RESUMO
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 mitochondrial ADP/ATP carrier (SLC25A4), also called the adenine nucleotide translocase, imports ADP into the mitochondrial matrix and exports ATP, which are key steps in oxidative phosphorylation. Historically, the carrier was thought to form a homodimer and to operate by a sequential kinetic mechanism, which involves the formation of a ternary complex with the two exchanged substrates bound simultaneously. However, recent structural and functional data have demonstrated that the mitochondrial ADP/ATP carrier works as a monomer and has a single substrate binding site, which cannot be reconciled with a sequential kinetic mechanism. Here, we study the kinetic properties of the human mitochondrial ADP/ATP carrier by using proteoliposomes and transport robotics. We show that the Km/Vmax ratio is constant for all of the measured internal concentrations. Thus, in contrast to earlier claims, we conclude that the carrier operates with a ping-pong kinetic mechanism in which substrate exchange across the membrane occurs consecutively rather than simultaneously. These data unite the kinetic and structural models, showing that the carrier operates with an alternating access mechanism.
Assuntos
Mitocôndrias , Translocases Mitocondriais de ADP e ATP , Humanos , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Cinética , Translocador 1 do Nucleotídeo Adenina/metabolismoRESUMO
The mitochondrial permeability transition (mPT) is a process that permits rapid exchange of small molecules across the inner mitochondrial membrane (IMM) and thus plays a vital role in mitochondrial function and cellular signaling. Formation of the pore that mediates this flux is well-documented in injury and disease but its regulation has also emerged as critical to the fate of stem cells during embryonic development. The precise molecular composition of the mPTP has been enigmatic, with far more genetic studies eliminating molecular candidates than confirming them. Rigorous studies in the recent decade have implicated central involvement of the F1Fo ATP synthase, or complex V of the electron transport chain, and continue to confirm a regulatory role for Cyclophilin D (CypD), encoded by Ppif, in modulating the sensitivity of the pore to opening. A host of endogenous molecules have been shown to trigger flux characteristic of mPT, including positive regulators such as calcium ions, reactive oxygen species, inorganic phosphate, and fatty acids. Conductance of the pore has been described as low or high, and reversibility of pore opening appears to correspond with the relative abundance of negative regulators of mPT such as adenine nucleotides, hydrogen ion, and divalent cations that compete for calcium-binding sites in the mPTP. Current models suggest that distinct pores could be responsible for differing reversibility and conductance depending upon cellular context. Indeed, irreversible propagation of mPT inevitably leads to collapse of transmembrane potential, arrest of ATP synthesis, mitochondrial swelling, and cell death. Future studies should clarify ambiguities in mPTP structure and reveal new roles for mPT in dictating specialized cellular functions beyond cell survival that are tied to mitochondrial fitness including stem cell self-renewal and fate. The focus of this review is to describe contemporary models of the mPTP and highlight how pore activity impacts stem cells and development.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Cálcio/metabolismo , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria , Trifosfato de Adenosina , Células-Tronco/metabolismo , PermeabilidadeRESUMO
The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.
Assuntos
Tartarugas , Animais , Cálcio/metabolismo , Peptidil-Prolil Isomerase F , Peróxido de Hidrogênio , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria , Permeabilidade , Ratos , Tartarugas/metabolismoRESUMO
Alzheimer's disease (AD), certainly the most widespread proteinopathy, has as classical neuropathological hallmarks, two groups of protein aggregates: senile plaques and neurofibrillary tangles. However, the research interest is rapidly gaining ground in a better understanding of other pathological features, first, of all the mitochondrial dysfunctions. Several pieces of evidence support the hypothesis that abnormal mitochondrial function may trigger aberrant processing of amyloid progenitor protein or tau and thus neurodegeneration. Here, our aim is to emphasize the role played by two 'bioenergetic' proteins inserted in the mitochondrial membranes, inner and outer, respectively, that is, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), in the progression of AD. To perform this, we will magnify the ANT and VDAC defects, which are measurable hallmarks of mitochondrial dysfunction, and collect all the existing information on their interaction with toxic Alzheimer's proteins. The pathological convergence of tau and amyloid ß-peptide (Aß) on mitochondria may finally explain why the therapeutic strategies used against the toxic forms of Aß or tau have not given promising results separately. Furthermore, the crucial role of ANT-1 and VDAC impairment in the onset/progression of AD opens a window for new therapeutic strategies aimed at preserving/improving mitochondrial function, which is suspected to be the driving force leading to plaque and tangle deposition in AD.
