Many cytochrome P450 (P450) enzymes and their associated drug metabolizing activities are down- regulated in inflammatory and infectious disease states, resulting in adverse reactions due to impaired drug clearance. In order to predict drug-disease interactions, it is important to understand the mechanisms involved. The mechanisms of this down-regulation are only partially understood, and research to date has focused on P450 gene transcription. Our laboratory has identified a novel mode of post-transcriptional down-regulation of drug-metabolizing P450s. Elevated production of NO in rat hepatocytes caused by inflammatory stimuli elicits the stimulated degradation of CYP2B enzymes via the ubiquitin-proteasome system, and there is evidence that a similar mechanism exists in human hepatocytes. Preliminary data indicates that at least two other P450 proteins are also regulated by this mechanism. This application has 4 specific aims: 1) To elucidate the mechanism by which NO regulates rat and human CYP2B. This will involve studies of wild type and mutated CYP2B enzymes in various cell lines. 2) To characterize the molecular machinery for NO-dependent degradation of CYP2B. A yeast two-hybrid strategy will be used to identify the ubiquitin E3 ligase and other proteins that interact with CYP2B enzymes in a NO-dependent manner. 3) To study the down-regulation of rat hepatic CYP2B enzymes in vivo, and its consequences for drug-drug interactions. Rats will be administered NO donors and we will study the down-regulation of CYP2B enzymes in association with changes in sedative-hypnotic drug action;4) To identify other proteins in rat hepatocytes that are targeted for degradation by physiologically-generated nitric oxide, using iTRAQ and other proteomic methodologies;These studies have implications not only for the function of CYP2B in metabolism of diverse therapeutic agents and in detoxification and bioactivation of environmental chemicals, but also for the toxicological, pharmacological, and physiological functions of other P450s that are regulated in the same way. They also have important implications for the broader field of NO and cytokine biology, since knowledge in the area of NO- and cytokine-stimulated protein degradation is limited.
People with ongoing infections or inflammatory diseases are more susceptible to the undesirable effects of drugs, because their livers cannot break down the drugs efficiently. This project will help us to understand what causes this change, and so allow us to predict what patients will need to have their drug doses adjusted to avoid this problem.
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