Gamma-secretase components and substrate interactions

A|342的产生增强被认为是PS1连锁常染色体的致病分子改变 占主导地位的时尚。本申请中提出的研究的总体目标是检查app-y-分泌酶。 相互作用，以更好地了解γ-分泌酶对A|342/40产生的调节。潜在的 假说是由四种膜相关蛋白组成的复合体--y-分泌酶可以采用不同的 不同地有利于Ap40或Ap42产生的构象。我们假设这些构象 通过核心y-组分的结构变化或变构修饰 很复杂。例如，使用基于FRET的检测，我们已经证明了FAD相关的PS1 增加A(342)的突变(Berezovska等人，2005年)导致PS1构象的特定改变。 Iwatsubo及其同事最近证明了Pen-2 N末端延伸(Isoo等人，2007年) 同样会导致y-复合体的结构变化，并提高Ap42/40比率。推定变构 调节剂，如一些非类固醇抗炎药，可降低A|342/4O比率(Lleo等人，2004年)， 或非诺贝特，增加A|342/40比值，以相反的方向改变PS1构象。因此，我们 假设FRET测量的Y-分泌酶复合体的构象反映了功能 导致A改变的y-复合体的改变(342/40比率。第二个相关的假设是 AP的量和A|342/40比率也可能取决于APP呈现给γ-分泌酶的方式和位置。淡出 APP中Y-裂解位点附近的突变，以及改变APP或PS1交易的相互作用项，以及 卵裂的细胞位置，可能以这种方式起作用。在这些平台上开发的高级成像方法 旨在监测活细胞中蛋白质构象和蛋白质相互作用的实验是 基于FRET、双分子互补和全内反射显微镜。与.一起 生化分析，敏感的ELISA，以及与PPG团队其他成员的互动，建议 实验将使我们能够探索亚细胞间隔和Pen2改变的影响(Aim1)， APP和底物操作(AIM2)和药物干预(AIM3)。重要的是， 这些分析的发展将使我们能够检验相互作用对y-分泌酶的影响。 从PPG努力中出现的分子、突变和新的底物。

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.