Illuminating Notch receptor-ligand selectivity through structure-guided protein engineering

通过结构引导的蛋白质工程阐明Notch受体-配体选择性

• 批准号: 10647862
• 负责人: Vincent Christopher Luca
• 金额: $ 43万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10647862
• 关键词: Affinity; Architecture; Area; Binding; Biological; Biology; Biophysics; Cell Communication; Cell Surface Receptors; Cells; Chemicals; Complex; Cues; Development; Directed Molecular Evolution; Discrimination; Electron Microscopy; Engineering; Environment; Funding; Goals; Kinetics; Laboratories; Language; Length; Ligands; Malignant Neoplasms; Mammals; Mediating; Molecular; Mutation; Outcome; Output; Pathway interactions; Post-Translational Protein Processing; Protein Engineering; Proteins; Research; Roentgen Rays; Series; Signal Transduction; Structure; Therapeutic; Tissues; Training; Vision; Visualization; Work; antagonist; aortic valve disorder; biophysical properties; cell type; design; experimental study; glycosylation; human disease; insight; interdisciplinary approach; mechanical force; mechanical properties; notch protein; novel; novel therapeutics; programs; receptor; reconstitution; response; structural biology; translational applications; transmission process

PROJECT SUMMARY Overall vision of the research program. My research is focused on understanding the “molecular language” of cell-to-cell communication. Cell surface receptors transmit signals in response to external cues, and receptor activity is regulated by extrinsic factors such as posttranslational modifications, mechanical force, and chemical environment. Using a multidisciplinary approach that leverages my training in structural biology, biophysics, and protein engineering, my group is attempting to solve difficult problems in the area of receptor biology. We hypothesize that the distinct structural and biophysical parameters governing receptor-ligand interactions control downstream signaling, and that ligands may be engineered to alter these parameters and reprogram signaling outputs. My long-term goal is to harness the insight obtained through our mechanistic studies to design proteins that can fine-tune receptor activity and function as powerful next-generation therapeutics. Overview of research in the laboratory. Notch signaling is initiated when a mechanosensory Notch receptor is “pulled on” by a Delta-like (DLL) or Jagged (Jag) ligand expressed on an adjacent cell. In mammals, the various Notch receptor (Notch1-4) and ligand (DLL1, DLL4, Jag1, Jag2) subtypes are able to induce distinct or even opposing cellular responses. Ligand selectivity is further regulated by Notch receptor glycosylation, which inhibits the activity of Jag ligands yet potentiates the activity of DLL ligands. We currently have a poor understanding of the molecular basis for ligand discrimination in the Notch pathway, and structural studies of Notch receptor-ligand interactions have been difficult because their nearly undetectable binding affinity precludes reconstitution of stable complexes. We recently overcame this obstacle by using directed evolution to strengthen DLL4 and Jag1 binding to Notch1, which allowed us to capture their interacting domains for x-ray crystal structure determination. My lab is now building upon these transformative results to explore how the structural, biophysical, and mechanical properties of various ligand subtypes contribute to their unique functions. Goals for the next five years. Over the five-year funding period, the goals of this project are to: (i) systematically characterize the structure, binding kinetics, and signaling potency of Notch receptor-ligand complexes; (ii) use electron microscopy to visualize the ultrastructural architecture of full-length Notch1-DLL4 and Notch1-Jag1 complexes; (iii) determine the biophysical basis by which Notch glycosylation regulates DLL and Jag activity, and (iv) design tissue-specific Notch antagonists. The successful completion of this work will establish the molecular mechanisms of Notch receptor-ligand selectivity and will guide the engineering of novel modulators of Notch signaling with expanded capabilities in a variety of biological and therapeutic contexts.

项目摘要 研究计划的总体构想。我的研究重点是理解“分子语言” 细胞间的交流。细胞表面受体响应外部信号传递信号，并且受体 活性受外源性因素如翻译后修饰、机械力和化学物质的调节。 环境使用多学科的方法，利用我在结构生物学，生物物理学， 蛋白质工程，我的小组正试图解决受体生物学领域的难题。我们 我假设，不同的结构和生物物理参数控制受体-配体相互作用控制 配体可以被工程化以改变这些参数并重新编程信号传导 产出我的长期目标是利用我们通过机械研究获得的洞察力来设计蛋白质 可以微调受体活性，并作为强大的下一代治疗药物发挥作用。 实验室研究概况。当机械感觉Notch受体被激活时， 通过在相邻细胞上表达的δ样（DLL）或锯齿状（Jag）配体“拉动”。在哺乳动物中， 各种Notch受体（Notch 1 -4）和配体（DLL 1、DLL 4、Jag 1、Jag 2）亚型能够诱导不同的或 甚至是相反的细胞反应配体选择性进一步受Notch受体糖基化调节， 抑制Jag配体的活性但增强DLL配体的活性。我们目前有一个贫穷的 理解Notch途径中配体识别的分子基础，以及 Notch受体-配体相互作用是困难的，因为它们几乎不可检测的结合亲和力排除了 稳定复合物的重建。我们最近克服了这个障碍，通过使用定向进化来加强 DLL 4和Jag 1与Notch 1结合，这使我们能够捕获它们的相互作用结构域用于X射线晶体结构 保持战略定力我的实验室现在正在这些变革性的结果的基础上，探索如何结构，生物物理， 各种配体亚型的机械性质有助于它们的独特功能。 未来五年的目标。在五年供资期内，该项目的目标是：㈠系统地 表征Notch受体-配体复合物的结构、结合动力学和信号传导效力;（ii）用途 用电子显微镜观察全长Notch 1-DLL 4和Notch 1-Jag 1的超微结构 （iii）确定Notch糖基化调节DLL和Jag活性的生物物理基础， 和（iv）设计组织特异性Notch拮抗剂。这项工作的顺利完成将建立 Notch受体-配体选择性的分子机制，并将指导新的Notch受体-配体选择性调节剂的工程化。 Notch信号传导在各种生物学和治疗环境中具有扩展的能力。