Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 945
Filtrar
1.
Cell Physiol Biochem ; 54(2): 211-229, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32100973

RESUMO

BACKGROUND/AIMS: Mitochondrial ATP synthase, in addition to being involved in ATP synthesis, is involved in permeability transition pore (PTP) formation, which precedes apoptosis in mammalian cells and programmed cell death in yeast. Mutations in genes encoding ATP synthase subunits cause neuromuscular disorders and have been identified in cancer samples. PTP is also involved in pathology. We previously found that in Saccharomyces cerevisiae, two mutations in ATP synthase subunit a (atp6-P163S and atp6-K90E, equivalent to those detected in prostate and thyroid cancer samples, respectively) in the OM45-GFP background affected ROS and calcium homeostasis and delayed yeast PTP (yPTP) induction upon calcium treatment by modulating the dynamics of ATP synthase dimer/oligomer formation. The Om45 protein is a component of the porin complex, which is equivalent to mammalian VDAC. We aimed to investigate yPTP function in atp6-P163S and atp6-K90E mutants lacking the e and g dimerization subunits of ATP synthase. METHODS: Triple mutants with the atp6-P163S or atp6-K90E mutation, the OM45-GFP gene and deletion of the TIM11 gene encoding subunit e were constructed by crossing and tetrad dissection. In spores capable of growing, the original atp6 mutations reverted to wild type, and two compensatory mutations, namely, atp6-C33S-T215C, were selected. The effects of these mutations on cellular physiology, mitochondrial morphology, bioenergetics and permeability transition (PT) were analyzed by fluorescence and electron microscopy, mitochondrial respiration, ATP synthase activity, calcium retention capacity and swelling assays. RESULTS: The atp6-C33S-T215C mutations in the OM45-GFP background led to delayed growth at elevated temperature on both fermentative and respiratory media and increased sensitivity to high calcium ions concentration or hydrogen peroxide in the medium. The ATP synthase activity was reduced by approximately 50% and mitochondrial network was hyperfused in these cells grown at elevated temperature. The atp6-C33S-T215C stabilized ATP synthase dimers and restored the yPTP properties in Tim11∆ cells. In OM45-GFP cells, in which Tim11 is present, these mutations increased the fraction of swollen mitochondria by up to 85% vs 60% in the wild type, although the time required for calcium release doubled. CONCLUSION: ATP synthase subunit e is essential in the S. cerevisiae atp6-P163S and atp6-K90E mutants. In addition to subunits e and g, subunit a is critical for yPTP induction and conduction. The increased yPTP conduction decrease the S. cerevisiae cell fitness.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Cobre/farmacologia , DNA Mitocondrial/metabolismo , Dimerização , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Mutagênese , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Temperatura Ambiente
2.
Biochim Biophys Acta Bioenerg ; 1861(1): 148091, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669489

RESUMO

F1FO ATP synthase, also known as complex V, is a key enzyme of mitochondrial energy metabolism that can synthesize and hydrolyze ATP. It is not known whether the ATP synthase and ATPase function are correlated with a different spatio-temporal organisation of the enzyme. In order to analyze this, we tracked and localized single ATP synthase molecules in situ using live cell microscopy. Under normal conditions, complex V was mainly restricted to cristae indicated by orthogonal trajectories along the cristae membranes. In addition confined trajectories that are quasi immobile exist. By inhibiting glycolysis with 2-DG, the activity and mobility of complex V was altered. The distinct cristae-related orthogonal trajectories of complex V were obliterated. Moreover, a mobile subpopulation of complex V was found in the inner boundary membrane. The observed changes in the ratio of dimeric/monomeric complex V, respectively less mobile/more mobile complex V and its activity changes were reversible. In IF1-KO cells, in which ATP hydrolysis is not inhibited by IF1, complex V was more mobile, while inhibition of ATP hydrolysis by BMS-199264 reduced the mobility of complex V. Taken together, these data support the existence of different subpopulations of complex V, ATP synthase and ATP hydrolase, the latter with higher mobility and probably not prevailing at the cristae edges. Obviously, complex V reacts quickly and reversibly to metabolic conditions, not only by functional, but also by spatial and structural reorganization.


