Gamma-secretase components and substrate interactions

A的产量增加|342被认为是PS1连锁常染色体显性遗传的致病性分子改变。 主导FAD。本申请中提出的研究的总体目标是检查APP-γ-分泌酶 为了更好地理解A的调节，|通过γ-分泌酶的342/40生产。底层 假设是γ-分泌酶，一种四种膜相关蛋白的复合物，可以采用不同的 不同的构象有利于Ap 40或Ap 42的产生。我们假设这些构象 通过核心y-组分的结构变化或核心y-组分的变构修饰而变得优选。 复杂.例如，使用基于FRET的测定，我们已经证明FAD相关的PS1 增加A（342）的突变（Berezovska等，2005）导致PS1构象的特异性改变。 Iwatsubo及其同事最近证明了Pen-2的N末端的延伸（Isoo等人，（2007年） 类似地导致y-复合物的结构变化，并提高Ap 42/40比率。假定变构 调节剂，如一些非甾体抗炎药，降低A| 342/40比率（Lleo等人，2004年）， 或非诺贝特，|342/40比例，在相反方向上改变PS1构象。因此我们 假设通过FRET测量的γ-分泌酶复合物的构象分布反映了功能性 导致A（342/40比率改变的y复合体的变化。第二个相关假设是， Ap和A的量|342/40比率还可能取决于APP如何以及在何处呈递给γ-分泌酶。FAD APP中y-切割位点附近的突变，以及修饰APP或PS1运输的相互作用物， 细胞分裂位点，可以以这种方式起作用。这些领域开发的先进成像方法 实验，旨在监测蛋白质构象和蛋白质-蛋白质相互作用在活细胞中， 基于FRET、双分子互补和全内反射显微镜。连同 生化测定，灵敏的ELISA，以及与PPG团队其他成员的相互作用， 实验将使我们能够探索亚细胞区室和Pen 2改变（Aim 1）的影响， APP和底物操作（Aim 2）和药理学干预（Aim 3）。重要的是 这些分析的发展将使我们能够检查相互作用对γ-分泌酶的影响。 分子，突变和新的底物，因为它们出现在PPG的努力。

Enhanced production of A|342is believed to be the pathogenic molecular alteration in PS1-linked autosomal dominant FAD. The overall goal of the studies proposed in this application is to examine APP-y-secretase interactions, in order to better understand the regulation of A|342/4o production by y-secretase. The underlying hypothesis is that y-secretase, a complex of four membrane associated proteins, can adopt different conformations which differentially favor Ap40 or Ap42 production. We postulate that these conformations become preferred either through structural changes in core y-components or allosteric modifications of the complex. For example, using FRET based assay we have demonstrated that FAD associated PS1 mutations that increase A(342 (Berezovska et al., 2005) lead to a specific alteration in PS1 conformation. Iwatsubo and colleagues recently demonstrated that extension of the N terminus of Pen-2 (Isoo et al., 2007) similarly leads to structural change in y-complex and enhances the Ap42/40 ratio. Presumed allosteric modulators, such as some nonsteroidal anti-inflammatory drugs, which lower A|342/4o ratio (Lleo et al., 2004), or fenofibrate, which increases A|342/4o ratio, alter PS1 conformation in opposite directions. We therefore postulate that the conformational profile of the y- secretase complex measured by FRET reflects functional changes in the y-complex that lead to alterations in A(342/40 ratios. A second, related hypothesis is that the amount of Ap and A|342/40 ratio may also depend on how and where APP is presented to y-secretase. FAD mutations near the y-cleavage site in APP, and interactors that modify either APP or PS1 trafficking, and the cellular site of cleavage, may act in this fashion. The advanced imaging methodologies developed in these experiments, designed to monitor protein conformation and protein - protein interactions in living cells, are based on FRET, bimolecular complementation, and total internal reflection microscopy. Together with biochemical assays, sensitive ELISA, and interactions with other members of the PPG team, the proposed experiments will allow us to explore the effects of subcellular compartments and Pen2 alterations (Aim1), APP and substrate manipulations (Aim2), and pharmacological interventions (Aim3). Importantly, the development of these assays will allow us to examine the consequences on y-secretase of interacting molecules, mutations, and novel substrates as they emerge from the PPG effort.