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1.
Cell Physiol Biochem ; 53(3): 465-479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31464387

RESUMO

BACKGROUND/AIMS: Cyclophilin D (CypD) mediates the mitochondrial permeability transition pore (mPTP) opening that contributes to mitochondrial dysfunction. CypD is regulated by its acetylation/deacetylation state that depends on Sirtuin-3 (SIRT3) mitochondrial deacetylase. Since obesity and metabolic syndrome decrease SIRT3 activity and expression, we tested the hypothesis that CypD hyperacetylation promotes mitochondrial dysfunction under this pathophysiological state, which is associated with ventricular dysfunction and heart failure. METHODS: Myocardial tissue samples from patients with left ventricular heart failure, with either obesity or normal weight, were processed for the expression of SIRT3 and acetylation profile by Western Blot (WB). In addition, a rat model of obesity and metabolic syndrome induced by 30% (w/v) of sucrose was conducted. The WB analysis was used to determine the levels of mitochondrial expression of SIRT3, Adenine Nucleotide Translocator (ANT), CypD and the acetylation profile, as well as immunoprecipitation to establish the acetylation levels of CypD. Mitochondrial function was assessed by oxygen consumption analysis and maximum Ca2+ retention capacity. Oxidative stress was assessed by aconitase activity, protein carbonyl and thiol groups content. RESULTS: SIRT3 expression in the biopsies of the failing human hearts showed a 46% decrease in the expression levels of obese patients in comparison to the non-obese patients (p=0.0219). Remarkably, body mass index was associated with protein acetylation (0.627; p = 0.035), suggesting that the acetylation profiles of the failing hearts of obese patients are partly mediated by a reduction in SIRT3, which is also associated with higher BNP levels, indicating a more severe ventricular dysfunction (-0.636; p = 0.043). Accordingly, obese rats demonstrated a SIRT3 mitochondrial expression decrease of 22% concomitantly with a hyperacetylated mitochondrial profile, including CypD. Cardiac mitochondria from obese animals were 2.5-fold more prone to mPTP opening than the controls. CONCLUSION: Our results indicate that obesity reduces SIRT3 expression and that CypD hyperacetylation increases mPTP opening, suggesting that the activation of SIRT3 might be a potential target to decrease ventricular dysfunction and slow the progression of heart failure.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Obesidade/metabolismo , Sirtuína 3/metabolismo , Acetilação , Adulto , Idoso , Animais , Índice de Massa Corporal , Cálcio/metabolismo , Ciclofilina D , Ciclofilinas/metabolismo , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Masculino , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Translocases Mitocondriais de ADP e ATP/metabolismo , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar
2.
Nature ; 571(7766): 515-520, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31341297

RESUMO

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.


Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Prótons , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Coenzimas/metabolismo , Ácidos Graxos/metabolismo , Transporte de Íons , Masculino , Camundongos , Consumo de Oxigênio
3.
J Biol Chem ; 294(31): 11654-11664, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31213526

RESUMO

Lysine methylation is a common posttranslational modification of nuclear and cytoplasmic proteins but is also present in mitochondria. The human protein denoted "family with sequence similarity 173 member B" (FAM173B) was recently uncovered as a mitochondrial lysine (K)-specific methyltransferase (KMT) targeting the c-subunit of mitochondrial ATP synthase (ATPSc), and was therefore renamed ATPSc-KMT. We here set out to investigate the biochemical function of its yet uncharacterized paralogue FAM173A. We demonstrate that FAM173A localizes to mitochondria, mediated by a noncanonical targeting sequence that is partially retained in the mature protein. Immunoblotting analysis using methyllysine-specific antibodies revealed that FAM173A knock-out (KO) abrogates lysine methylation of a single mitochondrial protein in human cells. Mass spectrometry analysis identified this protein as adenine nucleotide translocase (ANT), represented by two highly similar isoforms ANT2 and ANT3. We found that methylation occurs at Lys-52 of ANT, which was previously reported to be trimethylated. Complementation of KO cells with WT or enzyme-dead FAM173A indicated that the enzymatic activity of FAM173A is required for ANT methylation at Lys-52 to occur. Both in human cells and in rat organs, Lys-52 was exclusively trimethylated, indicating that this modification is constitutive, rather than regulatory and dynamic. Moreover, FAM173A-deficient cells displayed increased mitochondrial respiration compared with FAM173A-proficient cells. In summary, we demonstrate that FAM173A is the long-sought KMT responsible for ANT methylation at Lys-52, and point out the functional significance of Lys-52 methylation in ANT. Based on the established naming nomenclature for KMTs, we propose to rename FAM173A to ANT-KMT (gene name ANTKMT).


