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1.
FEBS Lett ; 594(4): 590-610, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31944285

RESUMO

Mitochondria provide the main source of energy for eukaryotic cells, oxidizing fatty acids and sugars to generate ATP. Mitochondrial fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are two key pathways involved in this process. Disruption of FAO can cause human disease, with patients commonly presenting with liver failure, hypoketotic glycaemia and rhabdomyolysis. However, patients with deficiencies in the FAO enzyme short-chain enoyl-CoA hydratase 1 (ECHS1) are typically diagnosed with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy that is normally associated with OXPHOS dysfunction. Furthermore, some ECHS1-deficient patients also exhibit secondary OXPHOS defects. This sequela of FAO disorders has long been thought to be caused by the accumulation of inhibitory fatty acid intermediates. However, new evidence suggests that the mechanisms involved are more complex, and that disruption of OXPHOS protein complex biogenesis and/or stability is also involved. In this review, we examine the clinical, biochemical and genetic features of all ECHS1-deficient patients described to date. In particular, we consider the secondary OXPHOS defects associated with ECHS1 deficiency and discuss their possible contribution to disease pathogenesis.


Assuntos
Enoil-CoA Hidratase/deficiência , Fosforilação Oxidativa , Animais , Ácidos Graxos/metabolismo , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia
2.
J Neurodev Disord ; 11(1): 29, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31839000

RESUMO

BACKGROUND: The mitochondrial aminoacyl-tRNA synthetase proteins (mt-aaRSs) are a group of nuclear-encoded enzymes that facilitate conjugation of each of the 20 amino acids to its cognate tRNA molecule. Mitochondrial diseases are a large, clinically heterogeneous group of disorders with diverse etiologies, ages of onset, and involved organ systems. Diseases related to mt-aaRS mutations are associated with specific syndromes that affect the central nervous system and produce highly characteristic MRI patterns, prototypically the DARS2, EARS, and AARS2 leukodystrophies, which are caused by mutations in mitochondrial aspartyl-tRNA synthetase, mitochondria glutamate tRNA synthetase, and mitochondrial alanyl-tRNA synthetase, respectively. BODY: The disease patterns emerging for these leukodystrophies are distinct in terms of the age of onset, nature of disease progression, and predominance of involved white matter tracts. In DARS2 and EARS2 disorders, earlier disease onset is typically correlated with more significant brain abnormalities, rapid neurological decline, and greater disability. In AARS2 leukodystrophy cases reported thus far, there is nearly invariable progression to severe disability and atrophy of involved brain regions, often within a decade. Although most mutations are compound heterozygous inherited in an autosomal recessive fashion, homozygous variants are found in each disorder and demonstrate high phenotypic variability. Affected siblings manifest disease on a wide spectrum. CONCLUSION: The syndromic nature and selective vulnerability of white matter tracts in these disorders suggests there may be a shared mechanism of mitochondrial dysfunction to target for study. There is evidence that the clinical variability and white matter tract specificity of each mt-aaRS leukodystrophy depend on both canonical and non-canonical effects of the mutations on the process of mitochondrial translation. Furthermore, different sensitivities to the mt-aaRS mutations have been observed based on cell type. Most mutations result in at least partial retention of mt-aaRS enzyme function with varied effects on the mitochondrial respiratory chain complexes. In EARS2 and AARS2 cells, this appears to result in cumulative impairment of respiration. Mt-aaRS mutations may also affect alternative biochemical pathways such as the integrated stress response, a homeostatic program in eukaryotic cells that typically confers cytoprotection, but can lead to cell death when abnormally activated in response to pathologic states. Systematic review of this group of disorders and further exploration of disease mechanisms in disease models and neural cells are warranted.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Encéfalo/enzimologia , Doenças Desmielinizantes/enzimologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/enzimologia , Transtornos do Neurodesenvolvimento/enzimologia , Animais , Encéfalo/patologia , Doenças Desmielinizantes/complicações , Humanos , Doenças Mitocondriais/complicações , Proteínas Mitocondriais/metabolismo
3.
Clin Chim Acta ; 496: 93-99, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31271740

RESUMO

BACKGROUND: Mitochondrial DNA depletion syndrome is a group of heterogeneous diseases with non-specific presentation. The common feature is the quantitative depletion of mitochondrial DNA without qualitative defects. Diagnosis of these diseases poses a challenge and whole exome sequencing is often needed for their diagnoses. CASE: Two siblings of a quartet family, presenting with hypotonia, microcephaly and severe intellectual disability, have been diagnosed to harbor two heterozygous variants in trans in the DTYMK gene of the thymidine biosynthesis pathway. Mitochondrial DNA depletion has been demonstrated in silico in the more severe sibling. CONCLUSIONS: We suggest the consideration of incorporating DTYMK as one of the associated genes of mitochondrial DNA depletion syndrome (MDDS). DTYMK may be the missing link in the mitochondrial nucleotide salvage pathway but further characterization and additional evidence would be needed.


