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Hyperhomocysteinemia (HHcy) is associated with increased diabetic cardiovascular diseases. However, the role of HHcy in atherogenesis associated with hyperglycemia (HG) remains unknown. To examine the role and mechanisms by which HHcy accelerates HG-induced atherosclerosis, we established an atherosclerosis-susceptible HHcy and HG mouse model. HHcy was established in mice deficient in cystathionine β-synthase (Cbs) in which the homocysteine (Hcy) level could be lowered by inducing transgenic human CBS (Tg-hCBS) using Zn supplementation. HG was induced by streptozotocin injection. Atherosclerosis was induced by crossing Tg-hCBS Cbs mice with apolipoprotein E-deficient (ApoE-/-) mice and feeding them a high-fat diet for 2 weeks. We demonstrated that HHcy and HG accelerated atherosclerosis and increased lesion monocytes (MCs) and macrophages (MØs) and further increased inflammatory MC and MØ levels in peripheral tissues. Furthermore, Hcy-lowering reversed circulating mononuclear cells, MC, and inflammatory MC and MC-derived MØ levels. In addition, inflammatory MC correlated positively with plasma Hcy levels and negatively with plasma s-adenosylmethionine-to-s-adenosylhomocysteine ratios. Finally, l-Hcy and d-glucose promoted inflammatory MC differentiation in primary mouse splenocytes, which was reversed by adenoviral DNA methyltransferase-1. HHcy and HG, individually and synergistically, accelerated atherosclerosis and inflammatory MC and MØ differentiation, at least in part, via DNA hypomethylation. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Previously, our laboratory developed a mouse model of CBS deficiency, TgI278T Cbs-/- (abbreviated as Cbs-/-), characterized by low weight, low adiposity, decreased Scd-1 expression, facial alopecia, and osteoporosis. To determine the potential benefit of a methionine-restricted diet (MRD), we fed Cbs-/- and Cbs+/- control mice either an MRD or a regular diet (RD) from weaning till 240 d of age. Cbs-/- mice fed the MRD had a 77% decrease in tHcy, 28% increase in weight, 130% increase in fat mass, 82% increase in Scd-1 expression, and 10.6% increase in bone density and entirely lacked the alopecia phenotype observed in age-matched Cbs-/- mice fed the RD. At the end of the study, Cbs-/- mice fed the MRD were phenotypically indistinguishable from Cbs+/- mice fed the RD. Notably, whereas the MRD diet was highly beneficial to Cbs-/- mice, it had nearly opposite effect on Cbs+/- mice. These studies show that a low-methionine diet can correct the phenotypic consequences of loss of CBS and provide a striking example of how genotype and diet can interact to influence phenotype in mammals. [ABSTRACT FROM AUTHOR]

Objective: The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−. Methods: Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of MtaplacZ/+ and Mtap+/+ mice. Results: Survival in both models was significantly decreased in MtaplacZ/+ compared to Mtap+/+ mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap+/+ mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc MtaplacZ/+ and Eµ-myc Mtap+/+ animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap+/+ and MtaplacZ/+ animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer. Conclusion: Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice. [ABSTRACT FROM AUTHOR]

Diet-induced high circulating levels of homocysteine, also known as hyper-homocysteinemia (HHcy), is associated with an acceleration of Alzheimer's disease-like amyloidosis. Herp is a homocysteine-responsive stress protein, which has been shown to increase the formation of amyloid-β (Aβ) via interaction with presenilins in vitro. The aim of our paper was to investigate the functional role that Herp plays in HHcy-induced amyloidosis. Amyloidosis secondary to diet-induced HHcy in Tg2576 mice is associated with an increase of Herp protein and mRNA levels. By contrast, no other stress-related proteins are altered by the same diet regimen. Compared to wild type animals, brains from a genetically induced HHcy mouse model did not manifest any significant change in Herp levels. Cells stably over-expressing human AβPP Swedish mutant incubated with high levels of homocysteine had an increase in Aβ formation, but no change in Herp level. Finally, over-expression of Herp did not result in any significant modification of Aβ levels. We conclude that the Herp protein pathway is unlikely to be directly involved in the pro-amyloidotic effect of HHcy. [ABSTRACT FROM AUTHOR]

