Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells
Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells
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DOI：
10.1073/pnas.0701185104
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发表时间：
2007-04-24
影响因子：
11.1
通讯作者：
Breakefield, Xandra O.
中科院分区：
文献类型：
--
作者：
Hewett, Jeffrey W.;Tannous, Bakhos;Breakefield, Xandra O.
TorsinA是一种AAA（+）蛋白，主要位于内质网（ER）和核膜的内腔中，负责早发性扭转肌张力障碍（DYT 1）。这种显性遗传性运动障碍的大多数病例是由torsinA羧基末端区域的谷氨酸缺失引起的。我们使用了一个敏感的报告，高斯荧光素酶（Gluc），以评估torsinA在处理蛋白质通过ER的作用。在对照组和DYT1患者的原代成纤维细胞中，大多数Gluc活性（95%）被释放到培养基中并通过分泌途径进行处理，这一点通过布雷菲德菌素A和诺克拉唑的抑制作用得到证实。Gluc与荧光蛋白的融合揭示了与ER蛋白的共配向和分馏以及Gluc与torsinA的关联。值得注意的是，发现来自DYT1患者的成纤维细胞与对照成纤维细胞相比分泌显著更少的Gluc活性。DYT1细胞中Gluc加工的这种减少似乎至少部分地由torsinA活性的丧失引起，因为与对照细胞相比，缺乏torsinA的小鼠胚胎成纤维细胞也具有减少的分泌。这些研究表明，该报告系统通过分泌途径的加工定量的灵敏度，并支持torsinA作为ER伴侣蛋白的作用。
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 nococlazole. 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.