FAD Mutations in the Amyloid Precursor Protein

描述（由申请人提供）：某些形式的阿尔茨海默病与γ -分泌酶产生的长形式β淀粉样肽相对于毒性较小的短形式（长形式= AB42；短形式= AB40）的增加直接相关。我们还知道，某些类似药物的小分子也会扰乱正常的AB42:AB40的产生比。在这个项目中，我们将首次提供蛋白质前体的结构表征，淀粉样蛋白b肽的长形式和短形式都是通过y分泌酶裂解产生的：淀粉样蛋白前体蛋白的N端672-770片段（APP672-770）。该蛋白的野生型APP672-770和已知的疾病相关突变形式都将被表征。通过分析、比较和对比不同形式的APP672-770的结构，我们将能够制定实验可测试的假设，准确地解释淀粉样蛋白- β肽的产生是如何以及为什么出错的。我们还将表征APP672-770与一个已知通过γ分泌酶调节其裂解的小分子之间形成的复合物。从这些研究中获得的见解可能最终被用作开发新的治疗药物的基础，这些药物被设计用来禁止或纠正某些形式的阿尔茨海默病中发生的淀粉样蛋白前体的异常加工。在初步数据中，我们成功地表达和纯化了APP672-770的野生型和疾病相关突变型，并表明可以获得高质量的核磁共振光谱。具体目的是：(1)利用核磁共振等方法表征野生型APP672-770的三维结构、动力学和膜相互作用。APP672-770是一种跨膜蛋白，作为γ -分泌酶裂解产生淀粉样β肽的底物。(2)表征与家族性阿尔茨海默病（FAD）相关的APP672-770突变形式的三维结构、动力学和膜相互作用。结果将与野生型蛋白的结果进行比较，以阐明突变如何促进阿尔茨海默病的结构生物物理基础。(3)表征一个小分子与APP672-770结合，既抑制γ -分泌酶裂解，又降低AB42:AB40的产生比。先前的研究表明，感兴趣的分子通过与APP672-770的预结合而不是通过与γ分泌酶的结合起作用。

DESCRIPTION (provided by applicant): Some forms of Alzheimer's disease are directly related to the enhanced production by gamma-secretase of a long form of the amyloid beta peptide relative to less toxic shorter forms (long form = AB42; short form = AB40). It is also known that certain small drug-like molecules can also perturb the normal AB42:AB40 production ratio. In this project we will provide a first-ever structural characterization of the protein precursor from which both long and short forms of the amyloid-B peptide are derived via y-secretase cleavage: the N- terminal 672-770 fragment of the amyloid precursor protein (APP672-770). Both wild type APP672-770 and known disease-linked mutant forms of this protein will be characterized. By analyzing, comparing, and contrasting the structures of the different forms of APP672-770 we will be able to formulate experimentally-testable hypotheses for exactly how and why amyloid-beta peptide production goes awry. We will also characterize the complex formed between APP672-770 and a small molecule known to modulate its cleavage by gamma-secretase. Insight from these studies may ultimately be used as the basis for developing novel therapeutic agents that are designed either to prohibit or to correct the aberrant processing of the amyloid precursor that occurs in some forms of Alzheimer's disease. In preliminary data we have succeeded in expressing and purifying both wild type and disease-linked mutant forms of APP672-770 and have shown that high quality NMR spectra can be obtained. The specific aims are: (1) Characterize the 3-D structure, dynamics, and membrane interactions of wild type APP672-770 using NMR spectroscopy and other methods. APP672-770 is a transmembrane protein that serves as the substrate for gamma- secretase cleavage to produce the amyloid-beta peptide. (2) Characterize the 3-D structures, dynamics, and membrane interactions of mutant forms of APP672-770 that have been linked to familial Alzheimer's disease (FAD). Results will be compared to those for the wild type protein in order to illuminate the structural biophysical basis for how mutations promote Alzheimer's disease. (3) Characterize the binding of a small molecule to APP672-770 that both inhibits gamma-secretase cleavage and lowers the AB42:AB40 production ratio. The molecule of interest has previously been shown to act through pre-association with APP672-770 rather than through association with gamma-secretase.