Assuntos
DNA Mitocondrial/metabolismo , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Núcleosídeo-Fosfato Quinase/genética , Criança , DNA Mitocondrial/genética , Humanos , Lactente , Masculino , Irmãos , Sequenciamento Completo do Exoma
4.
Int J Biochem Cell Biol ; 110: 140-142, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30885675

RESUMO

Some mechanisms of cellular stress, aging, and apoptosis are related to proteolysis. With respect to ClpP, little is known about the mechanical manner in which the substrate is hydrolyzed in and released from the degradation chamber. Furthermore, what would be the real influence of ClpP in mammalian UPRmt?


Assuntos
Endopeptidase Clp/metabolismo , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/enzimologia , Terapia de Alvo Molecular/métodos , Proteólise/efeitos dos fármacos , Animais , Humanos , Doenças Mitocondriais/metabolismo
5.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858161

RESUMO

Exome sequencing of two sisters with congenital cataracts, short stature, and white matter changes identified compound heterozygous variants in the PISD gene, encoding the phosphatidylserine decarboxylase enzyme that converts phosphatidylserine to phosphatidylethanolamine (PE) in the inner mitochondrial membrane (IMM). Decreased conversion of phosphatidylserine to PE in patient fibroblasts is consistent with impaired phosphatidylserine decarboxylase (PISD) enzyme activity. Meanwhile, as evidence for mitochondrial dysfunction, patient fibroblasts exhibited more fragmented mitochondrial networks, enlarged lysosomes, decreased maximal oxygen consumption rates, and increased sensitivity to 2-deoxyglucose. Moreover, treatment with lyso-PE, which can replenish the mitochondrial pool of PE, and genetic complementation restored mitochondrial and lysosome morphology in patient fibroblasts. Functional characterization of the PISD variants demonstrates that the maternal variant causes an alternative splice product. Meanwhile, the paternal variant impairs autocatalytic self-processing of the PISD protein required for its activity. Finally, evidence for impaired activity of mitochondrial IMM proteases suggests an explanation as to why the phenotypes of these PISD patients resemble recently described "mitochondrial chaperonopathies." Collectively, these findings demonstrate that PISD is a novel mitochondrial disease gene.


Assuntos
Carboxiliases/genética , Catarata/genética , Doenças Mitocondriais/enzimologia , Anormalidades Musculoesqueléticas/genética , Substância Branca/patologia , Adulto , Carboxiliases/metabolismo , Feminino , Fibroblastos/metabolismo , Genes Mitocondriais/genética , Células HEK293 , Homeostase/genética , Humanos , Mitocôndrias/enzimologia , Doenças Mitocondriais/sangue , Doenças Mitocondriais/patologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Fenótipo , Sítios de Splice de RNA/genética , Saccharomyces cerevisiae/enzimologia , Transfecção , Sequenciamento Completo do Exoma
6.
J Hum Genet ; 64(2): 73-85, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30401918

