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

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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