Intracellular localization of Wilson disease protein(ATP7B), a copper-transporting ATPase.

威尔逊病是一种遗传性疾病，其特征是由于肝细胞胆道铜排泄缺陷导致体内铜积聚。通过在培养细胞中表达带有绿色荧光蛋白（GFP）标记的ATP7B (GFP-ATP7B)，研究Wilson病基因产物ATP7B在细胞内的定位。荧光显微镜观察胞内细胞器。GFP-ATP7B与内核体晚期标记物共定位，但与高尔基体和溶酶体标记物不共定位。威尔森氏症患者有多种突变记录。患者的临床表现差异很大，但基因型-表型相关性的资料很少。我们通过表达带有GFP标记的突变体，研究了常见的ATP7B突变体His 1069Gl和突变体Asp l270Ser的分布。Asp l270Ser突变体定位于核内体晚期，而His 1069Gln突变体不定位于核内体晚期，被细胞质中的蛋白酶体降解。此外，他的1069Gl在微管组织中心形成了由降解物和中间丝组成的聚合体。在电子显微镜下，这些聚合体与Mallory小体相似。在铜耗尽和铜负载条件下，ATP7B都定位于后期核内体。ATP7B似乎将铜从细胞质转运到后期内体，铜可能通过溶酶体排泄到胆汁中。ATP7B突变引起的铜进入晚期核内体的紊乱一定是威尔森氏病的主要缺陷。ATP7B突变体的不同蛋白质特性可能部分解释了Wilson病患者的临床谱变化。

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion from hepatocytes. The intracellular localization of Wilson disease gene product, ATP7B, was investigated by expressing ATP7B tagged with green fluorescent protein (GFP)(GFP-ATP7B) in cultured cells. Intracellular organelles were visualized by fluorescence microscopy. GFP-ATP7B colocalized with the late endosome markers, but not with markers for the Golgi apparatus and lysosomes. Various mutations have been documented in patients with Wilson disease. The clinical manifestations vary greatly among the patients, however, there is little information on the genotype-phenotype correlation. We investigated the distribution of a common ATP7B mutant His 1069Gl and a mutant Asp l270Ser by expressing the mutants tagged with GFP. While Asp l270Ser mutant localized in the late endosomes, His 1069Gln mutant did not locate in the late endosomes and was degraded by the proteasomes in the cytoplasm. Furthermore, His 1069Gl formed aggresomes composed of the degradates and intermediate filamentsat the microtubule-organizing center. These aggresomes were similar to Mallory bodies on electron microscopy. ATP7B localized in the late endosomes in both copper-depleting and copper-loaded conditions. ATP7B seems to translocate copper from the cytosol into the late endosomes, and copper may be excreted to bile via lysosomes. The disturbed incorporation of copper into the late endosomes caused by mutated ATP7B must be the main defect in Wilson disease. The different protein properties of ATP7B mutants may in part explain the variety of clinical spectrums in patients with Wilson disease.