RESUMO
Cystathionine ß-synthase (CBS) deficiency has a wide clinical spectrum, ranging from neurodevelopmental problems, lens dislocation and marfanoid features in early childhood to adult onset disease with predominantly thromboembolic complications. We have analysed clinical and laboratory data at the time of diagnosis in 328 patients with CBS deficiency from the E-HOD (European network and registry for Homocystinurias and methylation Defects) registry. We developed comprehensive criteria to classify patients into four groups of pyridoxine responsivity: non-responders (NR), partial, full and extreme responders (PR, FR and ER, respectively). All groups showed overlapping concentrations of plasma total homocysteine while pyridoxine responsiveness inversely correlated with plasma/serum methionine concentrations. The FR and ER groups had a later age of onset and diagnosis and a longer diagnostic delay than NR and PR patients. Lens dislocation was common in all groups except ER but the age of dislocation increased with increasing responsiveness. Developmental delay was commonest in the NR group while no ER patient had cognitive impairment. Thromboembolism was the commonest presenting feature in ER patients, whereas it was least likely at presentation in the NR group. This probably is due to the differences in ages at presentation: all groups had a similar number of thromboembolic events per 1000 patient-years. Clinical severity of CBS deficiency depends on the degree of pyridoxine responsiveness. Therefore, a standardised pyridoxine-responsiveness test in newly diagnosed patients and a critical review of previous assessments is indispensable to ensure adequate therapy and to prevent or reduce long-term complications.
Assuntos
Cistationina beta-Sintase/deficiência , Homocistinúria/diagnóstico , Homocistinúria/tratamento farmacológico , Piridoxina/uso terapêutico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Diagnóstico Tardio , Europa (Continente) , Feminino , Homocistinúria/enzimologia , Humanos , Lactente , Modelos Lineares , Masculino , Metionina/sangue , Pessoa de Meia-Idade , Fenótipo , Sistema de Registros , Índice de Gravidade de Doença , Adulto JovemRESUMO
Classical homocystinuria (HCU) is an inborn error of metabolism caused by loss of cystathionine ß-synthase (CBS) activity with the concomitant buildup of homocysteine. In knockout (KO) mice, a mouse model of HCU, complete lack of CBS is neonatally lethal. Administration of OT-58, an enzyme therapy for HCU, during the first 5 weeks of life rescued KO mice survival by preventing liver disease. Here, we studied the impact of a long-term uninterrupted OT-58 treatment or its absence beyond the neonatal period on liver pathology and metabolism. Plasma and liver metabolites of KO mice on OT-58 treatment were substantially improved or normalized compared with those receiving vehicle. Increased plasma activities of alanine aminotransferase and aspartate aminotransferase of vehicle-injected KO mice suggested the progression of liver damage with age and lack of treatment. At 3 months of age, liver histology showed no signs of hepatopathy in both vehicle- and OT-58-treated KO mice. However, moderate to severe liver disease, characterized by steatosis, hepatocellular necroses, disorganized endoplasmic reticulum, and swollen mitochondria, developed in 6-month-old vehicle-injected KO mice. KO mice on OT-58 treatment remained asymptomatic and were indistinguishable from age-matched healthy controls. Long-term uninterrupted OT-58 treatment was essential to prevent severe liver disease in the KO mouse model of HCU.
