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

1.泛素偶联酶的生理底物：(1)发现泛素激活酶（E1）缺乏突变细胞（ts85）在细胞周期中核仁出现异常特征。(2)发现原癌基因产物Mos在爪蟾卵受精过程中被26S蛋白酶体通过在Ser3位点上的去磷酸化的Mos泛素化降解。(3)成功建立了多泛素化蛋白多泛素链特异性单克隆抗体。利用该抗体，进行了多泛素化蛋白的细胞内定位和定量研究。蛋白酶体的结构和功能：(1)从基因破坏研究中发现，蛋白酶体亚基Y13参与了26s蛋白酶体的atp依赖性蛋白水解活性。(2)揭示了26s蛋白酶体复合体存在两种异构体：一种是由20s蛋白酶体与调控亚基复合体以一比一的比例组成的复合体，另一种是一比二的复合体。(3)发现26s蛋白酶体的蛋白激酶活性与atp依赖性蛋白酶活性密切相关。(4)用电镜和透射电镜-原子力显微镜分析26s蛋白酶体的超微结构。(5)以氧化胰岛素b链作为底物分析20s蛋白酶体的底物特异性。(6)克隆青蛙26s蛋白酶体LMP7亚基cDNA并进行结构分析。(7)推测26 S（970kDa）蛋白酶体参与精子穿透卵磷脂被，20 S蛋白酶体参与精子与卵磷脂被结合。细胞周期调控机制：(1)atp依赖性蛋白酶体活性在海鞘胚胎细胞周期的前期和中期被短暂激活。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化，这是由细胞内钙动员引起的。(2)新发现了一个膜结合的蛋白酶体，其亚基组成与细胞质蛋白酶体不同。少

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