Cystathionine Beta-synthase Activity Research Articles

Potential Pharmacological Chaperones for Cystathionine Beta-Synthase-Deficient Homocystinuria.

Classical homocystinuria (HCU) is the most common loss-of-function inborn error of sulfur amino acid metabolism. HCU is caused by a deficiency in enzymatic degradation of homocysteine, a toxic intermediate of methionine transformation to cysteine, chiefly due to missense mutations in the cystathionine beta-synthase (CBS) gene. As with many other inherited disorders, the pathogenic mutations do not target key catalytic residues, but rather introduce structural perturbations leading to an enhanced tendency of the mutant CBS to misfold and either to form nonfunctional aggregates or to undergo proteasome-dependent degradation. Correction of CBS misfolding would represent an alternative therapeutic approach for HCU. In this review, we summarize the complex nature of CBS, its multi-domain architecture, the interplay between the three cofactors required for CBS function [heme, pyridoxal-5'-phosphate (PLP), and S-adenosylmethionine (SAM)], as well as the intricate allosteric regulatory mechanism only recently understood, thanks to advances in CBS crystallography. While roughly half of the patients respond to treatment with a PLP precursor pyridoxine, many studies suggested usefulness of small chemicals, such as chemical and pharmacological chaperones or proteasome inhibitors, rescuing mutant CBS activity in cellular and animal models of HCU. Non-specific chemical chaperones and proteasome inhibitors assist in mutant CBS folding process and/or prevent its rapid degradation, thus resulting in increased steady-state levels of the enzyme and CBS activity. Recent interest in the field and available structural information will hopefully yield CBS-specific compounds, by using high-throughput screening and computational modeling of novel ligands, improving folding, stability, and activity of CBS mutants.

Targeting Cystathionine Beta-Synthase Misfolding in Homocystinuria by Small Ligands: State of the Art and Future Directions

Classical homocystinuria (HCU) is the most common loss-of-function inborn error of sulfur amino acids metabolism. HCU is caused by a deficiency in enzymatic degradation of homocysteine, a toxic intermediate of methionine transformation to cysteine, chiefly due to missense mutations in the cystathionine betasynthase (CBS) gene. As with many other inherited disorders, the pathogenic mutations do not target key catalytic residues, but rather introduce structural perturbations leading to an enhanced tendency of the mutant CBS to misfold and either to form non-functional aggregates or to undergo proteasome-dependent degradation. Thus correction of CBS misfolding represents an alternative therapeutic approach for HCU. In this review, we summarize the complex nature of CBS, its multidomain architecture, the interplay between the three cofactors required for CBS function (heme, pyridoxal-5'-phosphate (PLP) and S-adenosyl-L-methionine) as well as the intricate allosteric regulatory mechanism only recently explained thanks to advances in CBS crystallography. While roughly half of the patients responds to treatment with a PLP precursor pyridoxine, many studies suggested usefulness of small chemicals, such as chemical and pharmacological chaperones or proteasome inhibitors, rescuing mutant CBS activity in cellular and animal models of HCU. Non-specific chemical chaperones and proteasome inhibitors assist in mutant CBS folding process and/or prevent its rapid degradation, thus resulting in increased steady state levels of the enzyme and CBS activity. Recent increased interest in the field and available structural information will hopefully yield CBS-specific compounds by using high-throughput screening and computational modeling of novel ligands improving folding, stability and activity of CBS.

Kinetic stability of cystathionine beta-synthase can be modulated by structural analogs of S-adenosylmethionine: Potential approach to pharmacological chaperone therapy for homocystinuria.

Many pathogenic missense mutations in human cystathionine beta-synthase (CBS) cause misfolding of the mutant enzyme resulting in aggregation or rapid degradation of the protein. Subsequent loss of CBS function leads to CBS-deficient homocystinuria (CBSDH). CBS contains two sets of binding sites for S-adenosylmethionine (SAM) that independently regulate the enzyme activity and kinetically stabilize its regulatory domain. In the present study, we examined the hypothesis that CBS activation may be decoupled from kinetic stabilization and thus CBS regulatory domain can serve as a novel drug target for CBSDH. We determined the effect of SAM and its close structural analogs on CBS activity, their binding to and stabilization of the regulatory domain in the absence and presence of competing SAM. Binding of S-adenosylhomocysteine and sinefungin lead to stabilization of the regulatory domains without activation of CBS. Direct titrations and competition experiments support specific binding of these two SAM analogs to the stabilizing sites. Binding of these two ligands also affects the enzyme proteolysis rate supporting the role of the stabilizing sites in CBS dynamics. Our results indicate that binding of SAM to regulatory and stabilizing sites in CBS may have evolved to display an exquisite thermodynamic and structural specificity towards SAM as well as the ability to transduce the allosteric signal responsible for CBS activation. Thus, ligands may be developed to function as kinetic stabilizers or pharmacological chaperones without interfering with the physiological activation of CBS by SAM.

