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Hemorragia , Infarto del Miocardio con Elevación del ST , Volumen Sistólico , Función Ventricular Izquierda , Humanos , Infarto del Miocardio con Elevación del ST/fisiopatología , Volumen Sistólico/fisiología , Masculino , Femenino , Persona de Mediana Edad , Función Ventricular Izquierda/fisiología , Factores de Riesgo , Recuento de Leucocitos , Anciano , Valor Predictivo de las Pruebas , Medición de Riesgo/métodos , Miocardio/patología , Intervención Coronaria PercutáneaRESUMEN
BACKGROUND: The recognition ability of right ventricular-pulmonary artery (RV-PA) coupling for coronary artery lesions (CAL) in children with Kawasaki disease (KD) has not been well characterized. This study aimed to determine whether RV-PA coupling is an independent the risk factors for CAL in children with KD. METHODS: Between October 2021 and August 2023, RV-PA coupling was assessed in 59 KD children using the ratio between echocardiographic tricuspid annular plane systolic excursion and pulmonary artery systolic pressure (PASP). Multivariable logistic regression analysis was used to identify the independent risk factors for CAL among the demographic, clinical, laboratory and echocardiographic data. RESULTS: Twenty-nine of 59 KD children had CAL according to the diagnostic criteria of echocardiography. There were significantly different white blood cell count, C-reactive protein, erythrocyte sedimentation rate, left ventricular ejection fraction, PASP and RV-PA coupling at admission, and significantly different acute/subacute phase ratio of RV-PA coupling between KD children with and without CAL ( P â <â 0.05). Multivariate logistic regression analysis identified that acute/subacute phase ratio of RV-PA coupling (ORâ =â 26.800; 95% CI, 1.276-562.668; P â =â 0.034) was an independent risk factor for CAL in children with KD. The area under receiver operating characteristic curve for the acute/subacute phase ratio of RV-PA coupling was 0.715 (95%CI: 0.624 - 0.825) to predict CAL in KD children ( P â <â 0.05), with a sensitivity of 81.25% and a specificity of 62.57% at the optimal cutoff value of 0.839. CONCLUSION: The acute/subacute phase ratio of RV-PA coupling was an independent risk factor for CAL in KD children.
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Síndrome Mucocutáneo Linfonodular , Arteria Pulmonar , Humanos , Síndrome Mucocutáneo Linfonodular/complicaciones , Síndrome Mucocutáneo Linfonodular/fisiopatología , Síndrome Mucocutáneo Linfonodular/diagnóstico , Masculino , Femenino , Arteria Pulmonar/fisiopatología , Arteria Pulmonar/diagnóstico por imagen , Factores de Riesgo , Preescolar , Lactante , Enfermedad de la Arteria Coronaria/fisiopatología , Enfermedad de la Arteria Coronaria/epidemiología , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/etiología , Ecocardiografía/métodos , Niño , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/fisiopatología , Función Ventricular Derecha/fisiología , Estudios RetrospectivosRESUMEN
OBJECTIVE: Pulmonary artery stiffness (PAS) is a strong and independent predictor of mortality in adult patients with pulmonary hypertension (PH). But the change in PAS during perinatal period remains unknown. Here, we aimed to explore the feasibility and performance of PAS on predicting persistent pulmonary hypertension of the newborn (PPHN). METHODS: 1325 fetuses underwent a dedicated echocardiography screening for fetal heart defects during second trimester, third-trimester and neonatal period with the measurement of acceleration time (PAAT) and maximal frequency shift (MFS) of pulmonary artery flow. PAS (MFS/PAAT ratio) was calculated. RESULTS: Six fetuses were diagnosed as PPHN. Compared with the normal fetuses, those with PH had greater values of PAS during each period of time (second trimester, 52.6(46.2-54.5) vs. 32.4(28.0-39.4) kHz/s, p = 0.0003; third trimester, 52.9(46.1-55.3) vs. 29.7(27.3-33.3) kHz/s, p = 0.0002; neonatal period, 127.4(85.2-150.8) vs. 26.6(22.7-35.0) kHz/s, p < 0.0001). There was a statistically significant correlation between PAS and mean pulmonary artery pressure (p < 0.05) but no correlation between PAS and gestational age (p > 0.05) whether in normal fetuses or not. The area under receiver operating characteristic curve (AUC) of 0.97 for PAS during third trimester was superior to that for PAS during second trimester (AUC, 0.94) in predicting PPHN. The optimal cutoff value of PAS during third trimester was 37.40 KHz/s, with a sensitivity of 100%, a specificity of 91%, and an accuracy of 92%. CONCLUSION: There was a significant difference in PAS between normal fetuses and those with PH. PAS has a power performance on predicting PPHN.
