RESUMO
A specific mutation (DeltaE) in torsinA underlies most cases of the dominantly inherited movement disorder, early-onset torsion dystonia (DYT1). TorsinA, a member of the AAA+ ATPase superfamily, is located within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). We investigated an association between torsinA and nesprin-3, which spans the outer nuclear membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin-3alpha co-immunoprecipitated with torsinA and this involved the C-terminal region of torsinA and the KASH domain of nesprin-3alpha. This association with human nesprin-3 appeared to be stronger for torsinADeltaE than for torsinA. TorsinA also associated with the KASH domains of nesprin-1 and -2 (SYNE1 and 2), which link to actin. In the absence of torsinA, in knockout mouse embryonic fibroblasts (MEFs), nesprin-3alpha was localized predominantly in the ER. Enrichment of yellow fluorescent protein (YFP)-nesprin-3 in the ER was also seen in the fibroblasts of DYT1 patients, with formation of YFP-positive globular structures enriched in torsinA, vimentin and actin. TorsinA-null MEFs had normal NE structure, but nuclear polarization and cell migration were delayed in a wound-healing assay, as compared with wild-type MEFs. These studies support a role for torsinA in dynamic interactions between the KASH domains of nesprins and their protein partners in the lumen of the NE, with torsinA influencing the localization of nesprins and associated cytoskeletal elements and affecting their role in nuclear and cell movement.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular/genética , Proteínas do Citoesqueleto , Distonia Muscular Deformante/genética , Distonia Muscular Deformante/metabolismo , Embrião de Mamíferos/metabolismo , Retículo Endoplasmático/genética , Fibroblastos/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Chaperonas Moleculares/genética , Mutação , Proteínas do Tecido Nervoso/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Plectina/genética , Plectina/metabolismo , Estrutura Terciária de Proteína/fisiologia , Vimentina/genética , Vimentina/metabolismo , Cicatrização/genéticaRESUMO
TorsinA is an AAA(+) protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope responsible for early onset torsion dystonia (DYT1). Most cases of this dominantly inherited movement disorder are caused by deletion of a glutamic acid in the carboxyl terminal region of torsinA. We used a sensitive reporter, Gaussia luciferase (Gluc) to evaluate the role of torsinA in processing proteins through the ER. In primary fibroblasts from controls and DYT1 patients most Gluc activity (95%) was released into the media and processed through the secretory pathway, as confirmed by inhibition with brefeldinA and nocodazole. Fusion of Gluc to a fluorescent protein revealed coalignment and fractionation with ER proteins and association of Gluc with torsinA. Notably, fibroblasts from DYT1 patients were found to secrete markedly less Gluc activity as compared with control fibroblasts. This decrease in processing of Gluc in DYT1 cells appear to arise, at least in part, from a loss of torsinA activity, because mouse embryonic fibroblasts lacking torsinA also had reduced secretion as compared with control cells. These studies demonstrate the exquisite sensitivity of this reporter system for quantitation of processing through the secretory pathway and support a role for torsinA as an ER chaperone protein.
Assuntos
Distonia/metabolismo , Distonia/patologia , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Contagem de Células , Retículo Endoplasmático/metabolismo , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Imunoprecipitação , Luciferases/metabolismo , Camundongos , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Solubilidade , Fatores de TempoRESUMO
Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms-recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA-the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.