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1.
Redox Biol ; 69: 103001, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38145589

RESUMO

Respiratory complex I plays a crucial role in the mitochondrial electron transport chain and shows promise as a therapeutic target for various human diseases. While most studies focus on inhibiting complex I at the Q-site, little is known about inhibitors targeting other sites within the complex. In this study, we demonstrate that diphenyleneiodonium (DPI), a N-site inhibitor, uniquely affects the stability of complex I by reacting with its flavin cofactor FMN. Treatment with DPI blocks the final stage of complex I assembly, leading to the complete and reversible degradation of complex I in different cellular models. Growing cells in medium lacking the FMN precursor riboflavin or knocking out the mitochondrial flavin carrier gene SLC25A32 results in a similar complex I degradation. Overall, our findings establish a direct connection between mitochondrial flavin homeostasis and complex I stability and assembly, paving the way for novel pharmacological strategies to regulate respiratory complex I.


Assuntos
Complexo I de Transporte de Elétrons , Riboflavina , Humanos , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Riboflavina/metabolismo , Mitocôndrias/metabolismo
2.
Sci Adv ; 6(26): eaba7509, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32637615

RESUMO

Mitochondrial respiratory complexes assemble into supercomplexes (SC). Q-respirasome (III2 + IV) requires the supercomplex assembly factor (SCAF1) protein. The role of this factor in the N-respirasome (I + III2 + IV) and the physiological role of SCs are controversial. Here, we study C57BL/6J mice harboring nonfunctional SCAF1, the full knockout for SCAF1, or the wild-type version of the protein and found that exercise performance is SCAF1 dependent. By combining quantitative data-independent proteomics, 2D Blue native gel electrophoresis, and functional analysis of enriched respirasome fractions, we show that SCAF1 confers structural attachment between III2 and IV within the N-respirasome, increases NADH-dependent respiration, and reduces reactive oxygen species (ROS). Furthermore, the expression of AOX in cells and mice confirms that CI-CIII superassembly segments the CoQ in two pools and modulates CI-NADH oxidative capacity.

3.
Cell Metab ; 30(6): 1120-1130.e5, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31588014

RESUMO

mtDNA is present in multiple copies in each cell derived from the expansions of those in the oocyte. Heteroplasmy, more than one mtDNA variant, may be generated by mutagenesis, paternal mtDNA leakage, and novel medical technologies aiming to prevent inheritance of mtDNA-linked diseases. Heteroplasmy phenotypic impact remains poorly understood. Mouse studies led to contradictory models of random drift or haplotype selection for mother-to-offspring transmission of mtDNA heteroplasmy. Here, we show that mtDNA heteroplasmy affects embryo metabolism, cell fitness, and induced pluripotent stem cell (iPSC) generation. Thus, genetic and pharmacological interventions affecting oxidative phosphorylation (OXPHOS) modify competition among mtDNA haplotypes during oocyte development and/or at early embryonic stages. We show that heteroplasmy behavior can fall on a spectrum from random drift to strong selection, depending on mito-nuclear interactions and metabolic factors. Understanding heteroplasmy dynamics and its mechanisms provide novel knowledge of a fundamental biological process and enhance our ability to mitigate risks in clinical applications affecting mtDNA transmission.


Assuntos
DNA Mitocondrial/genética , Desenvolvimento Embrionário/genética , Herança Materna/genética , Mitocôndrias/genética , Oogênese/genética , Animais , Linhagem Celular , Embrião de Mamíferos , Feminino , Fibroblastos , Haplótipos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos
4.
Cell Metab ; 25(1): 4-5, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28076765

RESUMO

Understanding the mammalian respiratory complex I assembly has been an arduous task due to the lack of appropriate techniques and the complexity of the process. In this issue, a new tour de force based on complexome profiling provides an encyclopedic description of the process (Guerrero-Castillo et al., 2017).


Assuntos
Complexo I de Transporte de Elétrons , Fosforilação Oxidativa , Animais , Humanos , Mitocôndrias
5.
Nature ; 539(7630): 579-582, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27775717

RESUMO

Respiratory chain complexes can super-assemble into quaternary structures called supercomplexes that optimize cellular metabolism. The interaction between complexes III (CIII) and IV (CIV) is modulated by supercomplex assembly factor 1 (SCAF1, also known as COX7A2L). The discovery of SCAF1 represented strong genetic evidence that supercomplexes exist in vivo. SCAF1 is present as a long isoform (113 amino acids) or a short isoform (111 amino acids) in different mouse strains. Only the long isoform can induce the super-assembly of CIII and CIV, but it is not clear whether SCAF1 is required for the formation of the respirasome (a supercomplex of CI, CIII2 and CIV). Here we show, by combining deep proteomics and immunodetection analysis, that SCAF1 is always required for the interaction between CIII and CIV and that the respirasome is absent from most tissues of animals containing the short isoform of SCAF1, with the exception of heart and skeletal muscle. We used directed mutagenesis to characterize SCAF1 regions that interact with CIII and CIV and discovered that this interaction requires the correct orientation of a histidine residue at position 73 that is altered in the short isoform of SCAF1, explaining its inability to interact with CIV. Furthermore, we find that the CIV subunit COX7A2 is replaced by SCAF1 in supercomplexes containing CIII and CIV and by COX7A1 in CIV dimers, and that dimers seem to be more stable when they include COX6A2 rather than the COX6A1 isoform.


