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

项目摘要/摘要 Rho家族小GTP酶是细胞迁移、基因转录、生长和生长等活动的关键调节因子。 生存，通过细胞因子、生长因子等多种分子发出信号而启动的过程， 和GPCR。DBL家族Rho鸟嘌呤核苷酸交换因子(RhoGEF)，包括约70个成员， 是Rac、CDC42和Rho等GTP酶信号转导的关键激活物。RhoGEF是多域的 蛋白质经常在细胞膜上起信号支架的作用，并在调节其他 信令路径，这是由其复杂的体系结构和每个成员都有一个 唯一的域组成。作为细胞迁移和分裂过程中的主要参与者，几个 RhoGEF与癌症有很强的相关性。尽管它们在临床上很重要，但这些蛋白质 在分子水平上从全分子、机械论的角度进行了深入的研究，而且没有 靶向这些酶的治疗性抑制剂。我们的实验室开创了全长RhoGEF研究的先河 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 是为了了解DBL Rhogef信号和调控背后复杂的、多组分的机制。 在这个家族中，依赖于磷脂酰肌醇3，4，5-三磷酸(PIP3)的RAC交换器(P-Rex) 包括P-Rex1和P-Rex2在内的亚家族是细胞迁移的重要调节因子。这两种异构体都是 与人类癌症有关，在这些癌症中，它们起到促进转移的作用。P-Rex2通常在 乳腺癌和黑色素瘤，突变分布在整个蛋白质中。一项研究确定它是 人类转移性黑色素瘤中突变最多的基因。总而言之，数据支持P-Rex是一个重要的 与疾病有关的信号分子和合适的治疗靶点。然而，尽管霸王龙是 15年前被发现，其调控机制的分子细节仍不完全清楚。 P-Rex蛋白被认为是在其不活跃的，非信号状态下被自动抑制，并在多种状态下被激活. 阶梯机构。它们由结合膜系留的G蛋白b和g亚基激活，位于 GPCR信号转导，并通过结合细胞膜脂质，包括脂质PIP3。此外，P-Rex还扮演着 通过与PKA和PTEN等信号蛋白结合，在各种细胞环境中构建信号支架，导致 P-Rex和结合伙伴活性的变化。我们的长期目标是了解 RhoGEF在基础状态和完全活性状态之间的转换以及如何调节这种转换，从 霸王龙亚家族。此外，我们将确定这些蛋白质如何在细胞中充当信号支架。 薄膜。利用纳米盘，我们将研究RhoGEF与脂质和调节因子的多价相互作用 分子，让我们对这些信号复合体有了前所未有的了解。机械论的假设将是 在体外和癌细胞系中进行了测试。一旦确定了重要的监管表面，我们将通过 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.