Deconstructing the molecular mechanisms of Notch receptor-ligand selectivity

 描述（由申请人提供）：在本K99提案中，Luca博士将研究分子识别如何影响Notch途径中的配体选择性，Notch途径是一种对细胞命运决定和许多癌症发展至关重要的信号系统。Luca博士将确定区分每类Notch配体的结构和生物物理决定因素，并将使用植根于结构和组合生物学的技术增强配体功能。本建议书中概述的五年培训计划从K教授指导下的辅导阶段开始。克里斯托弗加西亚在斯坦福大学，卢卡博士将扩大他的专业知识在蛋白质工程，生物物理学和结构生物学。这一经验将使卢卡博士成为一名独立的研究者，其实验范围将解决配体工程和癌症生物学中的难题。当细胞表面上的Notch受体与相对细胞上的配体接合时，Notch信号传导开始，从而触发激活所需的一系列蛋白水解裂解。虽然整体信号传导机制是保守的，但单个Notch受体和两类Notch配体（Jagged和Delta样）能够在正常细胞和癌细胞中诱导不同的细胞应答。有大量文献描述了发育和疾病中的多效性Notch信号传导，但我们对支配配体和受体特异性作用的事件知之甚少。Notch受体-配体相互作用的结构研究将阐明这一过程，但这些实验是困难的，因为它们几乎不可检测的亲和力排除了稳定复合物的重建。Luca博士最近利用定向进化克服了这一障碍，使Notch 1与Delta-like 4（DLL 4）的亲和力成熟相互作用，从而稳定了复合物的共结晶。由此产生的结构提供了Notch受体-配体界面的第一个也是唯一的可视化。在该提案的指导阶段，Luca博士将通过研究结构和生物物理参数如何影响差异配体活性来建立这一变革性结果。这将通过以下方式实现：1）进化以增强的亲和力结合Notch 1的锯齿蛋白1（Jag 1）变体，2）确定Notch 1-Jag 1相互作用结构域的高分辨率晶体结构，3）通过电子显微镜对全长Notch 1-Jag 1或Notch 1-DLL 4胞外结构域复合物进行成像，以及4）进行机械活化测定以确定Jag 1与DLL 4激活Notch所需的力。随着Luca博士过渡到独立职位，他将对DLL 4蛋白进行结构导向工程，以产生高亲和力Notch 3特异性DLL 4变体，并将其作为体内非小细胞肺癌肿瘤生长的抑制剂进行评估。然而，额外的受体特异性配体的产生将允许在未来几年内深入研究Notch生物学。的 这项工作的成功完成将为每类配体激活Notch建立结构和生物物理基础，并将指导在各种生物和治疗背景下具有扩展能力的Notch信号传导的新型调节剂的工程化。

 DESCRIPTION (provided by applicant): In this K99 proposal, Dr. Luca will investigate how molecular recognition influences ligand selectivity in the Notch pathway, a signaling system of fundamental importance to cell fate determination and the development of many cancers. Dr. Luca will determine the structural and biophysical determinants that distinguish each class of Notch ligand, and will enhance ligand function using techniques rooted in structural and combinatorial biology. The five year training plan outlined in this proposal begins with a mentored phase under the guidance of Prof. K. Christopher Garcia at Stanford, where Dr. Luca will expand his expertise in protein engineering, biophysics, and structural biology. This experience will establish Dr. Luca as an independent investigator with the experimental range to address difficult problems in ligand engineering and cancer biology. Notch signaling is initiated when a Notch receptor on the surface of a cell engages a ligand on an opposing cell, triggering a series of proteolytic cleavages required for activation. While the overall signaling mechanism is conserved, individual Notch receptors and the two classes of Notch ligands, Jagged and Delta- like, are able to induce distinct cellular responses in both normal and cancer cells. There is a large body of literature describing pleiotropic Notch signaling in development and disease, yet we poorly understand the events that govern ligand and receptor-specific effects. Structural studies of Notch receptor-ligand interactions would clarify this process, but these experiments are difficult because their nearly undetectable affinity precludes reconstitution of stable complexes. Dr. Luca recently overcame this obstacle using directed evolution to affinity-mature Notch1 interactions with Delta-like 4 (DLL4), which stabilized complexes for co-crystallization. The resulting structure provided the first and only visualization of a Notch receptor-ligand interface. During the mentored phase of this proposal, Dr. Luca will build upon this transformative result by investigating how structural and biophysical parameters influence differential ligand activities. This will be achieved by 1) evolving Jagged1 (Jag1) variants that bind Notch1 with enhanced affinity, 2) determining a high-resolution crystal structure of the Notch1-Jag1 interacting domains, 3) imaging full-length Notch1-Jag1 or Notch1-DLL4 extracellular domain complexes by electron microscopy and 4) performing mechanoactivation assays to determine the force requirements for Notch activation by Jag1 vs. DLL4. As Dr. Luca transitions to an independent position, he will perform structure-guided engineering of DLL4 proteins to generate high-affinity Notch3-specific DLL4 variants, and evaluate them as inhibitors of non-small cell lung cancer tumor growth in vivo. However, the generation of additional receptor-specific ligands will allow for deep interrogation of Notch biology for years to come. The successful completion of this work will establish the structural and biophysical basis for Notch activation by each class of ligand, and will instruct the engineering of novel modulators of Notch signaling with expanded capabilities in a variety of biological and therapeutic contexts.