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

为了探索ALG-2的生理功能，我们重点寻找与其相互作用的蛋白。此前，我们通过酵母双杂交的方法寻找ALG-2结合伙伴Alix的新的相互作用蛋白，并获得了一个候选蛋白CHMP4。我们通过免疫共沉淀实验和免疫荧光显微镜亚细胞共定位实验证明了Alix与该蛋白相互作用，提示Alix参与了内吞蛋白的内体分选。今年，我们证明了CHMP4的结合伙伴CHMP6在其N端是肉豆蔻酰化的，并直接与EAP20相互作用。在HeLa细胞中过表达的CHMP4b-或CHMP6-GFP蛋白在核周呈点状分布，并抑制摄取的EGF的消失，这可能是通过抑制内体向溶酶体转运的显性-负性效应所介导的。从alg-2相互作用蛋白的网络来看，alg-2 I…S更多地涉足膜上交通。事实上，Alix被认为通过多泡小体(MVB，其管腔中含有微泡的内体)的生物发生在蛋白质分选中发挥作用。我们还鉴定了一种新的Alix相互作用蛋白，其结构与RabGAP相似。该蛋白定位于细胞膜和细胞边缘附近，并与Alix共同定位于丝状伪足和片状伪足。另一方面，我们研究了Alix在单细胞和多细胞生物交界处的真核生物--盘状网柄菌中的功能。在钙限制条件下，Alix基因破坏的细胞不能发育成子实体。子实体由柄和孢子组成，发生细胞程序性死亡事件后发育为柄。因此，在Alix基因突变的细胞中观察到的异常可能是由于发育早期的细胞聚集功能受损所致，这意味着Alix参与了细胞黏附和细胞运动。较少

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