Assuntos
Trifosfato de Adenosina/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/genética , Células HeLa , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Translocadoras de Prótons/genética
3.
Biophys Chem ; 255: 106271, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31670160

RESUMO

Adenosine triphosphate (ATP) is the universal biological energy fuel, or nature's gasoline. The vast quantities of ATP required for sustenance of living processes in cells are synthesized by oxidative phosphorylation and photosynthesis. The chemiosmotic theory of energy coupling was proposed by Mitchell more than 50 years ago but has a contentious history. Part of the accumulated body of experimental evidence supports Mitchell's theory, and part of the evidence conflicts with the theory. Although Mitchell's theory was strongly criticized by several prominent scientists, the controversy was never resolved. Certain theoretical arguments and electrostatic calculations were originally made to justify the central tenet of the chemiosmotic theory of electrogenic proton transfer and violation of electrical neutrality in bulk aqueous phases by creation of a delocalized field. However, these calculations have not been scientifically scrutinized previously. Here it is proved from first principles that the original physical arguments and calculations made in support of steady state electrogenic ion transfer and chemiosmosis violate Gauss's law. Nath's two-ion theory of energy coupling in which the field is local, and ion translocation is dynamically electrogenic but overall electroneutral is shown to satisfactorily resolve the difficulties. Characterization of length scales in mitochondrial systems is shown to impose strong constraints on possible mechanisms of energy transduction. Some biological implications for energy coupling, transduction and ATP synthesis arising as a result of the above analysis are discussed. Examples of several other biological processes where the new theory is useful such as apoptosis, muscle contraction, the joint multisite regulation of oxidative phosphorylation and the Krebs cycle, and hindered protein aggregation arising from ATP's hydrotropic properties are outlined.


Assuntos
Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Íons/química , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Contração Muscular , Teoria Quântica , Termodinâmica
4.
Nat Commun ; 10(1): 4341, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554800

RESUMO

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Hidrólise , Magnésio/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
5.
J Microbiol Biotechnol ; 29(8): 1288-1298, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370116

RESUMO

Bacterial ATP synthases drive ATP synthesis by a rotary mechanism, and play a vital role in physiology and cell metabolism. Corynebacterium glutamicum is well known as an industrial workhorse for amino acid production, and its ATP synthase operon contains eight structural genes and two adjacent genes, cg1360 and cg1361. So far, the physiological functions of Cg1360 (GenBank CAF19908) and Cg1361 (GenBank CAF19909) remain unclear. Here, we showed that Cg1360 was a hydrophobic protein with four transmembrane helices (TMHs), while no TMH was found in Cg1361. Deletion of cg1360, but not cg1361, led to significantly reduced cell growth using glucose and acetic acid as carbon sources, reduced F1 portions in the membrane, reduced ATP-driven proton-pumping activity and ATPase activity, suggesting that Cg1360 plays an important role in ATP synthase function. The intracellular ATP concentration in the Δcg1360 mutant was decreased to 72% of the wild type, while the NADH and NADPH levels in the Δcg1360 mutant were increased by 29% and 26%, respectively. However, the Δcg1361 mutant exhibited comparable intracellular ATP, NADH and NADPH levels with the wild-type strain. Moreover, the effect of cg1360 deletion on L-valine production was examined in the L-valine-producing V-10 strain. The final production of L-valine in the V-10-Δcg1360 mutant reached 9.2 ± 0.3 g/l in shake flasks, which was 14% higher than that of the V-10 strain. Thus, Cg1360 can be used as an effective engineering target by altering energy metabolism for the enhancement of amino acid production in C. glutamicum.


Assuntos
Trifosfato de Adenosina/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Deleção de Genes , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Valina/biossíntese , Ácido Acético/metabolismo , Adenosina Trifosfatases , Carbono/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Metabolismo Energético , Fermentação , Ordem dos Genes , Glucose/metabolismo , NAD/metabolismo , NADP/metabolismo , Alinhamento de Sequência
6.
Anal Bioanal Chem ; 411(26): 6899-6911, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31407049

RESUMO

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 µM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.


Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Quadruplex G , Humanos , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neoplasias/metabolismo , Oligomicinas/farmacologia
7.
Redox Biol ; 26: 101258, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31234016

RESUMO

Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M-1 s-1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation-an established molecular correlate of impaired enzyme activity-in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50-60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.


