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

• 批准号: 10605227
• 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
• 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/10605227
• 关键词: Active Sites; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease therapeutic; Amino Acids; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Aspartate; Aspartic Endopeptidases; Binding; Brain; Carboxypeptidase; Carboxypeptidase A; Cerebrum; Chemicals; Complex; Computational Biology; Cryoelectron Microscopy; Deposition; Disease Pathway; Enzymes; Etiology; Functional disorder; 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; Proteolysis; Reporting; Senile Plaques; Stress; Structure; Substrate Domain; Substrate Interaction; Time; Transmembrane Domain; Variant; Visualization; Water; amyloid peptide; analog; beta secretase; crosslink; design; dominant genetic mutation; 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

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β淀粉样蛋白（Aβ）前体的显性突变引起的 蛋白（APP）和早老素-1和-2（PSEN 1，PSEN 2）。APP被β-分泌酶切割，然后在其单个 跨膜结构域（TMD）通过γ-分泌酶产生Aβ，其沉积为脑斑块。PSEN是 γ-分泌酶的催化组分，一种膜包埋的蛋白酶复合物。因此，FAD突变只是 在底物和蛋白酶产生Aβ;然而，致病机制仍然存在争议。 大多数PSEN FAD突变显示蛋白水解减少（功能丧失），但也增加蛋白水解的比例。 易于聚集的42-残基Aβ肽（Aβ42）（功能获得）。然而，γ-分泌酶具有多个 蛋白水解功能：APP底物的初始内切蛋白水解（ε）切割产生长Aβ，其通过 羧肽酶活性，而FAD突变型γ-分泌酶缺乏这种修剪功能。 γ-分泌酶沿着多种蛋白水解功能的新认识及冷冻电镜结构 蛋白酶-底物复合物的阐明使我们更深入地了解FAD的作用成为可能 突变。这里的目标是联合收割机化学，结构和计算生物学来阐明如何 早老素FAD突变改变γ-分泌酶的结构、动力学和功能。这样的理解， 深入了解这种膜包埋的蛋白酶复合物如何识别和促进蛋白水解 跨膜底物，为FAD的致病机制提供了重要线索，并提出了新的 预防FAD的策略。为此，我们建议： (1)开发化学探针以在底物相互作用的不同阶段捕获γ-分泌酶， 冷冻电镜分析。我们开发了完整的TMD底物模拟物，以将活性酶捕获在构象中， 用于膜内蛋白水解。设计的变化应允许ε的过渡态的可视化 蛋白水解、羧肽酶裂解、TMD螺旋解旋和底物的侧向门控。 (2)进行底物与FAD突变型γ-分泌酶相互作用的分子动力学（MD）模拟。 我们通过计算恢复了催化剂，模拟了水进入活性位点的过程，并捕获了 激活计算恢复的WT酶。我们将确定FAD PSEN 1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APP TMD γ-分泌酶加工的功能探针，并验证了它们的有效性。 方便和适当的APP底物的合成替代物。我们将使用这些和其他建议 底物探针，以确定FAD突变型γ-分泌酶对ε蛋白水解和特异性 羧肽酶修整步骤。