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.

Cystathionine-beta-synthase inhibition for colon cancer: Enhancement of the efficacy of aminooxyacetic acid via the prodrug approach.

Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

Small aminothiol compounds improve the function of Arg to Cys variant proteins: effect on the human cystathionine β-synthase p.R336C.

The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, ∼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.

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.

Novel Cancer Therapy: Targeting Methionine Metabolism

Methionine is an essential amino acid required in larger quantities during development. Albeit considered essential certain amounts of methionine can be synthesized through methionine synthase (MetS) activity from homocysteine using coenzymes methyl‐tetrahydrofolate (vitamin B9) and methylcobalamin (vitamin B12). Homocysteine can be reused to make methionine which is controlled by the levels of methyl‐tetrahydrofolate (vitamin B9) and methylcobalamin (vitamin B12). The other fate of homocysteine in mammals is to be channeled for cysteine synthesis which are controlled by serine, vitamin B12, and the activities of cystathionine betasynthase and cystathionine gammalyase. Methionine is activated into s‐adenosylmethionine (SAM) by methionine adenosyltransferase using ATP. Cytoplasmic and Nuclear MAT had been characterized, establishing the specific compartmentalized methylations. In the nucleus RNA, DNA and histones are modified by methylation, limited by SAM. In cytoplasm phosphatidyl choline, epinephrine, carnitine synthesis etc. requires SAM for methylation. In bacteria, methionine is catabolized to methylthiol and deaminated into alpha‐ketobutyrate by methionine gammalyase‐deaminase (MGLD). MGLD is absent in mammals. Molecularly cloned cytoplasmic‐GFP‐MGLD transfected into many cancer cells evinced either moderate cell death/severe aggregation depending on the cell types. With this priori, GFP‐MGLD gene with nuclear localization signal NLS‐GFP‐MGLD) was transfected into Hela, Ad293T, BHK, PC3 and DU145. In conclusion: total (quantitative) and site‐specific (qualitative) methylation pattern/signature is extremely crucial for cell progression that varies among cell types. Specific global methylation patterns/signatures studies are underway.

Marine natural products as inhibitors of cystathionine beta-synthase activity

A library consisting of characterized marine natural products as well as synthetic derivatives was screened for compounds capable of inhibiting the production of hydrogen sulfide (H2S) by cystathionine beta-synthase (CBS). Eight hits were validated and shown to inhibit CBS activity with IC50 values ranging from 83 to 187μM. The majority of hits came from a series of synthetic polyandrocarpamine derivatives. In addition, a modified fluorogenic probe for H2S detection with improved solubility in aqueous solutions is reported.

同型半胱氨酸尿症临床与实验室诊断

同型半胱氨酸尿症，常伴血浆同型半胱氨酸和蛋氨酸水平增高，是一种涉及胱硫醚β-合成酶活性改变的蛋氨酸代谢遗传性疾病。常导致心血管系统、中枢神经系统、肌肉和结缔组织等多系统功能障碍。此种代谢疾病的特点为血清同型半胱氨酸堆积和同型半胱氨酸在尿液中排出的增加。同型半胱氨酸的实验室分析复杂，综合分析对于确诊同型半胱氨酸尿症，及对疾病治疗疗效的监测有所裨益。 Homocystinuria with increased plasma homocysteine and methionine level is an inherited disorder of the metabolism of methionine, often involves the change of cystathionine beta synthase activity. This disorder leads to a multi-systemic disease of the cardiovascular system, CNS, muscles, and connective tissue. Homocystinuria represents a group of hereditary metabolic disorders characterized by the accumulation of homocysteine in serum and elevated excretion of homocysteine in urine. Despite the lab analysis of homocysteine is complicated, it gives a clue to make a definite diagnosis of homocystinuria and esepcially helps to monitor therapy efficacy.

Neuroprotection of up-regulated carbon monoxide by electrical acupuncture on perinatal hypoxic-ischemic brain damage in rats.

