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Jan P. Kraus - One of the best experts on this subject based on the ideXlab platform.
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Pharmacokinetics and pharmacodynamics of PEGylated truncated human Cystathionine Beta-Synthase for treatment of homocystinuria
2019Co-Authors: Tomas Majtan, Teodoro Bottiglieri, Erez M Bublil, Insun Park, Erland Arning, Frank Glavin, Jan P. KrausAbstract:Aims: PEGylated human truncated Cystathionine Beta-Synthase, lacking the C-terminal regulatory domain (PEGCBS),is a promising preclinical candidate for enzyme replacement therapy in homocystinuria (HCU). It wasdesigned to function as a metabolic sink to decrease the severely elevated plasma and tissue homocysteineconcentrations. In this communication, we evaluated pharmacokinetics (PK), pharmacodynamics (PD) and subchronictoxicity of PEG-CBS in homocystinuric mice, wild type rats and monkeys to estimate the minimumhuman efficacious dose for clinical trials.Main methods: Animal models received single or multiple doses of PEG-CBS. Activity of PEG-CBS and sulfuramino acid metabolites were determined in plasma and used to determine PK and PD.Key findings: The plasma half-lives of PEG-CBS after a single subcutaneous (SC) injection were approximately 20,44 and 73 h in mouse, rat and monkey, respectively. The SC administration of PEG-CBS resulted in a significantimprovement or full correction of metabolic imbalance in both blood and tissues of homocystinuric mice. The PDof PEG-CBS in mouse was dose-dependent, but less than dose-proportional, with the maximal efficacy achievedat 8 mg/kg. PEG-CBS was well-tolerated in mice and monkeys, but resulted in dose-dependent minimal-tomoderateinflammation at the injection sites and vacuolated macrophages in rats. Allometric scaling of animaldata was linear and the estimated human efficacious dose was determined as 0.66 mg/kg administered once aweek.Significance: These results provide critical preclinical data for the design of first-in-human PEG-CBS clinical trial.
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Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase
2019Co-Authors: Tomas Majtan, Jakub Krijt, Jitka Sokolová, Maria A. Ralat, Jana O. Kent, Jesse F. Gregory, Viktor Kožich, Michaela Krizkova, Jan P. KrausAbstract:Aims: The transsulfuration pathway enzymes Cystathionine Beta-Synthase (CBS) and Cystathionine gammalyaseare thought to be the major source of hydrogen sulfide (H2S). In this study, we assessed the role of CBS inH2S biogenesis.Results: We show that despite discouraging enzyme kinetics of alternative H2S-producing reactions utilizingcysteine compared with the canonical condensation of serine and homocysteine, our simulations of substratecompetitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine onCBS, thus leading to generation of appreciable amounts of H2S. The leading H2S-producing reaction is condensationof cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine ismore abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the maindeterminant of CBS H2S productivity. Abundance of cysteine over serine, for example, in plasma, allowed forup to 43% of CBS activity being responsible for H2S production, while excess of serine typical for intracellularlevels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine andcysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H2S pool.Innovation: Our study characterizes the H2S-producing potential of CBS under biologically relevant conditionsand highlights the serine-to-cysteine ratio as the main determinant of H2S production by CBS in vivo.Conclusion: Our data clarify the function of CBS in H2S biogenesis and the role of thioethers as surrogate H2Smarkers.
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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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Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase.
Antioxidants & redox signaling, 2017Co-Authors: Tomas Majtan, Jakub Krijt, Jitka Sokolová, Michaela Křížková, Maria A. Ralat, Jana O. Kent, Jesse F. Gregory, Viktor Kožich, Jan P. KrausAbstract:Abstract Aims: The transsulfuration pathway enzymes Cystathionine Beta-Synthase (CBS) and Cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (H2S). In this study, we a...
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Oligomeric status of human Cystathionine Beta-Synthase modulates AdoMet binding.
FEBS letters, 2016Co-Authors: Angel L. Pey, Jan P. Kraus, Luis Alfonso Martínez-cruz, Tomas MajtanAbstract:Cystathionine Beta-Synthase (CBS) plays a key role in the metabolism of sulfur-containing amino acids. CBS is a multidomain tetrameric enzyme allosterically activated by S-adenosylmethionine (AdoMet). Recent crystallographic analyses of engineered CBS lacking the loop made up of residues 516-525 revealed discrepancies in AdoMet binding compared to previous biophysical studies on a full-length CBS. Here, we show that removal of the loop 516-525 functionally eliminates the high affinity sites responsible for kinetic stabilization of the full-length enzyme and yields a dimeric AdoMet-inducible enzyme, in which kinetic stabilization is now exerted by AdoMet binding to the remaining low affinity sites.
