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Dieter P Reinhardt - One of the best experts on this subject based on the ideXlab platform.
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functional consequences of homocysteinylation of the elastic fiber proteins fibrillin 1 and tropoelastin
Journal of Biological Chemistry, 2010Co-Authors: Dirk Hubmacher, Judith T Cirulis, Fred W Keeley, Ming Miao, Dieter P ReinhardtAbstract:Homocystinuria caused by cystathionine-β-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains ∼13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-β-synthase-deficient homocystinuria.
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functional consequences of homocysteinylation of the elastic fiber proteins fibrillin 1 and tropoelastin
Journal of Biological Chemistry, 2010Co-Authors: Dirk Hubmacher, Judith T Cirulis, Fred W Keeley, Ming Miao, Dieter P ReinhardtAbstract:Homocystinuria caused by cystathionine-beta-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains approximately 13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-beta-synthase-deficient homocystinuria.
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modification of the structure and function of fibrillin 1 by homocysteine suggests a potential pathogenetic mechanism in homocystinuria
Journal of Biological Chemistry, 2005Co-Authors: Dirk Hubmacher, Kerstin Tiedemann, Rainer Bartels, Jurgen Brinckmann, Tillman Vollbrandt, B Batge, Holger Notbohm, Dieter P ReinhardtAbstract:Abstract Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-β-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-β binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-β binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.
Viktor Kožich - One of the best experts on this subject based on the ideXlab platform.
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Enzyme Replacement Therapy Ameliorates Multiple Symptoms of Murine Homocystinuria
2019Co-Authors: Tomas Majtan, Viktor Kožich, Erez M Bublil, Jakub Krijt, Wendell Jones, Insung Park, Warren D. Kruger, Steven Bassnett, Jan P. KrausAbstract:Classical homocystinuria (HCU) is the most common inheriteddisorder of sulfur amino acid metabolism caused by deficiencyin cystathionine beta-synthase (CBS) activity and characterizedby severe elevation of homocysteine in blood and tissues. Treatmentwith dietary methionine restriction is not optimal, andpoor compliance leads to serious complications. We developedan enzyme replacement therapy (ERT) and studied its efficacyin a severe form of HCU in mouse (the I278T model). Treatmentwas initiated before or after the onset of clinical symptomsin an effort to prevent or reverse the phenotype. ERTsubstantially reduced and sustained plasma homocysteine concentrationat around 100 mM and normalized plasma cysteinefor up to 9 months of treatment. Biochemical balance wasalso restored in the liver, kidney, and brain. Furthermore,ERT corrected liver glucose and lipid metabolism. The treatmentprevented or reversed facial alopecia, fragile and leanphenotype, and low bone mass. In addition, structurally defectiveciliary zonules in the eyes of I278T mice contained low densityand/or broken fibers, while administration of ERT frombirth partially rescued the ocular phenotype. In conclusion,ERT maintained an improved metabolic pattern and amelioratedmany of the clinical complications in the I278T mousemodel of HCU.
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Potential pharmacological chaperones for cystathionine beta-synthase-deficient homocystinuria
Handbook of experimental pharmacology, 2018Co-Authors: Tomas Majtan, Viktor Kožich, Angel L. Pey, Luis Alfonso Martínez-cruz, Paula Giménez-mascarell, Csaba Szabó, Jan P. KrausAbstract: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.
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Newborn screening for Homocystinurias and methylation disorders: systematic review and proposed guidelines
Journal of Inherited Metabolic Disease, 2015Co-Authors: Martina Huemer, Viktor Kožich, Matthias R. Baumgartner, Piero Rinaldo, Begoña Merinero, Elisabetta Pasquini, Antonia Ribes, Henk J BlomAbstract:Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for Homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency.
