Significance of CYP46A1 and other P450s in retinal function

描述（由申请人提供）：项目概述。细胞色素P450 （P450或CYP）酶构成血红素蛋白超家族，代谢各种内源性和外源性底物，在许多生物过程中发挥关键作用。直到最近才清楚，正常和病理的视觉功能也在其中。越来越多的证据表明，视网膜中的胆固醇对正常视力很重要。然而，目前对视网膜中的胆固醇转换和胆固醇羟基化p450在视网膜功能中的意义知之甚少。在眼外组织中，P450酶启动胆固醇降解的所有定量重要途径，对维持整体胆固醇稳态至关重要。最近发现两个胆固醇代谢p450 （27A1和46A1）在视网膜中高表达。CYP27A1是一种广泛存在的多功能甾醇27-羟化酶，被我们和其他人广泛表征。CYP46A1是最近克隆的，因此研究较少的酶。我们四年前才开始研究CYP46A1。众所周知，CYP46A1控制着大脑中胆固醇的消除，它的缺乏可能会导致阿尔茨海默病。我们已经确定CYP46A1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CYP27A1的底物特异性部分重叠，并且具有双重亚细胞定位。在特定细胞类型中的表达表明CYPs 46A1和27A1在视网膜中起着独特的作用。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CYP46A1，并对重叠底物进行两种酶的动力学表征。在这项新的拨款申请中，我们建议：1)表征CYP46A1的底物特异性，并将其与CYP27A1的底物特异性进行比较；2)确定CYP46A1活性位点；3)确定CYP46A1亚细胞定位在视网膜功能中的意义。这些结果将有助于预测CYP46A1的体内视网膜底物，并有助于了解CYP46A1和27A1是否在视网膜中发挥与在视网膜外组织中相同的作用。CYP46A1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，并将使我们更好地理解控制这组p450的底物特异性和催化效率的机制。研究胆固醇类似物，使用纯化酶进行动力学实验，计算机建模，定点诱变，晶体学和生物物理方法将用于实现该项目的目标。相关性（针对非专业观众）。这项研究将对理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.