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1.
Nat Commun ; 11(1): 4135, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811817

RESUMO

Complex I is the first and the largest enzyme of respiratory chains in bacteria and mitochondria. The mechanism which couples spatially separated transfer of electrons to proton translocation in complex I is not known. Here we report five crystal structures of T. thermophilus enzyme in complex with NADH or quinone-like compounds. We also determined cryo-EM structures of major and minor native states of the complex, differing in the position of the peripheral arm. Crystal structures show that binding of quinone-like compounds (but not of NADH) leads to a related global conformational change, accompanied by local re-arrangements propagating from the quinone site to the nearest proton channel. Normal mode and molecular dynamics analyses indicate that these are likely to represent the first steps in the proton translocation mechanism. Our results suggest that quinone binding and chemistry play a key role in the coupling mechanism of complex I.


Assuntos
Complexo I de Transporte de Elétrons/química , Simulação de Dinâmica Molecular , Quinonas/química , Thermus thermophilus/enzimologia , Regulação Alostérica , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Modelos Moleculares , NAD/química , NAD/metabolismo , Redes Neurais de Computação , Conformação Proteica , Prótons , Quinonas/metabolismo , Thermus thermophilus/genética
2.
PLoS One ; 15(6): e0234913, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574189

RESUMO

The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Animais , Metilação de DNA , Modelos Animais de Doenças , Regulação para Baixo , Complexo I de Transporte de Elétrons/genética , Metabolismo Energético/genética , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Histonas/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Fenilefrina/farmacologia , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , RNA-Seq , Ratos , Receptores Estrogênicos/genética
3.
Am J Physiol Heart Circ Physiol ; 319(1): H89-H99, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502376

RESUMO

Mitochondrial dysfunction occurs in most forms of heart failure. We have previously reported that Tead1, the transcriptional effector of Hippo pathway, is critical for maintaining adult cardiomyocyte function, and its deletion in adult heart results in lethal acute dilated cardiomyopathy. Growing lines of evidence indicate that Hippo pathway plays a role in regulating mitochondrial function, although its role in cardiomyocytes is unknown. Here, we show that Tead1 plays a critical role in regulating mitochondrial OXPHOS in cardiomyocytes. Assessment of mitochondrial bioenergetics in isolated mitochondria from adult hearts showed that loss of Tead1 led to a significant decrease in respiratory rates, with both palmitoylcarnitine and pyruvate/malate substrates, and was associated with reduced electron transport chain complex I activity and expression. Transcriptomic analysis from Tead1-knockout myocardium revealed genes encoding oxidative phosphorylation, TCA cycle, and fatty acid oxidation proteins as the top differentially enriched gene sets. Ex vivo loss of function of Tead1 in primary cardiomyocytes also showed diminished aerobic respiration and maximal mitochondrial oxygen consumption capacity, demonstrating that Tead1 regulation of OXPHOS in cardiomyocytes is cell autonomous. Taken together, our data demonstrate that Tead1 is a crucial transcriptional node that is a cell-autonomous regulator, a large network of mitochondrial function and biogenesis related genes essential for maintaining mitochondrial function and adult cardiomyocyte homeostasis.NEW & NOTEWORTHY Mitochondrial dysfunction constitutes an important aspect of heart failure etiopathogenesis and progression. However, the molecular mechanisms are still largely unknown. Growing lines of evidence indicate that Hippo-Tead pathway plays a role in cellular bioenergetics. This study reveals the novel role of Tead1, the downstream transcriptional effector of Hippo pathway, as a novel regulator of mitochondrial oxidative phosphorylation and in vivo cardiomyocyte energy metabolism, thus providing a potential therapeutic target for modulating mitochondrial function and enhancing cytoprotection of cardiomyocytes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Fatores de Transcrição/metabolismo , Animais , Respiração Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/genética , Transcriptoma
4.
Nat Commun ; 11(1): 2714, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483148

