The long-term objective of the proposed research is to elucidate the mechanisms of 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 neuronal NOS (nNOS) and lead to the covalent alteration, enhanced turnover, and loss of nNOS P450 protein via the ubiquitin proteasomal pathway. The loss of NOS is a mechanism of toxicity associated with these drugs. We have established that alteration of the active site conformation 'labilizes'the nNOS, which is then recognized by Hsp70 and Hsp90 chaperones, and is ubiquitinated by CHIP, a chaperone-associated ubiquitin ligase, resulting in the specific proteasomal degradation of the labilized nNOS. We plan on utilizing these discoveries and our recent ground-breaking success with electron microscopy (EM) studies on nNOS and nNOS?Hsp70?CHIP complexes to better understand how chaperones recognize labilized nNOS P450 through the following specific aims: (1) To characterize the structures of the stabilized and labilized states of nNOS with the use of single particle negative stain EM and cryogenic-EM techniques, (2) To characterize the structure of nNOS chaperone complexes with Hsp70 and Hsp90 by EM as well as LC-MS/MS techniques, (3) To isolate and characterize the chaperones, co-chaperones and other proteins that associate with labilized nNOS by use of a cell permeable thiol-cleavable crosslinker and LC- MS/MS methods. This work would be the first to elucidate the structure of full-length nNOS, nNOS?chaperone complexes, as well as determine the specific conformational states of nNOS that are recognized by chaperones. These studies should lead to a better understanding of how chaperones recognize labilized forms of nNOS and maintain protein quality. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. Moreover, understanding the mechanism of recognition of labilized nNOS and quality control may provide a new method to specifically remove proteins for therapeutic benefit. An example of such utility is our recent study on removal of protein aggregates through activation of chaperones in a neurodegenerative disease model (Nature Chemical Biology 9: 112-118, 2013).

Public Health Relevance

This research furthers our understanding of how the 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 #
2R01GM077430-09
Application #
8722771
Study Section
Special Emphasis Panel (ZRG1-DKUS-A (05))
Program Officer
Okita, Richard T
Project Start
2006-05-01
Project End
2018-04-30
Budget Start
2014-06-01
Budget End
2015-04-30
Support Year
9
Fiscal Year
2014
Total Cost
$388,750
Indirect Cost
$138,750
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
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
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
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
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
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
Peng, Hwei-Ming; Morishima, Yoshihiro; Pratt, William B et al. (2012) Modulation of heme/substrate binding cleft of neuronal nitric-oxide synthase (nNOS) regulates binding of Hsp90 and Hsp70 proteins and nNOS ubiquitination. J Biol Chem 287:1556-65
Morishima, Yoshihiro; Lau, Miranda; Peng, Hwei-Ming et al. (2011) Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft. Biochemistry 50:7146-56
Clapp, Kelly M; Peng, Hwei-Ming; Morishima, Yoshihiro et al. (2010) C331A mutant of neuronal nitric-oxide synthase is labilized for Hsp70/CHIP (C terminus of HSC70-interacting protein)-dependent ubiquitination. J Biol Chem 285:33642-51
Pratt, William B; Morishima, Yoshihiro; Peng, Hwei-Ming et al. (2010) Proposal for a role of the Hsp90/Hsp70-based chaperone machinery in making triage decisions when proteins undergo oxidative and toxic damage. Exp Biol Med (Maywood) 235:278-89

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