The mechanism of the Action of the calcium-binding protein ALG-2on the regulation of cell membrane receptor sorting

钙结合蛋白ALG-2调控细胞膜受体分选的作用机制

基本信息

批准号：
17380063
负责人：
MAKI Masatoshi
金额：
$ 10.11万
依托单位：
Nagoya 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 2007
项目状态：
已结题

项目摘要

ALG-2 is a penta-EF-hand calcium-binding protein. To explore physiological functions of ALG-2, we searched for new interacting proteins of ALG-2. We identified Sec31A, a component of COPII working in ER-Golgi transport, associates with ALG-2 in a calcium-dependent manner and it colocalized with ALG-2 in the ER exit site by immunofluorescence microscopic analysis. Surprisingly, the alternatively spliced deltaGF-122 isoform of ALG-2 does not bind Alix but retains the capacity of binding to Sec31A. We also found that phospholipid scramblase 3 (PLSCR3) possesses two ALG-2-binding sites : one (ABS1) is similar to Alix-binding site and the other (ABS2) is similar to the Sec31A-binding in that the deltaGF-122 isoform retains the binding capacity. The second ALG-2-binding site in PLSCR3 does not possess Tyr as an essential amino acid residue. Mutational analyses of ALG-2 reveal that ABS I and ABS2 may interact with different surfaces of ALG-2. We classified ALG-2-bidning proteins as two classes : Alix-type and non-Alix type. We elucidated the 3D-structures of ALG-2 in calcium-free form and Alix peptide complex form. We analyzed HD-PTP and Brox as paralogues of Alix that contain Brol domain. Both HD-PTP and Brox bind CHMP4 proteins. While HD-PTP possesses Pro-rich region that associate with ALG-2 and endophilin, Brox lacks such a domain but possesses a famesylation site at its C-terminus. Brox was found to be secreted as an exosome. During the course of ESCRT studies, we identified a novel ESCRT-III related protein and named it CHMP7. Overexpression of green fluorescent protein(GFP) fused CHMP7 suppressed release of virus-like particle (VLP) of murine leukemia virus (MuLV) gag protein, suggesting roles in endosomal sorting of cargo proteins

ALG-2是一种penta-ef-hand钙结合蛋白。为了探索ALG-2的生理功能，我们搜索了ALG-2的新相互作用蛋白。我们确定了Sec31a是ER-Golgi运输中工作的COPII的一个组成部分，以钙依赖性方式与ALG-2相连，并通过免疫荧光显微镜分析在ER出口位点与ALG-2共定位。令人惊讶的是，ALG-2的剪接剪接的Deltagf-122同工型不结合ALIX，而是保留与Sec31a结合的能力。我们还发现，磷脂cramblase 3（PLSCR3）具有两个ALG-2结合位点：一个（ABS1）类似于ALIX结合位点，另一个（ABS2）与SEC31A结合相似，因为Delttagf-122同一个同4oform保留了结合能力。 PLSCR3中的第二个ALG-2结合位点不具有TYR作为必需的氨基酸残基。 ALG-2的突变分析表明，ABS I和ABS2可能与ALG-2的不同表面相互作用。我们将ALG-2标准蛋白分类为两类：ALIX类型和非阿利克体类型。我们以无钙形式和ALIX肽复合物形式阐明了ALG-2的3D结构。我们将HD-PTP和BROX分析为包含BROL域的ALIX的旁系同源物。 HD-PTP和BROX都结合CHMP4蛋白。尽管HD-PTP拥有与ALG-2和内菲尔蛋白相关的亲富区域，但Brox缺乏这样的域，但在其C末端具有著名的位置。发现Brox被分泌为外泌体。在ESCRT研究过程中，我们确定了一种新型的ESCRT-III相关蛋白质，并将其命名为CHMP7。绿色荧光蛋白（GFP）融合CHMP7的过表达抑制了鼠白血病病毒（MULV）GAG蛋白的病毒样颗粒（VLP）的释放，这表明在货物体分类中作用