Role of the proteasome complex in degradation of human CYP2E1 in transfected HepG2 cells.
The aim of the present study was to characterize human CYP2E1 turnover and examine the possible role of the proteasome proteolytic pathway in the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Microsomes isolated from MVh2E1-9 cells catalyzed a slow degradation of the expressed CYP2E1, which was prevented by the addition of 4-methylpyrazole, a ligand which stabilizes CYP2E1. The addition of the cytosolic fraction of the HepG2 cells to the microsomes produced rapid degradation of CYP2E1. This rapid degradation required MgATP and was completely prevented by 4-methylpyrazole. Pulse-chase experiments after labeling CYP2E1 with [35S]-methionine and immunoprecipitation with anti-human CYP2E1 IgG indicated a biphasic turnover of CYP2E1 with half-lives of 2.5 and 6 hours. The addition of Czb-Ile-Glu(OtBu)-Ala-Leucinal(PSI) as a cell penetrating proteasome inhibitor, at concentrations ranging from 5 to 80 microM resulted in protection against the degradation of CYP2E1. PSI also increased the steady state accumulation of CYP2E1, consistent with its inhibition of CYP2E1 turnover. These results suggest that the proteasome complex plays a major role in the degradation of human CYP2E1 in the transfected HepG2 cells.
Yang
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Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Cederbaum
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AI
D000255:Adenosine Triphosphate / Q000378:metabolism; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D007700:Kinetics; D008113:Liver Neoplasms; D008715:Methionine / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D015853:Cysteine Proteinase Inhibitors; D007074:Immunoglobulin G; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008715:Methionine; D019392:Cytochrome P-450 CYP2E1; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex