RESUMO
The bactericidal activity of several antibiotics partially relies on the production of reactive oxygen species (ROS), which is generally linked to enhanced respiration and requires the Fenton reaction. Bacterial persister cells, an important cause of recurring infections, are tolerant to these antibiotics because they are in a dormant state. Here, we use Bacillus subtilis cells in stationary phase, as a model system of dormant cells, to show that pharmacological induction of membrane depolarization enhances the antibiotics' bactericidal activity and also leads to ROS production. However, in contrast to previous studies, this results primarily in production of superoxide radicals and does not require the Fenton reaction. Genetic analyzes indicate that Rieske factor QcrA, the iron-sulfur subunit of respiratory complex III, seems to be a primary source of superoxide radicals. Interestingly, the membrane distribution of QcrA changes upon membrane depolarization, suggesting a dissociation of complex III. Thus, our data reveal an alternative mechanism by which antibiotics can cause lethal ROS levels, and may partially explain why membrane-targeting antibiotics are effective in eliminating persisters.
Assuntos
Antibacterianos , Bacillus subtilis , Membrana Celular , Espécies Reativas de Oxigênio , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Superóxidos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view. SIGNIFICANCE: A crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)-1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.
Assuntos
Oxirredução , Cinética , Ligantes , Transporte de Elétrons , Fotossíntese , Luz , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/química , Bacterioclorofilas/metabolismo , Bacterioclorofilas/químicaRESUMO
Aerobic life is powered by membrane-bound redox enzymes that shuttle electrons to oxygen and transfer protons across a biological membrane. Structural studies suggest that these energy-transducing enzymes operate as higher-order supercomplexes, but their functional role remains poorly understood and highly debated. Here we resolve the functional dynamics of the 0.7 MDa III2IV2 obligate supercomplex from Mycobacterium smegmatis, a close relative of M. tuberculosis, the causative agent of tuberculosis. By combining computational, biochemical, and high-resolution (2.3 Å) cryo-electron microscopy experiments, we show how the mycobacterial supercomplex catalyses long-range charge transport from its menaquinol oxidation site to the binuclear active site for oxygen reduction. Our data reveal proton and electron pathways responsible for the charge transfer reactions, mechanistic principles of the quinone catalysis, and how unique molecular adaptations, water molecules, and lipid interactions enable the proton-coupled electron transfer (PCET) reactions. Our combined findings provide a mechanistic blueprint of mycobacterial supercomplexes and a basis for developing drugs against pathogenic bacteria.
Assuntos
Microscopia Crioeletrônica , Mycobacterium smegmatis , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/enzimologia , Transporte de Elétrons , Oxirredução , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Prótons , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Oxigênio/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Domínio Catalítico , Modelos MolecularesRESUMO
Hydrogen sulfide (H2S) has recently been recognized as an important gaseous transmitter with multiple physiological effects in various species. Previous studies have shown that H2S alleviated heat-induced ganoderic acids (GAs) biosynthesis, an important quality index of Ganoderma lucidum. However, a comprehensive understanding of the physiological effects and molecular mechanisms of H2S in G. lucidum remains unexplored. In this study, we found that heat treatment reduced the mitochondrial membrane potential (MMP) and mitochondrial DNA copy number (mtDNAcn) in G. lucidum. Increasing the intracellular H2S concentration through pharmacological and genetic means increased the MMP level, mtDNAcn, oxygen consumption rate level and ATP content under heat treatment, suggesting a role for H2S in mitigating heat-caused mitochondrial damage in G. lucidum. Further results indicated that H2S activates sulfide-quinone oxidoreductase (SQR) and complex III (Com III), thereby maintaining mitochondrial homeostasis under heat stress in G. lucidum. Moreover, SQR also mediated the negative regulation of H2S to GAs biosynthesis under heat stress. Furthermore, SQR might be persulfidated under heat stress in G. lucidum. Thus, our study reveals a novel physiological function and molecular mechanism of H2S signalling under heat stress in G. lucidum with broad implications for research on the environmental response of microorganisms.
