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Omega-3 (n3) is a polyunsaturated fatty acid well known for its anti-inflammatory and neuroprotective properties. Obesity is linked to chronic inflammation that disrupts metabolism, the intestine physiology and the central nervous system functioning. This study aims to determine if n3 supplementation can interfere with the effects of obesity on the mitochondrial activity, intestinal barrier, and neurotransmitter levels in the brain of Wistar rats that received cafeteria diet (CAF). We examined adipose tissue, skeletal muscle, plasma, intestine, and the cerebral cortex of four groups: CT (control diet), CTn3 (control diet with n3 supplementation), CAF, and CAFn3 (CAF and n3). Diets were offered for 13 weeks, with n3 supplementation in the final 5 weeks. Adipose tissue Electron Transport Chain complexes I, II, and III showed higher activity in CAF groups, as did complexes III and IV in skeletal muscle. Acetate levels in plasma were reduced in CAF groups, and Lipopolysaccharide (LPS) was higher in the CAF group but reduced in CAFn3 group. Claudin-5 in the intestine was lower in CAF groups, with no n3 supplementation effect. In the cerebral cortex, dopamine levels were decreased with CAF, which was reversed by n3. DOPAC, a dopamine metabolite, also showed a supplementation effect, and HVA, a diet effect. Serotonin levels increased in the CAF group that received supplementation. Therefore, we demonstrate disturbances in mitochondria, plasma, intestine and brain of rats submitted to CAF and the potential benefit of n3 supplementation in endotoxemia and neurotransmitter levels.
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Ácidos Graxos Ômega-3 , Obesidade , Ratos Wistar , Transmissão Sináptica , Animais , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-3/metabolismo , Obesidade/metabolismo , Masculino , Transmissão Sináptica/efeitos dos fármacos , Ratos , Suplementos Nutricionais , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacosRESUMO
Heat stress is an abiotic factor that affects the photosynthetic parameters of plants. In this study, we examined the photosynthetic mechanisms underlying the rapid response of tobacco plants to heat stress in a controlled environment. To evaluate transient heat stress conditions, changes in photochemical, carboxylative, and fluorescence efficiencies were measured using an infrared gas analyser (IRGA Licor 6800) coupled with chlorophyll a fluorescence measurements. Our findings indicated that significant disruptions in the photosynthetic machinery occurred at 45 °C for 6 h following transient heat treatment, as explained by 76.2% in the principal component analysis. The photosynthetic mechanism analysis revealed that the dark respiration rate (Rd and Rd*CO2) increased, indicating a reduced potential for carbon fixation during plant growth and development. When the light compensation point (LCP) increased as the light saturation point (LSP) decreased, this indicated potential damage to the photosystem membrane of the thylakoids. Other photosynthetic parameters, such as AMAX, VCMAX, JMAX, and ΦCO2, also decreased, compromising both photochemical and carboxylative efficiencies in the Calvin-Benson cycle. The energy dissipation mechanism, as indicated by the NPQ, qN, and thermal values, suggested that a photoprotective strategy may have been employed. However, the observed transitory damage was a result of disruption of the electron transport rate (ETR) between the PSII and PSI photosystems, which was initially caused by high temperatures. Our study highlights the impact of rapid temperature changes on plant physiology and the potential acclimatisation mechanisms under rapid heat stress. Future research should focus on exploring the adaptive mechanisms involved in distinguishing mutants to improve crop resilience against environmental stressors.
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BACKGROUND: Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. RESULTS: We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. CONCLUSION: Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.
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Mitocôndrias , Sêmen , Masculino , Humanos , Espécies Reativas de Oxigênio/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Desenvolvimento Embrionário , Estresse OxidativoRESUMO
BACKGROUND: Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. RESULTS: We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. CONCLUSION: Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.
