RESUMO
Lipid-protein interactions play essential roles in many biological phenomena. Lysophospholipid mediators, such as cyclic phosphatidic acid (cPA), have been recognized as secondary messengers, yet few cellular targets for cPA have been identified to date. Furthermore, the molecular mechanism that activates these downstream signaling events remains unknown. In this study, using metabolically stabilized cPA carba-derivative (2ccPA)-immobilized magnetic beads, we identified adenine nucleotide translocase 2 (ANT2) as a 2ccPA-interacting protein in microglial cells. 2ccPA was tested for its ability to inhibit apoptosis caused by phenylarsine oxide in microglial cells. This damage was significantly improved upon 2ccPA treatment, along with cell proliferation, apoptosis, reactive oxygen species production, and intracellular ATP levels. This is the first report to suggest the direct binding of 2ccPA to ANT2 in microglial cells and provides evidence for a new benefit of 2ccPA in protecting microglial cells from apoptotic death induced by the ANT2-mediated signaling pathway.
Assuntos
Microglia , Translocases Mitocondriais de ADP e ATP/fisiologia , Ácidos Fosfatídicos/metabolismo , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Camundongos , Microglia/citologia , Microglia/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.
Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Translocases Mitocondriais de ADP e ATP/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Ácido Bongcréquico/metabolismo , Citoplasma/metabolismo , Mitocôndrias/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/ultraestrutura , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.
Assuntos
Mitocôndrias Hepáticas/fisiologia , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Compostos de Sulfidrila/farmacologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Arsenicais/farmacologia , Atractilosídeo/análogos & derivados , Atractilosídeo/farmacologia , Cálcio/farmacologia , Ciclosporina/farmacologia , Etilmaleimida/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Conformação Proteica , Ratos Wistar , terc-Butil Hidroperóxido/farmacologiaRESUMO
In aerobic eukaryotic cells, the high energy metabolite ATP is generated mainly within the mitochondria following the process of oxidative phosphorylation. The mitochondrial ATP is exported to the cytoplasm using a specialized transport protein, the ADP/ATP carrier, to provide energy to the cell. Any deficiency or dysfunction of this membrane protein leads to serious consequences on cell metabolism and can cause various diseases such as muscular dystrophy. Described as a decisive player in the programmed cell death, it was recently shown to play a role in cancer. The objective of this review is to summarize the current knowledge of the involvement of the ADP/ATP carrier, encoded by the SLC25A4, SLC25A5, SLC25A6 and SLC25A31 genes, in human diseases and of the efforts made at designing different model systems to study this carrier and the associated pathologies through biochemical, genetic, and structural approaches.
Assuntos
Apoptose/fisiologia , Redes e Vias Metabólicas/fisiologia , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/fisiologia , Modelos Moleculares , Família Multigênica/genética , Neoplasias/fisiopatologia , Translocador 1 do Nucleotídeo Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/metabolismo , Translocador 3 do Nucleotídeo Adenina/metabolismo , Sequência de Aminoácidos , Apoptose/genética , Humanos , Redes e Vias Metabólicas/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias/genética , Conformação Proteica , Alinhamento de SequênciaRESUMO
Throughout our lifetime the F1Fo ATP synthase produces the majority of our biological energy, and plays central roles in the structure and organization of mitochondria, yet our understanding of its roles in human disease remain largely enigmatic. It seems logical that even intermittent impairment of this highly important enzyme could deprive the body's tissues of energy at crucial times, which may predispose or contribute to illness. Indeed, evidence is accumulating that there are dire consequences of energy depletion in acute lifethreatening conditions, such as heart attacks, as well as chronic diseases, including aging, cancer, diabetes and heart failure. Recent advances in our understanding of the expanding roles of F1Fo ATP synthase, and how it is regulated, combined with the development of novel strategies for manipulating its function, may provide renewed hope for therapeutic improvement of energy homeostasis, and mitochondrial integrity, in a host of human diseases. In this review we will highlight what is known about the molecular regulation of this amazing enzyme complex, discuss effects of physiological agonists and therapeutic drugs on its functions, and present evidence supporting its involvement in the ills of mankind. Finally, we will outline existing challenges, and promising new avenues for targeting the enzyme therapeutically.
