Nitric Oxide‐Dependent Proteasomal Degradation of Cytochrome P450 2B Proteins

Abstract

Inflammation and infection cause the down-regulation of cytochrome P450 enzymes in rodent and human hepatocytes. Although many of these effects are achieved via transcriptional suppression, we have found that CYP2B1 protein is rapidly down-regulated in primary cultures of rat hepatocytes by a NO-dependent mechanism that is independent of any transcriptional effects (Ferrari et al., 2001, Mol. Pharmacol. 60, 209-216). Here, we show that CYP2B proteins in primary rat hepatocyte cultures were suppressed more than 70% after 8hr treatment with interleukin-1 (IL-1β), in an NO-dependent manner. The calpain inhibtor E64-d or the lysosomal protease inhibitors NH4Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1, but treatment with the proteasome inhibitors MG132, clasto-Lactacystin β-lactone, or gliotoxin each abolished this suppression under conditions in which NO production was not affected. However, CYP2C11 was not down-regulated by IL-1 treatment and was not affected by the treatments of lysosomal, calpain, and proteasome inhibitors. Treatment of cells for 24 h with the NO-donors 3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18), S-nitrosoglutathione, and S-nitroso N-acetylpenicillamine (SNAP) also suppressed CYP2B proteins. Treatment of HeLa cells with 500 μM NOC-18 enhanced the ubiquitination of c-myc-tagged CYP2B at 6 hr. These data suggest that NO-dependent CYP2B down-regulation results from proteasomal degradation through the ubiquitination of CYP2B. (This research was supported by NIH Grant GM 069971)