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.

项目摘要/摘要 Rho家族小GTPases是细胞迁移，基因转录，生长和 生存，通过通过各种分子集合（包括细胞因子，生长因子，生长因子）发出信号引发的过程 和GPCR。 DBL家庭Rho Guanine核苷酸交换因子（Rhogefs），包括约70名成员， 是GTPases（例如RAC，CDC42和RHO）的信号传导的关键激活剂。 Rhogefs是多域 经常充当细胞膜的信号支架并在调节其他方面起作用的蛋白质 信号通路，这是由其复杂架构赋予的功能以及每个成员都有一个的事实 独特的域组成。作为细胞迁移和分裂的流程主要参与者，有几个 Rhogefs与癌症密切相关。尽管它们的临床重要性，但这些蛋白质非常重要 从整体分子，机械的角度研究在分子水平上，没有 靶向这些酶的治疗抑制剂。我们的实验室正在开创整个Rhogefs的研究 以及在脂质膜调节中的机制，使用冷冻EM作为主要方法。我们的长期目标 是要了解DBL RhoGEF信号传导和调节背后的复杂的多组分机制。 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） 包括P-REX1和P-REX2在内的亚家族充当细胞迁移的重要调节剂。两种同工型已经 与人类癌症相关，其中它们充当了促成 - 替代因素。 p-rex2通常在 乳腺癌和黑色素瘤，突变分布在整个蛋白质中。一项研究将其确定为 人类转移性黑色素瘤中最突变的基因。总共，数据支持P-Rex是重要的 信号分子与疾病有关和合适的治疗靶标。但是，即使P-Rex是 在15年前发现的调节机制的分子细节仍未得到充分了解。 假设P-Rex蛋白在其无活性，非信号状态下自身抑制 步骤机制。通过结合膜螺旋的G蛋白B和G亚基激活它们，下游 GPCR信号传导，并通过结合细胞膜脂质，包括脂质PIP3。另外，p-rex充当 通过与PKA和PTEN等信号蛋白结合，在各种细胞环境中的信号传导支架，导致 P-Rex和Binding合作伙伴的活动变化。我们的长期目标是了解与众不同 Rhogefs基础和完全活跃状态之间的转变，以及如何调节该转变 P-Rex亚家族。此外，我们将确定这些蛋白在细胞上如何充当信号支架 膜。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，使我们对这些信号传导复合物的前所未有。机械假设将是 在体外和癌细胞系中进行了测试。一旦确定了重要的调节表面，我们将通过 治疗分子的合理设计。