Assuntos
Química Click/métodos , Mitocôndrias/química , ATPases Mitocondriais Próton-Translocadoras/química , Óvulo/química , Polietilenoglicóis/química , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Trifosfato de Adenosina/biossíntese , Sequência de Aminoácidos , Animais , Dissulfetos/química , Embrião não Mamífero , Feminino , Fertilização In Vitro , Glutationa/metabolismo , Compostos Heterocíclicos com 1 Anel/química , Masculino , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Óvulo/citologia , Óvulo/enzimologia , Oxirredução , Filogenia , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Xenopus laevis/classificação , Xenopus laevis/embriologia , Xenopus laevis/metabolismo
8.
Sheng Li Xue Bao ; 71(3): 388-394, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31218329

RESUMO

The aim of this study was to determine the effects of extremely low frequency electromagnetic field (ELF-EMF) on energy metabolism and oxidative stress in Caenorhabditis elegans (C. elegans). Worms in three adult stages (young adult stage, egg-laying stage and peak egg-laying stage) were investigated under 50 Hz, 3 mT ELF-EMF exposure. ATP levels, ATP synthase activity in vivo, reactive oxygen species (ROS) content, and changes of total antioxidant capacity (TAC) were detected, and worms' oxidative stress responses were also evaluated under ELF-EMF exposure. The results showed that ATP levels were significantly increased under this ELF-EMF exposure, and mitochondrial ATP synthase activity was upregulated simultaneously. In young adult stage, worms' ROS level was significantly elevated, together with upregulated TAC but with a decreased ROS-TAC score indicated by principal component analysis. ROS level and TAC of worms had no significant changes in egg-laying and peak egg-laying stages. Based on these results, we concluded that ELF-EMF can enhance worm energy metabolism and elicit oxidative stress, mainly manifesting as ATP and ROS level elevation together with ATP synthase upregulation and ROS-TAC score decrease in young adult C. elegans.


Assuntos
Caenorhabditis elegans/efeitos da radiação , Radiação Eletromagnética , Metabolismo Energético , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Animais , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Espécies Reativas de Oxigênio/análise
9.
Oxid Med Cell Longev ; 2019: 8743257, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178976

RESUMO

The mitochondrial F-ATP synthase is the principal energy-conserving nanomotor of cells that harnesses the proton motive force generated by the respiratory chain to make ATP from ADP and phosphate in a process known as oxidative phosphorylation. In the energy-converting membranes, F-ATP synthase is a multisubunit complex organized into a membrane-extrinsic F1 sector and a membrane-intrinsic FO domain, linked by central and peripheral stalks. Due to its essential role in the cellular metabolism, malfunction of F-ATP synthase has been associated with a variety of pathological conditions, and the enzyme is now considered as a promising drug target for multiple disease conditions and for the regulation of energy metabolism. We discuss structural and functional features of mitochondrial F-ATP synthase as well as several conditions that partially or fully inhibit the coupling between the F1 catalytic activities and the FO proton translocation, thus decreasing the cellular metabolic efficiency and transforming the enzyme into an energy-dissipating structure through molecular mechanisms that still remain to be defined.


Assuntos
Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Metabolismo Energético , Humanos
10.
Biochim Biophys Acta Bioenerg ; 1860(7): 562-572, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31181185

RESUMO

Dozens of pathogenic mutations have been localized in the mitochondrial gene (MT-ATP6) that encodes the subunit a of ATP synthase. The subunit a together with a ring of identical subunits c moves protons across the mitochondrial inner membrane coupled to rotation of the subunit c-ring and ATP synthesis. One of these mutations, m.8851T>C, has been associated with bilateral striatal lesions of childhood (BSLC), a group of rare neurological disorders characterized by symmetric degeneration of the corpus striatum. It converts a highly conserved tryptophan residue into arginine at position 109 of subunit a (aW109R). We previously showed that an equivalent thereof in Saccharomyces cerevisiae (aW126R) severely impairs by an unknown mechanism the functioning of ATP synthase without any visible assembly/stability defect. Herein we show that ATP synthase function was recovered to varying degree by replacing the mutant arginine residue 126 with methionine, lysine or glycine or by replacing with methionine an arginine residue present at position 169 of subunit a (aR169). In recently described atomic structures of yeast ATP synthase, aR169 is at the center of a hydrophilic cleft along which protons are transported from the subunit c-ring to the mitochondrial matrix, in the proximity of the two residues known from a long time to be essential to the activity of FO (aR176 and cE59). We provide evidence that the aW126R change is responsible for electrostatic and steric hindrance that enables aR169 to engage in a salt bridge with cE59. As a result, aR176 cannot interact properly with cE5 and ATP synthase fails to effectively move protons across the mitochondrial membrane. In addition to insight into the pathogenic mechanism induced by the m.8851T>C mutation, the present study brings interesting information about the role of specific residues of subunit a in the energy-transducing activity of ATP synthase.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Humanos , Potencial da Membrana Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Fosforilação Oxidativa , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência
11.
Am J Physiol Endocrinol Metab ; 317(2): E284-E297, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31184932

