Kono Satoshi, Suzuki Hitoshi, Oda Toshiaki, Miyajima Hiroaki, Takahashi Yoshitomo, Shirakawa Kentaro, Ishikawa Kuniko, Kitagawa Masatoshi
First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan.
Neuromolecular Med. 2006;8(3):361-74. doi: 10.1385/NMM:8:3:361.
Aceruloplasminemia is a neurodegenerative disease characterized by parenchymal iron accumulation owing to mutations in the ceruloplasmin gene. Ceruloplasmin is expressed in the central nervous system in which most of the ceruloplasmin is located on the surface of astrocytes in a glycosylphosphatidylinositol (GPI)-anchored form. We herein describe the biochemical features of wild-type and mutant GPI-anchored ceruloplasmin. An overexpression of wild-type GPI-anchored ceruloplasmin in Chinese hamster ovary cells led to the formation of aggresome-like inclusions, especially in the presence of proteasome inhibitors. As expected from the properties of aggresomes, the inclusions were colocalized with gamma-tubulin and a disruption of microtubules using nocodazole blocked the formation of such inclusions. Aceruloplasminemia-linked mutant proteins failed to form such inclusions even after treatment with proteasomal inhibitors. An immunofluorescent analysis indicated that the mutant proteins were thus retained in the endoplasmic reticulum (ER), whereas the transfected cells showed a decreased viability. The expression of glucose-regulated protein 78 that is one of the ER stress sensor proteins, and the activity of glucose-regulated protein 78 promoter was upregulated in the cells transfected with the mutants. These findings indicated that when the overexpressed cytoplasmic wild-type ceruloplasmin was not subjected to degradation by the proteasome-ubiquitin system, then the wild-type protein was transported along the microtubules, thus forming inclusions at the microtubule organizing center, whereas the mutant ceruloplasmin failed to form any such inclusions, because the mutant protein might not have been translocated across the ER into the cytoplasm. Therefore, the mutant protein was considered to have accumulated in the ER thus leading to the ER stress, which resulted in cell death.
血浆铜蓝蛋白缺乏症是一种神经退行性疾病，其特征是由于血浆铜蓝蛋白基因突变导致实质铁蓄积。血浆铜蓝蛋白在中枢神经系统中表达，其中大部分血浆铜蓝蛋白以糖基磷脂酰肌醇（GPI）锚定形式位于星形胶质细胞表面。我们在此描述野生型和突变型GPI锚定血浆铜蓝蛋白的生化特征。在中国仓鼠卵巢细胞中过表达野生型GPI锚定血浆铜蓝蛋白导致形成聚集体样包涵体，尤其是在蛋白酶体抑制剂存在的情况下。正如从聚集体的特性所预期的那样，这些包涵体与γ-微管蛋白共定位，并且使用诺考达唑破坏微管会阻止此类包涵体的形成。即使在用蛋白酶体抑制剂处理后，与血浆铜蓝蛋白缺乏症相关的突变蛋白也未能形成此类包涵体。免疫荧光分析表明，突变蛋白因此保留在内质网（ER）中，而转染的细胞显示活力下降。在转染了突变体的细胞中，作为内质网应激传感器蛋白之一的葡萄糖调节蛋白78的表达以及葡萄糖调节蛋白78启动子的活性上调。这些发现表明，当过表达的细胞质野生型血浆铜蓝蛋白未被蛋白酶体-泛素系统降解时，野生型蛋白会沿微管运输，从而在微管组织中心形成包涵体，而突变型血浆铜蓝蛋白未能形成任何此类包涵体，因为突变蛋白可能未穿过内质网转运到细胞质中。因此，突变蛋白被认为在内质网中蓄积，从而导致内质网应激，进而导致细胞死亡。
