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3.
Nitric Oxide ; 88: 61-72, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30999001

RESUMO

This article reviews the interactions between nitric oxide (NO) and mitochondrial respiration. Mitochondrial ATP synthesis is responsible for virtually all energy production in mammals, and every other process in living organisms ultimately depends on that energy production. Furthermore, both necrosis and apoptosis, that summarize the main forms of cell death, are intimately linked to mitochondrial integrity. Endogenous and exogenous •NO inhibits mitochondrial respiration by different well-studied mechanisms and several nitrogen derivatives. Instantaneously, low concentrations of •NO, specifically and reversibly inhibit cytochrome c oxidase in competition with oxygen, in several tissues and cells in culture. Higher concentrations of •NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Peroxynitrite can cause opening of the permeability transition pore and opening of this pore causes loss of cytochrome c, which in turn might contribute to peroxynitrite-induced inhibition of respiration. Therefore, the inhibition of cytochrome c oxidase by •NO may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen, which depend on reactive nitrogen species formation, pH, proton motriz force and oxygen supply to tissues.


Assuntos
Respiração Celular/fisiologia , Mitocôndrias/metabolismo , Óxido Nítrico/fisiologia , Animais , Bactérias , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Ácido Peroxinitroso/fisiologia , Plantas
4.
Met Ions Life Sci ; 192019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30855115

RESUMO

Physiological metabolism of cyanide takes place by a single major pathway that forms non-toxic thiocyanate that is subsequently excreted. Rhodanese is the primary enzyme to execute metabolism of cyanide with minor pathways from other sulfurtransferases in vivo. The rhodanese enzyme depends on sulfur donor availability to metabolize cyanide and poisoning occurs at elevated cyanide concentrations in vivo. Cyanide interacts with over 40 metalloenzymes, but its lethal action is non-competitive inhibition of cytochrome c oxidase, halting cellular respiration and causing hypoxic anoxia. Only a handful of antidotes for treatment of cyanide poisoning are known; they are primarily inorganic compounds and metal complexes which are intended to intercept cyanide before it inhibits cellular respiration. The inorganic compounds manipulate hemoglobin, forming methemoglobin, or supply sulfur for the rhodanese enzyme. The metal complexes intercept the cyanide and bind it before reaching its target. Cobalt complexes of corrins and vitamin B12 derivatives are the state-of-the-art agents, while the longest employed complex, Co2EDTA, is designed to deliver "free" cobalt for binding of cyanide. Compounds that are in development are discussed from the point of how they are designed to intercept cyanide. The challenge of reversing the cyanide inhibition of cytochrome c oxidase is based on the catalytic active site structure and reactivity. General information about history and occurrence of poisoning and clinical symptoms is discussed and the challenges related to analytical methods available to analyze blood cyanide levels and to confirm the presence of cyanide poisoning.


Assuntos
Antídotos/farmacologia , Cianetos/envenenamento , Metais/farmacologia , Antídotos/química , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos
5.
Nat Commun ; 9(1): 5370, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560872

RESUMO

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc1:aa3. While the cytochrome bc1:aa3 is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc1:aa3 to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.


Assuntos
Antibióticos Antituberculose/farmacologia , Úlcera de Buruli/tratamento farmacológico , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mycobacterium ulcerans/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico , África , Animais , Antibióticos Antituberculose/uso terapêutico , Austrália , Úlcera de Buruli/microbiologia , Modelos Animais de Doenças , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium ulcerans/metabolismo , Doenças Negligenciadas/microbiologia , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Rifampina/farmacologia , Rifampina/uso terapêutico , Resultado do Tratamento
6.
mBio ; 9(6)2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30459190

