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1.
Trends Biochem Sci ; 43(11): 921-932, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30131192

RESUMO

Thousands of protein acyl modification sites have now been identified in vivo. However, at most sites the acylation stoichiometry is low, making functional enzyme-driven regulation in the majority of cases unlikely. As unmediated acylation can occur on the surface of proteins when acyl-CoA thioesters react with nucleophilic cysteine and lysine residues, slower nonenzymatic processes likely underlie most protein acylation. Here, we review how nonenzymatic acylation of nucleophilic lysine and cysteine residues occurs; the factors that enhance acylation at particular sites; and the strategies that have evolved to limit protein acylation. We conclude that protein acylation is an unavoidable consequence of the central role of reactive thioesters in metabolism. Finally, we propose a hypothesis for why low-stoichiometry protein acylation is selected against by evolution and how it might contribute to degenerative processes such as aging.


Assuntos
Acil Coenzima A/metabolismo , Cisteína/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Acil Coenzima A/química , Acilação , Animais , Cisteína/química , Humanos , Lisina/química , Proteínas/química
2.
Nature ; 532(7597): 112-6, 2016 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-27027295

RESUMO

Brown and beige adipose tissues can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1). Thermogenesis from these adipocytes can combat obesity and diabetes, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Here we show that acutely activated thermogenesis in brown adipose tissue is defined by a substantial increase in levels of mitochondrial reactive oxygen species (ROS). Remarkably, this process supports in vivo thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole-body energy expenditure. We further establish that thermogenic ROS alter the redox status of cysteine thiols in brown adipose tissue to drive increased respiration, and that Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine-nucleotide-inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify mitochondrial ROS induction in brown adipose tissue as a mechanism that supports UCP1-dependent thermogenesis and whole-body energy expenditure, which opens the way to improved therapeutic strategies for combating metabolic disorders.


Assuntos
Cisteína/química , Metabolismo Energético , Canais Iônicos/química , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Termogênese , Tecido Adiposo Marrom/química , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Respiração Celular , Cisteína/genética , Cisteína/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Canais Iônicos/deficiência , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução , Compostos de Sulfidrila/metabolismo , Termogênese/efeitos dos fármacos , Proteína Desacopladora 1
3.
Nucleic Acids Res ; 47(D1): D1225-D1228, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30398659

RESUMO

Increasing numbers of diseases are associated with mitochondrial dysfunction. This is unsurprising given mitochondria have major roles in bioenergy generation, signalling, detoxification, apoptosis and biosynthesis. However, fundamental questions of mitochondrial biology remain, including: which nuclear genes encode mitochondrial proteins; how their expression varies with tissue; and which are associated with disease. But experiments to catalogue the mitochondrial proteome are incomplete and sometimes contradictory. This arises because the mitochondrial proteome has tissue- and stage-specific variability, plus differences among experimental techniques and localization evidence types used. This leads to limitations in each technique's coverage and inevitably conflicting results. To support identification of mitochondrial proteins, we developed MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/), a database combining evidence of mitochondrial localization with information from public resources. Here we report upgrades to MitoMiner, including its re-engineering to be gene-centric to enable easier sharing of evidence among orthologues and support next generation sequencing, plus new data sources, including expression in different tissues, information on phenotypes and diseases of genetic mutations and a new mitochondrial proteome catalogue. MitoMiner is a powerful platform to investigate mitochondrial localization by providing a unique combination of experimental sub-cellular localization datasets, tissue expression, predictions of mitochondrial targeting sequences, gene annotation and links to phenotype and disease.


Assuntos
Biologia Computacional/métodos , Bases de Dados Factuais , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Gerenciamento de Dados/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Internet , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Fenótipo , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos
4.
Genet Mol Biol ; 44(4): e20210149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34807224

RESUMO

Mitochondrial complex I (CI) deficiency is the most common oxidative phosphorylation disorder described. It shows a wide range of phenotypes with poor correlation within genotypes. Herein we expand the clinics and genetics of CI deficiency in the brazilian population by reporting three patients with pathogenic (c.640G>A, c.1268C>T, c.1207dupG) and likely pathogenic (c.766C>T) variants in the NDUFV1 gene. We show the mutation c.766C>T associated with a childhood onset phenotype of hypotonia, muscle weakness, psychomotor regression, lethargy, dysphagia, and strabismus. Additionally, this mutation was found to be associated with headaches and exercise intolerance in adulthood. We also review reported pathogenic variants in NDUFV1 highlighting the wide phenotypic heterogeneity in CI deficiency.