Nathalie Janel - One of the best experts on this subject based on the ideXlab platform.
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alterations in the serotonin and dopamine pathways by Cystathionine Beta Synthase overexpression in murine brain
Molecular Neurobiology, 2019Co-Authors: Jacqueline London, Nathalie Janel, F Ndiaye, Linhchi Bui, Benoit Souchet, Fabrice Daubigney, Christophe Magnan, Serge Luquet, Julien Dairou, Claude RouchAbstract:Cystathionine Beta Synthase (CBS) is one of the 225 genes on chromosome 21 (HSA 21) that are triplicated in persons with trisomy 21 (Down syndrome). Although most triplicate HSA21 genes have their orthologous genes on murine chromosome 16, the murine ortholog of hCBS is on murine chromosome 17 and thus is not present in the well-studied Ts65Dn mouse model of trisomy 21. Persons with trisomy 21 (T21) present deficits in neurotransmission and exhibit early brain aging that can partially be explained by monoamine neurotransmitter alterations. We used transgenic mice for the hCBS gene, which overexpress the CBS protein in various brain regions, to study if CBS overexpression induces modifications in the monoamine neurotransmitters in the hypothalamus, thalamus, hippocampus, and striatum from transgenic and control female and male mice aged 3–4 months and 11–12 months. Sex, age, and brain area each influenced neurotransmitter levels. Briefly, the serotonin pathway was modified by CBS overexpression in various brain areas in female mice but not in male mice. The dopamine pathway was modified in brain regions according to sex and age. These results may allow us to better understand the role of the transsulfuration pathway and especially CBS overexpression in the metabolism of biogenic amines and the catecholamine catabolism in persons with trisomy 21.
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Cystathionine Beta Synthase deficiency induces catalase-mediated hydrogen peroxide detoxification in mice liver.
Biochimica et biophysica acta, 2008Co-Authors: Julien Hamelet, Virginie Seltzer, Emile Petit, Christophe Noll, Karine Andreau, Jean M Delabar, Nathalie JanelAbstract:Cystathionine Beta Synthase deficiency induces hyperhomocysteinemia which is considered as a risk factor for vascular diseases. Studies underlined the importance of altered cellular redox reactions in hyperhomocysteinemia-induced vascular pathologies. Nevertheless, hyperhomocysteinemia also induces hepatic dysfunction which may accelerate the development of vascular pathologies by modifying cholesterol homeostasis. The aim of the present study was to analyze the modifications of redox state in the liver of heterozygous Cystathionine Beta Synthase-deficient mice, a murine model of hyperhomocysteinemia. In this purpose, we quantified levels of reactive oxygen and nitrogen species and we assayed activities of main antioxidant enzymes. We found that Cystathionine Beta Synthase deficiency induced NADPH oxidase activation. However, there was no accumulation of reactive oxygen (superoxide anion, hydrogen peroxide) and nitrogen (nitrite, peroxynitrite) species. On the contrary, hepatic hydrogen peroxide level was decreased independently of an activation of glutathione-dependent mechanisms. In fact, Cystathionine Beta Synthase deficiency had no effect on glutathione peroxidase, glutathione reductase and glutathione S-transferase activities. However, we found a 50% increase in hepatic catalase activity without any variation of expression. These findings demonstrate that Cystathionine Beta Synthase deficiency initiates redox disequilibrium in the liver. However, the activation of catalase attenuates oxidative impairments.
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Homocysteine threshold value based on Cystathionine Beta Synthase and paraoxonase 1 activities in mice.