RESUMO

Mitochondrial fatty acid oxidation disorders (FAODs) are caused by defects in ß-oxidation enzymes, including very long-chain acyl-CoA dehydrogenase (VLCAD), trifunctional protein (TFP), carnitine palmitoyltransferase-2 (CPT2), carnitine-acylcarnitine translocase (CACT) and others. During prolonged fasting, infection, or exercise, patients with FAODs present with hypoglycemia, rhabdomyolysis, cardiomyopathy, liver dysfunction, and occasionally sudden death. This article describes the diagnosis, newborn screening, and treatment of long-chain FAODs with a focus on VLCAD deficiency. VLCAD deficiency is generally classified into three phenotypes based on onset time, but the classification should be comprehensively determined based on genotype, residual enzyme activity, and clinical course, due to a lack of apparent genotype-phenotype correlation. With the expansion of newborn screening for FAODs, several issues have arisen, such as missed detection, overdiagnosis (including detection of benign/asymptomatic type), and poor prognosis of the neonatal-onset form. Meanwhile, dietary management and restriction of exercise have been unnecessary for patients with the benign/asymptomatic type of VLCAD deficiency with a high fatty acid oxidation flux score. Although L-carnitine therapy for VLCAD/TFP deficiency has been controversial, supplementation with L-carnitine may be accepted for CPT2/CACT and multiple acyl-CoA dehydrogenase deficiencies. Recently, a double-blind, randomized controlled trial of triheptanoin (seven-carbon fatty acid triglyceride) versus trioctanoin (regular medium-chain triglyceride) was conducted and demonstrated improvement of cardiac functions on triheptanoin. Additionally, although the clinical efficacy of bezafibrate remains controversial, a recent open-label clinical trial showed efficacy of this drug in improving quality of life. These drugs may be promising for the treatment of FAODs, though further studies are required.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Ácidos Graxos/metabolismo , Hipolipemiantes/uso terapêutico , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/tratamento farmacológico , Programas de Rastreamento , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/tratamento farmacológico , Doenças Musculares/diagnóstico , Doenças Musculares/tratamento farmacológico , Síndrome Congênita de Insuficiência da Medula Óssea , Gerenciamento Clínico , Humanos , Erros Inatos do Metabolismo Lipídico/enzimologia , Doenças Mitocondriais/enzimologia , Doenças Musculares/enzimologia
7.
Mitochondrion ; 47: 179-187, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30423451

RESUMO

Diseases related to DNA polymerase gamma dysfunction comprise of heterogeneous clinical presentations with variable severity and age of onset. Molecular screening for the common POLG variants: p.Ala467Thr, p.Trp748Ser, p.Gly848Ser, and p.Tre251Ile has been conducted in a large population cohort (n = 3123) and in a clinically heterogeneous group of 1289 patients. Recessive pathogenic variants, including six novel ones were revealed in 22/26 patients. Infantile Alpers-Huttenlocher syndrome and adulthood ataxia spectrum were the most common found in our group. Distinct molecular profile identified in the Polish patients with significant predominance of p.Trp748Ser variant (50% of mutant alleles) reflected strikingly low population frequency of the three remaining variants and slightly higher p.Trp748Ser allele frequency in the general Polish population as compared to the non-Finish European population.


Assuntos
Ataxia/genética , Polimerase do DNA Mitocondrial/genética , Esclerose Cerebral Difusa de Schilder/genética , Genes Recessivos , Doenças Mitocondriais/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Substituição de Aminoácidos , Ataxia/enzimologia , Criança , Pré-Escolar , Esclerose Cerebral Difusa de Schilder/enzimologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/enzimologia , Polônia
8.
Nat Commun ; 9(1): 4065, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30283131

RESUMO

Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.


Assuntos
Cardiomiopatias/enzimologia , Cardiomiopatias/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Mutação/genética , Transferases de Grupos Nitrogenados/genética , Subunidades Proteicas/genética , Sequência de Aminoácidos , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Recém-Nascido , Lentivirus/metabolismo , Masculino , Modelos Moleculares , Miocárdio/patologia , Miocárdio/ultraestrutura , Transferases de Grupos Nitrogenados/química , Transferases de Grupos Nitrogenados/metabolismo , Fosforilação Oxidativa , Linhagem , Biossíntese de Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo
9.
Mol Genet Metab ; 125(3): 281-291, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30177229

RESUMO

An increasing number of mitochondrial diseases are found to be caused by pathogenic variants in nuclear encoded mitochondrial aminoacyl-tRNA synthetases. FARS2 encodes mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) which transfers phenylalanine to its cognate tRNA in mitochondria. Since the first case was reported in 2012, a total of 21 subjects with FARS2 deficiency have been reported to date with a spectrum of disease severity that falls between two phenotypes; early onset epileptic encephalopathy and a less severe phenotype characterized by spastic paraplegia. In this report, we present an additional 15 individuals from 12 families who are mostly Arabs homozygous for the pathogenic variant Y144C, which is associated with the more severe early onset phenotype. The total number of unique pathogenic FARS2 variants known to date is 21 including three different partial gene deletions reported in four individuals. Except for the large deletions, all variants but two (one in-frame deletion of one amino acid and one splice-site variant) are missense. All large deletions and the single splice-site variant are in trans with a missense variant. This suggests that complete loss of function may be incompatible with life. In this report, we also review structural, functional, and evolutionary significance of select FARS2 pathogenic variants reported here.