5.
Mol Genet Metab ; 129(1): 26-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31787496

RESUMO

NUBPL (Nucleotide-binding protein like) protein encodes a member of the Mrp/NBP35 ATP-binding proteins family, deemed to be involved in mammalian complex I (CI) assembly process. Exome sequencing of a patient presenting with infantile-onset hepatopathy, renal tubular acidosis, developmental delay, short stature, leukoencephalopathy with minimal cerebellar involvement and multiple OXPHOS deficiencies revealed the presence of two novel pathogenic compound heterozygous variants in NUBPL (p.Phe242Leu/p.Leu104Pro). We investigated patient's and control immortalised fibroblasts and demonstrated that both the peripheral and the membrane arms of complex I are undetectable in mutant NUBPL cells, resulting in virtually absent CI holocomplex and loss of enzyme activity. In addition, complex III stability was moderately affected as well. Lentiviral-mediated expression of the wild-type NUBPL cDNA rescued both CI and CIII assembly defects, confirming the pathogenicity of the variants. In conclusion, this is the first report describing a complex multisystemic disorder due to NUBPL defect. In addition, we confirmed the role of NUBPL in Complex I assembly associated with secondary effect on Complex III stability and we demonstrated a defect of mtDNA-related translation which suggests a potential additional role of NUBPL in mtDNA expression.


Assuntos
Variação Genética , Heterozigoto , Leucoencefalopatias/genética , Proteínas Mitocondriais/genética , Adolescente , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , DNA Mitocondrial , Feminino , Humanos , Lactente , Recém-Nascido , Leucoencefalopatias/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Mitocôndrias/patologia , Mutação , Adulto Jovem
6.
Nature ; 515(7527): 431-435, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25383517

RESUMO

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.


Assuntos
Isquemia/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Ácido Succínico/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Ácido Aspártico/metabolismo , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Fumaratos/metabolismo , Isquemia/enzimologia , Malatos/metabolismo , Masculino , Metabolômica , Camundongos , Mitocôndrias/enzimologia , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/metabolismo , Miocárdio/citologia , Miocárdio/enzimologia , Miocárdio/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , NAD/metabolismo , Traumatismo por Reperfusão/enzimologia , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/metabolismo , Succinato Desidrogenase/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(31): 8687-92, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27382158

RESUMO

The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme.


Assuntos
Cardiolipinas/metabolismo , Lisina/metabolismo , Simulação de Dinâmica Molecular , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cardiolipinas/química , Bovinos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lisina/química , Lisina/genética , Metilação , Ligação Proteica , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
8.
Biochim Biophys Acta Biomembr ; 1860(5): 1035-1045, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29366674

RESUMO

Cardiolipin in eukaryotes is found in the mitochondrial inner membrane, where it interacts with membrane proteins and, although not essential, is necessary for the optimal activity of a number of proteins. One of them is the mitochondrial ADP/ATP carrier, which imports ADP into the mitochondrion and exports ATP. In the crystal structures, cardiolipin is bound to three equivalent sites of the ADP/ATP carrier, but its role is unresolved. Conservation of residues at these cardiolipin binding sites across other members of the mitochondrial carrier superfamily indicates cardiolipin binding is likely to be important for the function of all mitochondrial carriers. Multiscale simulations were performed in a cardiolipin-containing membrane to investigate the dynamics of cardiolipin around the yeast and bovine ADP/ATP carriers in a lipid bilayer and the properties of the cardiolipin-binding sites. In coarse-grain simulations, cardiolipin molecules bound to the carriers for longer periods of time than phosphatidylcholine and phosphatidylethanolamine lipids-with timescales in the tens of microseconds. Three long-lived cardiolipin binding sites overlapped with those in the crystal structures of the carriers. Other shorter-lived cardiolipin interaction sites were identified in both membrane leaflets. However, the timescales of the interactions were of the same order as phosphatidylcholine and phosphatidylethanolamine, suggesting that these sites are not specific for cardiolipin binding. The calculation of lipid binding times and the overlap of the cardiolipin binding sites between the structures and simulations demonstrate the potential of multiscale simulations to investigate the dynamics and behavior of lipids interacting with membrane proteins.