RESUMO

Silver (Ag+) and copper (Cu+) ions have been used for centuries in industry, as well as antimicrobial agents in agriculture and health care. Nowadays, Ag+ is also widely used in the field of nanotechnology. Yet, the underlying mechanisms driving toxicity of Ag+ ions in vivo are poorly characterized. It is well known that exposure to excess metal impairs the photosynthetic apparatus of plants and algae. Here, we show that the light-harvesting complex II (LH2) is the primary target of Ag+ and Cu+ exposure in the purple bacterium Rubrivivax gelatinosus Ag+ and Cu+ specifically inactivate the 800-nm absorbing bacteriochlorophyll a (B800), while Ni2+ or Cd2+ treatment had no effect. This was further supported by analyses of CuSO4- or AgNO3-treated membrane proteins. Indeed, this treatment induced changes in the LH2 absorption spectrum related to the disruption of the interaction of B800 molecules with the LH2 protein. This caused the release of B800 molecules and subsequently impacted the spectral properties of the carotenoids within the 850-nm absorbing LH2. Moreover, previous studies have suggested that Ag+ can affect the respiratory chain in mitochondria and bacteria. Our data demonstrated that exposure to Ag+, both in vivo and in vitro, caused a decrease of cytochrome c oxidase and succinate dehydrogenase activities. Ag+ inhibition of these respiratory complexes was also observed in Escherichia coli, but not in Bacillus subtilis IMPORTANCE The use of metal ions represents a serious threat to the environment and to all living organisms because of the acute toxicity of these ions. Nowadays, silver nanoparticles are one of the most widely used nanoparticles in various industrial and health applications. The antimicrobial effect of nanoparticles is in part related to the released Ag+ ions and their ability to interact with bacterial membranes. Here, we identify, both in vitro and in vivo, specific targets of Ag+ ions within the membrane of bacteria. This include complexes involved in photosynthesis, but also complexes involved in respiration.


Assuntos
Burkholderiales/efeitos dos fármacos , Cobre/farmacologia , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Membrana/metabolismo , Fotossíntese/efeitos dos fármacos , Prata/farmacologia , Bacterioclorofila A/antagonistas & inibidores , Burkholderiales/fisiologia , Carotenoides/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexos de Proteínas Captadores de Luz/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Succinato Desidrogenase/antagonistas & inibidores
7.
Biochem Biophys Res Commun ; 506(1): 251-258, 2018 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-30348529

RESUMO

Researchers have shown that long noncoding RNAs (lncRNAs) are closely associated with the pathogenesis of colorectal cancer (CRC). In here, we aimed to explore the function of lncRNA MAFG-AS1 in tumorigenesis of CRC. Firstly, we found that the expression of MAFG-AS1 was upregulated in CRC tissues and positively correlated with the advanced tumor stage. A reciprocal repression was found between MAFG-AS1 and miR-147b. The expression of miR-147b was downregulated in CRC tissues and inversely correlated with MAFG-AS1. Both the low-expression of miR-147b expression and the advanced tumor stage were independent factor for poor survival probability. Furthermore, overexpression of MAFG-AS1 promoted cell proliferation, cell cycle progression, and invasion, and inhibited apoptosis, while transduction of miR-147b partially reversed the effect of MAFG-AS1 on cellular processes. Consistently, stable over-expression of MAFG-AS1 contributed to the growth of colon cancer cell xenografts in vivo. NDUFA4 was identified as a direct target of miR-147b and knockdown of NDUFA4 abolished the oncogenic role of miR-147b inhibitor. Besides, MAFG-AS1 contributed to cell glycolysis by sponging miR-147b and activation of NDUFA4, causing an upregulation of PDK1, PFK1 and PKM2. Taken together, our study suggested that MAFG-AS1 functions as a novel oncogenic lncRNA in the development of CRC by regulating miR-147b/NDUFA4.


Assuntos
Neoplasias Colorretais/patologia , Progressão da Doença , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fator de Transcrição MafG/genética , MicroRNAs/antagonistas & inibidores , RNA Longo não Codificante/fisiologia , Proteínas Repressoras/genética , Animais , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glicólise , Xenoenxertos , Humanos , Camundongos , MicroRNAs/fisiologia
8.
Cell Physiol Biochem ; 49(2): 717-727, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30165359

