RESUMO
Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, autoinhibited and an open, active conformation. Although the open conformation of mDia2 induces actin assembly thereby controlling many cellular processes, mDia2 possesses also actin-independent and conformation-insensitive scaffolding roles related to microtubules and p53, respectively. Thus, we hypothesize that mDia2 may have other unappreciated functions and regulatory modes. Here we identify and validate proteasome and Ubiquitin as mDia2-interacting partners using stable isotope labeling with amino acids in cell culture-based quantitative proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated, binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome substrate. Surprisingly, knockdown of mDia2 increases the activity of the proteasome in vitro, whereas mDia2 overexpression has opposite effects only when it adopts the open conformation and cannot induce actin assembly. Consistently, a combination of candidate and unbiased proteome-wide analyses indicates that mDia2 regulates the cellular levels of proteasome substrate ß-catenin and a number of ubiquitinated actin-regulatory proteins. Hence, these findings add more complexity to the mDia2 activity cycle by showing that the open conformation may control actin dynamics also through actin-independent regulation of the proteasome.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Actinas/metabolismo , Animais , Marcação por Isótopo , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/fisiologia , NADPH Desidrogenase/química , NADPH Desidrogenase/fisiologia , Conformação Proteica , Mapeamento de Interação de Proteínas , Ubiquitina/metabolismoRESUMO
Prior studies with Nicotiana and Arabidopsis described failed assembly of the chloroplastic NDH [NAD(P)H dehydrogenase] supercomplex by serial mutation of several subunit genes. We examined the properties of Zea mays leaves containing Mu and Ds insertions into nuclear gene exons encoding the critical o- and n-subunits of NDH, respectively. In vivo reduction of plastoquinone in the dark was sharply diminished in maize homozygous mutant compared to normal leaves but not to the extreme degree observed for the corresponding lesions in Arabidopsis. The net carbon assimilation rate (A) at high irradiance and saturating CO2 levels was reduced by one-half due to NDH mutation in maize although no genotypic effect was evident at very low CO2 levels. Simultaneous assessment of chlorophyll fluorescence and A in maize at low (2% by volume) and high (21%) O2 levels indicated the presence of a small, yet detectable, O2-dependent component of total linear photosynthetic electron transport in 21% O2 This O2-dependent component decreased with increasing CO2 level indicative of photorespiration. Photorespiration was generally elevated in maize mutant compared to normal leaves. Quantification of the proportion of total electron transport supporting photorespiration enabled estimation of the bundle sheath cell CO2 concentration (Cb) using a simple kinetic model of ribulose bisphosphate carboxylase/oxygenase function. The A versus Cb relationships overlapped for normal and mutant lines consistent with occurrence of strictly CO2-limited photosynthesis in the mutant bundle sheath cell. The results are discussed in terms of a previously reported CO2 concentration model [Laisk A, Edwards GE (2000) Photosynth Res 66: 199-224].
Assuntos
Dióxido de Carbono/metabolismo , NADPH Desidrogenase/metabolismo , NADPH Desidrogenase/fisiologia , NAD/metabolismo , Zea mays/metabolismo , Alelos , Arabidopsis/metabolismo , Carbono/metabolismo , Dióxido de Carbono/análise , Núcleo Celular , Clorofila , Cloroplastos/metabolismo , Escuridão , Transporte de Elétrons , Éxons , Genótipo , Mutação , NAD/genética , NADPH Desidrogenase/genética , Oxirredução , Oxigênio/análise , Oxigênio/metabolismo , Fotossíntese/efeitos dos fármacos , Pigmentos Biológicos/análise , Folhas de Planta/metabolismo , Plastoquinona/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Nicotiana/metabolismo , Zea mays/genética , Zea mays/efeitos da radiaçãoRESUMO
Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading-edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Recent work demonstrated, however, that, in the absence of the Arp2/3 complex, fibroblast cells adopt a leading edge with filopodia-like protrusions (FLPs) and maintain an ability to move, albeit with altered responses to different environmental signals. We show that formin-family actin nucleators are required for the extension of FLPs but are insufficient to produce a continuous leading edge in fibroblasts lacking Arp2/3 complex. Myosin II is concentrated in arc-like regions of the leading edge in between FLPs, and its activity is required for coordinated advancement of these regions with formin-generated FLPs. We propose that actomyosin contraction acting against membrane tension advances the web of arcs between FLPs. Predictions of this model are verified experimentally. The dependence of myosin II in leading-edge advancement helps explain the previously reported defect in directional movement in the Arpc3-null fibroblasts. We provide further evidence that this defect is cell autonomous during chemotaxis.
Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas de Transporte/fisiologia , Quimiotaxia , Fibroblastos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Miosina Tipo II/fisiologia , NADPH Desidrogenase/fisiologia , Pseudópodes/fisiologia , Actomiosina/metabolismo , Actomiosina/fisiologia , Animais , Proteínas de Transporte/metabolismo , Quimiotaxia/genética , Fibroblastos/metabolismo , Forminas , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , NADPH Desidrogenase/metabolismo , Pseudópodes/metabolismoRESUMO
Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)-deficient MV(D7) fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2(M/A) rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated filopodial morphology, dynamics, and function.
Assuntos
Moléculas de Adesão Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , NADPH Desidrogenase/fisiologia , Fosfoproteínas/fisiologia , Pseudópodes/fisiologia , Animais , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Transdução de SinaisRESUMO
Despite significant progress in clarifying the subunit compositions and functions of the multiple NADPH dehydrogenase (NDH-1) complexes in cyanobacteria, the subunit maturation and assembly of their NDH-1 complexes are poorly understood. By transformation of wild-type cells with a transposon-tagged library, we isolated three mutants of Synechocystis sp. PCC 6803 defective in NDH-1-mediated cyclic electron transfer and unable to grow under high light conditions. All the mutants were tagged in the same slr1097 gene, encoding an unknown protein that shares significant homology with the Arabidopsis protein chlororespiratory reduction 6 (CRR6). The slr1097 product was localized in the cytoplasm and was required for efficient assembly of NDH-1 complexes. Analysis of the interaction of Slr1097 with 18 subunits of NDH-1 complexes using a yeast two-hybrid system indicated a strong interaction with NdhI but not with other Ndh subunits. Absence of Slr1097 resulted in a significant decrease of NdhI in the cytoplasm, but not of other Ndh subunits including NdhH, NdhK and NdhM; the decrease was more evident in the cytoplasm than in the thylakoid membranes. In the ∆slr1097 mutant, NdhH, NdhI, NdhK and NdhM were hardly detectable in the NDH-1M complex, whereas almost half the wild-type levels of these subunits were present in NDH-1L complex; similar results were observed in the NdhI-less mutant. These results suggest that Slr1097 is involved in the maturation of NdhI, and that assembly of the NDH-1M complex is strongly dependent on this factor. Maturation of NdhI appears not to be crucial to assembly of the NDH-1L complex.
Assuntos
Proteínas de Bactérias/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Transporte de Elétrons/genética , Modelos Biológicos , NADPH Desidrogenase/metabolismo , NADPH Desidrogenase/fisiologia , Subunidades Proteicas/metabolismo , Synechocystis/genética , Tilacoides/metabolismoRESUMO
The role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow around photosystem I in photosynthetic regulation and plant growth at several temperatures was examined in rice (Oryza sativa) that is defective in CHLORORESPIRATORY REDUCTION 6 (CRR6), which is required for accumulation of sub-complex A of the chloroplast NDH complex (crr6). NdhK was not detected by Western blot analysis in crr6 mutants, resulting in lack of a transient post-illumination increase in chlorophyll fluorescence, and confirming that crr6 mutants lack NDH activity. When plants were grown at 28 or 35°C, all examined photosynthetic parameters, including the CO(2) assimilation rate and the electron transport rate around photosystems I and II, at each growth temperature at light intensities above growth light (i.e. 800 µmol photons m(-2) sec(-1)), were similar between crr6 mutants and control plants. However, when plants were grown at 20°C, all the examined photosynthetic parameters were significantly lower in crr6 mutants than control plants, and this effect on photosynthesis caused a corresponding reduction in plant biomass. The F(v)/F(m) ratio was only slightly lower in crr6 mutants than in control plants after short-term strong light treatment at 20°C. However, after long-term acclimation to the low temperature, impairment of cyclic electron flow suppressed non-photochemical quenching and promoted reduction of the plastoquinone pool in crr6 mutants. Taken together, our experiments show that NDH-dependent cyclic electron flow plays a significant physiological role in rice during photosynthesis and plant growth at low temperature.
