Studies of cholesterol maintenance in the retina and brain to delineate the importance of cholesterol-related pathways for retinal and brain functions and whether pharmacologic modulation of these pathways could lead to disease-modifying treatments for retinal and brain disorders.
Cholesterol is essential for life in mammals. However, if chronically in excess, it becomes a risk factor for cardiovascular and Alzheimer’s diseases and possibly age-related macular degeneration. The focus of this laboratory is on the four cytochrome P450 enzymes 7A1, 27A1, 46A1, and 11A1 that are necessary for cholesterol elimination from different organs.
We are trying to understand how cholesterol-metabolizing P450s function at the molecular level, what roles they play in the development of different diseases, and whether these enzymes could serve as targets for cholesterol lowering medications. Currently, there are two major projects in the laboratory. The first project is based on our previous structural and biochemical studies showing that the enzyme active site is conformationally flexible and can accommodate ligands other than sterols. The goal of this project is to identify marketed drugs that can either inhibit or stimulate the CYP46A1-mediated cholesterol hydroxylation in vivo. We use in-silico and in vitro screening of drug libraries, X-ray crystallography, and tests on animals.
The second project is based on studies by others showing that P450s 27A1, 11A1, and 46A1 are expressed in the retina. We know that in extraocular tissues, deficiency of cholesterol-metabolizing P450s is manifested clinically by cerebrotendinous xanthomatosis with premature atherosclerosis (P450 27A1), congenital lipoid adrenal hyperplasia (P450 11A1), and likely impairment of memory and learning and Alzheimer’s disease (as indicated phenotypically in P450 46A1 knockout mice and human studies of P450 46A1 polymorphisms). We hypothesize that in the retina P450s 27A1, 11A1, and 46A1 are important as well. The goal of this project is to delineate the roles of P450s 27A1, 11A1 and 46A1 in the retina. We are assessing whether the unique retinal environment affects catalytic properties of cholesterol-metabolizing CYPs, evaluating vision function in P450 knock out mice, and characterizing human retinal samples for the P450 expression and lipid content.
Mast N, Liao W-L, Pikuleva IA, Turko IV. Combined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase. Arch Bioch Biophys 2009;483:81-89.
Mast N, White MA, Bjorkhem I, Johnson EF, Stout CD, Pikuleva, IA. Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Aca. Sci. USA 2008;105:9546-9551.
White MA, Mast N, Bjorkhem I, Johnson EF, Stout CD, Pikuleva IA. Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1. Acta Cryst. D 2008;D64:487-495.
Pikuleva IA. Cholesterol-metabolizing cytochromes P450: implications for cholesterol lowering. Expert Opin Drug Metab Toxicol. 2008;11:1403-1414.
Pikuleva IA, Mast N, Liao W-L, Turko IV. Studies of membrane topology of mitochondrial cholesterol hydroxylases CYPs 27A1 and 11A1. Lipids 2008;43:1127-1132.
Mast N, Murtazina D, Liu H, Graham S, Bjorkhem I, Halpert J, Peterson J, Pikuleva IA. Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies. Biochemistry 2006;45:4398-4404.
Mast N, Graham S.E, Andersson U, Bjorkhem I, Hill C, Peterson J, Pikuleva IA. Cholesterol binding to cytochrome P450 7A1, a key enzyme in bile acid biosynthesis. Biochemistry 2005;44:3259-3271.