RESUMO

Electron transport chain (ETC) defects occurring from mitochondrial disease mutations compromise ATP synthesis and render cells vulnerable to nutrient and oxidative stress conditions. This bioenergetic failure is thought to underlie pathologies associated with mitochondrial diseases. However, the precise metabolic processes resulting from a defective mitochondrial ETC that compromise cell viability under stress conditions are not entirely understood. We design a whole genome gain-of-function CRISPR activation screen using human mitochondrial disease complex I (CI) mutant cells to identify genes whose increased function rescue glucose restriction-induced cell death. The top hit of the screen is the cytosolic Malic Enzyme (ME1), that is sufficient to enable survival and proliferation of CI mutant cells under nutrient stress conditions. Unexpectedly, this metabolic rescue is independent of increased ATP synthesis through glycolysis or oxidative phosphorylation, but dependent on ME1-produced NADPH and glutathione (GSH). Survival upon nutrient stress or pentose phosphate pathway (PPP) inhibition depends on compensatory NADPH production through the mitochondrial one-carbon metabolism that is severely compromised in CI mutant cells. Importantly, this defective CI-dependent decrease in mitochondrial NADPH production pathway or genetic ablation of SHMT2 causes strong increases in inflammatory cytokine signatures associated with redox dependent induction of ASK1 and activation of stress kinases p38 and JNK. These studies find that a major defect of CI deficiencies is decreased mitochondrial one-carbon NADPH production that is associated with increased inflammation and cell death.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Inflamação/metabolismo , Doenças Mitocondriais/metabolismo , Mutação , NADP/metabolismo , Animais , Morte Celular/genética , Linhagem Celular , Sobrevivência Celular/genética , Complexo I de Transporte de Elétrons/genética , Metabolismo Energético/genética , Glicólise/genética , Humanos , Inflamação/genética , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Fosforilação Oxidativa , Via de Pentose Fosfato/genética
5.
BMC Infect Dis ; 20(1): 262, 2020 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245373

RESUMO

BACKGROUND: Echinococcosis is a zoonotic parasitic disease causing serious health problems in both humans and animals in different endemic regions across the world. There are two different forms of human echinococcosis: Cystic Echinococcosis (CE) and Alveolar Echinococcosis (AE). CE is caused by the larval stage of Echinococcus granulosus sensu lato and AE by the larval stage of Echinococcus multilocularis. Geographically, CE is universally distributed, while AE is prevalent in the northern hemisphere. Although the disease is endemic in neighboring countries (China, Iran and India) of Pakistan, there are limited reports from that country. Besides, there are no comprehensive data on the genotyping of Echinococcus species in humans based on sequence analysis. This study aimed to detect the presence of human CE and to identify Echinococcus spp. in human isolates through genetic characterization of hydatid cysts in the Punjab Province of Pakistan. METHODS: Genetic analysis was performed on 38 human hydatid cyst samples collected from patients with echinococcosis using mitochondrial cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb) and NADH subunit 1 (nad1). Patient data including age, epidemiological history, sex, and location were obtained from hospital records. RESULTS: According to the sequence analysis we detected E. granulosus sensu stricto (n = 35), E. canadensis (G6/G7) (n = 2), and E. multilocularis (n = 1). Thus, the majority of the patients (92.1%, 35/38) were infected with E. granulosus s.s. This is the first molecular confirmation of E. canadensis (G6/G7) and E. multilocularis in human subjects from Pakistan. CONCLUSIONS: These findings suggested that E. granulosus s.s. is the dominant species in humans in Pakistan. In addition, E. canadensis (G6/G7) and E. multilocularis are circulating in the country. Further studies are required to explore the genetic diversity in both humans and livestock.


Assuntos
Equinococose/epidemiologia , Echinococcus granulosus/genética , Echinococcus multilocularis/genética , Análise de Sequência/métodos , Zoonoses/epidemiologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Ciclo-Oxigenase 1/genética , Citocromos b/genética , Equinococose/parasitologia , Echinococcus granulosus/isolamento & purificação , Echinococcus multilocularis/isolamento & purificação , Complexo I de Transporte de Elétrons/genética , Feminino , Genótipo , Humanos , Gado/parasitologia , Masculino , Pessoa de Meia-Idade , Paquistão/epidemiologia , Filogenia , Adulto Jovem , Zoonoses/parasitologia
6.
Nat Commun ; 11(1): 1643, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242014

RESUMO

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Animais , Complexo I de Transporte de Elétrons/genética , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
7.
Parasitol Res ; 119(2): 411-421, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31915912

