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

来自许多实验室的遗传学和细胞生物学证据表明， 早老素的功能和功能障碍在阿尔茨海默病的基本机制。 Aβ的进行性蓄积似乎比AD的其他重要致病特征早开始年 如神经炎症和tau蛋白缠结的形成，而早老素/γ-分泌酶复合物介导了 APP的最终裂解控制Aβ42/43至40的比例，有助于决定一个人的一生 倾向于AD。一项失败的非选择性γ-分泌酶抑制剂（semagacestat）临床试验导致一些 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 其它（例如，DeStrooper，Cell，2014）不相信，并寻求更深入的知识早老素 切割机制--以重新引起人们对靶向γ-分泌酶预防AD的兴趣。事实上， 已经描述了γ-分泌酶调节剂（与抑制剂相对）的种类，并且仅少数已经 开始进入临床试验。项目1对早老素生物学的长期兴趣使我们发表了 在目前的赠款期间，关于γ-分泌酶的几个新发现：a）存在一个生理性的 细胞中的α/γ-分泌酶复合物; B）精确定位Nicastrin作为空间上的看门人的功能 阻碍长底物的加工;和c）早老素/APP TMD相互作用的详细分析 γ-持续合成能力解释了PS裂解三肽的原因。现在，我们将建立在 这些进展深入研究了野生型和FAD突变体的基本机制， 早老素，进入某些γ-调节剂如何以及在何处变构影响其持续合成能力，并进入 我们最近发现的一种正常β/γ-分泌酶复合物的细胞生物学， 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（来自我们最近的论文- Bolduc et al，eLife， 2016）系统地分析PS1中的许多但不是所有家族性AD突变，以了解哪些PS1氨基酸 酸有助于不寻常的活性位点，该活性位点决定了γ- 分泌酶其次，我们将使用这些FAD突变体来研究其机制和PS1结合 一些最有前途的GSMs（γ-分泌酶调节剂）的位点，它们是非常有吸引力的候选者 减缓或预防AD。第三，我们将确认，然后功能分析一个意想不到的复杂 我们最近发现了两种制造Aβ肽的关键酶：β-分泌酶和γ-分泌酶。 根据当前资助期的进展和本文中广泛的初步数据，我们承诺 使用我们非常熟悉的先进的细胞生物学和生物化学方法来阐明 一种最不寻常和最令人着迷的蛋白质在AD中的正常生理学和病理学作用 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.