Vol. 61, Issue 4, 892-904, April 2002

Cytochrome P450 3A Conjugation to Ubiquitin in a Process Distinct from Classical Ubiquitination Pathway

R. C. Zangar, A. L. Kimzey, J. R. Okita, D. S. Wunschel, R. J. Edwards, H. Kim, and R. T. Okita

Pacific Northwest National Laboratory, Richland, Washington (R.C.Z., A.L.K., D.S.W.); Washington State University, Pullman, Washington (J.R.O., R.T.O.); Faculty of Medicine, Imperial College, Hammersmith Campus, London, England (R.J.E.); and Detroit R&D, Inc, Detroit, Michigan (H.K.)

We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg²⁺, or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.