Assuntos
Resposta ao Choque Térmico , Homeostase , Sulfeto de Hidrogênio , Potencial da Membrana Mitocondrial , Mitocôndrias , Reishi , Triterpenos , Sulfeto de Hidrogênio/metabolismo , Reishi/metabolismo , Reishi/genética , Triterpenos/metabolismo , Mitocôndrias/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Quinona Redutases/metabolismo , Quinona Redutases/genética , DNA Mitocondrial/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genéticaRESUMO
Root-knot nematodes (RKN; Meloidogyne species) are plant pathogens that introduce several effectors in their hosts to facilitate infection. The actual targets and functioning mechanism of these effectors largely remain unexplored. This study illuminates the role and interplay of the Meloidogyne javanica nematode effector ROS suppressor (Mj-NEROSs) within the host plant environment. Mj-NEROSs suppresses INF1-induced cell death as well as flg22-induced callose deposition and reactive oxygen species (ROS) production. A transcriptome analysis highlighted the downregulation of ROS-related genes upon Mj-NEROSs expression. NEROSs interacts with the plant Rieske's iron-sulfur protein (ISP) as shown by yeast-two-hybrid and bimolecular fluorescence complementation. Secreted from the subventral pharyngeal glands into giant cells, Mj-NEROSs localizes in the plastids where it interacts with ISP, subsequently altering electron transport rates and ROS production. Moreover, our results demonstrate that isp Arabidopsis thaliana mutants exhibit increased susceptibility to M. javanica, indicating ISP importance for plant immunity. The interaction of a nematode effector with a plastid protein highlights the possible role of root plastids in plant defense, prompting many questions on the details of this process.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Proteínas de Helminto , Proteínas Ferro-Enxofre , Imunidade Vegetal , Plastídeos , Espécies Reativas de Oxigênio , Animais , Arabidopsis/parasitologia , Arabidopsis/imunologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Mutação/genética , Doenças das Plantas/parasitologia , Doenças das Plantas/imunologia , Plastídeos/metabolismo , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Tylenchoidea/genética , Tylenchoidea/patogenicidadeRESUMO
Mitochondria play a pivotal part in ATP energy production through oxidative phosphorylation, which occurs within the inner membrane through a series of respiratory complexes1-4. Despite extensive in vitro structural studies, determining the atomic details of their molecular mechanisms in physiological states remains a major challenge, primarily because of loss of the native environment during purification. Here we directly image porcine mitochondria using an in situ cryo-electron microscopy approach. This enables us to determine the structures of various high-order assemblies of respiratory supercomplexes in their native states. We identify four main supercomplex organizations: I1III2IV1, I1III2IV2, I2III2IV2 and I2III4IV2, which potentially expand into higher-order arrays on the inner membranes. These diverse supercomplexes are largely formed by 'protein-lipids-protein' interactions, which in turn have a substantial impact on the local geometry of the surrounding membranes. Our in situ structures also capture numerous reactive intermediates within these respiratory supercomplexes, shedding light on the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. Structural comparison of supercomplexes from mitochondria treated under different conditions indicates a possible correlation between conformational states of complexes I and III, probably in response to environmental changes. By preserving the native membrane environment, our approach enables structural studies of mitochondrial respiratory supercomplexes in reaction at high resolution across multiple scales, from atomic-level details to the broader subcellular context.
Assuntos
Respiração Celular , Complexo III da Cadeia de Transporte de Elétrons , Complexo I de Transporte de Elétrons , Mitocôndrias , Animais , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/ultraestrutura , Modelos Moleculares , Fosforilação Oxidativa , Suínos , Ubiquinona/análogos & derivados , Ubiquinona/química , Ubiquinona/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismoRESUMO
Rieske non-heme iron oxygenases (ROs) are redox enzymes essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful biocatalysts due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H2O2) formation in the presence of a NADH cofactor regeneration system on the catalytic efficiency of CDO in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H2O2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20â¯mM) over 24â¯h (TTN of 1515) at a 3-fold increase in production rate.
