Mutant Cystathionine Beta-synthase Research Articles

Cellular turnover and degradation of the most common missense cystathionine beta-synthase variants causing homocystinuria.

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU.

Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants.

Homocystinuria (HCU), an inherited metabolic disorder caused by lack of cystathionine beta-synthase (CBS) activity, is chiefly caused by misfolding of single amino acid residue missense pathogenic variants. Previous studies showed that chemical, pharmacological chaperones or proteasome inhibitors could rescue function of multiple pathogenic CBS variants; however, the underlying mechanisms remain poorly understood. Using Chinese hamster DON fibroblasts devoid of CBS and stably overexpressing human WT or mutant CBS, we showed that expression of pathogenic CBS variant mostly dysregulates gene expression of small heat shock proteins HSPB3 and HSPB8 and members of HSP40 family. Endoplasmic reticulum stress sensor BiP was found upregulated with CBS I278T variant associated with proteasomes suggesting proteotoxic stress and degradation of misfolded CBS. Co-expression of the main effector HSP70 or master regulator HSF1 rescued steady-state levels of CBS I278T and R125Q variants with partial functional rescue of the latter. Pharmacological proteostasis modulators partially rescued expression and activity of CBS R125Q likely due to reduced proteotoxic stress as indicated by decreased BiP levels and promotion of refolding as indicated by induction of HSP70. In conclusion, targeted manipulation of cellular proteostasis may represent a viable therapeutic approach for the permissive pathogenic CBS variants causing HCU.

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

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

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.

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.

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.

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

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

The Nutrigenetics of Hyperhomocysteinemia: Quantitative Proteomics Reveals Differences in the Methionine Cycle Enzymes of Gene-Induced Versus Diet-Induced Hyperhomocysteinemia

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine beta-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p < or = 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS(+/-) mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS(+/-) genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.

Chemical chaperone rescue of mutant human cystathionine β-synthase

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.

Solvent-Accessible Cysteines in Human Cystathionine β-Synthase: Crucial Role of Cysteine 431 in S-Adenosyl-l-methionine Binding

Cystathionine beta-synthase (CBS) is a tetrameric heme protein that catalyzes the PLP-dependent condensation of serine and homocysteine to cystathionine. CBS occupies a crucial regulatory position between the methionine cycle and transsulfuration. Human CBS contains 11 cysteine residues that are highly conserved in mammals but completely absent in the yeast enzyme, which catalyzes an identical reaction, suggesting a possible regulatory role for some of these residues. In this report, we demonstrate that in both the presence and absence of the CBS allosteric regulator S-adenosyl-l-methionine (AdoMet), only C15 and C431 of human CBS are solvent accessible. Mutagenesis of C15 to serine did not affect catalysis or AdoMet activation but significantly reduced aggregation of the purified enzyme in vitro. Mutagenesis of C431 resulted in a constitutively activated form of CBS that could not be further activated by either AdoMet or thermal activation. We and others have previously reported a number of C-terminal CBS point mutations that result in a decreased or abolished response to AdoMet. In contrast to all of these previously investigated CBS mutants, the C431 mutant form of CBS was unable to bind AdoMet, indicating that either this residue is directly involved in AdoMet binding or its absence induces a conformational change that destroys the integrity of the binding site for this regulatory ligand.

Transcriptional Regulation of Cell-specific Expression of the Human Cystathionine β-Synthase Gene by Differential Binding of Sp1/Sp3 to the −1b Promoter

