RESUMO
Anaerobic biotechnology for wastewaters treatment can nowadays be considered as state of the art methods. Nonetheless, this technology exhibits certain inherent limitations when employed for industrial wastewater treatment, encompassing elevated substrate consumption, diminished electron transfer efficiency, and compromised system stability. To address the above issues, increasing interest is being given to the potential of using conductive non-biological materials, e,g., iron sulfide (FeS), as a readily accessible electron donor and electron shuttle in the biological decontamination process. In this study, Mackinawite nanoparticles (FeS NPs) were studied for their ability to serve as electron donors for p-chloronitrobenzene (p-CNB) anaerobic reduction within a coupled system. This coupled system achieved an impressive p-CNB removal efficiency of 78.3 ± 2.9% at a FeS NPs dosage of 1 mg/L, surpassing the efficiencies of 62.1 ± 1.5% of abiotic and 30.6 ± 1.6% of biotic control systems, respectively. Notably, the coupled system exhibited exclusive formation of aniline (AN), indicating the partial dechlorination of p-CNB. The improvements observed in the coupled system were attributed to the increased activity in the electron transport system (ETS), which enhanced the sludge conductivity and nitroaromatic reductases activity. The analysis of equivalent electron donors confirmed that the S2- ions dominated the anaerobic reduction of p-CNB in the coupled system. However, the anaerobic reduction of p-CNB would be adversely inhibited when the FeS NPs dosage exceeded 5 g/L. In a continuous operation, the p-CNB concentration and HRT were optimized as 125 mg/L and 40 h, respectively, resulting in an outstanding p-CNB removal efficiency exceeding 94.0% after 160 days. During the anaerobic reduction process, as contributed by the predominant bacterium of Thiobacillus with a 6.6% relative abundance, a mass of p-chloroaniline (p-CAN) and AN were generated. Additionally, Desulfomonile was emerged with abundances ranging from 0.3 to 0.7%, which was also beneficial for the reduction of p-CNB to AN. The long-term stable performance of the coupled system highlighted that anaerobic technology mediated by FeS NPs has a promising potential for the treatment of wastewater containing chlorinated nitroaromatic compounds, especially without the aid of organic co-substrates.
Assuntos
Compostos Ferrosos , Nitrobenzenos , Anaerobiose , Nitrobenzenos/metabolismo , Nitrobenzenos/química , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/química , Nanopartículas/química , Oxirredução , Eliminação de Resíduos Líquidos/métodos , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Águas Residuárias/química , Reatores BiológicosRESUMO
Mitochondrial reactive oxygen species (ROS) play important roles in cellular signaling; however, certain pathological conditions such as ischemia/reperfusion (I/R) injury disrupt ROS homeostasis and contribute to cell death. A major impediment to developing therapeutic measures against oxidative stress-induced cellular damage is the lack of a quantitative framework to identify the specific sources and regulatory mechanisms of mitochondrial ROS production. We developed a thermodynamically consistent, mass-and-charge balanced, kinetic model of mitochondrial ROS homeostasis focused on redox sites of electron transport chain complexes I, II, and III. The model was calibrated and corroborated using comprehensive data sets relevant to ROS homeostasis. The model predicts that complex I ROS production dominates other sources under conditions favoring a high membrane potential with elevated nicotinamide adenine dinucleotide (NADH) and ubiquinol (QH2) levels. In general, complex I contributes to significant levels of ROS production under pathological conditions, while complexes II and III are responsible for basal levels of ROS production, especially when QH2 levels are elevated. The model also reveals that hydrogen peroxide production by complex I underlies the non-linear relationship between ROS emission and O2 at low O2 concentrations. Lastly, the model highlights the need to quantify scavenging system activity under different conditions to establish a complete picture of mitochondrial ROS homeostasis. In summary, we describe the individual contributions of the electron transport system complex redox sites to total ROS emission in mitochondria respiring under various combinations of NADH- and Q-linked respiratory fuels under varying workloads.
Assuntos
Peróxido de Hidrogênio , Superóxidos , Superóxidos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transporte de Elétrons , Peróxido de Hidrogênio/metabolismo , Peróxidos/metabolismo , NAD/metabolismo , Complexo I de Transporte de Elétrons/metabolismoRESUMO
Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H2O2 (JH2O2) production. H2O2 is reduced to H2O by electrons supplied by NADPH. NADP+ is reduced back to NADPH by activation of mitochondrial membrane potential-dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH2O2 production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through ß-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP- and low-ADP-stimulated respiration that accelerating flux through ß-oxidation generates a corresponding increase in mitochondrial JH2O2 production, that the majority (â¼70-80%) of H2O2 produced is reduced to H2O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within ß-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH2O2 production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.