Assuntos
Terapia de Reposição de Enzimas , Homocistinúria/tratamento farmacológico , Hepatopatias/prevenção & controle , Animais , Modelos Animais de Doenças , Feminino , Fígado/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
The most common ureagenesis defect is X-linked ornithine transcarbamylase (OTC) deficiency which is a main target for novel therapeutic interventions. The spf ash mouse model carries a variant (c.386G>A, p.Arg129His) that is also found in patients. Male spf ash mice have a mild biochemical phenotype with low OTC activity (5%-10% of wild-type), resulting in elevated urinary orotic acid but no hyperammonemia. We recently established a dried blood spot method for in vivo quantification of ureagenesis by Gas chromatography-mass spectrometry (GC-MS) using stable isotopes. Here, we applied this assay to wild-type and spf ash mice to assess ureagenesis at different ages. Unexpectedly, we found an age-dependency with a higher capacity for ammonia detoxification in young mice after weaning. A parallel pattern was observed for carbamoylphosphate synthetase 1 and OTC enzyme expression and activities, which may act as pacemaker of this ammonia detoxification pathway. Moreover, high ureagenesis in younger mice was accompanied by elevated periportal expression of hepatic glutamine synthetase, another main enzyme required for ammonia detoxification. These observations led us to perform a more extensive analysis of the spf ash mouse in comparison to the wild-type, including characterization of the corresponding metabolites, enzyme activities in the liver and plasma and the gut microbiota. In conclusion, the comprehensive enzymatic and metabolic analysis of ureagenesis performed in the presented depth was only possible in animals. Our findings suggest such analyses being essential when using the mouse as a model and revealed age-dependent activity of ammonia detoxification.
Assuntos
Envelhecimento/fisiologia , Amônia/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/patologia , Ornitina Carbamoiltransferase/genética , Ureia/metabolismo , Fatores Etários , Animais , Modelos Animais de Doenças , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Doença da Deficiência de Ornitina Carbomoiltransferase/genéticaRESUMO
Classical homocystinuria (HCU) is the most common inherited disorder of sulfur amino acid metabolism caused by deficiency in cystathionine beta-synthase (CBS) activity and characterized by severe elevation of homocysteine in blood and tissues. Treatment with dietary methionine restriction is not optimal, and poor compliance leads to serious complications. We developed an enzyme replacement therapy (ERT) and studied its efficacy in a severe form of HCU in mouse (the I278T model). Treatment was initiated before or after the onset of clinical symptoms in an effort to prevent or reverse the phenotype. ERT substantially reduced and sustained plasma homocysteine concentration at around 100 µM and normalized plasma cysteine for up to 9 months of treatment. Biochemical balance was also restored in the liver, kidney, and brain. Furthermore, ERT corrected liver glucose and lipid metabolism. The treatment prevented or reversed facial alopecia, fragile and lean phenotype, and low bone mass. In addition, structurally defective ciliary zonules in the eyes of I278T mice contained low density and/or broken fibers, while administration of ERT from birth partially rescued the ocular phenotype. In conclusion, ERT maintained an improved metabolic pattern and ameliorated many of the clinical complications in the I278T mouse model of HCU.
Assuntos
Cistationina beta-Sintase/administração & dosagem , Terapia de Reposição de Enzimas , Homocistinúria/diagnóstico , Homocistinúria/terapia , Fenótipo , Aminoácidos Sulfúricos/sangue , Aminoácidos Sulfúricos/metabolismo , Animais , Cistationina beta-Sintase/química , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Glucose/metabolismo , Homocistinúria/metabolismo , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Estresse Oxidativo , Polietilenoglicóis/químicaRESUMO
BACKGROUND: The aim of our study was to identify the genetic background of thiopurine-induced toxicity in a patient with a wild-type thiopurine methyltransferase genotype and activity. A 38-year-old Caucasian woman presented with cutaneous necrotizing vasculitis pancytopenia one month after starting azathioprine therapy. METHODS: During a routine biochemical follow-up of the patient, undetectable serum uric acid (<10⯵l) was observed. A high performance liquid chromatography analysis of urinary purines revealed increased levels of xanthine (137â¯mmol/mol creatinine). The suspected diagnosis of hereditary xanthinuria, a rare autosomal recessive disorder of the last two steps of purine metabolism, was confirmed by sequence analysis. RESULTS: An analysis of XDH/XO and AOX1 revealed common polymorphisms, while analysis of the MOCOS gene identified a rare homozygous variant c.362Câ¯>â¯T. Dysfunction of this variant was confirmed by significantly decreased xanthine dehydrogenase/oxidase activity in the patient's plasma (<2% of control mean activity). CONCLUSIONS: We present a biochemical, enzymatic, and molecular genetic case study suggesting an important association between a hitherto undescribed dysfunction variant in the MOCOS gene and thiopurine-induced toxicity. The identified variant c.362Câ¯>â¯T results in slower thiopurine metabolism caused by inhibition of 6-mercaptopurine oxidation (catabolism) to 6-thioxanthine and 6-thiouric acid, which increases the formation of the nucleotide 6-thioguanine, which is toxic. This is the first clinical case to identify the crucial role of the MOCOS gene in thiopurine intolerance and confirm the impact of genetic variability of purine enzymes on different therapeutic outcomes in patients undergoing thiopurine treatment.
