RESUMO
Deep understanding of the pathophysiological role of the mitochondrial respiratory chain (MRC) relies on a well-grounded model explaining how its biogenesis is regulated. The lack of a consistent framework to clarify the modes and mechanisms governing the assembly of the MRC complexes and supercomplexes (SCs) works against progress in the field. The plasticity model was postulated as an attempt to explain the coexistence of mammalian MRC complexes as individual entities and associated in SC species. However, mounting data accumulated throughout the years question the universal validity of the plasticity model as originally proposed. Instead, as we argue here, a cooperative assembly model provides a much better explanation to the phenomena observed when studying MRC biogenesis in physiological and pathological settings.
Assuntos
Mitocôndrias , Membranas Mitocondriais , Animais , Transporte de Elétrons/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , MamíferosRESUMO
Mitochondrial respiratory chain (MRC) enzymes associate in supercomplexes (SCs) that are structurally interdependent. This may explain why defects in a single component often produce combined enzyme deficiencies in patients. A case in point is the alleged destabilization of complex I in the absence of complex III. To clarify the structural and functional relationships between complexes, we have used comprehensive proteomic, functional, and biogenetical approaches to analyze a MT-CYB-deficient human cell line. We show that the absence of complex III blocks complex I biogenesis by preventing the incorporation of the NADH module rather than decreasing its stability. In addition, complex IV subunits appeared sequestered within complex III subassemblies, leading to defective complex IV assembly as well. Therefore, we propose that complex III is central for MRC maturation and SC formation. Our results challenge the notion that SC biogenesis requires the pre-formation of fully assembled individual complexes. In contrast, they support a cooperative-assembly model in which the main role of complex III in SCs is to provide a structural and functional platform for the completion of overall MRC biogenesis.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Proteômica/métodos , Linhagem Celular , Complexo I de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Estabilidade Enzimática , Humanos , Mitocôndrias/metabolismo , Mutação , NAD/metabolismoRESUMO
Mitochondrial respiratory chain complexes I, III, and IV can associate into larger structures termed supercomplexes or respirasomes, thereby generating structural interdependences among the individual complexes yet to be understood. In patients, nonsense mutations in complex IV subunit genes cause severe encephalomyopathies randomly associated with pleiotropic complex I defects. Using complexome profiling and biochemical analyses, we have explored the structural rearrangements of the respiratory chain in human cell lines depleted of the catalytic complex IV subunit COX1 or COX2. In the absence of a functional complex IV holoenzyme, several supercomplex I+III2 species coexist, which differ in their content of COX subunits and COX7A2L/HIGD2A assembly factors. The incorporation of an atypical COX1-HIGD2A submodule attenuates supercomplex I+III2 turnover rate, indicating an unexpected molecular adaptation for supercomplexes stabilization that relies on the presence of COX1 independently of holo-complex IV formation. Our data set the basis for complex I structural dependence on complex IV, revealing the co-existence of alternative pathways for the biogenesis of "supercomplex-associated" versus individual complex IV, which could determine physiological adaptations under different stress and disease scenarios.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Linhagem Celular , HumanosRESUMO
We report a neonatal patient with hypertrophic cardiomyopathy (HCM), lactic acidosis and isolated complex I deficiency. Using a customized next-generation sequencing panel, we identified a novel hemizygous variant c.338G>A in the X-linked NDUFB11 gene that encodes the NADH: ubiquinone oxidoreductase subunit B11 of the mitochondrial respiratory chain (MRC) complex I (CI). Molecular and functional assays performed in the proband's target tissuesskeletal and heart muscleshowed biochemical disturbances of the MRC, suggesting a pathogenic role for this variant. In silico analyses initially predicted an amino acid missense change p.(Arg113Lys) in the NDUFB11 CI subunit. However, we showed that the molecular effect of the c.338G>A variant, which is located at the last nucleotide of exon 2 of the NDUFB11 gene in the canonical 'short' transcript (sized 462 bp), instead causes a splicing defect triggering the up-regulation of the expression of an alternative 'long' transcript (sized 492 bp) that can also be detected in the control individuals. Our results support the hypothesis that the canonical 'short' transcript is required for the proper NDUFB11 protein synthesis, which is essential for optimal CI assembly and activity, whereas the longer alternative transcript seems to represent a non-functional, unprocessed splicing intermediate. Our results highlight the importance of characterizing the molecular effect of new variants in the affected patient's tissues to demonstrate their pathogenicity and association with the clinical phenotypes.