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Vascular presentation of cystathionine beta-synthase deficiency in adulthood
Journal of Inherited Metabolic Disease, 2011Co-Authors: Martin Magner, Josef Hyánek, Lucie Krupková, Tomáš Honzík, Jiří Zeman, Viktor KožichAbstract:Several recent studies describing a solely vascular presentation of cystathionine beta-synthase (CBS) deficiency in adulthood prompted us to analyze the frequency of patients manifesting with vascular complications in the Czech Republic. Between 1980 and 2009, a total of 20 Czech patients with CBS deficiency have been diagnosed yielding an incidence of 1:311,000. These patients were divided into three groups based on symptoms leading to diagnosis: those with vascular complications, with connective tissue manifestation and with neurological presentation. A vascular event such as a clinical feature leading to diagnosis of homocystinuria was present in five patients, while two of them had no other symptoms typical for CBS deficiency at the time of diagnosis. All patients with the vascular manifestation were diagnosed only during the past decade. The median age of diagnosis was 29 years in the vascular, 11.5 years in the connective tissue and 4.5 years in the neurological group. The ratio of pyridoxine responsive to nonresponsive patients was higher in the vascular (4 of 5 patients) and connective tissue groups (6 of 7 patients) than in the neurological group (2 of 8 patients). Mutation c.833T>C (p.I278T) was frequent in patients with vascular (6/10 alleles) and connective tissue presentation (8/14 alleles), while it was not present in patients with neurological involvement (0/16 alleles). During the last decade, we have observed patients with homocystinuria diagnosed solely due to vascular events; this milder form of homocystinuria usually manifests at greater ages, has a high ratio of pyridoxine responsiveness/nonresponsiveness, and the mutation c.833T>C (p.I278T) is often present.
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cystathionine beta synthase null homocystinuric mice fail to exhibit altered hemostasis or lowering of plasma homocysteine in response to betaine treatment
Molecular Genetics and Metabolism, 2010Co-Authors: Kenneth N Maclean, Viktor Kožich, Jakub Sikora, Hua Jiang, Lori S Greiner, Eva Kraus, Jakub Krijt, Linda S Crnic, Robert H Allen, Sally P StablerAbstract:Cystathionine beta-synthase (CBS) deficient homocystinuria is an inherited metabolic defect that if untreated typically results in mental retardation, thromboembolism and a range of connective tissue disturbances. A knockout mouse model has previously been used to investigate pathogenic mechanisms in classical homocystinuria (Watanabe et al., PNAS 92 (1995) 1585–1589). This mouse model exhibits a semi-lethal phenotype and the majority of mice do not survive the early neonatal period. We report here that the birth incidence of cbs (−/−) mice produced from heterozygous crosses is non-Mendelian and not significantly improved by treatment with either the Hcy lowering compound betaine or the cysteine donor N-acetylcysteine. Betaine treatment did improve survival of cbs (−/−) mice and restored fertility to female cbs (−/−) mice but did so without significantly lowering Hcy levels. Surviving cbs (−/−) mice failed to show any alteration in coagulation parameters compared to wild-type controls. Moribund cbs (−/−) mice exhibited severe liver injury and hepatic fibrosis while surviving cbs (−/−) mice although less severely affected, still exhibited a level of severe liver injury that is not found in the human disease. The hepatopathy observed in this model may offer an explanation for the failure of cbs (−/−) mice to respond to betaine or exhibit a hypercoagulative phenotype. We conclude that although this model provides useful data on the biochemical sequelae of classical homocystinuria, it does not successfully recapitulate a number of important features of the human disease and its use for studying mechanisms in homocystinuria should be treated with caution as the hepatopathy produces changes which could influence the results.
Dirk Hubmacher - One of the best experts on this subject based on the ideXlab platform.
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functional consequences of homocysteinylation of the elastic fiber proteins fibrillin 1 and tropoelastin
Journal of Biological Chemistry, 2010Co-Authors: Dirk Hubmacher, Judith T Cirulis, Fred W Keeley, Ming Miao, Dieter P ReinhardtAbstract:Homocystinuria caused by cystathionine-β-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains ∼13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-β-synthase-deficient homocystinuria.
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functional consequences of homocysteinylation of the elastic fiber proteins fibrillin 1 and tropoelastin
Journal of Biological Chemistry, 2010Co-Authors: Dirk Hubmacher, Judith T Cirulis, Fred W Keeley, Ming Miao, Dieter P ReinhardtAbstract:Homocystinuria caused by cystathionine-beta-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains approximately 13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-beta-synthase-deficient homocystinuria.