RESUMO

BACKGROUND/AIMS: The phosphatidylinositol-3-kinase -AKT (PI3K-AKT) is an important intracellular signal pathway in regulating cell proliferation, differentiation and apoptosis. In previous studies, we've demonstrated that PI3K-AKT pathway protects cardiomyocytes from ischemic and hypoxic apoptosis through mitochondrial function. However, the molecular mechanisms underlying hypoxia-induced cardiomyocyte apoptosis via PI3K-AKT pathway remain ill-defined. Here, we addressed this question. METHODS: Cardiomyocytes were exposed to hypoxia, with/without different inhibitors and then protein levels were assessed by Western blotting. RESULTS: We found that the PI3K-AKT pathway was activated in cardiomyocytes that were exposed to hypoxia. Moreover, the phospho-AKT (pAKT) translocated from cytosol to mitochondria via mitochondrial adenosine triphosphate-dependent potassium (mitoKATP), leading to an increase in cytochrome c oxidase (CcO) activity to suppress apoptosis. On the other hand, the mitoKATP specific blocker, 5-hydroxydecanote (5-HD), or suppression of CcO using siRNA, inhibited the pAKT mitochondrial translocation to maintain the CcO activity, resulting in mitochondrial dysfunction and cellular apoptosis induced by hypoxia. CONCLUSION: These findings suggest that the anti-apoptotic effect of the PI3K-AKT pathway through pAKT translocation to mitochondrial via mitoKATP may be conducted through modification of CcO activity.


Assuntos
Apoptose , Hipóxia Celular , Fosfatidilinositol 3-Quinases/metabolismo , Canais de Potássio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Células Cultivadas , Cromonas/farmacologia , Ácidos Decanoicos/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidroxiácidos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Morfolinas/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Canais de Potássio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Sci Rep ; 8(1): 12734, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143716

RESUMO

Despite striking advances in the treatment of metastasized melanoma, the disease is often still fatal. Attention is therefore paid towards combinational regimens. Oxidants endogenously produced in mitochondria are currently targeted in pre-clinical and clinical studies. Cytotoxic synergism of mitochondrial cytochrome c oxidase (CcO) inhibition in conjunction with addition of exogenous oxidants in 2D and 3D melanoma cell culture models were examined. Murine (B16) and human SK-MEL-28 melanoma cells exposed to low-dose CcO inhibitors (potassium cyanide or sodium azide) or exogenous oxidants alone were non-toxic. However, we identified a potent cytotoxic synergism upon CcO inhibition and plasma-derived oxidants that led to rapid onset of caspase-independent melanoma cell death. This was mediated by mitochondrial dysfunction induced by superoxide elevation and ATP depletion. This observation was validated by siRNA-mediated knockdown of COX4I1 in SK-MEL-28 cells with cytotoxicity in the presence of exogenous oxidants. Similar effects were obtained with ADDA 5, a recently identified specific inhibitor of CcO activity showing low toxicity in vivo. Human keratinocytes were not affected by this combinational treatment, suggesting selective effects on melanoma cells. Hence, targeting mitochondrial CcO activity in conjunction with exogenous pro oxidant therapies may constitute a new and effective melanoma treatment modality.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Oxidantes/toxicidade , Gases em Plasma/química , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Melanoma/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , RNA Interferente Pequeno/metabolismo
10.
J Neonatal Perinatal Med ; 11(1): 79-86, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29689747

RESUMO

BACKGROUND: Hypoglycemia occurs frequently in the neonate and may result in neurologic dysfunction. Its impact on the kinetics of cellular respiration and bioenergetics in the neonatal brain remains to be explored. AIMS: Develop murine model to investigate the effects of hypoglycemia on neonatal brain bioenergetics. STUDY DESIGN: Forebrain fragments were excised from euthanized BALB/c pups aged <24 hours to 14 days. We measured cellular respiration (µM O2 min-1.mg-1) in phosphate-buffered saline with and without glucose, using phosphorescence oxygen analyzer, as well as cellular adenosine triphosphate (ATP, nmol.mg-1) using the luciferin-luciferase system. RESULTS: In the presence of glucose, although cellular respiration was 11% lower in pups ≤3 days compared to those 3- 14 days old (0.48 vs. 0.54), that difference was not statistically significant (p = 0.14). Respiration driven by endogenous metabolic fuels (without added glucose) was 16% lower in pups ≤3 days compared to those 3- 14 days (0.35 vs. 0.42, p = 0.03), confirming their increased dependency on exogenous glucose. Although cellular ATP was similar between the two age groups (14.9 vs. 11.2, p = 0.32), the ATP content was more severely depleted without added glucose in the younger pups, especially in the presence of the cytochrome c oxidase inhibitor cyanide. The first-order rate constant of cellular ATP decay (hydrolysis) was 44% lower in 2-day-old pups compared to 14-day-old mice (0.43 vs. 0.77 min-1, p = 0.03). CONCLUSIONS: Forebrain cellular respiration and ATP consumption are lower in young pups than older mice. In the absence of glucose, the support for these processes is reduced in young pups, explaining their brain hypersensitivity to hypoglycemia.


