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1.
Ecotoxicol Environ Saf ; 208: 111475, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33068975

RESUMO

Cocaine is one of the most widely used illicit drugs in the world, and as a result of incomplete removal by sewage treatment plants it is found in surface waters, where it represents a new potential risk for aquatic organisms. In this study we evaluated the influence of environmental concentrations of cocaine on the liver and the kidney of the European eel (Anguilla anguilla). The eels were exposed to 20 ng L-1 of cocaine for fifty days, after which, three and ten days after the interruption of cocaine exposure their livers and kidneys were compared to controls. The general morphology of the two organs was evaluated, as well as the following parameters: cytochrome oxidase (COX) and caspase-3 activities, as markers of oxidative metabolism and apoptosis activation, respectively; glucose-regulated protein (GRP)78 levels, as a marker of endoplasmic reticulum (ER)-stress; blood glucose level, as stress marker; serum levels of alanine aminotransferase (ALT), as a marker of liver injury and serum levels of C-reactive protein (CRP), as a marker of the inflammatory process. The liver showed morphologic alterations such as necrotic areas, karyolysis and pyknotic nuclei, while the kidneys had dilated glomeruli and the renal tubules showed pyknotic nuclei and karyolysis. In the kidney, the alterations persisted after the interruption of cocaine exposure. In the liver, COX and caspase-3 activities increased (COX: P = 0.01; caspase-3: P = 0.032); ten days after the interruption of cocaine exposure, COX activity returned to control levels (P = 0.06) whereas caspase-3 activity decreased further (P = 0.012); GRP78 expression increased only in post-exposure recovery specimens (three days: P = 0.007 and ten days: P = 0.008 after the interruption of cocaine exposure, respectively). In the kidney, COX and caspase-3 activities increased (COX: P = 0.02; caspase-3: P = 0.019); after the interruption of cocaine exposure, COX activity remained high (three days: P = 0.02 and ten days: P = 0.029 after the interruption of cocaine exposure, respectively) whereas caspase-3 activity returned to control values (three days: P = 0.69 and ten days: P = 0.67 after the interruption of cocaine exposure, respectively). Blood glucose and serum ALT and CRP levels increased (blood glucose: P = 0.01; ALT: P = 0.001; CRP: 0.015) and remained high also ten days after the interruption of cocaine exposure (blood glucose: P = 0.009; ALT: P = 0.0031; CRP: 0.036). These results suggest that environmental cocaine concentrations adversely affected liver and kidney of this species.


Assuntos
Anguilla/fisiologia , Cocaína/toxicidade , Poluentes Químicos da Água/toxicidade , Alanina Transaminase/metabolismo , Anguilla/sangue , Animais , Glicemia , Proteína C-Reativa/metabolismo , Caspase 3/metabolismo , Cocaína/análise , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Drogas Ilícitas , Rim/metabolismo , Fígado/metabolismo
2.
Sci Rep ; 10(1): 17012, 2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33046789

RESUMO

Statins are prescribed to treat hypercholesterolemia and to reduce the risk of cardiovascular disease. However, statin users frequently report myalgia, which can discourage physical activity or cause patients to discontinue statin use, negating the potential benefit of the treatment. Although a proposed mechanism responsible for Statin-Associated Myopathy (SAM) suggests a correlation with impairment of mitochondrial function, the relationship is still poorly understood. Here, we provide evidence that long-term treatment of hypercholesterolemic patients with Simvastatin at a therapeutic dose significantly display increased mitochondrial respiration in peripheral blood mononuclear cells (PBMCs), and platelets compared to untreated controls. Furthermore, the amount of superoxide is higher in mitochondria in PBMCs, and platelets from Simvastatin-treated patients than in untreated controls, and the abundance of mitochondrial superoxide, but not mitochondrial respiration trends with patient-reported myalgia. Ubiquinone (also known as coenzyme Q10) has been suggested as a potential treatment for SAM; however, an 8-week course of oral ubiquinone had no impact on mitochondrial functions or the abundance of superoxide in mitochondria from PBMCs, and platelets. These results demonstrate that long-term treatment with Simvastatin increases respiration and the production of superoxide in mitochondria of PBMCs and platelets.