Assuntos
Cardiomiopatias , Cardiomiopatia Hipertrófica , Doenças Mitocondriais , Humanos , Cardiomiopatias/genética , Doenças Mitocondriais/genética , Complexo I de Transporte de Elétrons/genética , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Mutação , LinhagemRESUMO
CBL is rapidly phosphorylated upon insulin receptor activation. Mice whole body CBL depletion improved insulin sensitivity and glucose clearance; however, the precise mechanisms remain unknown. We depleted either CBL or its associated protein SORBS1/CAP independently in myocytes and assessed mitochondrial function and metabolism compared to control cells. CBL- and CAP-depleted cells showed increased mitochondrial mass with greater proton leak. Mitochondrial respiratory complex I activity and assembly into respirasomes were reduced. Proteome profiling revealed alterations in proteins involved in glycolysis and fatty acid degradation. Our findings demonstrate CBL/CAP pathway couples insulin signaling to efficient mitochondrial respiratory function and metabolism in muscle.
Assuntos
Resistência à Insulina , Proteínas Proto-Oncogênicas c-cbl , Animais , Camundongos , Metabolismo Energético , Insulina/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Musculares/metabolismo , Células Musculares/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Respiração CelularRESUMO
Mitochondrial disorders (MD) comprise a group of heterogeneous clinical disorders for which non-invasive diagnosis remains a challenge. Two protein biomarkers have so far emerged for MD detection, FGF-21 and GDF-15, but the identification of additional biomarkers capable of improving their diagnostic accuracy is highly relevant. Previous studies identified Gelsolin as a regulator of cell survival adaptations triggered by mitochondrial defects. Gelsolin presents a circulating plasma isoform (pGSN), whose altered levels could be a hallmark of mitochondrial dysfunction. Therefore, we investigated the diagnostic performance of pGSN for MD relative to FGF-21 and GDF-15. Using ELISA assays, we quantified plasma levels of pGSN, FGF-21, and GDF-15 in three age- and gender-matched adult cohorts: 60 genetically diagnosed MD patients, 56 healthy donors, and 41 patients with unrelated neuromuscular pathologies (non-MD). Clinical variables and biomarkers' plasma levels were compared between groups. Discrimination ability was calculated using the area under the ROC curve (AUC). Optimal cut-offs and the following diagnostic parameters were determined: sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and efficiency. Comprehensive statistical analyses revealed significant discrimination ability for the three biomarkers to classify between MD and healthy individuals, with the best diagnostic performance for the GDF-15/pGSN combination. pGSN and GDF-15 preferentially discriminated between MD and non-MD patients under 50 years, whereas FGF-21 best classified older subjects. Conclusion: pGSN improves the diagnosis accuracy for MD provided by FGF-21 and GDF-15.
Assuntos
Fatores de Crescimento de Fibroblastos/sangue , Gelsolina/sangue , Fator 15 de Diferenciação de Crescimento/sangue , Doenças Mitocondriais/sangue , Doenças Mitocondriais/diagnóstico , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Over the past sixty years, researchers have made outmost efforts to clarify the structural organization and functional regulation of the complexes that configure the mitochondrial respiratory chain. As a result, the entire composition of each individual complex is practically known and, aided by notable structural advances in mammals, it is now widely accepted that these complexes stablish interactions to form higher-order supramolecular structures called supercomplexes and respirasomes. The mechanistic models and players that regulate the function and biogenesis of such superstructures are still under intense debate, and represent one of the hottest topics of the mitochondrial research field at present. Noteworthy, understanding the pathways involved in the assembly and organization of respiratory chain complexes and supercomplexes is of high biomedical relevance because molecular alterations in these pathways frequently result in severe mitochondrial disorders. The purpose of this review is to update the structural, biogenetic and functional knowledge about the respiratory chain supercomplexes and assembly factors involved in their formation, with special emphasis on their implications in mitochondrial disease. Thanks to the integrated data resulting from recent structural, biochemical and genetic approaches in diverse biological systems, the regulation of the respiratory chain function arises at multiple levels of complexity.
