Cholesterol and the Amyloid Precursor Protein

胆固醇和淀粉样前体蛋白

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/8839797
• 关键词: Alzheimer' s Disease; Amyloid; Amyloid beta-Protein Precursor; Avidity; Binding; Brain; C-terminal; Cell membrane; Cells; Cholesterol; Cleaved cell; Complex; Data; Development; Dissociation; Etiology; Foundations; Future; Goals; Health; Length; Link; Lipids; Literature; Measurement; Membrane; Membrane Microdomains; Membrane Proteins; Micelles; Neurons; Pathway interactions; Patients; Phase; Physiological; Production; Proteins; Relative (related person); Senile Plaques; Site; Solutions; Structure; Testing; Therapeutic; Vertebral column; Vesicle; Work; amyloidogenesis; base; cholesterol analog; cytotoxic; follow-up; insight; membrane model; molecular recognition; polypeptide; secretase; unilamellar vesicle

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）：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。在该途径中，全长淀粉样前体蛋白（APP）被β-分泌酶切割以释放称为“C99”的99个残基的跨膜C-末端结构域。然后C99被γ-分泌酶裂解以释放淀粉样蛋白-β（A β）多肽。有相当多的数据表明，神经元膜中胆固醇的升高促进了淀粉样蛋白生成途径，但还没有一个机制的解释。在我们最近的工作中，我们已经表明，C99形成一个特定的1：1复合物与胆固醇，与解离常数以及在哺乳动物细胞膜中的胆固醇的生理浓度范围内。这一观察结果，结合大量文献证据表明β-和γ-分泌酶往往与富含胆固醇的膜结构域（通常称为“脂筏”）相关，提出了胆固醇如何促进淀粉样蛋白生成的令人信服的假设。我们假设胆固醇和C99（或全长APP）之间形成复合物导致C99/APP向脂筏的分配增强，其中β-和γ-分泌酶驻留。相对于C99/APP不与胆固醇复合且蛋白质驻留在本体膜中的条件，这提高了淀粉样蛋白的产生速率。目标：目标1。确定本体膜和“脂筏”中C99/胆固醇复合物的结构。这一目标将提供C99的胆固醇分子识别的结构基础，也将提供有史以来第一次比较的膜蛋白的结构模型膜条件下，模拟脂筏与散装膜。目标2.阐明胆固醇中驱动其与C99相关的结构决定因素。这将涉及C99和各种胆固醇类似物/代谢物之间的结合研究，并将进一步阐明C99和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容易与C99结合的化合物提供起点。此外，我们将测试已知为“筏恐惧”的胆固醇类似物可以有效地与胆固醇竞争结合C99的可能性。目标3.确定胆固醇与C99和APP的结合是否增加了这些蛋白质向脂筏的分配。将采用巨单层囊泡和细胞衍生的囊泡。这些研究将检验胆固醇与C99和APP的关联驱动这些蛋白质分配到筏中的假设。此外，使用从Aim 2中选择的与胆固醇有效竞争但对筏没有亲合力的化合物，我们还将测试C99/APP的筏缔合是否可以被抑制。