Assuntos
Plaquetas/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Sinvastatina/farmacologia , Plaquetas/metabolismo , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Sinvastatina/uso terapêutico , Superóxidos/metabolismo
3.
Met Ions Life Sci ; 202020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32851825

RESUMO

CuA is a binuclear copper center acting as an electron transfer hub in terminal oxidases such as cytochrome c oxidase and nitrous oxide reductase. Its unique electronic structure is intimately linked to its function and has puzzled the community of biological inorganic chemistry for decades. Here we review the insights provided by different spectroscopic techniques of CuA centers, and the different experimental approaches to tackle its study, that encompass the synthesis of model compounds as well as protein engineering efforts. The contribution of the electronic structure to the thermodynamic and kinetic of electron transfer is extensively discussed. We also describe the proposed mechanism of CuAassembly in different organisms. The recent discovery of a novel CuA site opens new perspectives to this field.


Assuntos
Cobre/química , Sequência de Aminoácidos , Cobre/metabolismo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Análise Espectral
4.
J Neurosci ; 40(35): 6801-6811, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32747442

RESUMO

Limits on perceptual capacity result in various phenomena of inattentional blindness. Here we propose a neurophysiological account attributing these perceptual capacity limits directly to limits on cerebral cellular metabolism. We hypothesized that overall cerebral energy supply remains constant, regardless of overall mental processing demands; therefore, an attention mechanism is required to regulate limited cellular metabolism levels in line with attended task demands. Increased perceptual load in a task (imposing a greater demand on neural computations) should thus result in increased metabolism underlying attended processing, and reduced metabolism mediating unattended processing. We tested this prediction measuring oxidation states of cytochrome c oxidase (oxCCO), an intracellular marker of cellular metabolism. Broadband near-infrared spectroscopy was used to record oxCCO levels from human visual cortex while participants (both sexes) performed a rapid sequential visual search task under either high perceptual load (complex feature-conjunction search) or low load (feature pop-out search). A task-irrelevant, peripheral checkerboard was presented on a random half of trials. Our findings showed that oxCCO levels in visual cortex regions responsive to the attended-task stimuli were increased in high versus low perceptual load, whereas oxCCO levels related to unattended processing were significantly reduced. A negative temporal correlation of these load effects further supported our metabolism trade-off account. These results demonstrate an attentional compensation mechanism that regulates cellular metabolism levels according to processing demands. Moreover, they provide novel evidence for the widely held stipulation that overall cerebral metabolism levels remain constant regardless of mental task demand and establish a neurophysiological account for capacity limits in perception.SIGNIFICANCE STATEMENT We investigated whether capacity limits in perception can be explained by the effects of attention on the allocation of limited cellular metabolic energy for perceptual processing. We measured the oxidation state of cytochrome c oxidase, an intracellular measure of metabolism, in human visual cortex during task performance. The results showed increased levels of cellular metabolism associated with attended processing and reduced levels of metabolism underlying unattended processing when the task was more demanding. A temporal correlation between these effects supported an attention-directed metabolism trade-off. These findings support an account for inattentional blindness grounded in cellular biochemistry. They also provide novel evidence for the claim that cerebral processing is limited by a constant energy supply, which thus requires attentional regulation.


Assuntos
Atenção , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Córtex Visual/fisiologia , Percepção Visual , Adolescente , Adulto , Feminino , Humanos , Masculino , Córtex Visual/metabolismo
5.
Arterioscler Thromb Vasc Biol ; 40(10): 2376-2390, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787519

RESUMO

OBJECTIVES: Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD+) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD+-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD+ regeneration, pointing out to the key role of NAD+ depletion in the inhibition of platelet bioenergetics by CORM-A1. CONCLUSIONS: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD+ depletion.