Assuntos
Aldeído Oxidase/deficiência , Mercaptopurina/análogos & derivados , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Sulfurtransferases/genética , Xantina Desidrogenase/deficiência , Adulto , Aldeído Oxidase/genética , Feminino , Humanos , Mercaptopurina/efeitos adversos , Mercaptopurina/metabolismo , Metiltransferases/genética , Polimorfismo Genético/genética , Ácido Úrico/sangue , Xantina/urina , Xantina Desidrogenase/genética , Xantina Oxidase/genéticaRESUMO
Classical homocystinuria (HCU) is an inborn error of sulfur amino acid metabolism caused by deficient activity of cystathionine ß-synthase (CBS), resulting in an accumulation of homocysteine and a concomitant decrease of cystathionine and cysteine in blood and tissues. In mice, the complete lack of CBS is neonatally lethal. In this study, newborn CBS-knockout (KO) mice were treated with recombinant polyethyleneglycolylated human truncated CBS (PEG-CBS). Full survival of the treated KO mice, along with a positive impact on metabolite levels in plasma, liver, brain, and kidneys, was observed. The PEG-CBS treatment prevented an otherwise fatal liver disease characterized by steatosis, death of hepatocytes, and ultrastructural abnormalities of endoplasmic reticulum and mitochondria. Furthermore, treatment of the KO mice for 5 mo maintained the plasma metabolite balance and completely prevented osteoporosis and changes in body composition that characterize both the KO model and human patients. These findings argue that early treatment of patients with HCU with PEG-CBS may prevent clinical symptoms of the disease possibly without the need of dietary protein restriction.-Majtan, T., Hulková, H., Park, I., Krijt, J., Kozich, V., Bublil, E. M., Kraus, J. P. Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria.
Assuntos
Cistationina beta-Sintase/metabolismo , Cistationina beta-Sintase/uso terapêutico , Fígado Gorduroso/prevenção & controle , Homocistinúria/tratamento farmacológico , Homocistinúria/enzimologia , Hepatopatias/prevenção & controle , Osteoporose/prevenção & controle , Animais , Composição Corporal/efeitos dos fármacos , Cistationina beta-Sintase/genética , Modelos Animais de Doenças , Fígado Gorduroso/enzimologia , Feminino , Homocistinúria/metabolismo , Homocistinúria/patologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Hepatopatias/enzimologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Recombinantes/uso terapêuticoAssuntos
Anemia Megaloblástica/genética , Ácido Fólico/uso terapêutico , Hiper-Homocisteinemia/genética , Trocador 1 de Sódio-Hidrogênio/deficiência , Adolescente , Anemia Megaloblástica/tratamento farmacológico , Sistemas CRISPR-Cas , Células Cultivadas , Células Clonais , Mutação da Fase de Leitura , Técnicas de Inativação de Genes , Homozigoto , Humanos , Hiper-Homocisteinemia/tratamento farmacológico , Células K562 , Masculino , Recidiva , Deleção de Sequência , Trocador 1 de Sódio-Hidrogênio/genética , Vitamina B 12/uso terapêutico , Sequenciamento do ExomaRESUMO
BACKGROUND: Liver enzymes are released from hepatocytes into circulation and their activity can be measured in the blood. We examined whether the plasma activity of the liver enzyme ornithine carbamoyltransferase, determined by a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay, could be utilized for the detection of OTC deficiency (OTCD), an X-linked inborn error of the urea cycle. METHODS: The plasma ornithine carbamoyltransferase (OTC) activity was assayed in the reverse reaction using isotopically labeled citrulline-d4 as a substrate and by determination of the product, ornithine-d4, by LC-MS/MS analysis. RESULTS: The plasma OTC activity in the controls was in the range of 111-658 pkat/L (n=49, median 272 pkat/L), and the activity increased linearly with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in patients with hepatopathy. The OTC activity was subsequently determined in 32 individuals carrying mutations in the OTC gene, and OTC/ALT and OTC/AST ratios were calculated to account for the degree of hepatopathy, which is a common finding in OTCD. The OTC/ALT ratio enabled clear differentiation of OTCD hemizygotes (n=11, range 0-69×10-6) from controls (504-3440×10-6). This ratio also enabled the detection of 11 of 12 symptomatic heterozygotes (range 38-794×10-6), while this marker did not allow for reliable differentiation of asymptomatic heterozygotes (n=9) from controls. CONCLUSIONS: LC-MS/MS assay of plasma OTC activity enabled the detection of all hemizygous and the majority of symptomatic heterozygous OTCD patients in the tested cohort. This study demonstrates that non-invasive assay of enzymes expressed predominantly in the liver could be used as an alternative approach for diagnosing inborn errors of metabolism.
