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: 7227101
• 负责人: OKSANA BEREZOVSKA
• 金额: $ 24.21万
• 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/7227101
• 关键词: AP40; APP-PS1; Active Sites; Adopted; Affect; Age of Onset; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Appendix; Biochemical; Biological; Biological Assay; Biology; Brain; Catalytic Domain; Cell surface; Cells; Chromosome Pairing; Cleaved cell; Complex; Condition; Data; Dendritic Spines; Deposition; Disease; Drug Design; Elements; Elevation; Endopeptidases; Fluorescence; Fluorescence Resonance Energy Transfer; Future; Generations; Genetic; Goals; Green Fluorescent Proteins; 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; Proteins; 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; research study; response; secretase; therapeutic target; trafficking

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）中早老素（PS）相关Ap40和Ap42产生的分子基础。由于多通道跨膜蛋白结晶的复杂性和难度，对PS/y-分泌酶复合物的结构知之甚少。我们假设PS/y-secretase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。我们的观察，使用一种新的基于FRET的技术在完整的细胞以及生化方法；提供检测完整细胞中PS1/y分泌酶构象的方法，以及确定PS1和y分泌酶底物的接近性，包括APP和Notch。我们的观察结果支持一个具有以下重要可测试特征的模型：成熟的PS1与其他y分泌酶复合物成员结合，并采用N-和c -末端（NT和CT）紧密相连的构象；APP对y分泌酶活性位点的特异性呈现在有利于AB40和AB42的条件下发生变化。我们将从几个重要的临床角度来检验这个模型。fad相关的PS1突变导致ap42 / 40比值升高，但其机制尚不清楚。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，这代表了一种与PS1/y-分泌酶活性位点与APP对齐改变相关的致病性构象变化，有利于Ap42的切割。我们将测试几个模型来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，这是一种与FAD突变相反的表型。我们将验证一种假设，即选择性改变AB42/40比率的药物可以通过变构调节PS1/y-分泌酶和PS1/APP相互作用的构象，以与FAD突变相反的方式来实现。我们将探讨Ap42降低y分泌酶的变构调节剂是否可以“固定”致病性FAD突变体PS1构象（Aim2）。最后，我们提出验证激活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的假设。我们将利用新FRET技术的高空间分辨率，荧光寿命成像显微镜（FLIM），这是唯一适合监测完整和/或活细胞中蛋白质和复合物的相对构象变化，显示在完整细胞的亚细胞区室中发生这些变化（Aim3），并最终旨在将这些研究扩展到小鼠大脑。获得的结果将为PS1/y分泌酶复合物的构象变化及其与细胞内底物的相互作用提供重要的新见解，因此将有助于设计y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-底物接近度的试验将有助于开发可能被证明对理解疾病相关蛋白质构象变化有用的方法。