RESUMEN
INTRODUCTION: Sudden Unexplained Death in Childhood (SUDC) needs to be fully assessed considering its impact on the family, parents and siblings. Inborn Errors of Metabolism (IEM) such as Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD) should be taken into consideration when SUDC occurres. Our aim is to present a family with two successive SUDC and to discuss the post-mortem genetics investigations revealing an IEM implication. CASES REPORT: A complete autopsy with genetic testing was performed when the proband, a 4-year-old girl, died. A few years previously, her older brother had died at the same age and off the same condition. Years later, his exhumation was necessary in order to perform a post-mortem diagnosis.The two siblings were revealed to have had the same pathogenic genotype of the ACADM gene, heterozygous substitutions in ACADM (NM_000016.5): c.985â¯A>G p.(Lys329Glu) and c.347â¯G>A p.(Cys116Tyr). In addition, they also both carried a VUS in TECRL, a gene implicated in Catecholaminergic Polymorphic Tachycardia Ventricular (CPVT) and SUDC. CONCLUSION: We illustrate the importance of exome analyses for investigating unexplained sudden death, especially in children, with the possible impact for genetic counselling in the family. The finding of the implication of ACADM gene in this case, raises likely responsibility of the public health system in countries such as France, who delayed implementation of new born screening for these conditions. Exome analyses in this case detected unexpected complexity in interpretation linked to the identification of a second candidate gene for SUDC.
Asunto(s)
Acil-CoA Deshidrogenasa , Muerte Súbita , Humanos , Femenino , Preescolar , Muerte Súbita/etiología , Masculino , Acil-CoA Deshidrogenasa/genética , Acil-CoA Deshidrogenasa/deficiencia , Errores Innatos del Metabolismo Lipídico/genética , Linaje , Genotipo , Pruebas Genéticas , Hermanos , RecurrenciaRESUMEN
We describe two patients with mitochondrial DNA mutations in the gene encoding cytochrome b (m.15579A>G, p.Tyr278Cys and m.15045G>A p.Arg100Gln), which presented as a pure myopathic form (exercise intolerance), with an onset in childhood. Diagnosis was delayed, because acylcarnitine profile showed an increase in medium and long-chain acylcarnitines, suggestive of multiple acyl-CoA dehydrogenase deficiency, riboflavin transporter deficiency or FAD metabolism disorder. Implication of cytochrome b in fatty acid oxidation, and physiopathology of the mutations are discussed.
Asunto(s)
Citocromos b/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/diagnóstico , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Mutación Missense , Adulto , Anciano , ADN Mitocondrial/genética , Diagnóstico Diferencial , Tolerancia al Ejercicio/genética , Humanos , Masculino , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genéticaRESUMEN
The management of sporadic late-onset cerebellar ataxias represents a very heterogeneous group of patients and remains a challenge for neurologist in clinical practice. We aimed at describing the different causes of sporadic late-onset cerebellar ataxias that were diagnosed following standardized, exhaustive investigations and the population characteristics according to the aetiologies as well as at evaluating the relevance of these investigations. All patients consecutively referred to our centre due to sporadic, progressive cerebellar ataxia occurring after 40 years of age were included in the prospective, observational study. 80 patients were included over a 2 year period. A diagnosis was established for 52 patients (65%) corresponding to 18 distinct causes, the most frequent being cerebellar variant of multiple system atrophy (n = 29). The second most frequent cause was inherited diseases (including spinocerebellar ataxias, late-onset Friedreich's disease, SLC20A2 mutations, FXTAS, MELAS, and other mitochondrial diseases) (n = 9), followed by immune-mediated or other acquired causes. The group of patient without diagnosis showed a slower worsening of ataxia (p < 0.05) than patients with multiple system atrophy. Patients with later age at onset experienced faster progression of ataxia (p = 0.001) and more frequently parkinsonism (p < 0.05) than patients with earlier onset. Brain MRI, DaT scan, genetic analysis and to some extent muscle biopsy, thoracic-abdominal-pelvic tomodensitometry, and cerebrospinal fluid analysis were the most relevant investigations to explore sporadic late-onset cerebellar ataxia. Sporadic late-onset cerebellar ataxias should be exhaustively investigated to identify the underlying causes that are numerous, including inherited causes, but dominated by multiple system atrophy.