Assuntos
Doença de Alzheimer , Nucleotídeos de Adenina/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Humanos , Mitocôndrias/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Proteínas tau/metabolismoRESUMO
The mitochondrial adenine nucleotide translocator (ANT) plays the fundamental role of gatekeeper of cellular energy flow, carrying out the reversible exchange of ADP for ATP across the inner mitochondrial membrane. ADP enters the mitochondria where, through the oxidative phosphorylation process, it is the substrate of Fo-F1 ATP synthase, producing ATP that is dispatched from the mitochondrion to the cytoplasm of the host cell, where it can be used as energy currency for the metabolic needs of the cell that require energy. Long ago, we performed a method that allowed us to monitor the activity of ANT by continuously detecting the ATP gradually produced inside the mitochondria and exported in the extramitochondrial phase in exchange with externally added ADP, under conditions quite close to a physiological state, i.e., when oxidative phosphorylation takes place. More than 30 years after the development of the method, here we aim to put the spotlight on it and to emphasize its versatile applicability in the most varied pathophysiological conditions, reviewing all the studies, in which we were able to observe what really happened in the cell thanks to the use of the "ATP detecting system" allowing the functional activity of the ANT-mediated ADP/ATP exchange to be measured.
Assuntos
Translocases Mitocondriais de ADP e ATP/metabolismo , Doenças Mitocondriais/genética , Animais , Metabolismo Energético , Humanos , Translocases Mitocondriais de ADP e ATP/genética , Doenças Mitocondriais/metabolismoRESUMO
The permeability transition (PT) is an increased permeation of the inner mitochondrial membrane due to the opening of the PT pore (PTP), a Ca2+-activated high conductance channel involved in Ca2+ homeostasis and cell death. Alterations of the PTP have been associated with many pathological conditions and its targeting represents an incessant challenge in the field. Although the modulation of the PTP has been extensively explored, the lack of a clear picture of its molecular nature increases the degree of complexity for any target-based approach. Recent advances suggest the existence of at least two mitochondrial permeability pathways mediated by the F-ATP synthase and the ANT, although the exact molecular mechanism leading to channel formation remains elusive for both. A full comprehension of this to-pore conversion will help to assist in drug design and to develop pharmacological treatments for a fine-tuned PT regulation. Here, we will focus on regulatory mechanisms that impinge on the PTP and discuss the relevant literature of PTP targeting compounds with particular attention to F-ATP synthase and ANT.
Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , HumanosRESUMO
Tilapia piscidin (TP) 4 is an antimicrobial peptide derived from Nile tilapia (Oreochromis niloticus), which shows broad-spectrum antibacterial activity and excellent cancer-killing ability in vitro and in vivo. Like many other antimicrobial peptides, TP4 treatment causes mitochondrial toxicity in cancer cells. However, the molecular mechanisms underlying TP4 targeting of mitochondria remain unclear. In this study, we used a pull-down assay on A549 cell lysates combined with LC-MS/MS to discover that TP4 targets adenine nucleotide translocator (ANT) 2, a protein essential for adenine nucleotide exchange across the inner membrane. We further showed that TP4 accumulates in mitochondria and colocalizes with ANT2. Moreover, molecular docking studies showed that the interaction requires Phe1, Ile2, His3, His4, Ser11, Lys14, His17, Arg21, Arg24 and Arg25 residues in TP4 and key residues within the cavity of ANT2. These findings suggest a mechanism by which TP4 may induce mitochondrial dysfunction to disrupt cellular energy metabolism.
Assuntos
Translocador 2 do Nucleotídeo Adenina/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Ciclídeos/metabolismo , Proteínas de Peixes/farmacologia , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Células A549 , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/metabolismo , Humanos , Células MCF-7 , Microscopia Confocal , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Ligação ProteicaRESUMO
Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.