Genetic disorders of homocysteine (Hcy) or folate metabolism or high-methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy-thiolactone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine β-synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (N-Hcy-protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how N-Hcy-protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma N-Hcy-protein was elevated 10-fold in mice fed a high-methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene resulted in a 10- to 30-fold increase in plasma or serum N-Hcy-protein levels. Liver N-Hcy-protein was elevated 3.4-fold in severely and 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice, 3.6-fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr-deficient animals, suggesting that mice have a capacity to prevent accumulation of N-Hcy-protein in their organs. These findings provide evidence that N-Hcy-protein is an important metabolite associated with Hcy pathophysiology in the mouse. [ABSTRACT FROM AUTHOR]

Untreated cystathionine β-synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 µM), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 µM). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg-hCBS Cbs-/- mice, with a mean serum tHcy of 169 µM, and Tg-1278T Cbs-/- mice, with a mean tHcy of 296 µM. Only Tg-1278T Cbs-/- animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival rime. Metabolic profiling of serum and liver reveals that Tg-1278T Cbs-/- mice have significantly elevated levels of free oxidized homocysteine but not protein-bound homocysteine in serum and elevation of all forms of homocysteine and S-adenosyl homocysteine in the liver compared to Tg-hCBS Cbs-/- mice. RNA profiling of livers indicate that Tg-1278T Cbs-/- and Tg-hCBS Cbs-/- mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic. [ABSTRACT FROM AUTHOR]

Human cystathionine β–synthase (CBS) is an S-adenosylmethionine-regulated enzyme that plays a key role in the metabolism of homocysteine. Mutations in CBS are known to cause homocystinuria, an inborn error in metabolism. We previously developed a yeast functional assay for CBS and used it to characterize mutations found in homocystinuric patients. We discovered that many patient-derived mutations are functionally suppressed by deletion of the C‐terminal 142 amino acids, which contain a 53 amino acid motif known as the CBS domain. This domain is found in a wide variety of proteins of diverse biological function. Here we have used a genetic screen to identify missense mutations in the C-terminal region of CBS that can suppress the most common patient mutation, I278T. Seven suppressor mutations were identified, four of which map to the CBS domain. When combined in cis with another pathogenic mutation, V168M, six of seven of the suppressor mutations rescued the yeast phenotype. Enzyme activity analyses indicate that the suppressors restore activity from <2% to 17–64% of the wild-type levels. Analysis of the suppressor mutations in the absence of the pathogenic mutation shows that six of the seven suppressor alleles have lost enzymatic responsiveness to S-adenosylmethionine. Using homology modeling, we show that the suppressor mutations appear to map on one face of the CBS domain. Our results indicate that subtle changes to the C-terminus of CBS can restore activity to mutant proteins and provide a rationale for screening for compounds that can activate mutant CBS alleles. [ABSTRACT FROM PUBLISHER]

Classical homocystinuria is a rare disease caused by mutations in cystathionine β-synthase (CBS) gene (OMIM 613381). CBS catalyzes the first step of the transsulfuration pathway that converts homocysteine (Hcy) into cystathionine (Cysta) via a number of co-substrates and mechanisms. Formation of Cysta by condensation of Hcy and cysteine (Cys) produces a molar equivalent of hydrogen sulfide (H 2 S). H 2 S plays important roles in cognitive and vascular functions. Clinically, patients with CBS deficiency present with vascular, ocular, neurological and skeletal impairments. Biochemically, CBS deficiency manifests with elevated Hcy and reduced concentration of Cysta in plasma and urine. A number of pathogenic variants of human CBS have been characterized by their residual enzymatic activity, but very few studies have examined H 2 S production by pathogenic CBS variants, possibly due to technical hurdles in H 2 S detection and quantification. We describe a method for the real-time, continuous quantification of H 2 S formed by wild-type and pathogenic variants of human recombinant CBS, as well as by fibroblast extracts from healthy controls and patients diagnosed with CBS deficiency. The method takes advantage of the specificity and high affinity of hemoglobin I of the clam Lucina pectinata toward H 2 S and is based on UV–visible spectrophotometry. Comparison with the gold-standard, end-point H 2 S quantification method employing monobromobimane, as well as correlations with CBS enzymatic activity determined by LC-MS/MS showed agreement and correlation, and permitted the direct, time-resolved determination of H 2 S production rates by purified human recombinant CBS and by CBS present in fibroblast extracts. Rates of H 2 S production were highest for wild-type CBS, and lower for pathogenic variants. This method enables the examination of structural determinants of CBS that are important for H 2 S production and its possible relevance to the clinical outcome of patients. [Display omitted] • Hydrogen sulfide (H 2 S) is produced by transsulfuration enzymes and the microbiome. • Cystathionine beta-synthase (CBS) is the major intracellular source of H 2 S. • The chemical properties of H 2 S challenge its quantitation in biological specimens. • Hemoglobin I of Lucina pectinata serves as a specific biosensor to detect H 2 S. • H 2 S formed by purified CBS and in human fibroblasts are quantifiable in real time. [ABSTRACT FROM AUTHOR]