RESUMO

Hydrogen sulfide (H2S), a gaseous molecule, is involved in modulating multiple physiological functions, such as antioxidant, antihypertension, and the production of polysulfide cysteine. H2S may inhibit reactive oxygen species generation and ATP production through modulating respiratory chain enzyme activities; however, the mechanism of this effect remains unclear. In this study, db/db mice, neonatal rat cardiomyocytes, and H9c2 cells treated with high glucose, oleate, and palmitate were used as animal and cellular models of type 2 diabetes. The mitochondrial respiratory rate, respiratory chain complex activities, and ATP production were decreased in db/db mice compared with those in db/db mice treated with exogenous H2S. Liquid chromatography with tandem mass spectrometry analysis showed that the acetylation level of proteins involved in the mitochondrial respiratory chain were increased in the db/db mice hearts compared with those with sodium hydrosulfide (NaHS) treatment. Exogenous H2S restored the ratio of NAD+/NADH, enhanced the expression and activity of sirtuin 3 (SIRT3) and decreased mitochondrial acetylation level in cardiomyocytes under hyperglycemia and hyperlipidemia. As a result of SIRT3 activation, acetylation of the respiratory complexe enzymes NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1, and ATP synthase mitochondrial F1 complex assembly factor 1 was reduced, which enhanced the activities of the mitochondrial respiratory chain activity and ATP production. We conclude that exogenous H2S plays a critical role in improving cardiac mitochondrial function in diabetes by upregulating SIRT3.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Complexo II de Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , ATPases Mitocondriais Próton-Translocadoras/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Sirtuína 3/metabolismo , Acetilação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/metabolismo , NAD/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
12.
J Biol Chem ; 294(28): 10987-10997, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31160339

RESUMO

The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dimerização , Potencial da Membrana Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Mutagênese Sítio-Dirigida , Estabilidade Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
13.
Biochim Biophys Acta Bioenerg ; 1860(8): 659-678, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247171

RESUMO

Hypoxia causes mitochondrial cristae widening, enabled by the ~20% degradation of Mic60/mitofilin, with concomitant clustering of the MICOS complex, reflecting the widening of crista junctions (outlets) (Plecitá-Hlavatá et al. FASEB J., 2016 30:1941-1957). Attempting to accelerate metabolism by the addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to HepG2 cells pre-adapted to hypoxia, we found cristae narrowing by transmission electron microscopy. Glycolytic HepG2 cells, which downregulate hypoxic respiration, instantly increased respiration with dm2OG. Changes in intracristal space (ICS) morphology were also revealed by 3D super-resolution microscopy using Eos-conjugated ICS-located lactamase-ß. Cristae topology was resolved in detail by focused-ion beam/scanning electron microscopy (FIB/SEM). The spatial relocations of key cristae-shaping proteins were indicated by immunocytochemical stochastic 3D super-resolution microscopy (dSTORM), while analyzing inter-antibody-distance histograms: i) ATP-synthase dimers exhibited a higher fraction of shorter inter-distances between bound F1-α primary Alexa-Fluor-647-conjugated antibodies, indicating cristae narrowing. ii) Mic60/mitofilin clusters (established upon hypoxia) decayed, restoring isotropic random Mic60/mitofilin distribution (a signature of normoxia). iii) outer membrane SAMM50 formed more focused clusters. Less abundant fractions of higher ATP-synthase oligomers of hypoxic samples on blue-native electrophoresis became more abundant fractions at the high dm2OG load and at normoxia. This indicates more labile ATP-synthase dimeric rows established at crista rims upon hypoxia, strengthened at normoxia or dm2OG-substrate load. Hypothetically, the increased Krebs substrate load stimulates the cross-linking/strengthening of rows of ATP-synthase dimers at the crista rims, making them sharper. Crista narrowing ensures a more efficient coupling of proton pumping to ATP synthesis. We demonstrated that cristae morphology changes even within minutes.


