Deconstructing and Reconstructing the T Cell Signaling Network

描述(申请人提供)：T细胞-抗原呈递细胞相互作用的复杂性和目前用于研究它的简单生化方法需要不同的方法来理解TCR信号及其调节。共同的兴趣、现有的合作和独特的专业知识为我们(Chakraborty、Groves、Kuriyan、Roose和Weiss博士)提供了一个独特的机会，可以用更复杂和新颖的方法研究这个复杂的生物系统。我们提出了一个全面的程序来了解相互作用分子的特异性和调节，并开发了一个系统来研究TCR调节的脂质双层系统上的信号事件，以模拟基本上发生在质膜二维空间的事件。这种方法可能会对分子相互作用和动力学产生新的见解，这在复杂的细胞系统中是无法获得的，也不会被溶液中发生的反应所模仿，因为溶液中的扩散不限于二维。事实上，Groves博士和Kuriyan博士应用双层系统研究了在双层表面同时定位RAS和SOS蛋白的影响，得到了与溶液动力学相比催化活性显著提高的意想不到的结果。我们的总体目标是建立一个简单但强大的TCR信号的生化系统和计算模型，帮助我们了解调控的关键机制：(项目1)TCR相关免疫受体基于酪氨酸的激活基序(ITAM)和LAT的酪氨酸磷酸化；(项目2)由鸟嘌呤核苷酸交换因子(GEF)RasGRP和SOS激活LAT下游的RAS。通过研究二维系统中涉及的分子的特异性、调节和活性，我们试图模拟质膜内小叶的表面。我们将从最小的简单系统开始，迭代地增加复杂性。我们希望能够增加空间复杂性和分子复杂性。我们将研究定义明确和重要的输出，并不仅将采用生化和生物物理措施，而且将使用计算工具来表征这个系统。我们将使用建模来比较简单的系统和更复杂的系统，但也会研究这些简单的系统如何偏离在解决方案或更复杂的蜂窝系统中可以获得的系统。 公共卫生相关性(由申请人提供)：T细胞在几乎所有免疫反应和疾病中发挥关键作用。T细胞功能由T细胞抗原受体(TCR)介导的信号控制，但我们对这些信号是如何产生的了解相当初级，更准确地理解TCR信号是如何产生的，这些研究的目标，可能会导致T细胞介导的疾病的新疗法，包括关节炎和狼疮。 项目1： TCR途径蛋白酪氨酸激酶和磷酸酶的特异性和调控 项目负责人：韦斯，A 项目1描述(由申请人提供)：尽管参与TCR信号转导的大多数分子可能已经确定，但我们对基本信号转导与诱导信号状态的理解相当初级。酪氨酸激酶和磷酸酶维持着控制和维持基础状态的动态平衡，它们的功能活性或空间定位的变化对诱导酪氨酸磷酸化具有重要意义。这个项目的重点是了解这些酪氨酸激酶和磷酸酶在质膜上的特异性和复杂的调节。了解TCR胞浆链和LAT蛋白的磷酸化调节将是我们重点关注的终点。我们将使用：纯化的重组蛋白；新型基因控制的激酶抑制剂；包含确定数量的重组蛋白的模型二维脂质双层系统；以及用于描述简单双层系统并将其与现有研究充分的完整细胞或模型细胞系统的膜进行比较的计算模型。我们将以迭代的方式将这种二维模型的生化行为与细胞系统进行比较，增加组件的复杂性并应用计算建模。我们希望这样的分析能够揭示预期和意外的行为。我们还希望通过对简单系统和更复杂系统的比较，揭示以前可能没有意识到的调控途径的存在，例如正反馈电路和负反馈电路。我们清楚地认识到，我们不能模拟所有TCR信号的复杂性，但我们的目标是了解控制TCR链酪氨酸磷酸化的详细机制，以及这些事件如何导致LAT磷酸化。项目1的总体目标是了解TCR信号中涉及的蛋白酪氨酸激酶和磷酸酶的特异性和调节，这些信号导致膜表面的ITAM和LAT磷酸化。我们将：1)确定ITAM和LAT的Lck和ZAP-70的特异性的分子基础；2)开发Lck的模拟灵敏的抑制系统；3)确定Lck控制ITAM磷酸化的机制；4)确定ZAP-70控制LAT磷酸化的机制 公共卫生相关性(由申请人提供)：T细胞受体(TCR)为抗原控制T细胞反应的生化信号事件，但我们对这些信号事件的了解非常初级。这些研究旨在了解TCR控制的酶、激酶和磷酸酶是如何在表面模型膜上调节的，就像它们在细胞中一样。这些研究可能导致T细胞介导性疾病的新疗法

DESCRIPTION (provided by applicant): The complexity of the T cell - antigen presenting cell interaction and the current simple biochemical approaches being used to study it requires different approach to understand TCR signaling and its regulation. Common interests, existing collaborations and unique expertise, provide us (Drs. Chakraborty, Groves, Kuriyan, Roose and Weiss) with a unique opportunity to study this complex biological system with more sophisticated and novel approaches. We present here a comprehensive program to understand the specificity and regulation of the interacting molecules and the development of a system to study TCR regulated signaling events on a lipid bilayer system to simulate the events occurring essentially on the two dimensional space of the plasma membrane. Such an approach is likely to yield novel insights into molecular interactions and kinetics not obtainable in complex cellular systems and not mimicked by reactions that occur in solution, where diffusion is not limited to 2 dimensions. Indeed, unanticipated results, with marked increase in catalytic activity compared to solution kinetics, were obtained by Dr. Groves and Kuriyan who applied a bilayer system to study the influence of localizing Ras and SOS proteins together at the surface of a bilayer. Our overall objective is to develop a simple but robust biochemical system and computational model of TCR signaling that helps us understand the critical mechanisms that regulate: (Project #1) tyrosine phosphorylation of the TCR-associated immunoreceptor tyrosine-based activation motifs (ITAMs) and of LAT; and, (Project #2) the activation of Ras downstream of LAT by the guanine nucleotide exchange factors (GEFs) RasGRP and SOS. By studying the specificity, regulation and the activities of the molecules involved in a two-dimensional system we attempt to mimic the surface of the inner leaflet of the plasma membrane. We will start with a minimal simple system and iteratively add complexity. We hope to be able to add spatial complexity