Assuntos
Cardiolipinas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cardiolipinas/química , Cardiolipinas/genética , Bovinos , Sequência Conservada/genética , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae
9.
Genet Med ; 20(10): 1224-1235, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29517768

RESUMO

PURPOSE: To understand the role of the mitochondrial oxodicarboxylate carrier (SLC25A21) in the development of spinal muscular atrophy-like disease. METHODS: We identified a novel pathogenic variant in a patient by whole-exome sequencing. The pathogenicity of the mutation was studied by transport assays, computer modeling, followed by targeted metabolic testing and in vitro studies in human fibroblasts and neurons. RESULTS: The patient carries a homozygous pathogenic variant c.695A>G; p.(Lys232Arg) in the SLC25A21 gene, encoding the mitochondrial oxodicarboxylate carrier, and developed spinal muscular atrophy and mitochondrial myopathy. Transport assays show that the mutation renders SLC25A21 dysfunctional and 2-oxoadipate cannot be imported into the mitochondrial matrix. Computer models of central metabolism predicted that impaired transport of oxodicarboxylate disrupts the pathways of lysine and tryptophan degradation, and causes accumulation of 2-oxoadipate, pipecolic acid, and quinolinic acid, which was confirmed in the patient's urine by targeted metabolomics. Exposure to 2-oxoadipate and quinolinic acid decreased the level of mitochondrial complexes in neuronal cells (SH-SY5Y) and induced apoptosis. CONCLUSION: Mitochondrial oxodicarboxylate carrier deficiency leads to mitochondrial dysfunction and the accumulation of oxoadipate and quinolinic acid, which in turn cause toxicity in spinal motor neurons leading to spinal muscular atrophy-like disease.


Assuntos
Adipatos/metabolismo , DNA Mitocondrial/genética , Transportadores de Ácidos Dicarboxílicos/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Atrofia Muscular Espinal/genética , Adipatos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , DNA Mitocondrial/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Fibroblastos/efeitos dos fármacos , Homozigoto , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/fisiopatologia , Mutação , Ácidos Pipecólicos/metabolismo , Ácido Quinolínico/metabolismo
10.
J Med Genet ; 54(12): 815-824, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29079705

RESUMO

BACKGROUND: Hereditary myopathy with lactic acidosis and myopathy with deficiency of succinate dehydrogenase and aconitase are variants of a recessive disorder characterised by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, metabolic acidosis and rhabdomyolysis may occur. So far, this disease has been molecularly defined only in Swedish patients, all homozygous for a deep intronic splicing affecting mutation in ISCU encoding a scaffold protein for the assembly of iron-sulfur (Fe-S) clusters. A single Scandinavian family was identified with a different mutation, a missense change in compound heterozygosity with the common intronic mutation. The aim of the study was to identify the genetic defect in our proband. METHODS: A next-generation sequencing (NGS) approach was carried out on an Italian male who presented in childhood with ptosis, severe muscle weakness and exercise intolerance. His disease was slowly progressive, with partial recovery between episodes. Patient's specimens and yeast models were investigated. RESULTS: Histochemical and biochemical analyses on muscle biopsy showed multiple defects affecting mitochondrial respiratory chain complexes. We identified a single heterozygous mutation p.Gly96Val in ISCU, which was absent in DNA from his parents indicating a possible de novo dominant effect in the patient. Patient fibroblasts showed normal levels of ISCU protein and a few variably affected Fe-S cluster-dependent enzymes. Yeast studies confirmed both pathogenicity and dominance of the identified missense mutation. CONCLUSION: We describe the first heterozygous dominant mutation in ISCU which results in a phenotype reminiscent of the recessive disease previously reported.


Assuntos
Genes Dominantes , Proteínas Ferro-Enxofre/genética , Miopatias Mitocondriais/diagnóstico , Miopatias Mitocondriais/genética , Mutação , Sequência de Aminoácidos , Biomarcadores , Biópsia , Biologia Computacional/métodos , Eletroencefalografia , Eletromiografia , Fibroblastos/metabolismo , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Ferro-Enxofre/química , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Músculo Esquelético/patologia , Linhagem , Fenótipo , Análise de Sequência de DNA , Relação Estrutura-Atividade , Adulto Jovem
11.
Nucleic Acids Res ; 44(D1): D1258-61, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26432830

RESUMO

Mitochondrial proteins remain the subject of intense research interest due to their implication in an increasing number of different conditions including mitochondrial and metabolic disease, cancer, and neuromuscular degenerative and age-related disorders. However, the mitochondrial proteome has yet to be accurately and comprehensively defined, despite many studies. To support mitochondrial research, we developed MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk), a freely accessible mitochondrial proteomics database. MitoMiner integrates different types of subcellular localisation evidence with protein information from public resources, and so provides a comprehensive central resource for data on mitochondrial protein localisation. Here we report important updates to the database including the addition of subcellular immunofluorescent staining results from the Human Protein Atlas, computational predictions of mitochondrial targeting sequences, and additional large-scale mass-spectrometry and GFP tagging data sets. This evidence is shared across the 12 species in MitoMiner (now including Schizosaccharomyces pombe) by homology mapping. MitoMiner provides multiple ways of querying the data including simple text searches, predefined queries and custom queries created using the interactive QueryBuilder. For remote programmatic access, API's are available for several programming languages. This combination of data and flexible querying makes MitoMiner a unique platform to investigate mitochondrial proteins, with application in mitochondrial research and prioritising candidate mitochondrial disease genes.