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modification of the structure and function of fibrillin 1 by homocysteine suggests a potential pathogenetic mechanism in homocystinuria
Journal of Biological Chemistry, 2005Co-Authors: Dirk Hubmacher, Kerstin Tiedemann, Rainer Bartels, Jurgen Brinckmann, Tillman Vollbrandt, B Batge, Holger Notbohm, Dieter P ReinhardtAbstract:Abstract Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-β-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-β binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-β binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.
Brian Fowler - One of the best experts on this subject based on the ideXlab platform.
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Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system
Journal of Inherited Metabolic Disease, 2017Co-Authors: Patricie Burda, Brian Fowler, Terttu Suormala, D. Sean Froese, Dorothea Heuberger, Alexandra Schäfer, Matthias R. BaumgartnerAbstract:5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (
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gene identification for the cbld defect of vitamin b12 metabolism
The New England Journal of Medicine, 2008Co-Authors: David Coelho, Jordan P Lernerellis, David S Rosenblatt, Terttu Suormala, Matthias R. Baumgartner, Martin Stucki, Robert F Newbold, Brian FowlerAbstract:BACKGROUND: Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. METHODS: We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. RESULTS: The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. CONCLUSIONS: Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder.
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cble type of homocystinuria due to methionine synthase reductase deficiency functional correction by minigene expression
Human Mutation, 2005Co-Authors: Petra Zavadáková, Brian Fowler, Terttu Suormala, Zorka Novotna, Peter Mueller, Julia B. Hennermann, Jiri Zeman, Laura Vilarinho, Antonia M Vilaseca, Sven GutscheAbstract:The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T
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coexistence of hereditary homocystinuria and factor v leiden effect on thrombosis
The New England Journal of Medicine, 1996Co-Authors: Hanna Mandel, Brian Fowler, Benjamin Brenner, Moshe Berant, Nurith Rosenberg, Naomi Lanir, Cornelis Jakobs, Uri SeligsohnAbstract:Background Venous and arterial thromboembolism occurs in only about one third of patients homozygous for homocystinuria, which suggests that other, contributory factors are necessary for the development of thrombosis in these patients. Factor V Leiden, an R506Q mutation in the gene coding for factor V, is the most common cause of familial thrombosis and could be a potentiating factor. Methods We determined activated partial-thromboplastin times in the presence and absence of activated protein C and tested for the factor V Leiden mutation in 45 members of seven unrelated consanguineous kindreds in which at least 1 member was homozygous for homocystinuria. Results Thrombosis (venous, arterial, or both) occurred in 6 of 11 patients with homocystinuria (age, 0.2 to 8 years). All six also had the factor V Leiden mutation. One patient with prenatally diagnosed homocystinuria who was also heterozygous for factor V Leiden has received warfarin therapy since birth and has not had thrombosis (age, 18 months). Of fo...
Tomas Majtan - One of the best experts on this subject based on the ideXlab platform.
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classical homocystinuria from cystathionine beta synthase deficiency to novel enzyme therapies
Biochimie, 2020Co-Authors: Erez M Bublil, Tomas MajtanAbstract:Genetic defects in cystathionine beta-synthase (CBS), a key enzyme of organic sulfur metabolism, result in deficiency of CBS activity and a rare inborn error of metabolism called classical homocystinuria (HCU). HCU is characterized by massive accumulation of homocysteine, an intermediate of methionine metabolism, and multisystemic clinical symptoms. Current treatment options for HCU are very limited and often inefficient, partially due to a low patient compliance with very strict dietary regimen. Novel therapeutic approaches are needed to cope with the toxic accumulation of homocysteine and restoration of a healthy metabolic balance. Human CBS is a complex intracellular multimeric enzyme that relies on three cofactors (heme, pyridoxal-5'-phosphate and S-adenosylmethionine) for proper function. Engineering and chemical modification of human CBS yielded OT-58, a first-in-class enzyme therapy candidate for HCU. Pre-clinical testing of OT-58 showed its substantial efficacy in lowering plasma and tissue concentrations of homocysteine, improving metabolic balance and correcting clinical symptoms of HCU. In addition, OT-58 showed great safety and toxicity profile when administered to non-human primates. Overwhelmingly positive and extensive pre-clinical package propelled OT-58 into a first-in-human clinical trial, which started on January 2019. In a meantime, other enzyme therapies based on modified human cystathionine gamma-lyase or erythrocyte-encapsulated bacterial methionine gamma-lyase have shown efficacy in decreasing plasma homocysteine in HCU mice. In addition, gene therapy approaches using adenovirus or minicircle DNA have been evaluated in HCU. In this review, we summarize the current efforts developing novel therapies for HCU to address a high unmet medical need among HCU patients.