Classic homocystinuria is an inborn error of metabolism caused mainly by missense mutations leading to misfolded and/or unstable human cystathionine β-synthase (CBS) protein, causing the accumulation of excess total homocysteine (tHcy) in tissues. Previously, it has been shown that certain missense containing human CBS proteins can be functionally rescued in mouse models of CBS deficiency by treatment with proteasome inhibitors. The rescue by proteasome inhibitors is thought to work both by inhibiting the degradation of misfolded CBS protein and by inducing the levels of heat-shock chaperone proteins in the liver. Here we examine the effectiveness of two FDA approved protease inhibitors, carfilzomib and bortezomib, on various transgenic mouse models of human CBS deficiency. Our results show that although both drugs are effective in inducing the liver chaperone proteins Hsp70 and Hsp27, and are effective in inhibiting proteasome function, bortezomib was somewhat more robust in restoring the mutant CBS function. Moreover, there was no significant correlation between proteasome inhibition and CBS activity, suggesting that some of bortezomib's effects are via other mechanisms. We also test the use of low-doses of bortezomib and carfilzomib on various mouse models for extended periods of time and find that while low-doses are less toxic, they are also less effective at restoring CBS function. Overall, these results show that while restoration of mutant CBS function is possible with proteasome inhibitors, the exact mechanism is complicated and it will likely be too toxic for long-term patient treatment. [ABSTRACT FROM AUTHOR]

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5μM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca2+-activated potassium channel (KCa) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance KCa (SK) or intermediate-conductance KCa (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O2-), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO-) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O2- production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO- and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O2- production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms. [ABSTRACT FROM AUTHOR]

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 methioninesulfoxide 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. [ABSTRACT FROM AUTHOR]

Circadian disruption influences metabolic health. Metabolism modulates circadian function. However, the mechanisms coupling circadian rhythms and metabolism remain poorly understood. Here, we report that cystathionine β‐synthase (CBS), a central enzyme in one‐carbon metabolism, functionally interacts with the core circadian protein cryptochrome 1 (CRY1). In cells, CBS augments CRY1‐mediated repression of the CLOCK/BMAL1 complex and shortens circadian period. Notably, we find that mutant CBS‐I278T protein, the most common cause of homocystinuria, does not bind CRY1 or regulate its repressor activity. Transgenic CbsZn/Zn mice, while maintaining circadian locomotor activity period, exhibit reduced circadian power and increased expression of E‐BOX outputs. CBS function is reciprocally influenced by CRY1 binding. CRY1 modulates enzymatic activity of the CBS. Liver extracts from Cry1−/− mice show reduced CBS activity that normalizes after the addition of exogenous wild‐type (WT) CRY1. Metabolomic analysis of WT, CbsZn/Zn, Cry1−/−, and Cry2−/− samples highlights the metabolic importance of endogenous CRY1. We observed temporal variation in one‐carbon and transsulfuration pathways attributable to CRY1‐induced CBS activation. CBS‐CRY1 binding provides a post‐translational switch to modulate cellular circadian physiology and metabolic control. [ABSTRACT FROM AUTHOR]

