Mechanism of inhibition of APP processing and amyloid formation

描述(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聚。这些斑块主要由淀粉样前体蛋白(APP)在两种酶-和γ--分泌酶作用下的蛋白分解所产生的A？多肽组成。主要的切割产物是长度为40个残基的A？多肽(A？40)。然而，蛋白水解性不强，APP的裂解产物中~10%是含有两个额外氨基酸的多肽(A？42)。A？42比A？40的毒性更大，是大脑中淀粉样斑块的主要成分。这项研究的主要目标是建立针对神经毒性低聚物的小分子抑制剂的抑制机制，以便设计更有效的抑制剂。该方法是将结构方法与功能分析相结合，以确定三个特定目标中的A？42结构-功能关系。第一个目标是用包括溶液和固体核磁共振光谱、单触摸原子力显微镜和傅里叶变换红外光谱在内的一系列方法来确定Aç42的可溶低聚物和纤维的结构和毒性。第二个目标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被用来确定结合在膜双层上的A？42的缔合-解离速率和分布。FTIR光谱将被用来表征二级结构随膜组成的变化。溶液核磁共振和固态核磁共振波谱将用于跟踪目标1中确定的低聚物、原纤维和原纤维独有的特定结构标记。第三个目的是确定小分子、多肽和蛋白质抑制剂与低聚物和纤维相互作用的机制。小分子抑制剂包括天然产物姜黄素和白藜芦醇。多肽抑制剂是根据A？原纤维的结构设计的。这些蛋白抑制物来自髓鞘碱性蛋白的片段，我们已经证明，髓鞘碱性蛋白是脑白质中的一种天然的Aü抑制物。对A？-抑制剂相互作用的更好理解将影响针对可溶性低聚物的抑制剂的设计。我们的目标是确定1)神经毒性可溶低聚物与膜结合低聚物和A？42纤维的结构有何不同，2)与毒性较低的A？40形式相比，添加两种氨基酸如何改变A？42低聚物和纤维的结构，以及3)抑制剂如何与A？42结合并防止毒性。

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.