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Structure and Function of Gamma-Secretase in Familial Alzheimer's Disease

家族性阿尔茨海默病中伽玛分泌酶的结构和功能

基本信息

批准号：
10388359
负责人：
Michael S Wolfe
金额：
$ 58.21万
依托单位：
UNIVERSITY OF KANSAS LAWRENCE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10388359
关键词：
Active Sites Alzheimer&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos s disease therapeutic Amino Acids Amyloid beta-42 Amyloid beta-Protein Amyloid beta-Protein Precursor Aspartate Aspartic Endopeptidases Brain Carboxypeptidase Carboxypeptidase A Cerebrum Chemicals Complex Computational Biology Cryoelectron Microscopy Deposition Disease Pathway Enzymes Etiology Functional disorder G-substrate Generations Goals Induced Mutation Inherited Lateral Link Membrane Membrane Proteins Missense Mutation Modeling Molecular Molecular Conformation Mutation Neurodegenerative Disorders Pathogenesis Pathogenicity Peptide Hydrolases Peptides Prevention strategy Protein Precursors Proteolysis Reporting Senile Plaques Stress Structure Substrate Domain Substrate Interaction Time Transmembrane Domain Variant Visualization Water amyloid peptide analog base beta secretase crosslink design enzyme substrate complex familial Alzheimer disease gain of function gamma secretase inhibitor insight loss of function middle age mimetics molecular dynamics mutant peptidomimetics presenilin presenilin-1 presenilin-2 structural biology synthetic peptide therapeutically effective

项目摘要

Familial Alzheimer’s disease (FAD) is caused by dominant mutations in the amyloid-β (Aβ) precursor protein (APP) and presenilin-1 and -2 (PSEN1, PSEN2). APP is cleaved by β-secretase, then within its single transmembrane domain (TMD) by γ-secretase to produce Aβ, which deposits as cerebral plaques. PSEN is the catalytic component of γ-secretase, a membrane-embedded protease complex. Thus, FAD mutations are only in the substrate and protease that produce Aβ; nevertheless, pathogenic mechanisms remain controversial. Most PSEN FAD mutations show reduced proteolysis (loss of function) but also increase proportions of aggregation-prone 42-residue Aβ peptide (Aβ42) (gain of function). However, γ-secretase has multiple proteolytic functions: Initial endoproteolytic (ε) cleavage of APP substrate produces long Aβ that is trimmed via a carboxypeptidase activity, and FAD-mutant γ-secretases are deficient in this trimming function. New understanding of multiple proteolytic functions of γ-secretase along with recent cryo-EM structure elucidation of the protease-substrate complex now make possible a deeper understanding of effects of FAD mutations. The goal here is to combine chemical, structural, and computational biology to elucidate how presenilin FAD mutations alter γ-secretase structure, dynamics, and function. Such understanding should give insight into how this membrane-embedded protease complex recognizes and processively proteolyzes transmembrane substrates, provide critical clues to pathogenic mechanisms of FAD, and suggest new strategies for prevention of FAD. To this end, we propose to: (1) Develop chemical probes to trap γ-secretase in different stages of substrate interaction for structural analysis by cryo-EM. We developed full TMD substrate mimics to trap active enzyme in a conformation poised for intramembrane proteolysis. Designed variations should allow visualization of the transition states for ε proteolysis, carboxypeptidase cleavage, TMD helix unwinding, and lateral gating of substrate. (2) Perform molecular dynamics (MD) simulations of substrate interaction with FAD-mutant γ-secretase. We computationally restored catalytic aspartates, modeled entry of water to the active site, and captured activation of the computationally restored WT enzyme. We will determine effects of FAD PSEN1 mutations on γ-secretase structural dynamics and interaction with APP substrate and TMD mimics. (3) Develop synthetic substrate probes for analysis of proteolytic dysfunction of FAD-mutant γ-secretase. We developed a set of such functional probes of γ-secretase processing of APP TMD, validating them as convenient and appropriate synthetic surrogates for APP substrate. We will employ these and other proposed substrate probes to determine effects of FAD-mutant γ-secretases on ε proteolysis and specific carboxypeptidase trimming steps.

家族性阿尔茨海默病 (FAD) 是由淀粉样蛋白-β (Aβ) 前体的显性突变引起的蛋白 (APP) 和早老素-1 和 -2 (PSEN1、PSEN2)。 APP被β-分泌酶裂解，然后在其单一的跨膜结构域（TMD）通过γ-分泌酶产生Aβ，沉积为脑斑块。 PSEN 是 γ-分泌酶的催化成分，一种膜嵌入的蛋白酶复合物。因此，FAD 突变仅产生 Aβ 的底物和蛋白酶；然而，致病机制仍存在争议。大多数 PSEN FAD 突变表现出蛋白水解减少（功能丧失），但也增加了易于聚集的 42 残基 Aβ 肽 (Aβ42)（功能获得）。然而，γ-分泌酶具有多种蛋白水解功能：APP 底物的初始内蛋白水解 (ε) 裂解产生长 Aβ，并通过羧肽酶活性，而 FAD 突变型 γ-分泌酶缺乏这种修剪功能。对γ-分泌酶的多种蛋白水解功能以及最新冷冻电镜结构的新认识蛋白酶-底物复合物的阐明现在可以更深入地了解 FAD 的影响突变。这里的目标是结合化学、结构和计算生物学来阐明如何早老素 FAD 突变会改变 γ 分泌酶的结构、动力学和功能。这样的理解应该给予深入了解这种膜嵌入的蛋白酶复合物如何识别和进行蛋白水解跨膜底物，为 FAD 致病机制提供关键线索，并提出新的思路预防 FAD 的策略。为此，我们建议： (1) 开发化学探针在底物相互作用的不同阶段捕获γ-分泌酶以进行结构分析通过冷冻电镜分析。我们开发了完整的 TMD 底物模拟物，以在构象中捕获活性酶用于膜内蛋白水解。设计的变化应该允许 ε 的过渡态可视化蛋白水解、羧肽酶裂解、TMD 螺旋解旋和底物的横向门控。 (2) 对底物与 FAD 突变型 γ-分泌酶的相互作用进行分子动力学 (MD) 模拟。我们通过计算恢复了催化天冬氨酸，模拟了水进入活性位点的过程，并捕获了计算恢复的 WT 酶的激活。我们将确定 FAD PSEN1 突变对 γ-分泌酶结构动力学以及与 APP 底物和 TMD 模拟物的相互作用。 (3) 开发用于分析FAD突变型γ-分泌酶蛋白水解功能障碍的合成底物探针。我们开发了一套这样的 APP TMD γ-分泌酶加工功能探针，并验证它们为方便且合适的 APP 底物合成替代物。我们将采用这些和其他建议的底物探针以确定 FAD 突变体 γ 分泌酶对 ε 蛋白水解和特异性的影响羧肽酶修剪步骤。