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Células HeLa , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Humanos , Fígado/metabolismo , Lisina/metabolismo , Espectrometria de Massas , Metilação , Mitocôndrias/enzimologia , Peptídeos/análise , Ratos , Alinhamento de Sequência
4.
Cell ; 176(3): 435-447.e15, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30611538

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/metabolismo
5.
Mitochondrion ; 46: 380-392, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30391711

RESUMO

Cardiac ischemia and reperfusion (IR) injury induces excessive emission of deleterious reactive O2 and N2 species (ROS/RNS), including the non-radical oxidant peroxynitrite (ONOO-) that can cause mitochondria dysfunction and cell death. In this study, we explored whether IR injury in isolated hearts induces tyrosine nitration of adenine nucleotide translocase (ANT) and alters its interaction with the voltage-dependent anion channel 1 (VDAC1). We found that IR injury induced tyrosine nitration of ANT and that exposure of isolated cardiac mitochondria to ONOO- induced ANT tyrosine, Y81, nitration. The exposure of isolated cardiac mitochondria to ONOO- also led ANT to form high molecular weight proteins and dissociation of ANT from VDAC1. We found that IR injury in isolated hearts, hypoxic injury in H9c2 cells, and ONOO- treatment of H9c2 cells and isolated mitochondria, each decreased mitochondrial bound-hexokinase II (HK II), which suggests that ONOO- caused HK II to dissociate from mitochondria. Moreover, we found that mitochondria exposed to ONOO- induced VDAC1 oligomerization which may decrease its binding with HK II. We have reported that ONOO- produced during cardiac IR injury induced tyrosine nitration of VDAC1, which resulted in conformational changes of the protein and increased channel conductance associated with compromised cardiac function on reperfusion. Thus, our results imply that ONOO- produced during IR injury and hypoxic stress impeded HK II association with VDAC1. ONOO- exposure nitrated mitochondrial proteins and also led to cytochrome c (cyt c) release from mitochondria. In addition, in isolated mitochondria exposed to ONOO- or obtained after IR, there was significant compromise in mitochondrial respiration and delayed repolarization of membrane potential during oxidative (ADP) phosphorylation. Taken together, ONOO- produced during cardiac IR injury can nitrate tyrosine residues of two key mitochondrial membrane proteins involved in bioenergetics and energy transfer to contribute to mitochondrial and cellular dysfunction.


Assuntos
Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Ácido Peroxinitroso/metabolismo , Processamento de Proteína Pós-Traducional , Traumatismo por Reperfusão/fisiopatologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Cobaias , Mitocôndrias/efeitos dos fármacos , Miocárdio/patologia , Ligação Proteica/efeitos dos fármacos , Ratos
6.
Biofactors ; 45(1): 85-96, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30496631

RESUMO

Rhein, a monomeric anthraquinone obtained from the plant herb species Polygonum multiflorum and P. cuspidatum, has been proposed to have anticancer activity. This activity has been suggested to be associated with mitochondrial injury due to the induction of mitochondrial permeability transition pore (mPTP) opening. In this study, the effects of 5-80 µM rhein on cell viability, half-maximal inhibitory concentration (IC50 value), resistance index, and apoptosis were assessed in the liver cancer cell lines SMMC-7721 and SMMC-7721/DOX (doxorubicin-resistant cells). Rhein (10-80 µM) significantly reduced the viability of both cell lines; 20 µM rhein significantly increased sensitivity to DOX and increased apoptosis in SMMC-7721 cells, but reversed resistance to DOX by 7.24-fold in SMMC-7721/DOX cells. Treatment with rhein increased accumulation of DOX in SMMC-7721/DOX cells, inhibited mitochondrial energy metabolism, decreased cellular ATP, and ADP levels, and altered the ratio of ATP to ADP. These effects may result from the binding of rhein with voltage-dependent ion channels (VDACs), adenine nucleotide translocase (ANT), and cyclophilin D, affecting their function and leading to the inhibition of ATP transport by VDACs and ANT. ATP synthesis was greatly reduced and mitochondrial inner membrane potential decreased. Together, these results indicate that rhein could reverse drug resistance in SMMC-7721/DOX cells by inhibiting energy metabolism and inducing mPTP opening. © 2018 BioFactors, 45(1):85-96, 2019.