European journal of clinical investigation, 2007Co-Authors: Julien Hamelet, Christophe Noll, Jean M Delabar, Aït-yahya-graison E, Matulewicz E, Badel-chagnon A, Anne-claude Camproux, Demuth K, Jean-louis Paul, Nathalie JanelAbstract:Background Hyperhomocysteinaemia is a metabolic disorder associated with the development of premature atherosclerosis. Among the determinants which predispose to premature thromboembolic and atherothrombotic events, serum activity of paraoxonase 1, mainly synthesized in the liver, has been shown to be a predictor of cardiovascular disease and to be negatively correlated with serum homocysteine levels in human. Even though treatments of hyperhomocysteinaemic patients ongoing cardiovascular complications are commonly used, it still remains unclear above which homocysteine level a preventive therapy should be started. Materials and methods In order to establish a threshold of plasma homocysteine concentration we have analyzed the hepatic Cystathionine Beta Synthase and paraoxonase 1 activities in a moderate to intermediate murine model of hyperhomocysteinaemia. Using wild type and heterozygous Cystathionine Beta Synthase deficient mice fed a methionine enriched diet or a control diet, we first studied the link between Cystathionine Beta Synthase and paraoxonase 1 activities and plasma homocysteine concentration. Results Among the animals used in this study, we observed a negative correlation between plasma homocysteine level and Cystathionine Beta Synthase activity (ρ = –0·52, P = 0·0008) or paraoxonase 1 activity (ρ = –0·49, P = 0·002). Starting from these results, a homocysteine cut-off value of 15 µm has been found for both Cystathionine Beta Synthase (P = 0·0003) and paraoxonase 1 (P = 0·0007) activities. Conclusions Our results suggest that both Cystathionine Beta Synthase and paraoxonase 1 activities are significantly decreased in mice with a plasma homocysteine value greater than 15 µm. In an attempt to set up preventive treatment for cardiovascular disease our results indicate that treatments should be started from 15 µm of plasma homocysteine.
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Mice deficient in Cystathionine Beta Synthase display altered homocysteine remethylation pathway
Molecular genetics and metabolism, 2007Co-Authors: Jean-marc Alberto, Julien Hamelet, Christophe Noll, Sébastien Blaise, Jean-pierre Bronowicki, Jean-louis Guéant, Jean-maurice Delabar, Nathalie JanelAbstract:Cystathionine Beta Synthase (CBS) deficiency is a metabolic disorder that is biochemically characterized by severe hyperhomocysteinemia. In order to show the effects of CBS deficiency onto the activity of the enzymes involved in the remethylation pathway, we used the well characterized genetic model of severe hyperhomocysteinemia in mice. We showed that CBS deficiency in mice reduced hepatic methionine Synthase and Betaine-homocysteine methyltransferase activities, whereas 5,10-methylene tetrahydrofolate reductase activity was increased.
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Mice lacking Cystathionine Beta Synthase have lung fibrosis and air space enlargement.
Experimental and molecular pathology, 2007Co-Authors: Julien Hamelet, Jean-maurice Delabar, Nicole Maurin, Romain Fulchiron, Nathalie JanelAbstract:Cystathionine Beta Synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations, notably pulmonary thrombotic disease. However, the association between hyperhomocysteinemia and chronic obstructive pulmonary disease is not well understood. To investigate the role of hyperhomocysteinemia in lung injury and pulmonary fibrosis, we analyzed the lung of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. The degree of lung injury was assessed by histologic examination. Analysis of profibrogenic factors was performed by real-time quantitative reverse transcription-polymerase chain reaction. CBS-deficient mice develop fibrosis and air space enlargement in the lung, concomitant with an enhanced expression of heme oxygenase-1, pro(alpha)1 collagen type I, transforming growth factor-β1 and α-smooth muscle actin. However, lung fibrosis was found in the absence of increased inflammatory cell infiltrates as determined by histology, without changes in gene expression of proinflammatory cytokines TNFα and interleukin 6. The increased expression of α-smooth muscle actin and transforming growth factor-β1 emphasizes the role of myofibroblasts differentiation in case of lung fibrosis due to CBS deficiency in mice.
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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Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase
2019Co-Authors: Tomas Majtan, Jakub Krijt, Jitka Sokolová, Maria A. Ralat, Jana O. Kent, Jesse F. Gregory, Viktor Kožich, Michaela Krizkova, Jan P. KrausAbstract:Aims: The transsulfuration pathway enzymes Cystathionine Beta-Synthase (CBS) and Cystathionine gammalyaseare thought to be the major source of hydrogen sulfide (H2S). In this study, we assessed the role of CBS inH2S biogenesis.Results: We show that despite discouraging enzyme kinetics of alternative H2S-producing reactions utilizingcysteine compared with the canonical condensation of serine and homocysteine, our simulations of substratecompetitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine onCBS, thus leading to generation of appreciable amounts of H2S. The leading H2S-producing reaction is condensationof cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine ismore abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the maindeterminant of CBS H2S productivity. Abundance of cysteine over serine, for example, in plasma, allowed forup to 43% of CBS activity being responsible for H2S production, while excess of serine typical for intracellularlevels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine andcysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H2S pool.Innovation: Our study characterizes the H2S-producing potential of CBS under biologically relevant conditionsand highlights the serine-to-cysteine ratio as the main determinant of H2S production by CBS in vivo.Conclusion: Our data clarify the function of CBS in H2S biogenesis and the role of thioethers as surrogate H2Smarkers.