This study investigated the neuroprotection and potential mechanism of carbon monoxide (CO) against perinatal hypoxic-ischemic brain damage in rats by electrical acupuncture (EA). Animal behavior, morphological changes, cystathionine beta-synthase (CBS), hypoxia-inducible factor-1α (HIF-1α), and heme oxygenase-1 (HO-1) expression levels, and CO content in rat cortex cells were determined. Results demonstrated that EA treatment decreased the slope behavior and increased the overhang behavior of perinatal rats. The treatment also decreased the number of positive cells. The activator and inhibitor of CBS aggravated and remitted the hypoxic damage in cortex cells, respectively. EA treatment decreased CBS expression level and increased HO-1 and HIF-1α expression levels in perinatal rat cortex cells. Compared with the control groups, the CO content of cortex cells in the EA treatment group significantly increased (**p < 0.01). We hypothesized that EA treatment increases cortical CO content to protect against hypoxic damage via the hydrogen sulfide/CBS-CO/HO-1-HIF-1α system. This study provided a significant reference for EA therapy and cued a novel protective mechanism for cerebral palsy.

Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats

Homocystinuria is a neurometabolic disease caused by severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction, being that the pathomechanism is not fully understood. In the present study we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely 2'7'dichlorofluorescein (DCFH) oxidation, levels of thiobarbituric acid-reactive substances (TBARS), antioxidant enzyme activities (SOD, CAT and GPx), reduced glutathione (GSH), total sulfhydryl and carbonyl content, as well as nitrite levels in soleus skeletal muscle of young rats subjected to model of severe hyperhomocysteinemia. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3-0.6 μmol/g body weight), and/or creatine (50mg/kg body weight) from their 6th to the 28th days age. Controls and treated rats were decapitated at 12h after the last injection. Chronic homocysteine administration increased 2'7'dichlorofluorescein (DCFH) oxidation, an index of production of reactive species and TBARS levels, an index of lipoperoxidation. Antioxidant enzyme activities, such as SOD and CAT were also increased, but GPx activity was not altered. The content of GSH, sulfhydril and carbonyl were decreased, as well as levels of nitrite. Creatine concurrent administration prevented some homocysteine effects probably by its antioxidant properties. Our data suggest that the oxidative insult elicited by chronic hyperhomocystenemia may provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function. Creatine prevents some alterations caused by homocysteine.

One-carbon cycle alterations induced by Dyrk1a dosage

Hyperhomocysteinemia due to cystathionine beta synthase deficiency confers diverse clinical manifestations. It is characterized by elevated plasma homocysteine levels, a common amino acid metabolized by remethylation to methionine or transsulfuration to cysteine. We recently found a relationship between hepatic Dyrk1A protein expression, a serine/threonine kinase involved in signal transduction in biological processes, hepatic S-adenosylhomocysteine activity, and plasma homocysteine levels. We aimed to study whether there is also a relationship between Dyrk1a and cystathionine beta synthase activity. We used different murine models carrying altered gene coy numbers for Dyrk1a, and found a decreased cystathionine beta synthase activity in the liver of mice under-expressing Dyrk1a, and an increased in liver of mice over-expressing Dyrk1a. For each model, a positive correlation was found between cystathionine beta synthase activity and Dyrk1a protein expression in the liver of mice, which was confirmed in a non-modified genetic context. The positive correlation found between liver Dyrk1a protein expression and CBS activity in modified and non-modified genetic context strengthens the role of this kinase in one carbon metabolism.

PL02 Modulation of H2S levels in cells and tissues using pharmacological agents: Advances and pitfalls