Mutations in the cystathionine β-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The p.G307S mutation is the most frequent cause of CBS deficiency in Ireland, which has the highest prevalence of CBS deficiency in Europe. Individuals homozygous for this mutation tend to be severely affected and are pyridoxine nonresponsive, but the molecular basis for the strong effects of this mutation is unclear. Here, we characterized a transgenic mouse model lacking endogenous Cbs and expressing human p.G307S CBS protein from a zinc-inducible metallothionein promoter (Tg-G307S Cbs-/-). Unlike mice expressing other mutant CBS alleles, the Tg-G307S transgene could not efficiently rescue neonatal lethality of Cbs-/- in a C57BL/6J background. In a C3H/HeJ background, zinc-induced Tg-G307S Cbs-/- mice expressed high levels of p.G307S CBS in the liver, and this protein variant forms multimers, similarly to mice expressingWThuman CBS. However, the p.G307S enzyme had no detectable residual activity. Moreover, treating mice with proteasome inhibitors failed to significantly increase CBS-specific activity. These findings indicated that the G307S substitution likely affects catalytic function as opposed to causing a folding defect. Using molecular dynamics simulation techniques, we found that the G307S substitution likely impairs catalytic function by limiting the ability of the tyrosine at position 308 to assume the proper conformational state(s) required for the formation of the pyridoxal-cystathionine intermediate. These results indicate that the p.G307S CBS is stable but enzymatically inert and therefore unlikely to respond to chaperone-based therapy. [ABSTRACT FROM AUTHOR]

Classical homocystinuria (HCU) is a rare inborn error of amino acid metabolism characterized by accumulation of homocysteine, an intermediate product of methionine metabolism, leading to significant systemic toxicities, particularly within the vascular, skeletal, and ocular systems. Most patients require lifelong dietary therapy with severe restriction of natural protein to minimize methionine intake, and many patients still struggle to maintain healthy homocysteine levels. Since eliminating methionine from the diet reduces homocysteine levels, we hypothesized that an enzyme that can degrade methionine within the gastrointestinal (GI) tract could help HCU patients maintain healthy levels while easing natural protein restrictions. We describe the preclinical development of CDX-6512, a methionine gamma lyase (MGL) enzyme that was engineered for stability and activity within the GI tract for oral administration to locally degrade methionine. CDX-6512 is stable to low pH and intestinal proteases, enabling it to survive the harsh GI environment without enteric coating and to degrade methionine freed from dietary protein within the small intestine. Administering CDX-6512 to healthy non-human primates following a high protein meal led to a dose-dependent suppression of plasma methionine. In Tg - I278T Cbs −/− mice, an animal model that recapitulates aspects of HCU disease including highly elevated serum homocysteine levels, oral dosing of CDX-6512 after a high protein meal led to suppression in serum levels of both methionine and homocysteine. When animals received a daily dose of CDX-6512 with a high protein meal for two weeks, the Tg - I278T Cbs −/− mice maintained baseline homocysteine levels, whereas homocysteine levels in untreated animals increased by 39%. These preclinical data demonstrate the potential of CDX-6512 as an oral enzyme therapy for HCU. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia confers a high risk for thrombotic vascular events, but homocysteine-lowering therapies have been ineffective in reducing the incidence of secondary vascular outcomes, raising questions regarding the role of homocysteine as a mediator of cardiovascular disease. Therefore, to determine the contribution of elevated homocysteine to thrombosis susceptibility, we studied Cbs-/- mice conditionally expressing a zinc-inducible mutated human CBS (I278T) transgene. Tg-I278T Cbs-/- mice exhibited severe hyperhomocysteinemia and endothelial dysfunction in cerebral arterioles. Surprisingly, however, these mice did not display increased susceptibility to arterial or venous thrombosis as measured by photochemical injury in the carotid artery, chemical injury in the carotid artery or mesenteric arterioles, or ligation of the inferior vena cava. A survey of hemostatic and hemodynamic parameters revealed no detectible differences between control and Tg-I278T Cbs-/- mice. Our data demonstrate that severe elevation in homocysteine leads to the development of vascular endothelial dysfunction but is not sufficient to promote thrombosis. These findings may provide insights into the failure of homocysteine-lowering trials in secondary prevention from thrombotic vascular events. [ABSTRACT FROM AUTHOR]