Assuntos
Aminoacil-tRNA Sintetases/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Fenilalanina-tRNA Ligase/genética , Adolescente , Adulto , Aminoacil-tRNA Sintetases/deficiência , Criança , Pré-Escolar , Feminino , Deleção de Genes , Humanos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/deficiência , Mutação/genética , Paraplegia/genética , Paraplegia/patologia , Fenilalanina/genética , Fenilalanina/metabolismo , Fenilalanina-tRNA Ligase/química , Fenilalanina-tRNA Ligase/deficiência , Isoformas de Proteínas/genética , Relação Estrutura-Atividade , Adulto Jovem
10.
PLoS One ; 13(8): e0201811, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30096161

RESUMO

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.


Assuntos
Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Trifosfato de Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Atividade Motora/fisiologia , Mutação , Fenótipo , Reprodução/fisiologia
11.
Hum Mol Genet ; 27(21): 3710-3719, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30085106

RESUMO

Mitochondrial dynamics, including mitochondrial division, fusion and transport, are integral parts of mitochondrial and cellular function. DNM1L encodes dynamin-related protein 1 (Drp1), a member of the dynamin-related protein family that is required for mitochondrial division. Several de novo mutations in DNM1L are associated with a range of disease states. Here we report the identification of five patients with pathogenic or likely pathogenic variants of DNM1L, including two novel variants. Interestingly, all of the positions identified in these Drp1 variants are fully conserved among all members of the dynamin-related protein family that are involved in membrane division and organelle division events. This work builds upon and expands the clinical spectrum associated with Drp1 variants in patients and their impact on mitochondrial division in model cells.


Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas Associadas aos Microtúbulos/genética , Doenças Mitocondriais/enzimologia , Dinâmica Mitocondrial , Proteínas Mitocondriais/genética , Mutação , Linhagem Celular , Criança , Análise Mutacional de DNA , Dinaminas , Feminino , GTP Fosfo-Hidrolases/fisiologia , Humanos , Lactente , Masculino , Proteínas Associadas aos Microtúbulos/fisiologia , Doenças Mitocondriais/fisiopatologia , Proteínas Mitocondriais/fisiologia
12.
Nucleic Acids Res ; 46(16): 8630-8640, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30020492

RESUMO

Human polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3'-to-5' exoribonuclease principally located in mitochondria where it is responsible for RNA turnover and import. Mutations in PNPase impair structured RNA transport into mitochondria, resulting in mitochondrial dysfunction and disease. PNPase is a trimeric protein with a doughnut-shaped structure hosting a central channel for single-stranded RNA binding and degradation. Here, we show that the disease-linked human PNPase mutants, Q387R and E475G, form dimers, not trimers, and have significantly lower RNA binding and degradation activities compared to wild-type trimeric PNPase. Moreover, S1 domain-truncated PNPase binds single-stranded RNA but not the stem-loop signature motif of imported structured RNA, suggesting that the S1 domain is responsible for binding structured RNAs. We further determined the crystal structure of dimeric PNPase at a resolution of 2.8 Å and, combined with small-angle X-ray scattering, show that the RNA-binding K homology and S1 domains are relatively inaccessible in the dimeric assembly. Taken together, these results show that mutations at the interface of the trimeric PNPase tend to produce a dimeric protein with destructive RNA-binding surfaces, thus impairing both of its RNA import and degradation activities and leading to mitochondria disorders.


Assuntos
Mutação com Perda de Função , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Mutação de Sentido Incorreto , Mutação Puntual , Polirribonucleotídeo Nucleotidiltransferase/química , Estabilidade de RNA , RNA/metabolismo , Transporte Biológico , Cristalografia por Raios X , Dimerização , Humanos , Sequências Repetidas Invertidas , Doenças Mitocondriais/enzimologia , Modelos Moleculares , Polirribonucleotídeo Nucleotidiltransferase/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo
13.
Hum Mol Genet ; 27(21): 3697-3709, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29982452

