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1.
Hum Mutat ; 42(2): 135-141, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33169484

RESUMO

COX16 is involved in the biogenesis of cytochrome-c-oxidase (complex IV), the terminal complex of the mitochondrial respiratory chain. We present the first report of two unrelated patients with the homozygous nonsense variant c.244C>T(p. Arg82*) in COX16 with hypertrophic cardiomyopathy, encephalopathy and severe fatal lactic acidosis, and isolated complex IV deficiency. The absence of COX16 protein expression leads to a complete loss of the holo-complex IV, as detected by Western blot in patient fibroblasts. Lentiviral transduction of patient fibroblasts with wild-type COX16 complementary DNA rescued complex IV biosynthesis. We hypothesize that COX16 could play a role in the copper delivery route of the COX2 module as part of the complex IV assembly. Our data provide clear evidence for the pathogenicity of the COX16 variant as a cause for the observed clinical features and the isolated complex IV deficiency in these two patients and that COX16 deficiency is a cause for mitochondrial disease.


Assuntos
Acidose Láctica , Encefalopatias , Cardiomiopatias , Deficiência de Citocromo-c Oxidase , Hepatopatias , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Acidose Láctica/genética , Cardiomiopatias/genética , Deficiência de Citocromo-c Oxidase/genética , Humanos , Recém-Nascido , Proteínas Mitocondriais/metabolismo
2.
Am J Hum Genet ; 102(4): 685-695, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29576219

RESUMO

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.


Assuntos
Alelos , Perda Auditiva Neurossensorial/genética , Hipoglicemia/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Pré-Escolar , Análise Mutacional de DNA , DNA Mitocondrial/genética , Feminino , Fibroblastos/metabolismo , Perda Auditiva Neurossensorial/complicações , Humanos , Hipoglicemia/complicações , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/complicações , Proteínas Mitocondriais/química , Fosforilação Oxidativa , Subunidades Proteicas/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/química
3.
Am J Hum Genet ; 99(1): 208-16, 2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-27374773

RESUMO

Mitochondrial complex I deficiency results in a plethora of often severe clinical phenotypes manifesting in early childhood. Here, we report on three complex-I-deficient adult subjects with relatively mild clinical symptoms, including isolated, progressive exercise-induced myalgia and exercise intolerance but with normal later development. Exome sequencing and targeted exome sequencing revealed compound-heterozygous mutations in TMEM126B, encoding a complex I assembly factor. Further biochemical analysis of subject fibroblasts revealed a severe complex I deficiency caused by defective assembly. Lentiviral complementation with the wild-type cDNA restored the complex I deficiency, demonstrating the pathogenic nature of these mutations. Further complexome analysis of one subject indicated that the complex I assembly defect occurred during assembly of its membrane module. Our results show that TMEM126B defects can lead to complex I deficiencies and, interestingly, that symptoms can occur only after exercise.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Debilidade Muscular/genética , Mutação , Adolescente , Adulto , Criança , Complexo I de Transporte de Elétrons/genética , Exercício Físico , Exoma/genética , Teste de Complementação Genética , Heterozigoto , Humanos , Lactente , Masculino , Adulto Jovem
4.
J Pediatr ; 196: 309-313.e3, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29395179

RESUMO

We demonstrate that a heterozygous nuclear variant in the gene encoding mitochondrial complex I subunit NDUFV1 aggravates the cellular phenotype in the presence of a mitochondrial DNA variant in complex I subunit ND1. Our findings suggest that heterozygous variants could be more significant in inherited mitochondrial diseases than hitherto assumed.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Doenças Mitocondriais/genética , NADH Desidrogenase/genética , Criança , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/genética , Feminino , Testes Genéticos/métodos , Heterozigoto , Humanos , Recém-Nascido , Masculino , Doenças Mitocondriais/diagnóstico , Mutação , Fenótipo
5.
Hum Mutat ; 38(6): 692-703, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28247525

RESUMO

COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.