Assuntos
Trifosfato de Adenosina/metabolismo , Plaquetas/efeitos dos fármacos , Boranos/farmacologia , Monóxido de Carbono/farmacologia , Carbonatos/farmacologia , Glicólise/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , NAD/metabolismo , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Plaquetas/metabolismo , Respiração Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Masculino , Mitocôndrias/metabolismo
6.
Life Sci ; 258: 118195, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781073

RESUMO

AIMS: The estrogen-ERα axis participates in osteoblast maturation. This study was designed to further evaluated the roles of the estrogen-ERα axis in bone healing and the possible mechanisms. MAIN METHODS: Female ICR mice were created a metaphyseal bone defect in the left femurs and administered with methylpiperidinopyrazole (MPP), an inhibitor of ERα. Bone healing was evaluated using micro-computed tomography. Colocalization of ERα with alkaline phosphatase (ALP) and ERα translocation to mitochondria were determined. Levels of ERα, ERß, PECAM-1, VEGF, and ß-actin were immunodetected. Expression of chromosomal Runx2, ALP, and osteocalcin mRNAs and mitochondrial cytochrome c oxidase (COX) I and COXII mRNAs were quantified. Angiogenesis was measured with immunohistochemistry. KEY FINDINGS: Following surgery, the bone mass was time-dependently augmented in the bone-defect area. Simultaneously, levels of ERα were specifically upregulated and positively correlated with bone healing. Administration of MPP to mice consistently decreased levels of ERα and bone healing. As to the mechanisms, osteogenesis was enhanced in bone healing, but MPP attenuated osteoblast maturation. In parallel, expressions of osteogenesis-related ALP, Runx2, and osteocalcin mRNAs were induced in the injured zone. Treatment with MPP led to significant inhibition of the alp, runx2, and osteocalcin gene expressions. Remarkably, administration of MPP lessened translocation of ERα to mitochondria and expressions of mitochondrial energy production-related coxI and coxII genes. Furthermore, exposure to MPP decreased levels of PECAM-1 and VEGF in the bone-defect area. SIGNIFICANCE: The present study showed the contributions of the estrogen-ERα axis to bone healing through stimulation of energy production, osteoblast maturation, and angiogenesis.


Assuntos
Regeneração Óssea , Diferenciação Celular , Metabolismo Energético , Receptor alfa de Estrogênio/metabolismo , Neovascularização Fisiológica , Osteoblastos/citologia , Transdução de Sinais , Fosfatase Alcalina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Diferenciação Celular/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
7.
Sci Rep ; 10(1): 11643, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669663

RESUMO

The pathogenesis of declining bone mineral density, a universal feature of ageing, is not fully understood. Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in human tissues and mounting evidence suggests that they may be integral to the ageing process. To explore the potential effects of mtDNA mutations on bone biology, we compared bone microarchitecture and turnover in an ageing series of wild type mice with that of the PolgAmut/mut mitochondrial DNA 'mutator' mouse. In vivo analyses showed an age-related loss of bone in both groups of mice; however, it was significantly accelerated in the PolgAmut/mut mice. This accelerated rate of bone loss is associated with significantly reduced bone formation rate, reduced osteoblast population densities, increased osteoclast population densities, and mitochondrial respiratory chain deficiency in osteoblasts and osteoclasts in PolgAmut/mut mice compared with wild-type mice. In vitro assays demonstrated severely impaired mineralised matrix formation and increased osteoclast resorption by PolgAmut/mut cells. Finally, application of an exercise intervention to a subset of PolgAmut/mut mice showed no effect on bone mass or mineralised matrix formation in vitro. Our data demonstrate that mitochondrial dysfunction, a universal feature of human ageing, impairs osteogenesis and is associated with accelerated bone loss.


Assuntos
Envelhecimento/genética , Reabsorção Óssea/genética , Polimerase do DNA Mitocondrial/genética , DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Osteogênese/genética , Osteoporose/genética , Animais , Densidade Óssea/fisiologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/fisiopatologia , Calcificação Fisiológica , Contagem de Células , Polimerase do DNA Mitocondrial/deficiência , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Fêmur/metabolismo , Fêmur/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/patologia , Mutação , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/metabolismo , Osteoporose/fisiopatologia , Condicionamento Físico Animal
8.
Biochimie ; 176: 150-157, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32721502

RESUMO

Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H2O2 production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca2+-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promoted the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed.