Assuntos
Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Trifosfato de Adenosina/biossíntese , Corticosteroides/farmacologia , Envelhecimento/fisiologia , Albinismo/tratamento farmacológico , Alcoolismo/fisiopatologia , Animais , Doenças Autoimunes/fisiopatologia , Infecções Bacterianas/fisiopatologia , Restrição Calórica , Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus/fisiopatologia , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/efeitos dos fármacos , Modelos Moleculares , Neoplasias/fisiopatologia , Fosforilação Oxidativa , Subunidades Proteicas/químicaRESUMO
Calorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocase to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2 weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT-mediated 'energy-wasting' process during CR.
Assuntos
Restrição Calórica , Canais Iônicos/fisiologia , Mitocôndrias Musculares/metabolismo , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas Mitocondriais/fisiologia , Animais , Metabolismo Energético , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 3RESUMO
Mitochondrial ADP/ATP carrier (AAC) is a protein catalyzing the transport of adenine nucleotides across inner mitochondrial membrane. In this review article, we first briefly introduce structural and functional properties of this protein. Next, we describe the results of our recent studies on the difference in the C-terminal region between yeast type 2 AAC isoform and bovine type 1 AAC isoform. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier are discussed.
Assuntos
Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/fisiologia , Nucleotídeos de Adenina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Isoenzimas , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiaeRESUMO
The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.
Assuntos
Apoptose , Mitocôndrias/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Translocador 3 do Nucleotídeo Adenina/biossíntese , Translocador 3 do Nucleotídeo Adenina/genética , Translocador 3 do Nucleotídeo Adenina/fisiologia , Antineoplásicos/farmacologia , Caspase 9/metabolismo , Proliferação de Células , Forma Celular , Citoproteção , Células HeLa , Humanos , Peróxido de Hidrogênio/análise , Indazóis/farmacologia , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/fisiologia , Translocases Mitocondriais de ADP e ATP/biossíntese , Translocases Mitocondriais de ADP e ATP/sangue , Translocases Mitocondriais de ADP e ATP/genética , Fosforilação Oxidativa , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estaurosporina/farmacologia , Superóxidos/análiseRESUMO
ATP/ADP translocases transport ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate intracellular bacteria. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen "Candidatus Liberibacter asiaticus," the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The "Ca. Liberibacter asiaticus" gene (nttA) encoding the translocase was subsequently expressed in Escherichia coli and shown to enable E. coli to import ATP directly into the cell. Competition assays with the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in "Ca. Liberibacter solanacearum," the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from "Ca. Liberibacter asiaticus," indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment.
Assuntos
Proteínas de Bactérias/metabolismo , Translocases Mitocondriais de ADP e ATP/fisiologia , Doenças das Plantas/microbiologia , Rhizobiaceae/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biologia Computacional , Translocases Mitocondriais de ADP e ATP/genética , Modelos Biológicos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The ADP/ATP carrier catalyzes the exchange of ADP and ATP across the inner mitochondrial membrane. MATERIALS AND METHODS: The molecular dynamics of modeled yeast type 2 AAC (yAAC2) was analyzed and molecular parameters were determined. RESULTS: The yAAC2 C-terminal moved flexibly and a negative electrostatic potential field (ESP) was located in the C-terminal region. The ESP field is always located in the C-terminal area during C-terminal truncation (d1-d9). Further C-terminal truncation occurred on field invagination into the core region (d11, d14, d16). The 2-6 C-terminal amino acid truncation did not affect the biological activity. The d7-d9 truncated mutants lost their biological function. CONCLUSION: A critical point in yAAC2 function was shown between d6 and d7 C-terminal truncation. The C-terminal structure of yAAC2 is thought to be involved in biological function control.
