January 31, 2002
Journal Article

Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical ubiquitination pathway.

Abstract

Substrate binding to cytochrome P450 3A (CYP3A) has been reported to stabilize this P450 form but the molecular mechanisms involved have not been reported. In this study, we initially characterize a novel microsome system that exhibits CYP3A and CYP2E1 (but not CYP4A) ubiquitination activity in the absence of cytosol and then use this microsome system to examine substrate-mediated stabilization of CYP3A. The CYP3A ubiquitin ligase activity was present in microsomes isolated from rats treated with high doses (50 or 100 mg/kg/d) of nicardipine but not microsomes from control, dexamethasone-, nifedipine- or diltiazem-treated rats. An upward shift in the mass of total ubiquitinated proteins (detected with ubiquitin antibodies) was also present in microsomes that exhibited CYP3A ubiquitination. Addition of methylated ubiquitin blocked the formation of the high-molecular-weight CYP3A-ubiquitin bands and the concomitant loss of the intact 55 kDa CYP3A protein band. Addition of CYP3A substrates (e.g., clotrimazole, ketoconazole, erythromycin) inhibited CYP3A ubiquitination in the incubated microsomes but did not block ubiquitination of other proteins. The ubiquitination of CYP3A did not require the addition of NADPH, indicating that the inhibition of CYP3A ubiquitination by substrates was not associated with inhibition of NADPH-dependent CYP3A oxidase activity. Dimethylsulfoxide, which stabilizes CYP3A protein in vivo by a mechanism distinct from substrate-dependent stabilization, did not alter CYP3A ubiquitination in the microsomal fractions. These results provide the first evidence that substrates stabilize CYP3A protein by preventing ubiquitination and that this process is not dependent upon CYP3A oxidase activity.

Revised: July 22, 2010 | Published: January 31, 2002

Citation

Zangar R.C., A.L. Kimzey, J.R. Okita, D.S. Wunschel, R.J. Edwards, H.S. Kim, and R.T. Okita. 2002. Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical ubiquitination pathway. Molecular Pharmacology 61. PNWD-SA-5143.