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: 10615830
• 负责人: JAY T. GROVES
• 金额: $ 38.64万
• 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/10615830
• 关键词: Address; Agonist; Antigens; Autoantigens; Binding; Biology; Cell membrane; Cells; Chemicals; Complex; Data; Dimensions; Discrimination; Elements; Engineering; Event; Exhibits; Feedback; 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; Structure; System; T Cell Receptor Signaling Pathway; T-Cell Activation; 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 健康的免疫系统取决于 T 细胞检测外来激动剂 pMHC 分子的能力，以接近单一的速度 分子水平，在大量有时非常相似的自身抗原中。虽然T细胞还好 辨别能力已经通过实验建立起来，了解分子机制如何 TCR 信号系统实现这一点远非透明。噪声、变化和噪声等基本问题 信号保真度提出了严峻的挑战——无论是对于理解 T 细胞的工作原理还是对于 利用 T 细胞开发治疗策略。最近，一类被称为蛋白质的现象 凝结相变已经开始在生物学中出现。最初是在核背景下确定的 组织和基因表达，细胞膜上独特的二维蛋白质凝结具有 现在在涉及支架蛋白LAT的T细胞受体（TCR）信号系统中被发现。虽然 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被认为是 公认的实验认识表明，这种结构可以通过不同类型的相变形成 流程是较新的。蛋白质缩合相变可以表现出多种特性， 与更线性的分子聚类过程有很大不同，因此提供了多种不同的方法 调节分子信号系统的功能输出。项目 3 解决了总体问题 假设 LAT 蛋白质缩合相变的独特性质使得 T 细胞在抗原识别过程中表现出显着的敏感性和选择性。我们提出一系列 为了测试这一假设的各个方面而进行的调查结合了重构中的高度定量实验 在原代 T 细胞中进行精确单分子活细胞实验的分子系统。我们有初步的 数据表明活 T 细胞中的 LAT 凝结是由一种不寻常的动力学相变控制的 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 对这一发现的阐明将为我们更详细地研究 TCR 信号传导的相变。项目 1 和 2 重点关注近端信号反馈机制 导致 LAT 磷酸化和凝聚成核，项目 4 检查下游信号传导 从 LAT 凝析油中，项目 3 强调 LAT 的实验和计算表征 冷凝相变本身及其特性如何调制下游信号的测量 通过以下具体目标进行传播： 1. 定义控制 LAT 启动的因素 T细胞中的凝结； 2. 设计非冷凝LAT和类LAT系统； 3. 定义 LAT 的方式 冷凝物控制 Ras 和 Ca2+ 途径的下游信号传导。来自这项工作的见解 可能会解决 T 细胞受体信号通路机制功能的一些概念上的谜团 并强调了设计和操纵 T 细胞以获得治疗效果的替代角度。