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

描述(申请人提供)：仅在美国就有近500万人患有阿尔茨海默氏症，目前还没有预防或治疗这种疾病的手段。虽然这种疾病的分子基础尚不清楚，但大脑中淀粉样斑块的沉积是这种疾病的一个关键标志。这些斑块被广泛认为是致病的，巨大的努力集中在它们形成的机制上。淀粉样斑块是由42个残基的淀粉样多肽(A？)组成的，这种多肽是由淀粉样前体蛋白(APP)被？分泌酶降解而产生的。该酶定位于富含胆固醇的脂筏结构域中，而APP底物同时存在于细胞膜的液相和脂筏结构域中。因此，APP在RAFT膜结构域和非RAFT膜结构域之间的分布可能决定了A？多肽的生成效率。桑德斯实验室最近对APP(C99)的99个残基的C-末端片段进行的研究揭示了TM域(存在于APP中)中的胆固醇结合口袋。这些研究证实，C99与胆固醇以高亲和力结合在双层中，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。我们最近通过表征C99在相分离的巨大单层囊泡(GUV)中的定位来验证这一假说，GUV包含液体相(Lα)和液体有序(LO)结构域。结果表明，C99特异性定位于类RAFT LO结构域中。然而，携带可取消胆固醇结合的突变的C99变体强烈倾向于非RAFT的Lα期。这证实了胆固醇结合直接影响C99在膜内的分布。对这一现象最直观的解释是，由于胆固醇配体的高浓度，C99被驱动到LO结构域，这导致了有利的结合能。然而，对这种分配的热力学评估表明，结合能不能解释观察到的差异。因此，这种结合和分割的物理机制仍然不清楚。在接下来的实验中，我提出了一系列实验，旨在剖析膜和蛋白质在C99偶联结合和分配中的能量贡献。我将首先使用电子顺磁共振波谱来评估胆固醇在LO和L类α膜中的结合能。结果将表明胆固醇结合能是否对双分子层的变化敏感。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结构域的长度和刚性的差异如何影响其分割。这些研究将揭示C99的结构特征，这些结构特征对其在TE膜中的分选至关重要。最后，我将使用溶液核磁共振在LO和L类α双分子中研究游离和胆固醇结合的C99s的结构动力学，以确定双分子层如何影响其结合模式。总之，这些结果将为阿尔茨海默病的分子基础提供新的见解，并阐明膜内蛋白质分类的分子决定因素。

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.