Assuntos
Complexo II de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Mitocôndrias Hepáticas/metabolismo , Succinatos/farmacologia , Animais , Cálcio/metabolismo , Citocromos c/metabolismo , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , /metabolismo , Ratos , Ratos Wistar
9.
Biochim Biophys Acta Bioenerg ; 1861(11): 148262, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32673675

RESUMO

BACKGROUND: The electrochemical and spectroscopic investigation of bacterial electron-transfer proteins stabilized on solid state electrodes has provided an effective approach for functional respiratory enzyme studies. METHODS: We assess the biocompatibility of carboxylated graphene oxide (CGO) functionalized with Nickel nitrilotriacetic groups (CGO-NiNTA) ccordinating His-tagged cytochrome c oxidase (CcO) from Rhodobacter sphaeroides. RESULTS: Kinetic studies employing UV-visible absorption spectroscopy confirmed that the immobilized CcO oxidized horse-heart cytochrome c (Cyt c) albeit at a slower rate than isolated CcO. The oxygen reduction reaction as catalyzed by immobilized CcO could be clearly distinguished from that arising from CGO-NiNTA in the presence of Cyt c and dithiothreitol (DTT) as a sacrificial reducing agent. Our findings indicate that while the protein content is about 3.7‰ by mass with respect to the support, the contribution to the oxygen consumption activity averaged at 56.3%. CONCLUSIONS: The CGO-based support stabilizes the free enzyme which, while capable of Cyt c oxidation, is unable to carry out oxygen consumption in solution on its own under our conditions. The turnover rate for the immobilized CcO was as high as 240 O2 molecules per second per CcO unit. GENERAL SIGNIFICANCE: In vitro investigations of electron flow on isolated components of bacterial electron-transfer enzymes immobilized on the surface of CGO in suspension are expected to shed new light on microbial bioenergetic functions, that could ultimately contribute toward the improvement of performance in living organisms.


Assuntos
Proteínas de Bactérias/metabolismo , Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Grafite/química , Níquel/química , Oxigênio/química , Rhodobacter sphaeroides/enzimologia , Proteínas de Bactérias/química , Catálise , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , Cinética , Oxirredução , Suspensões
10.
Biochim Biophys Acta Bioenerg ; 1861(9): 148237, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32485159

RESUMO

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a3 (Fea3) and copper (CuB). Here, the influence of the ligation at the oxidized Fea33+-CuB2+ center on the electron-proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (Em(a)) for the cyanide (the low-spin Fea33+) and the formate-ligated CcO (the high-spin Fea33+). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of Em(a) were found for the CN- and the formate-ligated CcO with slopes of -13 mV/pH unit and -23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron-proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron-proton coupling at the physiological pH values is also substantiated by the UV-Vis absorption and electron-paramagnetic resonance spectroscopy investigations of the cyanide-ligated oxidized CcO. It is shown that the ligand exchange at Fea3+ between His-Fea3+-His and His-Fea3+-OH- occurs only at pH above 9.5 with the estimated pK >11.0.


Assuntos
Domínio Catalítico , Citocromos a/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Prótons , Animais , Biocatálise , Bovinos , Transporte de Elétrons , Oxirredução
11.
Biochim Biophys Acta Bioenerg ; 1861(10): 148251, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32598881

RESUMO

Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.


Assuntos
Metabolismo/efeitos dos fármacos , Saponinas/farmacologia , Animais , Calorimetria , Permeabilidade da Membrana Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Camundongos
12.
Biochim Biophys Acta Bioenerg ; 1861(10): 148239, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32531221

