Cholesterol and the Amyloid Precursor Protein

描述（由申请人提供）：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病密切相关。在这一途径中，全长淀粉样蛋白前体蛋白（APP）被分泌酶裂解，释放一个被称为“C99”的99残基跨膜c端结构域。然后C99被？-分泌酶释放淀粉样蛋白- A -多肽。有相当多的数据表明，神经元膜中胆固醇升高促进淀粉样蛋白生成途径，但尚未有一个机制的解释。在我们最近的工作中，我们已经证明C99与胆固醇形成特定的1:1复合物，其解离常数完全在哺乳动物膜中胆固醇的生理浓度范围内。这一观察结果，结合大量文献证据，证明了“和？”-分泌酶倾向于与富含胆固醇的膜结构域有关，通常被称为“脂筏”，这提出了一个关于胆固醇如何促进淀粉样蛋白形成的令人信服的假设。我们假设胆固醇和C99（或全长APP）之间复合物的形成导致C99/APP向脂筏的分配增强，其中¿-和？分泌酶。相对于C99/APP不与胆固醇络合且蛋白质驻留在体膜中的条件，这提高了淀粉样蛋白的生成速率。目标是：确定C99/胆固醇复合物在散装膜和脂筏中的结构。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大膜的模型膜条件下对膜蛋白的结构进行比较。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与多种胆固醇类似物/代谢物之间的结合研究，并将进一步阐明C99与胆固醇之间分子识别的基础。它还将为开发类似胆固醇的化合物提供一个起点，这些化合物与C99的结合更加紧密。此外，我们将测试已知的“筏恐惧症”胆固醇类似物与胆固醇结合C99的有效竞争的可能性。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进入脂质筏的分配。巨大的单层囊泡和细胞衍生的囊泡都将被使用。这些研究将验证胆固醇与C99和APP的关联驱动这些蛋白质分裂成筏的假设。此外，使用Aim 2中选择的与胆固醇有效竞争但对筏没有亲缘性的化合物，我们还将测试C99/APP的筏关联是否可以抑制。

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.