Objective: Elevated plasma total homocysteine (tHcy) is a risk factor for a variety of human diseases. Homocysteine is formed from methionine and has two primary metabolic fates: remethylation to form methionine or commitment to the transsulfuration pathway by the action of cystathionine β-synthase (CBS). We have examined the metabolic response in mice of a shift from a methionine-replete to a methionine-free diet. Methods and results: We found that shifting 3-mo-old C57BL6 mice to a methionine-free diet caused a transient increase in tHcy and an increase in the tHcy/methionine ratio. Because CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 d on the methionine-deficient diet, animals had a 50% reduction in the levels of liver CBS protein and enzyme activity. Examination of CBS mRNA and studies of transgenic animals that express CBS from a heterologous promoter indicated that this reduction is occurring post-transcriptionally. Loss of CBS protein was unrelated to intracellular levels of S-adenosylmethionine, a known regulator of CBS activity and stability. Conclusion: Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway by an S-adenosylmethionine–independent mechanism that signals a rapid downregulation of CBS protein. [Copyright &y& Elsevier]

Abstract: Betaine homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine (Hcy), forming dimethylglycine and methionine. We previously showed that inhibiting BHMT in mice by intraperitoneal injection of S-(α-carboxybutyl)-dl-homocysteine (CBHcy) results in hyperhomocysteinemia. In the present study, CBHcy was fed to rats to determine whether it could be absorbed and cause hyperhomocysteinemia as observed in the intraperitoneal administration of the compound in mice. We hypothesized that dietary administered CBHcy will be absorbed and will result in the inhibition of BHMT and cause hyperhomocysteinemia. Rats were meal-fed every 8 hours an l-amino acid–defined diet either containing or devoid of CBHcy (5 mg per meal) for 3 days. The treatment decreased liver BHMT activity by 90% and had no effect on methionine synthase, methylenetetrahydrofolate reductase, phosphatidylethanolamine N-methyltransferase, and CTP:phosphocholine cytidylyltransferase activities. In contrast, cystathionine β-synthase activity and immunodetectable protein decreased (56% and 26%, respectively) and glycine N-methyltransferase activity increased (52%) in CBHcy-treated rats. Liver S-adenosylmethionine levels decreased by 25% in CBHcy-treated rats, and S-adenosylhomocysteine levels did not change. Furthermore, plasma choline decreased (22%) and plasma betaine increased (15-fold) in CBHcy-treated rats. The treatment had no effect on global DNA and CpG island methylation, liver histology, and plasma markers of liver damage. We conclude that CBHcy-mediated BHMT inhibition causes an elevation in total plasma Hcy that is not normalized by the folate-dependent conversion of Hcy to methionine. Furthermore, metabolic changes caused by BHMT inhibition affect cystathionine β-synthase and glycine N-methyltransferase activities, which further deteriorate plasma Hcy levels. [Copyright &y& Elsevier]

Cystathionine β-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-methio- nine. 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. [ABSTRACT FROM AUTHOR]

Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Δtsc1 and Δtsc2, were up-regulated in Δbhd, and levels of specific intracellular amino acids known to be low in Δtsc1 and Δtsc2 were elevated in Δbhd. This "opposite" profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1+ dramatically increased permease expression levels in Δbhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Δbhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes. [ABSTRACT FROM AUTHOR]

