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

摘要 控制细胞行为的信号网络在空间和空间上高度动态和精确协调 时间到了。Rho家族GTP酶调控多种生物学过程，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，并被观察到 仅在单元的离散区域中具有精确定时。DBL家族的鸟嘌呤交换因子(GEF)是 RhoA GTP酶的主要激活物。通常在一个细胞中存在多个GEF，它们可以作用于 相同的GTP酶，某些GEF可以与不同的GTP酶相互作用。最近，有证据表明， DBL GEF的活性也分布在细胞内的离散区域，并受到精确的动力学调控。 因此，GEF和GTP酶形成复杂的信号网络，在空间和时间上受到严格控制。 传统的全球环境基金研究通常依赖于耗尽，通过击倒或敲除，或扩大，通过 全球环境基金具体活动的过度表达。虽然信息量大，但这些方法缺乏时空分辨率。 并可能引入生物人工制品，这是由于连接的全球环境基金/GTP酶可能产生的代偿效应 信令网络。因此，为了充分理解GEF的生物学作用，需要新的分子工具 这使得它们能够快速而精确地控制它们在活细胞中的活动。这项提议的目标是开发一种 高通量平台，可以很容易地应用于针对DBL家族设计光控抑制剂 全球创业投资基金。这些抑制剂将使内源性GEF的可逆抑制成为可能。 在活细胞中的动力学和微米分辨率。在目标1中，三种不同的方法依赖于计算 建模和高通量文库筛选将用于设计与高亲和力结合的分子 以及对DBL GEF的特异性，并阻止它们的GTPase关联。在AIM 2中，工程抑制剂将被融合 已知的光遗传模块，以便能够通过辐射精确控制它们的活动。在目标3中， 光遗传抑制剂将通过活细胞显微镜进行研究，以确定实验参数 为了在体内实现高效的全环基金抑制，需要进行微调。这个平台的效用将是 通过针对三种不同的DBL GEF设计光遗传抑制剂展示了这三种主要的 RhoA GTP酶、rac1、RhoA和CDC42。本文开发的平台通用性强，使用方便 为其他DBL GEF的研究开发分子工具。因此，这一建议将有助于DBL的研究 以前所未有的空间和时间分辨率跨越不同的生物系统。

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.