Assuntos
Temperatura Baixa , Transporte de Elétrons/fisiologia , NADPH Desidrogenase/fisiologia , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Genes de Plantas , Mutação , NADPH Desidrogenase/genética , Oryza/genéticaRESUMO
NADPH and NADH mediate reductant flow between cellular processes, linking central carbon and energy metabolism with intermediary metabolism, stress defence and development. Recent investigations have revealed paths of functional interactions, and have suggested that mitochondrial NADPH oxidation, especially together with the oxidative pentose phosphate pathway, is an important regulator of the cytosolic NADPH reduction level. Furthermore, stress-dependent metabolic pathways substantially affect the NADPH reduction level in particular physiological situations. The mitochondrial impact on the NADPH reduction level provides a model example of the physiological significance of the mitochondrial NAD(P)H dehydrogenase set-up, which is more complex in plants than in other organisms.
Assuntos
Mitocôndrias/fisiologia , NADP/metabolismo , Plantas/metabolismo , Plantas/ultraestrutura , Regulação para Baixo/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Mitocôndrias/metabolismo , Modelos Biológicos , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , NADPH Desidrogenase/fisiologia , Oxirredução , Plantas/genéticaRESUMO
Formins are potent actin assembly factors. Diaphanous formins, including mDia1, mDia2, and mDia3 in mammals, are implicated in mitosis and cytokinesis, but no chemical interactors have been reported. We developed an in vitro screen for inhibitors of actin assembly by mDia1 and identified an inhibitor of mDia1 and mDia2 that does not inhibit mDia3 at the concentrations tested. These results establish the druggability of mDia formins and introduce a first-generation inhibitor.
Assuntos
Actinas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Transporte/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , NADPH Desidrogenase/antagonistas & inibidores , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas Fetais/antagonistas & inibidores , Forminas , Humanos , Camundongos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/fisiologia , NADPH Desidrogenase/química , NADPH Desidrogenase/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Pirenos/química , Homologia de Sequência de AminoácidosRESUMO
In photosynthetic eukaryotes, nonphotochemical plastoquinone (PQ) reduction is important for the regulation of photosynthetic electron flow. In green microalgae where this process has been demonstrated, the chloroplastic enzyme that catalyses nonphotochemical PQ reduction has not been identified yet. Here, we show by an RNA interference (RNAi) approach that the NDA2 gene, belonging to a type II NAD(P)H dehydrogenases family in the green microalga Chlamydomonas reinhardtii, encodes a chloroplastic dehydrogenase that functions to reduce PQ nonphotochemically in this alga. Using a specific antibody, we show that the Nda2 protein is localized in chloroplasts of wild-type cells and is absent in two Nda2-RNAi cell lines. In both mutant cell lines, nonphotochemical PQ reduction is severely affected, as indicated by altered chlorophyll fluorescence transients after saturating illumination. Compared with wild type, change in light excitation distribution between photosystems ('state transition') upon inhibition of mitochondrial electron transport is strongly impaired in transformed cells because of inefficient PQ reduction. Furthermore, the amount of hydrogen produced by Nda2-RNAi cells under sulfur deprivation is substantially decreased compared with wild type, which supports previous assumptions that endogenous substrates serve as source of electrons for hydrogen formation. These results demonstrate the importance of Nda2 for nonphotochemical PQ reduction and associated processes in C. reinhardtii.
Assuntos
Chlamydomonas/metabolismo , Cloroplastos/metabolismo , NADPH Desidrogenase/fisiologia , Plastoquinona/metabolismo , Animais , Transporte de Elétrons , Hidrogênio , Luz , Oxirredução/efeitos da radiação , RNA Interferente Pequeno/farmacologiaRESUMO
The preprotein translocon at the inner envelope of chloroplasts (Tic complex) facilitates the import of nuclear-encoded preproteins into the organelle. Seven distinct subunits have been identified so far. For each of those, specific functions have been proposed based on structural prediction or experimental evidence. Three of those subunits possess modules that could act as redox-active regulatory components in the import process. To date, however, the mode of redox regulation of the import process remains enigmatic. To investigate how the chloroplast redox state influences translocon behavior and composition, we studied the Tic component and the putative redox sensor Tic62 in more detail. The experimental results provide evidence that Tic62 can act as a bona fide dehydrogenase in vitro, and that it changes its localization in the chloroplast dependent on the NADP+/NADPH ratio in the stroma. Moreover, the redox state influences the interactions of Tic62 with the translocon and the flavoenzyme ferredoxin-NADP+ oxidoreductase. Additionally, we give initial experimental insights into the Tic62 structure using circular dichroism measurements and demonstrate that the protein consists of two structurally different domains. Our results indicate that Tic62 possesses redox-dependent properties that would allow it to fulfill a role as redox sensor protein in the chloroplast.
