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1.
Cell ; 181(1): 168-188, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32220313

RESUMO

Mitochondrial diseases are clinically heterogeneous disorders caused by a wide spectrum of mutations in genes encoded by either the nuclear or the mitochondrial genome. Treatments for mitochondrial diseases are currently focused on symptomatic management rather than improving the biochemical defect caused by a particular mutation. This review focuses on the latest advances in the development of treatments for mitochondrial disease, both small molecules and gene therapies, as well as methods to prevent transmission of mitochondrial disease through the germline.


Assuntos
Mitocôndrias/genética , Doenças Mitocondriais/terapia , Animais , DNA Mitocondrial/genética , Terapia Genética , Genoma Mitocondrial/genética , Humanos , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Mutação , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo
2.
Cell ; 142(3): 456-67, 2010 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-20691904

RESUMO

RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3' --> 5' exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE-imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria.


Assuntos
Mitocôndrias/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Polirribonucleotídeo Nucleotidiltransferase/genética , Processamento Pós-Transcricional do RNA , Ribonuclease P/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
RNA Biol ; 21(1): 28-39, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39385590

RESUMO

The vast majority of oxygen-utilizing eukaryotes need to express their own mitochondrial genome, mtDNA, to survive. In comparison to size of their nuclear genome, mtDNA is minimal, even in the most exceptional examples. Having evolved from bacteria in an endosymbiotic event, it might be expected that the process of mtDNA expression would be relatively simple. The aim of this short review is to illustrate just how wrong this assumption is. The production of functional mitochondrial RNA across species evolved in many directions. Organelles use a dizzying array of RNA processing, modifying, editing, splicing and maturation events that largely require the import of nuclear-encoded proteins from the cytosol. These processes are sometimes driven by the unusual behaviour of the mitochondrial genome from which the RNA is originally transcribed, but in many examples the complex processes that are essential for the production of functional RNA in the organelle, are fascinating and bewildering.


Assuntos
RNA Mitocondrial , RNA , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , RNA/metabolismo , RNA/genética , Humanos , Animais , Mitocôndrias/genética , Mitocôndrias/metabolismo , Processamento Pós-Transcricional do RNA , Edição de RNA , Splicing de RNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Genoma Mitocondrial
4.
Mol Cell ; 62(1): 5-6, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058784

RESUMO

A paper from Jain et al. (2016) using whole-genome CRISPR knockout libraries in human cells and models of mitochondrial disease suggests chronic hypoxia could be an unexpected treatment for disorders of mitochondrial respiration.


Assuntos
Doença de Leigh/genética , Doença de Leigh/terapia , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Humanos
5.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526660

RESUMO

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multisubunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially encoded components are also integral members of these complexes, where does protein synthesis occur? As transcription, mRNA processing, maturation, and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites? We have adapted a click chemistry-based method coupled with stimulated emission depletion nanoscopy to address these questions. We report that, in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis is detected at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.


Assuntos
Compartimento Celular , Imageamento Tridimensional , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas , Alcinos , Células Cultivadas , DNA Mitocondrial/genética , Glicina/análogos & derivados , Humanos , Cinética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , RNA Mitocondrial/metabolismo , Transdução de Sinais
6.
J Cell Sci ; 133(1)2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896602

RESUMO

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use. The classic method for rescuing bacterial ribosomes is trans-translation. The key components of this system are absent from mammalian mitochondria; however, four members of a translation termination factor family are present, with some evidence of homology to members of a bacterial back-up rescue system. To date, there is no definitive demonstration of any other member of this family functioning in mitoribosome rescue. Here, we provide an overview of the processes and key players of canonical translation termination in both bacteria and mammalian mitochondria, followed by a perspective of the bacterial systems used to rescue stalled ribosomes. We highlight any similarities or differences with the mitochondrial translation release factors, and suggest potential roles for these proteins in ribosome rescue in mammalian mitochondria.


Assuntos
Bactérias/metabolismo , Ribossomos Mitocondriais/metabolismo , Animais
7.
EMBO Rep ; 21(9): e50964, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32852136

RESUMO

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?