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously characterized the CBS -1b minimal promoter (-3792 to -3667) and found that Sp1/Sp3, nuclear factor Y, and USF-1 were involved in the regulation of basal promoter activity (Ge, Y., Konrad, M. A., Matherly, L. H., Taub, J. W. (2001) Biochem. J. 357, 97-105). In this study, the critical cis-elements and transcription factors in the CBS -1b upstream region (-4046 to -3792) were examined in HT1080 and HepG2 cells, which differ approximately 10-fold in levels of CBS transcripts transcribed from the CBS -1b promoter. In DNase I footprint and gel shift analyses and transient transfections of mutant CBS -1b promoter constructs into HT1080 and HepG2 cells, transcriptionally important roles for Sp1/Sp3 binding to three GC boxes and one GT box and for binding of myeloid zinc finger 1-like proteins to two myeloid zinc finger 1 elements were indicated. In gel shift assays, very low levels of Sp1/Sp3 DNA-protein complexes were detected in HT1080 cells compared with HepG2 cells despite comparable levels of nuclear factor Y and USF-1 binding and similar levels of Sp1 and Sp3 proteins on Western blots. Mixing of HT1080 and HepG2 nuclear extracts resulted in no difference in total Sp factor binding in gel shift assays, thus excluding a role for an unknown activator or inhibitor in the disparate Sp1/Sp3 binding between the lines. Increased Sp1/Sp3 binding in gel shift assays was observed upon treatment of HT1080 nuclear extracts with protein kinase A, and decreased Sp1/Sp3 binding resulted from treatment of HepG2 nuclear extracts with calf alkaline phosphatase, suggesting a role for changes in Sp1/Sp3 phosphorylation in transcription factor binding and transactivation of the CBS -1b promoter. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down syndrome.

Impaired Heme Binding and Aggregation of Mutant Cystathionine β-Synthase Subunits in Homocystinuria

During the past 20 years, cystathionine beta-synthase (CBS) deficiency has been detected in the former Czechoslovakia with a calculated frequency of 1:349,000. The clinical manifestation was typical of homocystinuria, and about half of the 21 patients were not responsive to pyridoxine. Twelve distinct mutations were detected in 30 independent homocystinuric alleles. One half of the alleles carried either the c.833 T-->C or the IVS11-2A-->C mutation; the remaining alleles contained private mutations. The abundance of five mutant mRNAs with premature stop codons was analyzed by PCR-RFLP. Two mRNAs, c.828_931ins104 (IVS7+1G-->A) and c.1226 G-->A, were severely reduced in the cytoplasm as a result of nonsense-mediated decay. In contrast, the other three mRNAs-c.19_20insC, c.28_29delG, and c.210_235del26 (IVS1-1G-->C)-were stable. Native western blot analysis of 14 mutant fibroblast lines showed a paucity of CBS antigen, which was detectable only in aggregates. Five mutations-A114V (c.341C-->T), A155T (c.463G-->A), E176K (c.526G-->A), I278T (c.833T-->C), and W409_G453del (IVS11-2A-->C)-were expressed in Escherichia coli. All five mutant proteins formed substantially more aggregates than did the wild-type CBS, and no aggregates contained heme. These data suggest that abnormal folding, impaired heme binding, and aggregation of mutant CBS polypeptides may be common pathogenic mechanisms in CBS deficiency.

Mutations in the regulatory domain of cystathionine beta synthase can functionally suppress patient-derived mutations in cis.

Human cystathionine beta--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.

A yeast assay for functional detection of mutations in the human cystathionine beta-synthase gene.

Mutations in the human cystathionine beta-synthase (CBS) gene are known to cause homocystinuria and may also be a significant risk factor for premature atherosclerosis. We have previously shown that the human CBS protein can substitute for the endogenous yeast CBS protein in Saccharomyces cerevisiae. We now show that expression of three different CBS mutants known to be associated with reduced enzyme activity in humans fail to complement growth in the yeast assay. In addition, we have used the yeast CBS assay to identify eight mutant CBS alleles in cell lines from patients with CBS deficiency. These mutant alleles include two previously identified and five novel CBS mutations. Our results also demonstrate that the yeast CBS assay can detect a large percentage of individuals heterozygous for mutations in CBS. This system should be useful in determining the relationship between CBS mutations and human disease.

Studies on normal and mutant cystathionine beta-synthase from cultured skin fibroblasts.

Studies on normal and mutant cystathionine beta-synthase from cultured skin fibroblasts.