Assuntos
Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Animais , Bovinos , Modelos Químicos , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The organs and molecular mechanisms contributing to adaptive thermogenesis in marsupials are not known because some species apparently lack brown adipose tissue (BAT). The increased oxidative capacity and presence of uncoupling protein 3 (UCP3) in skeletal muscle led to speculations on whether uncoupled respiration sustains endothermy in the cold, as found for BAT. Here, we investigated the role of mitochondrial proton conductance in the small Australian marsupial Antechinus flavipes during cold exposure. Although there was a tendency toward higher oxidative capacity in skeletal muscle, indicating metabolic adjustments to the cold, we observed no change in basal proton conductance of isolated myotubular and liver mitochondria. In eutherians, 4-hydroxynonenal (HNE) is an activator of mitochondrial uncoupling mediated by UCP3 and ANT (adenine nucleotide translocase). In the marsupial A. flavipes, proton conductance in myotubular mitochondria could be induced by HNE selectively in the cold-acclimated group. Induced uncoupling activity could be attributed to the ANT as judged by inhibition with carboxyatractylate, while GDP, a putative inhibitor of rodent UCP3, had no detectable effects on marsupial UCP3. In contrast to previous expectations, basal proton conductance in the myotubular mitochondria of marsupials does not contribute to adaptive thermogenesis, as found for eutherian BAT. Increased sensitivity of proton conductance to HNE by the ANT suggests a greater requirement for mild uncoupling activity that may convey protection from lipid peroxidation and mitigate reactive oxygen species production during cold stress.
Assuntos
Marsupiais/fisiologia , Aclimatação , Tecido Adiposo Marrom/fisiologia , Aldeídos/farmacologia , Animais , Atractilosídeo/análogos & derivados , Atractilosídeo/farmacologia , Sequência de Bases , Clima Frio , Primers do DNA/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Canais Iônicos/genética , Canais Iônicos/fisiologia , Peroxidação de Lipídeos , Marsupiais/genética , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/fisiologia , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Prótons , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Termogênese/fisiologia , Proteína Desacopladora 3RESUMO
Overexpression of the adenine nucleotide translocase (ANT) has been shown to be cytotoxic in several cell types. Although ANT was originally proposed to be a critical component of the mitochondrial permeability transition (MPT) pore, recent data have suggested that this may not be the case. We therefore hypothesized that the cytotoxic actions of ANT are through an alternative mechanism, independent of the MPT pore. Infection of cultured neonatal cardiomyocytes with an ANT1-encoding adenovirus induced a gene dosage-dependent increase in cell death. However, ANT1 overexpression failed to induce MPT, and neither pharmacological nor genetic inhibition of the MPT pore was able to prevent ANT1-induced cell death. These data suggested that ANT1-induced death progressed through an MPT pore-independent pathway. Somewhat surprisingly, we observed that protein levels of Bax, a pro-apoptotic Bcl protein, were consistently elevated in ANT1-infected cardiomyocytes. Membranes isolated from ANT1-infected myocytes exhibited significantly increased amounts of membrane-inserted Bax, and immunocytochemistry revealed increased Bax activation in ANT1-infected myocytes. Co-expression with the Bax antagonist Bcl2 was able to greatly reduce the degree of ANT1-induced cell death. Furthermore, Bax/Bak-deficient fibroblasts were resistant to the cytotoxic effects of ANT1 overexpression. Interestingly, ANT1 overexpression was also associated with enhanced production of reactive oxygen species (ROS), and the antioxidant MnTBAP was able to significantly attenuate both the ANT1-induced upregulation of Bax and cell death. Taken together, these data indicate that ANT mediates cell death, not through the MPT pore, but rather via a ROS-dependent upregulation and activation of Bax.
