Structural and Functional Characterization of RhoGEF Regulation Using Nanodiscs to Assemble Membrane-associated Signaling Scaffolds

项目总结/摘要 Rho家族小GTP酶是诸如细胞迁移、基因转录、生长和增殖等活动的关键调节因子。 存活，通过多种分子包括细胞因子，生长因子， 和GPCR。Dbl家族Rho鸟嘌呤-核苷酸交换因子（RhoGEFs），包括大约70个成员， 是GTP酶如Rac、Cdc 42和Rho信号传导的关键激活剂。RhoGEF是多域的 这些蛋白质经常在细胞膜上充当信号支架，并在调节其他 信号通路，这是由它们复杂的结构和每个成员都有一个 独特的域组成。作为细胞迁移和分裂过程的主要参与者， RhoGEFs与癌症密切相关。尽管它们在临床上很重要，但这些蛋白质 从整个分子的角度，机械的角度，在分子水平上研究不足，并且没有 针对这些酶的治疗性抑制剂。我们的实验室是全长RhoGEFs研究的先驱 以及它们在脂膜上的调节机制，使用冷冻EM作为主要方法。我们的长期目标 了解Dbl RhoGEF信号传导和调节背后复杂的多组分机制。 在该家族中，磷脂酰肌醇3，4，5-三磷酸（PIP 3）依赖性Rac交换器（P-Rex） P-Rex亚家族包括P-Rex 1和P-Rex 2，是细胞迁移的重要调节因子。两种异构体都是 与人类癌症相关，其中它们充当促转移因子。P-Rex 2通常在 乳腺癌和黑色素瘤，突变分布在整个蛋白质中。一项研究将其确定为 人类转移性黑色素瘤中突变最多的基因。总之，数据支持P-雷克斯是一个重要的 与疾病有关的信号分子和合适的治疗靶点。尽管P-Rex 尽管在15年前发现，但其调控机制的分子细节仍不完全清楚。 假设P-Rex蛋白在其非活性、非信号传导状态下被自抑制，并在多信号传导状态下被激活。 步进机构它们通过结合下游的膜束缚G蛋白B和G亚基而被激活 GPCR信号传导，并通过结合细胞膜脂质，包括脂质PIP 3。此外，P-Rex还充当了 通过与PKA和PTEN等信号蛋白结合， P-Rex和结合伴侣的活性变化。我们的长期目标是了解 RhoGEFs在基础状态和完全活性状态之间的转变以及这种转变是如何调节的， 霸王龙亚科此外，我们将确定这些蛋白质如何作为细胞的信号支架 膜的使用纳米盘，我们将研究RhoGEFs与脂质和调节性的多价相互作用。 分子，让我们对这些信号复合物有了前所未有的了解。机械假说将是 在体外和癌细胞系中测试。一旦确定了重要的调控表面，我们将通过以下方式来靶向这些表面： 治疗分子的合理设计。

Project Summary/Abstract Rho family small GTPases are key regulators of activities such as cell migration, gene transcription, growth, and survival, processes initiated by signaling through diverse sets of molecules including cytokines, growth factors, and GPCRs. Dbl family Rho guanine-nucleotide exchange factors (RhoGEFs), including around 70 members, are critical activators of signaling by GTPases such as Rac, Cdc42, and Rho. RhoGEFs are multi-domain proteins that frequently function as signaling scaffolds at cell membranes and play roles in regulating other signaling pathways, a feature conferred by their complex architecture and the fact that each member has a unique domain composition. As major players in processes underlying cell migration and division, several RhoGEFs are strongly implicated in cancer. Despite their clinical importance, these proteins are vastly understudied at the molecular level from a whole-molecule, mechanistic perspective, and there are no therapeutic inhibitors that target these enzymes. Our laboratory is pioneering the study of full-length RhoGEFs and mechanisms in their regulation at lipid membranes, using cryo-EM as a major approach. Our long-term goal is to understand the complex, multi-component mechanisms behind Dbl RhoGEF signaling and regulation. Within this family, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger (P-Rex) subfamily, including P-Rex1 and P-Rex2, act as important regulators of cell migration. Both isoforms have been associated with human cancers, wherein they act as pro-metastatic factors. P-Rex2 is commonly mutated in breast cancer and melanoma, with mutations distributed throughout the protein. One study identified it as one of the most mutated genes in human metastatic melanomas. Altogether, data support that P-Rex is an important signaling molecule implicated in disease and a suitable therapeutic target. However, even though P-Rex was discovered over 15 years ago, the molecular details of its regulatory mechanisms are still not fully understood. P-Rex proteins are hypothesized to be autoinhibited in their inactive, non-signaling states and activated in multi- step mechanisms. They are activated by binding membrane-tethered G protein b and g subunits, downstream of GPCR signaling, and by binding cell membrane lipids, including the lipid PIP3. Additionally, P-Rex acts as a signaling scaffold in various cellular contexts by binding to signaling proteins like PKA and PTEN, resulting in changes in activities of P-Rex and the binding partner. Our long-term goal is to understand how different RhoGEFs transition between the basal and fully active states and how this transition is regulated, starting with the P-Rex subfamily. Furthermore, we will determine how these proteins act as signaling scaffolds at the cell membrane. Using nanodiscs, we will study the multi-valent interactions of RhoGEFs with lipids and regulatory molecules, giving us unprecedented insight into these signaling complexes. Mechanistic hypotheses will be tested in vitro and in cancer cell lines. Once important regulatory surfaces are identified, we will target these via rational design of therapeutic molecules.