Assuntos
Ensaios Enzimáticos/métodos , Fígado/enzimologia , Doença da Deficiência de Ornitina Carbomoiltransferase/sangue , Doença da Deficiência de Ornitina Carbomoiltransferase/diagnóstico , Ornitina Carbamoiltransferase/sangue , Calibragem , Cromatografia Líquida , Cromossomos Humanos X/genética , Estudos de Coortes , Estabilidade Enzimática , Feminino , Heterozigoto , Humanos , Modelos Lineares , Masculino , Mutação , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/enzimologia , Espectrometria de Massas em TandemRESUMO
Classical homocystinuria is caused by mutations in the cystathionine ß-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.
Assuntos
Arginina/farmacologia , Cistationina beta-Sintase/genética , Fibroblastos/efeitos dos fármacos , Heme/farmacologia , Homocistinúria/tratamento farmacológico , Chaperonas Moleculares/farmacologia , Mutação , Deficiências na Proteostase/tratamento farmacológico , Animais , Células CHO , Domínio Catalítico , Cricetulus , Cistationina beta-Sintase/metabolismo , Feminino , Fibroblastos/enzimologia , Predisposição Genética para Doença , Homocistinúria/diagnóstico , Homocistinúria/enzimologia , Homocistinúria/genética , Homozigoto , Humanos , Modelos Moleculares , Fenótipo , Conformação Proteica , Dobramento de Proteína , Deficiências na Proteostase/diagnóstico , Deficiências na Proteostase/enzimologia , Deficiências na Proteostase/genética , Relação Estrutura-Atividade , Especificidade por Substrato , TransfecçãoRESUMO
O-Acetylserine(thiol)lyases (OAS-TLs) play a pivotal role in a sulfur assimilation pathway incorporating sulfide into amino acids in microorganisms and plants, however, these enzymes have not been found in the animal kingdom. Interestingly, the genome of the roundworm Caenorhabditis elegans contains three expressed genes predicted to encode OAS-TL orthologs (cysl-1-cysl-3), and a related pseudogene (cysl-4); these genes play different roles in resistance to hypoxia, hydrogen sulfide and cyanide. To get an insight into the underlying molecular mechanisms we purified the three recombinant worm OAS-TL proteins, and we determined their enzymatic activities, substrate binding affinities, quaternary structures and the conformations of their active site shapes. We show that the nematode OAS-TL orthologs can bind O-acetylserine and catalyze the canonical reaction although this ligand may more likely serve as a competitive inhibitor to natural substrates instead of being a substrate for sulfur assimilation. In addition, we propose that S-sulfocysteine may be a novel endogenous substrate for these proteins. However, we observed that the three OAS-TL proteins are conformationally different and exhibit distinct substrate specificity. Based on the available evidences we propose the following model: CYSL-1 interacts with EGL-9 and activates HIF-1 that upregulates expression of genes detoxifying sulfide and cyanide, the CYSL-2 acts as a cyanoalanine synthase in the cyanide detoxification pathway and simultaneously produces hydrogen sulfide, while the role of CYSL-3 remains unclear although it exhibits sulfhydrylase activity in vitro. All these data indicate that C. elegans OAS-TL paralogs have distinct cellular functions and may play different roles in maintaining hydrogen sulfide homeostasis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Cisteína Sintase/metabolismo , Homeostase/fisiologia , Sulfeto de Hidrogênio/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Domínio Catalítico , Núcleo Celular/química , Núcleo Celular/enzimologia , Núcleo Celular/genética , Cianetos/metabolismo , Cisteína Sintase/química , Cisteína Sintase/genética , Sulfeto de Hidrogênio/química , Serina/análogos & derivados , Serina/química , Serina/genética , Serina/metabolismo , Especificidade por SubstratoRESUMO
The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine ß-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.