Assuntos
Morte Celular/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Antimutagênicos/farmacologia , Western Blotting , Morte Celular/genética , Células Cultivadas , Cobalto/farmacologia , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ciclofilinas/metabolismo , Fluoresceínas/farmacologia , Marcação In Situ das Extremidades Cortadas , Indicadores e Reagentes/farmacologia , Camundongos , Microscopia de Fluorescência , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
In Caenorhabditis elegans, the central cell-killing process is essentially controlled by the interplay of four apoptotic factors: EGL-1/BH3-only protein, CED-9/Bcl2, CED-4/Apaf1, and CED-3/caspase. In cells destined to die, EGL-1 binds to CED-9 and results in the release of CED-4 from the mitochondrion-tethered CED-9-CED-4 complex to the perinucleus, which facilitates processing of the CED-3 caspase to cause apoptosis. However, whether additional factors exist to regulate the cell-killing process remains largely unknown. We have identified here WAN-1, the C. elegans ortholog of mammalian adenine nucleotide translocator, as an important cell death regulator. Genetic inactivation of wan-1 significantly suppressed both somatic and germ line cell deaths in C. elegans. Consistently, chemical inhibition of WAN-1 activity also caused strong reduction of germ line apoptosis. WAN-1 localizes to mitochondria and can form complex with both CED-4 and CED-9. Importantly, the cell death initiator EGL-1 can disrupt the interaction between CED-9 and WAN-1. In addition, overexpression of WAN-1 induced ectopic cell killing dependently on the core cell death pathway. These findings suggest that WAN-1 is involved in the central cell-killing process and cooperates with the core cell death machinery to promote programmed cell death in C. elegans.
Assuntos
Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Animais , Apoptose/efeitos dos fármacos , Atractilosídeo/farmacologia , Ácido Bongcréquico/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Caspases/genética , Caspases/fisiologia , Linhagem Celular , Deleção de Genes , Expressão Gênica , Genes de Helmintos , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/genética , Complexos Multiproteicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologiaRESUMO
Like Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty.
Assuntos
Cardiopatias/etiologia , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Animais , Cardiomegalia/prevenção & controle , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/fisiologia , Insuficiência Cardíaca/etiologia , Humanos , Precondicionamento Isquêmico Miocárdico , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controleRESUMO
The mitochondrial ADP/ATP carrier is the paradigm of the mitochondrial carrier family (MCF), whose members are crucial for cross-talks between mitochondria, where cell energy is mainly produced, and the cytosol, where cell energy is mainly consumed. These carriers share structural and functional characteristics. Resolution of the 3D structure of the beef mitochondrial ADP/ATP carrier, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in substrate binding and transfer from the outside to the inside of mitochondria. To ascertain the role of these residues, namely, Y186, Y190, F191, and Y194, they were mutated into alanine in the yeast mitochondrial ADP/ATP carrier at equivalent positions (Y203, Y207, F208, and Y211). Two residues, Y203 and F208, appeared to be crucial for transport activity but not for substrate binding per se, indicating their involvement in the substrate transfer process through the carrier. Furthermore, it was possible to show that these mutations precluded conformational changes of the matrix loop m2, whose movements were demonstrated to participate in substrate transport by the wild-type carrier. Therefore, these aromatic residues may be involved in substrate gliding, and they may also confer specificity toward adenine nucleotides for the ADP/ATP carrier as compared with the MCF members.
Assuntos
Sequência Conservada , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/fisiologia , Nucleotídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Bovinos , Sequência Conservada/genética , Cristalografia por Raios X , Translocases Mitocondriais de ADP e ATP/genética , Dados de Sequência Molecular , Nucleotídeos/química , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The mitochondrial ADP/ATP carrier plays a central role in aerobic cell energetics by providing to the cytosol the ATP generated by oxidative phosphorylation. Though discovered around 40 years ago owing to the existence of unique inhibitors and in spite of numerous experimental approaches, this carrier, which stands as a model of the mitochondrial solute carriers keeps some long-standing mystery. There are still open challenging questions among them the precise ADP/ATP transport mechanism, the functional oligomeric state of the carrier and relationships between human ADP/ATP carrier dysfunctioning and pathologies. Deciphering the 3D structure of this carrier afforded a considerable progress of the knowledge but requires now additional data focused on molecular dynamics from this static picture. State of the art in this topic is reviewed and debated in this paper in view of better comprehending origin of the discrepancies in these questions and, finally, the multiple physiological roles of this carrier in eukaryotic cell economy.
