Correction of cystathionine ?-synthase deficiency in mice by treatment with proteasome inhibitors.
ABSTRACT: 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.
Project description:Cystathionine ?-synthase (CBS), a heme-containing pyridoxal-5-phosphate (PLP)-dependent enzyme, catalyzes the condensation of serine and homocysteine to yield cystathionine. Missense mutations in CBS, the most common cause of homocystinuria, often result in misfolded proteins. Arginine 266, where the pathogenic missense mutation R266K was identified, appears to be involved in the communication between heme and the PLP-containing catalytic center. Here, we assessed the effect of a short affinity tag (6xHis) compared to a bulky fusion partner (glutathione S-transferase - GST) on CBS wild type (WT) and R266K mutant enzyme properties. While WT CBS was successfully expressed either in conjunction with a GST or with a 6xHis tag, the mutant R266K CBS had no activity, did not form native tetramers and did not respond to chemical chaperone treatment when expressed with a GST fusion partner. Interestingly, expression of R266K CBS constructs with a 6xHis tag at either end yielded active enzymes. The purified, predominantly tetrameric, R266K CBS with a C-terminal 6xHis tag had ?82% of the activity of a corresponding WT CBS construct. Results from thermal pre-treatment of the enzyme and the denaturation profile of R266K suggests a lower thermal stability of the mutant enzyme compared to WT, presumably due to a disturbed heme environment.
Project description:Hydrogen sulfide (H2S), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of H2S or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and H2S regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or Na2S/GYY4137, a H2S donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas Na2S/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the Na2S-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/-, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for H2S-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.
Project description:Homocystinuria, which typically results from cystathionine ?-synthase (CBS) deficiency, is the most common defect of sulfur amino acid metabolism. CBS condenses homocysteine and serine to cystathionine that is then converted to cysteine. Individuals with homocystinuria have markedly elevated plasma levels of homocysteine and methionine and reduced concentrations of cystathionine and cysteine. Clinical disease manifestations include thromboembolism and neuropsychiatric, ocular, and skeletal complications. Here, we have shown that administration of PEGylated CBS into the circulation of homocystinuria model mice alters the extra- and intracellular equilibrium of sulfur amino acids, resulting in a decrease of approximately 75% in plasma total homocysteine (tHcy) and normalization of cysteine concentrations. Moreover, the decrease in homocysteine and the normalization of cysteine in PEGylated CBS-treated model mice were accompanied by improvement of histopathological liver symptoms and increased survival. Together, these data suggest that CBS enzyme replacement therapy (ERT) is a promising approach for the treatment of homocystinuria and that ERT for metabolic diseases may not necessitate introduction of the deficient enzyme into its natural intracellular compartment.
Project description:Cystathionine β-synthase (CBS) deficiency (Online Mendelian Inheritance in Man [OMIM] 236,200) is an autosomal recessive disorder that is caused by mutations in the CBS gene. It is the most common inborn error of sulfur metabolism and is the cause of classical homocystinuria, a condition characterized by very high levels of plasma total homocysteine and methionine. Although recognized as an inborn error of metabolism over 60years ago, these is still much we do not understand related to how this specific metabolic defect gives rise to its distinct phenotypes. To try and answer these questions, several groups have developed mouse models on CBS deficiency. In this article, we will review various mouse models of CBS deficiency and discuss how these mouse models compare to human CBS deficient patients.
Project description:Untreated cystathionine beta-synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 microM), 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 microM). 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 microM, and Tg-I278T Cbs(-/-) mice, with a mean tHcy of 296 microM. Only Tg-I278T 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 time. Metabolic profiling of serum and liver reveals that Tg-I278T 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-adenosylhomocysteine in the liver compared to Tg-hCBS Cbs(-/-) mice. RNA profiling of livers indicate that Tg-I278T 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.
Project description:Cystathionine beta-synthase (CBS) deficiency is a rare inherited disorder in the methionine catabolic pathway, in which the impaired synthesis of cystathionine leads to accumulation of homocysteine. Patients can present to many different specialists and diagnosis is often delayed. Severely affected patients usually present in childhood with ectopia lentis, learning difficulties and skeletal abnormalities. These patients generally require treatment with a low-methionine diet and/or betaine. In contrast, mildly affected patients are likely to present as adults with thromboembolism and to respond to treatment with pyridoxine. In this article, we present recommendations for the diagnosis and management of CBS deficiency, based on a systematic review of the literature. Unfortunately, the quality of the evidence is poor, as it often is for rare diseases. We strongly recommend measuring the plasma total homocysteine concentrations in any patient whose clinical features suggest the diagnosis. Our recommendations may help to standardise testing for pyridoxine responsiveness. Current evidence suggests that patients are unlikely to develop complications if the plasma total homocysteine concentration is maintained below 120 ?mol/L. Nevertheless, we recommend keeping the concentration below 100 ?mol/L because levels fluctuate and the complications associated with high levels are so serious.
