Alcohol potentiate xenobiotic toxicity is mediated in part by the induction of a unique isozyme of cytochrome P450, referred to as P450IIE1. This isozyme of P450 catalyzes the metabolic activation of compounds such as acetaminophen, CCI 4, phenol, benzene, and N-nitrosopyrrolidine. Thus, the concentration of reactive metabolites generated from these xenobiotics is dictated, in part, by the concentration of P450IIE1. Since the steady state concentration of an enzyme is a function of both the rate of synthesis and the rate of degradation, it is important to understand the regulation of both processes. Control of hepatic P450IIE1 is complex and is regulated at the level of transcription, mRNA stabilization, and protein stabilization depending on the metabolic state and age of the animal under investigation. Little is known about the mechanism of P450 degradation despite the important role degradation has in the regulation of enzyme concentration. The long range goal of our proposal is to determine the factors which target P450IIE1 for degradation and subsequently determine the proteolytic path of degradation. Ethanol could have an effect on both the targeting and the degradative path. This goal will be accomplished by the following specific aims: 1.Characterize stabilizers and labilizers for P450IIE1. Compounds interacting with P450IIE1 may act as either stabilizers, preventing degradation of the enzyme, or may act as labilizers, targeting the enzyme for degradation. Potential substrates and ligands for P450IIE1 will be screened labilizers and stabilizers by examining what effect they have on electron flux, oxygen activation and enzyme modification. 2.Characterize the time course of induction and degradation of P450IIE1 in mice. We have found that a single oral dose of ethanol or acetone leads to a rapid and significant increase in the concentration of P450IIE1 in CF-I mice. We will further characterize the induction time course by monitoring holoenzyme, apoprotein, heme, mRNA and cytosolic protein during the induction and degradation time course. Strain differences were observed which suggests a genetic difference in P450IIE1 regulation. We will examine various strains in more detail to determine whether the difference is at the level of synthesis or degradation. 3.Characterize the signal(s) which target P450IIE1 for degradation. There are a number of potential signals for the degradation of P450IIE1. These include, among others, phosphorylation ubiquitination, oxygen radical-dependent oxidation of critical amino acid residues, or other modification of the protein. Each of these events may be coupled with heme loss. Both whole animal and tissue culture models will be used to determine which signals may be involved in P450IIE1 degradation.
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