Assuntos
Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/fisiologia , Animais , Sequência Conservada , Evolução Molecular , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Translocases Mitocondriais de ADP e ATP/deficiência , Translocases Mitocondriais de ADP e ATP/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Modelos Moleculares , Mutação , Fosforilação Oxidativa , Estrutura Quaternária de ProteínaRESUMO
Defined mutations in the mitochondrial ADP/ATP carrier (AAC) are associated with certain types of progressive external ophthalmoplegia. AAC is required for oxidative phosphorylation (OXPHOS), and dysregulation of AAC has been implicated in apoptosis. Little is known about the AAC interactome, aside from a known requirement for the phospholipid cardiolipin (CL) and that it is thought to function as a homodimer. Using a newly developed dual affinity tag, we demonstrate that yeast AAC2 physically participates in several protein complexes of distinct size and composition. The respiratory supercomplex and several smaller AAC2-containing complexes, including other members of the mitochondrial carrier family, are identified here. In the absence of CL, most of the defined interactions are destabilized or undetectable. The absence of CL and/or AAC2 results in distinct yet additive alterations in respiratory supercomplex structure and respiratory function. Thus, a single lipid can significantly alter the functional interactome of an individual protein.
Assuntos
Cardiolipinas/fisiologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dimerização , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Eletroforese em Gel de Poliacrilamida , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Adenine nucleotide translocase (Ant) is the most abundant protein on the mitochondrial inner membrane (MIM) primarily involved in ADP/ATP exchange. Ant also possesses a discrete membrane uncoupling activity. Specific mis-sense mutations in the human Ant1 cause autosomal dominant Progressive External Ophthalmoplegia (adPEO), mitochondrial myopathy and cardiomyopathy, which are commonly manifested by fractional mitochondrial DNA (mtDNA) deletions. It is currently thought that the pathogenic mutations alter substrate preference (e.g. ATP versus ADP) thereby dominantly disturbing adenine nucleotide homeostasis in mitochondria. This may interfere with mtDNA replication, consequently affecting mtDNA stability and oxidative phosphorylation. Here, we showed that the adPEO-type A128P, A106D and M114P mutations in the yeast Aac2p share the following common dominant phenotypes: electron transport chain damage, intolerance to moderate over-expression, synthetic lethality with low Deltapsi(m) conditions, hypersensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and mtDNA instability. More interestingly, the aac2(A137D) allele mimicking ant1(A123D) in mitochondrial myopathy and cardiomyopathy exhibits similar dominant phenotypes. Because Aac2(A137D) is known to completely lack transport activity, it is strongly argued that the dominant mitochondrial damages are not caused by aberrant nucleotide transport. The four pathogenic mutations occur in a structurally dynamic gating region on the cytosolic side. We provided direct evidence that the mutant alleles uncouple mitochondrial respiration. The pathogenic mutations likely enhance the intrinsic proton-conducting activity of Ant, which excessively uncouples the MIM thereby affecting energy transduction and mitochondrial biogenesis. mtDNA disintegration is a phenotype co-lateral to mitochondrial damages. These findings provide mechanistic insights into the pathogenesis of the Ant1-induced diseases.
Assuntos
Substituição de Aminoácidos/genética , Genes Dominantes , Translocases Mitocondriais de ADP e ATP/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Membranas Mitocondriais/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Metabolismo Energético/genética , Humanos , Translocases Mitocondriais de ADP e ATP/metabolismo , Translocases Mitocondriais de ADP e ATP/fisiologia , Doenças Mitocondriais/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Oftalmoplegia Externa Progressiva Crônica/enzimologia , Oftalmoplegia Externa Progressiva Crônica/genética , Oftalmoplegia Externa Progressiva Crônica/metabolismo , Fenótipo , Transporte Proteico/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Desacopladores/metabolismoRESUMO
The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc(1))-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc(1)-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc(1)-COX supercomplex is perturbed, whereby a decrease in the III(2)-IV(2) assembly state relative to the III(2)-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc(1)-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc(1)-COX-TIM23 supercomplex are also discussed.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Apoptose , Potenciais da Membrana , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Oxigênio/química , Fenótipo , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/metabolismoRESUMO
In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.