Assuntos
Mitocôndrias , Doenças Mitocondriais , Humanos , Mitocôndrias/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/terapia
8.
Hum Mol Genet ; 27(10): 1743-1753, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29518248

RESUMO

LonP1 is a mitochondrial matrix protease whose selective substrate specificity is essential for maintaining mitochondrial homeostasis. Recessively inherited, pathogenic defects in LonP1 have been previously reported to underlie cerebral, ocular, dental, auricular and skeletal anomalies (CODAS) syndrome, a complex multisystemic and developmental disorder. Intriguingly, although classical mitochondrial disease presentations are well-known to exhibit marked clinical heterogeneity, the skeletal and dental features associated with CODAS syndrome are pathognomonic. We have applied whole exome sequencing to a patient with congenital lactic acidosis, muscle weakness, profound deficiencies in mitochondrial oxidative phosphorylation associated with loss of mtDNA copy number and MRI abnormalities consistent with Leigh syndrome, identifying biallelic variants in the LONP1 (NM_004793.3) gene; c.1693T > C predicting p.(Tyr565His) and c.2197G > A predicting p.(Glu733Lys); no evidence of the classical skeletal or dental defects observed in CODAS syndrome patients were noted in our patient. In vitro experiments confirmed the p.(Tyr565His) LonP1 mutant alone could not bind or degrade a substrate, consistent with the predicted function of Tyr565, whilst a second missense [p.(Glu733Lys)] variant had minimal effect. Mixtures of p.(Tyr565His) mutant and wild-type LonP1 retained partial protease activity but this was severely depleted when the p.(Tyr565His) mutant was mixed with the p.(Glu733Lys) mutant, data consistent with the compound heterozygosity detected in our patient. In summary, we conclude that pathogenic LONP1 variants can lead to a classical mitochondrial disease presentations associated with severe biochemical defects in oxidative phosphorylation in clinically relevant tissues.


Assuntos
Proteases Dependentes de ATP/genética , Anormalidades Craniofaciais/genética , Anormalidades do Olho/genética , Transtornos do Crescimento/genética , Luxação Congênita de Quadril/genética , Doença de Leigh/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Osteocondrodisplasias/genética , Anormalidades Dentárias/genética , Biópsia , Linhagem Celular , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/fisiopatologia , Exoma/genética , Anormalidades do Olho/metabolismo , Anormalidades do Olho/fisiopatologia , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/fisiopatologia , Luxação Congênita de Quadril/metabolismo , Luxação Congênita de Quadril/fisiopatologia , Humanos , Lactente , Doença de Leigh/metabolismo , Doença de Leigh/fisiopatologia , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/fisiopatologia , Músculo Esquelético/fisiopatologia , Mutação , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/fisiopatologia , Fosforilação Oxidativa , Anormalidades Dentárias/metabolismo , Anormalidades Dentárias/fisiopatologia , Sequenciamento do Exoma
9.
Am J Hum Genet ; 101(4): 525-538, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28942965

RESUMO

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp-/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp-/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.


Assuntos
Cardiomiopatias/genética , Proteínas de Transporte/genética , Transporte de Elétrons/fisiologia , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação , Adulto , Idade de Início , Idoso , Alelos , Sequência de Aminoácidos , Animais , Cardiomiopatias/complicações , Cardiomiopatias/patologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Células Cultivadas , Pré-Escolar , Estudos de Coortes , DNA Mitocondrial , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Recém-Nascido , Masculino , Camundongos , Pessoa de Meia-Idade , Doenças Mitocondriais/complicações , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Linhagem , Conformação Proteica , Homologia de Sequência , Índice de Gravidade de Doença , Adulto Jovem
10.
Biochem Soc Trans ; 47(5): 1429-1436, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31551356