RESUMO

Varroa destructor, a parasitic mite of the western honey bee, Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. Population genetics studies of the mite have provided information on two mitochondrial haplotypes infecting honey bee colonies, named K and J (after Korea and Japan, respectively, where they were originally identified). On the American continent, the K haplotype is much more prevalent, with the J haplotype only detected in some areas of Brazil. The aims of the present study were to assess the genetic diversity of V. destructor populations in the major beekeeping region of Argentina and to evaluate the presence of heteroplasmy at the nucleotide level. Phoretic mites were collected from managed A. mellifera colonies in ten localities, and four mitochondrial DNA (mtDNA) regions (COXI, ND4, ND4L, and ND5) were analyzed. Based on cytochrome oxidase subunit I (COXI) sequencing, exclusively the K haplotype of V. destructor was detected. Furthermore, two sub-haplotypes (KArg-N1 and KArg-N2) were identified from a variation in ND4 sequences and the frequency of these sub-haplotypes was found to significantly correlate with geographical latitude. The occurrence of site heteroplasmy was also evident for this gene. Therefore, ND4 appears to be a sensitive marker for detecting genetic variability in mite populations. Site heteroplasmy emerges as a phenomenon that could be relatively frequent in V. destructor.


Assuntos
Abelhas/parasitologia , DNA Mitocondrial/genética , Variação Genética/genética , Proteínas Mitocondriais/genética , Varroidae/genética , Animais , Argentina , Criação de Abelhas , Brasil , Complexo I de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Haplótipos , Japão , NADH Desidrogenase/genética , República da Coreia
8.
BMC Pediatr ; 20(1): 41, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996177

RESUMO

BACKGROUND: Maternally inherited complex I deficiencies due to mutations in MT-ND genes represent a heterogeneous group of multisystem mitochondrial disorders (MD) with a unfavourable prognosis. The aim of the study was to characterize the impact of the mutations in MT-ND genes, including the novel m.13091 T > C variant, on the course of the disease, and to analyse the activities of respiratory chain complexes, the amount of protein subunits, and the mitochondrial energy-generating system (MEGS) in available muscle biopsies and cultivated fibroblasts. METHODS: The respiratory chain complex activities were measured by spectrophotometry, MEGS were analysed using radiolabelled substrates, and protein amount by SDS-PAGE or BN-PAGE in muscle or fibroblasts. RESULTS: In our cohort of 106 unrelated families carrying different mtDNA mutations, we found heteroplasmic mutations in the genes MT-ND1, MT-ND3, and MT-ND5, including the novel variant m.13091 T > C, in 13 patients with MD from 12 families. First symptoms developed between early childhood and adolescence and progressed to multisystem disease with a phenotype of Leigh or MELAS syndromes. MRI revealed bilateral symmetrical involvement of deep grey matter typical of Leigh syndrome in 6 children, cortical/white matter stroke-like lesions suggesting MELAS syndrome in 3 patients, and a combination of cortico-subcortical lesions and grey matter involvement in 4 patients. MEGS indicated mitochondrial disturbances in all available muscle samples, as well as a significantly decreased oxidation of [1-14C] pyruvate in fibroblasts. Spectrophotometric analyses revealed a low activity of complex I and/or complex I + III in all muscle samples except one, but the activities in fibroblasts were mostly normal. No correlation was found between complex I activities and mtDNA mutation load, but higher levels of heteroplasmy were generally found in more severely affected patients. CONCLUSIONS: Maternally inherited complex I deficiencies were found in 11% of families with mitochondrial diseases in our region. Six patients manifested with Leigh, three with MELAS. The remaining four patients presented with an overlap between these two syndromes. MEGS, especially the oxidation of [1-14C] pyruvate in fibroblasts might serve as a sensitive indicator of functional impairment due to MT-ND mutations. Early onset of the disease and higher level of mtDNA heteroplasmy were associated with a worse prognosis.


Assuntos
DNA Mitocondrial , Complexo I de Transporte de Elétrons/deficiência , Doença de Leigh/genética , Síndrome MELAS/genética , Doenças Mitocondriais/genética , Mutação , Adolescente , Adulto , Idade de Início , Biópsia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Células Cultivadas , Criança , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Imagem por Ressonância Magnética , Músculo Esquelético/metabolismo
9.
EMBO J ; 39(3): e102817, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31912925

RESUMO

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/metabolismo
10.
DNA Cell Biol ; 39(1): 105-117, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31794266

