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。这些斑块主要由淀粉样前体蛋白（APP）通过两种蛋白酶（β-和γ-分泌酶）的蛋白水解产生的A β肽组成。初级切割产物是长度为40个残基的A β肽（A β 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（A β 42）的肽。A β 42肽比A β 40毒性更大，是脑中淀粉样斑块的主要成分。拟议研究的总体目标是建立靶向神经毒性Aesthetic寡聚体的小分子抑制剂的抑制机制，以设计更有效的抑制剂。该方法是将联合收割机结构分析与功能分析相结合，从三个方面确定A β 42的结构-功能关系。第一个目的是使用一套方法确定A β 42的可溶性低聚物和原纤维的结构和毒性，所述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。单分子全内反射荧光显微镜将用于确定结合至膜双层的A β 42的缔合-解离速率和分布。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Ablation寡聚体和原纤维的相互作用机制。小分子抑制剂包括天然产物姜黄素和白藜芦醇。肽抑制剂是根据黄芪原纤维的结构设计的。蛋白质抑制剂来源于髓鞘碱性蛋白的片段，我们已经证明髓鞘碱性蛋白是脑白色物质中的天然Ablation抑制剂。对AAPO 2-抑制剂相互作用的更好理解将影响可溶性低聚物的抑制剂的设计。目的是确定1）神经毒性可溶性低聚物在结构上如何不同于膜结合低聚物和A β 42原纤维，2）与毒性较小的A β 40形式相比，添加两个氨基酸如何改变A β 42低聚物和原纤维的结构，和3）抑制剂如何结合A β 42并防止毒性。

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 Aß peptides generated by proteolysis of the amyloid precursor protein (APP) by two proteases, ß- and γ-secretase. The primary cleavage product is an Aß peptide with a length of 40 residues (Aß40). However, proteolysis is not highly specific and ~10% of the cleavage products of APP are peptides with two additional amino acids (Aß42). The Aß42 peptide is more toxic than Aß40, 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 Aß oligomers in order to design more effective inhibitors. The approach is to combine structural methods with functional assays to determine Aß42 structure-function relationships in three specific aims. The first aim is to determine the structure and toxicity of the soluble oligomers and fibrils of Aß42 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 Aß42 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 Aß 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 Aß fibrils. The protein inhibitors are derived from fragments of the myelin basic protein, which we have shown is a natural Aß inhibitor in brain white matter. An improved understanding of Aß-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 Aß42 fibrils, 2) how the addition of two amino acids changes the structure of the Aß42 oligomers and fibrils compared to the less toxic Aß40 form, and 3) how inhibitors bind to Aß42 and prevent toxicity.