RESUMO
The regulation of protein synthesis is a vital and finely tuned process in cellular physiology. In neurons, this process is very precisely regulated, as which mRNAs undergo translation is highly dependent on context. One of the most prominent regulators of protein synthesis is the enzyme eukaryotic elongation factor kinase 2 (eEF2K) that regulates the elongation stage of protein synthesis. This kinase and its substrate, eukaryotic elongation factor 2 (eEF2) are important in processes such as neuronal development and synaptic plasticity. eEF2K is regulated by multiple mechanisms including Ca2+ -ions and the mTORC1 signaling pathway, both of which play key roles in neurological processes such as learning and memory. In such settings, the localized control of protein synthesis is of crucial importance. In this work, we sought to investigate how the localization of eEF2K is controlled and the impact of this on protein synthesis in neuronal cells. In this study, we used both SH-SY5Y neuroblastoma cells and mouse cortical neurons, and pharmacologically and/or genetic approaches to modify eEF2K function. We show that eEF2K activity and localization can be regulated by its binding partner Homer1b/c, a scaffolding protein known for its participation in calcium-regulated signaling pathways. Furthermore, our results indicate that this interaction is regulated by the mTORC1 pathway, through a known phosphorylation site in eEF2K (S396), and that it affects rates of localized protein synthesis at synapses depending on the presence or absence of this scaffolding protein.
Assuntos
Quinase do Fator 2 de Elongação/metabolismo , Proteínas de Arcabouço Homer/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Bicuculina/farmacologia , Células Cultivadas , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Camundongos , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The NMDA receptor is crucial to several functions in CNS physiology and some of its effects are mediated by promoting nitric oxide production from L-arginine and activation of signaling pathways and the transcription factor CREB. Our previous work demonstrated in retinal cells that increasing intracellular free L-arginine levels directly correlates to nitric oxide (NO) generation and can be promoted by protein synthesis inhibition and increase of free L-arginine concentration. Eukaryotic elongation factor 2 kinase (eEF2K), a calcium/calmodulin-dependent kinase, is also known to be activated by NMDA receptors leading to protein synthesis inhibition. Here we explored how does eEF2K participate in NMDA-induced NO signaling. We found that when this enzyme is inhibited, NMDA loses its ability to promote NO synthesis. On the other hand, when NO synthesis is increased by protein synthesis inhibition with cycloheximide or addition of exogenous L-arginine, eEF2K has no participation, showcasing a specific link between this enzyme and NMDA-induced NO signaling. We have previously shown that inhibition of the canonical NO signaling pathway (guanylyl cyclase/cGMP/cGK) blocks CREB activation by glutamate in retinal cells. Interestingly, pharmacological inhibition of eEF2K fully prevents CREB activation by NMDA, once again demonstrating the importance of eEF2K in NMDA receptor signaling. In summary, we demonstrated here a new role for eEF2K, directly controlling NMDA-dependent nitrergic signaling and modulating L-arginine availability in neurons, which can potentially be a new target for the study of physiological and pathological processes involving NMDA receptors in the central nervous system.
Assuntos
Sistema Nervoso Central/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , N-Metilaspartato/farmacologia , Óxido Nítrico/biossíntese , Animais , Arginina/farmacologia , Galinhas , Cicloeximida/farmacologia , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Indazóis/farmacologia , Masculino , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , RatosRESUMO
Attractive due to an alleged high biocompatibility, silica nanoparticles have been widely used in the field of nanomedicine; however, their proven capacity to induce the synthesis and release of pro-inflammatory cytokines in several cellular models has raised concern about their safety. Glutamate, the main excitatory amino acid transmitter triggers a wide variety of signal transduction cascades that regulate protein synthesis at transcriptional and translational levels. A stimulus-dependent dynamic change in the protein repertoire in neurons and glia cells is the molecular framework of higher brain functions. Within the cerebellum, Bergmann glia cells are the most abundant non-neuronal cells and span the entire molecular layer of the cerebellar cortex, wrapping the synapses in this structure. Taking into consideration the functional role of Bergmann glia in terms of the recycling of glutamate, lactate supply to neurons, and prevention of neurotoxic insults, we decided to investigate the possibility that silica nanoparticles affect Bergmann glia and by these means alter the major excitatory neurotransmitter system in the brain. To this end, we exposed cultured chick cerebellar Bergmann glia cells to silica nanoparticles and measured [35S]-methionine incorporation into newly synthesized polypeptides. Our results demonstrate that exposure of the cultured cells to silica nanoparticles exerts a time- and dose-dependent modulation of protein synthesis. Furthermore, altered patterns of eukaryotic initiation factor 2 alpha and eukaryotic elongation factor 2 phosphorylation were present upon nanoparticle exposure. These results demonstrate that glia cells respond to the presence of this nanomaterial modifying their proteome, presumably in an effort to overcome any plausible neurotoxic effect.
Assuntos
Nanopartículas/efeitos adversos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Dióxido de Silício/efeitos adversos , Animais , Embrião de Galinha , Relação Dose-Resposta a Droga , Quinase do Fator 2 de Elongação/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Metionina/metabolismo , Fosforilação , Cultura Primária de Células , Radioisótopos de Enxofre/metabolismo , Fatores de TempoRESUMO
The putative translation factor eIF5A is essential for cell viability and is highly conserved throughout evolution. Here, we describe genetic interactions between an eIF5A mutant and a translation initiation mutant (eIF4E) or a translation elongation mutant (eEF2). Polysome profile analysis of single and double mutants revealed that mutation in eIF5A reduces polysome run-off, contrarily to translation initiation mutants. Moreover, the polysome profile of an eIF5A mutant alone is very similar to that of a translation elongation mutant. Furthermore, depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosome transit time. Finally, we demonstrate that the formation of P bodies is inhibited in an eIF5A mutant, similarly to the effect of the translation elongation inhibitor cycloheximide. Taken together, these results not only reinforce a role for eIF5A in translation but also strongly support a function for eIF5A in the elongation step of protein synthesis.