Assuntos
Cisteína Dioxigenase/genética , Cisteína/metabolismo , Hepatócitos/metabolismo , Sulfeto de Hidrogênio/metabolismo , Tiossulfatos/metabolismo , Animais , Células Cultivadas , Cisteína/química , Cisteína Dioxigenase/deficiência , Feminino , Hepatócitos/enzimologia , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: In animals, dietary sulfur amino acid restriction (SAAR) improves metabolic health, possibly mediated by altering sulfur amino acid metabolism and enhanced anti-obesogenic processes in adipose tissue. AIM: To assess the effects of SAAR over time on the plasma and urine SAA-related metabolites (sulfurome) in humans with overweight and obesity, and explore whether such changes were associated with body weight, body fat and adipose tissue gene expression. METHODS: Fifty-nine subjects were randomly allocated to SAAR (â¼2 g SAA, n = 31) or a control diet (â¼5.6 g SAA, n = 28) consisting of plant-based whole-foods and supplemented with capsules to titrate contents of SAA. Sulfurome metabolites in plasma and urine at baseline, 4 and 8 weeks were measured using HPLC and LC-MS/MS. mRNA-sequencing of subcutaneous white adipose tissue (scWAT) was performed to assess changes in gene expression. Data were analyzed with mixed model regression. Principal component analyses (PCA) were performed on the sulfurome data to identify potential signatures characterizing the response to SAAR. RESULTS: SAAR led to marked decrease of the main urinary excretion product sulfate (p < 0.001) and plasma and/or 24-h urine concentrations of cystathionine, sulfite, thiosulfate, H2S, hypotaurine and taurine. PCA revealed a distinct metabolic signature related to decreased transsulfuration and H2S catabolism that predicted greater weight loss and android fat mass loss in SAAR vs. controls (all pinteraction < 0.05). This signature correlated positively with scWAT expression of genes in the tricarboxylic acid cycle, electron transport and ß-oxidation (FDR = 0.02). CONCLUSION: SAAR leads to distinct alterations of the plasma and urine sulfurome in humans, and predicted increased loss of weight and android fat mass, and adipose tissue lipolytic gene expression in scWAT. Our data suggest that SAA are linked to obesogenic processes and that SAAR may be useful for obesity and related disorders. TRIAL IDENTIFIER: https://clinicaltrials.gov/study/NCT04701346.
Assuntos
Tecido Adiposo , Aminoácidos Sulfúricos , Obesidade , Sobrepeso , Humanos , Obesidade/metabolismo , Obesidade/genética , Masculino , Feminino , Sobrepeso/metabolismo , Sobrepeso/genética , Adulto , Pessoa de Meia-Idade , Tecido Adiposo/metabolismo , Aminoácidos Sulfúricos/metabolismo , Aminoácidos Sulfúricos/sangue , Metaboloma , Regulação da Expressão GênicaRESUMO
BACKGROUND: Cystathionine ß-synthase (CBS)-deficient homocystinuria (HCU) is an inherited disorder of sulfur amino acid metabolism with varying severity and organ complications, and a limited knowledge about underlying pathophysiological processes. Here we aimed at getting an in-depth insight into disease mechanisms using a transgenic mouse model of HCU (I278T). METHODS: We assessed metabolic, proteomic and sphingolipidomic changes, and mitochondrial function in tissues and body fluids of I278T mice and WT controls. Furthermore, we evaluated the efficacy of methionine-restricted diet (MRD) in I278T mice. RESULTS: In WT mice, we observed a distinct tissue/body fluid compartmentalization of metabolites with up to six-orders of magnitude differences in concentrations among various organs. The I278T mice exhibited the anticipated metabolic imbalance with signs of an increased production of hydrogen sulfide and disturbed persulfidation of free aminothiols. HCU resulted in a significant dysregulation of liver proteome affecting biological oxidations, conjugation of compounds, and metabolism of amino acids, vitamins, cofactors and lipids. Liver sphingolipidomics indicated upregulation of the pro-proliferative sphingosine-1-phosphate signaling pathway. Liver mitochondrial function of HCU mice did not seem to be impaired compared to controls. MRD in I278T mice improved metabolic balance in all tissues and substantially reduced dysregulation of liver proteome. CONCLUSION: The study highlights distinct tissue compartmentalization of sulfur-related metabolites in normal mice, extensive metabolome, proteome and sphingolipidome disruptions in I278T mice, and the efficacy of MRD to alleviate some of the HCU-related biochemical abnormalities.