Assuntos
Acidose Láctica/genética , Ciclo-Oxigenase 1/genética , Grupo dos Citocromos c/genética , Insuficiência de Crescimento/genética , Hipertensão Pulmonar/genética , Acidose Láctica/patologia , Núcleo Celular/genética , Ciclo-Oxigenase 1/química , Grupo dos Citocromos c/química , Deficiência de Citocromo-c Oxidase , Complexo IV da Cadeia de Transporte de Elétrons , Insuficiência de Crescimento/patologia , Fibroblastos , Predisposição Genética para Doença , Homozigoto , Humanos , Hipertensão Pulmonar/patologia , Mitocôndrias/genética , Mutação , Subunidades Proteicas/genética
6.
Mol Genet Metab ; 120(3): 243-246, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27986404

RESUMO

NDUFAF3 is an assembly factor of mitochondrial respiratory chain complex I. Variants in NDUFAF3 have been identified as a cause of severe multisystem mitochondrial disease. In a patient presenting with Leigh syndrome, which has hitherto not been described as a clinical feature of NDUFAF3 deficiency, we identified a novel homozygous variant and confirmed its pathogenicity in patient fibroblasts studies. Furthermore, we present an analysis of complex I assembly routes representative of each functional module and, thereby, link NDUFAF3 to a specific step in complex I assembly. Therefore, our report expands the phenotype of NDUFAF3 deficiency and further characterizes the role of NDUFAF3 in complex I biogenesis.


Assuntos
Doença de Leigh/genética , Proteínas Mitocondriais/genética , Mutação , Análise de Sequência de DNA/métodos , Células Cultivadas , Exoma , Evolução Fatal , Feminino , Fibroblastos/citologia , Predisposição Genética para Doença , Homozigoto , Humanos , Lactente , Doença de Leigh/patologia , Fenótipo
7.
Hum Mol Genet ; 23(23): 6356-65, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25008109

RESUMO

Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.


Assuntos
Proteínas de Transporte/genética , Citocromos b/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Consanguinidade , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/genética , Estabilidade Enzimática , Feminino , Fibroblastos/metabolismo , Humanos , Recém-Nascido , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto
8.
PLoS Genet ; 9(12): e1004034, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24385928

RESUMO

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.


Assuntos
Citocromos b/biossíntese , Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Consanguinidade , Citocromos b/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Homozigoto , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/patologia , Doenças Mitocondriais/terapia , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Fosforilação Oxidativa , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Hum Mutat ; 36(1): 34-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25339201

RESUMO

COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6-deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady-state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6-deficient patients is provided.


Assuntos
Cardiomiopatia Hipertrófica/genética , Deficiência de Citocromo-c Oxidase/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/patologia , Cobre/administração & dosagem , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Células HEK293 , Humanos , Recém-Nascido , Mitocôndrias/metabolismo
10.
Hum Mol Genet ; 22(4): 656-67, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23125284

RESUMO

The mitochondrial respiratory chain complex IV (cytochrome c oxidase) is a multi-subunit enzyme that transfers electrons from cytochrome c to molecular oxygen, yielding water. Its biogenesis requires concerted expression of mitochondria- and nuclear-encoded subunits and assembly factors. In this report, we describe a homozygous missense mutation in FAM36A from a patient who displays ataxia and muscle hypotonia. The FAM36A gene is a remote, putative ortholog of the fungal complex IV assembly factor COX20. Messenger RNA (mRNA) and protein co-expression analyses support the involvement of FAM36A in complex IV function in mammals. The c.154A>C mutation in the FAM36A gene, a mutation that is absent in sequenced exomes, leads to a reduced activity and lower levels of complex IV and its protein subunits. The FAM36A protein is nearly absent in patient's fibroblasts. Cells affected by the mutation accumulate subassemblies of complex IV that contain COX1 but are almost devoid of COX2 protein. We observe co-purification of FAM36A and COX2 proteins, supporting that the FAM36A defect hampers the early step of complex IV assembly at the incorporation of the COX2 subunit. Lentiviral complementation of patient's fibroblasts with wild-type FAM36A increases the complex IV activity as well as the amount of holocomplex IV and of individual subunits. These results establish the function of the human gene FAM36A/COX20 in complex IV assembly and support a causal role of the gene in complex IV deficiency.