RESUMO

Septic cardiomyopathy (SC) is a rare and harmful cardiovascular disease with decreased left ventricular (LV) output and multiple organ failure, which poses a serious threat to human life. Despite the advances in SC, its diagnostic basis and treatment methods are limited, and the specific diagnostic biomarkers and its candidate regulatory targets have not yet been fully established. In this study, the GSE79962 gene expression profile was retrieved, with 20 patients with SC and 11 healthy donors as control. Weighted gene coexpression network analysis (WGCNA) was employed to investigate gene modules that were strongly correlated with clinical phenotypes. Blue module was found to be most significantly related to SC. Moreover, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the coexpression genes in blue module and showed that it was associated with metabolic pathways, oxidative phosphorylation, and cardiac muscle contraction. Furthermore, a total of 10 hub genes NDUFB5, TIMMDC1, VDAC3, COQ10A, MRPL16 (mitochondrial ribosomal protein L16), C3orf43, TMEM182, DLAT, NDUFA8, and PDHB (pyruvate dehydrogenase E1 beta subunit) in the blue module were identified at transcriptional level and further validated at translational level in myocardium of an lipopolysaccharide-induced septic cardiac dysfunction mouse model. Overall, the results of quantitative real-time polymerase chain reaction were consistent with most of the microarray analysis results. Intriguingly, we observed that the highest change was NDUFB5, TIMMDC1, and VDAC3. These identified and validated genes provided references that would advance the understanding of molecular mechanisms of SC. Taken together, using WGCNA, the hub genes NDUFB5, TIMMDC1, and VDAC3 might serve as potential biomarkers for diagnosis and/or therapeutic targets for precise treatment of SC in the future.


Assuntos
Cardiomiopatias/genética , Complexo I de Transporte de Elétrons/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Transporte da Membrana Mitocondrial/genética , Sepse/genética , Canais de Ânion Dependentes de Voltagem/genética , Idoso , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Biologia Computacional/métodos , Progressão da Doença , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Ontologia Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sepse/metabolismo , Sepse/patologia , Canais de Ânion Dependentes de Voltagem/metabolismo
11.
Biochim Biophys Acta Bioenerg ; 1861(2): 148133, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31825807

RESUMO

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII2, yielding a small amount of active CI+CIII2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH2-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII2, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII2 and CIV2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.


Assuntos
Trifosfato de Adenosina/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Consumo de Oxigênio , Animais , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Deleção de Genes , Mitocôndrias/genética , Oxirredução , Rotenona/farmacologia
12.
Biochim Biophys Acta Bioenerg ; 1861(2): 148137, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31825809

RESUMO

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc1 complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.


Assuntos
Aldeído Oxidase/metabolismo , Ciona intestinalis/genética , Expressão Gênica , Mitocôndrias Cardíacas/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Aldeído Oxidase/genética , Animais , Ciclo do Ácido Cítrico/genética , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , Mitocôndrias Cardíacas/genética , Consumo de Oxigênio/genética , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
13.
Sci Adv ; 5(12): eaax9484, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31844670

RESUMO

Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.


Assuntos
Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/ultraestrutura , Mitocôndrias/ultraestrutura , Yarrowia/ultraestrutura , Trifosfato de Adenosina/química , Complexo I de Transporte de Elétrons/genética , Humanos , Mitocôndrias/genética , Membranas Mitocondriais , Conformação Proteica , Yarrowia/genética
14.
Parasite ; 26: 73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31855174

RESUMO

Heterosentis holospinus Amin, Heckmann & Ha, 2011 (Arhythmacanthidae) was first described from the striped eel catfish, Plotosus lineatus (Plotosidae) in Halong Bay, Vietnam. New morphological information, scanning electron microscope images, molecular analysis, and Energy Dispersive X-ray analysis (EDXA) of hooks of specimens of H. holospinus from a new collection from the common ponyfish, Leiognathus equulus (Leiognathidae), in Quang Binh, Gulf of Tonkin, Vietnam are reported here for the first time. Additional details of the anterior trunk cone, proboscis hooks, wholly spined trunk, duck-bill-like spines with micropores, and micropore distribution, are described. The unique metal composition of hooks (EDXA) demonstrated a considerably higher level of calcium and phosphorus but lower level of sulfur at the hook basal arch than at the hook tip and edge. An analysis of our new sequences of cytochrome oxidase 1 (COI) showed that H. holospinus had low genetic variation and two haplotypes.


