta and carotid, indicating a role for H2S in this effect.In vivo data, first highlighted a significantly stronger effect of zofenopril vs enalapril treatment in lowering blood pressure (p l -cys was significantly improved in zofenopril vs enalapril treated animals (p Conclusion In conclusion, we show that sulfhydrylated ACE inhibitor zofenopril triggers H2S pathway and regulates different signal transduction cascades, other than inhibition of ACE. We suggest that this action could be, at least partly, explicative of the additional effects reported in the clinical literature about sulfhydrylated ACE inhibitors. Our data also imply the chance to understand, by using a drug already adopted in clinic, the role played by the l -Cys/H2S pathway in human hypertension.

Genetic studies of the cystathionine beta-synthase gene and myelomeningocele

Among infants born with spina bifida, the most common defect is myelomeningocele (MM). The prevention of MM by maternal periconceptional folic acid (FA) supplementation has been studied extensively. The protective effect provided by FA suggests that the genes involved in folate metabolism, such as cystathionine beta-synthase (CBS), warrant further investigation. This study sequenced the DNA from 96 patients with MM to identify novel potential disease-causing variants across the 17 exons of the CBS gene. The frequencies of known single nucleotide polymorphisms (SNPs) were identified, and sequences that differed from the reference sequences were considered novel variants. Statistical analysis was performed using two-sided Fisher's exact test to compare frequencies of SNPs between groups of patients and the known population frequencies. We found a new variant in exon 3 in one patient that results in a G/A change subsequently encoding a stop codon. In addition, we found a new variant in the 3'-UTR of exon 17. Allele frequencies for 10 known single nucleotide polymorphisms (SNPs) were determined: rs234706, rs72058776, rs1801181, rs6582281, rs71872941, rs12613, rs706208, rs706209, rs73906420, and rs9982921. Of the remaining 48 known SNPs, all tested DNAs were homozygous for the major allele. We identified a previously undescribed variant in exon 3 that encodes a stop codon, thus halting downstream translation of the CBS protein. According to the Human Splicing Finder, the 3'-UTR variant found in exon 17 is predicted to abolish the recognition sites for two splice binding factors, SRp40 and SF2/ASF. The functional significance of the 3'-UTR mutation needs to be investigated.

The Quantitative Significance of the Two Transsulfuration Enzymes for Tissue H2S Production

Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in various physiological processes such as regulation of blood pressure. Cystathionine beta‐synthase (CBS) and gamma‐cystathioninase (CSE) are the two enzymes that are responsible for the formation of H2S in the transsulfuration pathway. Quantitative Western blot analysis was used to determine the relative abundance of each enzyme in murine liver and kidney. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H2S generation in these tissues and in brain. Our results show that the level of CBS protein is significantly lower than CSE; 60‐fold and 20‐fold in liver and kidney respectively. Levels of both enzymes are higher in liver compared to kidney, 2‐fold and 6‐fold for CBS and CSE respectively. At high substrate concentrations (20 mM each cysteine and homocysteine), CBS and CSE contribute equally to H2S production in liver extract while CBS constitutes the major source of H2S in kidney and brain with CSE contributing approximately 20% and 6% respectively. Determination of tissue levels of these enzymes is important for therapeutic reasons that aim to specifically modulate H2S synthesis in tissue‐specific manner.

Rescue of Cystathionine β-Synthase (CBS) Mutants with Chemical Chaperones: PURIFICATION AND CHARACTERIZATION OF EIGHT CBS MUTANT ENZYMES

Missense mutations represent the most common cause of many genetic diseases including cystathionine beta-synthase (CBS) deficiency. Many of these mutations result in misfolded proteins, which lack biological function. The presence of chemical chaperones can sometimes alleviate or even restore protein folding and activity of mutant proteins. We present the purification and characterization of eight CBS mutants expressed in the presence of chemical chaperones such as ethanol, dimethyl sulfoxide, or trimethylamine-N-oxide. Preliminary screening in Escherichia coli crude extracts showed that their presence during protein expression had a significant impact on the amount of recovered CBS protein, formation of tetramers, and catalytic activity. Subsequently, we purified eight CBS mutants to homogeneity (P49L, P78R, A114V, R125Q, E176K, P422L, I435T, and S466L). The tetrameric mutant enzymes fully saturated with heme had the same or higher specific activities than wild type CBS. Thermal stability measurements demonstrated that the purified mutants are equally or more thermostable than wild type CBS. The response to S-adenosyl-L-methionine stimulation or thermal activation varied. The lack of response of R125Q and E176K to both stimuli indicated that their specific conformations were unable to reach the activated state. Increased levels of molecular chaperones in crude extracts, particularly DnaJ, indicated a rather indirect effect of the chemical chaperones on folding of CBS mutants. In conclusion, the chemical chaperones present in the expression medium were able to fully restore the activity of eight CBS mutants by improving their protein folding. This finding could have direct implications for the development of a therapeutical approach to pyridoxine unresponsive homocystinuria.

Human Cystathionine β-Synthase Is a Target for Sumoylation

Cystathionine beta-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits the toxic metabolite, homocysteine, to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. The molecular basis of the organ-specific pathologies associated with CBS deficiency is unknown as is the significance of the reported interaction between CBS and Huntingtin protein. In this study, we have used the yeast two-hybrid approach to screen for proteins that interact with CBS and have identified several components of the sumoylation pathway including Ubc9, PIAS1, PIAS3, Pc2, and RanBPM. We demonstrate that CBS is modified by the small ubiquitin-like modifier-1 protein (SUMO-I) under both in vitro and in vivo conditions. Deletion analysis of CBS indicates that the C-terminal regulatory domain is required for interaction with proteins in the sumoylation machinery. Sumoylated CBS is present in the nucleus where it is associated with the nuclear scaffold. The discovery that CBS is a target of sumoylation adds another layer to the complex regulation of this enzyme and reveals a previously unknown residence for this protein, i.e., in the nucleus.

Expression of mutant human cystathionine β-synthase rescues neonatal lethality but not homocystinuria in a mouse model

Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder in humans characterized by elevated levels of total plasma homocysteine (tHcy) and frequent thrombosis in humans. The I278T mutation is the most common mutation found in human CBS-deficient patients. The T424N mutation was identified as a mutation in human CBS that could restore function to I278T in Saccharomyces cerevisiae. In this report, we have engineered mice that express human I278T and I278T/T424N proteins from a metallotheinein-driven transgene. These transgene-containing mice were then bred to CBS knockout animals (Cbs-) to generate mice that express only human I278T or I278T/T424N protein. Both the I278T and the I278T/T424N transgenes are able to entirely rescue the previously described neonatal mortality phenotype despite the animals having a mean tHcy of 250 microm. The transgenic Cbs-/- animals exhibit facial alopecia, have moderate liver steatosis and are slightly smaller than heterozygous littermates. In contrast to human CBS deficiency, these mice do not exhibit extreme methioninemia. The mutant proteins are stable in the liver, kidney and colon, and liver extracts have only 2-3% of the CBS enzyme activity found in wild-type mice. Surprisingly, the I278T/T424N enzyme had exactly the same activity as the I278T enzyme indicating that T424N is unable to suppress I278T in mice. Our results show that elevated tHcy per se is not responsible for the neonatal lethality observed in Cbs-/- animals and suggests that CBS protein may have a function in addition to its role in homocysteine catabolism. These transgenic animals should be useful in the study of homocysteine related human disease.