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1.
Cell Mol Life Sci ; 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34524466

RESUMO

Mitochondria-the intracellular powerhouse in which nutrients are converted into energy in the form of ATP or heat-are highly dynamic, double-membraned organelles that harness a plethora of cellular functions that sustain energy metabolism and homeostasis. Exciting new discoveries now indicate that the maintenance of this ever changing and functionally pleiotropic organelle is particularly relevant in terminally differentiated cells that are highly dependent on aerobic metabolism. Given the central role in maintaining metabolic and physiological homeostasis, dysregulation of the mitochondrial network might therefore confer a potentially devastating vulnerability to high-energy requiring cell types, contributing to a broad variety of hereditary and acquired diseases. In this Review, we highlight the biological functions of mitochondria-localized enzymes from the perspective of understanding-and potentially reversing-the pathophysiology of inherited disorders affecting the homeostasis of the mitochondrial network and cellular metabolism. Using methylmalonic acidemia as a paradigm of complex mitochondrial dysfunction, we discuss how mitochondrial directed-signaling circuitries govern the homeostasis and physiology of specialized cell types and how these may be disturbed in disease. This Review also provides a critical analysis of affected tissues, potential molecular mechanisms, and novel cellular and animal models of methylmalonic acidemia which are being used to develop new therapeutic options for this disease. These insights might ultimately lead to new therapeutics, not only for methylmalonic acidemia, but also for other currently intractable mitochondrial diseases, potentially transforming our ability to regulate homeostasis and health.

2.
Am J Hum Genet ; 108(7): 1283-1300, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214447

RESUMO

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Análise Mutacional de DNA , Diploide , Biblioteca Gênica , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/fisiologia , Saccharomyces cerevisiae/genética
3.
Biochim Biophys Acta Mol Basis Dis ; 1867(10): 166201, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34147638

RESUMO

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.

4.
Biochimie ; 183: 100-107, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33476699

RESUMO

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.


Assuntos
Inibidores Enzimáticos/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , S-Adenosilmetionina/química , Regulação Alostérica , Humanos , Domínios Proteicos
5.
Biochimie ; 183: 35-43, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32659443

RESUMO

Cobalamin, commonly known as vitamin B12, is an essential micronutrient for humans because of its role as an enzyme cofactor. Cobalamin is one of over a dozen structurally related compounds - cobamides - that are found in certain foods and are produced by microorganisms in the human gut. Very little is known about how different cobamides affect B12-dependent metabolism in human cells. Here, we test in vitro how diverse cobamide cofactors affect the function of methylmalonyl-CoA mutase (MMUT), one of two cobalamin-dependent enzymes in humans. We find that, although cobalamin is the most effective cofactor for MMUT, multiple cobamides support MMUT function with differences in binding affinity (Kd), binding kinetics (kon), and concentration dependence during catalysis (KM, app). Additionally, we find that six disease-associated MMUT variants that cause cobalamin-responsive impairments in enzymatic activity also respond to other cobamides, with the extent of catalytic rescue dependent on the identity of the cobamide. Our studies challenge the exclusive focus on cobalamin in the context of human physiology, indicate that diverse cobamides can support the function of a human enzyme, and suggest future directions that will improve our understanding of the roles of different cobamides in human biology.


Assuntos
Coenzimas/química , Metilmalonil-CoA Mutase/química , Vitamina B 12/química , Coenzimas/metabolismo , Humanos , Cinética , Metilmalonil-CoA Mutase/metabolismo , Vitamina B 12/metabolismo
6.
Biochimie ; 183: 89-99, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33309754

RESUMO

Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as "heterozygous-only" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs.


Assuntos
Aminoácido N-Acetiltransferase , Carbamoil-Fosfato Sintase (Amônia) , Genes Dominantes , Mutação de Sentido Incorreto , Ornitina Carbamoiltransferase , Distúrbios Congênitos do Ciclo da Ureia , Substituição de Aminoácidos , Aminoácido N-Acetiltransferase/química , Aminoácido N-Acetiltransferase/genética , Aminoácido N-Acetiltransferase/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/química , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Ornitina Carbamoiltransferase/química , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Domínios Proteicos , Distúrbios Congênitos do Ciclo da Ureia/enzimologia , Distúrbios Congênitos do Ciclo da Ureia/genética
7.
Mol Genet Metab ; 130(3): 179-182, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32414565