Assuntos
Acantocéfalos/anatomia & histologia , Acantocéfalos/classificação , Peixes/parasitologia , Helmintíase Animal/parasitologia , Animais , Complexo I de Transporte de Elétrons/genética , Feminino , Doenças dos Peixes/parasitologia , Variação Genética , Haplótipos , Masculino , Microscopia Eletrônica de Varredura , Oceano Pacífico , Vietnã
15.
Biochem Soc Trans ; 47(6): 1963-1969, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31769488

RESUMO

Mitochondria control vitally important functions in cells, including energy production, cell signalling and regulation of cell death. Considering this, any alteration in mitochondrial metabolism would lead to cellular dysfunction and the development of a disease. A large proportion of disorders associated with mitochondria are induced by mutations or chemical inhibition of the mitochondrial complex I - the entry point to the electron transport chain. Subunits of the enzyme NADH: ubiquinone oxidoreductase, are encoded by both nuclear and mitochondrial DNA and mutations in these genes lead to cardio and muscular pathologies and diseases of the central nervous system. Despite such a clear involvement of complex I deficiency in numerous disorders, the molecular and cellular mechanisms leading to the development of pathology are not very clear. In this review, we summarise how lack of activity of complex I could differentially change mitochondrial and cellular functions and how these changes could lead to a pathology, following discrete routes.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Complexo I de Transporte de Elétrons/genética , Metabolismo Energético , Humanos , Mitocôndrias/metabolismo , Mutação , Doença de Parkinson/metabolismo , Espécies Reativas de Oxigênio/metabolismo
16.
Mol Cell Neurosci ; 101: 103417, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31678567

RESUMO

Mitochondria play essential metabolic roles in neural cells. Mitochondrial dysfunction has profound effects on the brain. In primary mitochondrial diseases, mutations that impair specific oxidative phosphorylation (OXPHOS) proteins or OXPHOS assembly factors lead to isolated biochemical defects and a heterogeneous group of clinical phenotypes, including mitochondrial encephalopathies. A broader defect of OXPHOS function, due to mutations in proteins involved in mitochondrial DNA maintenance, mitochondrial biogenesis, or mitochondrial tRNAs can also underlie severe mitochondrial encephalopathies. While primary mitochondrial dysfunction causes rare genetic forms of neurological disorders, secondary mitochondrial dysfunction is involved in the pathophysiology of some of the most common neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Many studies have investigated mitochondrial function and dysfunction in bulk central nervous system (CNS) tissue. However, the interpretation of these studies has been often complicated by the extreme cellular heterogeneity of the CNS, which includes many different types of neurons and glial cells. Because neurons are especially dependent on OXPHOS for ATP generation, mitochondrial dysfunction is thought to be directly involved in cell autonomous neuronal demise. Despite being metabolically more flexible than neurons, glial mitochondria also play an essential role in the function of the CNS, and have adapted specific metabolic and mitochondrial features to support their diversity of functions. This review analyzes our current understanding and the gaps in knowledge of mitochondrial properties of glia and how they affect neuronal functions, in health and disease.


Assuntos
Astrócitos/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Astrócitos/patologia , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Doenças Neurodegenerativas/genética , Fosforilação Oxidativa
17.
Environ Microbiol ; 21(12): 4724-4739, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31595636

RESUMO

Strategy of managing antibiotic-resistant Vibrio alginolyticus, a bacterial pathogen that threatens human health and animal farming, is not available due to the lack of knowledge about the underlying mechanism of antibiotic resistance. Here, we showed that gentamicin-resistant V. alginolyticus (VA-RGEN ) has four mutations on metabolism and one mutation on a two-component system by whole-genome and PCR-based sequencing, indicating the metabolic shift in VA-RGEN. Thus, metabolic profile was investigated by GC-MS based metabolomics. Glucose was identified as a crucial biomarker, whose abundance was decreased in VA-RGEN . Further analysis with iPath, and gene expression and enzyme activity of the pyruvate cycle (the P cycle) demonstrated a global depressed metabolic pathway network in VA-RGEN . Consistently, NADH, sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) system, membrane potential and intracellular gentamicin were decreased in VA-RGEN . These findings indicate that the reduced redox state contributes to antibiotic resistance. Interestingly, exogenous glucose potentiated gentamicin to efficiently kill VA-RGEN through the promotion of the P cycle, NADH, membrane potential and intracellular gentamicin. The potentiation was further confirmed in a zebrafish model. These results indicate that the gentamicin resistance reduces the P cycle and Na(+)-NQR system and thereby decreases redox state, membrane potential and gentamicin uptake, which can be reversed by exogenous glucose.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Gentamicinas/farmacologia , Glucose/metabolismo , Vibrio alginolyticus/metabolismo , Animais , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Gentamicinas/metabolismo , Oxirredução , Vibrio/metabolismo , Vibrio alginolyticus/efeitos dos fármacos , Vibrio alginolyticus/genética
18.
Int J Biochem Cell Biol ; 116: 105616, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31542429