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Hipertensión Pulmonar , Arteria Pulmonar , Adulto , Recién Nacido , Femenino , Humanos , Embarazo , Arteria Pulmonar/diagnóstico por imagen , Hipertensión Pulmonar/diagnóstico por imagen , Atención Prenatal , Feto , EcocardiografíaRESUMEN
BACKGROUND: Pulmonary transit time (PTT) and pulmonary pulse wave transit time (pPTT) are useful parameters for the evaluation of cardiopulmonary circulation and vascular alterations, but their relationship remains unknown. The aim of this study was to investigate the correlation between PTT and pPTT. METHODS: A total of 60 healthy volunteers were involved in this study. They were divided into two groups (30 participants per group): <50 years and >50 years. They all underwent Doppler echocardiography of pulmonary vein flow and contrast echocardiography with the measurement of pPTT and PTT, respectively. The correlation between PTT and pPTT was deduced. RESULTS: Compared with Group of <50 years, there was a significant increment in left atrial volume index, left atrial pressure and pulmonary artery stiffness but a significant reduction in acceleration times of pulmonary artery flow in Group of >50 years (p < 0.05). Group >50 years had longer PTT and but reduced normalized PTT by R-R interval (NPTT), reduced normalized pPTT by R-R interval (NpPTT) than Group <50 years (p < 0.05), while there was no significant difference in pPTT between the two groups (p > 0.05). PTT and NPTT were all negatively correlated with pPTT and NpPTT. The statistically significant strongest correlation was observed between PTT and NpPTT (r = -0.886, p < 0.0001). The regression equation for them was y = 7.4396-13.095x (R2 = 0.785; p < 0.001), where x and y represent NpPTT and PTT, respectively. CONCLUSION: PTT had close relation with pPTT in normal subjects. From the regression equation for them, we can get the value of PTT simply and easily by non-invasively measured pPTT.
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Ecocardiografía , Pulmón , Humanos , Ecocardiografía Doppler , Arteria Pulmonar/diagnóstico por imagen , Análisis de la Onda del PulsoRESUMEN
BACKGROUND: We aimed to evaluate the feasibility and performance of myocardial work echocardiography in assessing the severity of acute stroke and neurological deficits in patients with acute ischemic stroke. METHODS: A total of 176 patients were examined by echocardiography within 24-48 hours of symptom onset with the measurement of global and regional myocardial work. The National Institutes of Health Stroke Scale score of each patient was documented. RESULTS: With the increase of the National Institutes of Health Stroke Scale score, myocardial constructive work or positive work decreased (P 15 or not. The optimal cutoff value was 3.89, with a sensitivity of 100%, a specificity of 93.0%, a positive predictive value of 84.9%, a negative predictive value of 100%, and an accuracy of 95.7%. CONCLUSION: Noninvasive myocardial work is highly competent in assessing the severity of acute stroke and neurological deficits, which can be used as a powerful supplement to the conventional scoring system.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Isquemia Encefálica/complicaciones , Isquemia Encefálica/diagnóstico por imagen , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/diagnóstico por imagen , Valor Predictivo de las Pruebas , Ecocardiografía , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Right ventricle-pulmonary artery (RV-PA) coupling is an independent predictor of outcome in pulmonary arterial hypertension in adults. Here, we aimed to investigate the changes in RV-PA coupling during the perinatal period, and to evaluate its performance on predicting persistent pulmonary hypertension of the newborn (PPHN). METHODS: A total of 1196 fetuses underwent a dedicated echocardiography screening for foetal heart defects during second trimester (24-27 weeks' gestation), third trimester (34-37 weeks' gestation) and neonatal period (within 14 days after delivery) with the measurement of tricuspid annular plane systolic excursion (TAPSE) and mean pulmonary artery pressure (MPAP). The RV-PA coupling (TAPSE/MPAP ratio) was calculated. RESULTS: Six fetuses were diagnosed as persistent pulmonary hypertension of the newborn (PPHN). In normal fetuses, RV-PA coupling had been increasing from the second trimester to the third trimester and then to the neonatal period (0.12 ± 0.02 vs. 0.18 ± 0.05 vs. 0.23 ± 0.08 mm/mmHg, p < 0.05), while it had been decreasing during the same period of time in abnormal fetuses (0.18 ± 0.02 vs. 0.17 ± 0.02 vs. 0.17 ± 0.01 mm/mmHg, p < 0.05). There was a strong positive correlation between RV-PA coupling and gestational age (GA) in normal fetuses (r = 0.71, p < 0.0001). The area under receiver operating characteristic curve (AUC) of 0.989 for RV-PA coupling during second trimester was superior to that for RV-PA coupling during third trimester (AUC: 0.536) in predicting PPHN. The optimal cutoff value was 0.16 mm/mmHg, with a sensitivity of 100.00%, a specificity of 96.36% and an accuracy of 97.73%. CONCLUSION: RV-PA coupling had close relation with GA in normal fetuses. It was a strong predictor of PPHN.