Assuntos
Metabolismo Energético , Mitocôndrias Musculares/enzimologia , NADP Trans-Hidrogenase Específica para A ou B/metabolismo , Consumo de Oxigênio , Difosfato de Adenosina/metabolismo , Animais , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Proteínas Mitocondriais/metabolismo , OxirreduçãoRESUMO
Doxorubicin is an anthracycline-based chemotherapeutic that causes myotoxicity with symptoms persisting beyond treatment. Patients experience muscle pain, weakness, fatigue, and atrophy, but the underlying mechanisms are poorly understood. Studies investigating doxorubicin-induced myotoxicity have reported disrupted mitochondrial function. Mitochondria are responsible for regulating both cellular energy status and Ca2+ handling, both of which impact contractile function. Moreover, loss of mitochondrial integrity may initiate muscle atrophy. Skeletal muscle mitochondrial dysregulation may therefore contribute to an overall loss of skeletal muscle quality and performance that may be mitigated by appropriately targeted mitochondrial therapies. We therefore assessed the impact of doxorubicin on muscle performance and applied a multiplexed assay platform to diagnose alterations in mitochondrial respiratory control. Mice received a clinically relevant dose of doxorubicin delivered systemically and were euthanized 72 h later. We measured extensor digitorum longus and soleus muscle forces, fatigue, and contractile kinetics in vitro, along with Ca2+ uptake in isolated sarcoplasmic reticulum. Isolated skeletal muscle mitochondria were used for real-time respirometry or frozen for protein content and activity assays. Doxorubicin impaired muscle performance, which was indicated by reduced force production, fatigue resistance, and sarcoplasmic reticulum-Ca2+ uptake, which were associated with a substrate-independent reduction in respiration and membrane potential but no changes in the NAD(P)H/NAD(P)+ redox state. Protein content and dehydrogenase activity results corroborated these findings, indicating that doxorubicin-induced mitochondrial impairments are located upstream of ATP synthase within the electron transport system. Collectively, doxorubicin-induced lesions likely span mitochondrial complexes I-IV, providing potential targets for alleviating doxorubicin myotoxicity.
Assuntos
Doxorrubicina/farmacologia , Contração Muscular/efeitos dos fármacos , Fadiga Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Antraciclinas/farmacologia , Cálcio/metabolismo , Citrato (si)-Sintase/metabolismo , Transporte de Elétrons , Ferro/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Atrofia Muscular , Oxirredução , Retículo Sarcoplasmático/metabolismo , TermodinâmicaRESUMO
Photosynthetic organisms often experience extreme light conditions that can cause hyper-reduction of the chloroplast electron transport chain, resulting in oxidative damage. Accumulating evidence suggests that mitochondrial respiration and chloroplast photosynthesis are coupled when cells are absorbing high levels of excitation energy. This coupling helps protect the cells from hyper-reduction of photosynthetic electron carriers and diminishes the production of reactive oxygen species (ROS). To examine this cooperative protection, here we characterized Chlamydomonas reinhardtii mutants lacking the mitochondrial alternative terminal respiratory oxidases, CrAOX1 and CrAOX2. Using fluorescent fusion proteins, we experimentally demonstrated that both enzymes localize to mitochondria. We also observed that the mutant strains were more sensitive than WT cells to high light under mixotrophic and photoautotrophic conditions, with the aox1 strain being more sensitive than aox2 Additionally, the lack of CrAOX1 increased ROS accumulation, especially in very high light, and damaged the photosynthetic machinery, ultimately resulting in cell death. These findings indicate that the Chlamydomonas AOX proteins can participate in acclimation of C. reinhardtii cells to excess absorbed light energy. They suggest that when photosynthetic electron carriers are highly reduced, a chloroplast-mitochondria coupling allows safe dissipation of photosynthetically derived electrons via the reduction of O2 through AOX (especially AOX1)-dependent mitochondrial respiration.
