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是高度动态的，在空间上精确协调， 时间Rho家族GTP酶调节多种生物过程，例如细胞迁移、增殖和分化。 免疫激活这些分子的活性在亚细胞水平上受到严格控制， 仅在单元的离散区域中具有精确的定时。Dbl家族的鸟嘌呤交换因子（GEF）是 RhoA GTP酶的主要激活剂。细胞中通常存在多个GEF，它们可以作用于细胞内的 相同的GTP酶，并且某些GEF可以与不同的GTP酶相互作用。最近的研究表明， Db 1 GEF的活性也分布在细胞中的离散区域，并以精确的动力学进行调节。 因此，GEF和GTP酶形成了在空间和时间上受到严格控制的复杂信号网络。 传统的全球环境基金研究通常依赖于通过击倒或淘汰进行的消耗，或通过 全球环境基金的具体活动。虽然这些方法信息丰富，但缺乏时空分辨率 并且可能由于相关GEF/GTHBE中可能的补偿效应而引入生物伪影 信令网络因此，要充分了解GEFs的生物学作用，需要新的分子工具 从而可以快速精确地控制它们在活细胞中的活性。该提案的目标是制定一个 可容易地应用于工程化针对Dbl家族的光控抑制剂的高通量平台 的GEF。这些抑制剂将使内源性GEF的可逆抑制成为可能， 动力学和活细胞中的微米分辨率。在目标1中，三种不同的方法，依赖于计算 模型和高通量文库筛选，将进行测试，以工程化的分子，结合高亲和力 和特异性，并阻止其GT3结合。在目标2中，工程抑制剂将被融合 已知的光遗传学模块，以便允许通过照射精确控制它们的活性。在目标3中， 将通过活细胞显微镜研究光遗传学抑制剂，以确定 需要进行微调，以实现有效的GEF体内抑制。该平台的效用将是 通过针对三种不同的Dbl GEF工程化光遗传学抑制剂证明了这一点，所述Dbl GEF靶向三种主要的 RhoA GTP酶、Rac 1、RhoA和Cdc 42。本文所开发的平台具有通用性和实用性 开发用于研究其他Dbl GEF的分子工具。因此，这一建议将有助于对DBL的研究 全球环境基金在不同的生物系统中以前所未有的空间和时间分辨率。

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.