title: Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells.
abstract: 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.
authors.affiliation: Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Hewett
authors.forename: Bakhos
authors.initials: B
authors.lastname: Tannous
authors.forename: Brian P
authors.initials: BP
authors.lastname: Niland
authors.forename: Flavia C
authors.initials: FC
authors.lastname: Nery
authors.forename: Juan
authors.initials: J
authors.lastname: Zeng
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Li
authors.forename: Xandra O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D000818:Animals; D002452:Cell Count; D004421:Dystonia / Q000378:metabolism* / Q000473:pathology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012995:Solubility; D013997:Time Factors
chemical_list: C491548:Dyt1 protein, mouse; D018832:Molecular Chaperones; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; C108175:TOR1A protein, human; D008156:Luciferases; D019704:Protein Disulfide-Isomerases