RESUMO

MTHFD1 is a trifunctional protein containing 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase activities. It is encoded by MTHFD1 and functions in the cytoplasmic folate cycle where it is involved in de novo purine synthesis, synthesis of thymidylate and remethylation of homocysteine to methionine. Since the first reported case of severe combined immunodeficiency resulting from MTHFD1 mutations, seven additional patients ascertained through molecular analysis have been reported with variable phenotypes, including megaloblastic anemia, atypical hemolytic uremic syndrome, hyperhomocysteinemia, microangiopathy, infections and autoimmune diseases. We determined the level of MTHFD1 expression and dehydrogenase specific activity in cell extracts from cultured fibroblasts of three previously reported patients, as well as a patient with megaloblastic anemia and recurrent infections with compound heterozygous MTHFD1 variants that were predicted to be deleterious. MTHFD1 protein expression determined by Western blotting in fibroblast extracts from three of the patients was markedly decreased compared to expression in wild type cells (between 4.8 and 14.3% of mean control values). MTHFD1 expression in the fourth patient was approximately 44% of mean control values. There was no detectable methylenetetrahydrofolate dehydrogenase specific activity in extracts from any of the four patients. This is the first measurement of MTHFD1 function in MTHFD1 deficient patients and confirms the previous molecular diagnoses.


Assuntos
Fibroblastos/patologia , Deficiência de Ácido Fólico/diagnóstico , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Imunodeficiência Combinada Severa/diagnóstico , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/metabolismo , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/metabolismo , Humanos , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/metabolismo
8.
Biochim Biophys Acta Mol Basis Dis ; 1866(3): 165622, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31770620

RESUMO

Isolated methylmalonic aciduria (MMAuria) is primarily caused by deficiency of methylmalonyl-CoA mutase (MMUT or MUT). Biochemically, MUT deficiency results in the accumulation of methylmalonic acid (MMA), propionyl-carnitine (C3) and other metabolites. Patients often exhibit lethargy, failure to thrive and metabolic decompensation leading to coma or even death, with kidney and neurological impairment frequently identified in the long-term. Here, we report a hemizygous mouse model which combines a knock-in (ki) missense allele of Mut with a knock-out (ko) allele (Mut-ko/ki mice) that was fed a 51%-protein diet from day 12 of life, constituting a bespoke model of MMAuria. Under this diet, mutant mice developed a pronounced metabolic phenotype characterized by drastically increased blood levels of MMA and C3 compared to their littermate controls (Mut-ki/wt). With this bespoke mouse model, we performed a standardized phenotypic screen to assess the whole-body impairments associated with this strong metabolic condition. We found that Mut-ko/ki mice show common clinical manifestations of MMAuria, including pronounced failure to thrive, indications of mild neurological and kidney dysfunction, and degenerative morphological changes in the liver, along with less well described symptoms such as cardiovascular and hematological abnormalities. The analyses also reveal so far unknown disease characteristics, including low bone mineral density, anxiety-related behaviour and ovarian atrophy. This first phenotypic screening of a MMAuria mouse model confirms its relevance to human disease, reveals new alterations associated with MUT deficiency, and suggests a series of quantifiable readouts that can be used to evaluate potential treatment strategies.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Metilmalonil-CoA Mutase/deficiência , Metilmalonil-CoA Mutase/genética , Animais , Ansiedade/genética , Ansiedade/patologia , Densidade Óssea/genética , Modelos Animais de Doenças , Feminino , Rim/patologia , Masculino , Ácido Metilmalônico/metabolismo , Camundongos , Fenótipo
9.
J Inherit Metab Dis ; 42(2): 333-352, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30773687