Assuntos
Transporte de Elétrons/fisiologia , Membranas Mitocondriais/metabolismo , Humanos , Biogênese de OrganelasRESUMO
Uniparental disomy (UPD) is an underestimated cause of autosomal recessive disorders. In this study, we aim to raise awareness about the possibility of UPD in mitochondrial disorders - where it is a hardly described event -, by functionally characterizing a novel variant in a structural subunit of complex I (CI) of the mitochondrial oxidative phosphorylation system. Using next-generation sequencing, we identified a new intronic homozygous c.350â¯+â¯5Gâ¯>â¯A variant in the NDUFS4 gene in a one-year-old girl (being alive at the age of 7) belonging to a non-consanguineous family presenting with encephalopathy, psychomotor delay, lactic acidosis and a single CI deficiency, a less severe phenotype than those previously reported in most NDUFS4 patients. One parent lacked the variant, and microsatellite genotyping showed complete paternal uniparental isodisomy of the non-imprinted chromosome 5. We demonstrated in patient's skeletal muscle and fibroblasts splicing abnormalities, low expression of NDUFS4, undetectable NDUFS4 protein, defects in cellular respiration (decreased oxygen consumption and ATP production), and impaired assembly or stability of mitochondrial supercomplexes containing CI. Our findings support that c.350â¯+â¯5Gâ¯>â¯A variant is pathogenic, and reinforce that UPD, although rare, should be considered as a possible cause of mitochondrial diseases in order to provide accurate genetic counselling.
Assuntos
Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Doenças Mitocondriais/genética , Dissomia Uniparental/genética , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Predisposição Genética para Doença , Homozigoto , Humanos , Lactente , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Mutação/genética , Splicing de RNA/genética , Dissomia Uniparental/patologiaRESUMO
Despite considerable knowledge on the genetic basis of mitochondrial disorders, their pathophysiological consequences remain poorly understood. We previously used two-dimensional difference gel electrophoresis analyses to define a protein profile characteristic for respiratory chain complex III-deficiency that included a significant overexpression of cytosolic gelsolin (GSN), a cytoskeletal protein that regulates the severing and capping of the actin filaments. Biochemical and immunofluorescence assays confirmed a specific increase of GSN levels in the mitochondria from patients' fibroblasts and from transmitochondrial cybrids with complex III assembly defects. A similar effect was obtained in control cells upon treatment with antimycin A in a dose-dependent manner, showing that the enzymatic inhibition of complex III is sufficient to promote the mitochondrial localization of GSN. Mitochondrial subfractionation showed the localization of GSN to the mitochondrial outer membrane, where it interacts with the voltage-dependent anion channel protein 1 (VDAC1). In control cells, VDAC1 was present in five stable oligomeric complexes, which showed increased levels and a modified distribution pattern in the complex III-deficient cybrids. Downregulation of GSN expression induced cell death in both cell types, in parallel with the specific accumulation of VDAC1 dimers and the release of mitochondrial cytochrome c into the cytosol, indicating a role for GSN in the oligomerization of VDAC complexes and in the prevention of apoptosis. Our results demonstrate that respiratory chain complex III dysfunction induces the physiological upregulation and mitochondrial location of GSN, probably to promote cell survival responses through the modulation of the oligomeric state of the VDAC complexes.
