Mechanism of inhibition of APP processing and amyloid formation

描述（由申请人提供）：阿尔茨海默病（AD）是一种神经退行性疾病，其特征在于脑中淀粉样蛋白斑块的积累。这些斑块主要由A？淀粉样前体蛋白（APP）被两种蛋白酶水解产生的肽，然后呢？分泌酶主要裂解产物是A？肽的长度为40个残基（A？40）。然而，蛋白水解不是高度特异性的，APP的约10%的裂解产物是具有两个额外氨基酸的肽（A？42）。A？42肽比A？40，并且是脑中淀粉样斑块的主要成分。拟议的研究的总体目标是建立小分子抑制剂的抑制机制，针对神经毒性A？低聚物，以设计更有效的抑制剂。该方法是将结构方法与功能测定方法联合收割机相结合来确定A？三个具体目标中的42个结构-功能关系。第一个目的是确定的结构和毒性的可溶性低聚物和原纤维的A？42使用一套方法，包括溶液和固态核磁共振（NMR）光谱，单触原子力显微镜和傅里叶变换红外（FTIR）光谱。第二个目的是确定膜结合寡聚体的结构和寡聚体-膜相互作用的动力学。将使用单分子全内反射荧光显微镜来确定A？42结合到膜双层。FTIR光谱将用于表征作为膜组成函数的二级结构变化。溶液态NMR和固态NMR光谱将用于跟踪目标1中鉴定的寡聚体、原纤维和原纤维特有的特定结构标记物。第三个目的是确定小分子，肽和蛋白质抑制剂与A？低聚物和原纤维。小分子抑制剂包括天然产物姜黄素和白藜芦醇。肽抑制剂是根据A？纤维蛋白质抑制剂来自髓鞘碱性蛋白的片段，我们已经证明这是一种天然的A？脑白色物质中抑制剂。更好地理解A？抑制剂相互作用将影响可溶性低聚物的抑制剂的设计。我们的目标是建立1）如何神经毒性的可溶性低聚物不同的结构从膜结合的低聚物和A？42原纤维，2）如何添加两个氨基酸改变的A？42低聚物和原纤维相比，毒性较小的A？40的形式，和3）如何抑制剂绑定到A？42、防止中毒。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disease characterized by the accumulation of amyloid plaques in the brain. These plaques are composed of mostly A? peptides generated by proteolysis of the amyloid precursor protein (APP) by two proteases, ?- and ?-secretase. The primary cleavage product is an A? peptide with a length of 40 residues (A?40). However, proteolysis is not highly specific and ~10% of the cleavage products of APP are peptides with two additional amino acids (A?42). The A?42 peptide is more toxic than A?40, and is the principal component of amyloid plaques in the brain. The overarching goal of the proposed research is to establish the mechanism of inhibition for small molecule inhibitors that target neurotoxic A? oligomers in order to design more effective inhibitors. The approach is to combine structural methods with functional assays to determine A?42 structure-function relationships in three specific aims. The first aim is to determine the structure and toxicity of the soluble oligomers and fibrils of A?42 using a suite of methods including solution and solid-state nuclear magnetic resonance (NMR) spectroscopy, single touch atomic force microscopy and Fourier transform infrared (FTIR) spectroscopy. The second aim is to determine the structure of membrane-bound oligomers and the dynamics of oligomer- membrane interactions. Single molecule total internal reflection fluorescence microscopy will be used to establish the association-dissociation rates and distribution of A?42 bound to membrane bilayers. FTIR spectroscopy will be used to characterize the changes in secondary structure as a function of membrane composition. Solution-state NMR and solid-state NMR spectroscopy will be used to follow specific structural markers identified in Aim 1 that are unique to the oligomers, protofibrils and fibrils. The third aim is to determine the mechanism of interaction of small molecule, peptide and protein inhibitors with A? oligomers and fibrils. The small molecule inhibitors include the natural products, curcumin and resveratrol. The peptide inhibitors are designed on the basis of the structure of the A? fibrils. The protein inhibitors are derived from fragments of the myelin basic protein, which we have shown is a natural A? inhibitor in brain white matter. An improved understanding of A?-inhibitor interactions will impact the design of inhibitors to the soluble oligomers. The goal is to establish 1) how the neurotoxic soluble oligomers differ in structure from membrane-bound oligomers and A?42 fibrils, 2) how the addition of two amino acids changes the structure of the A?42 oligomers and fibrils compared to the less toxic A?40 form, and 3) how inhibitors bind to A?42 and prevent toxicity.