A novel proteomics approach to uncover Rho GTPases effector pathways

有问题的细胞过程概述，包括细胞迁移和黏附，依赖于细胞骨架动力学，即细胞对外部环境中的信号做出反应以动态方式重塑其形状的能力。例如，异常的细胞迁移可以导致癌症的扩散(称为转移)。我们正在研究22个Rho GTP酶，因为它们被认为是调节细胞骨架的中心分子。Rho GTP酶在外观上是调节细胞形状的简单的开/关分子开关。然而，至少有150种调控蛋白控制着Rho蛋白如何以及何时变得活跃，但只有少数几种蛋白具有良好的特性。哪些正性和负性调控Rho蛋白还没有定义。*此外，一旦Rho蛋白被“打开”，它就会与促进细胞内特定信号的效应器相互作用(例如，告诉细胞迁移)。同样，当我们认为每个Rho蛋白可以与多达40个效应器相互作用时，这个简单的分子系统变得高度复杂，而这些效应器还有待完全识别。事实上，我们已经知道，被称为GEF的Rho蛋白的激活剂并不能激活所有可能的效应器。我们预测，GEF提供了一个逻辑信号，只允许激活所需的效应器。由于技术的限制，GEF和Rho蛋白之间的相互作用从未被仔细研究过。目的最近，一种名为BioID的新技术正在出现，它是揭示蛋白质如何在细胞内相互作用(即物理接触)的有效方法。我们采用这项技术来研究Rho蛋白在细胞中的信号组织。我们对这个项目有三个主要目标：*1)我们想要以高分辨率系统地定位20个Rho GTP酶的所有相互作用的蛋白质。*2)我们将开发一种新的技术来识别单个Rho GTP酶/环境基金对的特定效应物。*3)我们将表征新的效应器和信号转导目标1-2的效应器的生物学功能。*工作的新颖性和预期意义*全球而言，这些相互作用筛选的完成将首次系统地定义Rho GTP酶与GEF和效应蛋白的相互作用。这将是第一次对Rho GTPase信号进行如此大规模的分析，这可能会改变对Rho GTPase信号的研究。这些方法将适用于分析所有150个RAS家族GTP酶的信号。该项目将为培养研究生培养能够理解细胞内高度复杂信号的下一代科学家提供一个出色的平台。未来，需要对信号通路的分子组织有如此深刻的了解，才能合理设计治疗人类疾病的强大药物。

OVERVIEW OF THE PROBLEMATIC******Cellular processes including cell migration and adhesion rely on cytoskeletal dynamics, i.e. the ability of the cells to remodel its shape in a dynamic manner in response to signals in the external environment. Aberrant cell migration, for example, can cause the spreading of cancers (known as metastasis). We are studying the 22 Rho GTPases as they are believed to be the central molecules to regulate the cytoskeleton. Rho GTPases are, in appearance, simple small on/off molecular switches that regulate cell shape. However, there are at least 150 regulatory proteins that control how and when Rho proteins become active but only a handful of them are well characterized. Which positive and negative regulate Rho proteins is not defined. ******In addition, once a Rho protein is turned “on”, it interacts with effectors that promote specific signals inside the cell (for example to tell to the cell to migrate). Again, this simple molecular system becomes highly complex when we think that each Rho protein can interact with up to 40 effectors and which remain to be fully identified. In reality, we already know that the activators of the Rho proteins, known as GEFs, do not activate all possible effectors. We predict that the GEFs provide a logistic signal that allows for the activation of only the required effectors. This interplay between GEFs and Rho proteins has never been explored carefully because of technical limitations.******OBJECTIVES******Recently, a novel technique, termed BioID, is emerging as a powerful approach to uncover how proteins interact (i.e. are in physical contact) inside the cell. We adapted this technology to study Rho protein signaling organization in cells. We have 3 major objectives for this project:******1) We want to systematically map, at high-resolution, all the interacting proteins of the 20 Rho GTPases.******2) We will develop a new technique to identify the specific effectors for individual Rho GTPase/GEF pairs.******3) We will characterize the biological function of the novel effectors and effectors signaling identified Aim 1-2. ******NOVELTY and EXPECTED SIGNIFICANCE OF THE WORK******Globally, completion of these interaction screens will systematically define, for the first time, Rho GTPases interactions with GEFs and with effector proteins. It will be the first time that such a large-scale analysis of the Rho GTPase signaling is conducted and it may transform the study of Rho GTPase signaling. These methods will be applicable to analyze signaling from all 150 Ras-family GTPases. This project will provide an outstanding platform for training graduate students toward the next generation of scientists that can understand highly complex signaling inside the cells. In the future, such a deep understanding of the molecular organization of signaling pathways is needed to rationally design powerful drugs to cure human diseases.