Assuntos
Cistationina beta-Sintase , Modelos Animais de Doenças , Homocistinúria , Fígado , Metabolômica , Camundongos Transgênicos , Proteômica , Esfingolipídeos , Animais , Camundongos , Homocistinúria/metabolismo , Homocistinúria/genética , Proteômica/métodos , Cistationina beta-Sintase/metabolismo , Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/genética , Fígado/metabolismo , Metabolômica/métodos , Esfingolipídeos/metabolismo , Mitocôndrias/metabolismo , Lipidômica/métodos , Proteoma/metabolismoRESUMO
CBSs (cystathionine ß-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins.
Assuntos
Caenorhabditis elegans/enzimologia , Cistationina beta-Sintase/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Sequência Conservada , Cistationina beta-Sintase/química , Cistationina beta-Sintase/genética , Citoplasma/enzimologia , Homeostase , Homocisteína/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
We report on the seventh known patient with S-adenosylhomocysteine hydrolase (SAHH) deficiency presenting at birth with features resembling phosphomannomutase 2 (PMM2-CDG Ia) deficiency. Plasma methionine and total homocysteine levels were normal at 2 months and increased only after the 8th month of age. SAHH deficiency was confirmed at 4.5 years of age by showing decreased SAHH activity (11% in both erythrocytes and fibroblasts), and compound heterozygosity for a known mutation c.145C>T (p.R49C) and a novel variant c.211G>A (p.G71S) in the AHCY gene. Retrospective analysis of clinical features revealed striking similarities between SAHH deficiency and the PMM2-CDG Ia.
Assuntos
Adenosil-Homocisteinase/deficiência , Adenosil-Homocisteinase/genética , Defeitos Congênitos da Glicosilação/diagnóstico , Mutação , Diagnóstico Diferencial , Eritrócitos/enzimologia , Eritrócitos/patologia , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Heterozigoto , Homocisteína/sangue , Humanos , Recém-Nascido , Metionina/sangue , Fosfotransferases (Fosfomutases)/deficiênciaRESUMO
Regulation of H2S homeostasis in humans is poorly understood. Therefore, we assessed the importance of individual enzymes in synthesis and catabolism of H2S by studying patients with respective genetic defects. We analyzed sulfur compounds (including bioavailable sulfide) in 37 untreated or insufficiently treated patients with seven ultrarare enzyme deficiencies and compared them to 63 controls. Surprisingly, we observed that patients with severe deficiency in cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) - the enzymes primarily responsible for H2S synthesis - exhibited increased and normal levels of bioavailable sulfide, respectively. However, an approximately 21-fold increase of urinary homolanthionine in CBS deficiency strongly suggests that lacking CBS activity is compensated for by an increase in CSE-dependent H2S synthesis from accumulating homocysteine, which suggests a control of H2S homeostasis in vivo. In deficiency of sulfide:quinone oxidoreductase - the first enzyme in mitochondrial H2S oxidation - we found normal H2S concentrations in a symptomatic patient and his asymptomatic sibling, and elevated levels in an asymptomatic sibling, challenging the requirement for this enzyme in catabolizing H2S under physiological conditions. Patients with ethylmalonic encephalopathy and sulfite oxidase/molybdenum cofactor deficiencies exhibited massive accumulation of thiosulfate and sulfite with formation of large amounts of S-sulfocysteine and S-sulfohomocysteine, increased renal losses of sulfur compounds and concomitant strong reduction in plasma total cysteine. Our results demonstrate the value of a comprehensive assessment of sulfur compounds in severe disorders of homocysteine/cysteine metabolism and provide evidence for redundancy and compensatory mechanisms in the maintenance of H2S homeostasis.