RESUMO

AIM: To explore the clinical presentation, course, treatment and impact of early treatment in patients with remethylation disorders from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) international web-based registry. RESULTS: This review comprises 238 patients (cobalamin C defect n = 161; methylenetetrahydrofolate reductase deficiency n = 50; cobalamin G defect n = 11; cobalamin E defect n = 10; cobalamin D defect n = 5; and cobalamin J defect n = 1) from 47 centres for whom the E-HOD registry includes, as a minimum, data on medical history and enrolment visit. The duration of observation was 127 patient years. In 181 clinically diagnosed patients, the median age at presentation was 30 days (range 1 day to 42 years) and the median age at diagnosis was 3.7 months (range 3 days to 56 years). Seventy-five percent of pre-clinically diagnosed patients with cobalamin C disease became symptomatic within the first 15 days of life. Total homocysteine (tHcy), amino acids and urinary methylmalonic acid (MMA) were the most frequently assessed disease markers; confirmatory diagnostics were mainly molecular genetic studies. Remethylation disorders are multisystem diseases dominated by neurological and eye disease and failure to thrive. In this cohort, mortality, thromboembolic, psychiatric and renal disease were rarer than reported elsewhere. Early treatment correlates with lower overall morbidity but is less effective in preventing eye disease and cognitive impairment. The wide variation in treatment hampers the evaluation of particular therapeutic modalities. CONCLUSION: Treatment improves the clinical course of remethylation disorders and reduces morbidity, especially if started early, but neurocognitive and eye symptoms are less responsive. Current treatment is highly variable. This study has the inevitable limitations of a retrospective, registry-based design.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Homocistinúria/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/metabolismo , Vitamina B 12/metabolismo , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Estudos Transversais , Progressão da Doença , Europa (Continente) , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Metilação , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Ácido Metilmalônico/urina , Fenótipo , Gravidez , Transtornos Psicóticos/metabolismo , Sistema de Registros , Estudos Retrospectivos , Adulto Jovem
10.
J Inherit Metab Dis ; 42(4): 673-685, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30693532

RESUMO

Vitamin B12 (cobalamin, Cbl) is a nutrient essential to human health. Due to its complex structure and dual cofactor forms, Cbl undergoes a complicated series of absorptive and processing steps before serving as cofactor for the enzymes methylmalonyl-CoA mutase and methionine synthase. Methylmalonyl-CoA mutase is required for the catabolism of certain (branched-chain) amino acids into an anaplerotic substrate in the mitochondrion, and dysfunction of the enzyme itself or in production of its cofactor adenosyl-Cbl result in an inability to successfully undergo protein catabolism with concomitant mitochondrial energy disruption. Methionine synthase catalyzes the methyl-Cbl dependent (re)methylation of homocysteine to methionine within the methionine cycle; a reaction required to produce this essential amino acid and generate S-adenosylmethionine, the most important cellular methyl-donor. Disruption of methionine synthase has wide-ranging implications for all methylation-dependent reactions, including epigenetic modification, but also for the intracellular folate pathway, since methionine synthase uses 5-methyltetrahydrofolate as a one-carbon donor. Folate-bound one-carbon units are also required for deoxythymidine monophosphate and de novo purine synthesis; therefore, the flow of single carbon units to each of these pathways must be regulated based on cellular needs. This review provides an overview on Cbl metabolism with a brief description of absorption and intracellular metabolic pathways. It also provides a description of folate-mediated one-carbon metabolism and its intersection with Cbl at the methionine cycle. Finally, a summary of recent advances in understanding of how both pathways are regulated is presented.


Assuntos
Deficiência de Ácido Fólico/metabolismo , Deficiência de Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Ácido Fólico/farmacologia , Humanos , Metilmalonil-CoA Mutase/metabolismo , Vitamina B 12/farmacologia
11.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1265-1272, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30682498

RESUMO

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of d-methylmalonyl-CoA and l-methylmalonyl-CoA in propionate catabolism. Autosomal recessive pathogenic variations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with pathogenic variations in MCEE. Nine patients were homozygous for the known nonsense variation p.Arg47* (c.139C > T), and one for the novel missense variation p.Ile53Arg (c.158T > G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described pathogenic variations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Šstructure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. We solved the structure of MCEE-Arg143Cys to 1.9 Šand found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this variation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Predisposição Genética para Doença/genética , Mutação , Racemases e Epimerases/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Códon sem Sentido , Cristalografia por Raios X , Homozigoto , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Dobramento de Proteína , Racemases e Epimerases/química , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo
12.
Nat Commun ; 9(1): 2261, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29891918

RESUMO

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Humanos , Cinética , Espectrometria de Massas , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , NADP/metabolismo , Fosforilação , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
13.
Nat Commun ; 9(1): 554, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29396438

RESUMO

The original version of this Article contained an error in the title, which was incorrectly given as 'APRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'. This has now been corrected in both the PDF and HTML versions of the Article to read 'A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'.

