Structural Basis for the Partitioning of C99 into Liquid-Ordered Membrane Domains

C99 划分为液序膜域的结构基础

• 批准号: 8717279
• 负责人: Jonathan Patrick Schlebach
• 金额: $ 5.33万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8717279
• 关键词: Accounting; Affect; Affinity; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Behavior; Binding; Biological; Brain; C-terminal; Cellular Membrane; Cholesterol; Coupled; Coupling; Deposition; Development; Disease; Electron Spin Resonance Spectroscopy; Engineering; Entropy; Enzymes; Evaluation; Exhibits; Fluorescence Microscopy; Glycine; Health; Lateral; Length; Ligands; Light; Lipids; Liquid substance; Measurement; Measures; Membrane; Membrane Microdomains; Membrane Proteins; Milk; Molecular; Mutation; Pathogenesis; Peptides; Phase; Phosphorylcholine; Play; Positioning Attribute; Protein Dynamics; Protein Engineering; Proteins; Relative (related person); Role; Senile Plaques; Series; Solutions; Sorting - Cell Movement; Sphingomyelins; Structure; Testing; Thermodynamics; Transmembrane Domain; Variant; Work; base; chemical property; design; flexibility; insight; novel; prevent; research study; restraint; secretase; therapeutic development; unilamellar vesicle

DESCRIPTION (provided by applicant): Nearly 5 million people in the US alone are afflicted with Alzheimer's disease, and there is currently no means to prevent or treat the disease. Though the molecular basis of the disease is unclear, the deposition of amyloid plaques in the brain is a key hallmark of the disease. These plaques are widely believed to be pathogenic, and tremendous efforts have focused on the mechanism governing their formation. Amyloid plaques are composed of the 42-residue amyloid ß peptide (Aß), which is generated through proteolytic cleavage of the amyloid precursor protein (APP) by ß-secretase. This enzyme is localized within cholesterol-rich lipid raft membrane domains, while the APP substrate exists in both the fluid-phase and lipid raft domains of cellular membranes. Therefore, the efficiency with which the Aß peptide is generated may be governed by the distribution of APP between the raft and non-raft membrane domains. Recent work in the Sanders lab on the 99-residue C-terminal fragment of APP (C99) has revealed a cholesterol binding pocket within the TM domain (present in APP). These studies have confirmed that C99 binds cholesterol with high affinity in bilayers, which suggests that the localization of C99 (and APP) may depend on the distribution of cholesterol in biological membranes. We recently tested this hypothesis by characterizing the localization of C99 within phase-separated giant unilamellar vesicles (GUVs), which contain both fluid phase (Lα) and liquid-ordered (Lo) domains. The results show that C99 is specifically localized within raft-like Lo domains. However, C99 variants carrying mutations that abolish cholesterol binding strongly prefer the non-raft Lα phase. This confirms cholesterol binding directly affects the distribution of C99 within the membrane. The most intuitive explanation for this phenomenon is that C99 is driven into the Lo domain due to the high concentration of the cholesterol ligand, which leads to favorable binding energetics. However, thermodynamic evaluations of this partitioning suggest that binding energetics cannot account for the observed differences. Thus, the physical mechanism for this coupled binding and partitioning remains unclear. In the following, I propose a series of experiments aimed at dissecting the energetic contributions of both the membrane and the protein in the coupled binding and partitioning of C99. I will first use EPR spectroscopy to assess the binding energetics of cholesterol in Lo and Lα like membranes. The results will suggest whether the cholesterol binding energetics are sensitive to changes in the bilayer. Next, I will use protein engineering and confocal fluorescence microscopy to determine how differences in the length and rigidity of the TM domain affect its partitioning. These studies will reveal the structural features of C99 that are critical for its sorting within te membrane. Finally, I will examine the structural dynamics of free and cholesterol-bound C99s using solution NMR in both Lo and Lα like bicelles in order to determine how bilayers affect its binding mode. Together, the results will provide novel insights into the molecular basis of Alzheimer's disease and elucidate the molecular determinants of protein sorting within the membrane.

描述（由申请人提供）：仅在美国就有近 500 万人患有阿尔茨海默病，目前尚无预防或治疗该疾病的方法，尽管该疾病的分子基础尚不清楚，但淀粉样斑块的沉积在体内。人们普遍认为这些斑块具有致病性，人们对淀粉样斑块由 42 个残基淀粉样蛋白组成的机制进行了巨大的研究。 β 肽 (Aß)，通过 β-分泌酶对淀粉样前体蛋白 (APP) 进行蛋白水解裂解而产生。该酶位于富含胆固醇的脂筏膜域内，而 APP 底物存在于液相和液相中。因此，Aß 肽的生成效率可能受 APP 在筏膜和非筏膜域之间的分布的影响。 APP (C99) 的 99 个残基 C 端片段揭示了 TM 结构域内的胆固醇结合口袋（存在于 APP 中）。这些研究证实，C99 在双层中以高亲和力结合胆固醇，这表明 C99 的定位。 （和 APP）可能取决于生物膜中胆固醇的分布，我们最近通过表征 C99 在相分离的巨型单层囊泡中的定位来测试这一假设。 (GUV)，同时包含液相 (Lα) 和液体有序强 (Lo) 结构域。结果表明，C99 特异性定位于筏状 Lo 结构域内。然而，携带废除胆固醇结合的突变的 C99 变体更喜欢非结构域。这证实了胆固醇结合直接影响C99在膜内的分布，对此现象最直观的解释是，由于高浓度的胆固醇配体，C99被驱动进入Lo结构域，从而导致有利的结合。然而，这种分配的热力学评估表明，结合能量学无法解释所观察到的差异，因此，这种耦合结合和分配的物理机制仍不清楚。在下文中，我提出了一系列旨在剖析能量贡献的实验。我将首先使用 EPR 光谱来评估 Lo 和 Lα 样膜中胆固醇的结合能量，结果将表明胆固醇结合能量是否对 C99 的变化敏感。接下来，我将使用蛋白质工程和共焦荧光显微镜来确定 TM 结构域的长度和刚性差异如何影响其划分。这些研究将揭示 C99 的结构特征，这些特征对于其在膜内的分选至关重要。最后，我将使用 Lo 和 Lα 等双分子中的溶液 NMR 检查游离和胆固醇结合的 C99 的结构动力学，以确定双层如何影响其结合模式，这些结果将为人们提供新的见解。阿尔茨海默病的分子基础，并阐明膜内蛋白质分类的分子决定因素。