RESUMO

Mitochondria are ubiquitous organelles present in the cytoplasm of all nucleated eukaryotic cells. These organelles are described as arising from a common ancestor but a comparison of numerous aspects of mitochondria between different organisms provides remarkable examples of divergent evolution. In humans, these organelles are of dual genetic origin, comprising ∼1500 nuclear-encoded proteins and thirteen that are encoded by the mitochondrial genome. Of the various functions that these organelles perform, it is only oxidative phosphorylation, which provides ATP as a source of chemical energy, that is dependent on synthesis of these thirteen mitochondrially encoded proteins. A prerequisite for this process of translation are the mitoribosomes. The recent revolution in cryo-electron microscopy has generated high-resolution mitoribosome structures and has undoubtedly revealed some of the most distinctive molecular aspects of the mitoribosomes from different organisms. However, we still lack a complete understanding of the mechanistic aspects of this process and many of the factors involved in post-transcriptional gene expression in mitochondria. This review reflects on the current knowledge and illustrates some of the striking differences that have been identified between mitochondria from a range of organisms.


Assuntos
Evolução Biológica , Mitocôndrias/metabolismo , Animais , Humanos , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo
11.
Proc Natl Acad Sci U S A ; 113(43): 12198-12201, 2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27729525

RESUMO

The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNAVal compared with the porcine use of mt-tRNAPhe We have explored this observation further. Our studies examine whether a range of mt-tRNAs are used by different mammals, or whether the mt-tRNA selection is strictly limited to only these two species of the 22 tRNAs encoded by the mitochondrial genome (mtDNA); whether there is tissue-specific variation within a single organism; and what happens to the human mitoribosome when levels of the mt-tRNAVal are depleted. Our data demonstrate that only mt-tRNAVal or mt-tRNAPhe are found in the mitoribosomes of five different mammals, each mammal favors the same mt-tRNA in all tissue types, and strikingly, when steady-state levels of mt-tRNAVal are reduced, human mitoribosome biogenesis displays an adaptive response by switching to the incorporation of mt-tRNAPhe to generate translationally competent machinery.


Assuntos
Ribossomos Mitocondriais/química , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , RNA de Transferência/ultraestrutura , Animais , Microscopia Crioeletrônica , DNA Mitocondrial/química , DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Humanos , Ribossomos Mitocondriais/ultraestrutura , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico 5S/genética , RNA de Transferência/genética , Suínos
12.
J Biol Chem ; 292(11): 4519-4532, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28082677

RESUMO

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.


Assuntos
Transferases Intramoleculares/metabolismo , RNA/metabolismo , Linhagem Celular , Humanos , Transferases Intramoleculares/análise , Transferases Intramoleculares/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Interferência de RNA , RNA Mitocondrial , RNA Ribossômico 16S/metabolismo , RNA Interferente Pequeno/genética , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Fenilalanina/metabolismo
13.
Biochem J ; 474(13): 2145-2158, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28512204

RESUMO

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.


Assuntos
Proteínas de Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA Ribossômico/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Homologia de Sequência de Aminoácidos
14.
Nucleic Acids Res ; 44(14): 6868-82, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27353330

RESUMO

LRPPRC is a protein that has attracted interest both for its role in post-transcriptional regulation of mitochondrial gene expression and more recently because numerous mutated variants have been characterized as causing severe infantile mitochondrial neurodegeneration. LRPPRC belongs to the pentatricopeptide repeat (PPR) protein family, originally defined by their RNA binding capacity, and forms a complex with SLIRP that harbours an RNA recognition motif (RRM) domain. We show here that LRPPRC displays a broad and strong RNA binding capacity in vitro in contrast to SLIRP that associates only weakly with RNA. The LRPPRC-SLIRP complex comprises a hetero-dimer via interactions by polar amino acids in the single RRM domain of SLIRP and three neighbouring PPR motifs in the second quarter of LRPPRC, which critically contribute to the LRPPRC-SLIRP binding interface to enhance its stability. Unexpectedly, specific amino acids at this interface are located within the PPRs of LRPPRC at positions predicted to interact with RNA and within the RNP1 motif of SLIRP's RRM domain. Our findings thus unexpectedly establish that despite the prediction that these residues in LRPPRC and SLIRP should bind RNA, they are instead used to facilitate protein-protein interactions, enabling the formation of a stable complex between these two proteins.