Abstract: Missense mutations in the cystathionine beta-synthase (CBS) gene, such as I278T, are responsible for CBS deficiency, the most common inherited disorder in sulfur metabolism. Expression of human mutant CBS proteins in Saccharomyces cerevisiae reveals that most disease causing mutations severely inhibit enzyme activity and cannot support growth of yeast on cysteine-free media. Here, we show that the osmolyte chemical chaperones glycerol, trimethylamine-N-oxide, dimethylsulfoxide, proline or sorbitol, when added to yeast media, allows growth on cysteine-free media and causes increased enzyme activity from I278T and three other mutant CBS proteins. Rescuable mutants are ones that are predicted to cause a decrease in solvent accessible surface area. The increase in enzyme activity is associated with stabilization of the tetramer form of the enzyme. This effect is not specific to yeast, as addition of the chaperone glycerol resulted in increased I278T activity when the enzyme is produced either in Escherichia coli or in a coupled in vitro transcription/translation reaction. However, no stimulation of specific activity was observed when chaperones were added directly to purified I278T indicating that the presence of chemical chaperones is required during translation. We also found that by mixing different chaperones we could achieve rescue at significantly lower chaperone concentrations. Taken together, our data show that chemical chaperones present during the initial folding process can facilitate proper folding of several mutant CBS proteins and suggest it may be possible to treat some inborn errors of metabolism with agents that enhance proper protein folding. [Copyright &y& Elsevier]

Abstract: 4-Methylthio-2-oxobutanoic acid (MTOB) is the final compound of the methionine salvage pathway that converts the polyamine byproduct methylthioadenosine to adenine and methionine. Here we find that MTOB inhibits growth of several human cell lines in a dose-dependent manner. Growth inhibition was specific for MTOB as we did not observe any inhibition with other chemically related compounds. MTOB treatment causes apoptosis and reduction of ornithine decarboxylase (ODC) activity but not ODC mRNA. To determine if MTOB exerts its effects primarily via ODC inhibition, we compared the effects of MTOB with the ODC-specific inhibitor difluoromethylornithine (DFMO). We found that MTOB was a more potent inducer of apoptosis than DFMO, lacked activation of caspase 3/7, and was able to induce apoptosis in cells lacking p53. Our results show that MTOB-induced growth inhibition and apoptosis is not simply secondary due to ODC inhibition and implies that MTOB activates apoptosis via other mechanisms. [Copyright &y& Elsevier]

Malignant mesotheliomas (MMs) are aggressive tumors derived from mesothelial cells lining the lungs, pericardium and peritoneum, and are often associated with occupational asbestos exposure. Suppression subtractive hybridization was used to identify genes differentially expressed in MM cells compared to normal mesothelial cells. A gene, SEP15, encoding a 15-kDa selenium-containing protein was isolated using this approach and was subsequently shown to be downregulated in ~60% of MM cell lines and tumor specimens. A SEP15 polymorphic variant, 1125A, resides in the SECIS recognition element in the 3'-UTR and may influence the efficiency of Sec incorporation into the protein during translation. Since previous studies have implicated a potential role of the trace element selenium as a chemopreventive agent in animal models and in several types of human cancer, we investigated the effect of selenium on MM cells and its dependence on SEP15 genotype. Selenium was shown to inhibit cell growth and induce apoptosis in a dose-dependent manner in MM cells but had minimal effect on normal mesothelial cells. However, MM cells with downregulated SEP15 or the 1125A variant were somewhat less responsive to the growth inhibitory and apoptotic effects of selenium than MM cells expressing wild-type protein. RNAi-based knockdown studies demonstrated that SEP15 inhibition makes sensitive MM cells more resistant to selenium. These data imply that selenium may be useful as a chemopreventive agent in individuals at high risk of MM due to asbestos exposure, although those with the 1125A polymorphism may be less responsive to the protective benefits of dietary selenium supplementation.Oncogene (2004) 23, 5032-5040. doi:10.1038/sj.onc.1207683 Published online 26 April 2004 [ABSTRACT FROM AUTHOR]

Cystathionine beta-synthase (CBS) is an evolutionarily conserved enzyme that plays a key role in mammalian sulfur amino acid biochemistry, mutations in which are the cause of classical homocystinuria (HCU), an inborn error of metabolism. Although there is agreement in the literature that CBS is a homomultimer, its precise structure is a source of confusion. Here, we performed a series of experiments examining the quaternary structure of various wild-type and mutant CBS enzymes using a combination of native gel electrophoresis, in situ activity assays, analytical ultracentrifugation, and gel filtration. Our data show that recombinantly expressed and purified full-length wild-type human CBS enzyme (hCBS) and HCU-causing variants (p.P422L, p.I435T, and p.R125Q CBS) form high molecular weight assemblies that are consistent with the properties expected of a filament. The filament is enzymatically active, and its size is sensitive to protein concentration. This behavior contrasts sharply with hCBS enzymes containing small deletions within the Bateman domain, which form stable tetramers and octamers regardless of concentration. Examination of liver lysates from humans and mice confirms the existence of enzymatically active high molecular weight aggregates in vivo, but also shows that these aggregates are specific to human CBS and do not occur in mice. Molecular modeling using AlphaFold2 suggests that these experimentally observed differences may be explained by subtle differences in the interaction mediated by the Bateman domains. Our results show that small differences in amino acid sequence can cause large differences in the size and shape of CBS multimers., (© 2024 The Protein Society.)

