Significance of CYP46A1 and other P450s in retinal function

CYP46A1 和其他 P450 在视网膜功能中的意义

基本信息

批准号：
7683260
负责人：
Irina A Pikuleva
金额：
$ 31.4万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7683260
关键词：
7-ketocholesterol Active Sites Affect Age Age related macular degeneration Alzheimer&apos s Disease Applications Grants Architecture Attention Bile Acids Binding Binding Sites Biochemical Biological Process Brain CYP46A1 gene CYP7A1 gene Catalysis Cell Nucleus Cholesterol Cholesterol Homeostasis Clinical Collaborations Comparative Study Complex Computer Simulation Cytochrome P450 Data Development Electron Transport Endoplasmic Reticulum Enzymes Eye diseases Goals Hemeproteins Homology Modeling Human Inorganic Sulfates Kinetics Knowledge Laboratories Lead Lipids Liver Location Maintenance Membrane Metabolic Metabolism Methods Mitochondria National Eye Institute Organ Organelles Oxidation-Reduction Oxidative Stress Pathway interactions Pattern Physiological Play Process Production Property Recombinants Recruitment Activity Research Research Personnel Retina Retinal Risk Factors Role Side Site-Directed Mutagenesis Steroid biosynthesis Steroids Sterols Structure Substrate Specificity Tissues United States National Institutes of Health Unspecified or Sulfate Ion Sulfates Vision Visual impairment Work base cell type cholesterol analog cholesterol control comparative enzyme deficiency in vivo insight interest mutant oxidation preference programs research study response steroid hormone vitamin D3 25-hydroxylase

项目摘要

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.

描述（申请人提供）：项目摘要。细胞色素P450（P450或CYP）酶构成血红素蛋白的超家族，可代谢各种内源性和外源性底物，并在许多生物过程中起关键作用。直到最近，正常的和病理视觉功能才变得很清楚。越来越多的证据表明，胆固醇在视网膜中对正常视力的重要性。然而，目前，关于视网膜中胆固醇更新以及胆固醇羟基P450在视网膜功能中的重要性知之甚少。在眼外组织中，P450酶启动了胆固醇降解的所有定量重要途径，并且对于维持总体胆固醇稳态至关重要。最近发现两种胆固醇代谢的P450（27a1和46a1）在视网膜中高度表达。 CYP27A1是一种多功能的普遍存在的固醇27-羟化酶，其广泛特征是我们和其他人。 CYP46A1是最近克隆的，因此研究的酶较少。我们仅在四年前就开始与CYP46A1合作。众所周知，CYP46A1控制大脑中消除胆固醇，其缺乏可能导致阿尔茨海默氏病。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，部分与CYP27A1的部分重叠，并且是双重亚细胞定位的。特定细胞类型中的表达表明CYPS 46A1和27A1在视网膜中起独特的作用。为了开始描述视网膜中胆固醇的这些作用以及胆固醇的代谢命运，我们需要更多地了解CYP46A1并对两种酶进行针对重叠底物的动力学表征。在这个新的赠款应用中，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）定义CYP46A1活动位点； 3）确定CYP46A1亚细胞位置在视网膜功能中的重要性。结果将允许预测CYP46A1的体内视网膜底物，并有助于了解CYPS 46A1和27A1是否在视网膜中与在视网膜外组织中起着相同的作用。 CYP46A1的表征还将为我们对胆固醇 - 代谢P450的持续比较分析提供宝贵的信息，并将更好地理解控制这套P450中底物特异性和催化效率的机制。使用胆固醇类似物，使用纯化酶的动力学实验，计算机建模，定向诱变，晶体学和生物物理'方法的研究将用于实现项目的目标。相关性（对于外行观众）。这项研究对于理解某些减少视力的眼病的发展至关重要。