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

Wilson病是一种遗传性疾病，其特征是由于肝细胞的胆汁铜排泄缺陷而导致铜在体内蓄积。通过在培养细胞中表达绿色荧光蛋白标记的肝豆状核变性基因产物ATP 7 B（GFP-ATP 7 B），研究其在细胞内的定位。通过荧光显微镜观察细胞内细胞器。GFP-ATP 7 B与晚期内体标记共定位，但不与高尔基体和溶酶体标记共定位。在Wilson病患者中已经记录了各种突变。临床表现差异很大的患者之间，但是，很少有信息的基因型-表型相关性。我们通过表达GFP标记的突变体，研究了ATP 7 B突变体His 1069 G1和突变体Asp 1270 Ser的分布。Asp 1270 Ser突变体定位于晚期内体，而His 1069 Gln突变体不定位于晚期内体，在胞浆中被蛋白酶体降解。此外，His 1069 Gl在微管组织中心形成由降解产物和中间产物组成的攻击体。电镜下可见类似于马洛里小体的侵袭小体。ATP 7 B定位于晚期内涵体在铜耗尽和铜负载条件。ATP 7 B似乎将铜从细胞质转运到晚期内体中，并且铜可以通过溶酶体排泄到胆汁中。由突变的ATP 7 B引起的铜掺入晚期内体的干扰必须是Wilson病的主要缺陷。ATP 7 B突变体的不同蛋白质特性可以部分解释Wilson病患者的临床谱的多样性。

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.