Structure and function in Notch Signaling

描述(由申请人提供)：本研究项目的主要目标是在结构和机制水平上阐明正常和致病的Notch信号转导的关键特征。在激活之前，Notch是静止的，对蛋白质降解具有抵抗力，但配体结合通过在紧邻膜外的膜旁位置刺激ADAM10(或ADAM17)金属蛋白酶对Notch的蛋白质水解性切割来触发“开”状态。本文提出的工作将解决Notch领域中尚未解决的关键问题，包括：i)Notch受体如何识别其配体，以及ii)Notch的ADAM10蛋白分解是如何实现和调控的。我们建议通过追求以下具体目标来解决信令中的这些关键问题： 1.确定Notch受体与其正则配体特异性结合的基础。在这个目标中，我们将结合结构、生化和基于细胞的分析来解决Notch信号转导中尚未解决的核心问题之一：Notch受体如何识别它们的配体？当务之急将是解决受体-配体复合体的X射线结构。在追求这一影响最大的长期目标的过程中，我们将确定各种Notch受体对不同Delta样配体的内在选择性，以及在分离和/或结合具有潜在治疗意义的阻断抗体的情况下确定单个受体和配体片段的结构。 2.确定ADAM家族金属蛋白水解酶在Notch受体配体依赖性蛋白分解中的催化特异性基础。这一目标的首要任务是确定不同Notch亚型的加工位点，阐明完整的ADAM10胞外区的结构，并通过比较分离的蛋白酶结构域和完整的胞外区在Notch衍生底物上的酶活性来确定ADAM调节区在Notch蛋白分解中的作用。 这些目标的成功完成将是我们对这一基本信号通路及其在正常发育过程中所起作用的理解的重大突破，也将在与Notch信号异常相关的疾病的病理生理方面取得重大突破。通过加深我们对配体参与和调节蛋白分解的结构和生化基础的了解，我们的研究将确定新的治疗策略，以管理与Notch相关的人类病理，包括发育障碍、神经退行性疾病、心血管疾病和癌症。

DESCRIPTION (provided by applicant): The overarching goal of this research project is to elucidate at a structural and mechanistic level critical features of both normal and pathogenic Notch signal transduction. Prior to activation, Notch is quiescent and resistant to proteolysis, bu ligand binding triggers the "on" state by stimulating proteolytic cleavage of Notch by the ADAM10 (or ADAM17) metalloprotease at a juxtamembrane site immediately external to the membrane. The work proposed here will address key unresolved questions in the Notch field, including: i) how Notch receptors specifically recognize their ligands, and ii) how ADAM10 proteolysis of Notch is achieved and regulated. We propose to address these key questions in signaling by pursuing the following specific aims: 1. Determine the basis for specific binding between Notch receptors and their canonical ligands. In this aim, we will combine structural, biochemical, and cell-based assays to address one of the central unresolved questions in Notch signal transduction: how do Notch receptors recognize their ligands? The top priority will be to solve an X-ray structure of a receptor-ligand complex. I the process of pursuing this highest-impact, long-term objective, we will determine the intrinsic selectivity of various Notch receptors for different Delta-like ligands, as well as determine structures of individual receptor and ligand fragments in isolation and/or in combination with blocking antibodies of potential therapeutic relevance. 2. Determine the basis for catalytic specificity of ADAM-family metalloproteases in ligand- dependent proteolysis of Notch receptors. Top priorities of this aim will be to identify the processing sites of the different Notch isoforms, to elucidate the structure of the full ADAM10 ectodomain, and to determine the role of the ADAM regulatory region on Notch proteolysis by comparing the enzymatic activities of the isolated protease domain and the full ectodomain on Notch-derived substrates. Successful completion of these aims will constitute a major breakthrough in our understanding of this fundamental signaling pathway and the role it plays during normal development and in the pathophysiology of diseases associated with aberrant Notch signaling. By deepening our knowledge about the structural and biochemical foundations underlying ligand engagement and regulated proteolysis, our studies will identify new therapeutic strategies for management of Notch-related human pathologies, which include developmental disorders, neurodegenerative diseases, cardiovascular diseases, and cancer.