The long-term objective of the proposed research is to elucidate the mechanisms of drug- and xenobiotic- mediated inactivation, degradation, and turnover of cytochrome P450 enzymes. Nitric oxide synthase (NOS), the most highly regulated cytochrome P450 enzyme, plays a key role in a variety of biological processes, including regulation of gastrointestinal motility and liver drug metabolism. We have discovered that drugs, such as guanabenz and tobacco, are metabolism-based inactivators of NOS and cause the covalent alteration, enhanced turnover, and loss of NOS protein. In that the loss of NOS function and protein may explain some of the toxicities associated with these drugs, we wondered how drugs cause the enhanced turnover of NOS. We have established that the drugs cause prosthetic heme alteration or tetrahydrobiopterin oxidation and that such alterations are triggers for degradation of NOS. Furthermore, we discovered that these alterations labilize NOS for ubiquitination and proteasomal degradation by a chaperone-dependent mechanism involving hsp90 and hsp70. We have also uncovered a potential repair pathway where cellular proteins, including chaperones, facilitate insertion of heme into heme-deficient apo-NOS. We can now utilize the discoveries to date to determine how chaperones select for repair or ubiquitination of drug-altered NOS. Thus, we propose the following specific aims: (1) To characterize the interaction of labilized forms of NOS with hsp70- and hsp90- chaperones and cochaperones, (2) To determine the role of hsp70 and hsp90 in the ubiquitination of NOS, (3) To isolate and characterize the heme insertion machinery that facilitates heme entry into apo-NOS. We will utilize siRNA, immunopurification, biochemical, and LC-MS/MS techniques in a variety of in vitro and cellular systems to address these aims. We will show how the molecular interactions between NOS and chaperones lead to defined and predictable biological responses that ultimately determine the pharmacological and toxicological profiles of drugs. This will aid in the design of safe and effective drugs to control NOS as well as strategies to decrease adverse drug effects related to NOS. We also address the fundamental biological processes of how the heme prosthetic group is inserted into NOS and how cells maintain NOS protein quality control. Taking advantage of this quality control mechanism may provide a new method to specifically remove proteins for therapeutic benefit. Overall, this work furthers our understanding of how the metabolism of drugs, especially those used chronically, can alter the normal biological processes to give rise to adverse as well as beneficial drug effects. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics.

Public Health Relevance

This research furthers our understanding of how metabolism of drugs alters biological processes that lead to adverse as well as beneficial drug effects. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. The discoveries made through this line of study indicate that a new way to target proteins for degradation may have important therapeutic uses.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
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Okita, Richard T
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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