Assuntos
Antraquinonas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Antraquinonas/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclofilinas/genética , Ciclofilinas/metabolismo , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Fallopia japonica/química , Fallopia multiflora/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Extratos Vegetais/química , Canais de Ânion Dependentes de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/metabolismo
7.
Mol Imaging Biol ; 21(4): 722-730, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30225759

RESUMO

PURPOSE: Although glucose transporter 1 (GLUT1) and hexokinase 2 (HK2) are known as major proteins involved in the molecular mechanisms for accumulating 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) in cancer cells, sometimes, [18F] FDG accumulation cannot be explained by the expression of these two proteins. We investigated the involvement of adenine nucleotide translocase 2 (ANT2), which catalyzes ADP/ATP exchange at the mitochondrial inner membrane, in [18F] FDG accumulation. PROCEDURES: ANT2 expression was evaluated in various cancer cell lines and human cancer tissues (microarrays) using western blot and immunohistochemical (IHC) staining, respectively. The expression levels of ANT2 were compared to [18F] FDG accumulation and pathologic findings, including differentiation grade. Additionally, we modulated ANT2 expression levels using ANT2 siRNA and an ANT2 expression vector in cancer cells and murine xenografted tumors. RESULTS: [18F] FDG accumulation correlated with ANT2 expression in various cancer cell lines; this was not explained by GLUT1 and/or HK2 expression. At both the cell and tissue levels, ANT2 expression was high in less-differentiated or more malignant type of cancers. [18F] FDG accumulation changed according to the modulation of the ANT2 expression level. CONCLUSION: In various cancer cells and tissues, the expression levels of ANT2 explained [18F] FDG accumulation better than those of GLUT1 and HK2. ANT2 can be used as a marker of dedifferentiated pathology and aggressiveness of cancer.


Assuntos
Fluordesoxiglucose F18/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/enzimologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Translocases Mitocondriais de ADP e ATP/genética , Neoplasias/genética , Tomografia por Emissão de Pósitrons
8.
Cell Biochem Biophys ; 76(4): 445-450, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30159781

RESUMO

Several studies have demonstrated that the mitochondrial membrane switches from selective to non-selective permeability because of its improved matrix Ca2+ accumulation and oxidative stress. This process, known as permeability transition, evokes severe dysfunction in mitochondria through the opening of a non-specific pore, whose chemical nature is still under discussion. There are some proposals regarding the components of the pore structure, e.g., the adenine nucleotide translocase and dimers of the F1 Fo-ATP synthase. Our results reveal that Ca2+ induces oxidative stress, which not only increases lipid peroxidation and ROS generation but also brings about both the collapse of the transmembrane potential and the membrane release of cytochrome c. Additionally, it is shown that Ca2+ increases the binding of the probe eosin-5-maleimide to adenine nucleotide translocase. Interestingly, these effects are diminished after the addition of ADP. It is suggested that pore opening is caused by the binding of Ca2+ to the adenine nucleotide translocase.


Assuntos
Cálcio/farmacologia , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Citocromos c/metabolismo , Rim/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/química , Ligação Proteica , Ratos , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/química , Succinato Desidrogenase/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo
9.
Cell Metab ; 28(3): 490-503.e7, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30043752

RESUMO

Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90 < 3 µM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.


