Cholesterol-metabolizing P450s and Alzheimer's disease

描述(申请人提供)：这是一份独立科学家奖的申请书(K02) 最近的研究结果表明，胆固醇代谢与阿尔茨海默病(AD)的病理生理机制有关，这为研究参与维持胆固醇稳态的蛋白质提供了理论基础。已知有四种细胞色素P450(P450)酶催化不同器官中胆固醇降解的第一步和关键步骤：脑中的P450 46A1，肝脏中的P450 7A1，类固醇合成组织和脑中的P450 11A1，以及所有其他肝外组织中的P450 27A1。该实验室的长期目标是：1)确定具有相同-lt；=25%序列同一性的四种酶如何结合完全相同的底物胆固醇，但却产生不同的产物，以及2)确定它们非常不同的催化效率的分子基础，允许P450 7A1每天代谢600毫克胆固醇，而P450 46A1仅6-7毫克。这一结果将为我们深入了解如何在体内调节胆固醇代谢P450的活性，从而将外周和大脑中的胆固醇维持在正常水平。我们目前的GM62882专注于P450的7A1和27A1，这两种酶对大脑外的胆固醇分解代谢至关重要。在此K02的研究计划中，我们建议扩大我们的结构/功能研究，并研究P450、46A1和11A1，这两种酶调节大脑中多余胆固醇的消除。职业发展计划包括与西奈山医学院阿尔茨海默病研究中心的研究人员合作进行研究，这将涉及私家侦探S研究项目中的两项新技术。用气相色谱-质谱法测定正常人和AD患者脑组织中胆固醇代谢产物的含量，用免疫细胞化学方法研究P450、46A1和11A1以及淀粉样蛋白前体和(3)蛋白在同一脑组织样本中的表达。新的方法和合作研究将有助于为P.I.S研究计划开辟新的方向，并促进基础科学知识在预防和治疗与胆固醇代谢失衡相关的疾病方面的应用。如果获得资金，释放时间和工资支持将显著提升P.I.S在基础科学研究方面的职业生涯，并使其更具医学导向。

DESCRIPTION (provided by applicant): 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.