RESUMO
Pathogenic variants in MMAB cause cblB-type methylmalonic aciduria, an autosomal-recessive disorder of propionate metabolism. MMAB encodes ATP:cobalamin adenosyltransferase, using ATP and cob(I)alamin to create 5'-deoxyadenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT). We identified bi-allelic disease-causing variants in MMAB in 97 individuals with cblB-type methylmalonic aciduria, including 33 different and 16 novel variants. Missense changes accounted for the most frequent pathogenic alleles (p.(Arg186Trp), N = 57; p.(Arg191Trp), N = 19); while c.700C > T (p.(Arg234*)) was the most frequently identified truncating variant (N = 14). In fibroblasts from 76 affected individuals, the ratio of propionate incorporation in the presence and absence of hydroxocobalamin (PI ratio) was associated to clinical cobalamin responsiveness and later disease onset. We found p.(Arg234*) to be associated with cobalamin responsiveness in vitro, and clinically with later onset; p.(Arg186Trp) and p.(Arg191Trp) showed no clear cobalamin responsiveness and early onset. Mapping these and novel variants onto the MMAB structure revealed their potential to affect ATP and AdoCbl binding. Follow-up biochemical characterization of recombinant MMAB identified its three active sites to be equivalent for ATP binding, determined by fluorescence spectroscopy (Kd = 21 µM) and isothermal calorimetry (Kd = 14 µM), but function as two non-equivalent AdoCbl binding sites (Kd1 = 0.55 µM; Kd2 = 8.4 µM). Ejection of AdoCbl was activated by ATP (Ka = 24 µM), which was sensitized by the presence of MMUT (Ka = 13 µM). This study expands the landscape of pathogenic MMAB variants, provides association of in vitro and clinical responsiveness, and facilitates insight into MMAB function, enabling better disease understanding.
Assuntos
Alquil e Aril Transferases , Erros Inatos do Metabolismo dos Aminoácidos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Alquil e Aril Transferases/metabolismo , Alelos , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Humanos , Mutação , Propionatos , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Vitamina B 12/metabolismoRESUMO
INTRODUCTION: Long-term outcome is postulated to be different in isolated methylmalonic aciduria caused by mutations in the MMAA gene (cblA type) compared with methylmalonyl-CoA mutase deficiency (mut), but case definition was previously difficult. METHOD: Cross-sectional analysis of data from the European Registry and Network for Intoxication type Metabolic Diseases (Chafea no. December 1, 2010). RESULTS: Data from 28 cblA and 95 mut patients in most cases confirmed by mutation analysis (including 4 new mutations for cblA and 19 new mutations for mut). Metabolic crisis is the predominant symptom leading to diagnosis in both groups. Biochemical disturbances during the first crisis were similar in both groups, as well as the age at diagnosis. Z scores of body height and body weight were similar in both groups at birth, but were significantly lower in the mut group at the time of last visit. Glomerular filtration rate was significantly higher in cblA; and as a consequence, chronic renal failure and related complications were significantly less frequent and renal function could be preserved even in older patients. Neurological complications were predominantly found in the mut subgroup. Methylmalonic acidemia (MMA) levels in urine and plasma were significantly lower in cblA. 27/28 cblA patients were reported to be responsive to cobalamin, only 86% of cblA patients were treated with i.m. hydroxocobalamin. In total, 73% of cblA and 98% of mut patients followed a calculated diet with amino acid supplements in 27% (cblA) and 69% (mut). During the study interval, six patients from the mut group died, while all cblA patients survived. CONCLUSION: Although similar at first, cblA patients respond to hydroxocobalamin treatment, subsequently show significantly lower levels of MMA and a milder course than mut patients.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo/genética , Metilmalonil-CoA Mutase/deficiência , Proteínas de Transporte da Membrana Mitocondrial/genética , Vitamina B 12/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/mortalidade , Criança , Estudos Transversais , Feminino , Taxa de Filtração Glomerular , Humanos , Falência Renal Crônica/etiologia , Masculino , Ácido Metilmalônico/sangue , Ácido Metilmalônico/urina , Metilmalonil-CoA Mutase/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , MutaçãoRESUMO
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 ProteicaRESUMO
Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte da Membrana Mitocondrial/química , Vitamina B 12/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Doenças Metabólicas/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Chaperonas Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Nitrorredutases/química , Oxirredutases , Fenótipo , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Erros Inatos do Metabolismo , Metilmalonil-CoA Mutase , Camundongos , Animais , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Glutamina , Multiômica , Erros Inatos do Metabolismo/genéticaRESUMO
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/metabolismoRESUMO
UNLABELLED: Non-CpG methylation is frequently present in stem cell DNA. We investigated the value of this epigenetic modification in cancerous DNA in order to establish the implications of CHH/CHG methylation for biomarker development. Therefore we used the restriction enzymes BstNI and PspGI within a combined multiplex PCR and targeted microarray approach for the elucidation of non-CpG (CCWGG) methylation. Targeting 544 CCWGG sites in 271 5' gene regions, the CHH/CHG methylation status of the MCF7 breast cancer cell line and blood from healthy volunteers and childhood ALL was analyzed. Statistical analysis of microarray data and subsequent SYBR green based qPCR on DNA digests was applied to confirm the results from the microarray screen. RESULT/CONCLUSION: The microarray experiments identified for the MCF7 cell line the genes MSH2 (p < 0.001), EREG (p < 0.001) and HSPA2 (p = 0.029) with CHH/CHG methylation, and in childhood ALL the genes HIST1H2AG (p = 0.003), PGF (p = 0.02), CPEB4 (p = 0.039) and TJP2 (p = 0.04). Validation using qPCR upon restriction digestion did not confirm the presence of CHH/CHG methylation in MCF7 DNA. In ALL samples only TJP2 was found harboring CHH/CHG methylation (p = 0.02). However, applying Bonferroni-correction for multiple testing that qPCR-result was not rated as statistically significant anymore. We concluded that non-CpG methylation in 544 CCWGG sites analyzed did not change in tumor cells. Thus any change of the CHH/CHG methylation pattern is a minor event in tumorigenesis, even if the stem cell markers OCT4, NANOG, STELLAR and GDF3 are expressed like in the MCF7 breast cancer cell line.