Studies of molecular mechanisms for proper maturation of secretory proteins

分泌蛋白正常成熟的分子机制研究

基本信息

批准号：
17370039
负责人：
WADA Ikuo
金额：
$ 9.41万
依托单位：
Fukushima Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Outline of this study to elucidate the molecular mechanisms for protein maturation in the secretory pathway is as follows. 1) Previously, we reported EDEM1 as a factor promoting selective degradation of aberrantly folded glycoproteins. We have now revealed that EDEM3, a soluble protein having similarly in sequence to EDEM1 also stimulates disposal of misfolded proteins in the endoplasmic reticulum. 2) We have shown that calreticulin plays regulatory roles in cell surface expression of CFTR, whose mutation is known to cause cystic fibrosis. 3) We have demonstrated that the endoplasmic reticulum also plays important roles in the process of phagocytosis, presumably by supplying enough amount of membranes during invagination. This issue is highly controversial and, until publication of this report, this hypothesis has been criticized by lack of solid evidences. We have provided evidences showign that ER SNARE protein positively regulates this process. 4) We have shown that EDEM1 prevents aggregation of terminally misfolded proteins while stimulating their disposal. 5) Our mobility analysis of meltrin beta using fluorescence correlation spectroscopy (FCS) indicated that shedding of lumenal domain of neuregulin beta 1 occurs in the Golgi region but not on the cell surface. 6) Sphingomyelinase, an lysosomal enzyme, is known to function in the extracellular space after secretion. We have shown that its efficient secretion is dependent on the presence of a disulfide bond at the carboxyl terminus. 7) By using siRNA library targeting ER proteins, we found more than 10 novel factors required for proper maturation of tyrosinase. 8) We have published our protocols to measure diffusion using FRAP and FCS in living cells.

这项研究的概述阐明了分泌途径中蛋白质成熟的分子机制如下。 1）以前，我们报道了EDEM1是促进异常折叠糖蛋白选择性降解的因素。我们现在已经透露，EDEM3是一种与EDEM1相似的可溶性蛋白，也刺激了内质网中错误折叠蛋白的处理。 2）我们已经表明，钙网蛋白在CFTR的细胞表面表达中起调节作用，该突变已知会引起囊性纤维化。 3）我们已经证明，内质网中也在吞噬作用过程中起着重要作用，大概是通过在内陷过程中提供足够数量的膜。这个问题引起了极大的争议，直到该报告发布之前，由于缺乏稳固的证据，该假设一直受到批评。我们提供的证据表明，ER核心蛋白会积极调节这一过程。 4）我们已经表明，EDEM1可以防止在刺激其处置的同时终止错误折叠的蛋白质的聚集。 5）我们使用荧光相关光谱（FC）对MELTRINβ的迁移率分析表明，神经蛋白β1的流体结构域的脱落发生在高尔基区域，但不在细胞表面。 6）已知糖体酶（一种溶酶体酶）在分泌后在细胞外空间中起作用。我们已经证明其有效分泌取决于羧基末端的二硫键。 7）通过使用针对ER蛋白的siRNA库，我们发现了酪氨酸酶正确成熟所需的10多个新因素。 8）我们已经发布了我们的方案，以在活细胞中使用FRAP和FC测量扩散。