Assuntos
Trifosfato de Adenosina/metabolismo , Animais Recém-Nascidos/fisiologia , Metabolismo Energético , Hipoglicemia/fisiopatologia , Consumo de Oxigênio/efeitos dos fármacos , Prosencéfalo/fisiopatologia , Fatores Etários , Animais , Respiração Celular/efeitos dos fármacos , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glucose/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Prosencéfalo/metabolismo , Cianeto de Sódio/farmacologia
11.
Metallomics ; 10(5): 735-744, 2018 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-29676768

RESUMO

Silver has long been used as an antimicrobial agent in general and medicinal use. Here, we observe that exposure of the Gram-positive, endospore-forming bacterium Bacillus subtilis to Ag(i) effects growth in a biphasic manner. In the first phase at Ag(i) concentrations below 50 µM B. subtilis growth is not affected, but activity of the respiratory enzyme cytochrome c oxidase is disrupted completely. Between 50 to 100 µM Ag(i) B. subtilis growth is drastically diminished and completely absent above 100 µM Ag(i). Synthesis of cytochrome c oxidase, or SCO proteins, have been shown to play a role in assembly of the CuA center of cytochrome c oxidase and we suppose that the effects observed here of silver on Bacillus subtilis in culture may be explained at least in part by the interaction of Bacillus SCO (BsSCO) with Ag(i). We find that Ag(i) forms a high affinity complex with BsSCO in vitro that blocks SCO's interaction with copper indicating competition between the metals for binding BsSCO. The interaction of BsSCO with Ag(i) exhibits multiple phases and is more complex than that observed for the high-affinity, 1 : 1 copper complex with BsSCO. We propose that the initial response of B. subtilis cultures is due to high affinity binding of Ag(i) to BsSCO that blocks the functionality of BsSCO required for assembly of cytochrome c oxidase. Our results provide evidence of a specific effect of silver on Bacillus subtilis cells and implies that SCO proteins play a role in sensitivity to Ag(i).


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Prata/toxicidade , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Cinética , Proteínas de Membrana/química , Ligação Proteica , Conformação Proteica
12.
Toxicol Lett ; 289: 1-13, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29501571

RESUMO

We performed a multiple 'omics study by integrating data on epigenomic, transcriptomic, and proteomic perturbations associated with mitochondrial dysfunction in primary human hepatocytes caused by the liver toxicant valproic acid (VPA), to deeper understand downstream events following epigenetic alterations in the mitochondrial genome. Furthermore, we investigated persistence of cross-omics changes after terminating drug treatment. Upon transient methylation changes of mitochondrial genes during VPA-treatment, increasing complexities of gene-interaction networks across time were demonstrated, which normalized during washout. Furthermore, co-expression between genes and their corresponding proteins increased across time. Additionally, in relation to persistently decreased ATP production, we observed decreased expression of mitochondrial complex I and III-V genes. Persistent transcripts and proteins were related to citric acid cycle and ß-oxidation. In particular, we identified a potential novel mitochondrial-nuclear signaling axis, MT-CO2-FN1-MYC-CPT1. In summary, this cross-omics study revealed dynamic responses of the mitochondrial epigenome to an impulse toxicant challenge resulting in persistent mitochondrial dysfunctioning. Moreover, this approach allowed for discriminating between the toxic effect of VPA and adaptation.


Assuntos
Anticonvulsivantes/efeitos adversos , DNA Mitocondrial/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Ácido Valproico/efeitos adversos , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Epigenômica , Perfilação da Expressão Gênica , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Cinética , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/agonistas , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteômica
13.
J Bioenerg Biomembr ; 50(1): 21-32, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29302769