RESUMO

Cytochrome c Oxidase (CcO) is the terminal electron acceptor in aerobic respiratory chain, reducing O2 to water. The released free energy is stored by pumping protons through the protein, maintaining the transmembrane electrochemical gradient. Protons are held transiently in a proton loading site (PLS) that binds and releases protons driven by the electron transfer reaction cycle. Multi-Conformation Continuum Electrostatics (MCCE) was applied to crystal structures and Molecular Dynamics snapshots of the B-type Thermus thermophilus CcO. Six residues are identified as the PLS, binding and releasing protons as the charges on heme b and the binuclear center are changed: the heme a3 propionic acids, Asp287, Asp372, His376 and Glu126B. The unloaded state has one proton and the loaded state two protons on these six residues. Different input structures, modifying the PLS conformation, show different proton distributions and result in different proton pumping behaviors. One loaded and one unloaded protonation states have the loaded/unloaded states close in energy so the PLS binds and releases a proton through the reaction cycle. The alternative proton distributions have state energies too far apart to be shifted by the electron transfers so are locked in loaded or unloaded states. Here the protein can use active states to load and unload protons, but has nearby trapped states, which stabilize PLS protonation state, providing new ideas about the CcO proton pumping mechanism. The distance between the PLS residues Asp287 and His376 correlates with the energy difference between loaded and unloaded states.


Assuntos
Grupo dos Citocromos b/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Prótons , Grupo dos Citocromos b/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Simulação de Dinâmica Molecular , Conformação Proteica , Thermus thermophilus/enzimologia
13.
PLoS One ; 15(6): e0234918, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579605

RESUMO

ZapE/Afg1 is a component of the inner cell membrane of some eubacteria and the inner mitochondrial membrane of eukaryotes. This protein is involved in FtsZ-dependent division of eubacteria. In the yeast and human mitochondrion, ZapE/Afg1 likely interacts with Oxa1 and facilitates the degradation of mitochondrion-encoded subunits of respiratory complexes. Furthermore, the depletion of ZapE increases resistance to apoptosis, decreases oxidative stress tolerance, and impacts mitochondrial protein homeostasis. It remains unclear whether ZapE is a multifunctional protein, or whether some of the described effects are just secondary phenotypes. Here, we have analyzed the functions of ZapE in Trypanosoma brucei, a parasitic protist, and an important model organism. Using a newly developed proximity-dependent biotinylation approach (BioID2), we have identified the inner mitochondrial membrane insertase Oxa1 among three putative interacting partners of ZapE, which is present in two paralogs. RNAi-mediated depletion of both ZapE paralogs likely affected the function of respiratory complexes I and IV. Consistently, we show that the distribution of mitochondrial ZapE is restricted only to organisms with Oxa1, respiratory complexes, and a mitochondrial genome. We propose that the evolutionarily conserved interaction of ZapE with Oxa1, which is required for proper insertion of many inner mitochondrial membrane proteins, is behind the multifaceted phenotype caused by the ablation of ZapE.


Assuntos
Deleção de Genes , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Biotinilação , Regulação para Baixo , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Eucariotos/genética , Genoma Mitocondrial , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fenótipo , Filogenia , Ligação Proteica
14.
Science ; 368(6491): 620-625, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32381719

RESUMO

Loss-of-function mutations in the copper (Cu) transporter ATP7A cause Menkes disease. Menkes is an infantile, fatal, hereditary copper-deficiency disorder that is characterized by progressive neurological injury culminating in death, typically by 3 years of age. Severe copper deficiency leads to multiple pathologies, including impaired energy generation caused by cytochrome c oxidase dysfunction in the mitochondria. Here we report that the small molecule elesclomol escorted copper to the mitochondria and increased cytochrome c oxidase levels in the brain. Through this mechanism, elesclomol prevented detrimental neurodegenerative changes and improved the survival of the mottled-brindled mouse-a murine model of severe Menkes disease. Thus, elesclomol holds promise for the treatment of Menkes and associated disorders of hereditary copper deficiency.


Assuntos
Cobre/metabolismo , Hidrazinas/uso terapêutico , Síndrome dos Cabelos Torcidos/tratamento farmacológico , Animais , Transporte Biológico/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Transportador de Cobre 1/genética , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidrazinas/farmacologia , Masculino , Síndrome dos Cabelos Torcidos/metabolismo , Síndrome dos Cabelos Torcidos/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/prevenção & controle , Ratos
15.
Am J Physiol Endocrinol Metab ; 319(1): E67-E80, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32396498