Assuntos
Sulfeto de Hidrogênio , Humanos , Sulfeto de Hidrogênio/metabolismo , Cisteína , Sulfetos/metabolismo , Homeostase , Enxofre , HomocisteínaRESUMO
Misfolding and aggregation of mutant enzymes have been proposed to play role in the pathogenesis of homocystinuria due to cystathionine ß-synthase (CBS) deficiency. Chemical chaperones have been recently shown to facilitate proper assembly of several CBS mutants. To asses the number of patients that may respond to chaperone therapy, we examined the effect of selected CBS ligands and osmolytes on assembly and activity of 27 CBS mutants that represent 70% of known CBS alleles. The mutant enzymes were expressed in a bacterial system, and their properties were assessed by native Western blotting and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, respectively. We studied the chaperoning activity of δ-aminolevulinic acid (δ-ALA)-a heme precursor-and of three osmolytes betaine, 2-aminoethanesulfonic acid (taurine), and glycerol. Fourteen mutants responded by at least 30% increase in the amount of correctly assembled tetramers and enzymatic activity to the coexpressional presence of either 0.5 mM δ-ALA, 100 mM betaine, and/or 750 mM glycerol. Eight of these mutants (p.R266K, p.P49L, p.R125Q, p.K102N, p.R369C, p.V180A, p.P78R, p.S466L) were rescuable by all of these three substances. Four mutants showed increased formation of tetramers that was not accompanied by changes in activity. Topology of mutations appeared to determine the chaperone responsiveness, as 11 of 14 solvent-exposed mutations were substantially more responsive than three of 13 buried mutations. This study identified chaperone-responsive mutants that represent 56 of 713 known patient-derived CBS alleles and may serve as a basis for exploring pharmacological approaches aimed at correcting misfolding in homocystinuria.
Assuntos
Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Homocistinúria/tratamento farmacológico , Ligantes , Chaperonas Moleculares/uso terapêutico , Dobramento de Proteína/efeitos dos fármacos , Alelos , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/uso terapêutico , Betaína/farmacologia , Betaína/uso terapêutico , Cistationina beta-Sintase/química , Cistationina beta-Sintase/efeitos dos fármacos , Escherichia coli/metabolismo , Glicerol/farmacologia , Homocistinúria/genética , Homocistinúria/metabolismo , Humanos , Chaperonas Moleculares/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Polimorfismo de Nucleotídeo Único/fisiologia , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Taurina/farmacologia , Taurina/uso terapêuticoRESUMO
Cystathionine ß-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 µL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.
Assuntos
Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/metabolismo , Homocistinúria/diagnóstico , Plasma/enzimologia , Espectrometria de Massas em Tandem/métodos , Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Calibragem , Estudos de Casos e Controles , Cromatografia Líquida , Estabilidade Enzimática , Homocistinúria/sangue , Homocistinúria/enzimologia , Humanos , Técnicas Imunoenzimáticas/métodos , Técnicas Imunoenzimáticas/normas , Plasma/química , Plasma/metabolismo , Fosfato de Piridoxal/farmacologia , S-Adenosilmetionina/farmacologia , Espectrometria de Massas em Tandem/normasRESUMO
Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B12 metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.
Assuntos
Oxirredutases/metabolismo , Retina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Feminino , Homocisteína/metabolismo , Homocistinúria/metabolismo , Humanos , Masculino , Ácido Metilmalônico/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação/genética , Oxirredutases/genética , Fenótipo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Vitamina B 12/metabolismo , Adulto JovemRESUMO
The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/- siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.