RESUMEN
The c-Jun N-terminal kinase (JNK) is an important mediator of physiological and pathophysiological processes in the central nervous system. Importantly, JNK not only is involved in neuronal cell death, but also plays a significant role in neuronal differentiation and regeneration. For example, nerve growth factor induces JNK-dependent neuronal differentiation in several model systems. The mechanism by which JNK mediates neuronal differentiation is not well understood. Here, we employed a proteomic strategy to better characterize the function of JNK during neuronal differentiation. We used SILAC-based quantitative proteomics to identify proteins that interact with JNK in PC12 cells in a nerve growth factor-dependent manner. Intriguingly, we found that JNK interacted with neuronal transport granule proteins such as Sfpq and Nono upon NGF treatment. We validated the specificity of these interactions by showing that they were disrupted by a specific peptide inhibitor that blocks the interaction of JNK with its substrates. Immunoprecipitation and Western blotting experiments confirmed the interaction of JNK1 with Sfpq/Nono and demonstrated that it was RNA dependent. Confocal microscopy indicated that JNK1 associated with neuronal granule proteins in the cytosol of PC12 cells, primary cortical neurons, and P19 neuronal cells. Finally, siRNA experiments confirmed that Sfpq was necessary for neurite outgrowth in PC12 cells and that it most likely acted in the same pathway as JNK. In summary, our data indicate that the interaction of JNK1 with transport granule proteins in the cytosol of differentiating neurons plays an important role during neuronal development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Ratones , Factores de Crecimiento Nervioso/farmacología , Células PC12 , Factor de Empalme Asociado a PTB , Estructura Terciaria de Proteína , Proteómica , Transporte de ARN , RatasRESUMEN
In response to fasting or hyperglycemia, the pancreatic ß-cell alters its output of secreted insulin; however, the pathways governing this adaptive response are not entirely established. Although the precise role of microRNAs (miRNAs) is also unclear, a recurring theme emphasizes their function in cellular stress responses. We recently showed that miR-184, an abundant miRNA in the ß-cell, regulates compensatory proliferation and secretion during insulin resistance. Consistent with previous studies showing miR-184 suppresses insulin release, expression of this miRNA was increased in islets after fasting, demonstrating an active role in the ß-cell as glucose levels lower and the insulin demand ceases. Additionally, miR-184 was negatively regulated upon the administration of a sucrose-rich diet in Drosophila, demonstrating strong conservation of this pathway through evolution. Furthermore, miR-184 and its target Argonaute2 remained inversely correlated as concentrations of extracellular glucose increased, underlining a functional relationship between this miRNA and its targets. Lastly, restoration of Argonaute2 in the presence of miR-184 rescued suppression of miR-375-targeted genes, suggesting these genes act in a coordinated manner during changes in the metabolic context. Together, these results highlight the adaptive role of miR-184 according to glucose metabolism and suggest the regulatory role of this miRNA in energy homeostasis is highly conserved.
Asunto(s)
Glucosa/metabolismo , Islotes Pancreáticos/fisiología , MicroARNs/fisiología , Animales , Proteínas Argonautas/metabolismo , Línea Celular , Homeostasis/fisiología , Islotes Pancreáticos/metabolismo , Ratones , MicroARNs/genética , Mitocondrias/metabolismoRESUMEN
The importin α:ß complex is responsible for the nuclear import of proteins bearing classical nuclear localization signals. In mammals, several importin α subtypes are known to exist that are suggested to have individual functions. Importin α 7 was shown to play a crucial role in early embryonic development in mice. Embryos from importin α 7-depleted females stop at the two-cell stage and show disturbed zygotic genome activation. As there is evidence that individual importin α subtypes possess cargo specificities, we hypothesized that importin α 7 binds a unique set of intracellular proteins. With the use of a collection of in vitro and in vivo binding assays, importin α 7 interaction partners were identified that differed from proteins found to bind to importin α 2 and 3. One of the proteins preferentially binding importin α 7 was the maternal effect protein Brg1. However, Brg1 was localized in oocyte nuclei in importin α 7-deficient embryos, albeit in reduced amounts, suggesting additional modes of nuclear translocation of this factor. An additional SILAC-based screening approach identified Ash2l, Chd3, Mcm3, and Smarcc1, whose nuclear import seems to be disturbed in importin α 7-deficient fibroblasts.
Asunto(s)
Oocitos/metabolismo , Proteómica/métodos , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Animales , Núcleo Celular/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Femenino , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Especificidad por Sustrato , Factores de Transcripción/metabolismoRESUMEN
Argonaute2 (Ago2) is an established component of the microRNA-induced silencing complex. Similar to miR-375 loss-of-function studies, inhibition of Ago2 in the pancreatic ß-cell resulted in enhanced insulin release underlining the relationship between these two genes. Moreover, as the most abundant microRNA in pancreatic endocrine cells, miR-375 was also observed to be enriched in Ago2-associated complexes. Both Ago2 and miR-375 regulate the pancreatic ß-cell secretome, and by using quantitative mass spectrometry, we identified the enhanced release of a set of proteins or secretion "signatures " in response to a glucose stimulus using the murine ß-cell line MIN6. In addition, the loss of Ago2 resulted in the increased expression of miR-375 target genes, including gephyrin and ywhaz. These targets positively contribute to exocytosis indicating they may mediate the functional role of both miR-375 and Ago proteins in the pancreatic ß-cell by influencing the secretory pathway. This study specifically addresses the role of Ago2 in the systemic release of proteins from ß-cells and highlights the contribution of the microRNA pathway to the function of this cell type.