Asunto(s)
Ataxia Cerebelosa/diagnóstico , Ataxia Cerebelosa/etiología , Atrofia de Múltiples Sistemas/complicaciones , Adulto , Edad de Inicio , Anciano , Encéfalo/diagnóstico por imagen , Canales de Calcio/genética , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/patología , Electromiografía , Femenino , Ataxia de Friedreich/complicaciones , Humanos , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Atrofia de Múltiples Sistemas/diagnóstico por imagen , Mutación/genética , Conducción Nerviosa/fisiología , Examen Neurológico , Proteínas Proto-Oncogénicas c-sis/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Ataxias Espinocerebelosas/complicaciones , Estadísticas no Paramétricas , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
The analysis of acylcarnitines (AC) in plasma/serum is established as a useful test for the biochemical diagnosis and the monitoring of treatment of organic acidurias and fatty acid oxidation defects. External quality assurance (EQA) for qualitative and quantitative AC is offered by ERNDIM and CDC in dried blood spots but not in plasma/serum samples. A pilot interlaboratory comparison between 14 European laboratories was performed over 3 years using serum/plasma samples from patients with an established diagnosis of an organic aciduria or fatty acid oxidation defect. Twenty-three different samples with a short clinical description were circulated. Participants were asked to specify the method used to analyze diagnostic AC, to give quantitative data for diagnostic AC with the corresponding reference values, possible diagnosis, and advice for further investigations.Although the reference and pathological concentrations of AC varied among laboratories, elevated marker AC for propionic acidemia, isovaleric acidemia, medium-chain acyl-CoA dehydrogenase, very long-chain acyl-CoA dehydrogenase, and multiple acyl-CoA dehydrogenase deficiencies were correctly identified by all participants allowing the diagnosis of these diseases. Conversely, the increased concentrations of dicarboxylic AC were not always identified, and therefore the correct diagnosis was not reach by some participants, as exemplified in cases of malonic aciduria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Misinterpretation occurred in those laboratories that used multiple-reaction monitoring acquisition mode, did not derivatize, or did not separate isomers. However, some of these laboratories suggested further analyses to clarify the diagnosis.This pilot experience highlights the importance of an EQA scheme for AC in plasma.
RESUMEN
Neonatal seizure incidence is approximately 3.5/1000 live births. Inborn metabolic diseases account for approximately 1-4% of neonatal seizure cases. Among them, the catabolism anomaly of sulfite to sulfate caused by sulfite oxidase or cofactor molybdenum deficiency (MoCD) is a rare metabolic disorder in which neurological damage is similar to that found in neonatal asphyxia. We report the case of a newborn child with a MoCD. Born of related parents, this child had intrauterine growth retardation predominating on size diagnosed in the third trimester of pregnancy. After an uneventful birth, he presented convulsions at the 12th hour of life, confirmed by an electroencephalogram. Anticonvulsants and adjuvant treatments were ineffective; the child then required intubation at day 5 of life. The initial biological assessment found an elevated blood lactate level and the chromatography of amino acids showed a significant decrease of cystine and the abnormal presence of sulfocysteine, suggestive of a lack of sulfite oxidase activity. The uric acid level measured secondarily was low, suggesting a MoCD. Brain MRI was performed at day 5 for diffuse ischemic injury of different ages. After limiting acute care, the child died at day 14 of life. The genetic study of the child found a homozygous mutation c.564+1G>A in the MOCS2 gene, confirming the diagnosis of MoCD, present in the heterozygous state in both parents. Investigations in a logical sequence quickly suggested the MoCD diagnosis in presence of a low plasma concentration of cysteine, the abnormal presence of sulfocysteine, and low uric acid levels. The diagnosis of sulfite oxidase deficiency was made. Until now, no treatment has proven effective but a new treatment appears to be effective in cases with a MOCS1 mutation.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/etiología , Encefalopatías/etiología , Errores Innatos del Metabolismo de los Metales/complicaciones , Sulfito-Oxidasa/deficiencia , Humanos , Recién Nacido , Masculino , Índice de Severidad de la EnfermedadRESUMEN
Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/normas , Quimera por Trasplante/genética , Europa (Continente) , Marcadores Genéticos , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Trasplante HomólogoRESUMEN
BACKGROUND: Deficiency of mitochondrial trifunctional protein (MTP) is caused by mutations in the HADHA and HADHB genes, which have been mostly delineated at the genomic DNA level and have not been always elucidated. AIM: To identify mutations in a French cohort of 52 MTP deficient patients and the susceptibility of mutations generating premature termination codons (PTCs) to the nonsense mRNA mediated decay (NMD). METHODS: Mutation screening in fibroblasts was performed at the cDNA level and real-time RT-PCR was used to compare the levels of the different PTC-bearing mRNAs before and after a treatment of fibroblasts by emetine, a translation inhibitor. RESULTS: A mutation detection rate of 100% was achieved. A total of 22 novel mutations were identified, including a large-sized genomic deletion in HADHB gene. A high proportion of all identified mutations were non-sense, frameshift and splicing mutations, generating (PTCs), distributed essentially on HADHA coding regions. We could demonstrate that the majority of mutations resulting in PTCs conform to the established rules governing the susceptibility to NMD. CONCLUSION: Our results emphasize the value of cDNA analysis in the characterization of HADHA and HADHB mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in MTP defects.