Assuntos
Ácidos Cetoglutáricos/farmacologia , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Respiração Celular , Dimerização , Células Hep G2 , Humanos , Hipóxia , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo
14.
Int J Biol Macromol ; 135: 582-590, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31121229

RESUMO

The shortcomings of conventional tuberculosis treatments resulting from the development of drug resistance in Mycobacterium tuberculosis drive a need for the formulation of novel therapeutic agents. The diarylquinoline class of drugs such as bedaquiline was recently approved for the treatment of multidrug-resistant strains of tuberculosis, primarily targeting c and ε subunits of the ATP synthases. Yet resistance to bedaquiline has already been reported. Therefore, Rv1311 was used as the target for the identification of possible inhibitors against the M. tuberculosis. The structure of Rv1311 was predicted and common feature pharmacophore models were generated which facilitated the identification of potential inhibitors in the ZINC database. The activities of the selected molecules were compared with known inhibitors of the ATP synthase using quantitative structure-activity relationship. The ZINC classified inhibitors showed comparable predicted activities with that of known inhibitors. Furthermore, the inhibitory behavior of the studied drug molecules was experimentally determined using in vitro techniques and showed the minimum inhibitory concentration as low as 25 µM. The resulted outcomes provide a deeper insight into the structural basis of Rv1311 inhibitions and can facilitate the process of drug design against tuberculosis.


Assuntos
Simulação por Computador , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Desenho de Drogas , Inibidores Enzimáticos/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica
15.
Nat Neurosci ; 22(6): 851-862, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086314

RESUMO

The GGGGCC repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is not known which dysregulated molecular pathways are primarily responsible for disease initiation or progression. We established an inducible mouse model of poly(GR) toxicity in which (GR)80 gradually accumulates in cortical excitatory neurons. Low-level poly(GR) expression induced FTD/ALS-associated synaptic dysfunction and behavioral abnormalities, as well as age-dependent neuronal cell loss, microgliosis and DNA damage, probably caused in part by early defects in mitochondrial function. Poly(GR) bound preferentially to the mitochondrial complex V component ATP5A1 and enhanced its ubiquitination and degradation, consistent with reduced ATP5A1 protein level in both (GR)80 mouse neurons and patient brains. Moreover, inducing ectopic Atp5a1 expression in poly(GR)-expressing neurons or reducing poly(GR) level in adult mice after disease onset rescued poly(GR)-induced neurotoxicity. Thus, poly(GR)-induced mitochondrial defects are a major driver of disease initiation in C9ORF72-related ALS/FTD.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Proteína C9orf72/genética , Demência Frontotemporal/fisiopatologia , Mitocôndrias/patologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Encéfalo/metabolismo , Expansão das Repetições de DNA , Modelos Animais de Doenças , Demência Frontotemporal/genética , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo
16.
Planta ; 250(2): 507-518, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31098709

RESUMO

MAIN CONCLUSION: 'Petaloid' cytoplasmic male sterility is commonly used as a stable genetic mechanism in carrot hybrid breeding. Its introgression in tropical carrot showed morphometric changes and molecular markers were identified for detection at early stage. Cytoplasmic male sterility (CMS) is the only genetic mechanism in carrot for commercial exploitation of heterosis and production of low cost affordable hybrid seeds. The 'petaloid' CMS system is stable and commonly used in hybrid breeding in temperate carrot but there is no information available on existence of natural CMS system in tropical Asiatic carrot. Therefore, the present study was aimed to investigate morphometric traits and organizational features of cytoplasmic atp9 gene sequences in newly converted CMS lines (BC4-7) of tropical carrot. The CMS lines had root traits at par with fertile counterparts while floral traits had variation. Petal colour and length, petaloids colour and shape and style length showed differences among the CMS lines and with their maintainers. Molecular markers are effective to establish male sterility at genetic level, for this, six fixed and stable CMS lines were screened with seven novel primer combinations. Out of which five pairs produced clearly distinguishable bands in CMS lines and their fertile counterparts. The study confirmed that the region between 3' end of atp9-1/atp9-3 gene and 5' end of region of homology to Arabidopsis thaliana mtDNA is ideal for developing the trait specific markers. These new CMS lines have potential to use in hybrid development and molecular markers will be useful to confirm male sterility to rogue out fertile plants.


