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，其特征是大脑中淀粉样斑块的积累。这些空斑主要由A？淀粉样前体蛋白（APP）被两种蛋白酶水解而产生的肽。-和？-分泌酶。主要裂解产物是A？长度为40个残基的肽（a ?40）。然而，蛋白水解并不是高度特异性的，APP的裂解产物中约有10%是含有两个额外氨基酸的肽（A?42）。一个吗?42肽比A？它是大脑中淀粉样斑块的主要成分。拟议研究的总体目标是建立针对神经毒性A？低聚物以设计更有效的抑制剂。方法是结合结构方法和功能分析来测定A？42结构-功能关系在三个特定的目标。第一个目的是确定A?的可溶性低聚物和原纤维的结构和毒性。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低聚物的结构和低聚物-膜相互作用的动力学。单分子全内反射荧光显微镜将用于建立A？42个与膜双层结合。FTIR光谱将用于表征二级结构的变化作为膜组成的函数。溶液态核磁共振和固态核磁共振波谱将用于跟踪Aim 1中鉴定的低聚物、原纤维和原纤维特有的特定结构标记。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。肽抑制剂是基于A？原纤维。蛋白质抑制剂来源于髓鞘碱性蛋白的片段，我们已经证明它是一种天然的a ？脑白质抑制剂。提高对A的理解？-抑制剂的相互作用将影响抑制剂对可溶性低聚物的设计。目的是建立1)神经毒性可溶性低聚物在结构上与膜结合低聚物和A？42原纤维，2)两个氨基酸的加入如何改变A的结构？42低聚物和原纤维与毒性较小的A？3)抑制剂如何与A结合？42、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 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. PUBLIC HEALTH RELEVANCE: Alzheimer's disease is associated with the deposition of fibrillar plaques in the human brain that are composed primarily of a 42-amino acid amyloid-? peptide (A?42). The proposed research is to investigate the molecular structure of A?42 and to develop inhibitors that reduce its toxicity.