Project 1: Structure-Function Relationships in Presenilin and Gamma-Secretase

来自多个实验室的遗传学和细胞生物学证据表明， 早老素在阿尔茨海默病基本发病机制中的作用和功能障碍。 Aβ的进行性积累似乎比AD的其他重要致病特征早几年开始 如神经炎症和tau缠结形成，而早老素/γ-分泌酶复合体介导 APP的最终乳沟，它控制着Aβ42/43到40的比率，有助于决定一个人的一生 有AD的倾向。一种非选择性γ分泌酶抑制剂(赛马西特)的临床试验失败，导致了一些 提示γ分泌酶可能不再是治疗开发的有价值的靶点，但我们和 其他人(例如，DeStrooper，Cell，2014)则持不同观点，并寻求对衰老的更深入了解 切割机制--为了重新引起人们对靶向γ-分泌酶预防AD的兴趣。事实上，有几个 已经描述了几类γ分泌酶调节剂(相对于抑制剂)，只有少数几种已经被描述 开始进入临床试验。项目1‘S对老年生物学的长期兴趣促使我们发表了 在当前的授权期内，关于γ分泌酶的几个新发现：a)存在一种生理的 细胞中的α/γ-分泌酶复合体；b)确定尼古丁作为守门人的功能--立体地 阻碍长衬底的加工；以及c)详细分析早老素/APP TMD相互作用 和γ加工性，解释了PS产生三肽裂解的原因。现在，我们将在 这些进展进一步深入分子细节研究野生型和fad突变体的基本机制。 研究某些γ调节剂如何以及在哪里影响它们的加工性，以及进入 我们最近发现的可能是AD中心的正常β/γ-分泌酶复合体的细胞生物学 发病机制。我们将追求三个相互关联但不重叠的具体目标，以收集这一新的 知识。首先，我们将使用一种新的策略(来自我们最新的论文-Bolduc等人，eLife， 2016)系统分析PS1中许多但不是所有家族性AD突变，以了解PS1的哪种氨基酸 酸对决定γ-三肽典型切割机制的不同寻常的活性部位有贡献- 分泌酶。其次，我们将使用这些FAD突变体来研究机制和PS1结合 一些最有希望的GSM(γ-分泌酶调节器)的位置，这些都是非常有吸引力的候选 用于减缓或预防AD。第三，我们将确认并从功能上分析意想不到的复杂情况 我们最近发现了产生Aβ多肽的两种关键酶：β-和γ-分泌酶。 基于当前授权期的进展和这里广泛的初步数据，我们致力于 用我们非常熟悉的先进的细胞生物学和生化方法来阐明 一种最不寻常和最吸引人的蛋白质在阿尔茨海默病中的正常生理和病理生物学作用 后生动物中的机器：早老素/γ-分泌酶复合体。

SUMMARY Genetic and cell biological evidence from many laboratories implicates both the normal function and the dysfunction of the presenilins in the fundamental mechanism of Alzheimer's disease. Progressive accumulation of Aβ appears to begin years before other important pathogenic features of AD such as neuroinflammation and tau tangle formation, and the presenilin/γ-secretase complex mediates the final cleavages of APP which control the Aβ42/43 to 40 ratios that help dictate a person's lifelong propensity to AD. A failed clinical trial of a non-selective γ-secretase inhibitor (semagacestat) has led some to suggest that γ-secretase may no longer be a worthy target for therapeutic development, but we and others (e.g., DeStrooper, Cell, 2014) believe otherwise and seek much deeper knowledge of the presenilin cleavage mechanism -- in order to renew interest in targeting γ-secretase to prevent AD. Indeed, several classes of γ-secretase modulators (as opposed to inhibitors) have been described, and just a few have begun to enter clinical trials. Project 1's long-standing interest in presenilin biology has led us to publish during the current grant period several novel findings about γ-secretase: a) the existence of a physiological α/γ-secretase complex in cells; b) pinpointing the function of Nicastrin as a gatekeeper -- sterically hindering the processing of long substrates; and c) a detailed analysis of presenilin/APP TMD interactions and γ-processivity that explains the reason for the tri-peptide cleavages made by PS. Now, we will build on these advances to delve in further molecular detail into the basic mechanisms of wild-type and FAD mutant presenilin, into how and where certain γ-modulators allosterically influence their processivity, and into the cell biology of a normal β/γ-secretase complex we recently discovered that could be central to AD pathogenesis. We will pursue 3 interrelated but non-overlapping Specific Aims to gather this new knowledge. First, we will use a novel strategy (emerging from our most recent paper – Bolduc et al, eLife, 2016) to analyze systematically many but not all familial AD mutations in PS1 to learn which PS1 amino acids contribute to the unusual active site that dictates the canonical tripeptide cleavage mechanism of γ- secretase. Second, we will use these FAD mutants to examine both the mechanisms and the PS1 binding sites of some of the most promising GSMs (γ-secretase modulators), which are highly attractive candidates for slowing or preventing AD. Third, we will confirm and then functionally analyze an unexpected complex we recently discovered between the two key enzymes that make Aβ peptides: β- and γ-secretase. Based on progress in the current grant period and extensive Preliminary Data herein, we are committed to using advanced cell biological and biochemical methods which we are deeply familiar with to elucidate the normal physiology and pathobiological role in AD of one of the most unusual and fascinating protein machines in metazoans: the presenilin/γ-secretase complex.