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Hipertensión Pulmonar , Disfunción Ventricular Derecha , Adulto , Ecocardiografía , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Hipertensión Pulmonar/diagnóstico por imagen , Recién Nacido , Embarazo , Arteria Pulmonar/diagnóstico por imagen , Función Ventricular DerechaRESUMEN
The importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme methionine synthase reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters' uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.
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Anomalías Congénitas/genética , Embrión de Mamíferos/metabolismo , Epigénesis Genética , Ferredoxina-NADP Reductasa/genética , Retardo del Crecimiento Fetal/genética , Ácido Fólico/metabolismo , Animales , Cruzamientos Genéticos , Metilación de ADN , Femenino , Ferredoxina-NADP Reductasa/metabolismo , Masculino , Ratones , MutaciónRESUMEN
Defects in the MMACHC gene represent the most common disorder of cobalamin (Cbl) metabolism, affecting synthesis of the enzyme cofactors adenosyl-Cbl and methyl-Cbl. The encoded MMACHC protein binds intracellular Cbl derivatives with different upper axial ligands and exhibits flavin mononucleotide (FMN)-dependent decyanase activity toward cyano-Cbl as well as glutathione (GSH)-dependent dealkylase activity toward alkyl-Cbls. We determined the structure of human MMACHC·adenosyl-Cbl complex, revealing a tailor-made nitroreductase scaffold which binds adenosyl-Cbl in a "base-off, five-coordinate" configuration for catalysis. We further identified an arginine-rich pocket close to the Cbl binding site responsible for GSH binding and dealkylation activity. Mutation of these highly conserved arginines, including a replication of the prevalent MMACHC missense mutation, Arg161Gln, disrupts GSH binding and dealkylation. We further showed that two Cbl-binding monomers dimerize to mediate the reciprocal exchange of a conserved "PNRRP" loop from both subunits, serving as a protein cap for the upper axial ligand in trans and required for proper dealkylation activity. Our dimeric structure is supported by solution studies, where dimerization is triggered upon binding its substrate adenosyl-Cbl or cofactor FMN. Together our data provide a structural framework to understanding catalytic function and disease mechanism for this multifunctional enzyme.
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Arginina/química , Proteínas Portadoras/química , Complejos Multienzimáticos/química , Multimerización de Proteína , Vitamina B 12/química , Vitamina B 12/fisiología , Arginina/genética , Proteínas Portadoras/genética , Catálisis , Cristalografía por Rayos X , Humanos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/fisiología , Mutación , Oxidorreductasas , Multimerización de Proteína/genética , Procesamiento Proteico-Postraduccional/genética , Estructura Terciaria de Proteína/genética , Vitamina B 12/genéticaRESUMEN
Vitamin B(12) (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.
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Cobamidas/química , Proteínas de Transporte de Membrana/química , Metilmalonil-CoA Mutasa/química , Proteínas Mitocondriales/química , Complejos Multiproteicos/química , Niño , Preescolar , Cobamidas/genética , Cobamidas/metabolismo , Cristalografía por Rayos X , Citosol/química , Citosol/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Errores Innatos del Metabolismo , Metilmalonil-CoA Mutasa/genética , Metilmalonil-CoA Mutasa/metabolismo , Mitocondrias/química , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación Missense , Estructura Cuaternaria de ProteínaRESUMEN
Holocarboxylase synthetase (HCS, human) and BirA (Escherichia coli) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases. Biotin attachment occurs on a highly conserved lysine residue within a consensus sequence (Ala/Val-Met-Lys-Met) that is found in carboxylases in most organisms. Numerous studies have indicated that HCS and BirA, as well as biotin protein ligases from other organisms, can attach biotin to apocarboxylases from different organisms, indicating that the mechanism of biotin attachment is well conserved. In this study, we examined the cross-reactivity of biotin attachment between human and bacterial biotin ligases by comparing biotinylation of p-67 and BCCP87, the biotin-attachment domain fragments from human propionyl-CoA carboxylase and E. coli acetyl-CoA carboxylase, respectively. While BirA has similar biotinylation activity toward the two substrates, HCS has reduced activity toward bacterial BCCP87 relative to its native substrate, p-67. The crystal structure of a digested form of p-67, spanning a sequence that contains a seven-residue protruding thumb loop in BCCP87, revealed the absence of a similar structure in the human peptide. Significantly, an engineered "thumbless" bacterial BCCP87 could be biotinylated by HCS, with substrate affinity restored to near normal. This study suggests that the thumb loop found in bacterial carboxylases interferes with optimal interaction with the mammalian biotin protein ligase. While the function of the thumb loop remains unknown, these results indicate a constraint on specificity of the bacterial substrate for biotin attachment that is not itself a feature of BirA.
