Molecular mechanism of Presenilin-1 (PS1) linked Alzheimer's Disease pathology

Presenilin-1 (PS1) 与阿尔茨海默病病理学相关的分子机制

• 批准号: 7097634
• 负责人: OKSANA BEREZOVSKA
• 金额: $ 27.04万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7097634
• 关键词: Alzheimer' s disease; active sites; allosteric site; aspartic endopeptidases; conformation; enzyme inhibitors; enzyme mechanism; enzyme substrate complex; fluorescence resonance energy transfer; genetically modified animals; laboratory mouse; molecular pathology; presenilin

DESCRIPTION (provided by applicant): The long term goal of this application is to understand the molecular basis of presenilin (PS)-linked production of Ap40 and Ap42 in familial Alzheimer's disease (FAD). Very little is known about the structure of the PS/y-secretase complex due to the complexity and difficulty of crystallizing multipass transmembrane proteins. We hypothesize that PS/y-secretase exists in different conformations, leading to different alignments of APP with the catalytic site, resulting in the production of different AB species. Our observations, using a novel FRET based technique in intact cells as well as biochemical approaches; provide methods to examine PS1/y-secretase conformation in intact cells as well as to determine proximity of PS1 and y-secretase substrates, including APP and Notch. Our observations support a model with the following important, testable features: mature PS1 associates with other y-secretase complex members and adopts a conformation with N- and C-termini (NT and CT) close together; and the specific presentation of APP to the y-secretase active site changes under conditions favoring AB40 vs. AB42. We will test this model from several clinically important perspectives. FAD-linked PS1 mutations lead to elevations in Ap42/4o ratio, but the mechanism is unknown. Our preliminary data suggest that FAD PS1 mutations bring PS1 NT and CT even closer together, and that this represents a pathogenic change in conformation associated with an alteration in the alignment of the active site of PS1/y-secretase with APP, favoring cleavage at Ap42. We will test several models to define the precise molecular mechanism that leads to this observed change in conformation (Aiml). Some nonsteroidal anti-inflammatory drugs have been shown to selectively lower AB42, a phenotype opposite to FAD mutations. We will test the hypothesis that pharmacological agents that selectively alter the AB42/40 ratio can do so by allosterically modulating the conformation of PS1/y-secretase and PS1/APP interactions in the opposite way to FAD mutations. We will explore whether Ap42 lowering allosteric modulators of the y-secretase can "fix" the pathogenic FAD mutant PS1 conformation (Aim2). Finally, we propose to test the hypothesis that physiologic stimuli that activate y-secretase and/or its substrate selectively change PS1 /y-secretase interactions with substrates in neurons. We will take advantage of the high spatial resolution of the new FRET technique, Fluorescence Lifetime Imaging Microscopy (FLIM), which is uniquely suited for monitoring relative conformational changes of proteins and complexes in intact and/or live cells, to show in what subcellular compartments in intact cells these changes occur (Aim3), and ultimately aim to extend these studies to the mouse brain. The results obtained will provide important new insights into conformational changes in the PS1/y-secretase complexes and its interaction with substrates within a cellular context, and thus will help in the design of allosteric modulators of y-secretase function as a therapeutic approach. More generally, the assay of monitoring PS1 conformation and y-secretase -substrate proximity will help to help develop methodologies that may prove useful in understanding disease-related protein conformation changes.

描述（由申请人提供）：本申请的长期目标是了解presenilin（PS）连接的AP40和AP42在家族性阿尔茨海默氏病（FAD）中的分子基础。由于晶体跨膜蛋白的复杂性和难度，对PS/Y - 分泌酶复合物的结构知之甚少。我们假设PS/Y - 分泌酶以不同的构型存在，从而导致APP与催化位点的不同对齐，从而导致不同的AB物种产生。我们的观察结果是使用完整细胞和生化方法中基于FRET的新技术；提供了检查完整细胞中PS1/Y-分泌酶构象的方法，以及确定PS1和Y-分泌酶底物（包括App和Notch）的接近度。我们的观察结果支持具有以下重要，可检验的特征的模型：成熟的PS1与其他Y-分泌酶复合构件相关联，并采用与N-和C-termini（NT和CT）构成的构象；在有利于AB40与AB42的条件下，将APP的特定呈现给Y-分泌酶的活动位点发生变化。我们将从几个临床上重要的角度测试该模型。 FAD连接的PS1突变导致AP42/4O比的升高，但该机制尚不清楚。我们的初步数据表明，FAD PS1突变使PS1 NT和CT更加近距离，这代表了与PS1/Y - 分泌酶与APP的活性位点变化有关的构象变化的致病变化，有利于AP42的裂解。我们将测试几个模型，以定义导致这种观察到的构象变化的精确分子机制（AIML）。一些非甾体类抗炎药已被证明可以选择性地降低AB42，这是与FAD突变相反的表型。我们将测试以下假设：通过对PS1/Y-分酶和PS1/APP相互作用的构象以与FAD突变相反的方式，可以选择性地改变AB42/40比率的药理学剂可以做到这一点。我们将探索Y-分泌酶的变构调节剂的AP42是否可以“固定”致病性FAD突变体PS1构象（AIM2）。最后，我们建议检验以下假设：激活Y-分泌酶和 /或其底物的生理刺激选择性地改变了与神经元中底物的PS1 /Y-分泌酶相互作用。我们将利用新的FRET技术，荧光寿命显微镜（FLIM）的高空间分辨率，该技术非常适合监测完整和/或活细胞中蛋白质和复合物的相对构象变化，以显示完整细胞中的亚细胞隔分段的完整细胞中这些变化的哪些变化（AIM3），并扩展了这些小鼠的大脑。获得的结果将为PS1/Y-分泌酶配合物的构象变化提供重要的新见解及其在细胞环境中与底物的相互作用，因此将有助于设计Y-分泌酶的变构调节剂作为治疗方法。更一般而言，监测PS1构象和Y-分泌酶 - 基底近端的测定将有助于开发可能证明有助于理解与疾病相关的蛋白质构象变化有用的方法。