Assuntos
Acil Coenzima A/fisiologia , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Homeostase/efeitos dos fármacos , Macrófagos , Mitocôndrias , Células 3T3 , Células A549 , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/metabolismo , Células HCT116 , Células Hep G2 , Humanos , Interleucina-4/metabolismo , Fígado/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
10.
Mol Pharm ; 15(6): 2413-2422, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29763317

RESUMO

Ethacrynic acid (EA) is a diuretic drug that is widely used to treat high-blood pressure and swelling caused by congestive heart failure or kidney failure. It acts through noncovalent inhibition of the Na+-K+-2Cl- cotransporter in the thick ascending limb of Henle's loop. Chemically, EA contains a Michael acceptor group that can react covalently with nucleophilic residues in proteins; however, the proteome reactivity of EA remains unexplored. Herein, we took a quantitative chemoproteomic approach to globally profile EA's targets in cancer cells. We discovered that EA induces impaired mitochondrial function accompanied by increased ROS production. Our profiling revealed that EA targets functional proteins on mitochondrial membranes, including adenine nucleotide translocases (ANTs). Site-specific mapping identified that EA covalently modifies a functional cysteine in ANTs, a mutation of which resulted in the rescuing effect on EA-induced mitochondrial dysfunction. The newly discovered modes of action offer valuable information to repurpose EA for cancer treatment.


Assuntos
Reposicionamento de Medicamentos , Ácido Etacrínico/farmacologia , Mitocôndrias/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Linhagem Celular Tumoral , Cisteína/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/metabolismo , Neoplasias/patologia , Proteoma/química , Proteoma/efeitos dos fármacos , Proteômica , Espécies Reativas de Oxigênio/metabolismo
11.
Eur J Clin Invest ; 48(6): e12932, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29603199

RESUMO

BACKGROUND: Fatty livers are considerably more susceptible to acute stressors, such as ischaemia/reperfusion (I/R). As the incidence of I/R is high due to surgical events and some pathologies, there is an urgent need to find strategies against I/R injury (I/RI) in fatty livers. We postulate that an acute pretreatment with indirubin-3'-oxime (Ind) or NAD+ prevents mitochondrial dysfunction associated with warm I/RI in fatty livers. MATERIALS AND METHODS: Zucker fatty rats were subjected to warm ischaemia and 12 hours of reperfusion. Ind or NAD+ was administered in the hepatic artery 30 minutes before ischaemia. Hepatic mitochondrial isolation was performed, and functional assays as well as molecular analysis were performed. RESULTS: Pretreatment decreased markers of liver injury while preserving mitochondrial cytochrome c content, which is related to the prevention of calcium-induced mitochondrial permeability transition (mPT), the decline in mitochondrial respiratory state 3 and ATP content. The generation of reactive oxygen species (ROS) was also diminished. Inhibition of GSK-3ß by Ind resulted in the prevention of cyclophilin-D (CypD) phosphorylation, unabling it to bind to the adenine nucleotide translocator (ANT), thus, preventing mPT induction. Furthermore, deacetylation of CypD at Lys residue by sirtuin 3 (SIRT3) caused its dissociation from ANT, contributing to an increase in mPT threshold in NAD+ -pretreated animals. CONCLUSIONS: Pretreatment with Ind or NAD+ protects fatty livers by maintaining mitochondrial calcium homoeostasis, thus, preserving mitochondrial function and energetic balance. As such, CypD might be a new protective target against I/RI in fatty livers.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Fígado Gorduroso/metabolismo , Indóis/farmacologia , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , NAD/farmacologia , Oximas/farmacologia , Traumatismo por Reperfusão/metabolismo , Isquemia Quente , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Ciclofilina D , Ciclofilinas/efeitos dos fármacos , Ciclofilinas/metabolismo , Citocromos c/efeitos dos fármacos , Citocromos c/metabolismo , Fígado Gorduroso/patologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Artéria Hepática , Fígado/metabolismo , Fígado/patologia , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Ratos , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas/metabolismo
12.
J Exp Bot ; 69(8): 1913-1924, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29538769

RESUMO

The importance of a plastidial soluble inorganic pyrophosphatase (psPPase) and an ATP/ADP translocator (NTT) for starch composition and tuber formation in potato (Solanum tuberosum) was evaluated by individual and simultaneous down-regulation of the corresponding endogenous genes. Starch and amylose content of the transgenic lines were considerably lower, and granule size substantially smaller, with down-regulation of StpsPPase generating the most pronounced effects. Single-gene down-regulation of either StpsPPase or StNTT resulted in increased tuber numbers per plant and higher fresh weight yield. In contrast, when both genes were inhibited simultaneously, some lines developed only a few, small and distorted tubers. Analysis of metabolites revealed altered amounts of sugar intermediates, and a substantial increase in ADP-glucose content of the StpsPPase lines. Increased amounts of intermediates of vitamin C biosynthesis were also observed. This study suggests that hydrolysis of pyrophosphate (PPi) by action of a psPPase is vital for functional starch accumulation in potato tubers and that no additional mechanism for consuming, hydrolysing, or exporting PPi exists in the studied tissue. Additionally, it demonstrates that functional PPi hydrolysis in combination with efficient ATP import is essential for tuber formation and development.