RESUMO

Complex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in NUBPL (nucleotide binding protein-like), encoding an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica. Leu102Pro destabilized the Ind1 protein, leading to a null-mutant phenotype. Asp103Tyr, Leu191Phe and Gly285Cys affected complex I assembly to varying degrees, whereas Gly136Asp substitution in Ind1 did not impact on complex I levels nor dNADH:ubiquinone activity. Blue-native polyacrylamide gel electrophoresis and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q module intermediate of complex I. In the Ind1 Asp103Tyr variant, the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 Gly285Cys variant was able to support complex I assembly at 28°C, but not at 10°C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Doenças Mitocondriais/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Yarrowia/metabolismo , Sequência de Aminoácidos , Complexo I de Transporte de Elétrons/genética , Humanos , Microrganismos Geneticamente Modificados , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Temperatura , Yarrowia/genética
14.
Biochim Biophys Acta Bioenerg ; 1859(8): 602-611, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29778688

RESUMO

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded FO domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS175N) prevents FO-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN175D, aN175K, aN175I, and aN175T), which, in light of a recently published atomic structure of yeast FO indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN175 and a nearby glutamate residue (aE172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS175N mutation can be suppressed by second-site suppressors (aP12S, aI171F, aI171N, aI239F, and aI200M), of which some are very distantly located (by 20-30 Å) from the original mutation. The possibility to compensate through long-range effects the aS175N mutation is an interesting observation that holds promise for the development of therapeutic molecules.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Mitocôndrias/metabolismo , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Conformação Proteica , Subunidades Proteicas , Prótons , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência
15.
J Vet Med Sci ; 80(3): 480-484, 2018 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-29311426

RESUMO

An 8-year-old male Japanese Shiba exhibited muscle wasting and a stiff gait. A low-amplitude myotonic discharge was recorded by needle electromyography (EMG). A histopathological examination on a tru-cut biopsy sample from the muscle revealed myofiber size variations. Internal nuclei and cytoplasmic vacuoles were observed in many fibers. A type 1 fiber predominance and many hybrid type fibers were observed immunohistochemically. On the basis of these EMG and histopathological findings, myotonic dystrophy (DM) was suspected as tentative diagnosis. The cytoplasm around the vacuoles was immunopositive for cytochrome c, tom 20, and SOD-1, suggesting that these vacuoles might occur within mitochondria. Collectively, these results indicate that a mitochondrial abnormality partly play the role on the pathogenesis of present case.


Assuntos
Doenças do Cão/fisiopatologia , Distrofia Miotônica/veterinária , Animais , Biópsia por Agulha/veterinária , Citocromos c/metabolismo , Doenças do Cão/diagnóstico , Doenças do Cão/patologia , Cães , Eletromiografia/veterinária , Masculino , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Doenças Mitocondriais/fisiopatologia , Doenças Mitocondriais/veterinária , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Doenças Musculares/veterinária , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/patologia , Distrofia Miotônica/fisiopatologia , Superóxido Dismutase-1/metabolismo
16.
Sci Rep ; 8(1): 1165, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29348607

RESUMO

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.


Assuntos
Acil-CoA Desidrogenases/genética , Complexo I de Transporte de Elétrons/deficiência , Fibroblastos/enzimologia , Doenças Mitocondriais/genética , NADH Desidrogenase/genética , Espécies Reativas de Oxigênio/metabolismo , Acil-CoA Desidrogenases/deficiência , Trifosfato de Adenosina/agonistas , Trifosfato de Adenosina/biossíntese , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Depuradores de Radicais Livres/farmacologia , Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , NADH Desidrogenase/deficiência , Óxidos de Nitrogênio/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , Espécies Reativas de Oxigênio/antagonistas & inibidores
17.
Environ Sci Pollut Res Int ; 25(6): 5779-5787, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29235020

RESUMO

Mitochondria play an essential role to supply the cell with metabolic energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). As a consequence, they are also the primary source of cellular reactive oxygen species (ROS) which can cause oxidative damage of individual respiratory chain complexes. Indeed, affected OXPHOS subunits result in decreases in ATP production and increases in ROS formation which generate oxidative phosphorylation deficiency leading to mitochondrial dysfunctions. It has been suggested that ROS play a vital role in the pathogenesis of mitochondrial diseases. To the best of our knowledge, this is the first study which aimed to investigate the genetic variant effect of the antioxidant enzymes GSTM1 and GSTT1 on mitochondrial disease among a Tunisian population. In this report, 109 patients with mitochondrial disease and 154 healthy controls were genotyped by multiplex PCR amplification, and data were analyzed by SPSS v20 software. The results showed that GSTM1 null genotype was found to be associated with mitochondrial disease with a protective effect; however, no significant association of GSTT1 polymorphism with mitochondrial disease risk was revealed. But, interestingly, our findings highlight that GSTM1 active and GSTT1 null genotype combination increased by three fold the risk of developing mitochondrial disease with p c = 0.020, notably mitochondrial myopathy with p c = 0.046 and Leigh syndrome with p c = 0.042. In conclusion, this study suggests that GSTM1 active and GSTT1 null genotype combination might be a risk factor in developing mitochondrial disease.


