CYP2E1 catalyzes the metabolism/metabolic activation of a wide variety of endogenous substrates, therapeutic agents, toxicants and procarcinogens. CYP2E1 is elevated in response to pathophysiological conditions (e.g. diabetes) and by inducers through either increased translational efficiency or by protection of the enzyme from ubiquitin-dependent proteolysis. To date, only ubiquitination of CCl4-damaged CYP2E1 has been reported. Preliminary results show ubiquitination of free CYP2E1 protein in rabbit reticulocyte cell-free translation system. Molecular modeling of the catalytic domain of CYP2E1 resulted in identification of a putative ubiquitination-target loop structure. An affinity-purified anti-peptide antibody reactive to this domain quenched CYP2E1 ubiquitination and the degradation of free CYP2E1 in the in vitro ubiquitination assay. The hypothesis of this research is that the rate of ubiquitin- dependent turnover of CYP2E1 protein occurs differentially for different forms of CYP2E1 and is a function of CYP2E1 conformation and the specific ubiquitination machinery for signaling its degradation.
The specific aims of this research are to: 1) examine CYP2E1 ubiquitination in vitro using a reconstituted ubiquitination assay; 2) to identify the lysyl residues that are likely involved in ubiquitination through site- directed mutagenesis; 3) biochemically identify the CYP2E1 ubiquitination machinery; 4) identify mammalian hepatic ubiquitin-dependent CYP2E1-turnover machinery and the other cellular factors that influence CY2E1 turnover by employing genetic screening methods using yeast. Rabbit reticulocyte lysate and rat liver lysate will be examined to assess the roles of enzymes which cause ubiquitination of the different forms of CY2E1 (e.g. """"""""free"""""""" form; microsomal form; xenobiotic-induced microsomal form; and finally, a microsomal form damaged by xenobiotic metabolism). Lysates will be fractionated to identify the enzymes required for ubiquitination of CYP2E1. Information from peptide sequences obtained from these ubiquitin-pathway enzymes will be used to clone cDNAs coding for these enzymes from a hepatic cDNA library. The domains and the lysyl residues targeted for ubiquitination in CYP2E1 will be mapped. Primary cultured rat hepatocyte model will be employed to assess the role(s) of these domains and lysyl residues in CYP2E1-turnover by expressing CYP2E1 mutants in the presence and absence of xenobiotics. Genetic screening will be performed to recover mammalian genes whose products can ubiquitinate CYP2E1. In addition to providing information on mechanisms regulating CYP2E1 turnover, this research will elucidate the mechanisms regulating turnover of the cytochrome P450s.