14.
Nat Commun ; 9(1): 67, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29302025

RESUMO

To date, epimutations reported in man have been somatic and erased in germlines. Here, we identify a cause of the autosomal recessive cblC class of inborn errors of vitamin B12 metabolism that we name "epi-cblC". The subjects are compound heterozygotes for a genetic mutation and for a promoter epimutation, detected in blood, fibroblasts, and sperm, at the MMACHC locus; 5-azacytidine restores the expression of MMACHC in fibroblasts. MMACHC is flanked by CCDC163P and PRDX1, which are in the opposite orientation. The epimutation is present in three generations and results from PRDX1 mutations that force antisense transcription of MMACHC thereby possibly generating a H3K36me3 mark. The silencing of PRDX1 transcription leads to partial hypomethylation of the epiallele and restores the expression of MMACHC. This example of epi-cblC demonstrates the need to search for compound epigenetic-genetic heterozygosity in patients with typical disease manifestation and genetic heterozygosity in disease-causing genes located in other gene trios.


Assuntos
Proteínas de Transporte/genética , Epistasia Genética , Erros Inatos do Metabolismo/genética , Mutação , Peroxirredoxinas/genética , Vitamina B 12/metabolismo , Alelos , Azacitidina/farmacologia , Sequência de Bases , Inibidores Enzimáticos/farmacologia , Saúde da Família , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Heterozigoto , Humanos , Masculino , Erros Inatos do Metabolismo/metabolismo , Oxirredutases , Linhagem , Sequenciamento Completo do Genoma
15.
J Biol Chem ; 292(28): 11980-11991, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28572511

RESUMO

Vitamin B12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Lisossomos/metabolismo , Erros Inatos do Metabolismo/genética , Modelos Moleculares , Mutação , Proteínas de Transporte Nucleocitoplasmático/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Substituição de Aminoácidos , Domínio Catalítico , Linhagem Celular Transformada , Células Cultivadas , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/enzimologia , Lisossomos/patologia , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Simulação de Acoplamento Molecular , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/deficiência , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Vitamina B 12/metabolismo
16.
Hum Mutat ; 38(8): 988-1001, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28497574

RESUMO

Mutations in the human MMAA gene cause the metabolic disorder cblA-type methylmalonic aciduria (MMA), although knowledge of the mechanism of dysfunction remains lacking. MMAA regulates the incorporation of the cofactor adenosylcobalamin (AdoCbl), generated from the MMAB adenosyltransferase, into the destination enzyme methylmalonyl-CoA mutase (MUT). This function of MMAA depends on its GTPase activity, which is stimulated by an interaction with MUT. Here, we present 67 new patients with cblA-type MMA, identifying 19 novel mutations. We biochemically investigated how missense mutations in MMAA in 22 patients lead to disease. About a third confer instability to the recombinant protein in bacterial and human expression systems. All 15 purified mutant proteins demonstrated wild-type like intrinsic GTPase activity and only one (p.Asp292Val), where the mutation is in the GTP binding domain, revealed decreased GTP binding. However, all mutations strongly decreased functional association with MUT by reducing GTPase activity stimulation upon incubation with MUT, while nine mutant proteins additionally lost the ability to physically bind MUT. Finally, all mutations interfered with gating the transfer of AdoCbl from MMAB to MUT. This work suggests loss of functional interaction between MMAA and MUT as a disease-causing mechanism that impacts processing and assembly of a cofactor to its destination enzyme.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Proteínas Mitocondriais/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Criança , Pré-Escolar , Cobamidas/metabolismo , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Proteínas Mitocondriais/genética , Mutação , Mutação de Sentido Incorreto/genética , Ligação Proteica
17.
Biochim Biophys Acta Mol Basis Dis ; 1863(1): 103-112, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27771510

RESUMO

An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG, cblE, and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs. We observed the known interactions of MS with MSR and with MMACHC as well as MMADHC with MMACHC, but we also observed novel interactions for MSR with MMACHC and with MMADHC and MS with MMADHC. Furthermore, we show that the absence of MS or MMACHC expression disrupts the interactions between the other interactome members, in cblC and cblG fibroblasts and in HepG2 cells transfected with siRNAs. Our data show that the processing of Cbl in cytoplasm occurs in a multiprotein complex composed of at least MS, MSR, MMACHC and MMADHC, which could contribute to shuttle safely and efficiently Cbl towards MS. Our data suggest that defective protein-protein interactions among key players of this pathway could contribute to the molecular mechanisms of the cblC, cblG and cblE genetic defects and provide novel insights into our understanding of the pathophysiology of inherited disorders of Cbl metabolism.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Proteínas de Transporte/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mapas de Interação de Proteínas , Vitamina B 12/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Oxirredutases , Mapeamento de Interação de Proteínas
18.
J Inherit Metab Dis ; 40(2): 297-306, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27743313

RESUMO

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20 % of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122 % of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.


