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The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma-derived cell lines, induced pluripotent stem cell hepatocytes (iPSC-Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma-derived, 2-week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC-Heps, which are known to exhibit batch-to-batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs.
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BACKGROUND AND AIMS: Patient-derived human-induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes (hiPSC-Heps) have facilitated the study of rare genetic liver diseases. Here, we aimed to establish an in vitro liver disease model of the urea cycle disorder ornithine transcarbamylase deficiency (OTCD) using patient-derived hiPSC-Heps. APPROACH AND RESULTS: Before modeling OTCD, we addressed the question of why hiPSC-Heps generally secrete less urea than adult primary human hepatocytes (PHHs). Because hiPSC-Heps are not completely differentiated and maintain some characteristics of fetal PHHs, we compared gene-expression levels in human fetal and adult liver tissue to identify genes responsible for reduced urea secretion in hiPSC-Heps. We found lack of aquaporin 9 (AQP9) expression in fetal liver tissue as well as in hiPSC-Heps, and showed that forced expression of AQP9 in hiPSC-Heps restores urea secretion and normalizes the response to ammonia challenge by increasing ureagenesis. Furthermore, we proved functional ureagenesis by challenging AQP9-expressing hiPSC-Heps with ammonium chloride labeled with the stable isotope [15 N] (15 NH4 Cl) and by assessing enrichment of [15 N]-labeled urea. Finally, using hiPSC-Heps derived from patients with OTCD, we generated a liver disease model that recapitulates the hepatic manifestation of the human disease. Restoring OTC expression-together with AQP9-was effective in fully correcting OTC activity and normalizing ureagenesis as assessed by 15 NH4 Cl stable-isotope challenge. CONCLUSION: Our results identify a critical role for AQP9 in functional urea metabolism and establish the feasibility of in vitro modeling of OTCD with hiPSC-Heps. By facilitating studies of OTCD genotype/phenotype correlation and drug screens, our model has potential for improving the therapy of OTCD.
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Aquaporinas/metabolismo , Células-Tronco Pluripotentes Induzidas , Hepatopatias , Doença da Deficiência de Ornitina Carbomoiltransferase , Adulto , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Hepatopatias/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/terapia , UreiaRESUMO
Deficiency of antiquitin (α-aminoadipic semialdehyde dehydrogenase), an enzyme involved in lysine degradation and encoded by ALDH7A1, is the major cause of vitamin B6 -dependent epilepsy (PDE-ALDH7A1). Despite seizure control with high dose pyridoxine (PN), developmental delay still occurs in approximately 70% of patients. We aimed to investigate metabolic perturbations due to possible previously unidentified roles of antiquitin, which may contribute to developmental delay, as well as metabolic effects of high dose pyridoxine supplementation reflecting the high doses used for seizure control in patients with PDE-ALDH7A1. Untargeted metabolomics by high resolution mass spectrometry (HRMS) was used to analyze plasma of patients with PDE-ALDH7A1 and two independently generated lines of cultured ReNcell CX human neuronal progenitor cells (NPCs) with CRISPR/Cas mediated antiquitin deficiency. Accumulation of lysine pathway metabolites in antiquitin-deficient NPCs and western-blot analysis confirmed knockdown of ALDH7A1. Metabolomics analysis of antiquitin-deficient NPCs in conditions of lysine restriction and PN supplementation identified changes in metabolites related to the transmethylation and transsulfuration pathways and osmolytes, indicating a possible unrecognized role of antiquitin outside the lysine degradation pathway. Analysis of plasma samples of PN treated patients with PDE-ALDH7A1 and antiquitin-deficient NPCs cultured in conditions comparable to the patient plasma samples demonstrated perturbation of metabolites of the gamma-glutamyl cycle, suggesting potential oxidative stress-related effects in PN-treated patients with PDE-ALDH7A1. We postulate that a model of human NPCs with CRISPR/Cas mediated antiquitin deficiency is well suited to characterize previously unreported roles of antiquitin, relevant to this most prevalent form of pyridoxine-dependent epilepsy.
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Epilepsia , Piridoxina , Humanos , Piridoxina/uso terapêutico , Lisina/metabolismo , Aldeído Desidrogenase , Epilepsia/metabolismo , Convulsões , MetabolômicaRESUMO
OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. METHODS: Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). RESULTS: The established method is precise (intra-day CV: 1.4-6.3%; inter-day CV: 1.3-15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61-121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: -5.6 to 7.4%), are stable in thymol (variation: -8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). CONCLUSIONS: The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.
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Purinas , Espectrometria de Massas em Tandem , Humanos , Criança , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Pirimidinas , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The metabolic defect in glycogen storage disease type I (GSDI) results in fasting hypoglycemia and typical secondary metabolic abnormalities (eg, hypertriglyceridemia, hyperlactatemia, hyperuricemia). The aim of this study was to assess further perturbations of the metabolic network in GSDI patients under ongoing treatment. METHODS: In this prospective observational study, plasma samples of 14 adult patients (11 GSDIa, 3 GSDIb. Mean age 26.4 years, range 16-46 years) on standard treatment were compared to a cohort of 31 healthy controls utilizing ultra-high performance liquid chromatography (UHPLC) in combination with high resolution tandem mass spectrometry (HR-MS/MS) and subsequent statistical multivariate analysis. In addition, plasma fatty acid profiling was performed by GC/EI-MS. RESULTS: The metabolomic profile showed alterations of metabolites in different areas of the metabolic network in both GSD subtypes, including pathways of fuel metabolism and energy generation, lipids and fatty acids, amino acid and methyl-group metabolism, the urea cycle, and purine/pyrimidine metabolism. These alterations were present despite adequate dietary treatment, did not correlate with plasma triglycerides or lactate, both parameters typically used to assess the quality of metabolic control in clinical practice, and were not related to the presence or absence of complications (ie, nephropathy or liver adenomas). CONCLUSION: The metabolic defect of GSDI has profound effects on a variety of metabolic pathways in addition to the known typical abnormalities. These alterations are present despite optimized dietary treatment, which may contribute to the risk of developing long-term complications, an inherent problem of GSDI which appears to be only partly modified by current therapy.
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Doença de Depósito de Glicogênio Tipo I , Hipoglicemia , Adolescente , Adulto , Cromatografia Líquida de Alta Pressão , Doença de Depósito de Glicogênio Tipo I/complicações , Humanos , Hipoglicemia/complicações , Metabolômica , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.
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Amônia , Citrulinemia , Camundongos , Animais , Nitrogênio , Citrulinemia/genética , Citrulinemia/terapia , Argininossuccinato Sintase/genética , Argininossuccinato Sintase/metabolismo , Terapia Genética , Ureia/metabolismoRESUMO
Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.
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Erros Inatos do Metabolismo dos Aminoácidos/genética , Encefalopatias Metabólicas/genética , Encéfalo/metabolismo , Gliose/genética , Glutaril-CoA Desidrogenase/deficiência , Glutaril-CoA Desidrogenase/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Arginina/metabolismo , Encéfalo/patologia , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/patologia , Creatina/sangue , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Gliose/metabolismo , Gliose/patologia , Glutaril-CoA Desidrogenase/metabolismo , Humanos , Lisina/metabolismo , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , RatosRESUMO
Deficiency of antiquitin (ATQ), an enzyme involved in lysine degradation, is the major cause of vitamin B6 -dependent epilepsy. Accumulation of the potentially neurotoxic α-aminoadipic semialdehyde (AASA) may contribute to frequently associated developmental delay. AASA is formed by α-aminoadipic semialdehyde synthase (AASS) via the saccharopine pathway of lysine degradation, or, as has been postulated, by the pipecolic acid (PA) pathway, and then converted to α-aminoadipic acid by ATQ. The PA pathway has been considered to be the predominant pathway of lysine degradation in mammalian brain; however, this was refuted by recent studies in mouse. Consequently, inhibition of AASS was proposed as a potential new treatment option for ATQ deficiency. It is therefore of utmost importance to determine whether the saccharopine pathway is also predominant in human brain cells. The route of lysine degradation was analyzed by isotopic tracing studies in cultured human astrocytes, ReNcell CX human neuronal progenitor cells and human fibroblasts, and expression of enzymes of the two lysine degradation pathways was determined by Western blot. Lysine degradation was only detected through the saccharopine pathway in all cell types studied. The enrichment of 15 N-glutamate as a side product of AASA formation through AASS furthermore demonstrated activity of the saccharopine pathway. We provide first evidence that the saccharopine pathway is the major route of lysine degradation in cultured human brain cells. These results support inhibition of the saccharopine pathway as a new treatment option for ATQ deficiency.
