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

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Abstract

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.

Introduction

Cytochromes P450 (CYP; P450), a superfamily of hemoproteins, are the major catalysts of the oxidative transformation of endogenous and exogenous compounds. The P450s are resident endoplasmic reticulum (ER) type I proteins anchored in the ER membrane by a single transmembrane domain (Porter and Coon, 1991). The concentrations of specific P450 forms present in a tissue will affect the metabolic fate of a xenobiotic. Factors that influence either enzyme synthesis or degradation will alter the steady-state concentration of the P450s and thus change the metabolic capacity of a tissue. The regulation of CYP2E1 is thought to involve multiple mechanisms including transcription, stabilization of mRNA, posttranslational modification, and substrate stabilization of the protein against degradation (Song, 1995). In contrast, CYP2B1 is regulated primarily by transcriptional mechanisms (Porter and Coon, 1991). In vivo the degradative pathways for CYP2E1 and CYP2B1 appear to be distinct and the two proteins exhibit different half-lives Correia, 2003, Parkinson et al., 1983, Ronis et al., 1991. There is evidence that the proteasome, the major soluble intracellular proteolytic complex, plays a major role in the degradation of CYP2E1 Banerjee et al., 2000, Goasduff and Cederbaum, 2000, Roberts, 1997, Yang and Cederbaum, 1997 and CYP3A4 Korsmeyer et al., 1999, Murray and Correia, 2001. In contrast, based on the presence of CYP2B1 in a lysosomal compartment, it was suggested that CYP2B1 is degraded primarily by a lysosomal pathway Masaki et al., 1987, Ronis et al., 1991.

Proteasome-dependent proteolysis is involved in many biologically important processes, such as the cell cycle, apoptosis, signal transduction, the immune response, and protein quality control within the secretory pathway Bonifacino and Weissman, 1998, Glickman and Ciechanover, 2002, Kostova and Wolf, 2003, McCracken and Brodsky, 2003. The proteasome is present in the cytosol and nucleus, and a significant fraction is associated with the ER Khan and Joseph, 2001, Rivett, 1998. While it is clear that the proteasome is involved in the degradation of many proteins, the requirement for ubiquitination is not absolute Grune et al., 2003, Liao et al., 2003, Orlowski and Wilk, 2003, Yu et al., 1997. In the present investigation, we used tetracycline (Tc)-controlled HeLa cell lines that inducibly and stably express rabbit CYP2E1 or rat CYP2B1 to characterize the intracellular proteolytic pathways involved in the degradation of these two proteins under steady-state conditions. The steady-state half-life of the two P450s is significantly different; CYP2E1 has a shorter half-life compared to CYP2B1 in the Tc-inducible HeLa cell line. Results with selective chemical inhibitors to different intracellular proteolytic pathways indicate that both CYP2E1 and CYP2B1 are susceptible to a proteasome-dependent degradative pathway. Mutagenesis of two Lys residues implicated as sites of ubiquitination on CYP2E1 (Banerjee et al., 2000) did not alter the degradation of CYP2E1. Results obtained examining the degradation of CYP2E1 in Chinese hamster E36ts20 cells that have a temperature-sensitive E1 ubiquitin-activating enzyme suggest that CYP2E1 may be degraded by the proteasome by a Ub-independent proteasomal pathway.

Section snippets

Materials

Full-length rabbit CYP2E1 and rat CYP2B1 cDNAs were provided by Drs. M.J. Coon (University of Michigan) and J. Halpert (University of Texas Medical School at Galveston), respectively. The expression vector pUHD10-3 was obtained from Dr. H. Bujard (University of Heidelberg, Heidelberg, Germany). tTA HeLa cells were provided by Dr. C.A. Enns (Oregon Health and Science University). Chinese hamster E36 and E36ts20 cells were provided by Dr. Alan L. Schwartz (Washington University School of

Characterization of CYP2E1 and CYP2B1 turnover in Tc-HeLa cells

The expression of CYP2E1 after removal of Tc from the medium was described (Huan and Koop, 1999). In preliminary studies, we found that the expression of CYP2B1 exhibited a similar time dependence when monitored both by the formation of 16α-hydroxytestosterone and protein levels by immunoblot analysis. Peak levels were observed 72 h after removal of Tc from the medium (data not shown). Previous in vivo studies suggested that the turnover of CYP2E1 and CYP2B1 was significantly different Masaki

Discussion

A number of studies suggest that CYP2E1 and CYP2B1 are degraded by different mechanisms. Results from in vivo and in vitro studies indicate CYP2E1 had a much shorter half-life (t1/2 about 6–7 h) McGehee et al., 1994, Song et al., 1989 than that of CYP2B1 (t1/2 about 25–30 h) Parkinson et al., 1983, Shiraki and Guengerich, 1984. The results from the current study are consistent with these results in that the half-life of CYP2E1 is shorter than CYP2B1 when expressed in Tc-HeLa cells. This

Acknowledgements

This work was supported by National Institute on Alcohol Abuse and Alcoholism grants AA08608 and AA05462. We are grateful to Dr. Jim Halpert for providing the CYP2B1 plasmid and antibody, Dr. Alan Schwartz for providing the E36ts20 and E36 cells, and Dr. Dirk Bohmann for providing the c-Jun and HA-Ub constructs. We would like to thank Julia Parazin and Tasha McDonald for excellent technical assistance.

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