Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation
Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation
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DOI：
10.1073/pnas.95.18.10854
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发表时间：
1998-09-01
影响因子：
11.1
通讯作者：
Gitlin, JD
中科院分区：
文献类型：
--
作者：
Payne, AS;Kelly, EJ;Gitlin, JD
Wilson病是一种常染色体隐性肝铜代谢疾病，由编码铜转运P型ATP酶的基因突变引起。为了阐明Wilson蛋白的功能，野生型和突变型Wilson cDNA在铜输出缺陷的Menkes铜转运蛋白缺陷斑驳成纤维细胞系中表达。野生型 cDNA 的表达证明了 Wilson 蛋白的跨高尔基体网络定位和铜依赖性运输，与之前对肝细胞内源表达蛋白的观察结果相同。此外，Wilson cDNA 的表达挽救了斑驳的表型，铜积累的减少和细胞活力的恢复证明了这一点。相反，H1069Q突变体Wilson cDNA的表达并不能挽救斑驳的表型，并且免疫荧光研究表明，这种突变体Wilson蛋白定位于内质网。与这些发现一致，脉冲追踪分析表明，与野生型蛋白相比，H1069Q突变体的半衰期缩短了5倍。将这些转染细胞系维持在 28 摄氏度，导致 H1069Q 蛋白定位于反式高尔基体网络中，这表明蛋白质折叠中的温度敏感性缺陷以及随后的降解构成了携带 H1069Q 突变的患者威尔逊病的分子基础。总而言之，这些研究描述了一个易于处理的表达系统，用于阐明哺乳动物细胞中铜转运 ATP 酶的功能和定位，并提供了令人信服的证据，表明 Wilson 蛋白可以在功能上替代 Menkes 蛋白，支持这些蛋白使用常见的生化机制来影响细胞铜稳态的概念。
Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase, To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum, Consistent with these findings, pulse-chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28 degrees C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.