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Ácido 2-Aminoadípico/análogos & derivados , Aldeído Desidrogenase/deficiência , Epilepsia/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Ácido 2-Aminoadípico/metabolismo , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Epilepsia/genética , Humanos , Redes e Vias Metabólicas , Ácidos Pipecólicos/metabolismo , Vitamina B 6/uso terapêuticoRESUMO
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031-4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187-3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
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Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Receptores de Fator de Acasalamento/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Micelas , Conformação Proteica , Domínios Proteicos , Homologia de Sequência de AminoácidosRESUMO
This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second, and third transmembrane (TM) domains and the contiguous loops of the α-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3, was investigated by solution NMR in trifluoroethanol/water. TM1-TM3 contains helical TM domains at the predicted locations, supported by continuous sets of medium-range NOEs. In addition, a short helix N-terminal to TM1 was detected, as well as a short helical stretch in the first extracellular loop. Two 161-residue polypeptides, [Ste2p(M1-R161), NT-TM1-TM3], that contain the entire N-terminal sequence, one with a single mutation, were directly expressed and isolated from Escherichia coli in yields as high as 30 mg/L. Based on its increased stability, the L11P mutant will be used in future experiments to determine long-range interactions. The study demonstrated that 3-TM domains of a yeast G protein-coupled receptor can be produced in isotopically labeled form suitable for solution NMR studies. The quality of spectra is superior to data recorded in micelles and allows more rapid data analysis. No tertiary contacts have been determined, and if present, they are likely transient. This observation supports earlier studies by us that secondary structure was retained in smaller fragments, both in organic solvents and in detergent micelles, but that stable tertiary contacts may only be present when the protein is imbedded in lipids.
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Receptores de Fator de Acasalamento/química , Proteínas de Saccharomyces cerevisiae/química , Trifluoretanol/química , Água/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Marcação por Isótopo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Fator de Acasalamento/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , TermodinâmicaRESUMO
BACKGROUND: Ornithine Transcarbamylase Deficiency (OTCD) is an X-linked urea cycle disorder characterized by acute hyperammonemic episodes. Hemizygous males are usually affected by a severe/fatal neonatal-onset form or, less frequently, by a late-onset form with milder disease course, depending on the residual enzymatic activity. Hyperammonemia can occur any time during life and patients could remain non- or mis-diagnosed due to unspecific symptoms. In heterozygous females, clinical presentation varies based on the extent of X chromosome inactivation. Maternal transmission in X-linked disease is the rule, but in late-onset OTCD, due to the milder phenotype of affected males, paternal transmission to the females is possible. So far, father-to-daughter transmission of OTCD has been reported only in 4 Japanese families. RESULTS: We identified in 2 Caucasian families, paternal transmission of late-onset OTCD with severe/fatal outcome in affected males and 1 heterozygous female. Furthermore, we have reassessed the pedigrees of other published reports in 7 additional families with evidence of father-to-daughter inheritance of OTCD, identifying and listing the family members for which this transmission occurred. CONCLUSIONS: Our study highlights how the diagnosis and pedigree analysis of late-onset OTCD may represent a real challenge for clinicians. Therefore, the occurrence of paternal transmission in OTCD should not be underestimated, due to the relevant implications for disease inheritance and risk of recurrence.
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Hiperamonemia , Doença da Deficiência de Ornitina Carbomoiltransferase , Masculino , Recém-Nascido , Humanos , Feminino , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Doença da Deficiência de Ornitina Carbomoiltransferase/diagnóstico , Núcleo Familiar , Hiperamonemia/genética , Heterozigoto , Pai , Ornitina Carbamoiltransferase/genéticaRESUMO
Vitamin B12 (B12) deficiency (B12D) can have detrimental effects on early growth and development. The Austrian newborn screening (NBS) program targets inborn errors of cobalamin metabolism and also detects B12D. Of 59 included neonates with B12D suspected by NBS, B12D was not further investigated in 16 (27%) retrospectively identified cases, not confirmed in 28 (48%), and confirmed in 15 (25%) cases. NBS and recall biomarkers were recorded. Age at sampling of the dried blood spots for NBS and the 1st-tier methionine/phenylalanine ratio were the strongest parameters to predict B12D (67.4% correct allocations). No differences between cases with confirmed, unconfirmed, or unknown B12D or differences to norms were observed for growth and psychomotor development (Vineland III scales, phone interviews with parents of children between months 10 and 14 of life). B12 intake was below recommendations in most mothers. NBS can detect reduced intracellular B12 activity. No advantage of NBS detection and treatment regarding infant cognitive development or growth could be proven. Since conspicuous NBS findings cannot be ignored, and to prevent exposing newborns to invasive diagnostics, assessment of maternal B12 status during pregnancy seems advisable.
