RESUMO
An uncoupling protein (cUCP) was identified in heart and skeletal muscle mitochondria of canary birds. cUCP was immunodetected using polyclonal antibodies raised against murine UCP2. Its molecular mass was similar to those of mammalian UCPs (32 kDa). The activity of cUCP was stimulated by palmitic acid (PA) and inhibited by GTP mainly in state 3 respiration. Additions of PA augmented state 4 respiration and lowered the ADP/O ratio. Thus, the activity of cUCP diverted energy from oxidative phosphorylation in state 3 respiration. cUCP in heart and skeletal muscles of canary birds might have implications in thermogenesis as well as protection against free radical production.
Assuntos
Canários/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Canais Iônicos/análise , Proteínas Mitocondriais/análise , Músculo Esquelético/metabolismo , Proteína Desacopladora 1RESUMO
Over the last decade, atypical myopathy (AM) in grazing horses has emerged in several European countries. An exploratory analysis was conducted to determine horse- and pasture-level indicators or factors associated with AM in Belgium. Belgian cases of AM confirmed by histology (n=57) were compared to their healthy co-grazing horses (n=77) and to pastured horses not involved with AM as controls (n=386). The pastures where confirmed cases were grazing (42 pastures; 38 sites; 44 incidences of AM) were compared with those of the controls (216 pastures; 96 sites; no incidence of AM). Statistically significant (P< or =0.05) exploratory variables, identified by means of adjusted odds ratios, suggested that indicators or factors associated with individual horses (young age, inactivity, body condition poor to normal), management practices (permanent pasturing, spreading of manure) and pasture characteristics (humid, sloping pastures, accumulated dead leaves, presence of waterway) may increase the risk of AM. Specific interventions based on these factors might help to reduce the incidence of AM.
Assuntos
Criação de Animais Domésticos/métodos , Doenças dos Cavalos/epidemiologia , Doenças Musculares/veterinária , Rabdomiólise/veterinária , Animais , Bélgica/epidemiologia , Estudos de Casos e Controles , Feminino , Doenças dos Cavalos/etiologia , Doenças dos Cavalos/prevenção & controle , Cavalos , Masculino , Doenças Musculares/epidemiologia , Doenças Musculares/etiologia , Doenças Musculares/prevenção & controle , Poaceae , Rabdomiólise/epidemiologia , Rabdomiólise/etiologia , Rabdomiólise/prevenção & controle , Fatores de RiscoRESUMO
Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Mitocôndrias Hepáticas/fisiologia , Proteínas Mitocondriais/fisiologia , Tecido Adiposo Marrom/fisiologia , Animais , Respiração Celular/fisiologia , Metabolismo Energético , Ácidos Graxos não Esterificados/metabolismo , Fígado Gorduroso/metabolismo , Canais Iônicos , Fosforilação Oxidativa , Estresse Oxidativo , Consumo de Oxigênio , Proteoma/metabolismo , Ubiquinona/metabolismo , Proteína Desacopladora 1 , Proteína Desacopladora 2RESUMO
BACKGROUND: The emergent nature of atypical myopathy or atypical myoglobinuria (AM) necessitates precise description of its clinical and epidemiologic features. PURPOSE: To define key features of AM to help practitioners recognize the disease and to advise owners to take preventive measures. ANIMALS: Belgian cases of AM confirmed by histology (CC horses; n = 57) from autumn 2000 to spring 2005 were included in the study. Co-grazing horses (Co-G horses; n = 77) that remained free of any abnormal clinical signs constituted a control group. METHODS: History, environmental characteristics, clinical signs, and laboratory results associated with AM were determined by a retrospective case series study. RESULTS: Young horses in poor or normal body condition were found to be at risk for AM. Pastures were characterized by poor natural drainage and vegetation of low nutritional value. Features of AM were seasonal occurrence, apparent link with weather conditions (ie, lack of solar radiation with no heavy frost and an excess of precipitation or relative humidity), sudden onset of clinical signs, and rapid death. Evaluation of serum creatine kinase activity indicated severe muscle destruction in CC horses and subclinical disease in a few Co-G horses. CONCLUSIONS: The association of AM with specific environmental conditions and individual animals suggests that young horses should not be pastured on bare premises subject to humidity when the weather has been very wet and cold for several days. Management of AM outbreaks should include control of Co-G horses who are apparently healthy.
