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

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手段。虽然该疾病的分子基础尚不清楚，但淀粉样斑块在大脑中的沉积是该疾病的一个关键标志。这些斑块被广泛认为是致病的，并且大量的努力集中在控制它们形成的机制上。淀粉样斑块由42-残基淀粉样肽(A)组成，A -残基淀粉样肽是通过分泌酶对淀粉样前体蛋白（APP）的蛋白水解裂解而产生的。该酶定位于富含胆固醇的脂筏膜结构域，而APP底物存在于细胞膜的液相和脂筏结构域。因此，A肽的生成效率可能取决于APP在筏膜和非筏膜结构域之间的分布。Sanders实验室最近对APP的99位残基c端片段（C99）的研究发现，在APP的TM结构域中存在一个胆固醇结合袋。这些研究证实了C99在双分子层中以高亲和力结合胆固醇，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。我们最近通过表征C99在相分离的巨型单层囊泡（GUVs）中的定位来验证这一假设，GUVs包含流体相（L -）和液体有序（Lo）结构域。结果表明，C99特异定位于筏状Lo结构域内。然而，携带消除胆固醇结合突变的C99变异体强烈倾向于非筏型L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，从而产生有利的结合能。然而，这种分配的热力学评价表明，结合能不能解释观察到的差异。因此，这种耦合绑定和分配的物理机制仍然不清楚。在下文中，我提出了一系列旨在剖析膜和蛋白质在C99的耦合结合和分配中的能量贡献的实验。我将首先使用EPR光谱来评估胆固醇在Lo和L样膜中的结合能。结果将提示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差异如何影响其分配。这些研究将揭示C99的结构特征，这对其在膜内的分选至关重要。最后，我将研究游离和胆固醇结合的c99的结构动力学，使用溶液核磁共振在Lo和L样双胞体中，以确定双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选的分子决定因素。

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 � 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.