Assuntos
Peróxido de Hidrogênio , Oxigênio , Oxigênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxirredução , Oxigenases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , NAD/metabolismo , Sistema Livre de Células , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismoRESUMO
Through a comprehensive molecular docking study, a unique series of naphthoquinones clubbed azetidinone scaffolds was arrived with promising binding affinity to Mycobacterial Cytbc1 complex, a drug target chosen to kill multi-drug resistant Mycobacterium tuberculosis (MDR-Mtb). Five compounds from series-2, 2a, 2c, 2g, 2h, and 2j, showcased significant in vitro anti-tubercular activities against Mtb H37Rv and MDR clinical isolates. Further, synergistic studies of these compounds in combination with INH and RIF revealed a potent bactericidal effect of compound 2a at concentration of 0.39 µg/mL, and remaining (2c, 2g, 2h, and 2j) at 0.78 µg/mL. Exploration into the mechanism study through chemo-stress assay and proteome profiling uncovered the down-regulation of key proteins of electron-transport chain and Cytbc1 inhibition pathway. Metabolomics corroborated these proteome findings, and heightened further understanding of the underlying mechanism. Notably, in vitro and in vivo animal toxicity studies demonstrated minimal toxicity, thus underscoring the potential of these compounds as promising anti-TB agents in combination with RIF and INH. These active compounds adhered to Lipinski's Rule of Five, indicating the suitability of these compounds for drug development. Particular significance of molecules NQ02, 2a, and 2h, which have been patented (Published 202141033473).
Assuntos
Antituberculosos , Complexo III da Cadeia de Transporte de Elétrons , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Antituberculosos/química , Antituberculosos/síntese química , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Simulação de Acoplamento Molecular , Benzoquinonas/química , Benzoquinonas/farmacologia , Animais , Humanos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Sinergismo FarmacológicoRESUMO
The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Complexo III da Cadeia de Transporte de Elétrons , Humanos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Microscopia Crioeletrônica , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Hidrólise , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Transporte ProteicoRESUMO
Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske iron-sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in α-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.
Assuntos
Proliferação de Células , Mitocôndrias Cardíacas , Miócitos Cardíacos , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Camundongos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Camundongos Knockout , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Glucose/metabolismoRESUMO
The functional and structural relationship among the individual components of the mitochondrial respiratory chain constitutes a central aspect of our understanding of aerobic catabolism. This interplay has been a subject of intense debate for over 50 years. It is well established that individual respiratory enzymes associate into higher-order structures known as respiratory supercomplexes, which represent the evolutionarily conserved organizing principle of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, supercomplexes are formed by a complex III homodimer flanked by one or two complex IV monomers, and their high-resolution structures have been recently elucidated. Despite the wealth of structural information, several proposed supercomplex functions remain speculative and our understanding of their physiological relevance is still limited. Recent advances in the field were made possible by the construction of yeast strains where the association of complex III and IV into supercomplexes is impeded, leading to diminished respiratory capacity and compromised cellular competitive fitness. Here, we discuss the experimental evidence and hypotheses relative to the functional roles of yeast respiratory supercomplexes. Moreover, we review the current models of yeast complex III and IV assembly in the context of supercomplex formation and highlight the data scattered throughout the literature suggesting the existence of cross talk between their biogenetic processes.
Assuntos
Mitocôndrias , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Transporte de Elétrons , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In the present study, we examined the mitochondrial hydrogen peroxide (mH2O2) generating capacity of α-ketoglutarate dehydrogenase (KGDH) and compared it to components of the electron transport chain using liver mitochondria isolated from male and female C57BL6N mice. We show for the first time there are some sex dimorphisms in the production of mH2O2 by electron transport chain complexes I and III when mitochondria are fueled with different substrates. However, in our investigations into these sex effects, we made the unexpected and compelling discovery that 1) KGDH serves as a major mH2O2 supplier in male and female liver mitochondria and 2) KGDH can form mH2O2 when liver mitochondria are energized with fatty acids but only when malate is used to prime the Krebs cycle. Surprisingly, 2-keto-3-methylvaleric acid (KMV), a site-specific inhibitor for KGDH, nearly abolished mH2O2 generation in both male and female liver mitochondria oxidizing palmitoyl-carnitine. KMV inhibited mH2O2 production in liver mitochondria from male and female mice oxidizing myristoyl-, octanoyl-, or butyryl-carnitine as well. S1QEL 1.1 (S1) and S3QEL 2 (S3), compounds that inhibit reactive oxygen species generation by complexes I and III, respectively, without interfering with OxPhos and respiration, had a negligible effect on the rate of mH2O2 production when pyruvate or acyl-carnitines were used as fuels. However, inclusion of KMV in reaction mixtures containing S1 and/or S3 almost abolished mH2O2 generation. Together, our findings suggest KGDH is the main mH2O2 generator in liver mitochondria, even when fatty acids are used as fuel.
