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

基本信息

批准号：
7617160
负责人：
OKSANA BEREZOVSKA
金额：
$ 23.72万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
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/7617160
关键词：
AP40 APP-PS1 Active Sites Adopted Affect Age of Onset Alzheimer&apos s Disease Amyloid beta-Protein Precursor Biochemical Biological Biological Assay Biology Brain Catalytic Domain Cell surface Cells Cleaved cell Complex Data Dendritic Spines Deposition Disease Drug Design Elements Fluorescence Fluorescence Resonance Energy Transfer Future Generations Genetic Goals Image Integral Membrane Protein Lead Life Link Measures Mediating Methodology Methods Microscopy Modeling Molecular Molecular Conformation Monitor Mus Mutation N-Methylaspartate Neurons Non-Steroidal Anti-Inflammatory Agents Pathology Peptide Hydrolases Peptides Pharmaceutical Preparations Phenotype Phosphotransferases Physiological Play Presynaptic Terminals Production Property Protein Conformation Relative (related person)Research Personnel Resolution Role Site Stimulus Structure Substrate Interaction Synapses Techniques Testing Therapeutic base design familial Alzheimer disease fluorophore insight interest member mutant neuronal cell body notch protein novel presenilin presenilin-1 presynaptic programs protein complex research study response secretase therapeutic target trafficking

项目摘要

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家族性阿尔茨海默病（FAD）中与早老素（PS）相关的Ap40和Ap42产生的分子基础。由于多次跨膜蛋白结晶的复杂性和难度，人们对 PS/γ 分泌酶复合物的结构知之甚少。我们假设 PS/γ-分泌酶以不同的构象存在，导致 APP 与催化位点的不同排列，从而产生不同的 AB 种类。我们在完整细胞中使用基于 FRET 的新型技术以及生化方法进行观察；提供了检查完整细胞中 PS1/y-分泌酶构象以及确定 PS1 和 y-分泌酶底物（包括 APP 和 Notch）的接近度的方法。我们的观察结果支持具有以下重要、可测试特征的模型：成熟的 PS1 与其他 y-分泌酶复合体成员结合，并采用 N 端和 C 端（NT 和 CT）靠近的构象； APP 对 γ-分泌酶活性位点的具体呈递在有利于 AB40 与 AB42 的条件下发生变化。我们将从几个临床重要的角度来测试这个模型。 FAD 相关的 PS1 突变导致 Ap42/4o 比率升高，但机制尚不清楚。我们的初步数据表明，FAD PS1 突变使 PS1 NT 和 CT 更加紧密地结合在一起，这代表了与 PS1/y-分泌酶活性位点与 APP 的排列变化相关的构象的致病性变化，有利于 Ap42 处的裂解。我们将测试几个模型来定义导致观察到的构象变化（Aiml）的精确分子机制。一些非甾体抗炎药已被证明可以选择性降低 AB42，这是一种与 FAD 突变相反的表型。我们将测试这样的假设：选择性改变 AB42/40 比例的药物可以通过以与 FAD 突变相反的方式变构调节 PS1/y-分泌酶的构象和 PS1/APP 相互作用来实现这一点。我们将探讨 Ap42 降低 y 分泌酶变构调节剂是否可以“修复”致病性 FAD 突变体 PS1 构象 (Aim2)。最后，我们建议测试以下假设：激活 y-分泌酶和/或其底物的生理刺激选择性地改变 PS1/y-分泌酶与神经元底物的相互作用。我们将利用新型 FRET 技术荧光寿命成像显微镜 (FLIM) 的高空间分辨率，该技术特别适合监测完整和/或活细胞中蛋白质和复合物的相对构象变化，以显示哪些亚细胞区室在完整的细胞中，这些变化会发生（目标3），最终目的是将这些研究扩展到小鼠大脑。获得的结果将为 PS1/γ-分泌酶复合物的构象变化及其与细胞环境内底物的相互作用提供重要的新见解，从而有助于设计 γ-分泌酶功能的变构调节剂作为治疗方法。更一般地说，监测 PS1 构象和 y-分泌酶底物接近度的测定将有助于开发可能有助于理解疾病相关蛋白质构象变化的方法。