Regulation of Cytochrome P450 2E1 by Heat Shock Protein 90-Dependent Stabilization and CHIP-Dependent Proteasomal Degradation
详细信息 查看全文
- 作者：Yoshihiro Morishima ; Hwei-Ming Peng ; Hsia-lien Lin ; Paul F. Hollenberg ; Roger K. Sunahara ; Yoichi Osawa ; and William B. Pratt
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 13, 2005
- 年：2005
- 卷：44
- 期：49
- 页码：16333 - 16340
- 全文大小：215K
- 年卷期：v.44,no.49(December 13, 2005)
- ISSN：1520-4995
文摘
Alcohol-inducible cytochrome P450 2E1 (CYP2E1) has the most rapid turnover of any memberof this large family of membrane-bound oxygenases, and its degradation rate is altered profoundly byvarious substrates, such as ethanol and CCl4. CYP2E1 is degraded by the ubiquitin-proteasome pathway,and because the hsp90/hsp70-based chaperone machinery is often involved in maintaining the balancebetween protein integrity and degradation by this pathway, we have asked whether CYP2E1 is regulatedby the chaperone machinery. We show here that treatment of transformed human skin fibroblasts stablyexpressing CYP2E1 with the hsp90 inhibitor radicicol results in CYP2E1 degradation that is inhibited bythe proteasome inhibitor lactacystin. Immunoadsorption of hsp90 from cytosol of HEK cells expressingthe truncated CYP2E1(
3-29) yields coadsorption of CYP2E1(3-29). Cotransfection of HEK cellswith both the truncated CYP2E1 and the hsp70-dependent E3 ubiquitin ligase CHIP results in CYP2E1(3-29) degradation, and CYP2E1(3-29) co-immunoadsorbs with myc-CHIP from cytosol ofcotransfected cells. Purified, bacterially expressed CYP2E1(3-29) is ubiquitylated in a CHIP-dependentmanner when it is incubated with a purified system containing the E1 ubiquitin activating enzyme, E2,and CHIP. CYP2E1 is the first P450 shown to be an hsp90 "client" protein that can be ubiquitylated bythe hsp70-dependent E3 ubiquitin ligase CHIP. Our observations lead to a general model of how substrates,such as ethanol, can regulate the interaction of CYP2E1 with the chaperones hsp90 and hsp70 to profoundlyalter enzyme turnover.
3-29) yields coadsorption of CYP2E1(3-29). Cotransfection of HEK cellswith both the truncated CYP2E1 and the hsp70-dependent E3 ubiquitin ligase CHIP results in CYP2E1(3-29) degradation, and CYP2E1(3-29) co-immunoadsorbs with myc-CHIP from cytosol ofcotransfected cells. Purified, bacterially expressed CYP2E1(3-29) is ubiquitylated in a CHIP-dependentmanner when it is incubated with a purified system containing the E1 ubiquitin activating enzyme, E2,and CHIP. CYP2E1 is the first P450 shown to be an hsp90 "client" protein that can be ubiquitylated bythe hsp70-dependent E3 ubiquitin ligase CHIP. Our observations lead to a general model of how substrates,such as ethanol, can regulate the interaction of CYP2E1 with the chaperones hsp90 and hsp70 to profoundlyalter enzyme turnover.