RESUMEN
Neurons fail to re-extend their processes within the central nervous system environment in vivo, and this is partly because of inhibitory proteins expressed within myelin debris and reactive astrocytes that actively signal to the injured nerve cells to limit their growth. The ability of the trans-acting activator of transcription (TAT) protein transduction domain (PTD) to transport macromolecules across biological membranes raises the possibility of developing it as a therapeutic delivery tool for nerve regeneration. Most studies have produced TAT PTD fusion protein in bacteria, which can result in problems such as protein solubility, the formation of inclusion bodies and the lack of eukaryotic posttranslational modifications. While some groups have investigated the production of TAT PTD fusion protein in mammalian cells, these strategies are focused on generating TAT PTD fusions that are targeted to the secretory pathway, where furin protease as well as other proteases can cleave the TAT PTD. As an alternative to mutating the furin cleavage site in the TAT PTD, we describe a novel method to generate cytosolic TAT PTD fusion proteins and purify them from cell lysates. Here, we use this method to generate TAT-C4RIP, a cell permeable competitive antagonist of binding between the small GTPase RhoA and the cytosolic phosphoprotein Collapsin response mediator protein 4 (CRMP4). We demonstrate that TAT-C4RIP transduces cells in vitro and in vivo and retains its biological activity to attenuate myelin inhibition in an in vitro neurite outgrowth assay.
Asunto(s)
Productos del Gen tat/genética , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Fragmentos de Péptidos/genética , Proteína de Unión al GTP rhoA/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Productos del Gen tat/metabolismo , Células HeLa , Humanos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuritas/efectos de los fármacos , Neuritas/fisiología , Fragmentos de Péptidos/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinéticaRESUMEN
Mutations in leucine-rich glioma inactivated (LGI1) are a genetic cause of autosomal dominant temporal lobe epilepsy with auditory features. LGI1 is a secreted protein that shares homology with members of the SLIT family, ligands that direct axonal repulsion and growth cone collapse, and we therefore considered the possibility that LGI1 may regulate neuronal process extension or growth cone collapse. Here we report that LGI1 does not affect growth directly but instead enhances neuronal growth on myelin-based inhibitory substrates and antagonizes myelin-induced growth cone collapse. We show that LGI1 mediates this effect by functioning as a specific Nogo receptor 1 (NgR1) ligand that antagonizes the action of myelin-based inhibitory cues. Finally, we demonstrate that NgR1 and ADAM22 physically associate to form a receptor complex in which NgR1 facilitates LGI1 binding to ADAM22.
Asunto(s)
Proteínas de la Mielina/metabolismo , Vaina de Mielina/fisiología , Neuronas/fisiología , Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas ADAM/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Células COS , Aumento de la Célula , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Proteínas Ligadas a GPI , Ganglios Espinales/crecimiento & desarrollo , Ganglios Espinales/fisiología , Conos de Crecimiento/fisiología , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nogo , Receptor Nogo 1 , Ratas , Ratas Sprague-DawleyRESUMEN
The sulfamide moiety has been utilized to design novel HDAC inhibitors. The potency and selectivity of these inhibitors were influenced both by the nature of the scaffold, and the capping group. Linear long-chain-based analogs were primarily HDAC6-selective, while analogs based on the lysine scaffold resulted in potent HDAC1 and HDAC6 inhibitors.