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Pharmacokinetics and pharmacodynamics of PEGylated truncated human Cystathionine Beta-Synthase for treatment of homocystinuria
2019Co-Authors: Tomas Majtan, Teodoro Bottiglieri, Erez M Bublil, Insun Park, Erland Arning, Frank Glavin, Jan P. KrausAbstract:Aims: PEGylated human truncated Cystathionine Beta-Synthase, lacking the C-terminal regulatory domain (PEGCBS),is a promising preclinical candidate for enzyme replacement therapy in homocystinuria (HCU). It wasdesigned to function as a metabolic sink to decrease the severely elevated plasma and tissue homocysteineconcentrations. In this communication, we evaluated pharmacokinetics (PK), pharmacodynamics (PD) and subchronictoxicity of PEG-CBS in homocystinuric mice, wild type rats and monkeys to estimate the minimumhuman efficacious dose for clinical trials.Main methods: Animal models received single or multiple doses of PEG-CBS. Activity of PEG-CBS and sulfuramino acid metabolites were determined in plasma and used to determine PK and PD.Key findings: The plasma half-lives of PEG-CBS after a single subcutaneous (SC) injection were approximately 20,44 and 73 h in mouse, rat and monkey, respectively. The SC administration of PEG-CBS resulted in a significantimprovement or full correction of metabolic imbalance in both blood and tissues of homocystinuric mice. The PDof PEG-CBS in mouse was dose-dependent, but less than dose-proportional, with the maximal efficacy achievedat 8 mg/kg. PEG-CBS was well-tolerated in mice and monkeys, but resulted in dose-dependent minimal-tomoderateinflammation at the injection sites and vacuolated macrophages in rats. Allometric scaling of animaldata was linear and the estimated human efficacious dose was determined as 0.66 mg/kg administered once aweek.Significance: These results provide critical preclinical data for the design of first-in-human PEG-CBS clinical trial.
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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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Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase.
Antioxidants & redox signaling, 2017Co-Authors: Tomas Majtan, Jakub Krijt, Jitka Sokolová, Michaela Křížková, Maria A. Ralat, Jana O. Kent, Jesse F. Gregory, Viktor Kožich, Jan P. KrausAbstract:Abstract Aims: The transsulfuration pathway enzymes Cystathionine Beta-Synthase (CBS) and Cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (H2S). In this study, we a...
Ruma Banerjee - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of Cystathionine Beta-Synthase with peroxynitrite.
Archives of biochemistry and biophysics, 2009Co-Authors: Laura Celano, Ruma Banerjee, Magdalena Gil, Sebastián Carballal, Rosario Durán, Ana Denicola, Beatriz AlvarezAbstract:Cystathionine Beta-Synthase (CBS) is a homocysteine metabolizing enzyme that contains pyridoxal phosphate (PLP) and a six-coordinate heme cofactor of unknown function. CBS was inactivated by peroxynitrite, the product of nitric oxide and superoxide radicals. The IC(50) was approximately 150microM for 5microM ferric CBS. Stopped-flow kinetics and competition experiments showed a direct reaction with a second-order rate constant of (2.4-5.0)x10(4)M(-1)s(-1) (pH 7.4, 37 degrees C). The radicals derived from peroxynitrite, nitrogen dioxide and carbonate radical, also inactivated CBS. Exposure to peroxynitrite did not modify bound PLP but led to nitration of Trp208, Trp43 and Tyr223 and alterations in the heme environment including loss of thiolate coordination, conversion to high-spin and bleaching, with no detectable formation of oxo-ferryl compounds nor promotion of one-electron processes. This study demonstrates the susceptibility of CBS to reactive oxygen/nitrogen species, with potential relevance to hyperhomocysteinemia, a risk factor for cardiovascular diseases.