Assuntos
Pirofosfatase Inorgânica/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos/crescimento & desenvolvimento , Plastídeos/enzimologia , Solanum tuberosum/enzimologia , Amido/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação da Expressão Gênica de Plantas , Pirofosfatase Inorgânica/genética , Translocases Mitocondriais de ADP e ATP/genética , Proteínas de Plantas/genética , Tubérculos/enzimologia , Tubérculos/genética , Tubérculos/metabolismo , Plastídeos/genética , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/metabolismo
13.
Biochemistry ; 57(6): 1031-1044, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29313673

RESUMO

Through the extensive screening of our chemical library, we found epoxycyclohexenedione (ECHD)-type compounds (AMM-59 and -120) as unique inhibitors of the bovine heart mitochondrial ADP/ATP carrier (AAC). This study investigated the mechanism of inhibition of AAC by ECHDs using submitochondrial particles (SMPs). Proteomic analyses of ECHD-bound AAC as well as biochemical characterization using different SH reagents showed that ECHDs inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160. Interestingly, AAC remarkably aggregated in SMPs upon being incubated with high concentrations of ECHDs for a long period of time. This aggregation was observed under both oxidative and reductive conditions of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SMP proteins, indicating that aggregation is not caused by intermolecular S-S linkages. ECHDs are the first chemicals, to the best of our knowledge, to induce prominent structural alteration in AAC without forming intermolecular S-S linkages. When all solvent-accessible cysteines (Cys57, Cys160, and Cys257) were previously modified by N-ethylmaleimide, the aggregation of AAC was completely suppressed. In contrast, when Cys57 or Cys160 is selectively modified by a SH reagent, the covalent binding of ECHDs to a residual free residue of the two cysteines is sufficient to induce aggregation. The aggregation-inducing ability of another ECHD analogue (AMM-124), which has an alkyl chain that is shorter than those of AMM-59 and -120, was significantly less efficient than that of the two compounds. On the basis of these results, the mechanism underlying the aggregation of AAC induced by ECHDs is discussed.


Assuntos
Cicloexanonas/química , Cicloexanonas/farmacologia , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Animais , Bovinos , Translocases Mitocondriais de ADP e ATP/metabolismo , Modelos Moleculares , Agregados Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
14.
Biochim Biophys Acta Biomembr ; 1860(5): 1035-1045, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29366674

RESUMO

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 cerevisiae
15.
Protein Expr Purif ; 144: 46-54, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29217202

RESUMO

Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded.


Assuntos
Translocases Mitocondriais de ADP e ATP/isolamento & purificação , Sistema Livre de Células , Expressão Gênica , Humanos , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Estrutura Secundária de Proteína
16.
Sci Rep ; 7(1): 15901, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29162845

RESUMO

Targeting mitochondria is a powerful strategy for pathogens to subvert cell physiology and establish infection. Helicobacter pylori is a bacterial pathogen associated with gastric cancer development that is known to target mitochondria directly and exclusively through its pro-apoptotic and vacuolating cytotoxin VacA. By in vitro infection of gastric epithelial cells with wild-type and VacA-deficient H. pylori strains, treatment of cells with purified VacA proteins and infection of a mouse model, we show that H. pylori deregulates mitochondria by two novel mechanisms, both rather associated with host cell survival. First, early upon infection VacA induces transient increase of mitochondrial translocases and a dramatic accumulation of the mitochondrial DNA replication and maintenance factors POLG and TFAM. These events occur when VacA is not detected intracellularly, therefore do not require the direct interaction of the cytotoxin with the organelle, and are independent of the toxin vacuolating activity. In vivo, these alterations coincide with the evolution of gastric lesions towards severity. Second, H. pylori also induces VacA-independent alteration of mitochondrial replication and import components, suggesting the involvement of additional H. pylori activities in mitochondria-mediated effects. These data unveil two novel mitochondrial effectors in H. pylori-host interaction with links on gastric pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Mitocondrial/metabolismo , Helicobacter pylori/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Polimerase do DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Camundongos , Translocases Mitocondriais de ADP e ATP/metabolismo , Modelos Biológicos , Transporte Proteico
17.
PLoS One ; 12(10): e0185691, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28977033

