Abstract
We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major ∼55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.
Footnotes
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This work was supported by National Institutes of Health Grants DK54812 (to R.C.Z., S.S., N.B.), HL50710, and P30-ES06639 (to T.A.K.). D.W.L. was supported by the Community College Initiative in Science and Technology, funded through U.S. Department of Energy's Office of Science.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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DOI: 10.1124/jpet.102.044628.
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ABBREVIATIONS: P450, cytochrome P450; DDEP, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine; HMM, high molecular mass; PI1, proteasome inhibitor 1 or Z-Ile-Glu(OtBu)-Ala-Leu-CHO; CFTR, cystic fibrosis transmembrane conductance regulator; S10, pellet and supernatant fractions formed from the 10,000g centrifugation of hepatocyte homogenates; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; RIPA, radioimmunoprecipitation assay.
- Received September 19, 2002.
- Accepted February 19, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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