Presenilin Biology and the Mechanisms of Alzheimer's Disease

摘要：自1995年克隆以来，根据这项赠款，它被鉴定为史无前例的 膜内天冬氨酸蛋白酶1999年，早老素参与了一系列显著的信号转导。 所有后生动物中的事件。多发性硬化症是通过对阿尔茨海默氏症的研究发现的，但很快就被证明 赋予生命所必需的功能，包括作为启动Notch核信号的蛋白酶。 因此，继续破译PS的结构、功能以及蛋白质和小分子调节因子是一项 优先考虑基础细胞生物学。同时，大脑中淀粉样蛋白b蛋白的不变积聚 (AB)早在症状出现之前的几十年，PS/g-分泌酶就成为机械和治疗的合理靶点 在公元后进修。尽管它在生物学上起着多效性的作用，但该酶的结构直到最近才被报道为3.4？， 留下了许多结构-功能细节尚未解决，以及可以安全而有效地调节的小分子 它对APP的裂解才刚刚进入人体试验。出于这些原因，三位经验丰富的合作者 在早老素的研究中希望将一系列的方法应用于细胞生物学、生物化学、细胞学 成像、遗传学、干细胞生物学和药物化学，以解决PS/g中的一些最棘手的问题- 分泌生物学。也就是说，基于我们的PS处理能力的新模型(Bolduc等人，eLife 2016)，我们能否插入 许多不同的导致FAD的PSI突变进入蛋白质中，并识别哪些残基对S1‘- 我们最近发现的介导三肽裂解的S2‘-S3’活性部位口袋？什么是生物学？ 在一个新的复合体中协调b-和g-分泌酶处理的机制？一种新的PS蛋白如何 在此发现的相互作用，即与GLT-1的相互作用，有助于调节星形胶质细胞对谷氨酸的吸收，或许 也改变g-分泌酶的功能吗？人们能否识别和验证具有足够效力和选择性的GSM 将g-分泌酶的裂解从有毒的AB42/43转移到保护性的Ab37/38肽？我们怎么能 了解更多关于PS/g-分泌酶参与中枢神经系统干细胞发育的信息？在这里，我们建议 许多相互关联的目标包含三个交叉主题，这些主题将我们的工作联系在一起。首先，所有3个项目 将建立在这项授权下发现的g-分泌酶处理的新解释机制：它的 控制结合的早老素酶中的3个口袋决定了三肽的连续切割。 具有催化位置的底物。其次是我们对小分子调节剂的强烈兴趣 PS/g-分泌酶复合体。我们的3个项目中的每一个都包括AIMS，它将检查GSM以评估其影响 我们正在单独研究的PS函数。第三个横切主题来自我们共享使用大型 PS1和APP质粒库、敏感的ELISA和高级显微镜试剂--所有这些都是在 这笔赠款。在分享这些方法和试剂时，我们的PPG不是相互关联的目标的集合，而是 而是一个高度集成的计划，其中我们使用共同主题和方法的变体来解决主要问题 关于g-分泌酶、RIP机制及其治疗和预防AD的安全调控方面的问题尚未解决。

SUMMARY: Since its cloning in 1995 and its identification (under this grant) as an unprecedented intramembrane aspartyl protease in 1999, Presenilin has been implicated in a remarkable array of signaling events in all metazoans. PS was discovered through research on Alzheimer’s disease, but it was soon shown to confer functions necessary for life, including as the protease that enables Notch nuclear signaling. Therefore, continuing to decipher the structure, functions, and protein and small-molecule regulators of PS is a priority for fundamental cell biology. At the same time, the invariant cerebral accumulation of amyloid b-protein (Ab) decades prior to symptoms has made PS/g-secretase a rational target for mechanistic and therapeutic study in AD. Despite its pleiotropic role in biology, the protease’s structure was only recently reported at 3.4Å, leaving many structure-function details unresolved, and small molecules that can safely and potently modulate its cleavage of APP are just entering human trials. For these reasons, three collaborators with deep experience in the study of Presenilin over 20 years wish to apply a range of methods in cell biology, biochemistry, cellular imaging, genetics, stem cell biology and medicinal chemistry to tackle some of the thorniest questions in PS/g- secretase biology. To wit, based on our new model of PS processivity (Bolduc et al., eLife 2016), can we insert many different FAD-causing PSI mutations into the protein and identify which residues contribute to the S1’- S2’-S3’ active-site pockets we recently found to mediate the tri-peptide cleavages? What is the biological mechanism of coordinated b- and g-secretase processing within a novel complex? How does a new PS protein interaction discovered here, namely with GLT-1, help modulate glutamate uptake by astrocytes and perhaps also change g-secretase function? Can one identify and validate GSMs that are sufficiently potent yet selective to chronically shift g-secretase cleavages from toxic Ab42/43 to protective Ab37/38 peptides? How can we learn more about the participation of PS/g-secretase in stem cell development in the CNS? Here, we propose numerous interrelated aims that incorporate three cross-cutting themes that unite our work. First, all 3 projects will build on a new explanatory mechanism of g-secretase processing discovered under this grant: that its processive tri-peptide cleavages are dictated by 3 pockets in the presenilin enzyme controlling the engagement of substrates with the catalytic site. Second is our strongly shared interest in small-molecule modulators of the PS/g-secretase complex. Each of our 3 projects includes aims that will examine GSMs to assess effects on the PS functions we are individually studying. A third cross-cutting theme comes from our shared use of a large library of PS1 and APP plasmids, sensitive ELISAs, and advanced microscopy reagents -- all developed under this grant. In sharing these approaches and reagents, our PPG is not a collection of marginally related aims but rather a highly integrated program in which we use variations on common themes & methods to address major unsolved questions about g-secretase, the RIP mechanism, and its safe modulation to treat and prevent AD.