RESUMO

Quercetin and dehydrosilybin are polyphenols which are known to behave like uncouplers of respiration in isolated mitochondria. Here we investigated whether the effect is conserved in whole cells. Following short term incubation, neither compound uncouples mitochondrial respiration in whole H9c2 cells below 50µM. However, following hypoxia, or long term incubation, leak (state IV with oligomycin) oxygen consumption is increased by quercetin. Both compounds partially protected complex I respiration, but not complex II in H9c2 cells following hypoxia. In a permeabilised H9c2 cell model, the increase in leak respiration caused by quercetin is lowered by increased [ADP] and is increased by adenine nucleotide transporter inhibitor, atractyloside, but not bongkrekic acid. Both quercetin and dehydrosilybin dissipate mitochondrial membrane potential in whole cells. In the case of quercetin, the effect is potentiated post hypoxia. Genetically encoded Ca++ sensors, targeted to the mitochondria, enabled the use of fluorescence microscopy to show that quercetin decreased mitochondrial [Ca++] while dehydrosilybin did not. Likewise, quercetin decreases accumulation of [Ca++] in mitochondria following hypoxia. Fluorescent probes were used to show that both compounds decrease plasma membrane potential and increase cytosolic [Ca++]. We conclude that the uncoupler-like effects of these polyphenols are attenuated in whole cells compared to isolated mitochondria, but downstream effects are nevertheless apparent. Results suggest that the effect of quercetin observed in whole and permeabilised cells may originate in the mitochondria, while the mechanism of action of cardioprotection by dehydrosilybin may be less dependent on mitochondrial uncoupling than originally thought. Rather, protective effects may originate due to interactions at the plasma membrane.


Assuntos
Quercetina/farmacologia , Silimarina/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Digitonina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Microscopia de Fluorescência , Translocases Mitocondriais de ADP e ATP/metabolismo
18.
Biophys J ; 113(11): 2311-2315, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29056231

RESUMO

Biophysical investigation of membrane proteins generally requires their extraction from native sources using detergents, a step that can lead, possibly irreversibly, to protein denaturation. The propensity of dodecylphosphocholine (DPC), a detergent widely utilized in NMR studies of membrane proteins, to distort their structure has been the subject of much controversy. It has been recently proposed that the binding specificity of the yeast mitochondrial ADP/ATP carrier (yAAC3) toward cardiolipins is preserved in DPC, thereby suggesting that DPC is a suitable environment in which to study membrane proteins. In this communication, we used all-atom molecular dynamics simulations to investigate the specific binding of cardiolipins to yAAC3. Our data demonstrate that the interaction interface observed in a native-like environment differs markedly from that inferred from an NMR investigation in DPC, implying that in this detergent, the protein structure is distorted. We further investigated yAAC3 solubilized in DPC and in the milder dodecylmaltoside with thermal-shift assays. The loss of thermal transition observed in DPC confirms that the protein is no longer properly folded in this environment.


Assuntos
Cardiolipinas/metabolismo , Mitocôndrias/enzimologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Ligação Proteica/efeitos dos fármacos
19.
PLoS One ; 12(7): e0181489, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28727843

RESUMO

The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle.


Assuntos
Translocases Mitocondriais de ADP e ATP/metabolismo , Simulação de Dinâmica Molecular , Difosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Ácido Bongcréquico/química , Translocases Mitocondriais de ADP e ATP/química , Ligação Proteica , Conformação Proteica
20.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(9): 832-841, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28504211

RESUMO

Ecto-F1-ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F1-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y13-mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F1-ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F1-ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F1-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Endocitose/fisiologia , Hepatócitos/metabolismo , Lipoproteínas HDL/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Apolipoproteína A-I/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Hep G2 , Humanos , Proteínas Mitocondriais/metabolismo , Transdução de Sinais/fisiologia
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