Assuntos
Membranas Mitocondriais/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Complexo IV da Cadeia de Transporte de Elétrons/química
6.
Cell Rep ; 15(1): 197-209, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27052170

RESUMO

Electrons feed into the mitochondrial electron transport chain (mETC) from NAD- or FAD-dependent enzymes. A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here, we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. Thus, CoQ redox status acts as a metabolic sensor that fine-tunes mETC configuration in order to match the prevailing substrate profile.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Elétrons , Ubiquinona/metabolismo , Animais , Linhagem Celular , Flavina-Adenina Dinucleotídeo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
J Cell Mol Med ; 20(5): 794-803, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26915330

RESUMO

Connexin 43 (Cx43), the gap junction protein involved in cell-to-cell coupling in the heart, is also present in the subsarcolemmal fraction of cardiomyocyte mitochondria. It has been described to regulate mitochondrial potassium influx and respiration and to be important for ischaemic preconditioning protection, although the molecular effectors involved are not fully characterized. In this study, we looked for potential partners of mitochondrial Cx43 in an attempt to identify new molecular pathways for cardioprotection. Mass spectrometry analysis of native immunoprecipitated mitochondrial extracts showed that Cx43 interacts with several proteins related with mitochondrial function and metabolism. Among them, we selected for further analysis only those present in the subsarcolemmal mitochondrial fraction and known to be related with the respiratory chain. Apoptosis-inducing factor (AIF) and the beta-subunit of the electron-transfer protein (ETFB), two proteins unrelated to date with Cx43, fulfilled these conditions, and their interaction with Cx43 was proven by direct and reverse co-immunoprecipitation. Furthermore, a previously unknown molecular interaction between AIF and ETFB was established, and protein content and sub-cellular localization appeared to be independent from the presence of Cx43. Our results identify new protein-protein interactions between AIF-Cx43, ETFB-Cx43 and AIF-ETFB as possible players in the regulation of the mitochondrial redox state.


Assuntos
Fator de Indução de Apoptose/metabolismo , Conexina 43/metabolismo , Flavoproteínas Transferidoras de Elétrons/metabolismo , Mitocôndrias Cardíacas/metabolismo , Subunidades Proteicas/metabolismo , Animais , Fator de Indução de Apoptose/genética , Conexina 43/genética , Flavoproteínas Transferidoras de Elétrons/genética , Feminino , Regulação da Expressão Gênica , Imunoprecipitação , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Oxirredução , Ligação Proteica , Mapeamento de Interação de Proteínas , Subunidades Proteicas/genética , Transdução de Sinais
8.
Cell Rep ; 10(7): 1110-21, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25704814

RESUMO

The mitochondrial genome relies heavily on post-transcriptional events for its proper expression, and misregulation of this process can cause mitochondrial genetic diseases in humans. Here, we report that a novel translational variant of Fas-activated serine/threonine kinase (FASTK) co-localizes with mitochondrial RNA granules and is required for the biogenesis of ND6 mRNA, a mitochondrial-encoded subunit of the NADH dehydrogenase complex (complex I). We show that ablating FASTK expression in cultured cells and mice results specifically in loss of ND6 mRNA and reduced complex I activity in vivo. FASTK binds at multiple sites along the ND6 mRNA and its precursors and cooperates with the mitochondrial degradosome to ensure regulated ND6 mRNA biogenesis. These data provide insights into the mechanism and control of mitochondrial RNA processing within mitochondrial RNA granules.


Assuntos
Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Microscopia Confocal , Complexos Multienzimáticos/metabolismo , Miocárdio/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , RNA Helicases/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
9.
Hum Mol Genet ; 22(6): 1233-48, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23255162

RESUMO

Coenzyme Q10 (CoQ(10)) or ubiquinone is a well-known component of the mitochondrial respiratory chain. In humans, CoQ(10) deficiency causes a mitochondrial syndrome with an unexplained variability in the clinical presentations. To try to understand this heterogeneity in the clinical phenotypes, we have generated a Coq9 Knockin (R239X) mouse model. The lack of a functional Coq9 protein in homozygous Coq9 mutant (Coq9(X/X)) mice causes a severe reduction in the Coq7 protein and, as consequence, a widespread CoQ deficiency and accumulation of demethoxyubiquinone. The deficit in CoQ induces a brain-specific impairment of mitochondrial bioenergetics performance, a reduction in respiratory control ratio, ATP levels and ATP/ADP ratio and specific loss of respiratory complex I. These effects lead to neuronal death and demyelinization with severe vacuolization and astrogliosis in the brain of Coq9(X/X) mice that consequently die between 3 and 6 months of age. These results suggest that the instability of mitochondrial complex I in the brain, as a primary event, triggers the development of mitochondrial encephalomyopathy associated with CoQ deficiency.


Assuntos
Encefalomiopatias Mitocondriais/enzimologia , Ubiquinona/deficiência , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Encefalomiopatias Mitocondriais/genética , Ubiquinona/genética , Ubiquinona/metabolismo
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