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（由申请人提供）：人们普遍认为淀粉样蛋白生成途径与大多数形式的阿尔茨海默病密切相关，在该途径中，全长淀粉样前体蛋白（APP）被β-分泌酶裂解，释放出99残基的跨膜C-。称为“C99”的末端结构域随后被 γ-分泌酶裂解以释放淀粉样蛋白-β (Aß) 多肽。有大量数据表明神经元膜中胆固醇升高会促进淀粉样蛋白生成途径，但尚未有机制解释。在我们最近的工作中，我们表明 C99 与胆固醇形成特定的 1:1 复合物，具有解离常数。这一观察结果与大量文献证据相结合，证明β-和γ-分泌酶往往与富含胆固醇的膜域（通常称为“脂质”）相关。筏”，提出了关于胆固醇如何促进淀粉样蛋白生成的令人信服的假设。我们研究了胆固醇和 C99（或全长 APP）之间复合物的形成导致 C99/APP 与脂筏的增强分配，其中 β- 和 γ-分泌酶驻留相对于 C99/APP 不与胆固醇复合并且蛋白质驻留在体膜中的条件，这提高了淀粉样蛋白-β 的产生率。目标是：目标 1。确定体膜和“脂筏”中 C99/胆固醇复合物的结构，这一目标将为 C99 对胆固醇的分子识别提供结构基础，并且还将提供膜蛋白结构的首次比较。模拟脂质筏与本体膜的膜条件。目标 2. 阐明胆固醇中驱动其与 C99 关联的结构决定因素这将涉及 C99 与各种胆固醇之间的结合研究。类似物/代谢物，并将进一步阐明 C99 和胆固醇之间的分子识别基础。它还将为模拟胆固醇但与 C99 结合更紧密的化合物提供一个起点。此外，我们将测试开发胆固醇类似物的可能性。已知具有“筏恐惧性”的蛋白可以与胆固醇有效竞争与 C99 的结合。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向脂筏的分配。这些研究将使用囊泡和细胞衍生的囊泡来检验胆固醇与 C99 和 APP 的关联驱动这些蛋白质分配成筏的假设。此外，使用来自 Aim 2 的选定化合物，这些化合物可以有效地与胆固醇竞争，但不具有这种作用。由于对raft的热情，我们还将测试是否可以抑制C99/APP的raft关联。