Assuntos
Chlamydomonas reinhardtii/crescimento & desenvolvimento , Regulação Enzimológica da Expressão Gênica , Luz , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Aclimatação , Sequência de Aminoácidos , Respiração Celular , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Transporte de Elétrons , Proteínas Mitocondriais/genética , Mutação , Oxirredução , Oxirredutases/genética , Fotossíntese , Proteínas de Plantas/genética , Homologia de SequênciaRESUMO
The mitochondrial cytochrome oxidase (CO) genes are involved in complex IV of the electron transport system, and dysfunction of CO genes leads to several diseases. However, no work has been reported on the codon usage pattern of these genes. We used bioinformatic methods to analyze the compositional properties and the codon usage pattern of the COI, COII, and COIII genes in fishes, birds, and mammals to understand the similarities and dissimilarities of codon usage in these genes, which gave an insight into the molecular biology of these genes. The effective number of codons (ENC) value of genes was high in different species of fishes, birds and mammals, which indicates that the codon bias of CO genes was low and the ENC values were significantly different among fishes, birds, and mammals, as revealed from the t test. The overall guanine and cytosine (GC) content in fishes, birds, and mammals was lower than 50% in all genes, indicating that the genes were AT-rich and significantly different among fishes, birds, and mammals. The TCA codon was overrepresented in fishes, birds, and mammals for the COI gene, in birds and mammals for the COII gene, but it was not overrepresented in others. Only three codons, namely CTA, CGA, and AAA, were overrepresented in all three groups for the COI, COII, and COIII genes, repectively. From the neutrality plot in fishes, birds, and mammals, it was observed that the slopes of the regression lines (regression coefficients) in the COI, COII, and COIII genes were <0.5, suggesting that natural selection played a major role, whereas mutation pressure played a minor role.
Assuntos
Uso do Códon/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Doenças Mitocondriais/genética , Seleção Genética/genética , Animais , Biologia Computacional , Genes Mitocondriais , HumanosRESUMO
Pesticides can easily reach surface waters via runoff and their potential to have detrimental impacts on freshwater organisms is high. Not much is known about how macroinvertebrates react to glyphosate contamination. In this study we investigated lethal and sublethal effects of the exposure of Gammarus fossarum to Roundup®, a glyphosate-based herbicide. The LC10 and LC50 values after 96 h were determined to be 0.65 ml/L Roundup® (230 mg/L glyphosate) and 0.96 ml/L Roundup® (340 mg/L glyphosate), respectively. As a sublethal measure of toxicity we conducted eight experiments with the feeding activity and the respiratory electron transport system (ETS) activity as endpoints. All experiments lasted seven days. Although the LC10 concentration of Roundup® was used for the feeding activity tests, 49% of the gammarids died before the end of the experiments, which is inconsistent with the calculated LC10-values. The feeding activity was significantly higher in Roundup®-enriched water (mean = 0.18 mg/mg x d) in comparison to pure spring water (mean = 0.079 mg/mg x d). No significant difference was observed between the ETS activity, which was determined after 24, 48 or 96 h after the start of the experiment, of the gammarids in Roundup® solution and in the control. The LC-values determined here are rather high, and exceed background glyphosate concentrations in most anthropogenically influenced surface waters. The increased feeding activity when exposed to Roundup® in combination with an unchanged ETS activity suggests effects on the metabolic efficiency of G. fossarum. We argue that Roundup® enhances the anabolic activity (feeding activity) in order to maintain the catabolic activity (ETS activity).
Assuntos
Anfípodes/fisiologia , Glicina/análogos & derivados , Herbicidas/toxicidade , Metabolismo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Glicina/toxicidade , Testes de Toxicidade , GlifosatoRESUMO
SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro, succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5-/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5-/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5-/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function.
Assuntos
Cardiolipinas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Hepatócitos/enzimologia , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Sirtuínas/metabolismo , Substituição de Aminoácidos , Animais , Cardiolipinas/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Células HEK293 , Hepatócitos/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Mutação , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sirtuínas/química , Sirtuínas/genéticaRESUMO
Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Malato Desidrogenase/metabolismo , Mitocôndrias Cardíacas/metabolismo , Espécies Reativas de Oxigênio/agonistas , Difosfato de Adenosina/metabolismo , Alameticina/farmacologia , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Biocatálise/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/metabolismo , Ionóforos/farmacologia , Magnésio/metabolismo , Malato Desidrogenase/química , Mitocôndrias Cardíacas/química , Mitocôndrias Cardíacas/efeitos dos fármacos , NAD/metabolismo , Oxirredução , Polienos/farmacologia , Porosidade/efeitos dos fármacos , Piridinas/farmacologia , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/metabolismoRESUMO
Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.