Assuntos
Predisposição Genética para Doença , Glutationa Transferase/genética , Doenças Mitocondriais/genética , Polimorfismo Genético , Adulto , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/enzimologia , Reação em Cadeia da Polimerase Multiplex , Fatores de Risco , Tunísia
18.
J Cell Mol Med ; 22(3): 1601-1613, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29265583

RESUMO

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA.


Assuntos
Trifosfato de Adenosina/biossíntese , Fibroblastos/enzimologia , Produtos do Gene tat/genética , Metilmalonil-CoA Mutase/genética , Mitocôndrias/enzimologia , Proteínas Recombinantes de Fusão/genética , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Sistemas CRISPR-Cas , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/patologia , Expressão Gênica , Produtos do Gene tat/metabolismo , Terapia Genética/métodos , Células Hep G2 , Humanos , Fígado/enzimologia , Fígado/patologia , Potencial da Membrana Mitocondrial , Ácido Metilmalônico/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Doenças Mitocondriais/terapia , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Engenharia de Proteínas/métodos , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
19.
FEBS J ; 285(3): 481-500, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29265728

RESUMO

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic ßcyto - or γcyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific ßcyto - and γcyto -actin KO mice. We found ßcyto - and γcyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking ßcyto - and/or γcyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific ßcyto - and γcyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old ßcyto - and γcyto -actin muscle-specific KO mice. Our data suggest that impaired Ca2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.


Assuntos
Actinas/metabolismo , Citoplasma/metabolismo , Mitocôndrias Musculares/metabolismo , Dinâmica Mitocondrial , Relaxamento Muscular , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Actinas/genética , Animais , Células Cultivadas , Citoplasma/patologia , Citoplasma/ultraestrutura , Embrião de Mamíferos/citologia , Técnicas In Vitro , Masculino , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Mitocôndrias Hepáticas/ultraestrutura , Mitocôndrias Musculares/patologia , Mitocôndrias Musculares/ultraestrutura , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Doenças Musculares/enzimologia , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Consumo de Oxigênio , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Retículo Sarcoplasmático/patologia , Retículo Sarcoplasmático/ultraestrutura
20.
Proc Natl Acad Sci U S A ; 114(52): E11285-E11292, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29237750

RESUMO

A single-nucleotide polymorphism in the human arylamine N-acetyltransferase 2 (Nat2) gene has recently been identified as associated with insulin resistance in humans. To understand the cellular and molecular mechanisms by which alterations in Nat2 activity might cause insulin resistance, we examined murine ortholog Nat1 knockout (KO) mice. Nat1 KO mice manifested whole-body insulin resistance, which could be attributed to reduced muscle, liver, and adipose tissue insulin sensitivity. Hepatic and muscle insulin resistance were associated with marked increases in both liver and muscle triglyceride (TAG) and diacylglycerol (DAG) content, which was associated with increased PKCε activation in liver and increased PKCθ activation in skeletal muscle. Nat1 KO mice also displayed reduced whole-body energy expenditure and reduced mitochondrial oxygen consumption in white adipose tissue, brown adipose tissue, and hepatocytes. Taken together, these studies demonstrate that Nat1 deletion promotes reduced mitochondrial activity and is associated with ectopic lipid-induced insulin resistance. These results provide a potential genetic link among mitochondrial dysfunction with increased ectopic lipid deposition, insulin resistance, and type 2 diabetes.


Assuntos
Arilamina N-Acetiltransferase/deficiência , Diabetes Mellitus Tipo 2 , Metabolismo Energético , Resistência à Insulina , Isoenzimas/deficiência , Mitocôndrias/enzimologia , Doenças Mitocondriais , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/patologia , Animais , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Diglicerídeos/genética , Diglicerídeos/metabolismo , Fígado/enzimologia , Fígado/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Consumo de Oxigênio/genética , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo , Triglicerídeos/genética , Triglicerídeos/metabolismo
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