Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells.

TitleProteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells.
Publication TypeJournal Article
Year of Publication2004
AuthorsHuan J-Y, Streicher JM, Bleyle LA, Koop DR
JournalToxicol Appl Pharmacol
Date Published2004 Sep 15
KeywordsAnimals, Calcium, Calpain, Cell Line, Cercopithecus aethiops, COS Cells, Cysteine Endopeptidases, Cytochrome P-450 CYP2B1, Cytochrome P-450 CYP2E1, Cytochrome P-450 CYP2E1 Inhibitors, Enzyme Inhibitors, Gene Expression Regulation, Enzymologic, Genetic Vectors, HeLa Cells, Hot Temperature, HSP90 Heat-Shock Proteins, Humans, Multienzyme Complexes, Mutagenesis, Proteasome Endopeptidase Complex, Rabbits, Rats, Tetracycline, Ubiquitin

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.

Alternate JournalToxicol. Appl. Pharmacol.
PubMed ID15364548
Grant ListAA05462 / AA / NIAAA NIH HHS / United States
AA08608 / AA / NIAAA NIH HHS / United States
Faculty Member Reference: 
John M. Streicher, PhD