THIS IS AN APPLICATION FOR AN INDEPENDENT SCIENTIST AWARD (K02) Recent results implicating cholesterol metabolism in the pathophysiology of Alzheimer's disease (AD) provide a rationale for studying the proteins involved in maintenance of cholesterol homeostasis. Four cytochrome P450 (P450) enzymes are known to catalyze the first and key steps in cholesterol degradation in different organs: P450 46A1 in the brain, P450 7A1 in the liver, P450 11A1 in steroidogenic tissues and the brain, and P450 27A1 in all other extrahepatic tissues. The long-term goals of this laboratory are: 1) to establish how the four enzymes that share <= 25% sequence identity bind the very same substrate, cholesterol, yet produce a different product, and 2) to determine the molecular basis for their very different catalytic efficiencies allowing P450 7A1 to metabolize 600 mg of cholesterol every day and P450 46A1 only 6-7 mg. The results will provide insight into how to modulate the activity of cholesterol-metabolizing P450s in vivo and thus maintain cholesterol at normal levels in the periphery and the brain. Our current GM62882 is focused on P450s 7A1 and 27A1, enzymes that are crucial for cholesterol catabolism outside the brain. In the Research Plan of this K02 we propose to expand our structure/function studies and investigate P450s 46A1 and 11A1, enzymes that regulate the elimination of excess cholesterol in the brain. The Career Development plan includes collaborative studies with investigators from the Alzheimer's Disease Research Center at Mount Sinai School of Medicine that will involve two techniques new to the P.I.'s research program. Gas chromatography-mass spectrometry will be used to quantify cholesterol metabolite levels in human brain samples of normal and AD-affected subjects, and immunocytochemistry to study the expression of P450s 46A1 and 11A1, as well as amyloid precursor and (3 proteins in the same brain samples. The new methodologies and collaborative studies will help in developing a new direction in the P.I.'s research program and foster application of the basic science knowledge to prevention and treatment of diseases associated with imbalances in cholesterol metabolism. If funded, the release time and salary support will significantly enhance the P.I.'s career in basic science research and make it more medically oriented.