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Humanos , Masculino , Sêmen/metabolismo , Mitocôndrias , Espermatozoides/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo , Desenvolvimento EmbrionárioRESUMO
Mitochondrial Complex I plays a crucial role in the proliferation, chemoresistance, and metastasis of breast cancer (BC) cells. This highlights it as an attractive target for anti-cancer drugs. Using submitochondrial particles, we identified FRV-1, an ortho-carbonyl quinone, which inhibits NADH:duroquinone activity in D-active conformation and reduces the 3ADP state respiration dependent on Complex I, causing mitochondrial depolarization, ATP drop, increased superoxide levels, and metabolic remodeling towards glycolysis in BC cells. Introducing methyl groups at FRV-1 structure produced analogs that acted as electron acceptors at the Complex I level or increased the inhibitory effect of FCCP-stimulated oxygen consumption rate, which correlated with their redox potential, but increased toxicity on RMF-621 human breast fibroblasts was observed. FRV-1 was inactive in the naphthoquinone oxidoreductase 1 (NOQ1)-positive BC cell line, MCF7, but the sensitivity was recovered by dicoumarol, a NOQ1 inhibitor, suggesting that FRV-1 is a NOQ1 substrate. Importantly, FRV-1 selectively inhibited the proliferation, migration, and invasion of NQO1 negative BC cell, MDA-MB-231, in an OXPHOS- and ROS-dependent manner and sensitized it to the BH3 mimetic drug venetoclax. Overall, FRV-1 is a novel Complex I inhibitor in D-active conformation, blocking possibly the re-activation to A-state, producing selective anti-cancer effects in NQO1-negative BC cell lines.
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Gallic (GA) and ellagic (EA) acids are present in foods, medicinal plants, teas, and dietary supplements. An acute toxicological study was conducted by oral administration of both compounds alone (200, 1000, and 2000 mg/kg) and combined (2000 mg/kg) and their effects on the electron transport chain (ETC) and the ROS production in kidney mitochondria further evaluated. All treatments induced a dose-dependent heart, lung, and kidney injury. However, the intensity of these effects varied according to the substance, with greater cardiac and renal toxicity for EA and pulmonary injury for GA, while the combination attenuated the toxicity of the isolated molecules. All substances inhibited the activity of complexes II, III, and IV of the ETC from renal mitochondria. However, no changes were observed regarding mitochondrial ROS production. These compounds have a non-negligible inherent deleterious potential, so their uncontrolled use at high doses (≥200 mg/kg) could cause undesirable effects.
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Ácido Gálico , Traumatismos Cardíacos , Ratos , Animais , Ratos Wistar , Ácido Gálico/farmacologia , Espécies Reativas de Oxigênio , Ácido Elágico/farmacologia , Rim , Pulmão , Administração OralRESUMO
Beyond the role of mitochondria in apoptosis initiation/execution, some mitochondrial adaptations support the metastasis and chemoresistance of cancer cells. This highlights mitochondria as a promising target for new anticancer strategies. Emergent evidence suggests that some snake venom toxins, both proteins with enzymatic and non-enzymatic activities, act on the mitochondrial metabolism of cancer cells, exhibiting unique and novel mechanisms that are not yet fully understood. Currently, six toxin classes (L-amino acid oxidases, thrombin-like enzymes, secreted phospholipases A2, three-finger toxins, cysteine-rich secreted proteins, and snake C-type lectin) that alter the mitochondrial bioenergetics have been described. These toxins act through Complex IV activity inhibition, OXPHOS uncoupling, ROS-mediated permeabilization of inner mitochondrial membrane (IMM), IMM reorganization by cardiolipin interaction, and mitochondrial fragmentation with selective migrastatic and cytotoxic effects on cancer cells. Notably, selective internalization and direct action of snake venom toxins on tumor mitochondria can be mediated by cell surface proteins overexpressed in cancer cells (e.g. nucleolin and heparan sulfate proteoglycans) or facilitated by the elevated Δψm of cancer cells compared to that non-tumor cells. In this latter case, selective mitochondrial accumulation, in a Δψm-dependent manner, of compounds linked to cationic snake peptides may be explored as a new anti-cancer drug delivery system. This review analyzes the effect of snake venom toxins on mitochondrial bioenergetics of cancer cells, whose mechanisms of action may offer the opportunity to develop new anticancer drugs based on toxin scaffolds.
