Cholesterol and the Amyloid Precursor Protein

描述(申请人提供)：淀粉样变通路被广泛认为与大多数形式的阿尔茨海默病密切相关。在这个途径中，全长的淀粉样前体蛋白(APP)被分泌酶切割，释放出一个99个残基的跨膜C-末端结构域，称为“C99”。然后C99被分泌酶切割，释放出淀粉样蛋白多肽。有相当多的数据表明，神经细胞膜中胆固醇的升高促进了淀粉样变的发生，但还没有一个机制上的解释。在我们最近的工作中，我们已经证明了C99与胆固醇形成了1：1的特异性络合物，其解离常数在哺乳动物细胞膜中胆固醇的生理浓度范围内。这一观察与大量文献证据相结合，表明分泌酶往往与富含胆固醇的膜域有关，通常被称为“脂筏”，这就提出了一个令人信服的假说，即胆固醇如何促进淀粉样蛋白的形成。我们假设胆固醇和C99(或全长APP)之间的复合体的形成导致C99/APP到脂筏的增强分配，其中？-和？-分泌酶驻留。与C99/APP不与胆固醇络合且蛋白质驻留在大块膜中的条件相比，这提高了淀粉样蛋白的产生速度。目的：目的1.确定C99/胆固醇复合体在体膜和“脂筏”中的结构。这一目标将为C99对胆固醇的分子识别提供结构基础，并将首次在模拟脂筏和整体膜的模拟膜条件下比较膜蛋白的结构。目的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与各种胆固醇类似物/代谢物之间的结合研究，并将进一步阐明C99与胆固醇之间的分子识别基础。它还将为开发模拟胆固醇但更能与C99结合的化合物提供一个起点。此外，我们将测试已知的“恐筏恐惧症”的胆固醇类似物能否有效地与胆固醇竞争与C99结合。目的3.确定胆固醇与C99和APP的结合是否增加了这些蛋白质在脂筏中的分配。巨大的单层囊泡和细胞衍生的囊泡都将被使用。这些研究将检验这样一种假设，即胆固醇与C99和APP的关联推动这些蛋白质分裂成木筏。此外，利用Aim 2中选择的与胆固醇有效竞争但对RAFT没有亲和力的化合物，我们还将测试是否可以抑制C99/APP的RAFT结合。

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 ¿-secretase to release a 99 residue transmembrane C-terminal domain known as "C99". C99 is then cleaved by ?-secretase to release the amyloid-¿ (A¿) 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 ¿- and ?-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 ¿- and ?-secretase reside. This enhances the rate of amyloid-¿ 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.