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Low frequency dynamics of Cystathionine Beta-Synthase
Biophysical Journal, 2009Co-Authors: Karunakaran Venugopal, Ruma Banerjee, Yuhan Sun, Zhenyu Zhang, Abdelkrim Benabbas, Sangita Singh, Paul M. ChampionAbstract:Femtosecond coherence spectroscopy is used to study the low frequency dynamics of Cystathionine Beta-Synthase (CBS). CBS is a pyridoxal-5′-phosphate-dependent heme enzyme with cysteine and histidine axial ligands that catalyzes the condensation of serine and homocysteine to form Cystathionine. Resonance excitation near the maximum of the ferric state Soret band reveals a mode near ∼40 cm−1 (phase ∼pi/2). The phase indicates that the initial non-equilibrium coherent wavepacket for this mode is dominated by a momentum displacement. This is consistent with doming of the ferric five-coordinate species and suggests photolysis of the histidine ligand. When exciting on the red side of the Soret band, a mode near ∼25 cm−1 is observed that exhibits a phase jump of ∼pi for blue-side excitation. This mode may involve the response of an unphotolyzed fraction of hot ferric six-coordinate species, subsequent to ultrafast non-radiative decay. A strong correlation between the “detuned” coherence spectrum (which reveals higher frequencies) and the Raman spectrum is also demonstrated. Normal coordinate structural decomposition of the ferric heme crystal structure predicts strong saddling, doming, and ruffling modes and they are observed in the coherence spectra. The relative intensities of these modes are monitored as a function of pH in order to explore the potential correlation between redox equilibria, pH, and protein-induced heme structural perturbations. The low frequency spectra of ferrous CBS and its NO-bound complex were also obtained, along with the CO rebinding kinetics. The geminate rebinding of CO to CBS was found to be unusually fast and similar to that of CooA.
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Dioxygen reactivity and heme redox potential of truncated human Cystathionine Beta-Synthase.
Biochemistry, 2008Co-Authors: Sebastián Carballal, Ruma Banerjee, Peter Madzelan, Carlos F Zinola, Martín Graña, Rafael Radi, Beatriz AlvarezAbstract:Cystathionine Beta-Synthase (CBS) catalyzes the condensation of serine and homocysteine to Cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.
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Reaction mechanism and regulation of Cystathionine Beta-Synthase.
Biochimica et biophysica acta, 2003Co-Authors: Ruma Banerjee, Ruby Evande, Omer Kabil, Sunil Ojha, Shin TaokaAbstract:In mammals, Cystathionine Beta-Synthase catalyzes the first step in the transsulfuration pathway which provides an avenue for the conversion of the essential amino acid, methionine, to cysteine. Cystathionine Beta-Synthase catalyzes a PLP-dependent condensation of serine and homocysteine to Cystathionine and is unique in also having a heme cofactor. In this review, recent advances in our understanding of the kinetic mechanism of the yeast and human enzymes as well as pathogenic mutants of the human enzyme and insights into the role of heme in redox sensing are discussed from the perspective of the crystal structure of the catalytic core of the human enzyme.
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Alleviation of intrasteric inhibition by the pathogenic activation domain mutation, D444N, in human Cystathionine Beta-Synthase.
Biochemistry, 2002Co-Authors: Ruby Evande, Henk J. Blom, Godfried H.j. Boers, Ruma BanerjeeAbstract:Human Cystathionine Beta-Synthase is a heme protein that catalyzes the condensation of serine and homocysteine to form Cystathionine in a pyridoxal phosphate-dependent reaction. Mutations in this enzyme are the leading cause of hereditary hyperhomocysteinemia with attendant cardiovascular and other complications. The enzyme is activated approximately 2-fold by the allosteric regulator S-adenosylmethionine (AdoMet), which is presumed to bind to the C-terminal regulatory domain. The regulatory domain exerts an inhibitory effect on the enzyme, and its deletion is correlated with a 2-fold increase in catalytic activity and loss of responsiveness to AdoMet. A mutation in the C-terminal regulatory domain, D444N, displays high levels of enzyme activity, yet is pathogenic. In this study, we have characterized the biochemical penalties associated with this mutation and demonstrate that it is associated with a 4-fold lower steady-state level of Cystathionine Beta-Synthase in a fibroblast cell line that is homozygous for the D444N mutation. The activity of the recombinant D444N enzyme mimics the activity of the wild-type enzyme seen in the presence of AdoMet and can be further activated approximately 2-fold in the presence of supraphysiolgical concentrations of the allosteric regulator. The mutation increases the K(act) for AdoMet from 7.4 +/- 0.2 to 460 +/- 130 microM, thus rendering the enzyme functionally unresponsive to AdoMet under physiological concentrations. These results indicate that the D444N mutation partially abrogates the intrasteric inhibition imposed by the C-terminal domain. We propose a model that takes into account the three kinetically distinguishable states that are observed with human Cystathionine Beta-Synthase: "basal" (i.e., wild-type enzyme as isolated), "activated" (wild-type enzyme + AdoMet or the D444N mutant as isolated), and superactivated (D444N mutant + AdoMet or wild-type enzyme lacking the C-terminal regulatory domain).
