A novel proteomics approach to uncover Rho GTPases effector pathways

问题概述 ** 包括细胞迁移和粘附在内的细胞过程依赖于细胞骨架动力学，即细胞响应外部环境信号以动态方式重塑其形状的能力。例如，异常的细胞迁移会导致癌症的扩散（称为转移）。我们正在研究22 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小开/关分子开关。然而，至少有150种调节蛋白控制Rho蛋白如何以及何时变得活跃，但只有少数被很好地表征。Rho蛋白的正、负调控机制尚不明确。** 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物相互作用（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个效应物相互作用时，这个简单的分子系统变得非常复杂，并且仍然有待完全鉴定。事实上，我们已经知道Rho蛋白的激活剂，称为GEF，并不能激活所有可能的效应物。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。由于技术限制，GEF和Rho蛋白之间的这种相互作用从未被仔细研究过。最近，一种名为BioID的新技术正在成为揭示蛋白质如何在细胞内相互作用（即物理接触）的强大方法。我们采用这种技术来研究细胞中的Rho蛋白信号组织。我们有三个主要目标：**1）我们希望系统地绘制高分辨率的20种Rho GTP酶的所有相互作用蛋白。2)我们将开发一种新的技术来识别单个Rho GTdR/GEF对的特异性效应子。** 3)我们将描述目标1-2所确定的新型效应子和效应子信号传导的生物学功能。** 工作的新奇和预期意义 ** 在全球范围内，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 GT3信号进行如此大规模的分析，它可能会改变Rho GT3信号的研究。这些方法将适用于分析来自所有150种Ras家族GTP酶的信号传导。该项目将为培养研究生提供一个优秀的平台，培养下一代能够理解细胞内高度复杂信号的科学家。在未来，需要对信号通路的分子组织有如此深刻的理解，才能合理设计治疗人类疾病的强效药物。

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.