Assuntos
Adenilato Quinase/metabolismo , Respiração Celular/fisiologia , Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Rim/metabolismo , Treonina/metabolismo , Adenilato Quinase/química , Animais , Apoptose , Cristalografia por Raios X , Citocromos c/química , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , Rim/citologia , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Oxirredução , Fosforilação , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Declining water quality is one of the main reasons of coral reef degradation in the Thousand Islands off the megacity Jakarta, Indonesia. Shifts in benthic community composition to higher soft coral abundances have been reported for many degraded reefs throughout the Indo-Pacific. However, it is not clear to what extent soft coral abundance and physiology are influenced by water quality. In this study, live benthic cover and water quality (i.e. dissolved inorganic nutrients (DIN), turbidity (NTU), and sedimentation) were assessed at three sites (< 20 km north of Jakarta) in Jakarta Bay (JB) and five sites along the outer Thousand Islands (20-60 km north of Jakarta). This was supplemented by measurements of photosynthetic yield and, for the first time, respiratory electron transport system (ETS) activity of two dominant soft coral genera, Sarcophyton spp. and Nephthea spp. Findings revealed highly eutrophic water conditions in JB compared to the outer Thousand Islands, with 44% higher DIN load (7.65 µM/L), 67% higher NTU (1.49 NTU) and 47% higher sedimentation rate (30.4 g m-2 d-1). Soft corals were the dominant type of coral cover within the bay (2.4% hard and 12.8% soft coral cover) compared to the outer Thousand Islands (28.3% hard and 6.9% soft coral cover). Soft coral abundances, photosynthetic yield, and ETS activity were highly correlated with key water quality parameters, particularly DIN and sedimentation rates. The findings suggest water quality controls the relative abundance and physiology of dominant soft corals in JB and may thus contribute to phase shifts from hard to soft coral dominance, highlighting the need to better manage water quality in order to prevent or reverse phase shifts.
RESUMO
Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Histidina Quinase/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/genética , Histidina Quinase/química , Histidina Quinase/genética , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Quimiotáticas Aceptoras de Metil/química , Proteínas Quimiotáticas Aceptoras de Metil/genética , Oxirredução , Domínios ProteicosRESUMO
Respiratory oxygen consumption rate (RO2) and potential respiration (Φ) has been monitored during a food deprivation period in the heterotrophic dinoflagellate Oxyrrhis marina. Φ was determined by measuring the activity of the enzymes from the electron transport system (ETS), the major contributor to the oxygen consumption in the cells. Additionally, we have quantified for the first time the concentration of pyridine nucleotides in this organism, both in their oxidized (NAD(P)(+)) and reduced forms (NAD(P)H). These molecules are the main electron donors at the beginning of the ETS. We observed a dramatic decrease in RO2 within the first days, whereas Φ steadily, but more gradually declined during the entire experiment. This led to a decrease of the RO2 /Φ with time. The intracellular total pool of NAD and NADP concentration, in turn, dropped exponentially in a manner parallel to the RO2. This strong decrease was mainly driven by a reduction in the concentration of the oxidized forms. The present work constitutes a first step in clarifying the role of intracellular NAD and NADP concentrations and the redox status in the control of in vivo RO2 in marine organisms.
Assuntos
Dinoflagellida/metabolismo , Transporte de Elétrons/fisiologia , NADP/metabolismo , Consumo de Oxigênio/fisiologia , Inanição/fisiopatologia , Zooplâncton/metabolismo , Animais , RespiraçãoRESUMO
In order to investigate the effects of phenol on activity and microfauna community structure of activated sludge, phenol with different concentrations were added into the wastewater for activated sludge culturing in a SBR reactor, then the activity parameters of TTC-ETS and INT-ETS of activated sludge were measured, and the dynamics of microfauna community was analyzed. The results indicated that TTC-ETS activity was more applicable to evaluate the effects of phenol on sludge activity than INT-ETS activity. The higher the concentration of phenol was, the more seriously the sludge activity was inhibited. When 50 mg·L-1 of phenol was added into the influent, the inhibition rate of sludge activity was (20.75±10.43)%, while it increased to (39.73±26.92)% with a great fluctuation at the phenol concentration of 100 mg·L-1. The inhibition rate of sludge activity fluctuated at about 40% in the later period of sludge culturing with 300 mg·L-1 of phenol. The inhibitory effects on microfauna community in activated sludge increased with increasing phenol concentration. Phenol had different effects on various groups of microfauna community, i.e., only one group (testate amoebae) was inhibited significantly at a low feeding concentration of phenol, many groups (sessile ciliates, testate amoebae, crawling ciliates, carnivorous ciliates) were inhibited at the phenol concentrations of 100 mg·L-1 and 300 mg·L-1, whereas a few groups (flagellates, nematodes, etc.) were promoted at the phenol concentration of 300 mg·L-1. There was a certain degree of relationship between sludge activity and microfauna under the influence of phenol, which means that Centropyxis aculeata, Plagiocampa mutabilis etc., may be bio-indicators for low sludge activity, and Epistylis lacustris, Bodo lens and Pleuromonas jaculans can be used as bio-indicators for high sludge activity in the activated sludge used to treat phenolic wastewater.