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BACKGROUND: Urushiols are pro-electrophilic haptens that cause severe contact dermatitis mediated by CD8+ effector T-cells and downregulated by CD4+ T-cells. However, the molecular mechanism by which urushiols stimulate innate immunity in the initial stages of this allergic reaction is poorly understood. Here we explore the sub-cellular mechanisms by which urushiols initiate the allergic response. RESULTS: Electron microscopy observations of mouse ears exposed to litreol (3-n-pentadecyl-10-enyl-catechol]) showed keratinocytes containing swollen mitochondria with round electron-dense inclusion bodies in the matrix. Biochemical analyses of sub-mitochondrial fractions revealed an inhibitory effect of urushiols on electron flow through the mitochondrial respiratory chain, which requires both the aliphatic and catecholic moieties of these allergens. Moreover, urushiols extracted from poison ivy/oak (mixtures of 3-n-pentadecyl-8,11,13 enyl/3-n-heptadecyl-8,11 enyl catechol) exerted a higher inhibitory effect on mitochondrial respiration than did pentadecyl catechol or litreol, indicating that the higher number of unsaturations in the aliphatic chain, stronger the allergenicity of urushiols. Furthermore, the analysis of radioactive proteins isolated from mitochondria incubated with 3H-litreol, indicated that this urushiol was bound to cytochrome c1. According to the proximity of cytochromes c1 and b, functional evidence indicated the site of electron flow inhibition was within complex III, in between cytochromes bL (cyt b566) and bH (cyt b562). CONCLUSION: Our data provide functional and molecular evidence indicating that the interruption of the mitochondrial electron transport chain constitutes an important mechanism by which urushiols initiates the allergic response. Thus, mitochondria may constitute a source of cellular targets for generating neoantigens involved in the T-cell mediated allergy induced by urushiols.
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Alérgenos , Citocromos b , Animais , Catecóis , Citocromos c , Citocromos c1 , Transporte de Elétrons , Camundongos , MitocôndriasRESUMO
Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.
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Apoptose , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Citocromos c/metabolismo , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Ionomicina , Mitocôndrias/patologia , Mitomicina , Oxigênio/metabolismo , Cultura Primária de CélulasRESUMO
Complications generated by hyperglycemia present in diabetes mellitus (DM) have been constantly related to oxidative stress and dysfunction in the mitochondrial electron transport chain (ETC). Sirtuin 3 (SIRT3), which is present in mitochondria, is responsible for regulating several proteins involved in metabolic homeostasis and oxidative stress. Studies have suggested alterations in the expression of SIRT3 in DM. The objective of this study was to evaluate the effects of phenolic compounds in jabuticaba (Plinia trunciflora), a berry native to Brazil, on the activity of mitochondrial ETC complexes, SIRT3 protein expression, and oxidative stress parameters in liver of diabetic rats induced by streptozotocin. After type 1 DM induction (streptozotocin 65 mg/kg), diabetic and healthy rats were treated with jabuticaba peel extract (JPE) by gavage (0.5 g/kg of weight) for 30 days. After treatments, those diabetic rats presented impaired activities of complexes I, II, and III of ETC along with an overexpression of SIRT3. In addition, an increase in lipid peroxidation and superoxide dismutase and catalase activities was observed in the diabetic group. The treatment with JPE was able to recover the activity of the mitochondrial complexes and reduce the expression of SIRT3. Furthermore, JPE treatment reduced oxidative damage to lipids and brought the antioxidants enzyme activities to basal levels in diabetic rats. Together, these results demonstrate that JPE can reduce oxidative stress related to DM by restoring mitochondrial complexes activity and regulating SIRT3 expression. Thus, JPE could become an alternative to reduce the development of complications related to DM.
