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

使用纳米圆盘组装膜相关信号支架的 RhoGEF 调节的结构和功能表征

• 批准号: 10794852
• 负责人: Jennifer Nicole Cash
• 金额: $ 1.7万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10794852
• 关键词: Address; Architecture; Binding; Biochemical; Breast Melanoma; Cancer cell line; Cell division; Cell membrane; Cell model; Cell physiology; Clinical; Complex; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Enzymes; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Genes; Genetic Transcription; Goals; Growth; Growth Factor; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Human; Image; Knowledge; Laboratories; Length; Lipids; Malignant Neoplasms; Membrane; Membrane Lipids; Membrane Proteins; Metastatic Melanoma; Methods; Molecular; Monomeric GTP-Binding Proteins; Mutate; Mutation; Output; PTEN gene; Peripheral; Physiology; Play; Principal Investigator; Process; Protein Isoforms; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Surface; Tertiary Protein Structure; Therapeutic; Therapeutic Uses; cell motility; cytokine; in vitro testing; inhibitor therapy; inorganic phosphate; insight; malignant breast neoplasm; member; membrane assembly; nanodisk; phosphatidylinositol 3,4,5-triphosphate; pressure; protein B; rational design; rho; scaffold; structural biology; therapeutic development; therapeutic target

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.

项目概要/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，通过不同分子组的信号启动的过程，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 (RhoGEF)，包括约 70 个成员， 是 Rac、Cdc42 和 Rho 等 GTPases 信号传导的关键激活剂。 RhoGEF 是多域的 蛋白质经常充当细胞膜上的信号支架并在调节其他 信号通路，其复杂的结构赋予的特征以及每个成员都有一个 独特的域组成。作为细胞迁移和分裂过程的主要参与者，一些 RhoGEF 与癌症密切相关。尽管它们具有临床重要性，但这些蛋白质在很大程度上 从全分子、机制的角度在分子水平上进行了研究，目前还没有 针对这些酶的治疗性抑制剂。我们的实验室正在开创全长 RhoGEF 的研究 及其在脂质膜上的调节机制，使用冷冻电镜作为主要方法。我们的长期目标 的目的是了解 Dbl RhoGEF 信号传导和调节背后复杂的多组分机制。 在这个家族中，磷脂酰肌醇 3,4,5-三磷酸 (PIP3) 依赖性 Rac 交换器 (P-Rex) 亚家族，包括 P-Rex1 和 P-Rex2，是细胞迁移的重要调节因子。两种亚型均已 与人类癌症相关，它们充当促转移因子。 P-Rex2 通常发生突变 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其中之一 人类转移性黑色素瘤中突变最多的基因。总而言之，数据支持霸王龙是一种重要的 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 15 年前就发现了它，但其调控机制的分子细节仍未完全了解。 据推测，P-Rex 蛋白在非活性、非信号传导状态下会自动抑制，并在多种状态下被激活。 阶梯机制。它们通过结合膜束缚的 G 蛋白 b 和 g 亚基（下游）而被激活。 GPCR 信号传导，并通过结合细胞膜脂质，包括脂质 PIP3。此外，P-Rex 还充当 通过与 PKA 和 PTEN 等信号蛋白结合，在各种细胞环境中形成信号支架，从而产生 霸王龙和结合伙伴的活动发生变化。我们的长期目标是了解差异有多大 RhoGEF 在基础状态和完全活跃状态之间转变以及如何调节这种转变，首先 霸王龙亚科。此外，我们将确定这些蛋白质如何充当细胞中的信号支架 膜。使用纳米圆盘，我们将研究 RhoGEF 与脂质和调节的多价相互作用 分子，使我们对这些信号复合物有了前所未有的了解。机制假设将是 在体外和癌细胞系中进行了测试。一旦确定了重要的监管面，我们将通过以下方式瞄准这些监管面： 治疗分子的合理设计。