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High throughput platform to engineer light-controlled inhibitors against guanine exchange factors of the Dbl family

用于设计针对 Dbl 家族鸟嘌呤交换因子的光控抑制剂的高通量平台

基本信息

批准号：
10706957
负责人：
Mihai Luchian Azoitei
金额：
$ 35.97万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10706957
关键词：
Affinity Area Binding Binding Sites Biological Biological Models Biological Process Cell physiology Cells Complex Computer Models Coupled Cues DH Domain Darkness Disease Engineering Ensure Environment Epitopes Face Family Frequencies GTPase-Activating Proteins Goals Guanine Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases Health Immune Kinetics Knock-out Libraries Light Location Methods Mission Molecular Molecular Probes Morphologic artifacts Outcome Phosphotransferases Play Proliferating Protein Engineering Proteins Research Resolution Role Scaffolding Protein Signal Pathway Signal Transduction Site Spatial Distribution Specificity Structure System Tertiary Protein Structure Testing Time United States National Institutes of Health Work biological systems cell behavior cell motility design experimental study immune activation improved in vivo inhibitor intersectin 1 irradiation knock-down live cell microscopy nanobodies new technology novel optogenetics overexpression prevent rho rho GTP-Binding Proteins scaffold screening spatiotemporal temporal measurement tool

项目摘要

ABSTRACT Signaling networks that control cellular behavior are highly dynamic and precisely coordinated in space and time. Rho family GTPases regulate diverse biological processes such as cell migration, proliferation and immune activation. The activity of these molecules is tightly controlled at the subcellular level and is observed with precise timing only in discrete regions of the cell. The Dbl family of guanine exchange factors (GEFs) are the main activators of RhoA GTPases. There are typically multiple GEFs present in a cell that can act on the same GTPase, and certain GEFs can interact with different GTPases. Recently, it has been shown that the activity of Dbl GEFs is also distributed at discrete regions in the cell and regulated with precise kinetics. Therefore, GEFs and GTPases form complex signaling networks that are tightly controlled in space and time. Traditional GEF studies typically rely on depletion, by knock down or knock out, or augmentation, by overexpression, of specific GEF activities. While informative, these approaches lack spatiotemporal resolution and could introduce biological artifacts due to possible compensatory effects in connected GEF/GTPase signaling networks. Therefore, to fully understand the biological roles of GEFs, new molecular tools are needed that allow the rapid and precise control of their activity in living cells. The goal of this proposal is to develop a high throughput platform that can be readily applied to engineer light-controlled inhibitors against the Dbl family of GEFs. These inhibitors will make possible the reversible inhibition of endogenous GEFs with second-level kinetics and at micron resolution in living cells. In Aim 1, three different approaches, that rely on computational modeling and high throughput library screening, will be tested to engineer molecules that bind with high affinity and specificity to Dbl GEFs and prevent their GTPase association. In Aim 2, engineered inhibitors will be fused to known optogenetic modules in order to allow the precise control of their activity by irradiation. In Aim 3, the optogenetic inhibitors will be studied by live cell microscopy to determine the experimental parameters that need to be fine-tuned in order to achieve efficient GEF inhibition in vivo. The utility of this platform will be demonstrated by engineering optogenetic inhibitors against three different Dbl GEFs that target the three major RhoA GTPases, Rac1, RhoA and Cdc42. The platform developed here is general and could be readily applied to develop molecular tools for the study of other Dbl GEFs. This proposal will thus facilitate the study of Dbl GEFs at unprecedent spatial and temporal resolution across diverse biological systems.

摘要