Assuntos
Anormalidades Múltiplas/genética , Ataxia/genética , Deficiência de Citocromo-c Oxidase/genética , Canais Iônicos/genética , Hipotonia Muscular/genética , Multimerização Proteica , Anormalidades Múltiplas/metabolismo , Sequência de Aminoácidos , Animais , Ataxia/metabolismo , Sequência de Bases , Células Cultivadas , Criança , Consanguinidade , Deficiência de Citocromo-c Oxidase/metabolismo , Análise Mutacional de DNA , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Expressão Gênica , Humanos , Canais Iônicos/metabolismo , Ácido Láctico/sangue , Ácido Láctico/líquido cefalorraquidiano , Masculino , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Hipotonia Muscular/metabolismo , Mutação de Sentido Incorreto , Proteínas de Saccharomyces cerevisiae/genética
11.
Hum Mol Genet ; 21(1): 115-20, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21965299

RESUMO

Mitochondrial complex I (CI) is a multi-subunit enzyme that forms the major entry point of nicotinamide adenine dinucleotide (NADH) electrons into the respiratory chain. Mutations in the NDUFS4 gene, encoding an accessory subunit of this complex, cause a Leigh-like phenotype in humans. To study the nature and penetrance of the CI defect in different tissues, we investigated the role of NDUFS4 in mice with fatal mitochondrial encephalomyopathy, caused by a systemic inactivation of the Ndufs4 gene. We report that the absence of NDUFS4 in different mouse tissues results in decreased activity and stability of CI. This CI instability leads to an increased disconnection of electron influx of the NADH dehydrogenase module from the holo-complex. However, the formation of respiratory supercomplexes still allows formation of active CI in these Ndufs4 knock-out mice. These results reveal the importance of these supramolecular interactions not only for stabilization but also for the assembly of CI, which becomes especially relevant in pathological conditions.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Encefalomiopatias Mitocondriais/enzimologia , Animais , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/química , Encefalomiopatias Mitocondriais/genética , Ligação Proteica , Estabilidade Proteica
12.
Am J Hum Genet ; 88(4): 488-93, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21457908

RESUMO

The assembly of mitochondrial respiratory chain complex IV (cytochrome c oxidase) involves the coordinated action of several assembly chaperones. In Saccharomyces cerevisiae, at least 30 different assembly chaperones have been identified. To date, pathogenic mutations leading to a mitochondrial disorder have been identified in only seven of the corresponding human genes. One of the genes for which the relevance to human pathology is unknown is C2orf64, an ortholog of the S. cerevisiae gene PET191. This gene has previously been shown to be a complex IV assembly factor in yeast, although its exact role is still unknown. Previous research in a large cohort of complex IV deficient patients did not support an etiological role of C2orf64 in complex IV deficiency. In this report, a homozygous mutation in C2orf64 is described in two siblings affected by fatal neonatal cardiomyopathy. Pathogenicity of the mutation is supported by the results of a complementation experiment, showing that complex IV activity can be fully restored by retroviral transduction of wild-type C2orf64 in patient-derived fibroblasts. Detailed analysis of complex IV assembly intermediates in patient fibroblasts by 2D-BN PAGE revealed the accumulation of a small assembly intermediate containing subunit COX1 but not the COX2, COX4, or COX5b subunits, indicating that C2orf64 is involved in an early step of the complex IV assembly process. The results of this study demonstrate that C2orf64 is essential for human complex IV assembly and that C2orf64 mutational analysis should be considered for complex IV deficient patients, in particular those with hypertrophic cardiomyopathy.