Assuntos
Ácidos Graxos , Peróxido de Hidrogênio , Complexo Cetoglutarato Desidrogenase , Mitocôndrias Hepáticas , Animais , Feminino , Masculino , Camundongos , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Peróxido de Hidrogênio/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , OxirreduçãoRESUMO
It is well known that in the heart and kidney mitochondria, more than 95% of ATP production is supported by the ß-oxidation of long-chain fatty acids. However, the ß-oxidation of fatty acids by mitochondria has been studied much less than the substrates formed during the catabolism of carbohydrates and amino acids. In the last few decades, several discoveries have been made that are directly related to fatty acid oxidation. In this review, we made an attempt to re-evaluate the ß-oxidation of long-chain fatty acids from the perspectives of new discoveries. The single set of electron transporters of the cardiac mitochondrial respiratory chain is organized into three supercomplexes. Two of them contain complex I, a dimer of complex III, and two dimers of complex IV. The third, smaller supercomplex contains a dimer of complex III and two dimers of complex IV. We also considered other important discoveries. First, the enzymes of the ß-oxidation of fatty acids are physically associated with the respirasome. Second, the ß-oxidation of fatty acids creates the highest level of QH2 and reverses the flow of electrons from QH2 through complex II, reducing fumarate to succinate. Third, ß-oxidation is greatly stimulated in the presence of succinate. We argue that the respirasome is uniquely adapted for the ß-oxidation of fatty acids. The acyl-CoA dehydrogenase complex reduces the membrane's pool of ubiquinone to QH2, which is instantly oxidized by the smaller supercomplex, generating a high energization of mitochondria and reversing the electron flow through complex II, which reverses the electron flow through complex I, increasing the NADH/NAD+ ratio in the matrix. The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes a hydride (H-, a proton plus two electrons) transfer across the inner mitochondrial membrane, reducing the cytosolic pool of NADP(H), thus providing the heart with ATP for muscle contraction and energy and reducing equivalents for the housekeeping processes.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Ácidos Graxos , Ácidos Graxos/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Oxirredução , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ácido Succínico/metabolismo , Succinatos/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Mitochondrial electron transport chain complexes organize into supramolecular structures called respiratory supercomplexes (SCs). The role of respiratory SCs remains largely unconfirmed despite evidence supporting their necessity for mitochondrial respiratory function. The mechanisms underlying the formation of the I1III2IV1 "respirasome" SC are also not fully understood, further limiting insights into these processes in physiology and diseases, including neurodegeneration and metabolic syndromes. NDUFB4 is a complex I accessory subunit that contains residues that interact with the subunit UQCRC1 from complex III, suggesting that NDUFB4 is integral for I1III2IV1 respirasome integrity. Here, we introduced specific point mutations to Asn24 (N24) and Arg30 (R30) residues on NDUFB4 to decipher the role of I1III2-containing respiratory SCs in cellular metabolism while minimizing the functional consequences to complex I assembly. Our results demonstrate that NDUFB4 point mutations N24A and R30A impair I1III2IV1 respirasome assembly and reduce mitochondrial respiratory flux. Steady-state metabolomics also revealed a global decrease in citric acid cycle metabolites, affecting NADH-generating substrates. Taken together, our findings highlight an integral role of NDUFB4 in respirasome assembly and demonstrate the functional significance of SCs in regulating mammalian cell bioenergetics.
Assuntos
Complexo I de Transporte de Elétrons , Mitocôndrias , Transporte de Elétrons , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Humanos , Células HEK293RESUMO
Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart.