Assuntos
Proteínas de Neoplasias/metabolismo , Motivo de Reconhecimento de RNA , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/genética , Sequência Conservada , Reagentes de Ligações Cruzadas/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Mutação/genética , Proteínas de Neoplasias/química , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/química
15.
Cell Tissue Res ; 367(1): 5-20, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27411691

RESUMO

Oxidative phosphorylation (OXPHOS) is the mechanism whereby ATP, the major energy source for the cell, is produced by harnessing cellular respiration in the mitochondrion. This is facilitated by five multi-subunit complexes housed within the inner mitochondrial membrane. These complexes, with the exception of complex II, are of a dual genetic origin, requiring expression from nuclear and mitochondrial genes. Mitochondrially encoded mRNA is translated on the mitochondrial ribosome (mitoribosome) and the recent release of the near atomic resolution structure of the mammalian mitoribosome has highlighted its peculiar features. However, whereas some aspects of mitochondrial translation are understood, much is to be learnt about the presentation of mitochondrial mRNA to the mitoribosome, the biogenesis of the machinery, the exact role of the membrane, the constitution of the translocon/insertion machinery and the regulation of translation in the mitochondrion. This review addresses our current knowledge of mammalian mitochondrial gene expression, highlights key questions and indicates how defects in this process can result in profound mitochondrial disease.


Assuntos
Mamíferos/metabolismo , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas , Animais , Humanos , Ribossomos Mitocondriais/metabolismo , Modelos Biológicos
16.
J Inherit Metab Dis ; 40(1): 121-130, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27696117

RESUMO

Mitochondrial diseases collectively represent one of the most heterogeneous group of metabolic disorders. Symptoms can manifest at any age, presenting with isolated or multiple-organ involvement. Advances in next-generation sequencing strategies have greatly enhanced the diagnosis of patients with mitochondrial disease, particularly where a mitochondrial aetiology is strongly suspected yet OXPHOS activities in biopsied tissue samples appear normal. We used whole exome sequencing (WES) to identify the molecular basis of an early-onset mitochondrial syndrome-pathogenic biallelic variants in the HTRA2 gene, encoding a mitochondria-localised serine protease-in five subjects from two unrelated families characterised by seizures, neutropenia, hypotonia and cardio-respiratory problems. A unifying feature in all affected children was 3-methylglutaconic aciduria (3-MGA-uria), a common biochemical marker observed in some patients with mitochondrial dysfunction. Although functional studies of HTRA2 subjects' fibroblasts and skeletal muscle homogenates showed severely decreased levels of mutant HTRA2 protein, the structural subunits and complexes of the mitochondrial respiratory chain appeared normal. We did detect a profound defect in OPA1 processing in HTRA2-deficient fibroblasts, suggesting a role for HTRA2 in the regulation of mitochondrial dynamics and OPA1 proteolysis. In addition, investigated subject fibroblasts were more susceptible to apoptotic insults. Our data support recent studies that described important functions for HTRA2 in programmed cell death and confirm that patients with genetically-unresolved 3-MGA-uria should be screened by WES with pathogenic variants in the HTRA2 gene prioritised for further analysis.


Assuntos
Variação Genética/genética , Serina Peptidase 2 de Requerimento de Alta Temperatura A/genética , Erros Inatos do Metabolismo/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Morte Celular/genética , Células Cultivadas , Criança , Exoma/genética , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/genética , Músculo Esquelético/metabolismo , Serina Proteases/genética , Síndrome
17.
Hum Mol Genet ; 23(4): 949-67, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24092330

RESUMO

Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named heterologous inferential analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA, we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritization of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool.


Assuntos
RNA Ribossômico/genética , RNA/genética , Simulação por Computador , Sequência Conservada , Análise Mutacional de DNA , Estudos de Associação Genética , Humanos , Modelos Moleculares , Mutação , Neoplasias/genética , Conformação de Ácido Nucleico , RNA/química , RNA Mitocondrial , RNA Ribossômico/química
18.
Hum Mol Genet ; 23(23): 6345-55, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25008111

RESUMO

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.


