Visualizing ligand-induced signal propagation in the TCR-signaling complex

在这项提案中，要求为NIH资助的纽约大学学院项目的费用延期提供资金 医疗挽回因飓风桑迪造成的损失和研究时间延误救灾 《拨款法案》。这项要求的目的是完成我们在#年提出的目标 家长资助GM085586。家长拨款中描述的这个项目旨在了解如何动态 T细胞受体-肽-MHC(TCR-pMHC)界面的改变和/或构象变化 有助于T细胞激活，并确定负责传递配体的机制 TCR与CD3信号亚基的结合。这个应用的中心假设是构象 TCR在配体结合时引起的变化和/或灵活性可以通过膜传递到 暴露CD3ε/ζ链中的细胞质结构域，以及构象变化和/或 灵活性是T细胞信号在数量和/或质量上的差异的原因。测试我们的 假设我们结合了X射线结晶学和核磁共振(核磁共振)实验 非合成氨基酸掺入与交联实验和荧光共振相结合 能量转移(FRET)分析TCR整体结构组织和构象的变化 当与不同的pMHC结合时。我们期望这些研究将提供对分子的洞察。 配体诱导的pMHC-TCR界面构象变化向CD3转移的机制 以前所未有的灵敏度和分辨率影响T细胞激活结果的信号复杂 以前是可能的。加深了对蛋白质-蛋白质相互作用的结构生物物理学和 结构发生构象变化的倾向将是至关重要的，特别是在 受体参与细胞信号传递的案例。此外，这样的生物物理研究将提供基本的 对蛋白质结构和动力学的洞察，解释这些特征是如何用于特定信号的 目的以及蛋白质如何在不同的细胞环境中发挥作用。这些结果预计将是 对受体信号感兴趣的科学界。此外，这些基本知识将 最终允许我们设计多肽或其他可以用来监测和操纵细胞的试剂 指导癌症和自身免疫性疾病的治疗和疫苗设计的信号事件 折磨着成千上万的人。

In this proposal funds are requested for a for-cost extension for an NIH funded project at NYU School of Medicine to recover losses and delay in research time due to Hurricane Sandy under the Disaster Relief appropriations Act. This request is with the purpose to complete our proposed aims and objectives proposed in parent grant GM085586. This project described in the parent grant aims to understand how dynamic alterations and/or conformational changes at the T-cell receptor-peptide-MHC (TCR-pMHC) interface contribute to T-cell activation and to identify the mechanism that is responsible for communicating ligand engagement of TCR to CD3 signaling subunits. The central hypothesis of this application is that conformational changes and/or flexibility induced in the TCR upon ligand binding can be transmitted across the membrane to expose cytoplasmic domains in the CD3ε/ζ chains, and that differences in conformational changes and/or flexibility are responsible for quantitative and/or qualitative differences in T-cell signaling. To test our hypothesis we have combined X-ray crystallography and nuclear magnetic resonance (NMR) experiments and non-synthetic amino acid incorporation combined with cross-linking experiments and fluorescence resonance energy transfer (FRET) to analyze the changes in the overall structural organization and conformation of TCR when binding to different pMHC. We expect that these studies will provide insight into the molecular mechanism of how ligand induced conformational changes at the pMHC-TCR interface translocate to the CD3 signaling complex to influence T-cell activation outcomes with a sensitivity and resolution that have not been possible before. An increased understanding of the structural biophysics of protein-protein interactions and of the propensity of structures to undergo conformational change will be of critical importance, particularly in the case of receptors involved in cell signaling. Furthermore, such biophysical studies will provide fundamental insights into protein structure and dynamics, explain how these features are used for specific signaling purposes and how the proteins function in distinct cellular environments. These results are expected to be of interest for the scientific community interested in receptor signaling. In addition, this basic knowledge will eventually allow us to design polypeptides or other agents that can be used to monitor and manipulate cell signaling events to guide the design of therapeutics and vaccines for cancer and autoimmune disease which afflict thousands of people.