A novel proteomics approach to uncover Rho GTPases effector pathways

有问题的概述******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中的信号以动态方式重塑其形状的能力。例如，异常的细胞迁移可能导致癌症扩散（称为转移）。我们正在研究 22 种 Rho GTPases，因为它们被认为是调节细胞骨架的中心分子。从外观上看，Rho GTPases 是调节细胞形状的简单小型开/关分子开关。然而，至少有 150 种调节蛋白可以控制 Rho 蛋白如何以及何时变得活跃，但其中只有少数得到了充分的表征。哪些正向和负向调节 Rho 蛋白尚未明确。 ******此外，一旦 Rho 蛋白“开启”，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 个效应器相互作用并且这些效应器仍有待完全识别时，这个简单的分子系统就变得高度复杂。事实上，我们已经知道 Rho 蛋白的激活剂（称为 GEF）不会激活所有可能的效应器。我们预测 GEF 提供逻辑信号，仅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探索过。******目标****** 最近，一种称为 BioID 的新技术正在兴起，它是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。我们采用这项技术来研究细胞中的 Rho 蛋白信号传导组织。我们这个项目有 3 个主要目标：*****1) 我们希望以高分辨率系统地绘制 20 个 Rho GTPase 的所有相互作用蛋白图谱。******2) 我们将开发一种新技术来识别单个 Rho GTPase/GEF 对的特定效应器。******3) 我们将表征目标 1-2 确定的新型效应器和效应器信号传导的生物学功能。 ******这项工作的新颖性和预期意义****** 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 GTPases 与 GEF 和效应蛋白的相互作用。这将是首次对 Rho GTPase 信号传导进行如此大规模的分析，它可能会改变 Rho GTPase 信号传导的研究。这些方法将适用于分析所有 150 个 Ras 家族 GTP 酶的信号传导。该项目将为培养研究生提供一个出色的平台，培养下一代科学家，使他们能够理解细胞内高度复杂的信号传导。未来，需要对信号通路的分子组织有如此深入的了解，才能合理设计有效的药物来治疗人类疾病。

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.