Yanagimoto Chikatoshi, Harada Masaru, Kumemura Hiroto, Koga Hironori, Kawaguchi Takumi, Terada Kunihiko, Hanada Shinichiro, Taniguchi Eitaro, Koizumi Yukio, Koyota Souichi, Ninomiya Haruaki, Ueno Takato, Sugiyama Toshihiro, Sata Michio
Division of Gastroenterology, Department of Medicine and Research Center for Innovative Cancer Therapy of the 21st Century COE Program for Medical Science, Kurume University School of Medicine, Kurume, Japan.
Exp Cell Res. 2009 Jan 15;315(2):119-26. doi: 10.1016/j.yexcr.2008.10.022. Epub 2008 Oct 31.
Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.
威尔逊病是一种遗传性疾病，其特征是由于胆汁铜排泄缺陷导致体内铜蓄积。威尔逊病基因产物（ATP7B）在铜掺入铜蓝蛋白（Cp）和胆汁铜排泄过程中发挥作用。然而，肝细胞中的铜代谢仍不清楚。尼曼-匹克病C型（NPC）是一种脂质贮积病，最常发生突变的基因是NPC1，其基因产物NPC1是一种晚期内体蛋白，可调节细胞内囊泡运输。在本研究中，我们诱导了NPC表型，并检测了ATP7B的定位以及铜蓝蛋白（Cp）的铜结合成熟形式——全铜蓝蛋白（holo-Cp）的分泌情况。使用可诱导NPC表型的U18666A以及通过RNA干扰敲低NPC1来调节囊泡运输。ATP7B与晚期内体标志物共定位，但不与反式高尔基体网络标志物共定位。U18666A和NPC1敲低可减少全铜蓝蛋白向培养基中的分泌，但不影响其他分泌蛋白的分泌。用U18666A处理后，铜在细胞中蓄积。这些发现表明，ATP7B定位于晚期内体，晚期内体中的铜通过NPC1依赖途径转运至分泌区室，并掺入脱辅基铜蓝蛋白以形成全铜蓝蛋白。