Assuntos
Proteínas Mitocondriais/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Proteínas Mitocondriais/genética , Mutação , Proteínas de Neoplasias/metabolismo , Fosforilação Oxidativa , Polinucleotídeo Adenililtransferase/genética , Cultura Primária de Células , Processamento Pós-Transcricional do RNA , RNA Mitocondrial , Proteínas de Ligação a RNA/metabolismo
19.
Chembiochem ; 17(14): 1312-6, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27124570

RESUMO

Mitochondria are central to health and disease, hence there is considerable interest in developing mitochondria-targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondria- targeted cyclooctyne (MitoOct) that accumulates several- hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential-dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion-targeted molecule reaches the matrix. This "ClickIn" approach will facilitate development of mitochondria-targeted therapies.


Assuntos
Química Click/métodos , Sistemas de Liberação de Medicamentos/métodos , Mitocôndrias/metabolismo , Azidas/análise , Azidas/química , Azidas/farmacocinética , Ciclo-Octanos/química , Ciclo-Octanos/farmacocinética , Portadores de Fármacos/química , Humanos , Espectrometria de Massas , Membranas Mitocondriais/metabolismo , Terapia de Alvo Molecular/métodos
20.
Brain ; 138(Pt 12): 3503-19, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26510951

RESUMO

Mitochondrial Complex IV [cytochrome c oxidase (COX)] deficiency is one of the most common respiratory chain defects in humans. The clinical phenotypes associated with COX deficiency include liver disease, cardiomyopathy and Leigh syndrome, a neurodegenerative disorder characterized by bilateral high signal lesions in the brainstem and basal ganglia. COX deficiency can result from mutations affecting many different mitochondrial proteins. The French-Canadian variant of COX-deficient Leigh syndrome is unique to the Saguenay-Lac-Saint-Jean region of Québec and is caused by a founder mutation in the LRPPRC gene. This encodes the leucine-rich pentatricopeptide repeat domain protein (LRPPRC), which is involved in post-transcriptional regulation of mitochondrial gene expression. Here, we present the clinical and molecular characterization of novel, recessive LRPPRC gene mutations, identified using whole exome and candidate gene sequencing. The 10 patients come from seven unrelated families of UK-Caucasian, UK-Pakistani, UK-Indian, Turkish and Iraqi origin. They resemble the French-Canadian Leigh syndrome patients in having intermittent severe lactic acidosis and early-onset neurodevelopmental problems with episodes of deterioration. In addition, many of our patients have had neonatal cardiomyopathy or congenital malformations, most commonly affecting the heart and the brain. All patients who were tested had isolated COX deficiency in skeletal muscle. Functional characterization of patients' fibroblasts and skeletal muscle homogenates showed decreased levels of mutant LRPPRC protein and impaired Complex IV enzyme activity, associated with abnormal COX assembly and reduced steady-state levels of numerous oxidative phosphorylation subunits. We also identified a Complex I assembly defect in skeletal muscle, indicating different roles for LRPPRC in post-transcriptional regulation of mitochondrial mRNAs between tissues. Patient fibroblasts showed decreased steady-state levels of mitochondrial mRNAs, although the length of poly(A) tails of mitochondrial transcripts were unaffected. Our study identifies LRPPRC as an important disease-causing gene in an early-onset, multisystem and neurological mitochondrial disease, which should be considered as a cause of COX deficiency even in patients originating outside of the French-Canadian population.


Assuntos
Deficiência de Citocromo-c Oxidase/genética , Doenças Mitocondriais/genética , Proteínas de Neoplasias/genética , Proteínas/genética , Canadá , Células Cultivadas , Pré-Escolar , Deficiência de Citocromo-c Oxidase/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Proteínas de Repetições Ricas em Leucina , Masculino , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Mutação , Linhagem , Proteínas/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial
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