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A？淀粉样前体蛋白(APP)被两种酶，？-和？-分泌酶蛋白分解而产生的多肽。主要的切割产物是A？多肽，长度为40个残基(A？40)。然而，蛋白水解性不强，APP的裂解产物中约有10%是含有两个附加氨基酸(A42)的多肽。A？42肽比A？40毒性更大，是大脑中淀粉样斑块的主要成分。这项研究的首要目标是建立针对神经毒素A？的小分子抑制剂的抑制机制。低聚物才能设计出更有效的抑制剂。方法是将结构方法和功能分析相结合，在三个特定的目标中确定A？42结构-功能关系。第一个目的是用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等一系列方法确定A？42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A42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A？低聚物和纤维。小分子抑制剂包括天然产物姜黄素和白藜芦醇。在此基础上设计了多肽抑制剂。纤维。这些蛋白抑制剂来自髓鞘碱性蛋白的片段，我们已经证明了它是一种天然的A？脑白质中的抑制物。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。目的是确定1)神经毒性可溶低聚物与膜结合低聚物和A42纤维在结构上有何不同，2)与毒性较低的A40形式相比，两个氨基酸的添加如何改变A42低聚物和纤维的结构，以及3)抑制剂如何与A42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 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.