Assuntos
Doenças Mitocondriais , Miócitos Cardíacos , Adulto , Camundongos , Humanos , Animais , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Antioxidantes/metabolismo , Trifosfato de Adenosina/metabolismo , Doenças Mitocondriais/metabolismo , Mamíferos/metabolismoRESUMO
Respiratory complex III (a.k.a., the bc1 complex) plays a key role in the electron transport chain in aerobic cells. The bc1 complex exhibits multiple unique electron tunneling (ET) processes, such as ET-bifurcation at the Qo site and movement of the Rieske domain. Moreover, we previously discovered that electron tunneling in the low potential arm of the bc1 complex is regulated by a key phenylalanine residue (Phe90). The main goal of the current work is to study the dynamics of the key Phe90 residue in the electron tunneling reaction between heme bL and heme bH as a function of the occupancy of the Qo and Qi binding sites in the bc1 complex. We simulated the molecular dynamics of four model systems of respiratory complex III with different ligands bound at the Qo and Qi binding sites. In addition, we calculated the electron tunneling rate constants between heme bL and heme bH along the simulated molecular dynamics trajectories. The binding of aromatic ligands at the Qo site induces a conformational cascade that properly positions the Phe90 residue, reducing the through-space ET distance from â¼7 to â¼5.5 Å and thus enhancing the electron transfer rate between the heme bL and the heme bH redox pair. Also, the binding of aromatic ligands at the Qi site induces conformational changes that stabilize the Phe90 conformational variation from â¼1.5 to â¼0.5 Å. Hence, our molecular dynamics simulation results show an on-demand two-step conformational connection between the occupancy of the Qo and Qi binding sites and the conformational dynamics of the Phe90 residue. Additionally, our dynamic electron tunneling results confirm our previously reported findings that the Phe90 residue acts as an electron-tunneling gate or switch, controlling the electron transfer rate between the heme bL and heme bH redox systems.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Elétrons , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Ligantes , Transporte de Elétrons , Oxirredução , Sítios de Ligação , Simulação de Dinâmica Molecular , Heme/químicaRESUMO
Due to the limitations of the present risk genes in understanding the etiology of amyotrophic lateral sclerosis (ALS), it is necessary to find additional causative genes utilizing novel approaches. In this study, we conducted a two-stage proteome-wide association study (PWAS) using ALS genome-wide association study (GWAS) data (N = 152,268) and two distinct human brain protein quantitative trait loci (pQTL) datasets (ROSMAP N = 376 and Banner N = 152) to identify ALS risk genes and prioritized candidate genes with Mendelian randomization (MR) and Bayesian colocalization analysis. Next, we verified the aberrant expression of risk genes in multiple tissues, including lower motor neurons, skeletal muscle, and whole blood. Six ALS risk genes (SCFD1, SARM1, TMEM175, BCS1L, WIPI2, and DHRS11) were found during the PWAS discovery phase, and SARM1 and BCS1L were confirmed during the validation phase. The following MR (p = 2.10 × 10-7) and Bayesian colocalization analysis (ROSMAP PP4 = 0.999, Banner PP4 = 0.999) confirmed the causal association between SARM1 and ALS. Further differential expression analysis revealed that SARM1 was markedly downregulated in lower motor neurons (p = 7.64 × 10-3), skeletal muscle (p = 9.34 × 10-3), and whole blood (p = 1.94 × 10-3). Our findings identified some promising protein candidates for future investigation as therapeutic targets. The dysregulation of SARM1 in multiple tissues provides a new way to explain ALS pathology.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Estudo de Associação Genômica Ampla , Teorema de Bayes , Encéfalo/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , 17-Hidroxiesteroide Desidrogenases/metabolismoRESUMO
The respiratory chain in aerobic organisms is composed of a number of membrane-bound protein complexes that link electron transfer to proton translocation across the membrane. In mitochondria, the final electron acceptor, complex IV (CIV), receives electrons from dimeric complex III (CIII2), via a mobile electron carrier, cytochrome c. In the present study, we isolated the CIII2CIV supercomplex from the fission yeast Schizosaccharomyces pombe and determined its structure with bound cyt. c using single-particle electron cryomicroscopy. A respiratory supercomplex factor 2 was found to be bound at CIV distally positioned in the supercomplex. In addition to the redox-active metal sites, we found a metal ion, presumably Zn2+, coordinated in the CIII subunit Cor1, which is encoded by the same gene (qcr1) as the mitochondrial-processing peptidase subunit ß. Our data show that the isolated CIII2CIV supercomplex displays proteolytic activity suggesting a dual role of CIII2 in S. pombe. As in the supercomplex from S. cerevisiae, subunit Cox5 of CIV faces towards one CIII monomer, but in S. pombe, the two complexes are rotated relative to each other by ~45°. This orientation yields equal distances between the cyt. c binding sites at CIV and at each of the two CIII monomers. The structure shows cyt. c bound at four positions, but only along one of the two symmetrical branches. Overall, this combined structural and functional study reveals the integration of peptidase activity with the CIII2 respiratory system and indicates a two-dimensional cyt. c diffusion mechanism within the CIII2-CIV supercomplex.
