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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077430-07
Application #
8251225
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
2006-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
7
Fiscal Year
2012
Total Cost
$384,741
Indirect Cost
$137,319
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Morishima, Yoshihiro; Zhang, Haoming; Lau, Miranda et al. (2016) Improved method for assembly of hemeprotein neuronal NO-synthase heterodimers. Anal Biochem 511:24-6
Zhang, Haoming; Lauver, D Adam; Wang, Hui et al. (2016) Significant Improvement of Antithrombotic Responses to Clopidogrel by Use of a Novel Conjugate as Revealed in an Arterial Model of Thrombosis. J Pharmacol Exp Ther 359:11-7
Lee, Sung Ki; Tatiyaborworntham, Nantawat; Grunwald, Eric W et al. (2015) Myoglobin and haemoglobin-mediated lipid oxidation in washed muscle: observations on crosslinking, ferryl formation, porphyrin degradation, and haemin loss rate. Food Chem 167:258-63
Pratt, William B; Gestwicki, Jason E; Osawa, Yoichi et al. (2015) Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases. Annu Rev Pharmacol Toxicol 55:353-71
Pratt, William B; Morishima, Yoshihiro; Gestwicki, Jason E et al. (2014) A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases. Exp Biol Med (Maywood) 239:1405-13
Yokom, Adam L; Morishima, Yoshihiro; Lau, Miranda et al. (2014) Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain. J Biol Chem 289:16855-65
Wang, Adrienne M; Miyata, Yoshinari; Klinedinst, Susan et al. (2013) Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation. Nat Chem Biol 9:112-8
Zhang, Haoming; Gay, Sean C; Shah, Manish et al. (2013) Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis. Biochemistry 52:355-64
Ingelman-Sundberg, Magnus; Zhong, Xiao-Bo; Hankinson, Oliver et al. (2013) Potential role of epigenetic mechanisms in the regulation of drug metabolism and transport. Drug Metab Dispos 41:1725-31
Ahsan, Aarif; Ray, Dipankar; Ramanand, Susmita G et al. (2013) Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization. J Biol Chem 288:26879-86

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