Assuntos
Bases de Dados de Proteínas , Proteínas Mitocondriais/análise , Animais , Bovinos , Proteínas de Fluorescência Verde/análise , Humanos , Espectrometria de Massas , Camundongos , Proteínas Mitocondriais/química , Sinais Direcionadores de Proteínas , Proteômica , Ratos
12.
Nucleic Acids Res ; 44(16): 7804-16, 2016 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-27466392

RESUMO

Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.


Assuntos
DNA Mitocondrial/genética , Endonucleases/metabolismo , Mitocôndrias/metabolismo , Mutação/genética , Dedos de Zinco , Linhagem Celular Tumoral , Citometria de Fluxo , Dosagem de Genes , Humanos , RNA Catalítico/metabolismo
14.
J Med Genet ; 53(12): 846-849, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27683825

RESUMO

BACKGROUND: Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. METHODS: Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. RESULTS: We found compound heterozygous mutations in COA7: a paternal c.410A>G, p.Y137C, and a maternal c.287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. CONCLUSIONS: We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested.


Assuntos
Deficiência de Citocromo-c Oxidase/metabolismo , Leucoencefalopatias/metabolismo , Proteínas Mitocondriais/genética , Mutação , Sequência de Aminoácidos , Deficiência de Citocromo-c Oxidase/genética , Análise Mutacional de DNA , Feminino , Humanos , Leucoencefalopatias/genética , Mitocôndrias , Proteínas Mitocondriais/química , Alinhamento de Sequência , Adulto Jovem
15.
Am J Hum Genet ; 93(3): 482-95, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23993194

RESUMO

Whole-exome sequencing and autozygosity mapping studies, independently performed in subjects with defective combined mitochondrial OXPHOS-enzyme deficiencies, identified a total of nine disease-segregating FBXL4 mutations in seven unrelated mitochondrial disease families, composed of six singletons and three siblings. All subjects manifested early-onset lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy consistently associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures. A wide range of other multisystem features were variably seen, including dysmorphism, skeletal abnormalities, poor growth, gastrointestinal dysmotility, renal tubular acidosis, seizures, and episodic metabolic failure. Mitochondrial respiratory chain deficiency was present in muscle or fibroblasts of all tested individuals, together with markedly reduced oxygen consumption rate and hyperfragmentation of the mitochondrial network in cultured cells. In muscle and fibroblasts from several subjects, substantially decreased mtDNA content was observed. FBXL4 is a member of the F-box family of proteins, some of which are involved in phosphorylation-dependent ubiquitination and/or G protein receptor coupling. We also demonstrate that FBXL4 is targeted to mitochondria and localizes in the intermembrane space, where it participates in an approximately 400 kDa protein complex. These data strongly support a role for FBXL4 in controlling bioenergetic homeostasis and mtDNA maintenance. FBXL4 mutations are a recurrent cause of mitochondrial encephalomyopathy onset in early infancy.


Assuntos
Predisposição Genética para Doença , Encefalomiopatias Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Idade de Início , Criança , Pré-Escolar , Cromossomos Humanos Par 6/genética , DNA Complementar/genética , Proteínas F-Box/química , Proteínas F-Box/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Genes Recessivos/genética , Células HEK293 , Humanos , Lactente , Recém-Nascido , Masculino , Mitocôndrias/metabolismo , Encefalomiopatias Mitocondriais/epidemiologia , Músculo Esquelético/patologia , Proteínas Mutantes/metabolismo , Fosforilação Oxidativa , Linhagem , Transporte Proteico , Frações Subcelulares/metabolismo , Síndrome , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
16.
Nucleic Acids Res ; 41(4): 2354-69, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23275553

RESUMO

Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.