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Cadmium (Cd) inhibits soybean root growth, but its exact mode of action is still not completely understood. We evaluated the effects of Cd on growth, mitochondrial respiration, lipid peroxidation, total phenols, glutathione, and activities of lipoxygenase (LOX), superoxide dismutase (SOD), and catalase (CAT) in soybean roots. In primary roots, Cd stimulated KCN-insensitive respiration and KCN-SHAM-insensitive respiration, indicating the involvement of the alternative oxidase (AOX) pathway, while it decreased KCN-sensitive respiration, suggesting an inhibition of the cytochrome oxidase pathway (COX). In isolated mitochondria, Cd uncoupled the oxidative phosphorylation since it decreased state III respiration (coupled respiration) and ADP/O and respiratory control ratios, while it increased state IV respiration (depletion of exogenously added ADP). The uncoupling effect increased extramitochondrial LOX activity, lipid peroxidation, and oxidized and reduced glutathione, which induced an antioxidant response with enhanced SOD and CAT activities. In brief, our findings reveal that Cd acts as an uncoupler of the mitochondrial oxidative phosphorylation in soybean roots, disturbing cellular respiration and inducing oxidative cellular stress.
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Cádmio , Fosforilação Oxidativa , Antioxidantes/metabolismo , Cádmio/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Raízes de Plantas/metabolismo , Glycine max/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Mammalian cells synthesize H2S from sulfur-containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H2S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H2S signals. In this study, we report that H2S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H2S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H2S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while downregulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H2S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H2S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H2S synthesis) and fat mass.
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Glutamina , Sulfeto de Hidrogênio , NAD , Metabolismo Energético , Lipogênese , Mitocôndrias/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.
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Acetilcoenzima A/metabolismo , Mitocôndrias Musculares/metabolismo , Doenças Mitocondriais/diagnóstico , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Adolescente , Adulto , Biópsia , Respiração Celular , Criança , Técnicas de Laboratório Clínico/métodos , Criopreservação , Transporte de Elétrons , Feminino , Humanos , Síndrome MELAS/diagnóstico , Síndrome MELAS/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Doenças Mitocondriais/metabolismo , Membranas Mitocondriais/metabolismo , Músculo Esquelético/patologia , Oftalmoplegia Externa Progressiva Crônica/diagnóstico , Oftalmoplegia Externa Progressiva Crônica/metabolismo , Fosforilação Oxidativa , Permeabilidade , Manejo de Espécimes , Adulto JovemRESUMO
BACKGROUND: Urushiols are pro-electrophilic haptens that cause severe contact dermatitis mediated by CD8+ effector T-cells and downregulated by CD4+ T-cells. However, the molecular mechanism by which urushiols stimulate innate immunity in the initial stages of this allergic reaction is poorly understood. Here we explore the sub-cellular mechanisms by which urushiols initiate the allergic response. RESULTS: Electron microscopy observations of mouse ears exposed to litreol (3-n-pentadecyl-10-enyl-catechol]) showed keratinocytes containing swollen mitochondria with round electron-dense inclusion bodies in the matrix. Biochemical analyses of sub-mitochondrial fractions revealed an inhibitory effect of urushiols on electron flow through the mitochondrial respiratory chain, which requires both the aliphatic and catecholic moieties of these allergens. Moreover, urushiols extracted from poison ivy/oak (mixtures of 3-n-pentadecyl-8,11,13 enyl/3-n-heptadecyl-8,11 enyl catechol) exerted a higher inhibitory effect on mitochondrial respiration than did pentadecyl catechol or litreol, indicating that the higher number of unsaturations in the aliphatic chain, stronger the allergenicity of urushiols. Furthermore, the analysis of radioactive proteins isolated from mitochondria incubated with 3H-litreol, indicated that this urushiol was bound to cytochrome c1. According to the proximity of cytochromes c1 and b, functional evidence indicated the site of electron flow inhibition was within complex III, in between cytochromes bL (cyt b566) and bH (cyt b562). CONCLUSION: Our data provide functional and molecular evidence indicating that the interruption of the mitochondrial electron transport chain constitutes an important mechanism by which urushiols initiates the allergic response. Thus, mitochondria may constitute a source of cellular targets for generating neoantigens involved in the T-cell mediated allergy induced by urushiols.