Assuntos
Schizosaccharomyces , Schizosaccharomyces/metabolismo , Saccharomyces cerevisiae/metabolismo , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Elétrons , Peptídeo Hidrolases/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismoRESUMO
Metabolic pathways and proteins responsible for maintaining mitochondrial dynamics and homeostasis in the Plasmodium parasite, the causative agent of malaria, remain to be elucidated. Here, we identified and functionally characterized a novel OPA3-like domain-containing protein in P. falciparum (PfOPA3). We show that PfOPA3 is expressed in the intraerythrocytic stages of the parasite and localizes to the mitochondria. Inducible knock-down of PfOPA3 using GlmS ribozyme hindered the normal intraerythrocytic cycle of the parasites; specifically, PfOPA3-iKD disrupted parasite development as well as parasite division and segregation at schizont stages, which resulted in a drastic reduction in the number of merozoites progenies. Parasites lacking PfOPA3 show severe defects in the development of functional mitochondria; the mitochondria showed reduced activity of mtETC but not ATP synthesis, as evidenced by reduced activity of complex III of the mtETC, and increased sensitivity for drugs targeting DHODH as well as complex III, but not to the drugs targeting complex V. Further, PfOPA3 downregulation leads to reduction in the level of mitochondrial proton transport uncoupling protein (PfUCP) to compensate reduced activity of complex III and maintain proton efflux across the inner membrane. The reduced activity of DHODH, which is responsible for pyrimidine biosynthesis required for nuclear DNA synthesis, resulted in a significant reduction in parasite nuclear division and generation of progeny. In conclusion, we show that PfOPA3 is essential for the functioning of mtETC and homeostasis required for the development of functional mitochondria as well as for parasite segregation, and thus PfOPA3 is crucial for parasite survival during blood stages.
Assuntos
Malária Falciparum , Parasitos , Animais , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Parasitos/metabolismo , Di-Hidro-Orotato Desidrogenase , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Prótons , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Malária Falciparum/metabolismo , Mitocôndrias/metabolismo , Homeostase , Proliferação de Células , Eritrócitos/metabolismoRESUMO
Aims: Mitochondrial dysfunction is closely associated with the development of diabetic complications. In diabetic retinopathy, electron transport chain is compromised and mitochondrial DNA (mtDNA) is damaged, downregulating transcription of mtDNA-encoded cytochrome B (CYTB) and its antisense long noncoding RNA, long noncoding RNA cytochrome B (LncCytB). Our goal was to investigate the role of LncCytB in the regulation of CYTB and mitochondrial function in diabetic retinopathy. Methods: Using human retinal endothelial cells, genetically manipulated for LncCytB (overexpression or silencing), the effect of high glucose (20 mM d-glucose) on LncCytB-CYTB interactions (by chromatin isolation by RNA purification), CYTB gene expression (by real-time quantitative polymerase chain reaction), complex III activity, mitochondrial free radicals, and oxygen consumption rate (OCR, by Seahorse XF analyzer) was investigated. Key results were confirmed in the retinal microvessels from streptozotocin-induced diabetic mice. Results: High glucose decreased LncCytB-CYTB interactions, and while LncCytB overexpression ameliorated glucose-induced decrease in CYTB gene transcripts, complex III activity and OCR and increase in mitochondrial reactive oxygen species, LncCytB-siRNA further attenuated CYTB gene transcription, complex III activity, and OCR. Similar decrease in LncCytB-CYTB interactions and CYTB transcription was observed in diabetic mice. Furthermore, maintenance of mitochondrial homeostasis by overexpressing superoxide dismutase or sirtuin 1 in mice ameliorated diabetes-induced decrease in LncCytB-CYTB interactions and CYTB gene transcripts, and also improved complex III activity and mitochondrial respiration. Innovation and Conclusion: LncCytB downregulation in hyperglycemic milieu downregulates CYTB transcription, which inhibits complex III activity and compromises mitochondrial stability and OCR. Thus, preventing LncCytB downregulation in diabetes has potential of inhibiting the development of diabetic retinopathy, possibly via maintaining mitochondrial respiration. Antioxid. Redox Signal. 39, 817-828.