Significance of CYP46A1 and other P450s in retinal function

描述（由申请人提供）：项目摘要。细胞色素P450（Cytochrome P450，P450）是血红素蛋白的一个超家族，参与多种内源性和外源性底物的代谢，在许多生物学过程中发挥重要作用。直到最近才清楚正常和病理性视觉功能都在其中。越来越多的证据表明视网膜中胆固醇对正常视力的重要性。然而，目前对视网膜中的胆固醇周转以及胆固醇羟化P450在视网膜功能中的意义知之甚少。在眼外组织中，P450酶启动所有重要的胆固醇降解途径，对维持整体胆固醇稳态至关重要。最近发现两种胆固醇代谢P450（27 A1和46 A1）在视网膜中高度表达。CYP 27 A1是一种普遍存在的多功能甾醇27-羟化酶，我们和其他人对其进行了广泛的研究。CYP 46 A1是最近克隆的酶，因此研究较少。我们在四年前才开始研究CYP 46 A1。已知CYP 46 A1控制胆固醇从大脑中的消除，其缺乏可能导致阿尔茨海默病。我们已经确定CYP 46 A1是一种不寻常的胆固醇代谢P450：它似乎具有非常广泛的底物特异性，与CYP 27 A1和双重亚细胞定位的底物特异性部分重叠。在特定细胞类型中的表达表明CYP 46 A1和27 A1在视网膜中发挥独特的作用。为了开始描述这些作用以及胆固醇在视网膜中的代谢命运，我们需要更多地了解CYP 46 A1，并对重叠底物进行两种酶的动力学表征。在这项新的授权申请中，我们建议：1）表征CYP 46 A1的底物特异性，并将其与CYP 27 A1的底物特异性进行比较; 2）定义CYP 46 A1活性位点; 3）确定CYP 46 A1亚细胞定位在视网膜功能中的意义。这些结果将允许预测CYP 46 A1的体内视网膜底物，并有助于了解CYP 46 A1和27 A1在视网膜中是否发挥与在视网膜外组织中相同的作用。CYP 46 A1的表征也将为我们正在进行的胆固醇代谢P450的比较分析提供宝贵的信息，并将导致更好地理解控制底物特异性和催化效率的机制，在这组P450。胆固醇类似物的研究、使用纯化酶的动力学实验、计算机建模、定点诱变、晶体学和生物物理学方法将用于实现该项目的目标。相关性（对于外行观众）。这项研究对于了解某些降低视力的眼病的发展至关重要。

DESCRIPTION (provided by applicant): Project Summary. Cytochrome P450 (P450 or CYP) enzymes constitute a superfamily of heme proteins that metabolize a wide variety of both endogenous and exogenous substrates and play key roles in many biological processes. Only recently has it become clear that normal and pathological visual functions are among these. A growing body of evidence suggests the importance of cholesterol in the retina for normal vision. However, little is currently known about cholesterol turnover in the retina and the significance of cholesterol-hydroxylating P450s in retinal function. In extraocular tissues, P450 enzymes initiate all quantitatively important pathways of cholesterol degradation and are crucial for maintenance of overall cholesterol homeostasis. Two cholesterol-metabolizing P450s (27A1 and 46A1) were recently found to be highly expressed in the retina. CYP27A1 is a polyfunctional ubiquitous sterol 27-hydroxylase extensively characterized by us and others. CYP46A1 is a recently cloned and therefore much less studied enzyme. We began to work with CYP46A1 only four years ago. It is known that CYP46A1 controls cholesterol elimination from the brain, and its deficiency may contribute to Alzheimer's disease. We have already established that CYP46A1 is an unusual cholesterol-metabolizing P450: it seems to have very broad substrate specificities that overlap in part with those of CYP27A1 and a dual subcellular localization. Expression in specific cell types suggests that CYPs 46A1 and 27A1 play unique roles in the retina. To begin to delineate these roles as well as the metabolic fate of cholesterol in the retina, we need to know more about CYP46A1 and perform kinetic characterization of the two enzymes against overlapping substrates. In this new grant application we propose to: 1) characterize substrate specificities of CYP46A1 and compare them with those of CYP27A1; 2) define the CYP46A1 active site; and 3) ascertain the significance of CYP46A1 subcellular location in retinal function. The results will allow prediction of the in vivo retinal substrates for CYP46A1 and help to understand whether CYPs 46A1 and 27A1 play the same roles in the retina as they do in extraretinal tissues. Characterization of CYP46A1 will also provide invaluable information for our on-going comparative analysis of cholesterol-metabolizing P450s and will lead to a better understanding of the mechanisms that control substrate specificity and catalytic efficiency in this set of P450s. Studies with cholesterol analogs, kinetic experiments using purified enzymes, computer modeling, site-directed mutagenesis, crystallographic, and biophysical'methods will be used to achieve the goals of the project. Relevance (for lay audience). This research will be critical for understanding the development of certain eye diseases that reduce vision.