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.

本研究旨在阐明分泌途径中蛋白质成熟的分子机制，其概要如下。 1）之前，我们报道了 EDEM1 作为促进异常折叠糖蛋白选择性降解的因子。我们现在发现，EDEM3（一种与 EDEM1 具有相似序列的可溶性蛋白）也能刺激内质网中错误折叠蛋白的处理。 2）我们已经证明，钙网蛋白在 CFTR 的细胞表面表达中发挥调节作用，CFTR 的突变已知会导致囊性纤维化。 3）我们已经证明，内质网在吞噬过程中也发挥着重要作用，大概是通过在内陷过程中提供足够量的膜来实现的。这个问题极具争议性，直到本报告发表之前，这一假设一直因缺乏确凿证据而受到批评。我们提供的证据表明 ER SNARE 蛋白正向调节这一过程。 4) 我们已经证明 EDEM1 可以防止最终错误折叠蛋白质的聚集，同时刺激它们的处理。 5）我们使用荧光相关光谱（FCS）对meltrin beta的迁移率分析表明，神经调节蛋白β1的腔域脱落发生在高尔基体区域，而不是在细胞表面。 6) 鞘磷脂酶是一种溶酶体酶，已知在分泌后在细胞外空间发挥作用。我们已经证明，其有效分泌取决于羧基末端二硫键的存在。 7) 通过使用针对 ER 蛋白的 siRNA 文库，我们发现了酪氨酸酶正常成熟所需的 10 多个新因子。 8) 我们已经发布了使用 FRAP 和 FCS 在活细胞中测量扩散的方案。