Assuntos
Regulação da Expressão Gênica de Plantas , NADPH Desidrogenase/metabolismo , NADP/química , Pisum sativum/metabolismo , Sequência de Aminoácidos , Cloroplastos/metabolismo , Dicroísmo Circular , Ferredoxinas/química , Modelos Biológicos , Dados de Sequência Molecular , NADPH Desidrogenase/fisiologia , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Homologia de Sequência de AminoácidosRESUMO
NAD(P)H:quinone oxidoreductase 1(-/-) (NQO1(-/-)), NQO1(+/-) along with NRH:quinone oxidoreductase 2(-/-) (NQO2(-/-)), and wild-type (WT) mice were exposed to five once weekly doses of mitomycin C. The mice were euthanized 15 weeks after the first dose. Blood cell counts and histologic analyses were done. WT and NQO2(-/-) mice showed hypocellularity and a significant increase in adipocytes in bone marrow. They also showed anemia because of the loss of RBC and hemoglobin. The neutrophils and platelets were reduced, whereas other blood cell types and tissues were normal. Interestingly, NQO1(-/-) mice showed a complete resistance to mitomycin C-induced bone marrow cytotoxicity and reduction in RBC, hemoglobin, and neutrophils. NQO1(+/-) mice also showed limited resistance to mitomycin C-induced bone marrow cytotoxicity. These data show a major in vivo role of NQO1 in metabolic activation of mitomycin C with implications in mitomycin C chemotherapy.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Mitomicina/efeitos adversos , Mitomicina/metabolismo , NADPH Desidrogenase/fisiologia , Animais , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/metabolismo , Biotransformação , Células da Medula Óssea/citologia , Contagem de Eritrócitos , Feminino , Hemoglobinas/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/genéticaRESUMO
Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 approximately 30 nmol/L; calcium mobility assay) and to be 10,000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 approximately 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.
Assuntos
Toxina Diftérica/química , Toxina Diftérica/toxicidade , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/patologia , Neurotoxinas/química , Neurotoxinas/toxicidade , Sistema Nervoso Parassimpático/efeitos dos fármacos , Ponte/patologia , Tegmento Mesencefálico/patologia , Urotensinas/química , Urotensinas/toxicidade , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Toxina Diftérica/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Masculino , NADPH Desidrogenase/metabolismo , NADPH Desidrogenase/fisiologia , Neurônios/patologia , Sistema Nervoso Parassimpático/patologia , Plasmídeos/genética , RatosRESUMO
Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.
Assuntos
Apoptose/fisiologia , Pâncreas/citologia , Espécies Reativas de Oxigênio/metabolismo , Vitamina K 3/química , Vitamina K 3/metabolismo , Animais , Camundongos , Mitocôndrias/metabolismo , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/fisiologia , NADP/metabolismo , NADPH Desidrogenase/antagonistas & inibidores , NADPH Desidrogenase/fisiologia , Oxirredução , Pâncreas/enzimologia , Pâncreas/metabolismo , Vitamina K 3/antagonistas & inibidoresRESUMO
After incubation at 42 degrees C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (deltandhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of deltandhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42 degrees C for 6 h, the photochemical activity declined more markedly in deltandhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (deltapH), while the consumption of deltapH was highly inhibited in deltandhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO2 assimilation by providing extra deltapH, thus reducing the photooxidative damage.