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Animais , Camundongos , Alérgenos , Citocromos b , Catecóis , Citocromos c1 , Citocromos c , Transporte de Elétrons , MitocôndriasRESUMO
Nutritional imbalance in early life may disrupt the hypothalamic control of energy homeostasis and increase the risk of metabolic disease. The hypothalamic serotonin (5-hydroxytryptamine; 5-HT) system based in the hypothalamus plays an important role in the homeostatic control of energy balance, however the mechanisms underlying the regulation of energy metabolism by 5-HT remain poorly described. Several crucial mitochondrial functions are altered by mitochondrial stress. Adaptations to this stress include changes in mitochondrial multiplication (i.e, mitochondrial biogenesis). Due to the scarcity of evidence regarding the effects of serotonin reuptake inhibitors (SSRI) such as fluoxetine (FLX) on mitochondrial function, we sought to investigate the potential contribution of FLX on changes in mitochondrial function and biogenesis occurring in overfed rats. Using a neonatal overfeeding model, male Wistar rats were divided into 4 groups between 39 and 59 days of age based on nutrition and FLX administration: normofed + vehicle (NV), normofed + FLX (NF), overfed + vehicle (OV) and overfed + FLX (OF). We found that neonatal overfeeding impaired mitochondrial respiration and increased oxidative stress biomarkers in the hypothalamus. FLX administration in overfed rats reestablished mitochondrial oxygen consumption, increased mitochondrial uncoupling protein 2 (Ucp2) expression, reduced total reactive species (RS) production and oxidative stress biomarkers, and up-regulated mitochondrial biogenesis-related genes. Taken together our results suggest that FLX administration in overfed rats improves mitochondrial respiratory chain activity and oxidative balance and increases the transcription of genes employed in mitochondrial biogenesis favoring mitochondrial energy efficiency in response to early nutritional imbalance.
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Fármacos Antiobesidade/farmacologia , Metabolismo Energético/efeitos dos fármacos , Fluoxetina/farmacologia , Hipotálamo/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Biogênese de Organelas , Hipernutrição/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Fatores Etários , Fenômenos Fisiológicos da Nutrição Animal , Animais , Animais Recém-Nascidos , Animais Lactentes , Hipotálamo/metabolismo , Hipotálamo/patologia , Hipotálamo/fisiopatologia , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Estado Nutricional , Hipernutrição/metabolismo , Hipernutrição/patologia , Hipernutrição/fisiopatologia , Oxirredução , Consumo de Oxigênio , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismoRESUMO
Flavodoxins are electron carrier flavoproteins present in bacteria and photosynthetic microorganisms which duplicate the functional properties of iron-sulphur containing ferredoxins and replace them under adverse environmental situations that lead to ferredoxin decline. When expressed in plant chloroplasts, flavodoxin complemented ferredoxin deficiency and improved tolerance to multiple sources of biotic, abiotic and xenobiotic stress. Analysis of flavodoxin-expressing plants grown under normal conditions, in which the two carriers are present, revealed phenotypic effects unrelated to ferredoxin replacement. Flavodoxin thus provided a tool to alter the chloroplast redox poise in a customized way and to investigate its consequences on plant physiology and development. We describe herein the effects exerted by the flavoprotein on the function of the photosynthetic machinery. Pigment analysis revealed significant increases in chlorophyll a, carotenoids and chlorophyll a/b ratio in flavodoxin-expressing tobacco lines. Results suggest smaller antenna size in these plants, supported by lower relative contents of light-harvesting complex proteins. Chlorophyll a fluorescence and P700 spectroscopy measurements indicated that transgenic plants displayed higher quantum yields for both photosystems, a more oxidized plastoquinone pool under steady-state conditions and faster plastoquinone dark oxidation after a pulse of saturating light. Many of these effects resemble the phenotypes exhibited by leaves adapted to high irradiation, a most common environmental hardship faced by plants growing in the field. The results suggest that flavodoxin-expressing plants would be better prepared to cope with this adverse situation, and concur with earlier observations reporting that hundreds of stress-responsive genes were induced in the absence of stress in these lines.
