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Studies on structure and the biological functions of a large multi-molecular complex "the proteasome"

大型多分子复合物“蛋白酶体”的结构和生物学功能研究

基本信息

批准号：
03102006
负责人：
ICHIHARA Akira
金额：
$ 108.8万
依托单位：
Institute for Enzyme Research, The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

(1) Structural Analysis of Proteasomes at the Molecular Level : We demonstrated that proteasomes are found as two isoforms in cells. One is the ATP-independent 20S form and the other is an ATP-dependent 26S form. Various physical analyzes showed that molecular masses of these two forms are determined to be approximately 750 kDa and 2000 kDa, respectively. The primary structures of 7 alpha-type and 9 beta-type subunits of the 20S proteasome complex in human, rat and yeast were determined by their cDNA cloning. Based on the homology analysis, we proposed the novel term "the proteasome gene family" for these highly homologous gene groups. Furthermore, we have prepared the large crystals of the bovine 20S proteasome and carried out its preliminary X-ray analysis. By electron microscopy, we proposed the "caterpillar-shape" model for the 26S proteasome, consisting of approximately 50 non-identical subunits. The 26S proteasome was found to be the unusually large supra-molecular complex.(2) Molecular Functions of Proteasomes : The 26S proteasome was demonstrated to be an ATP-dependent protease catalyzing selective degradation of target proteins conjugated with multiple ubiquitins as the degradation signal. Moreover, we found for the first time that ornithine decarboxylase, a most short-lived enzyme in cells responsible for biosynthesis of polyamines, was degraded ATP-dependently by the 26S proteasome without ubiquitination.(3) Physiological Functions of Proteasomes : The 26S proteasome degraded various nuclear oncoproteins, such as Mos, Fos and Myc, closely related with cell cycle progression in an ATP-, ubiquitin-dependent fashion, suggesting possible involvement of the proteasome in cell cycle regulation. In addition, we found that gamma-interferon induced proteasomes with different subunit organizations for acquirement of the functional diversity and suggested that proteasoms play an essential role for the MHC class-I restricted antigen processing pathway.

(1)蛋白酶体在分子水平上的结构分析：我们证明了蛋白酶体在细胞中有两种同工异构体。一种是不依赖atp的20S型，另一种是不依赖atp的26S型。各种物理分析表明，这两种形式的分子质量分别约为750 kDa和2000 kDa。用cDNA克隆法测定了人、大鼠和酵母20S蛋白酶体复合物的7个α型亚基和9个β型亚基的初级结构。基于同源性分析，我们将这些高度同源的基因群命名为“蛋白酶体基因家族”。此外，我们还制备了牛20S蛋白酶体的大晶体，并进行了初步的x射线分析。通过电子显微镜，我们提出了26S蛋白酶体的“毛毛虫形状”模型，由大约50个不相同的亚基组成。发现26S蛋白酶体是异常大的超分子复合物。(2)蛋白酶体的分子功能：26S蛋白酶体是一种atp依赖的蛋白酶，催化以多种泛素偶联作为降解信号的靶蛋白的选择性降解。此外，我们首次发现鸟氨酸脱羧酶是细胞中负责多胺生物合成的最短命的酶，它被26S蛋白酶体降解atp依赖，而不发生泛素化。(3)蛋白酶体的生理功能：26S蛋白酶体降解多种核癌蛋白，如Mos、Fos和Myc，这些蛋白以ATP、泛素依赖的方式与细胞周期进程密切相关，提示该蛋白酶体可能参与细胞周期调节。此外，我们发现γ -干扰素诱导具有不同亚基组织的蛋白酶体获得功能多样性，并提示蛋白酶体在MHC i类限制性抗原加工途径中发挥重要作用。