Structure and function in Notch Signaling

Notch 信号传导的结构和功能

• 批准号: 8927540
• 负责人: Stephen C. Blacklow
• 金额: $ 40.24万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-16 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8927540
• 关键词: Acute; Acute T Cell Leukemia; Address; Adult; Award; Binding; Biochemical; Biological Assay; Blocking Antibodies; CADASIL; Cardiovascular Diseases; Cell Nucleus; Cell surface; Cells; Cleaved cell; Complex; Credentialing; DNA; Defect; Development; Disease; Enzymes; Event; Family; Foundations; Functional disorder; Gene Targeting; Goals; Health; Homeostasis; Human; Human Biology; Human Pathology; Immune response; Individual; Inherited; Integral Membrane Protein; Knowledge; Ligand Binding; Ligands; Malignant Neoplasms; Membrane; Metalloproteases; Molecular; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Notch Signaling Pathway; Nucleic Acid Regulatory Sequences; Oncogenic; Organism; Peptide Hydrolases; Play; Process; Protease Domain; Protein Isoforms; Proteolysis; Receptor Activation; Recurrence; Regulation; Research Project Grants; Resistance; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Site; Specificity; Stroke; Structure; Syndrome; T-Lymphocyte; Therapeutic; Tissues; Transcriptional Activation; Work; angiogenesis; base; cell growth; developmental disease; fly; gamma secretase; human disease; leukemia; leukemia/lymphoma; loss of function mutation; malignant breast neoplasm; neoplastic cell; notch protein; novel therapeutics; premature; receptor; receptor binding; response; therapeutic target; tumor

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.

描述（由申请人提供）：本研究项目的总体目标是在结构和机制水平上阐明正常和致病性Notch信号转导的关键特征。在活化之前，Notch是静止的并且对蛋白水解具有抗性，但是配体结合通过在紧邻膜外部的质膜位点处刺激ADAM 10（或ADAM 17）金属蛋白酶对Notch的蛋白水解切割而触发“开启”状态。本文提出的工作将解决Notch领域尚未解决的关键问题，包括：i）Notch受体如何特异性识别其配体，以及ii）Notch的ADAM 10蛋白水解如何实现和调节。我们建议通过追求以下具体目标来解决信号方面的这些关键问题： 1.确定Notch受体与其典型配体之间特异性结合的基础。在这个目标中，我们将结合联合收割机的结构，生物化学和细胞为基础的分析，以解决一个中心的Notch信号转导未解决的问题：如何做Notch受体识别其配体？首要任务将是解决受体-配体复合物的X射线结构。在追求这一最高影响力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，以及确定单独和/或与潜在治疗相关性的阻断抗体组合的单个受体和配体片段的结构。 2.确定ADAM家族金属蛋白酶在Notch受体的配体依赖性蛋白水解中的催化特异性的基础。这个目标的首要任务将是确定不同的Notch亚型的加工位点，阐明完整的ADAM 10胞外域的结构，并通过比较分离的蛋白酶结构域和完整的胞外域对Notch衍生底物的酶活性来确定ADAM调节区对Notch蛋白水解的作用。 这些目标的成功完成将构成我们对这一基本信号通路及其在正常发育过程中和与异常Notch信号相关的疾病的病理生理学中所起作用的理解的重大突破。通过加深我们对配体参与和调节蛋白水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，用于管理Notch相关的人类病理学，包括发育障碍，神经退行性疾病，心血管疾病和癌症。