Assuntos
Cloroplastos/enzimologia , NADPH Desidrogenase/fisiologia , Nicotiana/enzimologia , Transporte de Elétrons/fisiologia , Temperatura Alta , Estresse Oxidativo/fisiologia , FenótipoRESUMO
NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) are cytosolic enzymes that catalyze metabolic reduction of quinones and derivatives. NQO1-null and NQO2-null mice were generated that showed decreased lymphocytes in peripheral blood, myeloid hyperplasia, and increased sensitivity to skin carcinogenesis. In this report, we investigated the in vivo role of NQO1 and NQO2 in immune response and autoimmunity. Both NQO1-null and NQO2-null mice showed decreased B-cells in blood, lower germinal center response, altered B cell homing, and impaired primary and secondary immune responses. NQO1-null and NQO2-null mice also showed susceptibility to autoimmune disease as revealed by decreased apoptosis in thymocytes and pre-disposition to collagen-induced arthritis. Further experiments showed accumulation of NADH and NRH, cofactors for NQO1 and NQO2, indicating altered intracellular redox status. The studies also demonstrated decreased expression and lack of activation of immune-related factor NF-kappaB. Microarray analysis showed altered chemokines and chemokine receptors. These results suggest that the loss of NQO1 and NQO2 leads to altered intracellular redox status, decreased expression and activation of NF-kappaB, and altered chemokines. The results led to the conclusion that NQO1 and NQO2 are endogenous factors in the regulation of immune response and autoimmunity.
Assuntos
Formação de Anticorpos , Autoimunidade , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , NADPH Desidrogenase/fisiologia , Quinona Redutases/fisiologia , Animais , Apoptose , Artrite/metabolismo , Células da Medula Óssea/metabolismo , Citosol/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/metabolismo , NF-kappa B/metabolismo , Oxirredução , Quinona Redutases/metabolismoRESUMO
Formin proteins are regulators of actin dynamics, mediating assembly of unbranched actin filaments. These multidomain proteins are defined by the presence of a Formin Homology 2 (FH2) domain. Previous work has shown that FH2 domains bind to filament barbed ends and move processively at the barbed end as the filament elongates. Here we report that two FH2 domains, from mammalian FRL1 and mDia2, also bundle filaments, whereas the FH2 domain from mDia1 cannot under similar conditions. The FH2 domain alone is sufficient for bundling. Bundled filaments made by either FRL1 or mDia2 are in both parallel and anti-parallel orientations. A novel property that might contribute to bundling is the ability of the dimeric FH2 domains from both FRL1 and mDia2 to dissociate and recombine. This property is not observed for mDia1. A difference between FRL1 and mDia2 is that FRL1-mediated bundling is competitive with barbed end binding, whereas mDia2-mediated bundling is not. Mutation of a highly conserved isoleucine residue in the FH2 domain does not inhibit bundling by either FRL1 or mDia2, but inhibits barbed end activities. However, the severity of this mutation varies between formins. For mDia1 and mDia2, the mutation strongly inhibits all effects of barbed end binding, but affects FRL1 much less strongly. Furthermore, our results suggest that the Ile mutation affects processivity. Taken together, our data suggest that the bundling activities of FRL1 and mDia2, while producing phenotypically similar bundles, differ in mechanistic detail.
Assuntos
Actinas/química , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , NADPH Desidrogenase/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Ligação Competitiva , DNA/química , Dimerização , Forminas , Peptídeos e Proteínas de Sinalização Intracelular/química , Ponto Isoelétrico , Camundongos , Proteínas Associadas aos Microtúbulos , Mutação , NADPH Desidrogenase/química , Ligação Proteica , Estrutura Terciária de Proteína , CoelhosRESUMO
Many histochemical investigations indicated that the oxytocin (OXY), the arginine vasopressin (AVP) and the nitric oxide synthase (NOS) have been synthesized in the supraoptic nucleus (SON) neurons. The objective of this study was to examine the age-related expression of the OXY, the AVP and the NOS in the SON of the young adult (2-month-old) and the aged (24-month-old) rats. The histochemistry for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d; marker for the NOS) and the double labeling histochemistry for the OXY/NADPH-d or the AVP/NADPH-d were employed, and the quantitative analysis was performed with a computer-assisted image processing system. In comparison of the young adult and the aged group, the cell number, the cell size and the reactive density of the NOS-expressing neurons showed a significant increase along with age, and these evidences suggested the age-related increase of the nitric oxide (NO) production. The age-related significant increase was not detected in the number of the OXY/NOS-expressing neurons in the dorsal part, but was detected in the number of the AVP/NOS-expressing neurons in the ventral part. Based on our histochemical findings and reports demonstrated by other authors, we attempted to discuss the physiological role of NOS for the secretion of posterior pituitary hormones along with age.
