A novel proteomics approach to uncover Rho GTPases effector pathways

一种揭示 Rho GTPases 效应通路的新型蛋白质组学方法

• 批准号: RGPIN-2016-04808
• 负责人: Côté, JeanFrançois
• 金额: $ 3.93万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=709512
• 关键词: novel; proteomics; approach; uncover; Rho

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.

有问题的概述 细胞过程包括细胞迁移和依从性，依赖于细胞骨架动力学，即细胞以动态方式重塑其形状的能力，以响应外部环境中的信号。例如，异常细胞迁移会导致癌症的扩散（称为转移）。我们正在研究22个Rho GTPase，因为它们被认为是调节细胞骨架的中心分子。在外观上，Rho GTPase是调节细胞形状的简单小开/关。但是，至少有150种调节蛋白可以控制Rho蛋白如何以及何时活跃，但只有少数蛋白质的表征很好。阳性和阴性调节RHO蛋白的定义。 另外，一旦将RHO蛋白“打开”，它就会与促进细胞内特定信号的效果相互作用（例如，告诉细胞迁移）。同样，当我们认为每种Rho蛋白可以与多达40个效果相互作用并保持充分鉴定时，这种简单的分子系统就变得高度复杂。实际上，我们已经知道，Rho蛋白（称为GEFS）的活化剂并未激活所有可能的效果。我们预测GEF提供了一个逻辑信号，该信号仅允许激活所需的效果。由于技术局限性，GEFS和RHO蛋白之间的这种相互作用从未仔细探索。 目标 最近，一种被称为生物类的新技术正在成为一种有力的方法，可以发现细胞内部蛋白质如何相互作用（即在物理接触中）。我们调整了这项技术来研究细胞中的Rho蛋白信号组织。我们有3个主要目标： 1）我们想在高分辨率上系统地绘制20种Rho GTPases的所有相互作用蛋白。 2）我们将开发一种新技术，以确定单个Rho GTPase/GEF对的特定效果。 3）我们将表征新型效应和效应信号传导确定目标1-2的生物学功能。 工作的新颖性和期望的意义 在全球范围内，这些相互作用屏幕的完成将首次系统地定义Rho GTPases与GEFS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分析，并可能改变Rho GTPase信号传导的研究。这些方法将适用于分析所有150个RAS家庭GTPase的信号传导。该项目将为培训研究生提供一个出色的平台，以了解下一代科学家，这些科学家可以理解细胞内部的高度复杂信号。将来，需要对信号通路的分子组织进行如此深刻的理解，以合理地设计强大的药物来治愈人类疾病。