Asunto(s)
ADN Complementario/genética , Trastornos del Metabolismo de los Lípidos/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Complejos Multienzimáticos/genética , Mutación , Secuencia de Bases , Estudios de Cohortes , Femenino , Francia , Haploinsuficiencia , Humanos , Masculino , Proteína Trifuncional Mitocondrial , Subunidad alfa de la Proteína Trifuncional Mitocondrial , Subunidad beta de la Proteína Trifuncional Mitocondrial , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNAsunto(s)
Síndromes Mielodisplásicos/genética , Cromosomas Humanos Par 5 , Proteínas de Unión al ADN/genética , Dioxigenasas , Genes p16 , Histona Demetilasas , Humanos , Mutación , Síndromes Mielodisplásicos/etiología , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genéticaRESUMEN
3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder due to a deficiency of the 3-hydroxy-3-methylglutarylCoA lyase (HMG-CoA lyase), a mitochondrial enzyme involved in ketogenesis and in the final step of l-leucine catabolism. HMG-CoA lyase deficiency can lead, in particular circumstances, such as fever, prolonged fasting or digestive disorders, to brutal and severe hypoglycemia with metabolic acidosis and sometimes fatal coma. We report on a new case of 3-hydroxy-3-methylglutaric aciduria particular by its late onset in a 3-year-old patient. Molecular investigation identified two new sequence modifications in the HMGCL gene: c.494G>A (p.Arg165Gln) and c.820G>A (p.Gly274Arg). We remind about this case report that the therapeutical is mainly preventive and allows a very good prognosis for this disease. Long-term treatment consists in limited fasting time, continuous low protein diet and l-carnitine supplementation. Preventive measures are essential: prevention of fasting and emergency treatment during intercurrent infections.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Aberraciones Cromosómicas , Genes Recesivos/genética , Hipoglucemia/genética , Meglutol/orina , Oxo-Ácido-Liasas/deficiencia , Oxo-Ácido-Liasas/genética , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Carnitina/administración & dosificación , Preescolar , Terapia Combinada , Análisis Mutacional de ADN , Dieta con Restricción de Proteínas , Exones/genética , Humanos , Hipoglucemia/orina , Leucina/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Enfermedades Raras/terapia , Análisis de Secuencia de ADNAsunto(s)
Trasplante de Médula Ósea , ADN/metabolismo , Electroforesis Capilar/métodos , Repeticiones de Minisatélite , Reacción en Cadena de la Polimerasa , Quimera por Trasplante/genética , Adolescente , Alelos , Médula Ósea/patología , Niño , Preescolar , ADN/sangre , Cartilla de ADN/química , Electroforesis Capilar/economía , Estudios de Evaluación como Asunto , Femenino , Fluorescencia , Humanos , Lactante , Leucemia/genética , Leucemia/terapia , Masculino , Paris , Reacción en Cadena de la Polimerasa/economía , Polimorfismo Genético/genética , Estudios Prospectivos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Application fields of RT-PCR (reverse transcription-polymerase chain reaction) in clinical diagnosis comprises the assessment of viral load for RNA viruses and the analysis of gene transcription products. RT-PCR is also helpful when large genes have to be sequenced. Developments of quantitative approaches using real-time PCR recently led to a major widening of RT-PCR applications in clinical diagnosis. However, RT reaction is delicate due to its lack of reproducibility and to RNA lability and frequent contamination by DNA. In some cases additional difficulties come from the need to obtain a specific amplification in the presence of homologous sequences which might be present in higher amounts than the sequence of interest. These caveats have to be taken into account, when designing the RT protocol, and when choosing PCR primers and internal and/or external references. This review is aimed at helping the experimental setup of a RT-PCR based assay according to the objectives.