Assuntos
Doenças dos Cavalos/diagnóstico , Doenças Musculares/veterinária , Ração Animal , Criação de Animais Domésticos , Animais , Bélgica/epidemiologia , Feminino , Doenças dos Cavalos/epidemiologia , Cavalos , Masculino , Doenças Musculares/diagnóstico , Doenças Musculares/epidemiologia , Mioglobinúria/diagnóstico , Mioglobinúria/epidemiologia , Mioglobinúria/veterinária , Estudos Retrospectivos , Tempo (Meteorologia)RESUMO
Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/metabolismo , Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Western Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Eletroforese em Gel Bidimensional/métodos , Radicais Livres/metabolismo , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Canais Iônicos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/efeitos dos fármacos , Fenótipo , Proteômica/métodos , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteína Desacopladora 1RESUMO
Cardiogenic shock is the leading cause of death among patients hospitalized with acute myocardial infarction (MI). Understanding the mechanisms for acute pump failure is therefore important. The aim of this study is to examine in an acute MI dog model whether mitochondrial bio-energetic function within non-ischemic wall regions are associated with pump failure. Anterior MI was produced in dogs via ligation of left anterior descending (LAD) coronary artery, that resulted in an infract size of about 30% of the left ventricular wall. Measurements of hemodynamic status, mitochondrial function, free radical production and mitochondrial uncoupling protein 3 (UCP3) expression were determined over 24 h period. Hemodynamic measurements revealed a > 50% reduction in cardiac output at 24 h post infarction when compared to baseline. Biopsy samples were obtained from the posterior non-ischemic wall during acute infarction. ADP/O ratios for isolated mitochondria from non-ischemic myocardium at 6 h and 24 h were decreased when compared to the ADP/O ratios within the same samples with and without palmitic acid (PA). GTP inhibition of (PA)-stimulated state 4 respiration in isolated mitochondria from the non-ischemic wall increased by 7% and 33% at 6 h and 24 h post-infarction respectively when compared to sham and pre-infarction samples. This would suggest that the mitochondria are uncoupled and this is supported by an associated increase in UCP3 expression observed on western blots from these same biopsy samples. Blood samples from the coronary sinus measured by electron paramagnetic resonance (EPR) methods showed an increase in reactive oxygen species (ROS) over baseline at 6 h and 24 h post-infarction. In conclusion, mitochondrial bio-energetic ADP/O ratios as a result of acute infarction are abnormal within the non-ischemic wall. Mitochondria appear to be energetically uncoupled and this is associated with declining pump function. Free radical production may be associated with the induction of uncoupling proteins in the mitochondria.
Assuntos
Débito Cardíaco/fisiologia , Ventrículos do Coração/fisiopatologia , Mitocôndrias Cardíacas/fisiologia , Infarto do Miocárdio/fisiopatologia , Difosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Cães , Canais Iônicos , Masculino , Proteínas Mitocondriais , Isquemia Miocárdica/metabolismo , Espécies Reativas de Oxigênio/sangue , Proteína Desacopladora 3RESUMO
In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ácido Palmítico/farmacologia , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Respiração Celular/efeitos dos fármacos , Dactinomicina/farmacologia , Elétrons , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos não Esterificados/farmacologia , Guanosina Trifosfato/farmacologia , Membranas Intracelulares/metabolismo , Malonatos/farmacologia , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa , Ácido Palmítico/metabolismo , Tubérculos/efeitos dos fármacos , Tubérculos/metabolismo , Prótons , Solanum tuberosum/efeitos dos fármacos , Ubiquinona/metabolismoRESUMO
The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCP1-His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.
Assuntos
Proteínas de Transporte/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/isolamento & purificação , Bovinos , Cromatografia de Afinidade , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Canais Iônicos , Proteínas de Membrana/isolamento & purificação , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/química , Translocases Mitocondriais de ADP e ATP/química , Proteínas Mitocondriais , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Desacopladora 1RESUMO
In this study we show that mitochondria of Dictyostelium discoideum contain both alternative oxidase (AOX) and uncoupling protein (UCP). AOX was stimulated by purine mononucleoside and was monomeric. UCP was stimulated by free fatty acids and was poorly sensitive to GTP. Both proteins collaborated in energy dissipation when activated together. AOX expression in free-living ameboid cells decreased strongly from exponential to stationary phase of growth but much less during starvation-induced aggregation. In contrast, UCP expression was constant in all conditions indicating permanent need. Our results suggest that AOX could play a role in cell differentiation, mainly by protecting prespore cells from programmed cell death.
Assuntos
Proteínas de Transporte/metabolismo , Dictyostelium/enzimologia , Proteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Animais , Divisão Celular , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/metabolismo , Guanosina Monofosfato/metabolismo , Hidrogênio , Immunoblotting , Canais Iônicos , Potenciais da Membrana , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Oxigênio/metabolismo , Consumo de Oxigênio , Proteínas de Plantas , Termodinâmica , Fatores de Tempo , Proteína Desacopladora 1RESUMO
HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.
