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: 8856220
• 负责人: Jonathan Patrick Schlebach
• 金额: $ 5.6万
• 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/8856220
• 关键词: Accounting; Affect; Affinity; Alzheimer' s Disease; 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; Phase; Phosphorylcholine; Play; Positioning Attribute; Protease Domain; Protein Dynamics; Protein Engineering; Proteins; Relative (related person); Role; Senile Plaques; Series; Solutions; Sorting - Cell Movement; Sphingomyelins; Structure; Testing; Thermodynamics; Transmembrane Domain; Variant; Work; amyloid peptide; base; chemical property; design; flexibility; insight; novel; peptide A; 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 � peptide (A�), which is generated through proteolytic cleavage of the amyloid precursor protein (APP) by �-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 A� 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 (L�) 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 L� 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 L� 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 L� 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（由申请人证明）：仅在美国，阿尔茨海默氏病或​​治疗该疾病的近500万人。这些斑块被广泛认为是致病性的淀粉样蛋白斑。 S> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> </s> PID筏膜结构域，而APP底物的两个 - 流体型脂质脂质筏结构域则存在于细胞膜的脂质脂质结构域中。在Sanders实验室中，APP的99个残留C末端片段（C99）揭示了与TM结构域的胆固醇结合口袋（APP中存在）。表明C99（和APP）的定位可能取决于胆固醇在生物膜中的分布，我们最近通过表征C99在相位分离的巨型Unilamellar囊泡（GUVS）中的定位来对此进行了测试。 - 订购（LO）结构域。结果表明，C99特定于木筏样lo iver中，C99变体的calying突变强烈偏爱非抗发射液的胆固醇。膜的最直观的解释是促进胆固醇的胆固醇配体，即绑定无法观察到的差异。 C99的耦合结合和分配中的蛋白。结合模式。