Assuntos
Cryptosporidium parvum/metabolismo , Cisteína/metabolismo , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/metabolismo , Nucleotídeos/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Sequência de Aminoácidos , Atractilosídeo/análogos & derivados , Atractilosídeo/química , Ácido Bongcréquico/química , Lactococcus lactis/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Filogenia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Cardiolipin in eukaryotes is found in the mitochondrial inner membrane, where it interacts with membrane proteins and, although not essential, is necessary for the optimal activity of a number of proteins. One of them is the mitochondrial ADP/ATP carrier, which imports ADP into the mitochondrion and exports ATP. In the crystal structures, cardiolipin is bound to three equivalent sites of the ADP/ATP carrier, but its role is unresolved. Conservation of residues at these cardiolipin binding sites across other members of the mitochondrial carrier superfamily indicates cardiolipin binding is likely to be important for the function of all mitochondrial carriers. Multiscale simulations were performed in a cardiolipin-containing membrane to investigate the dynamics of cardiolipin around the yeast and bovine ADP/ATP carriers in a lipid bilayer and the properties of the cardiolipin-binding sites. In coarse-grain simulations, cardiolipin molecules bound to the carriers for longer periods of time than phosphatidylcholine and phosphatidylethanolamine lipids-with timescales in the tens of microseconds. Three long-lived cardiolipin binding sites overlapped with those in the crystal structures of the carriers. Other shorter-lived cardiolipin interaction sites were identified in both membrane leaflets. However, the timescales of the interactions were of the same order as phosphatidylcholine and phosphatidylethanolamine, suggesting that these sites are not specific for cardiolipin binding. The calculation of lipid binding times and the overlap of the cardiolipin binding sites between the structures and simulations demonstrate the potential of multiscale simulations to investigate the dynamics and behavior of lipids interacting with membrane proteins.
Assuntos
Cardiolipinas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cardiolipinas/química , Cardiolipinas/genética , Bovinos , Sequência Conservada/genética , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMO
The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas Fúngicas/química , Translocases Mitocondriais de ADP e ATP/química , Saccharomycetales/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Cinética , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/enzimologia , TermodinâmicaRESUMO
The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Assuntos
Translocador 2 do Nucleotídeo Adenina/deficiência , Cardiopatias Congênitas/genética , Insuficiência Cardíaca/genética , Ventrículos do Coração/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Animais , Transporte Biológico , Proliferação de Células , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Insuficiência Cardíaca/embriologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/anormalidades , Ventrículos do Coração/embriologia , Integrases , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/patologia , Dilatação Mitocondrial/genética , Miócitos Cardíacos/patologia , Organogênese , FenótipoRESUMO
The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Membranas Mitocondriais/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Ácido Bongcréquico/farmacologia , Cardiolipinas/metabolismo , Bovinos , Sequência Consenso , Humanos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/química , Modelos Moleculares , Proteínas de Transporte de Fosfato/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explores the effects of extracellular ADP on low [K(+)]-induced apoptosis in rat cerebellar granule cells. ADP, released into the extracellular space in brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine. The findings reported in this paper demonstrate that ADP inhibits the proapoptotic stimulus supposedly via: i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s. This conclusion is validated by the increase in SOD and catalase activities as well as by the GSSG/GSH ratio value decrease, in conjunction with the drop of ROS level and the prevention of the ADP protective effect by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a novel functionally selective antagonist of purine receptor; ii) safeguard of the functionality of the mitochondrial adenine nucleotide-1 translocator (ANT-1), which is early impaired during apoptosis. This effect is mediated by its plausible internalization into cell occurring as such or after its hydrolysis, by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell. Moreover, the findings that ADP also protects ANT-1 from the toxic action of the two Alzheimer's disease peptides, i.e. Aß1-42 and NH2htau, which are known to be produced in apoptotic cerebellar neurons, further corroborate the molecular mechanism of neuroprotection by ADP, herein proposed.
Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Difosfato de Adenosina/metabolismo , Antioxidantes/metabolismo , Mitocôndrias/efeitos dos fármacos , Translocador 1 do Nucleotídeo Adenina/genética , Peptídeos beta-Amiloides/toxicidade , Animais , Apoptose/genética , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Humanos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Cultura Primária de Células , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Proteínas tau/toxicidadeRESUMO
Inflammation contributes significantly to cardiac dysfunction. Although the initial phase of inflammation is essential for repair and healing, excessive proinflammatory cytokines are detrimental to the heart. We found that adenine nucleotide translocator isoform-1 (ANT1) protein levels were significantly decreased in the inflamed heart of C57BL/6 mice following cecal ligation and puncture. To understand the molecular mechanisms involved, we performed small-interfering RNA-mediated knockdown of ANT1 and studied tumor necrosis factor-α (TNFα)-induced inflammatory responses in myocardium-derived H9c2 cells and cardiomyocytes. ANT1 knockdown significantly increased swollen mitochondria and mitochondrial reactive oxygen species, concomitant with increased TNFα-induced NF-κB reporter gene activity and interleukin-6 and TNFα expression. A mitochondrial-targeted antioxidant mito-TEMPO attenuated TNFα-induced mitochondrial reactive oxygen species, NF-κB reporter gene activity, and cytokine expression in ANT1 knockdown cells. Interestingly, TNFα or lipopolysaccharide (LPS) treatment significantly decreased ANT1 protein levels, suggesting a feed-forward regulation of proinflammatory cytokine expression activated by ANT1 downregulation. These data suggest that ANT1 downregulation contributes to cardiac inflammation post-cecal ligation and puncture. Preventing ANT1 downregulation could provide a novel molecular target to temper cardiac inflammation.
Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Miócitos Cardíacos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Translocador 1 do Nucleotídeo Adenina/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Genes Reporter , Inflamação/sangue , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/imunologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Dilatação Mitocondrial , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
In mammalian cells, mitochondria receive most incoming iron, yet no entry pathway for iron at the outer mitochondrial membrane (OMM) has been characterized. Our results show that the divalent metal transporter 1 (DMT1) occurs in the OMM. Immunoblots detected DMT1 in mitochondria from a pneumocyte cell model in their OMM. Using the split-ubiquitin yeast 2-hybrid system, we found that cytochrome c oxidase subunit II (COXII) and the translocase of OMM 6-kDa subunit (Tom6) homologue interact with DMT1. COXII coimmunoprecipitates with DMT1. There are 4 DMT1 isoforms that differ at the N and C termini. Using HEK293 cells that inducibly express all of the 4 ends of DMT1, we found all of them in the OMM, as detected by immunoblots after cell fractionation, and in isolated mitochondria, as detected by immunofluorescence. Immunoblot analysis of purified cell fractions from rat renal cortex confirmed and extended these results to the kidney, which expressed high levels of DMT1. Immunogold labeling detected DMT1 colocalization in mitochondria with the voltage-dependent anion-selective channel protein-1, which is expressed in the OMM. We suggest that DMT1 not only exports iron from endosomes, but also serves to import the metal into the mitochondria.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Células Epiteliais Alveolares/citologia , Animais , Ânions , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Endossomos/metabolismo , Células HEK293 , Humanos , Córtex Renal/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Plasmídeos/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The cell-toxic bile salt glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA) are responsible for hepatocyte demise in cholestatic liver diseases, while tauroursodeoxycholic acid (TUDCA) is regarded hepatoprotective. We demonstrate the direct mitochondrio-toxicity of bile salts which deplete the mitochondrial membrane potential and induce the mitochondrial permeability transition (MPT). The bile salt mediated mechanistic mode of destruction significantly differs from that of calcium, the prototype MPT inducer. Cell-toxic bile salts initially bind to the mitochondrial outer membrane. Subsequently, the structure of the inner boundary membrane disintegrates. And it is only thereafter that the MPT is induced. This progressive destruction occurs in a dose- and time-dependent way. We demonstrate that GCDCA and TCDCA, but not TUDCA, preferentially permeabilize liposomes containing the mitochondrial membrane protein ANT, a process resembling the MPT induction in whole mitochondria. This suggests that ANT is one decisive target for toxic bile salts. To our knowledge this is the first report unraveling the consecutive steps leading to mitochondrial destruction by cell-toxic bile salts.
Assuntos
Ácido Glicoquenodesoxicólico/toxicidade , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/agonistas , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Lipossomos/química , Fígado/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/química , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Translocases Mitocondriais de ADP e ATP/isolamento & purificação , Proteínas de Transporte da Membrana Mitocondrial/agonistas , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Miocárdio/química , Ratos , Ácido Tauroquenodesoxicólico/toxicidade , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/isolamento & purificaçãoRESUMO
Functional as well as structural alterations in mitochondria size, shape and distribution are precipitating, early events in progression of Alzheimer's Disease (AD). We reported that a 20-22kDa NH2-tau fragment (aka NH2htau), mapping between 26 and 230 amino acids of the longest human tau isoform, is detected in cellular and animal AD models and is neurotoxic in hippocampal neurons. The NH2htau -but not the physiological full-length protein- interacts with Aß at human AD synapses and cooperates with it in inhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange. Here we show that the NH2htau also adversely affects the interplay between the mitochondria dynamics and their selective autophagic clearance. Fragmentation and perinuclear mislocalization of mitochondria with smaller size and density are early found in dying NH2htau-expressing neurons. The specific effect of NH2htau on quality control of mitochondria is accompanied by (i) net reduction in their mass in correlation with a general Parkin-mediated remodeling of membrane proteome; (ii) their extensive association with LC3 and LAMP1 autophagic markers; (iii) bioenergetic deficits and (iv) in vitro synaptic pathology. These results suggest that NH2htau can compromise the mitochondrial biology thereby contributing to AD synaptic deficits not only by ANT-1 inactivation but also, indirectly, by impairing the quality control mechanism of these organelles.
Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Linhagem Celular Tumoral , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Humanos , Mitocôndrias/ultraestrutura , Neurônios/ultraestrutura , Sinapses/metabolismoRESUMO
Osteopenia and osteoporosis are among the most common metabolic bone diseases and represent major public health problems, with sufferers having an increased fracture risk. Diabetes is one of the most common diseases contributing to osteopenia and osteoporosis. However, the mechanisms underlying diabetes-induced osteopenia and osteoporosis remain unclear. Bone reconstruction, including bone formation and absorption, is a dynamic process. Large-conductance Ca2+-activated K+ channels (BK channels) regulate the function of bone marrow-derived mesenchymal stem cells, osteoblasts, and osteoclasts. Our previous studies revealed the relationship between BK channels and the function of osteoblasts via various pathways under physiological conditions. In this study, we reported a decrease in the expression of BK channels in mice with diabetes-induced osteopenia. BK deficiency enhanced mitochondrial Ca2+ and activated classical PINK1 (PTEN induced putative kinase 1)-PRKN/Parkin (parkin RBR E3 ubiquitin protein ligase)-dependent mitophagy, whereas the upregulation of BK channels inhibited mitophagy in osteoblasts. Moreover, SLC25A5/ANT2 (solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 5), a critical inner mitochondrial membrane protein participating in PINK1-PRKN-dependent mitophagy, was also regulated by BK channels. Overall, these data identified a novel role of BK channels in regulating mitophagy in osteoblasts, which might be a potential target for diabetes-induced bone diseases.Abbreviations: AGE, advanced glycation end products; Baf A1, bafilomycin A1; BK channels, big-conductance Ca2+-activated K+ channels; BMSCs, bone marrow-derived mesenchymal stem cells; BSA, bovine serum albumin; FBG, fasting blood glucose; IMM, inner mitochondrial membrane; ITPR1, inositol 1,4,5-trisphosphate receptor 1; MAM, mitochondria-associated ER membrane; OMM, outer mitochondrial membrane; PINK1, PTEN induced putative kinase 1; PPID/CyP-D, peptidylprolyl isomerase D (cyclophilin D); PRKN/PARK2, parkin RBR E3 ubiquitin protein ligase; ROS, reactive oxygen species; SLC25A5/ANT2, solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 5; STZ, streptozotocin.
RESUMO
The appendix of the Sauromatum senosum inflorescence is a striking example of thermogenesis in plants. On the day of opening, the Sauromatum appendix becomes hot, reaching up to 32 °C. Aspirin, salicylic acid and 2,6-dihydroxybenzoic acid, a subclass of NSAIDs, induce a temperature rise from three mitochondrial sources: alternative oxidase, F1 FO -ATP synthase and adenine nucleotide translocator. This temperature rise is synchronized and compounded under various light/dark regimes. We studied the effect of different subgroups of NSAIDs on the temperature rise. Tissue slices of appendix of Sauromatum and Arum italicum inflorescences at a pre-mature stage were treated with the three inducers in combination with one NSAID under constant light or darkness and under different photoperiods. Temperature rise generated by the three heat sources in the presence of inducers and different non-selective NSAIDs were not compounded and occurred at three different times. Under constant light, DuP-697, ibuprofen, flurbiprofen, acetaminophen and diclofenac suppressed the temperature rise induced by the three salicylates. Desynchronization and delayed temperature rise were detected with 6/42-h light/ dark and 15/33-h light/dark regimes in the presence of celecoxib and ibuprofen. With a 24/24-h light/dark regime, temperature rise was suppressed in the presence of ibuprofen. There were differences in response to individual NSAIDs between appendix tissue of A. italicum and S. venosum. Mitochondrial energy balance is affected by NSAIDs. There is an interaction between light/dark regime and temperature rise and a relationship between timing mechanism and temperature rise.