Assuntos
Metabolismo Basal , Glucose/metabolismo , Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/deficiência , Animais , Dióxido de Carbono/metabolismo , Isótopos de Carbono/metabolismo , Radioisótopos de Carbono/metabolismo , Embrião de Mamíferos , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Fibroblastos/metabolismo , Glutamatos/metabolismo , Homozigoto , Imuno-Histoquímica , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Músculo Esquelético/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Consumo de Oxigênio/genética , Consumo de Oxigênio/fisiologia , Pró-Colágeno-Prolina Dioxigenase/genética , Tomografia Computadorizada por Raios XRESUMO
Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Mitocôndrias/metabolismo , Proteômica/métodos , Complexos de ATP Sintetase/biossíntese , Aclimatação , Animais , Regulação da Temperatura Corporal , Temperatura Baixa , Eletroforese em Gel Bidimensional/métodos , Canais Iônicos/biossíntese , Masculino , Proteínas Mitocondriais/biossíntese , Ratos , Ratos Wistar , Proteína Desacopladora 1RESUMO
Superoxide anion generation and the impairment of oxidative phosphorylation yield were studied in rat skeletal muscle mitochondria submitted to anoxia/reoxygenation in vitro. Production of superoxide anion was detected after several cycles of anoxia/reoxygenation. Concomitantly, a decrease of state 3 respiration and phosphorylation yield (ADP/O) were observed. The latter resulted from a proton leak. The presence of palmitic acid during anoxia/reoxygenation cycles led to a dose-dependent inhibition of superoxide anion production together with a partial protection of the ADP/O ratio measured after anoxia/reoxygenation. The ADP/O decrease was shown to be due to a permeability transition pore-sustained proton leak, as it was suppressed by cyclosporine A. The permeability transition pore activation was induced during anoxia/reoxygenation by superoxide anion, as it was cancelled by the spin trap (POBN), which scavenges superoxide anion and by palmitic acid, which induces mitochondrial uncoupling. It can be proposed that the palmitic acid-induced proton leak cancels the production of superoxide anion by mitochondria during anoxia/reoxygenation and therefore prevents the occurrence of the superoxide anion-induced permeability transition pore-mediated proton leak after anoxia/reoxygenation.
Assuntos
Mitocôndrias Musculares/metabolismo , Oxigênio/metabolismo , Prótons , Espécies Reativas de Oxigênio/metabolismo , Animais , Hipóxia Celular , Ciclosporina/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Técnicas In Vitro , Transporte de Íons , Masculino , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Óxidos de Nitrogênio/química , Fosforilação Oxidativa , Consumo de Oxigênio , Ácido Palmítico/farmacologia , Piridinas , Ratos , Ratos Wistar , Superóxidos/metabolismoRESUMO
The energy-dissipating alternative oxidase (AOX) from Hansenula anomala was expressed in Saccharomyces cerevisiae. The recombinant AOX was functional. A comparative analysis by two-dimensional differential in-gel electrophoresis (2D-DIGE) of mitochondrial protein patterns found in wild-type and recombinant AOX strains was performed. 60 proteins exhibiting a significant difference in their abundance were identified. Interestingly, proteins implicated in major metabolic pathways such as Krebs cycle and amino acid biosynthesis were up-regulated. Surprisingly, an up-regulation of the respiratory-chain complex III was associated with a down-regulation of the ATP synthase complex.
Assuntos
Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Oxirredutases/metabolismo , Saccharomyces cerevisiae/enzimologia , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Espectrometria de Massas , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredutases/genética , Consumo de Oxigênio , Pichia/enzimologia , Pichia/genética , Proteínas de Plantas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein.
Assuntos
Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Canais Iônicos , Lectina de Ligação a Manose/metabolismo , Proteínas de Membrana/química , Proteínas Mitocondriais , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Proteína Desacopladora 1RESUMO
Steatosis encompasses the accumulation of droplets of fats into hepatocytes. In this work, we performed a comparative analysis of mitochondrial protein patterns found in wild-type and steatosis-affected liver using the novel technique two-dimensional differential in-gel electrophoresis (2D-DIGE). A total of 56 proteins exhibiting significant difference in their abundances were unambiguously identified. Interestingly, major proteins that regulate generation and consumption of the acetyl-CoA pool were dramatically changed during steatosis. Many proteins involved in the response to oxidative stress were also affected.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteômica/métodos , Acetilcoenzima A/química , Trifosfato de Adenosina/química , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Fígado Gorduroso/patologia , Processamento de Imagem Assistida por Computador , Peroxidação de Lipídeos , Fígado/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Modelos Biológicos , Estresse Oxidativo , Peroxissomos/metabolismoRESUMO
We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using blue native gel electrophoresis coupled to denaturating electrophoresis and immunodetection have indicated associations between alternative oxidase and oxidative phosphorylation complexes in both amoeba and tomato mitochondria. These associations are dependent on the expression level of alternative oxidase according to the physiological state in both organisms. Alternative oxidase associates broadly with large complexes of the respiratory chain when it is expressed in large amount, i.e., in ripe tomato and exponentially growing amoeba. On the contrary, alternative oxidase interacts specifically with complex III even if expression of the oxidase is low, i.e., in green tomato and stationary phase amoeba. This specific interaction represents a higher level of regulation driven by protein-protein interactions leading to a direct kinetic interplay between the cytochrome pathway and alternative oxidase in both plant and amoeba mitochondria.