RESUMO

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower hindlimb oxygen consumption rates (OCRs), indicating depressed mitochondrial oxidative phosphorylation capacity in their skeletal muscle. We hypothesized that OCRs are lower in skeletal muscle mitochondria from IUGR fetuses, due to reduced electron transport chain (ETC) activity and lower abundances of tricarboxylic acid (TCA) cycle enzymes. IUGR sheep fetuses (n = 12) were created with mid-gestation maternal hyperthermia and compared with control fetuses (n = 12). At 132 ± 1 days of gestation, biceps femoris muscles were collected, and the mitochondria were isolated. Mitochondria from IUGR muscle have 47% lower State 3 (Complex I-dependent) OCRs than controls, whereas State 4 (proton leak) OCRs were not different between groups. Furthermore, Complex I, but not Complex II or IV, enzymatic activity was lower in IUGR fetuses compared with controls. Proteomic analysis (n = 6/group) identified 160 differentially expressed proteins between groups, with 107 upregulated and 53 downregulated mitochondria proteins in IUGR fetuses compared with controls. Although no differences were identified in ETC subunit protein abundances, abundances of key TCA cycle enzymes [isocitrate dehydrogenase (NAD+) 3 noncatalytic subunit ß (IDH3B), succinate-CoA ligase ADP-forming subunit-ß (SUCLA2), and oxoglutarate dehydrogenase (OGDH)] were lower in IUGR mitochondria. IUGR mitochondria had a greater abundance of a hypoxia-inducible protein, NADH dehydrogenase 1α subcomplex 4-like 2, which is known to incorporate into Complex I and lower Complex I-mediated NADH oxidation. Our findings show that mitochondria from IUGR skeletal muscle adapt to hypoxemia and hypoglycemia by lowering Complex I activity and TCA cycle enzyme concentrations, which together, act to lower OCR and NADH production/oxidation in IUGR skeletal muscle.


Assuntos
Ciclo do Ácido Cítrico/fisiologia , Complexo I de Transporte de Elétrons/metabolismo , Retardo do Crescimento Fetal/metabolismo , Mitocôndrias Musculares/metabolismo , Animais , Regulação para Baixo , Complexo II de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Retardo do Crescimento Fetal/enzimologia , Músculos Isquiossurais/enzimologia , Músculos Isquiossurais/metabolismo , Hipoglicemia/enzimologia , Hipoglicemia/metabolismo , Hipóxia/enzimologia , Hipóxia/metabolismo , Isocitrato Desidrogenase/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Mitocôndrias Musculares/enzimologia , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Insuficiência Placentária/enzimologia , Insuficiência Placentária/metabolismo , Gravidez , Proteômica , Ovinos , Succinato-CoA Ligases/metabolismo , Regulação para Cima
16.
PLoS One ; 15(5): e0233177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413073

RESUMO

Mitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX). Pulse-chase experiments indicated a correlation between the loss of newly translated Atp9 complexed to Cox6 and an increase of newly formed Atp9 ring, but did not exclude the possibility of an alternate source of Atp9 for ring formation. Here we have extended studies on the functions and structure of this complex, referred to as Atco. We show that Atco is the exclusive source of Atp9 for the ATP synthase assembly. Pulse-chase experiments show that newly translated Atp9, present in Atco, is converted to a ring, which is incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship. Even though Atco does not contain the ring form of Atp9, cross-linking experiments indicate that it is oligomeric and that the inter-subunit interactions are similar to those of the bona fide ring. We propose that, by providing Atp9 for biogenesis of ATP synthase, Atco complexes free Cox6 for assembly of COX. This suggests that Atco complexes may play a role in coordinating assembly and maintaining proper stoichiometry of the two oxphos enzymes.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Mitocondrial/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
17.
Life Sci ; 254: 117785, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32416167

