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

• 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
• 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

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.

抽象的 控制细胞行为的信号网络是高度动态的，并且在空间和 时间。 Rho家族GTP酶调节各种生物学过程，例如细胞迁移，增殖和 免疫激活。这些分子的活性在亚细胞水平上受到严格控制，并观察到 仅在单元格的离散区域中进行精确的时间。 DBL家族的鸟嘌呤交换因子（GEF）是 RhoA GTPases的主要激活剂。单元中通常存在多个GEF，可以作用于 相同的GTPase和某些GEF可以与不同的GTPase相互作用。最近，已经表明 DBL GEF的活性也分布在细胞中的离散区域，并用精确的动力学调节。 因此，GEF和GTPases形成了在空间和时间上紧密控制的复杂信号网络。 传统的GEF研究通常依赖于耗尽，击倒或淘汰或扩大。 过表达，特定的GEF活动。虽然信息丰富，但这些方法缺乏时空分辨率 并且可能由于连接的GEF/GTPase可能的补偿作用而引入生物伪影 信号网络。因此，为了充分了解GEF的生物学作用，需要新的分子工具 这允许快速，精确地控制它们在活细胞中的活性。该提议的目的是开发 高吞吐量平台，可以容易地应用于针对DBL家族的工程师光控制抑制剂 GEFS。这些抑制剂将使具有第二级内源性GEF的可逆抑制作用可逆抑制 活细胞中的动力学和微分分辨率。在AIM 1中，依赖计算的三种不同方法 建模和高吞吐量库筛选，将测试与高亲和力结合的工程师分子 以及对DBL GEF的特异性，并防止其GTPase关联。在AIM 2中，工程抑制剂将被融合 已知的光遗传学模块，以便通过辐射精确控制其活性。在AIM 3中 活细胞显微镜将研究光遗传学抑制剂，以确定实验参数 为了在体内实现有效的GEF抑制，需要进行微调。这个平台的实用性将是 通过工程光遗传学抑制剂针对针对三个主要的三个不同的DBL GEF证明 Rhoa GTPases，Rac1，Rhoa和Cdc42。这里开发的平台是一般的，可以很容易地应用 开发用于研究其他DBL GEF的分子工具。因此，该建议将有助于研究DBL 在不同生物系统的空间和时间分辨率方面，GEF在不同的空间和时间分辨率上。