Hackam A S, Singaraja R, Zhang T, Gan L, Hayden M R
Centre for Molecular Medicine and Therapeutics, 980 West 28th Avenue, Vancouver, BC, Canada V5Z 4H4 and Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
Hum Mol Genet. 1999 Jan;8(1):25-33. doi: 10.1093/hmg/8.1.25.
A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have lead to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system that compared the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. We first assessed the mode of nuclear transport of N-terminal fragments of huntingtin, and show that the predicted endogenous NLS is not functional, providing data in support of passive nuclear transport. This result suggests that proteolysis is a necessary step for nuclear entry of huntingtin. Additionally, insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. The findings of nuclear and cytoplasmic aggregates in affected brains, together with these in vitro data, support the nucleus and cytosol as subcellular sites for pathogenesis in HD.
CAG 重复扩增疾病的一个共同特征是由突变的多聚谷氨酰胺扩增蛋白形成细胞内聚集体。尽管在受影响的患者中存在聚集体，但聚集体与疾病发病机制之间的确切关系仍未解决。对突变型亨廷顿蛋白的体内和体外研究结果导致了这样一种假设，即聚集体的核定位对于亨廷顿病（HD）的病理学至关重要。我们使用 293T 细胞培养模型系统测试了这一假设，该系统比较了细胞质和细胞核中亨廷顿蛋白聚集体的频率和毒性。我们首先评估了亨廷顿蛋白 N 端片段的核运输模式，并表明预测的内源性核定位信号无功能，为被动核运输提供了支持数据。这一结果表明蛋白水解是亨廷顿蛋白进入细胞核的必要步骤。此外，将核输入或输出序列插入含有 548 或 151 个氨基酸的亨廷顿蛋白片段中，以逆转这些蛋白质的正常定位。改变片段的亚细胞定位并未影响其总聚集体频率。与细胞质聚集体相比，核聚集体的存在在毒性方面也没有显著差异。在受影响的大脑中发现的细胞核和细胞质聚集体，以及这些体外数据，支持细胞核和细胞质作为 HD 发病机制的亚细胞位点。