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Aclimatação/efeitos da radiação , Flavodoxina/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Nicotiana/metabolismo , Fotossíntese/efeitos da radiação , Folhas de Planta/genética , Relação Dose-Resposta à Radiação , Fenótipo , Folhas de Planta/efeitos da radiação , Nicotiana/genética , Nicotiana/fisiologia , Nicotiana/efeitos da radiaçãoRESUMO
The mitochondrion has emerged as a promising therapeutic target for novel cancer treatments because of its essential role in tumorigenesis and resistance to chemotherapy. Previously, we described a natural compound, 10-((2,5-dihydroxybenzoyl)oxy)decyl) triphenylphosphonium bromide (GA-TPP+C10), with a hydroquinone scaffold that selectively targets the mitochondria of breast cancer (BC) cells by binding to the triphenylphosphonium group as a chemical chaperone; however, the mechanism of action remains unclear. In this work, we showed that GA-TPP+C10 causes time-dependent complex inhibition of the mitochondrial bioenergetics of BC cells, characterized by (1) an initial phase of mitochondrial uptake with an uncoupling effect of oxidative phosphorylation, as previously reported, (2) inhibition of Complex I-dependent respiration, and (3) a late phase of mitochondrial accumulation with inhibition of α-ketoglutarate dehydrogenase complex (αKGDHC) activity. These events led to cell cycle arrest in the G1 phase and cell death at 24 and 48 h of exposure, and the cells were rescued by the addition of the cell-penetrating metabolic intermediates l-aspartic acid ß-methyl ester (mAsp) and dimethyl α-ketoglutarate (dm-KG). In addition, this unexpected blocking of mitochondrial function triggered metabolic remodeling toward glycolysis, AMPK activation, increased expression of proliferator-activated receptor gamma coactivator 1-alpha (pgc1α) and electron transport chain (ETC) component-related genes encoded by mitochondrial DNA and downregulation of the uncoupling proteins ucp3 and ucp4, suggesting an AMPK-dependent prosurvival adaptive response in cancer cells. Consistent with this finding, we showed that inhibition of mitochondrial translation with doxycycline, a broad-spectrum antibiotic that inhibits the 28 S subunit of the mitochondrial ribosome, in the presence of GA-TPP+C10 significantly reduces the mt-CO1 and VDAC protein levels and the FCCP-stimulated maximal electron flux and promotes selective and synergistic cytotoxic effects on BC cells at 24 h of treatment. Based on our results, we propose that this combined strategy based on blockage of the adaptive response induced by mitochondrial bioenergetic inhibition may have therapeutic relevance in BC.
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Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Quinases Proteína-Quinases Ativadas por AMP , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Doxiciclina/farmacologia , Sinergismo Farmacológico , Feminino , Gentisatos/química , Gentisatos/farmacologia , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Complexo Cetoglutarato Desidrogenase/genética , Mitocôndrias/patologia , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Quinases/genética , Ribossomos/efeitos dos fármacosRESUMO
Leaf senescence is a developmental process critical for plant fitness, which involves genetically controlled cell death and ordered disassembly of macromolecules for reallocating nutrients to juvenile and reproductive organs. While natural leaf senescence is primarily associated with aging, it can also be induced by environmental and nutritional inputs including biotic and abiotic stresses, darkness, phytohormones and oxidants. Reactive oxygen species (ROS) are a common thread in stress-dependent cell death and also increase during leaf senescence. Involvement of chloroplast redox chemistry (including ROS propagation) in modulating cell death is well supported, with photosynthesis playing a crucial role in providing redox-based signals to this process. While chloroplast contribution to senescence received less attention, recent findings indicate that changes in the redox poise of these organelles strongly affect senescence timing and progress. In this review, the involvement of chloroplasts in leaf senescence execution is critically assessed in relation to available evidence and the role played by environmental and developmental cues such as stress and phytohormones. The collected results indicate that chloroplasts could cooperate with other redox sources (e.g., mitochondria) and signaling molecules to initiate the committed steps of leaf senescence for a best use of the recycled nutrients in plant reproduction.
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Helminths use an alternative mitochondrial electron transport chain (ETC) under hypoxic conditions, such as those found in the gastrointestinal tract. In this alternative ETC, fumarate is the final electron acceptor and rhodoquinone (RQ) serves as an electron carrier. RQ receives electrons from reduced nicotinamide adenine dinucleotide through complex I and donates electrons to fumarate through complex II. In this latter reaction, complex II functions in the opposite direction to the conventional ETC (i.e., as fumarate reductase instead of succinate dehydrogenase). Studies in Ascaris suum indicate that this is possible due to changes in complex II, involving alternative succinate dehydrogenase (SDH) subunits SDHA and SDHD, derived from duplicated genes. We analyzed helminth genomes and found that distinct lineages have different gene duplications of complex II subunits (SDHA, SDHB, SDHC, and SDHD). Similarly, we found lineage-specific duplications in genes encoding complex I subunits that interact with quinones (NDUF2 and NDUF7). The phylogenetic analysis of ETC subunits revealed a complex history with independent evolutionary events involving gene duplications and losses. Our results indicated that there is not a common evolutionary event related to ETC subunit genes linked to RQ. The free-living nematode Caenorhabditis elegans uses RQ and has two genes encoding SDHA (sdha-1 and sdha-2) and two genes encoding NDUF2 (nduf2-1 and nduf2-2). sdha-1 and nduf2-1 are essential genes and have a similar expression pattern during C. elegans lifecycle. Using knockout strains, we found that sdha-2 and nduf2-2 are not essential, even in hypoxia. Yet, sdha-2 and nduf2-2 expression is increased in the early embryo and in dauer larvae, stages where there is low oxygen tension. Strikingly, sdha-1 and sdha-2 as well as nduf2-1 and nduf2-2 showed inverted expression profiles during the C. elegans life cycle. Finally, we found that sdha-2 and nduf2-2 knockout mutant strain progeny is affected. Our results indicate that different complex I and II subunit gene duplications provide increased fitness to worms.
RESUMO
The combined effects of exposure to increasing temperature and copper (Cu) concentrations were evaluated in the zooxanthellate scleractinian coral Mussismilia harttii. Endpoints analyzed included activity of enzymes involved in glycolysis (pyruvate kinase, PK; lactate dehydrogenase, LDH), Krebs cycle (citrate synthase, CS; isocitrate dehydrogenase; IDH), electron transport chain (electron transport system, ETS) and pentose phosphate pathway (glucose-6-phosphate dehydrogenase, G6PDH). Coral polyps were kept under control conditions (25.0⯱â¯0.1⯰C; 2.9⯱â¯0.7⯵g/L Cu) or exposed to combined treatments of increasing temperature (26.6⯱â¯0.1⯰C and 27.3⯱â¯0.1⯰C) and concentrations of dissolved Cu (5.4⯱â¯0.9 and 8.6⯱â¯0.3⯵g/L) for 4 and 12 days using a mesocosm system. PK activity was not affected by stressors. LDH, CS, IDH, ETS and G6PDH activities were temporally inhibited by stressors alone. CS, ETS and G6PDH activities remained inhibited by the combination of stressors after 12 days. Furthermore, all combinations between increasing temperature and exposure Cu were synergistic after prolonged exposure. Taken together, stressors applied alone led to temporary inhibitory effects on energy metabolism enzymes of the coral M. harttii, however, prolonged exposure reveals strong deleterious effects over the metabolism of corals due to the combination of stressors. The present study is the first one to give insights into the combined effects of increasing temperature and Cu exposure in the energy metabolism enzymes of a scleractinian coral. Findings suggest that moderate Cu contamination in future increasing temperature scenarios can be worrying for aerobic and oxidative metabolism of M. harttii.