Assuntos
Cardiomiopatias/enzimologia , Cardiomiopatias/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Consanguinidade , Análise Mutacional de DNA , Eletroforese em Gel Bidimensional , Evolução Fatal , Feminino , Fibroblastos/enzimologia , Teste de Complementação Genética , Homozigoto , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta , Linhagem , Multimerização Proteica , Homologia de Sequência de Aminoácidos
13.
Nucleic Acids Res ; 40(9): 4040-51, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22238375

RESUMO

In a comparative genomics study for mitochondrial ribosome-associated proteins, we identified C7orf30, the human homolog of the plant protein iojap. Gene order conservation among bacteria and the observation that iojap orthologs cannot be transferred between bacterial species predict this protein to be associated with the mitochondrial ribosome. Here, we show colocalization of C7orf30 with the large subunit of the mitochondrial ribosome using isokinetic sucrose gradient and 2D Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis. We co-purified C7orf30 with proteins of the large subunit, and not with proteins of the small subunit, supporting interaction that is specific to the large mitoribosomal complex. Consistent with this physical association, a mitochondrial translation assay reveals negative effects of C7orf30 siRNA knock-down on mitochondrial gene expression. Based on our data we propose that C7orf30 is involved in ribosomal large subunit function. Sequencing the gene in 35 patients with impaired mitochondrial translation did not reveal disease-causing mutations in C7orf30.


Assuntos
Proteínas Mitocondriais/fisiologia , Biossíntese de Proteínas , Proteínas Ribossômicas/fisiologia , Subunidades Ribossômicas Maiores de Eucariotos/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Genes Bacterianos , Células HEK293 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Óperon , Filogenia , Estrutura Terciária de Proteína , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Análise de Sequência de DNA
14.
Biochim Biophys Acta ; 1822(2): 168-75, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22036843

RESUMO

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.


Assuntos
Complexo I de Transporte de Elétrons/genética , Doença de Leigh/genética , Mitocôndrias/enzimologia , Mutação , NADH Desidrogenase/genética , Sequência de Aminoácidos , Animais , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Catálise , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Lactente , Recém-Nascido , Doença de Leigh/enzimologia , Doença de Leigh/metabolismo , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , NADH Desidrogenase/metabolismo , Conformação Proteica , Transdução Genética/métodos , Ubiquinona/metabolismo
15.
Am J Hum Genet ; 84(6): 718-27, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19463981

RESUMO

Mitochondrial complex I deficiency is the most prevalent and least understood disorder of the oxidative phosphorylation system. The genetic cause of many cases of isolated complex I deficiency is unknown because of insufficient understanding of the complex I assembly process and the factors involved. We performed homozygosity mapping and gene sequencing to identify the genetic defect in five complex I-deficient patients from three different families. All patients harbored mutations in the NDUFAF3 (C3ORF60) gene, of which the pathogenic nature was assessed by NDUFAF3-GFP baculovirus complementation in fibroblasts. We found that NDUFAF3 is a genuine mitochondrial complex I assembly protein that interacts with complex I subunits. Furthermore, we show that NDUFAF3 tightly interacts with NDUFAF4 (C6ORF66), a protein previously implicated in complex I deficiency. Additional gene conservation analysis links NDUFAF3 to bacterial-membrane-insertion gene cluster SecF/SecD/YajC and to C8ORF38, also implicated in complex I deficiency. These data not only show that NDUFAF3 mutations cause complex I deficiency but also relate different complex I disease genes by the close cooperation of their encoded proteins during the assembly process.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Complexo I de Transporte de Elétrons/genética , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Consanguinidade , Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida , Evolução Fatal , Feminino , Teste de Complementação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Linhagem , Homologia de Sequência de Aminoácidos
16.
J Med Genet ; 48(11): 737-40, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21617257