and molecular complexity. We will study well-defined and important outputs, and will bring to bear not only biochemical and biophysical measures but also computational tools to characterize this system. We will use modeling to compare simple to more complex systems but also study how these simple systems deviate from those obtainable in solution or in more complex cellular systems. PUBLIC HEALTH RELEVANCE (provided by applicant): T cells play critical roles in nearly all immune responses and diseases. T cell function is controlled by signals mediated by the T cell antigen receptor (TCR), but our understanding of how these signals are generated is rather rudimentary, A more precise understanding of how TCR signals are generated, the goal of these studies, could lead to novel therapies of T cell-mediated disease, including arthritis and lupus. PROJECT 1: Title: - Specificity and Regulation TCR Pathway Protein Tyrosine Kinases and Phosphatases Project Leader: WEISS, A PROJECT 1 DESCRIPTION (provided by applicant): Although most of the molecules involved in TCR signaling have likely been identified, our understanding of the basal signaling versus the induced signaling states are rather rudimentary. Tyrosine kinases and phosphatases maintain the dynamic equilibrium that controls and maintains the both the basal state and changes in their functional activities or spatial localization are important for Induced tyrosine phosphorylation. This project focuses on understanding the specificity and complex regulation of these tyrosine kinases and phosphatases on at the plasma membrane. Understanding the regulation of phosphorylation of the TCR cytoplasmic ?-chain as well as of LAT proteins will be the endpoints we will focus on. We will use: purified recombinant proteins; novel genetically controlled kinase inhibitors; a model two dimensional lipid bilayer system containing defined quantities of recombinant proteins; and, computational modeling to describe and compare the simple bilayer system to existing well-studied intact cells or membranes of model cellular systems. We will compare the biochemical behavior of such two dimensional models to cellular systems in an iterative way, increasing component complexity and by applying computational modeling. We expect such analyses to reveal expected as well as unanticipated behaviors. We also expect the comparison of the simple system to more complex systems to reveal the presence of regulatory pathways that may not have previously been appreciated, such as positive and negative feedback circuitry. We clearly recognize that we cannot model all of the complexity of TCR signaling but we aim to understand the detailed mechanisms controlling tyrosine phosphorylation of the TCR ?-chain and how these events lead to LAT phosphorylation. The overall goal of project #1 is to understand the specificity and regulation of protein tyrosine kinases and phosphatases involved In TCR signaling that lead to ITAM and LAT phosphorylation at the membrane surface. We will: 1) define the molecular basis for specificity for Lck and ZAP-70 for ITAMs and LAT, respectively; 2) develop an analog sensitive inhibitor system for Lck; 3) define the mechanisms that control ITAM phosphorylation by Lck; and, 4) define the mechanisms that control LAT phosphorylation by ZAP-70 PUBLIC HEALTH RELEVANCE (provided by applicant): Biochemical signaling events by the T cell receptor (TCR) for antigen controls T cell responses but our understanding of these signaling events Is very rudimentary. These studies aim to understand how the enzymes, kinases and phosphatases, that the TCR controls are regulated on a surface model membrane, as they would be in a cell. These studies could lead to novel therapies of T cells mediated diseases