Asunto(s)
Medicina Clínica/métodos , Técnicas y Procedimientos Diagnósticos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , HumanosRESUMEN
c-Fos proto-oncoprotein is rapidly and transiently expressed in cells undergoing the G(0)-to-S phase transition in response to stimulation for growth by serum. Under these conditions, the rapid decay of the protein occurring after induction is accounted for by efficient recognition and degradation by the proteasome. PEST motifs are sequences rich in Pro, Glu, Asp, Ser and Thr which have been proposed to constitute protein instability determinants. c-Fos contains three such motifs, one of which comprises the C-terminal 20 amino acids and has already been proposed to be the major determinant of c-Fos instability. Using site-directed mutagenesis and an expression system reproducing c-fos gene transient expression in transfected cells, we have analysed the turnover of c-Fos mutants deleted of the various PEST sequences in synchronized mouse embryo fibroblasts. Our data showed no role for the two internal PEST motifs in c-Fos instability. However, deletion of the C-terminal PEST region led to only a twofold stabilization of the protein. Taken together, these data indicate that c-Fos instability during the G0-to-S phase transition is governed by a major non-PEST destabilizer and a C-terminal degradation-accelerating element. Further dissection of c-Fos C-terminal region showed that the degradation-accelerating effect is not contributed by the whole PEST sequence but by a short PTL tripeptide which cannot be considered as a PEST motif and which can act in the absence of any PEST environment. Interestingly, the PTL motif is conserved in other members of the fos multigene family. Nevertheless, its contribution to protein instability is restricted to c-Fos suggesting that the mechanisms whereby the various Fos proteins are broken down are, at least partially, different. MAP kinases-mediated phosphorylation of two serines close to PTL, which are both phosphorylated all over the G(0)-to-S phase transition, have been proposed by others to stabilize c-Fos protein significantly. We, however, showed that the PTL motif does not exert its effect by counteracting a stabilizing effect of these phosphorylations under our experimental conditions.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Péptidos/química , Proteínas Proto-Oncogénicas c-fos/metabolismo , Células 3T3 , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Northern Blotting , Western Blotting , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Ratas , Fase de Descanso del Ciclo Celular , Fase S , Homología de Secuencia de Aminoácido , Elemento de Respuesta al Suero , Factores de Tiempo , TransfecciónRESUMEN
Mutations in the MUT locus encoding for the methylmalonyl-CoA mutase (MCM) apoenzyme are responsible for the mut forms of methylmalonic acidemia (MMA). To date, 49 different mutations have been identified in mut MMA. Only two frequent mutations have been reported in the Japanese population and in African-Americans. Here we report a new missense mutation N219Y (731 A-->T) which we found in five unrelated families of French and Turkish descent. All the patients exhibited a severe mut(degree) phenotype and three of them were homozygotes for N219Y. Direct involvement of the mutation in the loss of enzyme activity was demonstrated by mutagenesis and transient expression study. Mapping of the mutation onto a three-dimensional model of human MCM constructed by homology with the Propionibacterium shermanii enzyme shows that it lies in a highly conserved secondary structure motif and might suggest impaired folding and/or poor stability compatible with the mut(degree) phenotype. Finally, a 1% N219Y carrier frequency was observed in a French anonymous control population. Thus, N219Y is the first frequent mut mutation to be reported in the Caucasian population.