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Acetil-CoA Carboxilasa/química , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas Portadoras/química , Proteínas de Escherichia coli/química , Metilmalonil-CoA Descarboxilasa/química , Proteínas Represoras/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II , Humanos , Metilmalonil-CoA Descarboxilasa/genética , Metilmalonil-CoA Descarboxilasa/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Especificidad por Sustrato/genéticaRESUMEN
MMAB (methylmalonic aciduria type B) is a mitochondrial enzyme involved in the metabolism of vitamin B(12). It functions as the ATP:cob(I)alamin adenosyltransferase for the generation of adenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MCM). Impaired MMAB activity leads to the inherited disorder methylmalonic aciduria and is responsible for the cblB complementation group. In this study, the effects on substrate binding of two catalytically inactive patient mutations, R190H and R186W, were investigated using intrinsic fluorescence quenching of MMAB as a measure of ligand-binding. We report the dissociation constant (K(d)) of wild-type MMAB for HOCbl is 51 microM and for ATP is 365 microM and show that cobalamin enhances the affinity of MMAB for ATP, while ATP does not show detectable effects on cobalamin binding. We confirm that residue Arg190 plays a role in the formation of the ATP-binding site as described previously [H.L. Schubert, C.P. Hill, Structure of ATP-bound human ATP:cobalamin adenosyltransferase, Biochemistry 45 (2006) 15188-15196]. Unexpectedly, mutation R186W does not disrupt the binding of HOCbl to MMAB as predicted; instead, both R190H and R186W significantly disrupt the affinity between MMAB and AdoCbl. We surmise that these two residues may be critical for the transfer of the 5'-deoxyadenosyl group from ATP to cob(I)alamin, possibly by contributing to the precise positioning of the two substrates to permit catalysis to occur. Characterization of ligand-binding by MMAB provides insight into the mechanism of cobalamin adenosylation and the effect of patient mutations in the inherited disorder.
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Adenosina Trifosfato/metabolismo , Transferasas Alquil y Aril/genética , Errores Innatos del Metabolismo/enzimología , Mutación , Adenosina Trifosfato/química , Transferasas Alquil y Aril/metabolismo , Sitios de Unión , Catálisis , Humanos , Ligandos , Espectrometría de Fluorescencia , Vitamina B 12/metabolismoRESUMEN
Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of methionine synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying methionine synthase reductase, an enzyme essential for the activity of methionine synthase. This model is a hypomorph, with reduced methionine synthase reductase activity, thus avoiding the lethality associated with the absence of methionine synthase activity. Mtrr(gt/gt) mice have increased plasma homocyst(e)ine, decreased plasma methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrr(gt/gt) mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocyst(e)inemia may be useful in understanding biological effects of elevated homocyst(e)ine.
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Ferredoxina-NADP Reductasa/genética , Ferredoxina-NADP Reductasa/metabolismo , Ácido Fólico/metabolismo , Metionina/metabolismo , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Embrión de Mamíferos/metabolismo , Femenino , Corazón/embriología , Homocisteína/sangre , Homocisteína/metabolismo , Hiperhomocisteinemia/enzimología , Hiperhomocisteinemia/metabolismo , Riñón/embriología , Riñón/metabolismo , Hígado/embriología , Hígado/metabolismo , Masculino , Metionina/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Methylmalonyl-CoA epimerase (MCE) catalyzes the interconversion of D- and L-methylmalonyl-CoA in the pathway responsible for the degradation of branched chain amino acids, odd chain-length fatty acids, and other metabolites. Despite the occurrence of metabolic disorders in the enzymatic step occurring immediately upstream of MCE (propionyl-CoA carboxylase) and downstream of MCE (adenosylcobalamin-dependent methylmalonyl-CoA mutase), no disease-causing mutations have been described affecting MCE itself. A patient, formerly identified as belonging to the cblA complementation group of vitamin B12 disorders but lacking mutations in the affected gene, MMAA, was tested for mutations in the MCEE gene. The patient's fibroblasts had normal levels of adenosylcobalamin compared to controls, whereas other cblA cell lines typically had reduced levels of the cofactor. As well, this patient had a milder form of methylmalonic aciduria than usually observed in cblA patients. The patient was found to be homozygous for a c.139C>T (p.R47X) mutation in MCEE by sequence analysis that was confirmed by restriction digestion of PCR products. One sibling, also with mild methylmalonic aciduria, was homozygous for the mutation. Both parents and one other sibling were heterozygous. A nearby insertion polymorphism, c.41-160_161insT, heterozygous in both parents, showed the wild-type configuration on the mutant alleles. To assess the impact of isolated MCE deficiency in cultured cells, HeLa cells were transfected with a selectable vector containing MCEE-specific small interfering RNA (siRNA) to suppress gene expression. The reduced level of MCEE mRNA resulted in the reduction of [14C]-propionate incorporation into cellular macromolecules. However, siRNA only led to a small reduction in pathway activity, suggesting that previously postulated non-enzymatic conversion of D- to L-methylmalonyl-CoA may contribute to some flux through the pathway. We conclude that the patient's MCEE defect was responsible for the mild methylmalonic aciduria, confirming a partial requirement for the enzymatic activity in humans.