Assuntos
Envelhecimento/fisiologia , Arginina Vasopressina/biossíntese , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo I/biossíntese , Ocitocina/biossíntese , Núcleo Supraóptico/enzimologia , Animais , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , NADPH Desidrogenase/fisiologia , Ratos , Ratos Sprague-Dawley , Núcleo Supraóptico/citologia , Núcleo Supraóptico/crescimento & desenvolvimentoRESUMO
NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones is suggested to be involved in cancer prevention. In the present study, using transfected CHO cells, it was demonstrated that the relation between NQO1 activity and the resulting protection against the cytotoxicity of menadione shows a steep dose-response curve revealing a 'lower protection threshold' of 0.5mumol DCPIP/min/mg protein and an 'upper protection threshold' at 1mumol DCPIP/min/mg protein. In an additional in vivo experiment it was investigated how both in vitro critical activity levels of NQO1, relate to NQO1 activities in mice and man, either without or upon induction of the enzyme by butylated hydroxyanisol (BHA) or indole-3-carbinol (I(3)C). Data from an experiment with CD1 mice revealed that base-line NQO1 levels in liver, kidney, small intestine, colon and lung are generally below the observed 'lower protection threshold' in vitro, this also holds for most human tissue S-9 samples. To achieve NQO1 levels above this 'lower protection threshold' will require 5-20 fold NQO1 induction. Discussion focuses on the relevance of the in vitro NQO1 activity thresholds for the in vivo situation. We conclude that increased protection against menadione toxicity can probably not be achieved by NQO1 induction but should be achieved by other mechanisms. Whether this conclusion also holds for other electrophiles and the in vivo situation awaits further definition of their NQO1 protection thresholds.
Assuntos
Citoproteção , NAD(P)H Desidrogenase (Quinona)/fisiologia , NADPH Desidrogenase/fisiologia , Vitamina K 3/toxicidade , Animais , Células CHO , Linhagem Celular , Cricetinae , Feminino , Humanos , Camundongos , NADPH Desidrogenase/análiseRESUMO
A 410-nm absorbing species which enhanced the reduction rate of cytochrome c by Old Yellow Enzyme (OYE) with NADPH was found in Saccharomyces cerevisiae. It was solubilized together with OYE by the treatment of yeast cells with 10% ethyl acetate. The purified species showed visible absorption spectra in both oxidized and reduced forms, which were the same as those of the yeast microsomal cytochrome b5. At least 14 amino acid residues of the N-terminal region coincided with those of yeast microsomal b5, but the protein had a lower molecular weight determined to be 12,600 by SDS-PAGE and 9775 by mass spectrometry. The cytochrome b5-like protein enhanced the reduction rate of cytochrome c by OYE, and a plot of the reduction rates against its concentration showed a sigmoidal curve with an inflexion point at 6x10(-8) M of the protein.
Assuntos
Citocromos b5/química , Citocromos c/metabolismo , Citosol/enzimologia , NADPH Desidrogenase/fisiologia , NADP/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Catálise , Citocromos b5/isolamento & purificação , Citocromos b5/metabolismo , Transporte de Elétrons , Oxirredução , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Aging is associated with a functional decline and change in the phenotypic distribution of T cell subsets. The free radical theory of aging is widely promoted as the mechanistic basis for cellular senescence, including the immune system. Although the exact molecular explanation for the role of oxidative stress in cellular senescence is unclear, there is a connection to altered mitochondrial function, both as a contributor and as a target of oxidative stress. In this study we demonstrate that splenic T lymphocytes from old C57BL/6 mice exhibit a significant decline in mitochondrial membrane potential (deltapsi(m)). However, despite this change, there is a lower rate of withdrawal apoptosis in the memory CD4+ and CD8+ T cells. To explain the survival of these long-lived cells against a background of increased oxidative stress, we demonstrate increased glutathione production and phase II enzyme expression, which combine to protect memory T cells against oxidative stress, mitochondrial dysfunction, and cell death. The accumulation of memory T cells with aging explains higher phase II enzyme expression in CD4+ and CD8+ T cells from old mice. Compared with wild-type mice, mice lacking the expression of NF-E2-related factor-2, the transcription factor that regulates phase II enzyme expression, had a significantly enhanced rate of apoptosis in the presence of an oxidative stress stimulus. NF-E2-related factor-2-deficient T cells exhibit a bigger decline in deltapsi(m) and increased reactive oxygen species production than cells from wild-type animals. Taken together, we suggest that phase II enzyme expression and the accompanying increase in intracellular thiol levels protect memory T cells from mitochondrial dysfunction and spontaneous apoptosis.