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Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury, but how metabolism controls their regenerative potential is poorly understood. We describe that primary metabolic changes can determine murine MuSC fate decisions. We found that glutamine anaplerosis into the tricarboxylic acid (TCA) cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and fusion, combined with loss of self-renewal in vitro and in vivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation and inhibited the malate-aspartate shuttle (MAS). The resulting defect in transporting NADH-reducing equivalents into the mitochondria induced compartment-specific NAD+/NADH ratio shifts. MAS activity restoration or directly altering NAD+/NADH ratios normalized myogenesis. In conclusion, GLUD1 prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS in proliferating MuSCs. It thereby acts as a compartment-specific metabolic brake on MuSC differentiation.
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Methylmalonic aciduria (MMA) is an inborn error of metabolism with multiple monogenic causes and a poorly understood pathogenesis, leading to the absence of effective causal treatments. Here we employ multi-layered omics profiling combined with biochemical and clinical features of individuals with MMA to reveal a molecular diagnosis for 177 out of 210 (84%) cases, the majority (148) of whom display pathogenic variants in methylmalonyl-CoA mutase (MMUT). Stratification of these data layers by disease severity shows dysregulation of the tricarboxylic acid cycle and its replenishment (anaplerosis) by glutamine. The relevance of these disturbances is evidenced by multi-organ metabolomics of a hemizygous Mmut mouse model as well as through identification of physical interactions between MMUT and glutamine anaplerotic enzymes. Using stable-isotope tracing, we find that treatment with dimethyl-oxoglutarate restores deficient tricarboxylic acid cycling. Our work highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA.
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Erros Inatos do Metabolismo , Metilmalonil-CoA Mutase , Camundongos , Animais , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Glutamina , Multiômica , Erros Inatos do Metabolismo/genéticaRESUMO
Osteogenesis imperfecta (OI) is an inherited skeletal dysplasia characterized by low bone density, bone fragility and recurrent fractures. The characterization of its heterogeneous genetic basis has allowed the identification of novel players in bone development. In 2016, we described the first X-linked recessive form of OI caused by hemizygous MBTPS2 missense variants resulting in moderate to severe phenotypes. MBTPS2 encodes site-2 protease (S2P), which activates transcription factors involved in bone (OASIS) and cartilage development (BBF2H7), ER stress response (ATF6) and lipid metabolism (SREBP) via regulated intramembrane proteolysis. In times of ER stress or sterol deficiency, the aforementioned transcription factors are sequentially cleaved by site-1 protease (S1P) and S2P. Their N-terminal fragments shuttle to the nucleus to activate gene transcription. Intriguingly, missense mutations at other positions of MBTPS2 cause the dermatological spectrum condition Ichthyosis Follicularis, Atrichia and Photophobia (IFAP) and Keratosis Follicularis Spinulosa Decalvans (KFSD) without clinical overlap with OI despite the proximity of some of the pathogenic variants. To understand how single amino acid substitutions in S2P can lead to non-overlapping phenotypes, we aimed to compare the molecular features of MBTPS2-OI and MBTPS2-IFAP/KFSD, with the ultimate goal to unravel the pathomechanisms underlying MBTPS2-OI. RNA-sequencing-based transcriptome profiling of primary skin fibroblasts from healthy controls (n = 4), MBTPS2-OI (n = 3), and MBTPS2-IFAP/KFSD (n = 2) patients was performed to identify genes that are differentially expressed in MBTPS2-OI and MBTPS2-IFAP/KFSD individuals compared to controls. We observed that SREBP-dependent genes are more downregulated in OI than in IFAP/KFSD. This is coupled to alterations in the relative abundance of fatty acids in MBTPS2-OI fibroblasts in vitro, while no consistent alterations in the sterol profile were observed. Few OASIS-dependent genes are suppressed in MBTPS2-OI, while BBF2H7- and ATF6-dependent genes are comparable between OI and IFAP/KFSD patients and control fibroblasts. Importantly, we identified genes involved in cartilage physiology that are differentially expressed in MBTPS2-OI but not in MBTPS2-IFAP/KFSD fibroblasts. In conclusion, our data provide clues to how pathogenic MBTPS2 mutations cause skeletal deformities via altered fatty acid metabolism or cartilage development that may affect bone development, mineralization and endochondral ossification.
