Ethanol Induces CYP2E1 by Protein Stabilization

Abstract

In the present study, we demonstrate that ethanol induces CYP2E1 by protein stabilization in vivo. The control half-life of CYP2E1 was determined to be 6-7 h followed by a slower secondary phase. The half-life of ethanol-stabilized CYP2E1 was calculated to be 38 h. The mechanism underlying the rapid degradation of CYP2E1 was also investigated and appears to involve the ubiquitin-proteasome proteolytic pathway. An in vitro assay using the cytosolic fraction was developed to further characterize CYP2E1 degradation. Using this assay, 40-50% loss of CYP2E1 was observed in 1 h, coincident with the formation of high M r ubiquitin-CYP2E1 conjugates. At concentrations approximating those found in vivo, ethanol protects CYP2E1 from cytosolic degradation. No loss of CYP2B1/2 was observed under identical conditions, suggesting that this reaction is specific for certain P-450s which are rapidly turned over.A member of the microsomal P-450 family of proteins, P-450 2E1 (CYP2E1), 1 is induced by a variety of chemicals and physiological states, some of which have been linked to the development of liver disease and carcinogenesis via oxidative stress (1). The most significant chemical inducer of CYP2E1 is ethanol, a compound which is also a substrate for the protein. Debate still exists as to the precise mechanism of CYP2E1 induction by ethanol, with some reports indicating that ethanol stabilizes CYP2E1 in vitro (2) and another group suggesting that ethanol induces CYP2E1 by increasing protein synthesis (3). In the present study, we have attempted to define the nature of CYP2E1 regulation by ethanol in vivo using controls, chronic ethanol-treated, and ethanol-withdrawing animals. Results from CYP2E1 pulse-labeling show that ethanol induces CYP2E1 predominantly by protein stabilization, with the administration of ethanol attenuating the rapid phase of CYP2E1 degradation. We have also characterized the pathway responsible for this degradation, and present data in vivo and in vitro that suggest ubiquitin conjugation may target CYP2E1 for rapid proteolysis.

Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

“…Other chemicals, such as ethanol, can regulate CYP2E1 activity by altering protein stabilization without affecting transcriptional activity (Roberts et al 1995). Ethanol has also been shown to increase CYP2E1 gene expression at high doses (> 300 mg/dL blood) (Badger et al 1993).…”

The Role of CYP2E1 in the Drug Metabolism or Bioactivation in the Brain

“…The induction of CYP2E1 seems to be regulated at the posttranscriptional or posttranslational levels by the stabilization of mRNA [84] or by protection against the rapid degradation of protein [85] in the liver. The posttranscriptional regulation would be responsible for not only the inducible, but also the constitutive expression of CYP2E1 in liver [86].…”

Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame

“…Thus, although all CYPs 3A as well as all structurally/functionally inactivated P450s are predominantly ERAD/UPD targets (Correia et al 1987, 1992a, 1992b, 2005; Correia, 2003; Tierney et al, 1992; Sohn et al, 1991; Dai & Cederbaum, 1995; Roberts, 1997; Schmiedlin-Ren, 1997; Korsmeyer et al, 1997; Wang et al, 1999; Murray & Correia, 2001; Morishima et al, 2005; Liao et al, 2006; Correia & Liao, 2007; Faouzi et al, 2007; Lee et al, 2008), native P450s such as rat liver CYPs 2B1 and 2C11 are predominantly ERAD-II substrates requiring ALD instead of UPD, henceforth referred to as the ERAD/ALD pathway (Masaki et al, 1987; Ronis & Ingelman-Sundberg, 1989; Ronis et al, 1991; Murray et al, 2002; Liao et al, 2005). On the other hand, human or rat liver CYP2E1 utilizes both ERAD/UPD and ERAD/ALD pathways, depending on whether it is suicidally inactivated or native/substrate-free, or native/substrate-bound, respectively (Song et al, 1989; Roberts et al, 1995; Bardag-Gorce, 2002; Morishima et al, 2005; Wang et al, 2011). Thus, although a preferential P450 routing into either pathway apparently exists, the pathway not chosen may simply represent a convenient alternative backup, in eventualities such as ER-stress when ERAD/UPD is overloaded and clogged with misfolded/aggregated proteins awaiting clearance, and competition for the ERAD/UPD cellular machinery is insurmountable.…”

“…This is also true when CPR function is chemically inhibited in cell culture (Goasduff & Cederbaum, 1999; Zhukov & Ingelman-Sundberg, 1999). Furthermore, just substrate (EtOH or acetone)-binding to the CYP2E1 active site and catalysis, aborts its futile cycling to ROS, thereby prolonging CYP2E1 half-life and stabilizing the protein (Song et al, 1989; Roberts et al, 1995; Chien et al, 1997; Bardag-Gorce et al, 2006). Additionally, quasi-irreversible P450 inactivators such as TAO that coordinate tightly to the CYP3A-ferrous heme and abort its catalytic turnover, also dramatically prolong CYP3A half-life, resulting in the elevation of hepatic CYP3A content (Watkins et al, 1986), another valid example of P450 induction via protein stabilization.…”