The biological roles of endogenous H 2 S are multiple and rapidly expanding. Hydrogen sulfide exerts its effects in most organ systems including the central and peripheral nervous system, the cardiovascular, gastrointestinal and respiratory systems; H 2 S also participates in the regulation of cellular metabolism and immunological/inflammatory responses. Much of the enzymatically generated H 2 S is derived from two pyridoxal-5′-phosphate (PLP)-dependent enzymes responsible for the metabolism of l -cysteine: cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE); a third pathway that catalyzes the production of H 2 S from l -cysteine via the combined action of 3-mercaptopyruvate sulphurtransferase and cysteine aminotransferase (3MST/CAT) has also been described. CSE and CBS catalyze several H 2 S-generating reactions using cysteine and/or homocysteine as substrates. CBS is the predominant H 2 S-generating enzyme in the brain and nervous system and is highly expressed in liver and kidney, while CSE is mainly expressed in the liver and in vascular and non-vascular smooth muscle. To study the role of this novel gasotransmitter, investigators have relied on genetic models, knock down of H 2 S-producing enzymes by siRNA, and on pharmacological inhibitors. As genetic and siRNA approaches require additional expertise and access to specialized facilities, most researchers prefer to use pharmacological inhibitors. The most commonly used agents to inhibit H 2 S biosynthesis include propargylglycine (PAG), β -cyanoalanine (BCA), aminooxyacetic acid (AOAA), trifluoroalanine and hydroxylamine. Using recombinant human H 2 S-producing enzymes we have found that PAG exhibits selectivity towards CSE vs. CBS, but its potency is lower than that of l -aminoethoxyvinylglycine (AVG). BCA, although selective for CSE, it also inhibits CBS when used at high concentrations. In contrast, none of the compounds tested exhibited significant selectivity towards CBS. In addition, the above-mentioned compounds did not inhibit 3MST. It should also be stressed that even the inhibitors that were found to be selective for CSE have been shown to inhibit other PLP-dependent enzymes. Inhibiting CSE or CBS activity will affect the levels of several metabolites and intermediates in the transulfuration pathway. For example, CBS inhibition leads to increased homocysteine levels. To avoid this unwanted side effect when long term inhibition of CBS or CSE is desired (such as in conditions where H 2 S production is up regulated contributing to disease progression), scavenging of H 2 S would be the preferred approach. Our efforts to develop H 2 S scavengers will be presented during the meeting. We conclude that design and development of new inhibitors for each of the H 2 S-synthesizing enzymes, as well as H 2 S scavengers is an unmet need for the field, so that the role of H 2 S-regulated pathways in the development and treatment of disease can be evaluated.

P23 CBS gene variants are associated with conditions characterized by ischemia

The CBS gene regulates the expression of cystathionine beta-synthase (CBS), a key enzyme in the production of hydrogen sulfide (H 2 S) by conversion of homocysteine (Hcy). H 2 S is a gasotransmitter that protects cells against ischemic damage by exerting vasodilatory, anti-inflammatory and antioxidant effects. Both renal transplantation and preeclampsia (PE) are conditions marked by ischemia. Renal ischemia/reperfusion impairs CBS activity and consequently H 2 S production. Partial restoration of CBS activity causes an increase in H 2 S levels and a reduction of ischemia/reperfusion damage. Also, H 2 S is involved in placental vascular tone regulation. Furthermore, preeclamptic women have hyperhomocysteinemia and a decreased placental expression of the CBS gene. We therefore hypothesize that variations in the CBS gene caused by single nucleotide polymorphisms (SNPs) affect both susceptibility to ischemic damage in renal transplantation and the onset of PE. The genotype of 1271 renal transplantation donor and recipient pairs in the University Medical Centre Groningen were determined for seven tag SNPs in the CBS locus. These SNPs were analyzed for association with primary non-function (PNF), delayed graft function, first years biopsy proven acute rejection, death-censored graft survival and patient survival. In the Oslo University Hospital, 99 controls and 60 severe and 39 mild cases of PE were genotyped for six of these seven tag SNPs. Severe and mild cases of PE were subdivided into early- (<34 weeks) and late-onset (>34 weeks of pregnancy). The SNPs were then analyzed for association with the onset of PE. Univariate analysis showed improved graft survival in kidney transplant recipients that homozygously carry the minor allele of rs11203172 (HR[95%CI] = 0.124[0.017–0.882], p = 0.013 (homozygous carriers of the minor allele versus others)). Furthermore, no PNF was seen in these patients. Multivariate analyses showed no significant associations, most probably due to the relatively low number of patients in this group and the number of parameters studied. Univariate analysis also showed patients carrying the minor allele of rs11203172 to have a reduced risk to develop severe PE (OR[95%CI] = 0.54[0.21–0.94], p = 0.023). Conversely, the minor allele of rs234706, which is known to be associated with low Hcy, increased the risk to develop mild, late-onset PE (2.10[1.15–3.85], p = 0.016). SNPs in the CBS gene are associated with both kidney graft survival and the risk to develop PE. Changes of the function or expression of CBS caused by SNPs may affect ischemic conditions through decreased H 2 S production or Hcy accumulation. Since CBS is markedly reduced under ischemic conditions we assume that these findings are of considerable importance. Further characterization of the effects of these CBS gene variants and their alleged role in ischemia is warranted.

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.

Homocysteine induces energy imbalance in rat skeletal muscle: Is creatine a protector?