Asunto(s)
Sustitución de Aminoácidos/genética , Errores Innatos del Metabolismo Lipídico/genética , Ácido Metilmalónico/sangre , Mutación Missense/genética , Población Blanca/genética , Secuencia de Aminoácidos , Asparagina/genética , Niño , Preescolar , Femenino , Humanos , Lactante , Errores Innatos del Metabolismo Lipídico/sangre , Errores Innatos del Metabolismo Lipídico/enzimología , Masculino , Ácido Metilmalónico/metabolismo , Metilmalonil-CoA Mutasa/genética , Metilmalonil-CoA Mutasa/metabolismo , Datos de Secuencia Molecular , Tirosina/genéticaRESUMEN
c-Fos proto-oncoprotein is a short-lived transcription factor degraded by the proteasome in vivo. Its mutated forms expressed by the mouse osteosarcomatogenic retroviruses, FBJ-MSV and FBR-MSV, are stabilized two- and threefold, respectively. To elucidate the mechanisms underlying v-Fos(FBJ) and v-Fos(FBR) protein stabilization, we conducted a genetic analysis in which the half-lives and the sensitivities to various cell-permeable protease inhibitors of a variety of cellular and viral protein mutants were measured. Our data showed that the decreased degradation of v-Fos(FBJ) and v-Fos(FBR) is not simply explained by the deletion of a c-Fos destabilizing C-terminal domain. Rather, it involves a complex balance between opposing destabilizing and stabilizing mutations which are distinct and which include virally-introduced peptide motifs in both cases. The mutations in viral Fos proteins conferred both total insensitivity to proteasomal degradation and sensitivity to another proteolytic system not naturally operating on c-Fos, explaining the limited stabilization of the two proteins. This observation is consistent with the idea that FBR-MSV and FBJ-MSV expression machineries have evolved to ensure controlled protein levels. Importantly, our data illustrate that the degradation of unstable proteins does not necessarily involve the proteasome and provide support to the notion that highly related proteins can be broken down by different proteolytic systems in living cells.
Asunto(s)
Proteínas Oncogénicas v-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Cisteína Endopeptidasas/metabolismo , Mutación del Sistema de Lectura , Semivida , Complejos Multienzimáticos/metabolismo , Ácido Mirístico/metabolismo , Proteínas Oncogénicas v-fos/genética , Mutación Puntual , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Virus del Sarcoma Murino/genética , Eliminación de SecuenciaRESUMEN
c-Fos proto-oncoprotein is a short-lived transcription factor with oncogenic potential. We have shown that it is massively degraded by the proteasome in vivo under various experimental conditions. Other proteolytic systems including lysosomes and calpains, might, however, also marginally operate on it. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for its addressing to the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome- and ubiquitin-dependent in vivo. Interestingly, several lines of evidence indicate that the addressing of c-Fos and c-Jun to the proteasome is, at least in part, governed by different mechanisms. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of c-Fos main destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to an unidentified proteolytic system. This observation is consistent with the idea that Fos-expressing viruses have evolved to ensure control protein levels to avoid high protein accumulation-linked apoptosis. In conclusion, the unveiling of the complex mechanism network responsible for the degradation of AP-1 family members is still at its beginning and a number of issues regarding the regulation of this process and the addressing to the proteasome are still unresolved.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Proteínas Oncogénicas v-fos/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ubiquitinas/metabolismo , Animales , Fibroblastos/metabolismo , Genes fos/genética , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Retroviridae/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Inherited defects in the gene encoding the methylmalonyl-CoA mutase (MCM) result in the mut forms of methylmalonic aciduria (MMA). Twelve mutations have been identified associated with the mut(-) phenotype. We report two novel mutations (K621N and D156N) in a compound heterozygote mut(-) patient. These two mutations and three previously published ones (H627N, A191E, Y231N) were mapped onto a three-dimensional homology model of the human MCM constructed from the crystal structure of the Propionibacterium shermanii enzyme.
Asunto(s)
Acilcoenzima A/química , Acilcoenzima A/deficiencia , Acilcoenzima A/genética , Mutación Missense , Mutación , Preescolar , Femenino , Genotipo , Heterocigoto , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Propionibacterium/enzimología , Estructura Terciaria de ProteínaRESUMEN
Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection. Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations. Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some "imprint" in the sequence of bacterial genomes. Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators. The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.