Assuntos
Daucus carota/genética , Infertilidade das Plantas/genética , Proteínas de Plantas/genética , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Quimera , Citoplasma/genética , DNA Mitocondrial/genética , Daucus carota/anatomia & histologia , Daucus carota/fisiologia , Marcadores Genéticos/genética , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas
17.
Int J Oncol ; 55(1): 7-20, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115496

RESUMO

Mitotane is used for the treatment of adrenocortical cancer and elicits its anticancer effects via inhibition of mitochondrial respiration. Targeting mitochondria­dependent metabolism has emerged as a promising strategy for thyroid cancer (TC) treatment. We hypothesized that mitotane targets mitochondria and induces apoptosis in TC cells. Cell lines representative of the major histological variants of TC were chosen: Follicular (FTC­133), poorly differentiated (BCPAP), anaplastic (SW1736 and C643) and medullary (TT) TC cells, and were treated with mitotane (0­100 µM). Mitochondrial membrane potential, cell viability and apoptosis were examined by JC­1 staining and by western blot analysis using an antibody against caspase­3. The expression of mitochondrial molecules and DNA damage markers and the activation of endoplasmic reticulum (ER) stress were determined by western blotting. The expression of mitochondrial ATP synthase subunit ß (ATP5B) was examined by immunostaining in 100 human TC tissue samples. Treatment with mitotane (50 µM for 24 h) decreased the viability of FTC­133, BCPAP, SW1736, C643 and TT cells by 12, 59, 54, 31 and 66%, respectively. Morphological evidence of ER stress and overexpression of ER markers was observed in TC cells following exposure to mitotane. The treatment led to increased expression of histone γH2AX, indicating DNA damage, and to caspase­3 cleavage. Consistent with the results of the cell viability assays, the overexpression of pro­apoptotic genes following treatment with mitotane was more prominent in TC cells harboring mutations in the serine/threonine­protein kinase B­raf gene and proto­oncogene tyrosine­protein kinase receptor Ret. Treatment with mitotane was associated with loss of mitochondrial membrane potential and decreased expression of ATP5B, particularly in the medullary TC (MTC)­derived TT cells. Immunohistochemical analysis of mitochondrial ATP5B in human TC specimens demonstrated its overexpression in cancer compared with normal thyroid tissue. The level of ATP5B expression was higher in MTC compared with the follicular, papillary or anaplastic types of TC. Mitotane elicited pleiotropic effects on TC cells, including induction of ER stress, inhibition of mitochondrial membrane potential and induction of apoptosis. The results of the present study suggest that mitotane could be considered as a novel agent for the treatment of aggressive types of TC.


Assuntos
Antineoplásicos Hormonais/farmacologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mitotano/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética
18.
FEBS Open Bio ; 9(4): 571-581, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30984533

RESUMO

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine-123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF-1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo-ATPase was inhibited with oligomycin-A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.


Assuntos
Mitocôndrias/química , ATPases Mitocondriais Próton-Translocadoras/genética , Canais de Potássio/metabolismo , Células Piramidais/fisiologia , Proteínas de Répteis/genética , Tartarugas/fisiologia , Anaerobiose , Animais , Concentração de Íons de Hidrogênio , Potencial da Membrana Mitocondrial/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Proteínas de Répteis/metabolismo
19.
Chem Biodivers ; 16(6): e1900085, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30990952