Asunto(s)
Proteínas Argonautas/fisiología , Células Secretoras de Insulina/metabolismo , Proteoma/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Insulina/metabolismo , Secreción de Insulina , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Proteoma/genética , Interferencia de ARNRESUMEN
Stable isotope labeling by amino acids in cell culture (SILAC) is widely used to quantify protein abundance in tissue culture cells. Until now, the only multicellular organism completely labeled at the amino acid level was the laboratory mouse. The fruit fly Drosophila melanogaster is one of the most widely used small animal models in biology. Here, we show that feeding flies with SILAC-labeled yeast leads to almost complete labeling in the first filial generation. We used these "SILAC flies" to investigate sexual dimorphism of protein abundance in D. melanogaster. Quantitative proteome comparison of adult male and female flies revealed distinct biological processes specific for each sex. Using a tudor mutant that is defective for germ cell generation allowed us to differentiate between sex-specific protein expression in the germ line and somatic tissue. We identified many proteins with known sex-specific expression bias. In addition, several new proteins with a potential role in sexual dimorphism were identified. Collectively, our data show that the SILAC fly can be used to accurately quantify protein abundance in vivo. The approach is simple, fast, and cost-effective, making SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics.
Asunto(s)
Drosophila melanogaster/química , Proteínas/análisis , Animales , Cromatografía Liquida , Femenino , Lisina/química , Masculino , Modelos Animales , Mapeo Peptídico , Espectrometría de Masas en TándemRESUMEN
Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.
Asunto(s)
Apoptosis/efectos de los fármacos , Hipocampo/efectos de los fármacos , Meningitis Neumocócica/tratamiento farmacológico , Degeneración Nerviosa/tratamiento farmacológico , Compuestos Organometálicos/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Modelos Animales de Enfermedad , Hipocampo/enzimología , Hipocampo/patología , Meningitis Neumocócica/enzimología , Meningitis Neumocócica/patología , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/microbiología , Compuestos Organometálicos/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas WistarRESUMEN
Pneumococcal meningitis is associated with caspase 3-dependent apoptosis of recently post-mitotic immature neurons in the dentate gyrus of the hippocampus. The death of these cells is implicated in the learning and memory deficits in patients surviving the disease. The stress-activated protein kinase c-Jun N-terminal kinase (JNK) has been shown to be an important mediator of caspase 3-dependent neuronal apoptosis. However, whether JNK is involved in hippocampal apoptosis caused by pneumococcal meningitis has so far not been investigated. Here we show in a neonatal rat model of pneumococcal meningitis that JNK3 but not JNK1 or JNK2 is activated in the hippocampus during the acute phase of infection. At the cellular level, JNK3 activation was accompanied in the dentate gyrus by markedly increased phosphorylation of its major downstream target c-Jun in early immature (Hu-positive) neurons, but not in migrating (doublecortin-positive) neurons, the cells that do undergo apoptosis. These findings suggested that JNK may not be involved in pneumococcal meningitis-induced hippocampal apoptosis. Indeed, although intracerebroventricular administration of D-JNKI-1 or AS601245 (two highly specific JNK inhibitors) inhibited c-Jun phosphorylation and protein expression in the hippocampus, hippocampal apoptosis was unaffected. Collectively, these results demonstrate that JNK does not mediate hippocampal apoptosis in pneumococcal meningitis, and that JNK may be involved in processes unrelated to apoptosis in this disease.
Asunto(s)
Apoptosis/fisiología , Hipocampo/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Meningitis Neumocócica/enzimología , Neuronas/enzimología , Animales , Animales Recién Nacidos , Proteína Doblecortina , Activación Enzimática/fisiología , Hipocampo/patología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Meningitis Neumocócica/patología , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Neuronas/patología , Ratas , Ratas WistarRESUMEN
N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.
Asunto(s)
Acetilcisteína/farmacología , Encéfalo/metabolismo , Endotelina-1/metabolismo , Endotelio Vascular/metabolismo , Depuradores de Radicales Libres/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Encéfalo/citología , Línea Celular , Endotelina-1/genética , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , FN-kappa B/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Fosforilación , Transporte de Proteínas , Ratas , Regulación hacia ArribaRESUMEN
The fruit fly Drosophila melanogaster is one of the most widely used and well-studied model organisms in biology and therefore a promising tool for quantitative proteomics. Here, we describe a method to label D. melanogaster with stable isotope labeled amino acids in vivo. Feeding flies with heavy lysine labeled yeast cells leads to virtually complete heavy labeling already in the first filial generation. The approach is simple, fast, and cost-effective, which makes SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics.