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

家族性阿尔茨海默病(FAD)是由淀粉样蛋白-β(Aβ)前体基因的显性突变引起的 蛋白质(APP)和早老素-1和-2(PSEN1、PSEN2)。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A-β，以脑内斑块的形式沉积。PSEN是 γ-分泌酶的催化成分，是一种膜包埋的蛋白酶复合体。因此，FAD的突变只是 在产生Aβ的底物和蛋白酶中；然而，致病机制仍然存在争议。 大多数PSEN FAD突变显示蛋白分解减少(功能丧失)，但也增加了 易于聚集的42个残基的Aβ肽(Aβ42)(功能增强)。然而，γ-分泌酶有多个 蛋白分解功能：APP底物的初始内蛋白分解(ε)裂解产生长β，通过 羧肽酶活性和FAD突变的γ分泌酶缺乏这种修剪功能。 对γ-分泌酶多重蛋白分解功能的新认识及最新冷冻-EM结构 蛋白水解酶-底物复合体的阐明使我们有可能更深入地了解FAD的作用。 突变。这里的目标是结合化学、结构和计算生物学来阐明 早老素FAD突变会改变γ分泌酶的结构、动力学和功能。这样的理解应该给人 深入了解这种膜包埋的蛋白酶复合体是如何识别和处理蛋白降解的 跨膜底物，为FAD的发病机制提供了关键线索，并提出了新的 预防时尚的策略。为此，我们建议： (1)开发化学探针捕获底物相互作用不同阶段的γ-分泌酶 用低温电子显微镜进行分析。我们开发了完整的TMD底物模拟物来捕获构象稳定的活性酶 用于膜内蛋白分解。设计的变体应允许可视化ε的过渡态 蛋白质分解、羧基肽酶裂解、TMD螺旋解旋和底物的横向门控。 (2)FAD突变体γ-分泌酶与底物相互作用的分子动力学模拟。 我们通过计算恢复了催化天冬氨酸，模拟了水进入活性部位，并捕获了 通过计算恢复的WT酶的激活。我们将确定FAD PSEN1突变对 γ-分泌酶的结构动力学以及与APP底物和TMD模拟物的相互作用。 (3)合成底物探针，用于分析FAD突变型γ-分泌酶的蛋白分解功能障碍。 我们开发了一套APP TMD的γ分泌酶处理的功能探针，验证了它们的有效性 方便、合适的APP基材合成替代物。我们将采用这些和其他建议 底物探针法测定FAD突变型γ分泌酶对ε蛋白降解和特异性的影响 羧基肽酶修剪步骤。

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.