Premium
Nitric oxide regulation of CYP2B proteins expressed in HepG2 cells
Author(s) -
Smith Tarik Jamar,
Lee Choonmyung,
Aitken Alison,
Kitchen Chad,
Murphy T.J.,
Morgan Edward
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb113-b
Subject(s) - microbiology and biotechnology , transfection , cell culture , mutant , tyrosine , hek 293 cells , biology , cysteine , nitric oxide , biochemistry , enzyme , chemistry , gene , genetics , endocrinology
We previously found that reactive Nitrogen species (RNS) down‐regulate cytochrome P450 2B enzymes in rat hepatocytes. The purpose of this project was to establish a human hepatoma (HepG2) cell model for studying the regulation of CYP2B proteins by RNS. We hypothesized that modifications of specific cysteine or tyrosine residues on CYP2B proteins by NO, or RNS derived from NO, target the enzymes for proteolytic degradation. cDNAs from 2B1 and 2C11 were cloned into pCL retroviral vectors. Recombinant viruses were generated by transfection of Phoenix 293 cells with the plasmids, and HepG2 cells were infected with the viral supernatants. A control virus expressing Green Fluorescent Protein was used for evaluating the efficiency of HepG2 cell transduction. Wild type and mutant CYP proteins were readily detectable by Western blotting in the transduced cells. Treatment of cells expressing CYP2B1 with the NO donors S‐nitrosoglutathione or S‐nitroso‐N‐acetyl‐penicillamine for 24 h caused reductions of CYP2B1 protein content to 40% and 55% of control levels, respectively. These effects were attenuated in cells expressing a CYP2B1 mutant containing a tyrosine to phenylalanine substitution at position 244, and no effect was seen in cells expressing CYP2C11. These results suggest that Y244 modification may be a necessary step in the NO‐dependent regulation of CYP2B1. Supported by NIH grant GM069971.