RESUMO

The discovery of J147 represented a significant milestone in the treatment of age-related disorders, which was further augmented by the recent identification of mitochondrial ATP synthase as the therapeutic target. However, the underlying molecular events associated with the modulatory activity of J147 have remained unresolved till date. Herein, we present, for the first time, a dynamical approach to investigate the allosteric regulation of mATP synthase by J147, using a reliable human αÎ³ß protein model. The highlight of our findings is the existence of the J147-bound protein in distinct structural associations at different MD simulation periods coupled with concurrent open↔close transitions of the ß catalytic and α allosteric (ATP5A) sites as defined by Cα distances (d), TriCα (Θ) and dihedral (φ) angular parameters. Firstly, there was an initial pairing of the αγ subunits away from the ß subunit followed by the formation of the 'non-catalytic' αß pair at a distance from the γ subunit. Interestingly, J147-induced structural arrangements were accompanied by the systematic transition of the ß catalytic site from a closed to an open state, while there was a concurrent transition of the allosteric site from an open αE conformation to a closed state. Consequentially, J147 reduced the structural activity of the whole αÎ³ß complex, while the unbound system exhibited high atomistic deviations and structural flexibility. Furthermore, J147 exhibited favorable binding at the allosteric site of mATP synthase with considerable electrostatic energy contributions from Gln215, Gly217, Thr219, Asp312, Asp313, Glu371 and Arg406. These findings provide details on the possible effects of J147 on mitochondrial bioenergetics, which could facilitate the structure-based design of novel small-molecule modulators of mATP synthase in the management of Alzheimer's disease and other neurodegenerative disorders.


Assuntos
Curcumina/análogos & derivados , Hidrazinas/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Regulação Alostérica , Doença de Alzheimer/tratamento farmacológico , Sítios de Ligação , Domínio Catalítico , Curcumina/farmacologia , Humanos , Hidrazinas/metabolismo , Hidrazinas/uso terapêutico , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , Simulação de Acoplamento Molecular , Análise de Componente Principal , Eletricidade Estática , Termodinâmica
20.
Breast Cancer Res Treat ; 176(2): 271-289, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31006104

RESUMO

PURPOSE: Primary breast and prostate cancers can be cured, but metastatic disease cannot. Identifying cell factors that predict metastatic potential could guide both prognosis and treatment. METHODS: We used Cell-SELEX to screen an RNA aptamer library for differential binding to prostate cancer cell lines with high vs. low metastatic potential. Mass spectroscopy, immunoblot, and immunohistochemistry were used to identify and validate aptamer targets. Aptamer properties were tested in vitro, in xenograft models, and in clinical biopsies. Gene expression datasets were queried for target associations in cancer. RESULTS: We identified a novel aptamer (Apt63) that binds to the beta subunit of F1Fo ATP synthase (ATP5B), present on the plasma membrane of certain normal and cancer cells. Apt63 bound to plasma membranes of multiple aggressive breast and prostate cell lines, but not to normal breast and prostate epithelial cells, and weakly or not at all to non-metastasizing cancer cells; binding led to rapid cell death. A single intravenous injection of Apt63 induced rapid, tumor cell-selective binding and cytotoxicity in MDA-MB-231 xenograft tumors, associated with endonuclease G nuclear translocation and DNA fragmentation. Apt63 was not toxic to non-transformed epithelial cells in vitro or adjacent normal tissue in vivo. In breast cancer tissue arrays, plasma membrane staining with Apt63 correlated with tumor stage (p < 0.0001, n = 416) and was independent of other cancer markers. Across multiple datasets, ATP5B expression was significantly increased relative to normal tissue, and negatively correlated with metastasis-free (p = 0.0063, 0.00039, respectively) and overall (p = 0.050, 0.0198) survival. CONCLUSION: Ecto-ATP5B binding by Apt63 may disrupt an essential survival mechanism in a subset of tumors with high metastatic potential, and defines a novel category of cancers with potential vulnerability to ATP5B-targeted therapy. Apt63 is a unique tool for elucidating the function of surface ATP synthase, and potentially for predicting and treating metastatic breast and prostate cancer.


Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Neoplasias da Mama/patologia , Membrana Celular/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neoplasias da Próstata/patologia , Administração Intravenosa , Animais , Aptâmeros de Nucleotídeos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Detecção Precoce de Câncer , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Masculino , Camundongos , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Estadiamento de Neoplasias , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Técnica de Seleção de Aptâmeros , Resultado do Tratamento , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA