Cholesterol and the Amyloid Precursor Protein

胆固醇和淀粉样前体蛋白

基本信息

批准号：
8839797
负责人：
CHARLES R SANDERS
金额：
$ 29.83万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2016-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The amyloidogenic pathway is widely believed to be closely linked to most forms of Alzheimer's disease. In this pathway the full length amyloid precursor protein (APP) is cleaved by ß-secretase to release a 99 residue transmembrane C-terminal domain known as "C99". C99 is then cleaved by γ-secretase to release the amyloid-ß (Aß) polypeptides. There is a considerable body of data that elevated cholesterol in neuronal membranes promotes the amyloidogenic pathway, but there has not been a mechanistic explanation. In our recent work we have shown that C99 forms a specific 1:1 complex with cholesterol, with a dissociation constant well within the physiological concentration range of cholesterol in mammalian membranes. This observation, combined with a large body of literature evidence that the ß- and γ-secretases tend to be associated with cholesterol-rich membrane domains often referred to as "lipid rafts", suggests a compelling hypothesis for how cholesterol promotes amyloidogenesis. We hypothesize that formation of a complex between cholesterol and C99 (or full length APP) results in enhanced partitioning of C99/APP to lipid rafts, where ß- and γ-secretase reside. This enhances the rate of amyloid-ß production relative to conditions in which C99/APP is not complexed with cholesterol and the protein resides in bulk membranes. Aims are: Aim 1. Determine the structure of the C99/cholesterol complex in both bulk membranes and in "lipid rafts". This aim will provide the structural basis for molecular recognition of cholesterol by C99 and will also provide the first ever comparison of the structure of a membrane protein under model membrane conditions that mimic lipid rafts versus bulk membranes. Aim 2. Elucidate the structural determinants in cholesterol that drive its association with C99. This will involve binding studies between C99 and a variety of cholesterol analogs/metabolites and will further illuminate the basis for molecular recognition between C99 and cholesterol. It will also provide a starting point for developing compounds that mimic cholesterol but that bind even more avidly to C99. Moreover, we will test the possibility that cholesterol analogs known to be "raft-phobic" can compete effectively with cholesterol for binding to C99. Aim 3. Determine whether binding of cholesterol to C99 and APP increases partitioning of these proteins into lipid rafts. Both giant unilamellar vesicles and cell-derived vesicles will be employed. These studies will test the hypothesis that association of cholesterol with the C99 and APP drives partitioning of these protein into rafts. Moreover, using selected compounds from Aim 2 that compete effectively with cholesterol but that have no avidity for rafts, we will also test whether raft association of C99/APP can be suppressed.

描述（由适用提供）：淀粉样蛋白生成途径被广泛认为与大多数形式的阿尔茨海默氏病紧密相关。在此途径中，全长淀粉样蛋白前体蛋白（APP）被ß-分泌酶裂解以释放99个居住的跨膜C末端结构域，称为“ C99”。然后，C99被γ-分泌酶裂解以释放淀粉样蛋白（Aß）多肽。有一个考虑到神经元机制中胆固醇升高的数据体可促进淀粉样蛋白生成途径，但没有机械解释。在我们最近的工作中，我们表明C99与胆固醇形成了特定的1：1复合物，在哺乳动物机制中胆固醇的物理浓度范围内离解良好。这一观察结果与大量文献证据相结合，表明β-分泌酶和γ-分泌酶倾向于与富含胆固醇的膜结构域相关，通常称为“脂质筏”，这表明胆固醇如何促进淀粉样蛋白生成。我们假设胆固醇和C99（或全长应用）之间的复合物的形成导致C99/APP对脂质筏的分配增强，其中ß-和γ-分泌酶驻留在其中。相对于C99/APP没有与胆固醇和块状蛋白质住宅相络合的条件，这提高了淀粉样蛋白 - ß产生的速率。目的是：目标1。确定散装膜和“脂质筏”中C99/胆固醇复合物的结构。这个目标将为C99对胆固醇的分子识别提供结构基础，并将在模型膜条件下对膜蛋白的结构进行首次比较，以模拟脂质筏与散装膜相比。目标2。阐明胆固醇中的结构决定剂，使其与C99相关。这将涉及C99与多种胆固醇类似物/代谢产物之间的结合研究，并将进一步阐明C99和胆固醇之间分子识别的基础。它还将为开发模仿胆固醇的化合物提供一个起点，但它更加热烈地与C99结合。此外，我们将测试称为“筏恐惧症”的胆固醇类似物可以与胆固醇有效竞争以与C99结合的可能性。目标3。确定胆固醇与C99和APP的结合是否会增加这些蛋白质的分配到脂质筏中。将雇用巨大的Unilamelar蔬菜和细胞来源的蔬菜。这些研究将检验以下假设：胆固醇与C99和APP的关联将这些蛋白质分配为筏。此外，使用来自AIM 2的选定化合物，这些化合物与胆固醇有效竞争但没有木筏的同性恋，我们还将测试是否可以抑制C99/APP的筏关联。