In mammals, excess cholesterol is removed mainly through conversion to bile acids, and only a small portion is utilized for production of steroid hormones. In the liver, the major site of cholesterol degradation, bile acid biosynthesis is initiated and controlled by cytochrome P450 7A1 (CYP7A1), in the brain by CYP46A1, and in most extrahepatic tissues by CYP27A1. Steroid hormone biosynthesis is initiated by CYP11A1. Thus, CYPs 7A1, 27A1, and 46A1 play key roles in cholesterol homeostasis, whereas CYP11A1 is essential for steroidogenesis. The long-term goal of this laboratory continues to be elucidation of the mechanisms that control substrate specificity and catalytic efficiency of cholesterol-metabolizing P450s. During the previous award period we have intensively studied CYPs 27A1 and 7A1 and also began characterization of CYP46A1, an enzyme whose normal function could be important for memory and learning as well as for prevention of Alzheimer's disease. In this competing renewal, we propose to focus solely on CYP46A1. We have determined the substrate-bound (cholesterol sulfate) and ligand-free X-ray structures of CYP46A1. We also established that, unlike the other cholesterol hydroxylases, CYP46A1 has a broad substrate specificity and can oxidize a number of structurally different compounds. CYP46A1 appeared to have the lowest catalytic efficiency among the four cholesterol-metabolizing P450s and unusual ability to interact with both, microsomal and mitochondrial P450 redox partners. We found that redox partners have differential effects on CYP46A1 substrate specificity. The most unexpected finding, however, was that some marketed drugs inhibit the CYP46A1 activity in vitro, whereas others stimulate the cholesterol hydroxylation mediated by CYP46A1. We propose to further explore our findings and 1) elucidate the structural basis of steroid and drug binding to CYP46A1;2) delineate structure/function relationships in the CYP46A1 active site, putative water channel and sites of redox partner interaction;and 3) evaluate CYP46A1 as a drug target. Crystallization of CYP46A1 in complex with different ligands, site-directed mutagenesis, various biochemical assays, and computational analysis will be used to achieve the goals of the project. The results will provide important insight into how CYP46A1 functions and consequently how cholesterol homeostasis is maintained in the brain. The information obtained will also be of pharmacological relevance: it will help to improve safety assessment of marketed drugs, be possibly used in the future drug design, and could result in a principally new direction in prevention of Alzheimer's disease and in treatment of age-associated deterioration of memory and learning. Characterization of CYP4A1 proposed here is an important part of our comparative analysis of cholesterol-metabolizing P450s. These studies will also lead to a better understandi g of general features of substrate- and redox partner-binding and catalysis in the P450 superfamily. Public Health Relevance: This research is focused on the main enzyme involved in cholesterol elimination from the human brain. An understanding of how this enzyme works will contribute to prevention and treatment of diseases associated with cholesterol imbalance in the brain.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062882-09
Application #
7841881
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
2001-05-01
Project End
2012-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
9
Fiscal Year
2010
Total Cost
$325,114
Indirect Cost
Name
Case Western Reserve University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Mast, Natalia; Anderson, Kyle W; Lin, Joseph B et al. (2017) Cytochrome P450 27A1 Deficiency and Regional Differences in Brain Sterol Metabolism Cause Preferential Cholestanol Accumulation in the Cerebellum. J Biol Chem 292:4913-4924
Mast, Natalia; Anderson, Kyle W; Johnson, Kevin M et al. (2017) In vitro cytochrome P450 46A1 (CYP46A1) activation by neuroactive compounds. J Biol Chem 292:12934-12946
Saeed, Ahmed A; Edström, Erik; Pikuleva, Irina et al. (2017) On the importance of albumin binding for the flux of 7?-hydroxy-3-oxo-4-cholestenoic acid in the brain. J Lipid Res 58:455-459
Mast, Natalia; Saadane, Aicha; Valencia-Olvera, Ana et al. (2017) Cholesterol-metabolizing enzyme cytochrome P450 46A1 as a pharmacologic target for Alzheimer's disease. Neuropharmacology 123:465-476
Mast, Natalia; Lin, Joseph B; Anderson, Kyle W et al. (2017) Transcriptional and post-translational changes in the brain of mice deficient in cholesterol removal mediated by cytochrome P450 46A1 (CYP46A1). PLoS One 12:e0187168
Anderson, Kyle W; Mast, Natalia; Hudgens, Jeffrey W et al. (2016) Mapping of the Allosteric Site in Cholesterol Hydroxylase CYP46A1 for Efavirenz, a Drug That Stimulates Enzyme Activity. J Biol Chem 291:11876-86
Anderson, Kyle W; Chen, Junjun; Wang, Meiyao et al. (2015) Quantification of histone deacetylase isoforms in human frontal cortex, human retina, and mouse brain. PLoS One 10:e0126592
Anderson, Kyle W; Mast, Natalia; Pikuleva, Irina A et al. (2015) Histone H3 Ser57 and Thr58 phosphorylation in the brain of 5XFAD mice. FEBS Open Bio 5:550-6
Mast, Natalia; Lin, Joseph B; Pikuleva, Irina A (2015) Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy. Mol Pharmacol 88:428-36
van Lier, Johan E; Mast, Natalia; Pikuleva, Irina A (2015) Cholesterol hydroperoxides as substrates for cholesterol-metabolizing cytochrome?P450 enzymes and alternative sources of 25-hydroxycholesterol and other oxysterols. Angew Chem Int Ed Engl 54:11138-42

Showing the most recent 10 out of 46 publications