Homocystinuria is a neurometabolic disease caused by a severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction. In this study, we investigated the effect of chronic hyperhomocysteinemia on the cell viability of the mitochondrion, as well as on some parameters of energy metabolism, such as glucose oxidation and activities of pyruvate kinase, citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, respiratory chain complexes and creatine kinase in gastrocnemius rat skeletal muscle. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 µmol/g body weight) and/or creatine (50 mg/kg body weight) from the 6th to the 28th days of age. The animals were decapitated 12 h after the last injection. Homocysteine decreased the cell viability of the mitochondrion and the activities of pyruvate kinase and creatine kinase. Succinate dehydrogenase was increased other evaluated parameters were not changed by this amino acid. Creatine, when combined with homocysteine, prevented or caused a synergistic effect on some changes provoked by this amino acid. Creatine per se or creatine plus homocysteine altered glucose oxidation. These findings provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function, more studies are needed to elucidate them. Although creatine prevents some alterations caused by homocysteine, it should be used with caution, mainly in healthy individuals because it could change the homeostasis of normal physiological functions.

Blood biomarkers of methylation in Down syndrome and metabolic simulations using a mathematical model

The study tests the metabolites of the methylation cycle in individuals with Down syndrome (DS) and applies a mathematical model in order to change this cycle by nutritional factors. We measured concentrations of the metabolites related to the methylation cycle in the blood of 35 young individuals with DS and 47 controls of comparable age. Moreover, we applied a mathematical model to learn more about the regulation of the methylation cycle in DS. Concentrations of cystathionine, cysteine, betaine, choline, dimethylglycine, S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), and holotranscobalamin were significantly higher in DS compared to the controls. The median SAM/SAH ratio was lower in DS and that of methionine and reduced glutathione did not differ significantly between the groups. The mathematical model showed that enhanced methionine turnover and accelerated Hcy-remethylation might explain the shift in the methylation cycle in DS. In addition to the DS-related excess of cystathionine beta synthase (CBS) activity, increases in the activities of MS and betaine homocysteine methyl transferase, and in methionine input were necessary to account for the changes in metabolite levels observed in DS. A low-methionine diet might offer a perspective for reversing the metabolic imbalance in DS, but this awaits clinical investigations.

NO- and H2S-synthesizing neurons in the thoracic ganglion of crabs Eriocheir japonicus and Pugettia quadridens (Decapoda)

By methods of histochemical marking of NADPH-diaphorase and immunohistochemical marking of cystathionine-beta-synthase (CBS), the presence and distribution of NO and H2S-synthesizing elements in thoracic ganglion are shown in two crab species Eriocheir japonicus and Pugettia quadreidens. In the thoracic ganglion, NO and H2S have been established to be synthesized by different neurons. The high CBS activity in efferent elements--segmentary motoneurons, interneurons, and nerve fibers in the extremity neuropil--indicates involvement of hydrogen sulfide in organization of the crustacean motor behavior. The presence of H2S-synthesizing neurons in the wall of sternal artery in E. japonicus allows suggesting participation of H2S in regulation of vascular tonus.

Induction of VMAT-1 and TPH-1 Expression Induces Vesicular Accumulation of Serotonin and Protects Cells and Tissue from Cooling/Rewarming Injury

DDT1 MF-2 hamster ductus deferens cells are resistant to hypothermia due to serotonin secretion from secretory vesicles and subsequent cystathionine beta synthase (CBS) mediated formation of H2S. We investigated whether the mechanism promoting resistance to hypothermia may be translationally induced in cells vulnerable to cold storage. Thus, VMAT-1 (vesicular monoamino transferase) and TPH-1 (tryptophan hydroxylase) were co-transfected in rat aortic smooth muscle cells (SMAC) and kidney tissue to create a serotonin-vesicular phenotype (named VTSMAC and VTkidney, respectively). Effects on hypothermic damage were assessed. VTSMAC showed a vesicular phenotype and an 8-fold increase in serotonin content and 5-fold increase in its release upon cooling. Cooled VTSMAC produced up to 10 fold higher concentrations of H2S, and were protected from hypothermia, as shown by a 50% reduction of caspase 3/7 activity and 4 times higher survival compared to SMAC. Hypothermic resistance was abolished by the inhibition of CBS activity or blockade of serotonin re-uptake. In VTkidney slices, expression of CBS was 3 fold increased in cold preserved kidney tissue, with two-fold increase in H2S concentration. While cooling induced substantial damage to empty vector transfected kidney as shown by caspase 3/7 activity and loss of FABP1, VTkidney was fully protected and comparable to non-cooled control. Thus, transfection of VMAT-1 and TPH-1 induced vesicular storage of serotonin which is triggered release upon cooling and has protective effects against hypothermia. The vesicular serotonergic phenotype protects against hypothermic damage through re-uptake of serotonin inducing CBS mediated H2S production both in cells and kidney slices.