Background: Cystathionine β-synthase (CBS)-deficient homocystinuria (HCU) is an inherited disorder of sulfur amino acid metabolism with varying severity and organ complications, and a limited knowledge about underlying pathophysiological processes. Here we aimed at getting an in-depth insight into disease mechanisms using a transgenic mouse model of HCU (I278T)., Methods: We assessed metabolic, proteomic and sphingolipidomic changes, and mitochondrial function in tissues and body fluids of I278T mice and WT controls. Furthermore, we evaluated the efficacy of methionine-restricted diet (MRD) in I278T mice., Results: In WT mice, we observed a distinct tissue/body fluid compartmentalization of metabolites with up to six-orders of magnitude differences in concentrations among various organs. The I278T mice exhibited the anticipated metabolic imbalance with signs of an increased production of hydrogen sulfide and disturbed persulfidation of free aminothiols. HCU resulted in a significant dysregulation of liver proteome affecting biological oxidations, conjugation of compounds, and metabolism of amino acids, vitamins, cofactors and lipids. Liver sphingolipidomics indicated upregulation of the pro-proliferative sphingosine-1-phosphate signaling pathway. Liver mitochondrial function of HCU mice did not seem to be impaired compared to controls. MRD in I278T mice improved metabolic balance in all tissues and substantially reduced dysregulation of liver proteome., Conclusion: The study highlights distinct tissue compartmentalization of sulfur-related metabolites in normal mice, extensive metabolome, proteome and sphingolipidome disruptions in I278T mice, and the efficacy of MRD to alleviate some of the HCU-related biochemical abnormalities., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cystathionine beta-synthase (CBS)-deficient homocystinuria (HCU) is the most common inborn error of sulfur amino acid metabolism. The pyridoxine non-responsive form of the disease manifests itself by massively increasing plasma and tissue concentrations of homocysteine, a toxic intermediate of methionine metabolism that is thought to be the major cause of clinical complications including skeletal deformities, connective tissue defects, thromboembolism and cognitive impairment. The current standard of care involves significant dietary interventions that, despite being effective, often adversely affect quality of life of HCU patients, leading to poor adherence to therapy and inadequate biochemical control with clinical complications. In recent years, the unmet need for better therapeutic options has resulted in development of novel enzyme and gene therapies and exploration of pharmacological approaches to rescue CBS folding defects caused by missense pathogenic mutations. Here, we review scientific evidence and current state of affairs in development of recent approaches to treat HCU., (© 2022 The British Pharmacological Society.)

Inborn errors of metabolism (IEM) comprise a large class of recessive genetic diseases involving disorders of cellular metabolism that tend to be caused by missense mutations in which a single incorrect amino acid is substituted in the polypeptide chain. Cystathionine beta-synthase (CBS) deficiency is an example of an IEM that causes large elevations of blood total homocysteine levels, resulting in phenotypes in several tissues. Current treatment strategies involve dietary restriction and vitamin therapy, but these are only partially effective and do not work in all patients. Over 85% of the described mutations in CBS-deficient patients are missense mutations in which the mutant protein fails to fold into an active conformation. The ability of CBS to achieve an active conformation is affected by a variety of intracellular protein networks including the chaperone system and the ubiquitin/proteasome system, collectively referred to as the proteostasis network. Proteostasis modulators are drugs that perturb various aspects of these networks. In this article, we will review the evidence that modulation of the intracellular protein folding environment can be used as a potential therapeutic strategy to treat CBS deficiency and discuss the pros and cons of such a strategy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)