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

描述(由申请人提供)：T细胞在对入侵病原体和癌症的适应性抗原特异性免疫反应中发挥核心作用，同时在很大程度上避免自我反应(自身免疫)。这些细胞功能是通过T细胞受体(TCR)与多肽-MHC(PMHC)复合体的相互作用而介导的，从而通过TCR-CD3信号复合体传递信号。大量的证据已经被用来理解TCR激活的分子机制，并且有证据支持TCR在识别pMHC后发生构象变化。尽管如此，TCR识别中的分子和结构事件如何转化为细胞内信号的差异仍然是一个谜。这一应用的中心假设是，配体结合时TCR中诱导的构象变化可以跨膜传递，暴露CD3中的细胞质结构域？构象变化的差异是T细胞信号的数量和/或质量差异的原因。为了验证我们的假设，我们将结合X射线结晶学、核磁共振(NMR)和一种新的荧光能量转移(FRET)分析方法来分析TCR与不同的pMHC结合时整体结构组织和构象的变化。我们期望这些研究将深入了解配体如何诱导pMHC-TCR界面的构象变化转移到CD3信号复合体，以前所未有的灵敏度和分辨率影响T细胞激活结果的分子机制。提高对蛋白质-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的理解将是至关重要的，特别是在参与细胞信号传递的受体的情况下。此外，这样的生物物理研究将提供对蛋白质结构和动力学的基本见解，解释这些特征如何用于特定的信号目的，以及蛋白质如何在不同的细胞环境中发挥作用。这些结果有望引起对受体信号转导感兴趣的科学界的兴趣。此外，这些基础知识最终将使我们能够设计多肽或其他试剂，可以用来监测和操纵细胞信号事件，以指导癌症和自身免疫性疾病的治疗和疫苗的设计，这些疾病困扰着成千上万的人。我们希望我们的研究将以前所未有的灵敏度和分辨率深入了解配体诱导的T细胞受体构象变化如何影响T细胞激活结果的分子机制。这些信息将被证明是有用的，既有助于理解受体介导的信号是如何启动的，也将从治疗的角度通过T细胞受体从药物上调节信号，从而提高癌症或艾滋病毒患者的T细胞的敏感性，或降低自身免疫性疾病(多发性硬化症或糖尿病)患者的敏感性。

DESCRIPTION (provided by applicant): T cells play a central role in the adaptive antigen-specific immune response to invading pathogens and cancer while largely avoiding self-reactivity (autoimmunity). These cellular functions are mediated by interaction of the T-cell receptor (TCR) with peptide-MHC (pMHC) complexes which leads to signaling through the TCR-CD3 signaling complex. A great body of evidence has been generated to understand the molecular mechanisms of TCR-activation and evidence supports that the TCR undergoes conformational change upon recognition of pMHC. Still, it remains an enigma how molecular and structural events in TCR-recognition translate into differences in intracellular signaling. The central hypothesis of this application is that conformational changes induced in the TCR upon ligand binding can be transmitted across the membrane to expose cytoplasmic domains in the CD3??? chains, and that differences in conformational changes are responsible for quantitative and/or qualitative differences in T-cell signaling. To test our hypothesis we will combine X-ray crystallography, nuclear magnetic resonance (NMR) and a novel fluorescence energy transfer (FRET) assay 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. Proposal narrative We expect that our studies will provide insight into the molecular mechanism of how ligand-induced conformational changes in the T cell receptor influence T-cell activation outcomes with a sensitivity and resolution that has not been possible before. This information will prove useful in both understanding how receptor-mediated signaling is initiated and from a therapeutic point of view to modulate signaling through the T cell receptor pharmacologically to either increase the sensitivity of T-cells in patients with cancer or HIV or decrease the sensitivity in patients with autoimmune diseases (multiple sclerosis or diabetes).