The role of LAT protein condensation phase transitions in T cell signaling

LAT 蛋白缩合相变在 T 细胞信号传导中的作用

• 批准号: 10428140
• 负责人: JAY T. GROVES
• 金额: $ 46.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10428140
• 关键词: Address; Agonist; Antigens; Autoantigens; Binding; Biology; Cell membrane; Cells; Chemicals; Complex; Data; Dimensions; Discrimination; Elements; Engineering; Event; Exhibits; Feedback; Forms Controls; Foundations; Gene Expression; Image; Imaging Techniques; Immune system; Individual; Investigation; Kinetics; MAP Kinase Gene; Measurement; Modification; Molecular; Mutation; Noise; Nuclear; Output; Pathway interactions; Peptide/MHC Complex; Phase Transition; Phosphorylation; Physical condensation; Process; Property; Proteins; Reaction; Receptor Signaling; Resolution; Role; Scaffolding Protein; Series; Signal Transduction; Signaling Molecule; Son of Sevenless Proteins; Structure; System; T Cell Receptor Signaling Pathway; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; Variant; Work; cellular imaging; dimer; engineered T cells; experimental study; insight; mutant; novel; receptor binding; reconstitution; response; scaffold; single molecule; therapeutic development; two-dimensional

ABSTRACT - Project 3 A healthy immune system depends on T cells’ abilities to detect foreign agonist pMHC molecules, at near single molecule levels, among vast numbers of sometimes very similar self-antigens. While the T cell’s fine discrimination capabilities are experimentally well established, understanding how the molecular machinery of the TCR signaling system achieves this is far from transparent. Fundamental issues of noise, variation, and signal fidelity present serious challenges—both for understanding how the T cell works as well as for development of therapeutic strategies utilizing T cells. Recently, a class of phenomena known as protein condensation phase transitions have begun to emerge in biology. Originally identified in the context of nuclear organization and gene expression, a distinct two-dimensional protein condensation on the cell membrane has now been discovered in the T cell receptor (TCR) signaling system involving the scaffold protein LAT. While the role of LAT as a scaffold for the clustering of downstream signaling molecules in T cells has long been recognized, experimental realization that this structure can form through distinct types of phase transition processes is more recent. Protein condensation phase transitions can exhibit a wide range of properties that differ substantially from more linear molecular clustering processes, and thus offer a variety of different ways to regulate the functional output of molecular signaling systems. Project 3 addresses the overarching hypothesis that unique properties of the LAT protein condensation phase transition enable the remarkable sensitivity and selectivity T cells exhibit during antigen recognition. We propose a series of investigations to test aspects of this hypothesis that combine highly quantitative experiments in reconstituted molecular systems with precision single-molecule live cell experiments in primary T cells. We have preliminary data indicating that LAT condensation in live T cells is controlled by an unusual type of kinetic phase transition and that specific molecular features of proximal TCR signaling are tuned to take advantage of this. Confirmation and elucidation of this discovery will form the foundation of our more detailed investigation into the role of the phase transition in TCR signaling. While Projects 1 and 2 focus on proximal signaling feedback mechanisms leading up to LAT phosphorylation and condensate nucleation, and Project 4 examines signaling downstream from the LAT condensate, Project 3 emphasizes experimental and computational characterization of the LAT condensation phase transition itself and measurements of how its properties modulate downstream signal propagation through the following specific aims: 1. Define the factors controlling initiation of LAT condensation in T cells; 2. Engineer non-condensing LAT and LAT-like systems; 3. Define how LAT condensates control downstream signaling to Ras and Ca2+ pathways. Insights originating from this work will likely resolve some conceptual mysteries on mechanistic function of the T cell receptor signaling pathway and highlight alternative angles to engineer and manipulate T cells for therapeutic benefit.

摘要-项目3 一个健康的免疫系统依赖于T细胞检测外源激动剂pMHC分子的能力， 分子水平，在大量的有时非常相似的自身抗原中。T细胞没事的时候 辨别能力是实验上建立良好的，了解如何分子机制， TCR信号系统实现这一点远非透明。噪声、变化和 信号保真度提出了严重的挑战-无论是理解T细胞如何工作， 开发利用T细胞的治疗策略。最近，一类被称为蛋白质的现象 凝结相变已经开始出现在生物学中。最初是在核问题的背景下确定的， 组织和基因表达，一个独特的二维蛋白质凝聚在细胞膜上， 现在在涉及支架蛋白LAT的T细胞受体（TCR）信号传导系统中发现。而 LAT作为T细胞中下游信号分子聚集的支架的作用长期以来一直是 公认的，实验性的认识，这种结构可以形成通过不同类型的相变 过程是最近的。蛋白质缩合相变可以表现出广泛的性质， 基本上不同于更线性的分子聚类过程，因此提供了各种不同的方法来 调节分子信号系统的功能输出。项目3解决了 假设LAT蛋白质缩合相变独特性质使得 T细胞在抗原识别过程中表现出显著的敏感性和选择性。我们提出了一系列 为了检验这一假设的各个方面，联合收割机结合了重组的高定量实验， 分子系统与原代T细胞中的精确单分子活细胞实验。我们有初步的 数据表明，活T细胞中的LAT凝聚是由一种不寻常的动力学相变类型控制的， 并且近端TCR信号传导的特定分子特征被调整以利用这一点。确认 这一发现的阐明将成为我们对这一发现的作用进行更详细研究的基础。 TCR信号传导中的相变。而项目1和项目2则侧重于近端信号反馈机制 导致LAT磷酸化和冷凝物成核，项目4检查下游信号传导 从LAT冷凝物，项目3强调LAT的实验和计算表征 冷凝相变本身及其性质如何调制下游信号的测量 通过以下具体目标进行宣传：1.定义控制LAT启动的因素 T细胞中的凝聚; 2.工程师非冷凝LAT和LAT类系统; 3.定义LAT如何 缩合物控制Ras和Ca 2+途径的下游信号传导。源自这项工作的见解 将可能解决一些概念上的奥秘，机械功能的T细胞受体信号通路 并强调了工程和操纵T细胞以获得治疗益处的替代角度。