RESUMO

Neonicotinoids have high agonistic affinity to insect nicotinic acetylcholine receptors (nAChR) and are frequently used as insecticides against most devastating lepidopteran insect pests. Imidacloprid influenced dose-dependent decline in the state III and IV respiration, respiration control index (RCI), and P/O ratios, in vitro and in vivo. The bioassay indicated its LD50 value to be 531.24 µM. The insecticide exhibited a dose-dependent inhibition on F0F1-ATPase and complex IV activity. At 600 µM, the insecticide inhibited 83.62 and 27.13% of F0F1-ATPase and complex IV activity, respectively, and induced the release of 0.26 nmoles/min/mg protein of cytochrome c. A significant dose- and time-dependent increase in oxidative stress was observed; at 600 µM, the insecticide correspondingly induced lipid peroxidation, LDH activity, and accumulation of H2O2 content by 83.33, 31.51 and 223.66%. The stress was the maximum at 48 h of insecticide treatment (91.58, 35.28, and 189.80%, respectively). In contrast, catalase and superoxide dismutase were reduced in a dose- and time-dependent manner in imidacloprid-fed larvae. The results therefore suggest that imidacloprid impedes mitochondrial function and induces oxidative stress in H. armigera, which contributes to reduced growth of the larvae along with its neurotoxic effect.


Assuntos
Larva/crescimento & desenvolvimento , Mitocôndrias/metabolismo , Mariposas/efeitos dos fármacos , Neonicotinoides/toxicidade , Nitrocompostos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inseticidas/toxicidade , Larva/efeitos dos fármacos , Mariposas/metabolismo , Mariposas/ultraestrutura , Síndromes Neurotóxicas/etiologia , ATPases Translocadoras de Prótons/antagonistas & inibidores
14.
Chem Res Toxicol ; 30(12): 2197-2208, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29116760

RESUMO

In aqueous media at neutral pH, the binding of two cyanide molecules per cobinamide can be described by two formation constants, Kf1 = 1.1 (±0.6) × 105 M-1 and Kf2 = 8.5 (±0.1) × 104 M-1, or an overall cyanide binding constant of ∼1 × 1010 M-2. In comparison, the cyanide binding constants for cobalamin and a fully oxidized form of cytochrome c oxidase, each binding a single cyanide anion, were found to be 7.9 (±0.5) × 104 M-1 and 1.6 (±0.2) × 107 M-1, respectively. An examination of the cyanide-binding properties of cobinamide at neutral pH by stopped-flow spectrophotometry revealed two kinetic phases, rapid and slow, with apparent second-order rate constants of 3.2 (±0.5) × 103 M-1 s-1 and 45 (±1) M-1 s-1, respectively. Under the same conditions, cobalamin exhibited a single slow cyanide-binding kinetic phase with a second-order rate constant of 35 (±1) M-1 s-1. All three of these processes are significantly slower than the rate at which cyanide is bound by complex IV during enzyme turnover (>106 M-1 s-1). Overall, it can be understood from these findings why cobinamide is a measurably better cyanide scavenger than cobalamin, but it is unclear how either cobalt corrin can be antidotal toward cyanide intoxication as neither compound, by itself, appears able to out-compete cytochrome c oxidase for available cyanide. Furthermore, it has also been possible to unequivocally show in head-to-head comparison assays that the enzyme does indeed have greater affinity for cyanide than both cobalamin and cobinamide. A plausible resolution of the paradox that both cobalamin and cobinamide clearly are antidotal toward cyanide intoxication, involving the endogenous auxiliary agent nitric oxide, is suggested. Additionally, the catalytic consumption of oxygen by the cobalt corrins is demonstrated and, in the case of cobinamide, the involvement of cytochrome c when present. Particularly in the case of cobinamide, these oxygen-dependent reactions could potentially lead to erroneous assessment of the ability of the cyanide scavenger to restore the activity of cyanide-inhibited cytochrome c oxidase.


Assuntos
Cobalto/metabolismo , Corrinoides/metabolismo , Cianetos/metabolismo , Cianetos/toxicidade , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Oxigênio/metabolismo , Cobalto/química , Corrinoides/química , Cianetos/química , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/química , Estrutura Molecular , Oxigênio/química
15.
Biochim Biophys Acta Bioenerg ; 1858(12): 982-990, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28866381

