Identification of a ubiquitination-Target/Substrate-interaction domain of cytochrome P-450 (CYP) 2E1.
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.
Banerjee
A
A
Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201-2654, USA. abanerj@cmb.biosci.wayne.edu
Kocarek
T A
TA
Novak
R F
RF
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D007120:Immunochemistry; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000201:enzymology / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
D014452:Ubiquitins; D019392:Cytochrome P-450 CYP2E1