Asunto(s)
Adenosina Trifosfatasas , Disparidad de Par Base , Reparación del ADN , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Evolución Molecular , Alelos , Proteínas Bacterianas/genética , Escherichia coli/genética , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Genotipo , Proteínas MutL , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mutación , Fenotipo , Monoéster Fosfórico Hidrolasas/genética , Filogenia , Reacción en Cadena de la Polimerasa , Pirofosfatasas , Recombinación Genética , Salmonella typhimurium/genéticaRESUMEN
According to our current knowledge, protein ubiquitination involves three steps: activation of ubiquitin through formation of an energy-rich bond with an E1 ubiquitin-activating enzyme; and transfer of activated ubiquitin onto E2 ubiquitin-conjugating enzymes, which, in turn, alone, or in combination with E3 ubiquitin-protein ligase enzymes, transfer ubiquitin onto target proteins. A31N-ts20 cells are mouse embryo fibroblasts, thermosensitive for E1. We show here that: (a) the enzymatic activity of the enzyme is heat-inactivatable in vitro; and (b) a major mechanism responsible for E1 inactivation in vivo consists of accelerated destruction. Surprisingly, a >90% reduction in E1 abundance little alters the formation of the bulk of protein-ubiquitin conjugates when A31N-ts20 cells are grown at the nonpermissive temperature, indicating that cautious interpretation of results is required when studying ubiquitination of specific substrates using this cell line. Surprisingly, our data also indicate that, in vivo, ubiquitination of the various protein substrates in A31N-ts20 cells requires different amounts of E1, indicating that this mutant cell line can be used for unveiling the existence of differences in the intimate mechanisms responsible for the ubiquitination of the various cell proteins in vivo, and for providing criteria of reliability when developing in vitro ubiquitination assays for specific proteins.
Asunto(s)
Ligasas/genética , Ligasas/metabolismo , Animales , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Fibroblastos , Histonas/metabolismo , Calor , Leupeptinas/farmacología , Ligasas/antagonistas & inhibidores , Ratones , Mutación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Temperatura , Proteína p53 Supresora de Tumor/metabolismo , Enzimas Activadoras de Ubiquitina , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismoRESUMEN
c-Fos and c-Jun proteins are highly unstable transcription factors that heterodimerize within the AP-1 transcription complex. Their accumulation is transiently induced at the beginning of the G0-to-S phase transition in quiescent cells stimulated for growth. To address the mechanisms responsible for rapid clearance of c-Fos and c-Jun proteins under these experimental conditions, we have used the ts20 mouse embryo fibroblasts which express a thermosensitive mutant of the E1 enzyme of the ubiquitin pathway. The use of cell-permeant protease inhibitors indicates that both proteins are degraded by the proteasome and excludes any major contribution for calpains and lysosomes during the G0-to-S phase transition. Synchronisation of ts20 cells at the non permissive temperature blocks the degradation of c-Jun, indicating that this process is E1-dependent. In contrast, c-Fos is broken down according to an apparently E1-independent pathway in ts20 cells, although a role for ubiquitinylation in this process cannot be formally ruled out. Interestingly, c-Jun is highly unstable in c-Fos-null mouse embryo fibroblasts stimulated for growth. Taken together, these observations show that in vivo during a G0-to-S phase transition (i) the precise mechanisms triggering c-Fos and c-Jun directing to the proteasome are not identical, (ii) the presence of c-Fos is not an absolute prerequisite for the degradation of c-Jun and (iii) the degradation of c-Jun is not required for that of c-Fos.
Asunto(s)
Ciclo Celular , Cisteína Endopeptidasas/metabolismo , Ligasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Células 3T3 , Animales , Sangre , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Bovinos , Células Cultivadas , Medios de Cultivo , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente Indirecta , Ligasas/biosíntesis , Ratones , Complejo de la Endopetidasa Proteasomal , Fase de Descanso del Ciclo Celular , Fase S , Temperatura , Ubiquitina-Proteína LigasasRESUMEN
We have previously shown that NF-kappaB nuclear translocation can be observed upon human immunodeficiency virus type 1 (HIV-1) binding to cells expressing the wild-type CD4 molecule, but not in cells expressing a truncated form of CD4 that lacks the cytoplasmic domain (M. Benkirane, K.-T. Jeang, and C. Devaux, EMBO J. 13:5559-5569, 1994). This result indicated that the signaling cascade which controls HIV-1-induced NF-kappaB activation requires the integrity of the CD4 cytoplasmic tail and suggested the involvement of a second protein that binds to this portion of the molecule. Here we investigate the putative role of p56(lck) as a possible cellular intermediate in this signal transduction pathway. Using human cervical carcinoma HeLa cells stably expressing CD4, p56(lck), or both molecules, we provide direct evidence that expression of CD4 and p56(lck) is required for HIV-1-induced NF-kappaB translocation. Moreover, the fact that HIV-1 stimulation did not induce nuclear translocation of NF-kappaB in cells expressing a mutant form of CD4 at position 420 (C420A) and the wild-type p56(lck) indicates the requirement for a functional CD4-p56(lck) complex.