Assuntos
Transporte de Elétrons/fisiologia , Gelsolina/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Antimicina A/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Citocromos c/metabolismo , Fibroblastos/metabolismo , Gelsolina/genética , Células HeLa , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Membranas Mitocondriais/metabolismo , Eletroforese em Gel Diferencial Bidimensional/métodos , Canal de Ânion 1 Dependente de Voltagem/fisiologiaRESUMO
Lethal neonatal encephalopathies are heterogeneous congenital disorders that can be caused by mitochondrial dysfunction. Biallelic large deletions in the contiguous ATAD3B and ATAD3A genes, encoding mitochondrial inner membrane ATPases of unknown function, as well as compound heterozygous nonsense and missense mutations in the ATAD3A gene have been recently associated with fatal neonatal cerebellar hypoplasia. In this work, whole exome sequencing (WES) identified the novel homozygous variant c.1217â¯Tâ¯>â¯G in ATAD3A, predicting a p.(Leu406Arg) substitution, in four siblings from a consanguineous family presenting with fatal neonatal cerebellar hypoplasia, seizures, axial hypotonia, hypertrophic cardiomyopathy, hepatomegaly, congenital cataract, and dysmorphic facies. Biochemical phenotypes of the patients included hyperlactatemia and hypocholesterolemia. Healthy siblings and parents were heterozygous for this variant, which is predicted to introduce a polar chain within the catalytic domain of ATAD3A that shortens its beta-sheet structure, presumably affecting protein stability. Accordingly, patient's fibroblasts with the homozygous variant displayed a specific reduction in ATAD3A protein levels associated with profound ultrastructural alterations of mitochondrial cristae and morphology. Our findings exclude the causative role of ATAD3B on this severe phenotype, expand the phenotypical spectrum of ATAD3A pathogenic variants and emphasize the vital role of ATAD3A in mitochondrial biogenesis.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Cerebelo/anormalidades , Genes Recessivos , Predisposição Genética para Doença , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , ATPases Associadas a Diversas Atividades Celulares/química , Alelos , Substituição de Aminoácidos , Cerebelo/diagnóstico por imagem , Cerebelo/patologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Lactente , Masculino , Proteínas de Membrana/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/química , Modelos Moleculares , Malformações do Sistema Nervoso/diagnóstico por imagem , Linhagem , Conformação Proteica , Relação Estrutura-Atividade , Ultrassonografia/métodos , Sequenciamento do ExomaRESUMO
Oxidative phosphorylation system (OXPHOS) deficiencies are rare diseases but constitute the most frequent inborn errors of metabolism. We analyzed the autophagy route in 11 skin fibroblast cultures derived from patients with well characterized and distinct OXPHOS defects. Mitochondrial membrane potential determination revealed a tendency to decrease in 5 patients' cells but reached statistical significance only in 2 of them. The remaining cells showed either no change or a slight increase in this parameter. Colocalization analysis of mitochondria and autophagosomes failed to show evidence of increased selective elimination of mitochondria but revealed more intense autophagosome staining in patients' fibroblasts compared with controls. Despite the absence of increased mitophagy, Parkin recruitment to mitochondria was detected in both controls' and patients' cells and was slightly higher in cells harboring complex I defects. Western blot analysis of the autophagosome marker LC3B, confirmed significantly higher levels of the protein bound to autophagosomes, LC3B-II, in patients' cells, suggesting an increased bulk autophagy in OXPHOS defective fibroblasts. Inhibition of lysosomal proteases caused significant accumulation of LC3B-II in control cells, whereas in patients' cells this phenomenon was less pronounced. Electron microscopy studies showed higher content of late autophagic vacuoles and lysosomes in OXPHOS defective cells, accompanied by higher levels of the lysosomal marker LAMP-1. Our findings suggest that in OXPHOS deficient fibroblasts autophagic flux could be partially hampered leading to an accumulation of autophagic vacuoles and lysosomes.
Assuntos
Autofagia/fisiologia , Mitocôndrias/fisiologia , Doenças Mitocondriais/patologia , Mitofagia/fisiologia , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Lisossomos/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/fisiopatologia , Fagossomos/metabolismo , Fagossomos/fisiologiaRESUMO
Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington's disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD.
Assuntos
Encéfalo , Proteínas de Ligação a DNA , Proteína Huntingtina , Doença de Huntington , Células-Tronco Pluripotentes Induzidas , Mitocôndrias , Proteínas Mitocondriais , Organoides , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Organoides/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Encéfalo/metabolismo , Encéfalo/patologia , Doença de Huntington/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mitocôndrias/metabolismo , Mutação , Dinâmica Mitocondrial/genéticaRESUMO
In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5'-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56(D6) and ccdc56(D11) alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/assembly.
Assuntos
Proteínas de Drosophila/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Imuno-Histoquímica , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
Mutations in GBA1, the gene encoding the lysosomal enzyme ß-glucocerebrosidase (GCase), which cause Gaucher's disease, are the most frequent genetic risk factor for Parkinson's disease (PD). Here, we employ global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration.