Assuntos
Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Espasticidade Muscular/genética , Mutação de Sentido Incorreto/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Domínio Catalítico/genética , Fibroblastos/metabolismo , Genótipo , Humanos , Hiper-Homocisteinemia/genética , Cinética , Proteínas Mutantes/genética , NADP/genética , Polimorfismo de Nucleotídeo Único/genética , Transtornos Psicóticos/genética , Tetra-Hidrofolatos/genética
19.
J Biol Chem ; 291(39): 20563-73, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27519416

RESUMO

Methylmalonic aciduria (MMAuria), caused by deficiency of methylmalonyl-CoA mutase (MUT), usually presents in the newborn period with failure to thrive and metabolic crisis leading to coma or even death. Survivors remain at risk of metabolic decompensations and severe long term complications, notably renal failure and neurological impairment. We generated clinically relevant mouse models of MMAuria using a constitutive Mut knock-in (KI) allele based on the p.Met700Lys patient mutation, used homozygously (KI/KI) or combined with a knockout allele (KO/KI), to study biochemical and clinical MMAuria disease aspects. Transgenic Mut(ki/ki) and Mut(ko/ki) mice survive post-weaning, show failure to thrive, and show increased methylmalonic acid, propionylcarnitine, odd chain fatty acids, and sphingoid bases, a new potential biomarker of MMAuria. Consistent with genetic dosage, Mut(ko/ki) mice have lower Mut activity, are smaller, and show higher metabolite levels than Mut(ki/ki) mice. Further, Mut(ko/ki) mice exhibit manifestations of kidney and brain damage, including increased plasma urea, impaired diuresis, elevated biomarkers, and changes in brain weight. On a high protein diet, mutant mice display disease exacerbation, including elevated blood ammonia, and catastrophic weight loss, which, in Mut(ki/ki) mice, is rescued by hydroxocobalamin treatment. This study expands knowledge of MMAuria, introduces the discovery of new biomarkers, and constitutes the first in vivo proof of principle of cobalamin treatment in mut-type MMAuria.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Dosagem de Genes , Metilmalonil-CoA Mutase , Fenótipo , Característica Quantitativa Herdável , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Amônia/metabolismo , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Encéfalo/patologia , Carnitina/análogos & derivados , Carnitina/sangue , Proteínas na Dieta/efeitos adversos , Proteínas na Dieta/farmacologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Rim/metabolismo , Rim/patologia , Ácido Metilmalônico/sangue , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Camundongos Knockout
20.
Hum Mutat ; 37(8): 745-54, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27167370

RESUMO

Isolated methylmalonic aciduria (MMA) is an autosomal-recessive disorder of propionate metabolism that is most commonly caused by mutations in the methylmalonyl-CoA mutase (MUT) gene (mut-type MMA). We investigated a cohort of 151 patients, classifying 114 patients as mut(0) and 32 as mut(-) (five not defined). As per the definition, mut(-) patients showed a higher propionate incorporation ratio in vitro, which was correlated to a considerably later age of onset compared with mut(0) patients. In all patients, we found a total of 110 different mutations, of which 41 were novel. While the missense alleles p.Asn219Tyr, p.Arg369His, and p.Arg694Trp recurred in >10 alleles, 47 mutations were identified only once, suggesting many patients carry private mutations. Deficient alleles in the mut(-) subclass were almost exclusively caused by missense mutations, found disproportionately in the C-terminal cofactor binding domain. On the contrary, only half of the mut(0) mutations were of the missense type. Western blot analysis revealed reduced MUT protein for all 34 cell lines (27 mut(0) , seven mut(-) ) tested, suggesting protein instability as a major mechanism of deficiency in mut-type MMA. This large-scale evaluation helps to characterize the landscape of MUT mutations and their relationship to dysfunction and disease.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Mutação , Idade de Início , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Sítios de Ligação , Linhagem Celular , Regulação para Baixo , Humanos , Mutação INDEL , Metilmalonil-CoA Mutase/química , Modelos Moleculares , Mutação de Sentido Incorreto , Estabilidade Proteica
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