Viktor Kožich - One of the best experts on this subject based on the ideXlab platform.
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Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase
2019Co-Authors: Tomas Majtan, Jakub Krijt, Jitka Sokolová, Maria A. Ralat, Jana O. Kent, Jesse F. Gregory, Viktor Kožich, Michaela Krizkova, Jan P. KrausAbstract:Aims: The transsulfuration pathway enzymes Cystathionine Beta-Synthase (CBS) and Cystathionine gammalyaseare thought to be the major source of hydrogen sulfide (H2S). In this study, we assessed the role of CBS inH2S biogenesis.Results: We show that despite discouraging enzyme kinetics of alternative H2S-producing reactions utilizingcysteine compared with the canonical condensation of serine and homocysteine, our simulations of substratecompetitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine onCBS, thus leading to generation of appreciable amounts of H2S. The leading H2S-producing reaction is condensationof cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine ismore abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the maindeterminant of CBS H2S productivity. Abundance of cysteine over serine, for example, in plasma, allowed forup to 43% of CBS activity being responsible for H2S production, while excess of serine typical for intracellularlevels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine andcysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H2S pool.Innovation: Our study characterizes the H2S-producing potential of CBS under biologically relevant conditionsand highlights the serine-to-cysteine ratio as the main determinant of H2S production by CBS in vivo.Conclusion: Our data clarify the function of CBS in H2S biogenesis and the role of thioethers as surrogate H2Smarkers.
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A proactive genotype-to-patient-phenotype map for Cystathionine Beta-Synthase
2018Co-Authors: Song Sun, Jochen Weile, Marta Verby, Atina G. Cote, Iosifina Fotiadou, Julia Kitaygorodsky, Jasper Rine, Pavel Ješina, Viktor KožichAbstract:Success in precision medicine depends on our ability to determine which rare human genetic variants have functional effects. Classical homocystinuria, characterized by elevated homocyst(e)ine in plasma and urine, is caused by primarily-rare variants in the Cystathionine Beta-Synthase (CBS) gene. About half of patients respond to vitamin B6 therapy. With early detection in newborns, existing therapies are highly effective. Functional CBS variants, especially those that respond to vitamin B6, can be detected based on their ability to restore growth in yeast cells lacking CYS4 (the yeast ortholog of CBS). This assay has previously been carried out only reactively after first observation of a variant in patients. Here we describe a proactive comprehensive missense variant effect map for human CBS. Together, saturation codon-replacement mutagenesis, en masse growth selection at different vitamin B6 levels, and sequencing yielded a look-up table for CBS missense variant function and vitamin B6-remediability in yeast. The CBS variant effect map identified disease variants and predicted both disease severity (r = 0.82) and human clinical response to vitamin B6 (r = 0.89). Thus, highly-multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.
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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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Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase.
Antioxidants & redox signaling, 2017Co-Authors: Tomas Majtan, Jakub Krijt, Jitka Sokolová, Michaela Křížková, Maria A. Ralat, Jana O. Kent, Jesse F. Gregory, Viktor Kožich, Jan P. KrausAbstract:Abstract Aims: The transsulfuration pathway enzymes Cystathionine Beta-Synthase (CBS) and Cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (H2S). In this study, we a...
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Guidelines for the diagnosis and management of Cystathionine Beta-Synthase deficiency
Journal of Inherited Metabolic Disease, 2017Co-Authors: Andrew A. M. Morris, Viktor Kožich, Generoso Andria, Saikat Santra, Tawfeg I. M. Ben-omran, Anupam B. Chakrapani, Ellen Crushell, Mick J. Henderson, Michel Hochuli, Martina HuemerAbstract: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 μmol/L. Nevertheless, we recommend keeping the concentration below 100 μmol/L because levels fluctuate and the complications associated with high levels are so serious.