Assuntos
Glucosilceramidase , Doença de Parkinson , Humanos , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Proteômica , Doença de Parkinson/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Metabolismo Energético/genética , Mutação , Lisossomos/metabolismo , alfa-Sinucleína/metabolismo , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/metabolismoRESUMO
The structural and functional organization of the mitochondrial respiratory chain (MRC) remains intensely debated. Here, we show the co-existence of two separate MRC organizations in human cells and postmitotic tissues, C-MRC and S-MRC, defined by the preferential expression of three COX7A subunit isoforms, COX7A1/2 and SCAFI (COX7A2L). COX7A isoforms promote the functional reorganization of distinct co-existing MRC structures to prevent metabolic exhaustion and MRC deficiency. Notably, prevalence of each MRC organization is reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC). Under oxidative conditions, the C-MRC is bioenergetically more efficient, whereas the S-MRC preferentially maintains oxidative phosphorylation (OXPHOS) upon metabolic rewiring toward glycolysis. We show a link between the metabolic signatures converging at the PDC and the structural and functional organization of the MRC, challenging the widespread notion of the MRC as a single functional unit and concluding that its structural heterogeneity warrants optimal adaptation to metabolic function.
Assuntos
Glicólise , Fosforilação Oxidativa , Humanos , Transporte de Elétrons , Membranas Mitocondriais/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
The regulatory role of actin cytoskeleton on mitochondrial function is a growing research field, but the underlying molecular mechanisms remain poorly understood. Specific actin-binding proteins (ABPs), such as Gelsolin, have also been shown to participate in the pathophysiology of mitochondrial OXPHOS disorders through yet to be defined mechanisms. In this mini-review, we will summarize the experimental evidence supporting the fundamental roles of actin cytoskeleton and ABPs on mitochondrial trafficking, dynamics, biogenesis, metabolism and apoptosis, with a particular focus on Gelsolin involvement in mitochondrial disorders. The functional interplay between the actin cytoskeleton, ABPs and mitochondrial membranes for the regulation of cellular homeostasis thus emerges as a new exciting field for future research and therapeutic approaches.
RESUMO
The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III2 + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFIKO) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III2 + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFIKO cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Marcação por Isótopo/métodos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Sistemas CRISPR-Cas , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Espectrometria de MassasRESUMO
Complexome profiling is an emerging 'omics' approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the "minimum information required for a complexome profiling experiment" (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
Assuntos
Bases de Dados Factuais , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , Proteoma/metabolismo , Software , Humanos , Proteoma/análiseRESUMO
Mutations in BCS1L, an assembly factor that facilitates the insertion of the catalytic Rieske Iron-Sulfur subunit into respiratory chain complex III, result in a wide variety of clinical phenotypes that range from the relatively mild Björnstad syndrome to the severe GRACILE syndrome. To better understand the pathophysiological consequences of such mutations, we studied fibroblasts from six complex III-deficient patients harboring mutations in the BCS1L gene. Cells from patients with the most severe clinical phenotypes exhibited slow growth rates in glucose medium, variable combined enzyme deficiencies, and assembly defects of respiratory chain complexes I, III, and IV, increased H(2)O(2) levels, unbalanced expression of the cellular antioxidant defenses, and apoptotic cell death. In addition, all patients showed cytosolic accumulation of the BCS1L protein, suggestive of an impaired mitochondrial import, assembly or stability defects of the BCS1L complex, fragmentation of the mitochondrial networks, and decreased MFN2 protein levels. The observed structural alterations were independent of the respiratory chain function and ROS production. Our results provide new insights into the role of pathogenic BCS1L mutations in mitochondrial function and dynamics.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/genética , Fibroblastos/patologia , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Mutação/genética , ATPases Associadas a Diversas Atividades Celulares , Antioxidantes/metabolismo , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Pré-Escolar , DNA Complementar/genética , Evolução Fatal , Feminino , Fibroblastos/enzimologia , Humanos , Lactente , Recém-Nascido , Masculino , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Frações Subcelulares/metabolismoRESUMO
Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. We investigated the effect of mtDNA haplogroups on the assembly of oxidative phosphorylation (OXPHOS) complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially delayed assembly rates of respiratory chain complexes I, III and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.