RESUMO

BACKGROUND: This study investigated a girl with Leigh syndrome born to first-cousin parents of Pakistani descent with an isolated respiratory chain complex I deficiency in muscle and fibroblasts. Her early development was delayed, and from age 2 years she started losing motor abilities. Cerebral MRI showed basal ganglia lesions typical of Leigh syndrome. METHODS AND RESULTS: A genome-wide search for homozygosity was performed with the Affymetrix GeneChip 50K Xba array. The analysis revealed several homozygous regions. Three candidate genes were identified, and in one of the genes, NDUFA12, a homozygous c.178C→T mutation leading to a premature stop codon (p.Arg60X) was found. Western blot analysis showed absence of NDUFA12 protein in patient fibroblasts and functional complementation by a baculovirus system showed restoration of complex I activity. CONCLUSION: NDUFA12 mutations are apparently not a frequent cause of complex I deficiency, since mutations were not found by screening altogether 122 complex I deficient patients in two different studies. NDUFA12 encodes an accessory subunit of complex I and is a paralogue of NDUFAF2. Despite the complete absence of NDUFA12 protein, a fully assembled and enzymatically active complex I could be found, albeit in reduced amounts. This suggests that NDUFA12 is required either at a late step in the assembly of complex I, or in the stability of complex I.


Assuntos
Códon sem Sentido , Complexo I de Transporte de Elétrons/deficiência , Fibroblastos/enzimologia , Doença de Leigh/genética , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Músculos/enzimologia , Western Blotting , Criança , Consanguinidade , Análise Mutacional de DNA , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Feminino , Fibroblastos/patologia , Teste de Complementação Genética , Estudo de Associação Genômica Ampla , Homozigoto , Humanos , Doença de Leigh/diagnóstico , Doença de Leigh/enzimologia , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , Músculos/patologia , Análise de Sequência com Séries de Oligonucleotídeos
17.
Anal Biochem ; 407(2): 287-9, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20705045

RESUMO

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an essential tool for investigating mitochondrial respiratory chain complexes. However, with current BN-PAGE protocols for Caenorhabditis elegans (C. elegans), large worm amounts and high quantities of mitochondrial protein are required to yield clear results. Here, we present an efficient approach to isolate mitochondrial complex I (NADH:ubiquinone oxidoreductase) from C. elegans, grown on agar plates. We demonstrate that considerably lower amounts of mitochondrial protein are sufficient to isolate complex I and to display clear in-gel activity results. Moreover, we present the first complex I assembly profile for C. elegans, obtained by two-dimensional BN/SDS-PAGE.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Mitocôndrias/metabolismo , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/isolamento & purificação , Complexo I de Transporte de Elétrons/isolamento & purificação , NAD/química , Oxirredutases/química
18.
Biochim Biophys Acta Bioenerg ; 1861(8): 148202, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275929

RESUMO

Protein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexome profiling with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Biotinilação , Evolução Molecular , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , Ligação Proteica
19.
Biochim Biophys Acta Bioenerg ; 1861(8): 148213, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32335026

RESUMO

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4-/- mouse tissues. Ndufs4-/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4-/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4-/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4-/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Deleção de Genes , Doença de Leigh/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , NADPH Desidrogenase/metabolismo , Animais , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Doença de Leigh/metabolismo , Camundongos , Fosforilação Oxidativa , Estabilidade Proteica
20.
Eur J Hum Genet ; 25(11): 1273-1277, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28853723

RESUMO

Mitochondrial respiratory chain complex I consists of 44 different subunits and can be subgrouped into three functional modules: the Q-, the P- and the N-module. NDUFAF4 (C6ORF66) is an assembly factor of complex I that associates with assembly intermediates of the Q-module. Via exome sequencing, we identified a homozygous missense variant in a complex I-deficient patient with Leigh syndrome. Supercomplex analysis in patient fibroblasts revealed specifically altered stoichiometry. Detailed assembly analysis of complex I, indicative of all of its assembly routes, showed an accumulation of parts of the P- and the N-module but not the Q-module. Lentiviral complementation of patient fibroblasts with wild-type NDUFAF4 rescued complex I deficiency and the assembly defect, confirming the causal role of the variant. Our report on the second family affected by an NDUFAF4 variant further characterizes the phenotypic spectrum and sheds light into the role of NDUFAF4 in mitochondrial complex I biogenesis.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Doença de Leigh/genética , Mutação de Sentido Incorreto , Proteínas de Ligação a Calmodulina/metabolismo , Células Cultivadas , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Homozigoto , Humanos , Lactente , Doença de Leigh/patologia , Masculino , Multimerização Proteica
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