Abstract
Cytochrome P-450 (CYP) 2E1, the alcohol-inducible form of CYP, metabolizes a wide variety of endogenous substrates, therapeutic agents, protoxicants, and procarcinogens. CYP2E1 levels are post-transcriptionally elevated in response to certain xenobiotic inducers (e.g., pyridine), and proposed mechanisms include increased translational efficiency and protection of the enzyme from ubiquitin-dependent proteolysis. Molecular modeling of a predicted cytosolic domain of CYP2E1 resulted in identification of a putative ubiquitination-target/substrate-interaction structure (residues 317–340). An affinity-purified antibody reactive to this domain quenched CYP2E1 ubiquitination in a concentration-dependent manner in a rabbit reticulocyte lysate-based ubiquitination assay. The same antibody also inhibited rat liver microsomal chlorzoxazone 6-hydroxylase activity, a marker of CYP2E1 catalytic activity, in an equivalent concentration-dependent manner. These two observations suggest an association between the CYP2E1 cytosolic domain involved in catalysis and its serving as a target for ubiquitination. Thus, these results provide a plausible mechanistic explanation for the observation that substrate binding shields the CYP2E1 protein from turnover by the ubiquitin-proteasome-dependent machinery.
Footnotes
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Send reprint requests to: Amit Banerjee, Ph.D., Institute of Chemical Toxicology, Wayne State University, 2727 Second Ave., Room 4000, Detroit, MI 48201-2654]. E. mail:abanerj{at}cmb.biosci.wayne.edu
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A.B. is supported by Institutional start-up funds and by National Institutes of Health Grant GM59467, T.A.K. by National Institutes of Health Grant HL50710, and R.F.N. by National Institutes of Health Grant ES03656. We would also like to acknowledge support from the National Institute of Environmental Health Sciences Center Grant P30-ES06639 and services from its Cell Culture and Cytometry and Imaging Facility Cores.
- Abbreviations used are::
- CYP
- cytochrome P-450
- CFTR
- cystic fibrosis transmembrane conductance regulator
- ER
- endoplasmic reticulum
- Received July 20, 1999.
- Accepted November 29, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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