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Enzyme Replacement Therapy Ameliorates Multiple Symptoms of Murine Homocystinuria
2019Co-Authors: Tomas Majtan, Viktor Kožich, Erez M Bublil, Jakub Krijt, Wendell Jones, Insung Park, Warren D. Kruger, Steven Bassnett, Jan P. KrausAbstract:Classical homocystinuria (HCU) is the most common inheriteddisorder of sulfur amino acid metabolism caused by deficiencyin cystathionine beta-synthase (CBS) activity and characterizedby severe elevation of homocysteine in blood and tissues. Treatmentwith dietary methionine restriction is not optimal, andpoor compliance leads to serious complications. We developedan enzyme replacement therapy (ERT) and studied its efficacyin a severe form of HCU in mouse (the I278T model). Treatmentwas initiated before or after the onset of clinical symptomsin an effort to prevent or reverse the phenotype. ERTsubstantially reduced and sustained plasma homocysteine concentrationat around 100 mM and normalized plasma cysteinefor up to 9 months of treatment. Biochemical balance wasalso restored in the liver, kidney, and brain. Furthermore,ERT corrected liver glucose and lipid metabolism. The treatmentprevented or reversed facial alopecia, fragile and leanphenotype, and low bone mass. In addition, structurally defectiveciliary zonules in the eyes of I278T mice contained low densityand/or broken fibers, while administration of ERT frombirth partially rescued the ocular phenotype. In conclusion,ERT maintained an improved metabolic pattern and amelioratedmany of the clinical complications in the I278T mousemodel of HCU.
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Potential pharmacological chaperones for cystathionine beta-synthase-deficient homocystinuria
Handbook of experimental pharmacology, 2018Co-Authors: Tomas Majtan, Viktor Kožich, Angel L. Pey, Luis Alfonso Martínez-cruz, Paula Giménez-mascarell, Csaba Szabó, Jan P. KrausAbstract: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.
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structural basis of regulation and oligomerization of human cystathionine β synthase the central enzyme of transsulfuration
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: June Erenoorbea, Tomas Majtan, Jan P. Kraus, Iker Oyenarte, Luis Alfonso MartinezcruzAbstract:Cystathionine β-synthase (CBS) controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H2S. CBS condenses serine and homocysteine to cystathionine with the help of three cofactors, heme, pyridoxal-5'-phosphate, and S-adenosyl-l-methionine. Inherited deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur metabolism. We present the structure of the human enzyme, discuss the unique arrangement of the CBS domains in the C-terminal region, and propose how they interact with the catalytic core of the complementary subunit to regulate access to the catalytic site. This arrangement clearly contrasts with other proteins containing the CBS domain including the recent Drosophila melanogaster CBS structure. The absence of large conformational changes and the crystal structure of the partially activated pathogenic D444N mutant suggest that the rotation of CBS motifs and relaxation of loops delineating the entrance to the catalytic site represent the most likely molecular mechanism of CBS activation by S-adenosyl-l-methionine. Moreover, our data suggest how tetramers, the native quaternary structure of the mammalian CBS enzymes, are formed. Because of its central role in transsulfuration, redox status, and H2S biogenesis, CBS represents a very attractive therapeutic target. The availability of the structure will help us understand the pathogenicity of the numerous missense mutations causing inherited homocystinuria and will allow the rational design of compounds modulating CBS activity.