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Mechanisms of molecular recognition and cell cycle control in ubiquitin-proteasome system

泛素-蛋白酶体系统的分子识别和细胞周期调控机制

基本信息

批准号：
05304054
负责人：
YOKOSAWA Hideyoshi
金额：
$ 2.69万
依托单位：
Hokkaido University, Faculty of Pharm.Sci.
依托单位国家：
日本
项目类别：
Grant-in-Aid for Co-operative Research (A)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05304054/
关键词：
Proteasome Ubiquitin Protease Molecular recognition Cell cycle Development Fertilization Oocyte maturation

项目摘要

1.Physiological Substrates of Ubiquitin-conjugating Enzymes : (1) Ubiquitin-activating enzyme (E1)-defficient mutant cell (ts85) was found to undergo the abnormal features in nucleolus during cell cycle. (2) Proto-oncogene product, Mos, was found to be degraded by the 26S proteasome via ubiquitination of the dephosphorylated Mos at Ser3 on fertilization of the Xenopus eggs. (3) We succeeded to establish the several monoclonal antibodies specific to the multi-ubiquitin chains of the polyubiquitinated proteins. By using this antibody, studies on intracellular localization and quantification of the multi-ubiquitinated proteins were performed.Structure and Function of Proteasomes : (1) From the gene-desruption studies, it was suggested that a proteasome subunit, Y13, is involved in the ATP-dependent proteolytic activity of the 26 S proteasome. (2) It was revealed that there are two isofoms in the 26 S proteasome complex : one is a complex made up by the 20 S proteasome and the regulatory s … More ubunit complex at a ratio of one, while the other is a complex a ratio of one two. (3) The 26 S proteasome was found to have a protein kinase activity closely coupled to ATP-dependent protease activity. (4) Ultrastructure of the 26 S proteasome was analyzed by EM and TM-AFM.(5) Substrate specificity of the 20 S proteasome was analyzed by using oxidized insulim B-chain as a substrate. (6) cDNA of the LMP7 subunit of the frog 26 S proteasome was cloned and its structural analysis was performed. (7) It was suggested that the 26 S(970kDa) proteasome is involved in the sperm penetration through the vitelline coat, while the 20 S proteasome is involved in the sperm binding to the vitelline coat.3.Mechanisms of Cell Cycle Control : (1) ATP-dependent proteasome activity was transiently activated at a prophase and metaphase during the cell cycle of ascidian embryos. It was demonstrated that this activation is due to the interconversion between 20 S proteasome and 26 S proteasome, which is induced by intracellular calcium mobilization. (2) A membrane-bound from of the proteasome, witch is distinct from the cytosol proteasome in the subunit compositions, was newly identified. Less

1.泛素结合酶的生理底物：（1）发现泛素激活酶（E1）缺失突变体细胞（ts 85）在细胞周期中出现核仁异常特征。(2)原癌基因产物Mos在非洲爪蟾卵受精过程中被26 S蛋白酶体降解，并通过泛素化去磷酸化的Mos的丝氨酸3位。(3)我们成功地建立了几个单克隆抗体特异性的多泛素化蛋白质的多泛素链。蛋白酶体的结构与功能：（1）通过对26 S蛋白酶体的基因组学研究，发现26 S蛋白酶体中存在一个蛋白酶体亚基Y13，它参与了26 S蛋白酶体的ATP依赖性蛋白水解活性。(2)26 S蛋白酶体复合物有两种异构体：一种是20 S蛋白酶体与调节蛋白酶体组成的复合物，另一种是20 S蛋白酶体与调节蛋白酶体组成的复合物 ...更多信息亚基复合物的比率为1，而另一个是比率为1/2的复合物。(3)发现26 S蛋白酶体具有与ATP依赖性蛋白酶活性密切相关的蛋白激酶活性。(4)用透射电镜（EM）和透射电镜-原子力显微镜（TM-AFM）观察26 S蛋白酶体的超微结构。(5)以氧化的胰岛素B链为底物，分析了20 S蛋白酶体的底物特异性。(6)克隆了蛙26 S蛋白酶体LMP 7亚基的cDNA，并进行了结构分析。(7)推测26 S（970 kDa）蛋白酶体参与精子穿透卵黄膜的过程，而20 S蛋白酶体参与精子与卵黄膜的结合过程。3.细胞周期调控机制：（1）在海鞘胚胎细胞周期的前期和中期，ATP依赖的蛋白酶体活性被短暂激活。结果表明，这种激活是由于20 S蛋白酶体和26 S蛋白酶体之间的相互转换，这是由细胞内钙动员引起的。(2)新发现了一种与胞浆蛋白酶体在亚基组成上不同的膜结合型蛋白酶体。少