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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是一种五EF手型钙结合蛋白。为了探索ALG-2的生理功能，我们寻找新的ALG-2相互作用蛋白。我们确定Sec 31 A，COPII的一个组成部分，在ER-高尔基体运输工作，与ALG-2在钙依赖性的方式，它与ALG-2共定位在ER出口网站的免疫荧光显微镜分析。令人惊讶的是，ALG-2的选择性剪接的deltaGF-122亚型不结合阿利克斯，但保留了结合Sec 31 A的能力。我们还发现，磷脂乱序酶3（PLSCR 3）具有两个ALG-2结合位点：一个（ABS 1）类似于Alix结合位点，另一个（ABS 2）类似于Sec 31 A结合位点，因为deltaGF-122亚型保留了结合能力。PLSCR 3中的第二ALG-2结合位点不具有Tyr作为必需氨基酸残基。ALG-2的突变分析显示ABS I和ABS 2可能与ALG-2的不同表面相互作用。我们将ALG-2结合蛋白分为Alix型和非Alix型两类。我们阐明了无钙形式和阿利克斯肽复合物形式的ALG-2的三维结构。我们分析了HD-PTP和Brox作为含有Brol结构域的阿利克斯的旁系同源物。HD-PTP和Brox均结合CHMP 4蛋白。而HD-PTP具有与ALG-2和内啡肽相关的Pro富集区，Brox缺乏这样的结构域，但在其C-末端具有法尼基化位点。发现Brox作为外泌体分泌。在ESCRT研究过程中，我们发现了一种新的ESCRT-III相关蛋白，并将其命名为CHMP 7。过表达绿色荧光蛋白（GFP）融合的CHMP 7抑制小鼠白血病病毒（MuLV）gag蛋白的病毒样颗粒（VLP）的释放，表明其在货物蛋白的内体分选中的作用