RESUMO

Cytochrome c oxidase (CcO) from mammalian mitochondria binds Ca2+ and Na+ in a special cation binding site. Binding of Ca2+ brings about partial inhibition of the enzyme while Na+ competes with Ca2+ for the binding site and protects the enzyme from the inhibition [Vygodina, T., Kirichenko, A. and Konstantinov, A.A. (2013). Direct Regulation of Cytochrome c oxidase by Calcium Ions. PLoS One 8(9): e74436]. In the original studies, the inhibition was found to depend significantly on the ionic composition of the buffer. Here we describe inhibition of CcO by Ca2+ in media containing the main ionic components of cytoplasm (150mM KCl, 12mM NaCl and 1mM MgCl2). Under these conditions, Ca2+ inhibits CcO with effective Ki of 20-26µM, that is an order of magnitude higher than determined earlier in the absence of Na+. At physiological value of ionic strength, the inhibition can be observed at any turnover number of CcO, rather than only at low TN (<10s-1) as found previously. The inhibition requires partially oxidized state of cytochrome c and is favored by high ionic strength with a sharp transition at 0.1-0.2M. The high Ki=20-26µM found for CcO inhibition by calcium matches closely the known value of "Km" for Ca2+-induced activation of the mitochondrial calcium uniporter. The inhibition of CcO by Ca2+ is proposed to modulate mitochondrial Ca2+-uptake via the mitochondrial calcium uniporter, promote permeability transition pore opening and induce reduction of Mia40 in the mitochondrial intermembrane space.


Assuntos
Sítios de Ligação , Cálcio/química , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mitocôndrias/enzimologia , Apoptose/efeitos dos fármacos , Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/química , Mitocôndrias/genética , Concentração Osmolar , Oxirredução/efeitos dos fármacos , Ligação Proteica
16.
Cell Death Dis ; 8(6): e2853, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28569778

RESUMO

The detection of intracellular molecular oxygen (O2) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O2-independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O2. Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at 'physiological' tissue O2 levels (5% O2). Multiplexing also allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O2 despite a decrease in mitochondrial membrane potential.


Assuntos
Citocromos c/metabolismo , Mitocôndrias/metabolismo , Sondas Moleculares/química , Oxigênio/análise , Análise de Célula Única/métodos , Antimicina A/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fluorenos/química , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metaloporfirinas/química , Mitocôndrias/efeitos dos fármacos , Oxigênio/metabolismo , Polímeros/química , Rodaminas/química , Azida Sódica/farmacologia , Imagem com Lapso de Tempo/métodos
17.
Oncotarget ; 8(23): 37568-37583, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28455961

RESUMO

Patients with glioblastoma have one of the lowest overall survival rates among patients with cancer. Standard of care for patients with glioblastoma includes temozolomide and radiation therapy, yet 30% of patients do not respond to these treatments and nearly all glioblastoma tumors become resistant. Chlorpromazine is a United States Food and Drug Administration-approved phenothiazine widely used as a psychotropic in clinical practice. Recently, experimental evidence revealed the anti-proliferative activity of chlorpromazine against colon and brain tumors. Here, we used chemoresistant patient-derived glioma stem cells and chemoresistant human glioma cell lines to investigate the effects of chlorpromazine against chemoresistant glioma. Chlorpromazine selectively and significantly inhibited proliferation in chemoresistant glioma cells and glioma stem cells. Mechanistically, chlorpromazine inhibited cytochrome c oxidase (CcO, complex IV) activity from chemoresistant but not chemosensitive cells, without affecting other mitochondrial complexes. Notably, our previous studies revealed that the switch to chemoresistance in glioma cells is accompanied by a switch from the expression of CcO subunit 4 isoform 2 (COX4-2) to COX4-1. In this study, chlorpromazine induced cell cycle arrest selectively in glioma cells expressing COX4-1, and computer-simulated docking studies indicated that chlorpromazine binds more tightly to CcO expressing COX4-1 than to CcO expressing COX4-2. In orthotopic mouse brain tumor models, chlorpromazine treatment significantly increased the median overall survival of mice harboring chemoresistant tumors. These data indicate that chlorpromazine selectively inhibits the growth and proliferation of chemoresistant glioma cells expressing COX4-1. The feasibility of repositioning chlorpromazine for selectively treating chemoresistant glioma tumors should be further explored.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Clorpromazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glioma/tratamento farmacológico , Animais , Antineoplásicos Alquilantes/farmacologia , Antipsicóticos/farmacologia , Neoplasias Encefálicas/metabolismo , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Antagonistas de Dopamina/farmacologia , Reposicionamento de Medicamentos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glioma/metabolismo , Humanos , Camundongos Nus , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Bull Exp Biol Med ; 162(4): 433-435, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28239789