Assuntos
Acanthamoeba castellanii/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Oxirredutases/metabolismo , Mapeamento de Interação de Proteínas/métodos , Solanum lycopersicum/metabolismo , Membrana Celular/metabolismo , Proteínas Mitocondriais , Proteínas de Plantas , Especificidade da EspécieRESUMO
Alternative oxidase (AOX) and uncoupling protein (UCP) are present simultaneously in tomato fruit mitochondria. In a previous work, it has been shown that protein expression and activity of these two energy-dissipating systems exhibit large variations during tomato fruit development and ripening on the vine. It has been suggested that AOX and UCP could be responsible for the respiration increase at the end of ripening and that the cytochrome pathway could be implicated in the climacteric respiratory burst before the onset of ripening. In this study, the use of tomato mutants that fail normal ripening because of deficiencies in ethylene perception or production as well as the treatment of one selected mutant with a chemical precursor of ethylene have revealed that the bioenergetics of tomato fruit development and ripening is under the control of this plant hormone. Indeed, the evolution pattern of bioenergetic features changes with the type of mutation and with the introduction of ethylene into an ethylene-synthesis-deficient tomato fruit mutant during its induced ripening.
Assuntos
Proteínas de Transporte/metabolismo , Respiração Celular/fisiologia , Transferência de Energia/fisiologia , Etilenos/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Oxirredutases/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Canais Iônicos , Solanum lycopersicum/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais , Mutação , Oxirredutases/genética , Proteínas de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Proteína Desacopladora 1RESUMO
Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, non-climacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-microM linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.
Assuntos
Solanum lycopersicum/metabolismo , Trifosfato de Adenosina/biossíntese , Proteínas de Transporte/metabolismo , Metabolismo Energético , Ácidos Graxos não Esterificados/metabolismo , Canais Iônicos , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Mutação , Oxirredutases/metabolismo , Consumo de Oxigênio , Proteínas de Plantas/metabolismo , Proteína Desacopladora 1RESUMO
The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in reconstituted systems. On the contrary in isolated mitochondria, the protonophoretic action of muscle UCPs had failed to be demonstrated in the absence of superoxide production. We showed here for the first time that muscle UCPs were activated in state 3 respiration by linoleic acid and dissipated energy from oxidative phosphorylation by decreasing the ADP/O ratio. The efficiency of UCPs in mitochondrial uncoupling increased when the state 3 respiratory rate decreased. The inhibition of the linoleic acid-induced uncoupling by a purine nucleotide (GTP), was not observed in state 4 respiration, in uninhibited state 3 respiration, as well as in state 3 respiration inhibited by complex III inhibitors. On the contrary, the progressive inhibition of state 3 respiration by n -butyl malonate, which inhibits the uptake of succinate, led to a full inhibitory effect of GTP. Therefore, as the inhibitory effect of GTP was observed only when the reduced state of coenzyme Q was decreased, we propose that the coenzyme Q redox state could be a metabolic sensor that modulates the purine nucleotide inhibition of FFA-activated UCPs in muscle mitochondria.
Assuntos
Guanosina Trifosfato/metabolismo , Ácido Linoleico/farmacologia , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Ubiquinona/antagonistas & inibidores , Ubiquinona/metabolismo , Desacopladores/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Cinética , Masculino , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Chronic inflammation through foam cells and macrophages is important in atherosclerosis development, and can be considered as therapeutic targets. Cyclooxygenase and NADPH-oxidase were expressed within atherosclerotic lesions. Reactive oxygen species produced by NADPH oxidase were found to trigger the cyclooxygenase-2 expression. The effects of preferential COX-2 inhibitors on ROS produced by Chlamydia-primed human monocytes (THP-1 cells) were evaluated by fluorescence, chemiluminescence, oxymetry, and EPR spin trapping. Fluorescence assays showed an increased production of ROS with Chlamydia versus cells primed by 10(-8)M PMA. COX-2 inhibitors inhibited in a dose-dependent manner the luminol-enhanced CL while ibuprofen and diclofenac increased the chemiluminescence response. By EPR spin trapping, COX-2 inhibitors, ibuprofen, and diclofenac, exhibited a dose-dependent inhibiting effect (10 and 100muM) on the EPR signal appearance. Our cell model combining EPR, chemiluminescence, and oxymetry appeared relevant to study the modulating effects of preferential COX-2 inhibitors on the cell oxidant activity and chronic inflammatory diseases.