Regulation of cytochrome P450 2E1 by heat shock protein 90-dependent stabilization and CHIP-dependent proteasomal degradation.
Alcohol-inducible cytochrome P450 2E1 (CYP2E1) has the most rapid turnover of any member of this large family of membrane-bound oxygenases, and its degradation rate is altered profoundly by various substrates, such as ethanol and CCl(4). CYP2E1 is degraded by the ubiquitin-proteasome pathway, and because the hsp90/hsp70-based chaperone machinery is often involved in maintaining the balance between protein integrity and degradation by this pathway, we have asked whether CYP2E1 is regulated by the chaperone machinery. We show here that treatment of transformed human skin fibroblasts stably expressing CYP2E1 with the hsp90 inhibitor radicicol results in CYP2E1 degradation that is inhibited by the proteasome inhibitor lactacystin. Immunoadsorption of hsp90 from cytosol of HEK cells expressing the truncated CYP2E1(Delta3-29) yields coadsorption of CYP2E1(Delta3-29). Cotransfection of HEK cells with both the truncated CYP2E1 and the hsp70-dependent E3 ubiquitin ligase CHIP results in CYP2E1(Delta3-29) degradation, and CYP2E1(Delta3-29) co-immunoadsorbs with myc-CHIP from cytosol of cotransfected cells. Purified, bacterially expressed CYP2E1(Delta3-29) is ubiquitylated in a CHIP-dependent manner 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 by the 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 profoundly alter enzyme turnover.
Morishima
Yoshihiro
Y
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA. ymo@umich.edu
Peng
Hwei-Ming
HM
Lin
Hsia-lien
HL
Hollenberg
Paul F
PF
Sunahara
Roger K
RK
Osawa
Yoichi
Y
Pratt
William B
WB
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002460:Cell Line; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007783:Lactones / Q000378:metabolism; D018942:Macrolides; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D007783:Lactones; D018942:Macrolides; D025801:Ubiquitin; C067713:lactacystin; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D046988:Proteasome Endopeptidase Complex; C035359:monorden; D000111:Acetylcysteine