RESUMO

As the most important bioactive substance in Garcinia cambogia, (-)-hydroxycitric acid (HCA) is widely used in food additives to regulate obesity and diabetes in animals or humans, while the mechanism is poorly understood. The purpose of this study was to elucidate the regulatory effect and mechanism of (-)-HCA in regulating glucose and lipid metabolism in chicken primary hepatocytes. The results showed that (-)-HCA obviously decreased triglyceride content through inhibiting the fatty acid synthase protein level, and enhancing the protein level of phosphorylated acetyl CoA carboxylase, enoyl coenzyme A hydratase short chain 1 and carnitine palmitoyltransferase 1A in hepatocytes. Moreover, (-)-HCA markedly enhanced the protein level of phosphofructokinase-1, pyruvate dehydrogenase, succinate dehydrogenase A and complex IV, and which led to the enhancing of glucose uptake and catabolism in hepatocytes. Importantly, the regulation of (-)-HCA on these key factors associated with lipid and glucose metabolism in hepatocytes was mainly achieved through activation of AMP-activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1α-nuclear respiratory factor 1 signaling pathway. These results convincingly demonstrated the mechanism of (-)-HCA's regulating on glucose and lipid metabolism, and provided a strategy in prevention of diseases associated with glycolipid metabolic abnormalities in animals, even in humans.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Citratos/farmacologia , Metabolismo Energético/fisiologia , Hepatócitos/metabolismo , Fator 1 Relacionado a NF-E2/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Galinhas , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Enoil-CoA Hidratase/metabolismo , Glucose/metabolismo , Fosfofrutoquinase-1/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Piruvato Desidrogenase (Lipoamida)/metabolismo , Transdução de Sinais/fisiologia , Succinato Desidrogenase/metabolismo , Triglicerídeos/metabolismo
18.
Nat Commun ; 11(1): 2676, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32472090

RESUMO

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Elétrons/fisiologia , Hydrogenophilaceae/enzimologia , Cristalografia por Raios X , Conformação Proteica
19.
Biochim Biophys Acta Mol Basis Dis ; 1866(10): 165843, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32454166

RESUMO

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 µM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1ß and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1ß mRNA showed an increase in choroid-RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.


Assuntos
Cobre/metabolismo , Células Epiteliais/metabolismo , Degeneração Macular/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Corioide/metabolismo , Cobre/farmacologia , Transportador de Cobre 1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Degeneração Macular/patologia , Masculino , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia , Pigmentos da Retina/genética , Fatores de Transcrição/metabolismo
20.
PLoS One ; 15(5): e0232360, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379772

RESUMO

The free radical nitric oxide (NO) is a powerful metabolic regulator in vertebrates and invertebrates. At cellular concentrations in the nanomolar range, and simultaneously reduced internal oxygen partial pressures (pO2), NO completely inhibits cytochrome-c-oxidase (CytOx) activity and hence mitochondrial- and whole-tissue respiration. The infaunal clam Arctica islandica regulates pO2 of hemolymph and mantle cavity water to mean values of <5 kPa, even in a completely oxygen-saturated environment of 21 kPa. These low internal pO2 values support a longer NO lifespan and NO accumulation in the body fluids and can thus trigger a depression of metabolic rate in the clams. Measurable amounts of NO formation were detected in hemocyte cells (~110 pmol NO 100-1 hemocytes h-1 at 6 kPa), which was not prevented in the presence of the NO synthase inhibitor L-NAME, and in the gill filaments of A. islandica. Adding a NO donor to intact gills and tissue homogenate significantly inhibited gill respiration and CytOx activity below 10 kPa. Meanwhile, the addition of the NO-oxidation product nitrite did not affect metabolic rates. The high nitrite levels found in the hemolymph of experimental mussels under anoxia do not indicate cellular NO production, but could be an indication of nitrate reduction by facultative anaerobic bacteria associated with tissue and/or hemolymph biofilms. Our results suggest that NO plays an important role in the initiation of metabolic depression during self-induced burrowing and shell closure of A. islandica. Furthermore, NO appears to reduce mitochondrial oxygen radical formation during surfacing and cellular reoxygenation after prolonged periods of hypoxia and anoxia.


Assuntos
Bivalves/metabolismo , Hipóxia/metabolismo , Óxido Nítrico/metabolismo , Animais , Antioxidantes/metabolismo , Bivalves/fisiologia , Respiração Celular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Radicais Livres/metabolismo , Brânquias/metabolismo , Glutationa/metabolismo , Hemócitos/metabolismo , Hemolinfa/metabolismo , Longevidade/fisiologia , Mitocôndrias/metabolismo , Nitritos/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
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