Project description:Homocystinuria is an inborn error of metabolism due to the deficiency in cystathionine beta-synthase (CBS) enzyme activity. It leads to the elevation of both homocysteine and methionine levels in the blood and urine. Consequently, this build-up could lead to several complications such as nearsightedness, dislocated eye lenses, a variety of psychiatric and behavioral disorders, as well as vascular system complications. The prevalence of homocystinuria is around 1/200,000 births worldwide. However, its prevalence in the Gulf region, notably Qatar, is exceptionally high and reached 1:1800. To date, more than 191 pathogenic CBS mutations have been documented. The majority of these mutations were identified in Caucasians of European ancestry, whereas only a few mutations from African-Americans or Asians were reported. Approximately 87% of all CBS mutations are missense and do not target the CBS catalytic site, but rather result in unstable misfolded proteins lacking the normal biological function, designating them for degradation. The early detection of homocystinuria along with low protein and methionine-restricted diet is the best treatment approach for all types of homocystinuria patients. Yet, less than 50% of affected individuals show a significant reduction in plasma homocysteine levels after treatment. Patients who fail to lower the elevated homocysteine levels, through high protein-restricted diet or by B6 and folic acid supplements, are at higher risk for cardiovascular diseases, neurodegenerative diseases, neural tube defects, and other severe clinical complications. This review aims to examine the mutations spectrum of the CBS gene, the disease management, as well as the current and potential treatment approaches with a greater emphasis on studies reported in the Middle East and North Africa (MENA) region.
Project description: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.
Project description:Missense mutations in the cystathionine beta-synthase (CBS) gene are the most common cause of clinical homocystinuria in humans. The p.S466L mutation was identified in a homocystinuric patient, but enzymatic studies with recombinant protein show this mutant to be highly active. To understand how this mutation causes disease in vivo, we have created mice lacking endogenous mouse CBS and expressing either wild-type (Tg-hCBS) or p.S466L (Tg-S466L) human CBS under control of zinc inducible metallothionein promoter. In the presence of zinc, we found that the mean serum total homocysteine (tHcy) of Tg-S466L mice was 142+/-55 microM compared to 16+/-13 microM for hCBS mice. Tg-S466L mice also had significantly higher levels of total free homocysteine and S-adenosylhomocysteine in liver and kidney. Only 48% of Tg-S466L mice had detectable CBS protein in the liver, whereas all the Tg-hCBS animals had detectable protein. Surprisingly, CBS mRNA was significantly elevated in Tg-S466L animals compared to Tg-hCBS, implying that the reduction in p.S466L protein was occurring due to posttranscriptional mechanisms. In Tg-S466L animals with detectable liver CBS, the enzyme formed tetramers and was active, but lacked inducibility by S-adenosylmethionine (AdoMet). However, even in Tg-S466L animals that had in vitro liver CBS activity equivalent to Tg-hCBS animals there was significant elevation of serum tHcy. Our results show that p.S466L causes homocystinuria by affecting both the steady state level of CBS protein and by reducing the efficiency of the enzyme in vivo.
Project description:Cystathionine beta synthase (CBS) is the rate-limiting enzyme responsible for the de novo synthesis of cysteine. Patients with CBS deficiency have greatly elevated plasma total homocysteine (tHcy), decreased levels of plasma total cysteine (tCys), and often a marfanoid appearance characterized by thinness and low body-mass index (BMI). Here, we characterize the growth and body mass characteristics of CBS deficient TgI278T Cbs(-/-) mice and show that these animals have significantly decreased fat mass and tCys compared to heterozygous sibling mice. The decrease in fat mass is accompanied by a 34% decrease in liver glutathione (GSH) along with a significant decrease in liver mRNA and protein for the critical fat biosynthesizing enzyme Stearoyl CoA desaturase-1 (Scd-1). Because plasma tCys has been positively associated with fat mass in humans, we tested the hypothesis that decreased tCys in TgI278T Cbs(-/-) mice was the cause of the lean phenotype by placing the animals on water supplemented with N-acetyl cysteine (NAC) from birth to 240 days of age. Although NAC treatment in TgI278T Cbs(-/-) mice caused significant increase in serum tCys and liver GSH, there was no increase in body fat content or in liver Scd-1 levels. Our results show that lack of CBS activity causes loss of fat mass, and that this effect appears to be independent of low serum tCys.