RESUMO

Administration of simvastatin was followed by a decrease in activities of superoxide dismutase and cytochrome oxidase in rat mitochondria, which attested to dysfunction of the respiratory chain. The decrease in total ATPase and Ca2+-ATPase activities in muscle tissue homogenates reflected impaired transport of active cations essential for muscle contraction. We conclude that abnormal relationships in the system of energy synthetic and energy-dependent processes in myocytes serve as the molecular basis for the formation of statin-induced degenerative changes.


Assuntos
Anticolesterolemiantes/efeitos adversos , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Doenças Musculares/induzido quimicamente , Fosforilação Oxidativa/efeitos dos fármacos , Sinvastatina/efeitos adversos , Animais , Animais não Endogâmicos , Transporte de Elétrons/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Mitocôndrias/metabolismo , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Doenças Musculares/fisiopatologia , Ratos , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo
19.
Biol Chem ; 398(7): 737-750, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-27926476

RESUMO

In the past, divergent results have been reported based on different methods and conditions used for enzymatic activity measurements of cytochrome c oxidase (CytOx). Here, we analyze in detail and show comparable and reproducible polarographic activity measurements of ATP-dependent inhibition of CytOx kinetics in intact and non-intact rat heart mitochondria and mitoplasts. We found that this mechanism is always present in isolated rat heart mitochondria and mitoplasts; however, it is measurable only at high ATP/ADP ratios using optimal protein concentrations. In the kinetics assay, measurement of this mechanism is independent of presence or absence of Tween-20 and the composition of measuring buffer. Furthermore, the effect of atractyloside on intact rat heart mitochondria confirms that (i) ATP inhibition occurs under uncoupled conditions [in the presence of carbonly cyanide m-chlorophenyl hydrazone (CCCP)] when the classical respiratory control is absent and (ii) high ATP/ADP ratios in the matrix as well as in the cytosolic space are required for full ATP inhibition of CytOx. Additionally, ATP inhibition measured in intact mitochondria extends in the presence of oligomycin, thus indicating further that the problem to measure the inhibitory effect of ATP on CytOx is apparently due to the lack of very high ATP/ADP ratios in isolated mitochondria.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/enzimologia , Animais , Cinética , Mitocôndrias Cardíacas/metabolismo , Ratos
20.
J Biol Chem ; 291(46): 24188-24199, 2016 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-27679486

RESUMO

The enzyme cytochrome c oxidase (CcO) or complex IV (EC 1.9.3.1) is a large transmembrane protein complex that serves as the last enzyme in the respiratory electron transport chain of eukaryotic mitochondria. CcO promotes the switch from glycolytic to oxidative phosphorylation (OXPHOS) metabolism and has been associated with increased self-renewal characteristics in gliomas. Increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure, and patients with primary glioblastoma multiforme and high tumor CcO activity have worse clinical outcomes than those with low tumor CcO activity. Therefore, CcO is an attractive target for cancer therapy. We report here the characterization of a CcO inhibitor (ADDA 5) that was identified using a high throughput screening paradigm. ADDA 5 demonstrated specificity for CcO, with no inhibition of other mitochondrial complexes or other relevant enzymes, and biochemical characterization showed that this compound is a non-competitive inhibitor of cytochrome c When tested in cellular assays, ADDA 5 dose-dependently inhibited the proliferation of chemosensitive and chemoresistant glioma cells but did not display toxicity against non-cancer cells. Furthermore, treatment with ADDA 5 led to significant inhibition of tumor growth in flank xenograft mouse models. Importantly, ADDA 5 inhibited CcO activity and blocked cell proliferation and neurosphere formation in cultures of glioma stem cells, the cells implicated in tumor recurrence and resistance to therapy in patients with glioblastoma. In summary, we have identified ADDA 5 as a lead CcO inhibitor for further optimization as a novel approach for the treatment of glioblastoma and related cancers.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glioma , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glioma/tratamento farmacológico , Glioma/enzimologia , Humanos , Camundongos , Proteínas de Neoplasias/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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