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Analyses of protein-activating enzyme (Apg7p) complex essential for autophagy.

自噬必需的蛋白质激活酶 (Apg7p) 复合物的分析。

基本信息

批准号：
12680639
负责人：
UENO Takashi
金额：
$ 2.18万
依托单位：
Juntendo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Autophagy, a major degradative pathway of cell constituents via lysosomal/vacuolar system, is a dynamic process accompanying continuous formation and changes of membrane structures. In order to better understand the mechanism of mammalian autophagy, we have pursued our studies on unique ubiquitin-like protein conjugation system indispensable for autophagy. There are two ubiquitin-like modifiers ; Apg12p and Apg8p. The two modifiers are activated by Apg7p, the common E1 enzyme, in ATP-dependent manner. Then, Apg12p is transferred to Apg10p, an E2 enzyme, and finally conjugated with Apg5p. Apg8p is transferred to Apg3p, another E2 enzyme, and then conjugated with phosphatidylethanolamine. The two conjugation reactions, which operate independently but concertedly, are necessary for autophagosome formation. During the course of this study, we have focused our attention to the interaction between Apg7p and other proteins. Our new findings are summarized as follows. 1) Apg7p exists in homodimer as revealed by yeast two-hybrid analysis and chemical cross-linking experiments. The deletion of its carboxyl 40 amino acids results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an acticve-site cysteine. 2) In mammalian cells, three Apg8p homologs (MAP-LC3, GABARAP, and GATE-16) exist. We showed that all the three homologs can be activated by Apg7p and subsequently transferred to Apg3p. It has been also found that lipidation form of MAP-LC3 is enriched in rat liver autophagosomal/autolysosomal membranes, indicating that at least MAP-LC3 is a definitive Apg8p homolog that can be targeted onto autophagosomes. 3) Overexpression of both Apg7p and Apg3p, which enhances lipidation of Apg8ps, promotes the formation of Apg12p-Apg5p conjugate. Thus, there are intimate crosstalks between the two protein conjugation pathways.

自噬是伴随膜结构不断形成和改变的动态过程，是细胞成分通过溶酶体/空泡系统降解的主要途径。为了更好地了解哺乳动物自噬的机制，我们对自噬不可或缺的独特泛素样蛋白偶联系统进行了研究。有两种类泛素修饰剂；Apg12p和Apg8p。这两种修饰剂由常见的E1酶Apg7p以atp依赖的方式激活。然后，将Apg12p转移到E2酶Apg10p上，最终与Apg5p偶联。Apg8p被转移到另一种E2酶Apg3p上，然后与磷脂酰乙醇胺结合。这两种偶联反应是自噬体形成所必需的，它们独立而协调地起作用。在研究过程中，我们将重点放在了Apg7p与其他蛋白的相互作用上。我们的新发现总结如下。1)通过酵母双杂交分析和化学交联实验发现，Apg7p存在于同二聚体中。其羧基40个氨基酸的缺失不仅导致Apg7p二聚化缺陷，而且导致Apg7p与Apg12p、Apg8p和Apg12p- apg5p结合两种底物的相互作用缺陷，而突变的Apg7p同时含有ATP结合域和活性位点半胱氨酸。2)在哺乳动物细胞中，存在3个Apg8p同源物（MAP-LC3、GABARAP和GATE-16）。我们发现这三个同源物都可以被Apg7p激活并随后转移到Apg3p上。研究还发现，脂化形式的MAP-LC3在大鼠肝脏自噬体/自溶酶体膜中富集，这表明至少MAP-LC3是Apg8p的明确同源物，可以靶向自噬体。3)过表达Apg7p和Apg3p，增强Apg8ps的脂化，促进Apg12p-Apg5p偶联物的形成。因此，两种蛋白质偶联途径之间存在密切的串扰。