Assuntos
Amoeba , Cilióforos , Fenol/química , Esgotos , Reatores Biológicos , Águas ResiduáriasRESUMO
The specialised fauna of freshwater springs is affected by contamination of the water with xenobiotics from human activities in the surrounding landscape. We assessed the effects of exposure to toxins in laboratory and field experiments by using copper sulphate as a model substance and Gammarus fossarum Koch, 1836, as the model organism. This amphipod is a common representative of the European spring fauna and copper is a widespread contaminant, mainly from agricultural practice. The experiments were conducted in test chambers placed in flow channels and directly in a spring. The gammarids were fed with conditioned beech leaf discs, which had been exposed to a 0.8 mg Cu/L solution for 96 h. The feeding activity of the amphipods was quantified on the level of the organism; and the respiratory electron transport system (ETS) assay was conducted in order to determine changes on the cellular level in the test organisms. The results show that the feeding activity, when the leaf discs were contaminated with copper, was not significantly different from the control. The ETS activity of the gammarids, which had been feeding on the copper contaminated leaf discs was however significantly reduced. The results followed the same pattern for gammarids from both the laboratory and the spring. By conducting the experiments not only in a laboratory but also directly in a spring in the field, we took a crucial step towards a more realistic approach when examining environmental pollutants on an organism. Our findings demonstrate the importance of conducting experiments out in the field, in natural conditions, as well as in the laboratory.
Assuntos
Anfípodes/efeitos dos fármacos , Sulfato de Cobre/toxicidade , Animais , Transporte de Elétrons/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Exposição Ambiental , Água Doce , Poluentes Químicos da Água/toxicidadeRESUMO
In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the ß-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and ß-subunits. Lysine residues 199 and 202 of mature ETFß are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFß is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFß lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFß are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFß in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the ß-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria.
Assuntos
Flavoproteínas/metabolismo , Lisina/metabolismo , Metiltransferases/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Cromatografia de Afinidade , Primers do DNA , Transporte de Elétrons , Humanos , Espectrometria de Massas , Metilação , Metiltransferases/química , Dados de Sequência MolecularRESUMO
α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca(2+) oscillations that burden mitochondria, we examined mitochondrial Ca(2+) stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca(2+), promoted Ca(2+)-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca(2+). Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca(2+) homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca(2+)-handling requirements.
Assuntos
Biopolímeros/fisiologia , Cálcio/fisiologia , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/fisiologia , alfa-Sinucleína/fisiologia , Animais , Benzotiazóis , Biopolímeros/química , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Tiazóis/metabolismo , alfa-Sinucleína/químicaRESUMO
Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044GâC), compound heterozygous patients with severe myopathy have been identified to carry the c.149GâA missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms.
Assuntos
Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Miopatias Mitocondriais/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Respiração Celular , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Heterozigoto , Humanos , Ferro/química , Potenciais da Membrana , Miopatias Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutagênese , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Enxofre/químicaRESUMO
The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (NLys(395)) is conserved in NuoL (LLys(399)) but is replaced by glutamic acid (MGlu(407)) in NuoM. Our mutation study on NLys(395) suggests that this residue participates in the proton translocation. Furthermore, we found that MGlu(407) is also essential and most likely interacts with conserved LArg(175). Glutamic acids, NGlu(133), MGlu(144), and LGlu(144), are corresponding residues. Unlike mutants of MGlu(144) and LGlu(144), mutation of NGlu(133) scarcely affected the energy-transducing activities. However, a double mutant of NGlu(133) and nearby KGlu(72) showed significant inhibition of these activities. This suggests that NGlu(133) bears a functional role similar to LGlu(144) and MGlu(144) but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that NLys(158) is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of NTM14 interacts with the Mß sheet located on the opposite side of helix HL. Taken together, the mechanism of H(+) translocation in NDH-1 is discussed.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Subunidades Proteicas/metabolismo , Substituição de Aminoácidos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Transporte de Íons/fisiologia , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , PrótonsRESUMO
Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.