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（由适用提供）：仅在美国，就有近500万人患有阿尔茨海默氏病，目前没有预防或治疗这种疾病的能力。尽管该疾病的分子基础尚不清楚，但在大脑中淀粉样蛋白斑块的沉积是该疾病的关键标志。这些斑块被普遍认为是致病性的，并且巨大的努力集中在控制其形成的机制上。淀粉样蛋白斑块由42个残留淀粉样β辣椒（Aß）组成，该淀粉样蛋白（Aß）是通过淀粉样蛋白前体蛋白（APP）通过β-分泌酶产生的。该酶位于富含胆固醇的脂质筏膜结构域中，而APP底物均存在于细胞膜的流体相和脂质筏结构域中。因此，产生Aß胡椒的效率可以受筏和非羊膜域之间的应用分布的控制。在Sanders Lab的最新工作中，APP的99个残留C末端片段（C99）揭示了TM域内的胆固醇结合口袋（APP中）。这些研究证实，C99在双层中结合具有高亲和力的胆固醇，这表明C99（和APP）的定位可能取决于胆固醇在生物膜中的分布。最近，我们通过表征了C99在相分离的巨型Unilamelar蔬菜（GUVS）中的定位，该假设既包含流体相（Lα）和液体排序（LO）域。结果表明，C99专门定位在类似筏的LO域中。然而，携带废除胆固醇结合的突变的C99变体强烈偏爱非抗活的Lα相。这证实胆固醇结合直接影响膜内C99的分布。对此现象的最直观的解释是，C99是由于胆固醇配体的高浓度而进入LO结构域的，这导致了有利的结合能。但是，对该分区的热力学评估表明，结合能量不能解释观察到的差异。这是这种耦合结合和分配的物理机制尚不清楚。在下文中，我提出了一系列实验，旨在解剖膜和蛋白质在C99的结合和分配中的能量贡献。我将首先使用EPR光谱来评估LO和Lα（如膜）中胆固醇的结合能。结果将表明胆固醇结合能量是否对双层变化敏感。接下来，我将使用蛋白质工程和共聚焦荧光显微镜来确定TM结构域长度和刚性的差异如何影响其分配。这些研究将揭示C99的结构特征，这些特征对于在TE膜内的分类至关重要。最后，我将使用溶液NMR在LO和Lα（如双丝）中使用溶液NMR检查自由和胆固醇结合的C99的结构动力学，以确定双层如何影响其结合模式。总之，结果将为阿尔茨海默氏病的分子基础提供新的见解，并阐明膜内蛋白质排序的分子决定剂。