Nitric oxide (NO) acts as an intra- and inter-cellular signaling molecule in many tissues, where it has important physiological and pathophysiological functions. NO regulates the activities and functions of cellular proteins by a diversity of mechanisms. Modification of critical amino acids on proteins, such as S-nitrosylation and oxidation of cysteines, or nitration of tyrosines, by NO or reactive nitrogen species derived from NO, is emerging as an important mechanism by which NO regulates cellular function. We have recently obtained compelling evidence that NO generated during an inflammatory response causes a rapid, NO-dependent down-regulation of rat liver CYP2B proteins (members of the cytochrome P450 superfamily). Here, we propose to investigate the hypothesis that modification of CYP2B proteins by reactive nitrogen species results in their targeted proteolytic degradation, and to identify the specific amino acids targeted on human CYP2B6. This will be accomplished by pulse-chase studies on CYP2B enzyme turnover in rat hepatocytes, and on degradation of wild-type and mutant CYP2B6 enzymes expressed in human hepatoma cells via retroviral transduction. Amino acid adducts will be identified by a variety of methods including immunoptecipitation with adduct-specific antibodies, chemical modification, protein chip adsorption and mas spectrometry. Since little is known about the regulation of other human CYP enzymes by NO, we will also investigate the regulation of human CYP3A4, 2D6, 2E1 and 2C enzymes by NO in cultured human hepatocytes. These studies investigate a highly novel mechanism of regulation of CYP proteins. As such, they have implications not only for the function of CYP2B enzymes in metabolism of diverse therapeutic agents, detoxification and bioactivation of environmental chemicals, and in gene therapy, but also for the toxicological, pharmacological, and physiological functions of other CYPs that are regulated in the same way. They also have important implications for the broader field of NO biology, since knowledge in the area of NO-stimulated protein degradation is limited.
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