RESUMO
The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Glucose/metabolismo , Quinases da Glicogênio Sintase/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Aspergillus nidulans/enzimologia , Repressão Catabólica/genética , Fungos/genética , Fungos/metabolismo , Glicerol/metabolismo , Concentração Osmolar , Fosforilação/genética , Mapas de Interação de Proteínas/genética , Proteínas Repressoras/genética , Xilose/metabolismoRESUMO
BACKGROUND: Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3ß, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3ß during Dengue virus-2 (DENV-2) infection was investigated. METHODS: GSK3ß participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS: Phosphorylation of GSK3ß-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3ß reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3ß. CONCLUSIONS: The results suggest that GSK3ß participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.
Assuntos
Vírus da Dengue/enzimologia , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/fisiologia , Replicação Viral/fisiologia , Aedes/citologia , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Microscopia de Fluorescência , Fosforilação/fisiologia , Transdução de SinaisRESUMO
BACKGROUND Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3β, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3β during Dengue virus-2 (DENV-2) infection was investigated. METHODS GSK3β participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS Phosphorylation of GSK3β-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3β reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3β. CONCLUSIONS The results suggest that GSK3β participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.
Assuntos
Animais , Replicação Viral/fisiologia , Vírus da Dengue/enzimologia , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/fisiologia , Fosforilação/fisiologia , Transdução de Sinais , Western Blotting , Apoptose/fisiologia , Aedes/citologia , Linhagem Celular Tumoral , Microscopia de FluorescênciaRESUMO
Valproic acid (VA) is an antiepileptic that is also used for the treatment of bipolar disorders. The objective was to evaluate the neuroprotective effects of VA on a brain ischemia model. The groups of male Wistar rats were: SO (sham-operated), ischemic and ischemic treated with VA (25, 50 and 100â¯mg/kg, p.o.). After anesthesia with ketamine and xilazine, the animals were subjected to clamping of carotid arteries (30â¯min) and reperfusion. Except for the carotid clamping, the SO group was submitted to the same procedure. On the 7th day, the animals were behaviorally evaluated, euthanized and had their brain dissected for neurochemical and immunohistochemical assays. The data were analyzed by ANOVA and Tukey as the post hoc test. The results showed that VA reversed partly or completely the behavioral (locomotor activity and memory deficits), neurochemical (striatal DA and DOPAC levels, brain nitrite and lipid peroxidation) and immunohistochemical alterations (iNOS, COX-2, HDAC and GSK3) observed in the untreated ischemic group. VA neuroprotective effects are probably related to its anti-inflammatory and antioxidant properties, as well as to HDAC and GSK3 inhibitory effects. These findings stimulate translational studies focusing on VA as a neuroprotective drug to be potentially used in the clinic for several neurological conditions.
Assuntos
Isquemia Encefálica/prevenção & controle , Quinases da Glicogênio Sintase/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Fármacos Neuroprotetores/farmacologia , Ácido Valproico/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Encéfalo/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Peroxidação de Lipídeos , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , RatosRESUMO
RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT)/Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.
Assuntos
Técnicas de Silenciamento de Genes , RNA de Cadeia Dupla/genética , Rhipicephalus/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Eletroporação , Feminino , Expressão Gênica , Quinases da Glicogênio Sintase/genética , Quinases da Glicogênio Sintase/metabolismo , Heptanos/química , Óvulo/química , Óvulo/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Solventes/química , Técnicas de Cultura de Tecidos , Ceras/químicaRESUMO
Muscarinic agonists alter the metabolism of amyloid precursor protein, leading to an increase in alpha-secretase cleavage and a decreased production of amyloidogenic peptides; suggesting that these compounds might modify the Alzheimer's disease process. A second therapeutic target in AD is the accumulation of stably phosphorylated tau into neurofibrillary tangles; an early event correlating with cognitive impairment. Glycogen synthase kinase-3 (GSK-3beta) phosphorylates tau and is inhibited via protein kinase C (PKC). As certain muscarinic receptors are linked to PKC, we examined the effect of a range of agonists on GSK-3beta phosphorylation of tau. In neurons a nonspecific muscarinic agonist, carbachol, reduced tau phosphorylation. In nonneuronal cells expressing the ml receptor a range of ml agonists reduced transiently-expressed tau phosphorylation and altered its microtubulebinding properties. These findings link the two pathological process of AD-APP metabolism and tau phosphorylation - and suggest that muscarinic and other cholinergic compounds might have disease-modifying properties.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Proteínas tau/efeitos dos fármacos , Acetilcolina/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carbacol/farmacologia , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Feto , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Cloreto de Lítio/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/metabolismo , Tetrazóis/farmacologia , Tiadiazóis/farmacologia , Proteínas tau/metabolismoRESUMO
Neuroblastoma cells are used as a model system to study neuronal differentiation. Here we describe the induction of morphological differentiation of mouse neuroblastoma Neuro 2a (N2a) cells by treatments with either chemical inhibitors of cyclin-dependent kinases or lithium, which inhibits glycogen synthase kinase-3. Cyclin-dependent kinase inhibitors cause a rapid cell cycle block as well as the extension of multiple neurites per cell. These multipolar differentiated cells then undergo a massive death. However, lithium promotes a delayed mitotic arrest and the extension of one or two long neurites per cell. This differentiation is maximal after 48 hours of lithium treatment and the differentiated cells remain viable for long periods of time. Neuronal differentiation in lithium-treated cells is preceded by the accumulation of beta-catenin, a protein which is efficiently proteolyzed when it is phosphorylated by glycogen synthase kinase-3. Both neuronal differentiation and beta-catenin accumulation are observed in lithium-treated cells either in the absence or in the presence of supraphysiological concentrations of inositol. The results are consistent with the hypothesis that inhibition of glycogen synthase kinase-3 by lithium triggers the differentiation of neuroblastoma N2a cells.