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Early-onset epileptic encephalopathy (EE) and combined developmental and epileptic encephalopathies (DEE) are clinically and genetically heterogeneous severely devastating conditions. Recent studies emphasized de novo variants as major underlying cause suggesting a generally low-recurrence risk. In order to better understand the full genetic landscape of EE and DEE, we performed high-resolution chromosomal microarray analysis in combination with whole-exome sequencing in 63 deeply phenotyped independent patients. After bioinformatic filtering for rare variants, diagnostic yield was improved for recessive disorders by manual data curation as well as molecular modeling of missense variants and untargeted plasma-metabolomics in selected patients. In total, we yielded a diagnosis in â¼42% of cases with causative copy number variants in 6 patients (â¼10%) and causative sequence variants in 16 established disease genes in 20 patients (â¼32%), including compound heterozygosity for causative sequence and copy number variants in one patient. In total, 38% of diagnosed cases were caused by recessive genes, of which two cases escaped automatic calling due to one allele occurring de novo. Notably, we found the recessive gene SPATA5 causative in as much as 3% of our cohort, indicating that it may have been underdiagnosed in previous studies. We further support candidacy for neurodevelopmental disorders of four previously described genes (PIK3AP1, GTF3C3, UFC1, and WRAP53), three of which also followed a recessive inheritance pattern. Our results therefore confirm the importance of de novo causative gene variants in EE/DEE, but additionally illustrate the major role of mostly compound heterozygous or hemizygous recessive inheritance and consequently high-recurrence risk.
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Variações do Número de Cópias de DNA , Epilepsia/genética , Sequenciamento do Exoma/métodos , Taxa de Mutação , Adolescente , Adulto , Criança , Pré-Escolar , Epilepsia/diagnóstico , Exoma , Feminino , Genes Recessivos , Humanos , Lactente , MasculinoRESUMO
Cotranslational protein folding is a vectorial process, and for membrane proteins, N-terminal helical segments are the first that become available for membrane insertion. While structures of many G-protein coupled receptors (GPCRs) in various states have been determined, the details of their folding pathways are largely unknown. The seven transmembrane (TM) helices of GPCRs often contain polar residues within the hydrophobic core, and some of the helices in isolation are predicted to be only marginally stable in a membrane environment. Here we review our efforts to describe how marginally hydrophobic TM helices of GPCRs integrate into the membrane in absence of all compensating interhelical contacts, ideally capturing early biogenesis events. To this end, we use truncated GPCRs, here referred to as fragments. We present data from the human Y4 and the yeast Ste2p receptors in detergent micelles derived from solution NMR techniques. We find that secondary structure in the fragments is similar to corresponding parts of the entire receptors. However, uncompensated polar or charged residues destabilize the helices, and prevent proper integration into the lipid bilayer, in agreement with the biophysical scales from Wimley and White for the partitioning of amino acids into the membrane-interior. We observe that the stability and integration of single TM helices is improved by adding neighboring helices. We describe a topology study, in which all possible forms of the Y4 receptor were made so that the entire receptor is truncated from the N-terminus by one TM helix at a time. We discover that proteins with an increasing number of helices assume a more defined topology. In a parallel study, we focused on the role of extracellular loops in ligand recognition. We demonstrate that transferring all loops of the human Y1 receptor onto the E. coli outer membrane protein OmpA in a suitable topology results in a chimeric receptor that displays, albeit reduced, affinity and specificity for the cognate ligand. Our data indicate that not all TM helices will spontaneously insert into the helix, and we suggest that at least for some GPCRs, N-terminal segments might remain associated with the translocon until their interacting partners are biosynthesized.