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Codón sin Sentido , Errores Innatos del Metabolismo/genética , Ácido Metilmalónico/orina , Racemasas y Epimerasas/genética , Técnicas de Cultivo de Célula , Línea Celular , Niño , Femenino , Fibroblastos , Prueba de Complementación Genética , Células HeLa , Homocigoto , Humanos , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/genética , Reacción en Cadena de la Polimerasa , Interferencia de ARN , ARN Interferente Pequeño , TransfecciónRESUMEN
ATP:cob(I)alamin adenosyltransferase (MMAB protein; methylmalonic aciduria type B) is an enzyme of vitamin B(12) metabolism that converts reduced cob(I)alamin to the adenosylcobalamin co-factor required for the functional activity of methylmalonyl-CoA mutase. Mutations in the human MMAB gene result in a block in adenosylcobalamin synthesis and are responsible for the cblB complementation group of inherited vitamin B(12) disorders. In this study, we examined the impact of several mutations, previously identified in cblB patients and clustered within a small, highly conserved region in MMAB. We confirmed mitochondrial expression of MMAB in human cells and showed that two mutations, R186W and E193K, were associated with absent protein by Western blot, while one, R191W, coupled with another point mutation, produced a protein in patient fibroblasts. Wild type MMAB and all four mutant proteins were stably expressed at high level as GST-fusion proteins, but only the R191W protein was enzymatically active. It showed an elevated K(m) of 320 microM (vs 6.8 microM for wild type enzyme) for ATP and 60 microM (vs 3.7 microM) for cob(I)alamin, with a reduction in k(cat) for both substrates. Circular dichroism spectroscopy revealed that three mutant proteins examined retained a alpha-helical structure as for the wild type protein. Characterization of MMAB will contribute to our understanding of cobalamin processing in mammalian cells and of disease mechanisms in the genetic disorders.
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Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Errores Innatos del Metabolismo/enzimología , Vitamina B 12/metabolismo , Transferasas Alquil y Aril/química , Línea Celular , Dicroismo Circular , Humanos , Cinética , Mutación Puntual , Estructura Secundaria de ProteínaRESUMEN
Vitamin B(12) (cobalamin) is an essential cofactor of two enzymes, methionine synthase and methylmalonyl-CoA mutase. The conversion of the vitamin to its coenzymes requires a series of biochemical modifications for which several genetic diseases are known, comprising eight complementation groups (cblA through cblH). The objective of this study was to clone the gene responsible for the cblA complementation group thought to represent a mitochondrial cobalamin reductase. Examination of bacterial operons containing genes in close proximity to the gene for methylmalonyl-CoA mutase and searching for orthologous sequences in the human genome yielded potential candidates. A candidate gene was evaluated for deleterious mutations in cblA patient cell lines, which revealed a 4-bp deletion in three cell lines, as well as an 8-bp insertion and point mutations causing a stop codon and an amino acid substitution. These data confirm that the identified gene, MMAA, corresponds to the cblA complementation group. It is located on chromosome 4q31.1-2 and encodes a predicted protein of 418 aa. A Northern blot revealed RNA species of 1.4, 2.6, and 5.5 kb predominating in liver and skeletal muscle. The deduced amino acid sequence reveals a domain structure, which belongs to the AAA ATPase superfamily that encompasses a wide variety of proteins including ATP-binding cassette transporter accessory proteins that bind ATP and GTP. We speculate that we have identified a component of a transporter or an accessory protein that is involved in the translocation of vitamin B(12) into mitochondria.