Mechanism of regulation of programmed cell death by intracellular calcium-binding protein ALG-2

细胞内钙结合蛋白ALG-2调控程序性细胞死亡的机制

• 批准号: 14360051
• 负责人: MAKI Masatoshi
• 金额: $ 9.41万
• 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: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360051/
• 关键词: Alix; ALG-2; multivesicular endosome; protein sorting; endocytosis; calcium; membrane traffic; interaction; CHMP; EF-ハンド; 多胞体; peflin; アネキシン; 二量体

In order to explore the physiological functions of ALG-2, we focused on searching for its interacting proteins. Previously, we searched for novel interacting proteins of Alix, an ALG-2 binding partner, by the yeast two-hybrid method, and obtained a candidate protein named CHMP4. We demonstrated that Alix interacts with this protein by co-immunoprecipitation assay and by subcellular co-localization by immunofluorescence microscopic analysis, suggesting involvement of Alix in endosomal sorting of endocytosed proteins. This year we demonstrated that CHMP6,an binding partner of CHMP4,is myristoylated at its N-terminus and directly interacts with EAP20. Overexpressed CHMP4b-or CHMP6-GFP protein in HeLa cells exhibited a punctate distribution in the perinuclear region, and inhibited disappearance of up-taken EGF, which is probably mediated by dominant-negative effect on suppression of transport from endosomes to lysosomes. From the network of ALG-2 interacting proteins, it seems that ALG-2 i … More s involved in membrane traffic. Indeed, Alix is suggested to play roles in protein sorting by biogenesis of multivesicular bodies (MVB, endosomes containing microvesicles in its lumen). We also identified a novel Alix interacting protein, which has a structure similar to RabGAP. This protein is localized in the vicinity of plasma membrane and cell edges, and colocalized with Alix in the filopodia and lamellipodia. On the other hand, we investigated the function of Alix in the cellular slime mold Dictyostelium discoideum, which is a model eukaryote in the boundary of unicellular and multicellular organism. Alix-gene-disrupted cells did not develop into fruiting bodies under Ca^<2+>-restricted conditions. Fruiting bodies are composed of stalk and spore, where event of programmed cell death occurs to develop into stalk. Thus, abnormalities observed in the Alix-gene-disrupted cells may be caused by impairment in cell aggregation in the early stage of development, implying that Alix is involved in cell adhesion and cell movement. Less

为了探索ALG-2的物理功能，我们专注于寻找其相互作用的蛋白质。以前，我们通过酵母两杂交方法搜索了ALG-2结合伴侣Alix的新型相互作用蛋白，并获得了一种名为CHMP4的候选蛋白。我们证明，ALIX通过共免疫沉淀测定法和通过免疫荧光显微镜分析通过亚蛋白质共定位与该蛋白质相互作用，这表明今年我们证明了CHMP6的CHMP6是CHMP4的结合伴侣，在其N-末端与N-termoylylyus肉毒肉化相互作用，并与N-terminus直接相互作用，并与EAP20相互作用。 Overexpressed CHMP4b-or CHMP6-GFP protein in HeLa cells exposed a punctate distribution in the perinuclear region, and inhibited disappearance of up-taken EGF, which is probably mediated by dominant-negative effect on suppression of transport From the network of ALG-2 interacting proteins, it seems that ALG-2 i … More s involved in membrane traffic.实际上，建议ALIX通过多种体的生物发生（MVB，内体内包含微泡中的微泡）在蛋白质分类中起作用。我们还确定了一种新型的ALIX相互作用蛋白，该蛋白具有与RabGap相似的结构。该蛋白质位于质膜和细胞边缘附近，并与丝状和薄片中的Alix共定位。另一方面，我们研究了ALIX在细胞模具dictyostelium discoideum中的功能，这是单细胞和多细胞生物边界中的模型真核生物。在CA^<2+> - 限制条件下，Alix-Gene中断的细胞没有发展成为结果。果实的体由茎和散射组成，在此发生了程序性细胞死亡的事件，以形成茎。这是，在ALIX-GEN-GENEDESCRUPT的细胞中观察到的异常可能是由于发育早期细胞聚集的损害引起的，这意味着ALIX参与细胞粘附和细胞运动。较少的

项目成果

H.Satoh 他4名: "ALG-2 Interacts with the amino-terminal domain of annexin XI in a Ca^<2+-> dependent manner"Biochemical and Biophysical Research Communications. 291. 1166-1172 (2002)

H. Satoh 和其他 4 人：“ALG-2 以 Ca ^ 2+- 依赖性方式与膜联蛋白 XI 的氨基末端结构域相互作用”，生物化学和生物物理研究通讯。 291. 1166-1172 (2002)

Katoh 他7名: "The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting"Journal of Biological Chemistry. 278. 39104-39113 (2003)

Katoh 和其他 7 人：“ALG-2 相互作用蛋白 Alix 与 CHMP4b 结合，CHMP4b 是参与多囊泡体分选的酵母 Snf7 的人类同源物”《生物化学杂志》278. 39104-39113 (2003)。

柴田秀樹, 牧正敏: "細胞内物質輸送の選別オルガネラMVB:エンドソームのダイナミックな膜構造の変化とその制御因子"化学と生物. 41. 352-354 (2003)

Hideki Shibata，Masatoshi Maki：“MVB，细胞内物质运输的分选细胞器：内体的动态膜结构变化及其调节因素”化学与生物学，41. 352-354 (2003)。

Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting.

• DOI: 10.1042/bj20041227
• 发表时间: 2005-04
• 期刊: The Biochemical journal
• 作者: Chiharu Yorikawa;H. Shibata;S. Waguri;Kazumi Hatta;Mio Horii;Keiichi Katoh;Toshihide Kobayashi;Y. Uchiyama;M. Maki

H.Satoh 他3名: "The penta-EF-hand domain of ALG-2 interacts with amino-terminal domain of both annexin VII and annexin XI in a Ca^<2+-> dependent manner"Biochimica et Biophysica Acta. 1600. 61-67 (2002)

H. Satoh 和其他 3 人：“ALG-2 的五-EF-手结构域以 Ca^2+- 依赖性方式与膜联蛋白 VII 和膜联蛋白 XI 的氨基末端结构域相互作用”Biochimica et Biophysicala Acta 1600。 .61-67 (2002)