Presenilin Biology and the Mechanism of Alzheimer's Disease

描述（由申请人提供）：早老素作为后生动物中普遍存在的膜内蛋白酶的发现证实了生物学中的一个新兴趋势。基础研究和应用研究之间的传统区别已经变得越来越模糊，因为严格以疾病为导向的研究揭示了基本的生物学机制。在这个更新的应用程序中，10名研究人员谁合作成功，在过去十年中的正常和病理生物学早老素（PS）希望延长他们的生产相互作用到新的实验，将进一步阐明PS/？- 分泌酶在正常生物学中的作用以及这种不寻常的多蛋白复合物和各种相关基因产物在阿尔茨海默病发生中的作用。基于我们四个相互关联的项目中每个项目的大量初步数据，并得到三个重要核心的支持，我们将追求许多具体目标，包括：1。试图确定PS/？-通过对纯化的活性复合物进行冷冻电子显微镜检查以及对各个组分进行X射线晶体学检查来鉴定分泌酶; 2.扩展我们最近的发现，胆固醇显着提高催化活性的纯化？- 分泌酶和膜脂质一般似乎代表最有效的调节剂的生产和Notch裂解; 3.进行广泛的基因组、功能遗传和蛋白质分析，寻找可能与早老蛋白相关途径有关的新的迟发性AD候选基因; 4.使用先进的显微镜方法在活细胞（双分子荧光互补; FLIM）的图像和定量的构象和相互作用的早老素与APP，与新的候选基因出现（2），与脂质和？- 本文鉴定的调节化合物; 5.鉴定NaJ、APP和APLP 2的β 2亚基的共同结合配偶体和共同亚细胞加工途径，这三种蛋白质被β-和γ-分泌酶相同地加工。6.进行无偏见的蛋白质组学筛选，以更全面地定义“？- 分泌组”，即，未知的程度？印刷受体.这些目标和许多额外的目标和子目标将在我们的4个项目中协同进行，一个项目的进展将修改另一个项目的实验计划。简而言之，我们渴望继续我们的密切互动，以更清楚地了解PS及其相关蛋白质和脂质如何在健康和疾病中处理膜内的许多底物。敷设总结：十位经验丰富的科学家在过去十年中进行了富有成效的合作，他们希望解决一种不寻常的蛋白质切割酶的结构和功能，这种酶是所有多细胞动物生命所必需的，并与阿尔茨海默病的病因有关。

DESCRIPTION (provided by applicant): The discovery of presenilins as ubiquitous intramembrane proteases in metazoans exemplifies an emerging trend in biology. The traditional distinction between basic and applied research has become increasingly blurred, as studies initiated with a strictly disease-oriented focus uncover fundamental biological mechanisms. In this renewal application, ten investigators who have collaborated successfully during the last decade on the normal and pathological biology of presenilin (PS) wish to extend their productive interactions into new experiments that will further illuminate the structure-function relationships of PS/ ? -secretase in normal biology and the role of this unusual multi-protein complex and various related gene products in the genesis of Alzheimer's disease. Based on extensive preliminary data in each of our four interrelated projects and supported by three vital cores, we will pursue numerous Specific Aims that include: 1. attempting to determine the structure of PS/ ?-secretase by performing cryo-electron microscopy of the purified, active complex as well as x-ray crystallography of the individual components; 2. extending our recent discovery that cholesterol dramatically enhances the catalytic activity of purified ? -secretase and that membrane lipids in general appear to represent the most potent regulators of both A¿ production and Notch cleavage; 3. performing extensive genomic, functional genetic and protein analyses searching for novel lateonset AD candidate genes potentially implicated in presenilin-related pathways; 4. using advanced microscopy approaches in living cells (bimolecular fluorescence complementation; FLIM) to image and quantify the conformations and interactions of presenilin with APP, with new candidate genes emerging from (2), and with lipids and ? -modulating compounds identified herein; 5. identifying common binding partners and common subcellular processing pathways for the (32 subunit of NaJ, APP and APLP2, three proteins that are processed identically by both (3- and y-secretase. 6. performing an unbiased proteomics screen to more fully define the " ? -secretome", i.e., the extent of unknown ?-substrates. These and numerous additional aims and sub-aims will be pursued collaboratively across our 4 projects, with progress in one project modifying experimental plans in another. In short, we are anxious to continue our close interaction to bring greater clarity to exactly how PS and its associated proteins and lipids process a host of substrates within membranes in health and disease. Lay summary: Ten experienced scientists who have collaborated productively for the past decade wish to address the structure and the functions of an unusual protein-cutting enzyme that is required for life in all multi-cellular animals and implicated in the causation of Alzheimer's disease.