Assuntos
Códon de Iniciação , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteoma/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase gama , DNA Mitocondrial/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , DNA Polimerase Dirigida por DNA/isolamento & purificação , Humanos , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutação , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/isolamento & purificação , Proteínas de Ligação a Poli(A)/metabolismo , Polinucleotídeo Adenililtransferase/isolamento & purificação , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteoma/metabolismo
17.
Nucleic Acids Res ; 40(Database issue): D1160-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22121219

RESUMO

MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/) is a data warehouse for the storage and analysis of mitochondrial proteomics data gathered from publications of mass spectrometry and green fluorescent protein tagging studies. In MitoMiner, these data are integrated with data from UniProt, Gene Ontology, Online Mendelian Inheritance in Man, HomoloGene, Kyoto Encyclopaedia of Genes and Genomes and PubMed. The latest release of MitoMiner stores proteomics data sets from 46 studies covering 11 different species from eumetazoa, viridiplantae, fungi and protista. MitoMiner is implemented by using the open source InterMine data warehouse system, which provides a user interface allowing users to upload data for analysis, personal accounts to store queries and results and enables queries of any data in the data model. MitoMiner also provides lists of proteins for use in analyses, including the new MitoMiner mitochondrial proteome reference sets that specify proteins with substantial experimental evidence for mitochondrial localization. As further mitochondrial proteomics data sets from normal and diseased tissue are published, MitoMiner can be used to characterize the variability of the mitochondrial proteome between tissues and investigate how changes in the proteome may contribute to mitochondrial dysfunction and mitochondrial-associated diseases such as cancer, neurodegenerative diseases, obesity, diabetes, heart failure and the ageing process.


Assuntos
Bases de Dados de Proteínas , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Animais , Bovinos , Proteínas de Fluorescência Verde/análise , Humanos , Espectrometria de Massas , Camundongos , Proteínas Mitocondriais/genética , Proteômica , Ratos , Interface Usuário-Computador
18.
J Biol Chem ; 287(11): 7925-34, 2012 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-22262851

RESUMO

Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the C(α) carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.


Assuntos
Substituição de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/genética , Sítios de Ligação , Transporte Biológico Ativo/fisiologia , Humanos , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Ornitina/química , Ornitina/genética , Ornitina/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
19.
Exp Cell Res ; 318(11): 1316-23, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22326460

RESUMO

Recent studies report mitochondrial carrier homolog 2 (MTCH2) as a novel and uncharacterized protein that acts as a receptor-like protein for the truncated BH3-interacting domain death agonist (tBID) protein in the outer membrane of mitochondria. These studies, using mouse embryonic stem cells and fibroblasts as well as mice with a conditional knockout of MTCH2 in the liver, showed that deletion of MTCH2 hindered recruitment of tBID to the mitochondria with subsequent reductions in the activation of pro-apoptotic proteins, mitochondrial outer membrane permeabilization and apoptosis. Sequence analysis shows that MTCH2 is present in all examined multicellular Metazoa as well as unicellular Choanoflagellata, and is a highly derived member of the mitochondrial carrier family. Mitochondrial carriers are monomeric transport proteins that are usually found in the inner mitochondrial membrane, where they exchange small substrates between the mitochondrial matrix and intermembrane space. There are extensive differences between the protein sequences of MTCH2 and other mitochondrial carriers that may explain the ability of MTCH2 to associate with tBID and thus its role in apoptosis. We review the experimental evidence for the role of MTCH2 in apoptosis and suggest that the original transport function of the ancestral MTCH2 mitochondrial carrier has been co-opted by the apoptotic machinery to provide a receptor and signaling mechanism.


Assuntos
Apoptose , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Animais , Coanoflagelados , Evolução Molecular , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Transporte Proteico
20.
Proc Natl Acad Sci U S A ; 107(5): 1930-5, 2010 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-20133838

RESUMO

In oxidative phosphorylation, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. Complex I contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters, in several subunits, to transfer the electrons to quinone. Understanding coupled electron transfer in complex I requires a detailed knowledge of the properties of individual clusters and of the cluster ensemble, and so it requires the correlation of spectroscopic and structural data: This has proved a challenging task. EPR studies on complex I from Bos taurus have established that EPR signals N1b, N2 and N3 arise, respectively, from the 2Fe cluster in the 75 kDa subunit, and from 4Fe clusters in the PSST and 51 kDa subunits (positions 2, 7, and 1 along the seven-cluster chain extending from the flavin). The other clusters have either evaded detection or definitive signal assignments have not been established. Here, we combine double electron-electron resonance (DEER) spectroscopy on B. taurus complex I with the structure of the hydrophilic domain of Thermus thermophilus complex I. By considering the magnetic moments of the clusters and the orientation selectivity of the DEER experiment explicitly, signal N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3). The implications of our assignment for the mechanisms of electron transfer and energy transduction by complex I are discussed.


Assuntos
Complexo I de Transporte de Elétrons/química , Animais , Proteínas de Bactérias/química , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Transferência de Energia , Modelos Moleculares , Estrutura Molecular , Estrutura Terciária de Proteína , Thermus thermophilus/química
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