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  • 02월 28일 19시 이후 : 초록수정 불가능, 일정확인 및 검색만 가능

제111회 대한화학회 학술발표회, 총회 및 기기전시회 Purification and Characterization of Mammalian Proteasomes [Reaxys포스터상]

2013년 2월 22일 11시 58분 38초
BIO.P-612 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
4월 17일 (수요일) 16:00~19:00
저자 및
최원훈, 신승균, 황민혜, JiangYanxialei, 이민재*
경희대학교 응용화학과, Korea
The ubiquitin-proteasome system (UPS) is a major cellular mechanism that regulates most short-lived intracellular protein levels. Protein substrates labeled with polyubiquitin chains are mostly degraded by 26S proteasomes into small peptides of 3 to 24 amino acids. The proteasome is a ~2.5 MDa holoenzyme complex consisting of at least 33 distinct subunits with, structurally and functionally distinctive, two distinguishable the 28-subunit core particle (CP, also known as the 20S) and the 19-subunit regulatory particle (RP, also known as the 19S). Here we present a novel and more efficient method to purify mammalian proteasome from 293 cells using a tandem-affinity tag, which is stably incorporated into beta4/Pre1 subunit of the proteasome. The proteasome showed good proteolytic characteristics toward chymotrypsin-like and trypsin-like activities. Moreover, we observed that Usp14, a deubiquitinating enzyme (DUB) is significantly associated with the proteasomes. Through a biochemical assays using purified mammalian proteasomes and recombinant Usp14 proteins from yeast, we discovered that 1) the Usp14 DUB activity is required proteasomal activation through unidentified mechanism, 2) a proteasome inhibitor MG132 diminished the DUB activity, and, however, 3) a Usp14 inhibitor IU1 abolished the effect of MG132. These results indicate that our proteasome purification system is an invaluable tool to understand the regulatory mechanisms of the UPS-mediated protein degradation.