RESUMO

Type 2 diabetes has become an epidemic disease largely explained by the dramatic increase in obesity in recent years. Mitochondrial dysfunction is suggested as an underlying factor in obesity and type 2 diabetes. In this study, we evaluated changes in oxidative phosphorylation and mitochondrial biogenesis in a new human obesity and type 2 diabetes model, TALLYHO/Jng mice. We hypothesized that the sequence variants identified in the whole genome analysis of TALLYHO/Jng mice would affect oxidative phosphorylation and contribute to obesity and insulin resistant phenotypes. To test this hypothesis, we investigated differences in the expression and activity of oxidative phosphorylation complexes, including the transcription and translation of nuclear- and mitochondrial-encoded subunits and enzymatic activities, in the liver and kidney of TALLYHO/Jng and C57BL/6 J mice. A significant decrease was observed in the expression of nuclear- and mitochondrial-encoded subunits of complex I and IV, respectively, in TALLYHO/Jng mice, which coincided with significant reductions in their enzymatic activities. Furthermore, sequence variants were identified in oxidative phosphorylation complex subunits, a mitochondrial tRNA synthetase, and mitochondrial ribosomal proteins. Our data suggested that the lower expression and activity of oxidative phosphorylation complexes results in the diminished energy metabolism observed in TALLYHO/Jng mice. Sequence variants identified in mitochondrial proteins accentuated a defect in mitochondrial protein synthesis which also contributes to impaired biogenesis and oxidative phosphorylation in TALLYHO/Jng mice. These results demonstrated that the identification of factors contributing to mitochondrial dysfunction will allow us to improve the disease prognosis and treatment of obesity and type 2 diabetes in humans.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Obesidade/metabolismo , Fosforilação Oxidativa , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/genética , Obesidade/genética , Obesidade/patologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
19.
Int J Mol Sci ; 20(18)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31491876

RESUMO

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.


Assuntos
Complexo I de Transporte de Elétrons/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , alfa-Galactosidase/biossíntese , Linhagem Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Doença de Fabry/genética , Doença de Fabry/metabolismo , Dosagem de Genes , Expressão Gênica , Humanos , Células Jurkat , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Transdução Genética , Transgenes , alfa-Galactosidase/genética
20.
Genes (Basel) ; 10(9)2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500202

RESUMO

The buffalo (Bubalus bubalis.L) is prevalent in China and the increasing demand for meat production has changed its role from being a beast of burden to a meat source. The low fat deposition level has become one of the main barriers for its use in meat production. It is urgent to reveal factors involved in fat deposition in buffalo. This study performed RNA sequencing to investigate both long noncoding RNAs (lncRNAs) and mRNAs of adipose tissues in young and adult buffalos. A total of 124 lncRNAs and 2008 mRNAs showed differential expression patterns between young and adult samples. Coexpression analysis and functional enrichment revealed 585 mRNA-lncRNA pairs with potential function in fat deposition. After validation by qRT-PCR, we focused on a lncRNA transcribed from the ubiquinone oxidoreductase subunit C2 (NDUFC2) antisense (AS) strand which showed high correlation with thyroid hormone responsive protein (THRSP). NDUFC2-AS lncRNA is highly expressed in adipose tissue and maturation adipocytes and mainly exists in the nucleus. Functional assays demonstrated that NDUFC2-AS lncRNA promotes adipogenic differentiation by upregulating the expression levels of THRSP and CCAAT enhancer binding protein alpha (C/EBPα) in buffalo. These results indicate that NDUFC2-AS lncRNA promotes fat deposition in buffalo.


Assuntos
Adipócitos/metabolismo , Adipogenia , Búfalos/genética , RNA Longo não Codificante/genética , Adipócitos/citologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Complexo I de Transporte de Elétrons/genética , Feminino , Masculino
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