Mechanistic Insights into Activation and Regulation of Interferon-inducible GTPase GBP2

干扰素诱导型 GTP 酶 GBP2 激活和调节的机制见解

• 批准号: 10166764
• 负责人: Qian Yin
• 金额: $ 37.3万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-18 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10166764
• 关键词: Address; Attention; Bacteria; Bacteriolysis; Binding; Binding Proteins; Biochemical; Biological Assay; Biophysics; Cells; Complex; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Crystallography; Cytolysis; Data; Detection; Development; Disease; Dynamin; Electron Microscopy; Enzyme Kinetics; Exposure to; Extravasation; Family; Fluorescence; Glean; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Higher Order Chromatin Structure; Human; Hydrolysis; Immune; Immunity; Individual; Infection; Inflammation; Interferons; Knowledge; Lead; Length; Light; Lipids; Liposomes; Maps; Measures; Membrane; Modeling; Molecular; Molecular Conformation; Nucleotides; Parasites; Pathogen detection; Pattern recognition receptor; Play; Process; Protein Activation Pathway; Proteins; Regulation; Research; Role; Rupture; Structure; System; Techniques; Toxoplasma gondii; Vacuole; Work; X-Ray Crystallography; analog; antimicrobial; autoinflammatory; base; biophysical techniques; cell growth regulation; cryogenics; cytokine; design; enzyme activity; fight against; guanylate; insight; light scattering; microbial; novel therapeutic intervention; novel therapeutics; pathogen; pathogen exposure; pathogenic microbe; protein activation; prototype; reconstitution; recruit; response; unilamellar vesicle

PROJECT SUMMARY Interferon-inducible GTPases are among the most potent effectors in cell-autonomous immunity. They are dynamin-like large GTPases highly induced by proinflammatory cytokines, especially type I and II interferons. Interferon-inducible GTPases eliminate or restrict intracellular bacteria and protozoan parasites through a variety of strategies. The most prominent strategies are rupture of pathogen-containing vacuoles (PCVs) to expose pathogens to cytosolic pattern recognition receptors (PRRs), and direct attack and lysis of bacterial membranes. On the other hand, interferon-inducible GTPases are under tight control to avoid indiscriminate attack on host cell endomembranes. The critical roles of IFN-inducible GTPases are gaining attention and appreciation recently, yet the molecular mechanisms of their activation, their effector functions on target membranes, and their regulation by cellular and microbial factors remain evasive. In this proposal, we study the activation and regulation mechanisms of interferon-inducible GTPases using guanylate-binding protein 2 (GBP2) as the prototype. In our preliminary studies, we purified GBP2, determined its crystal structures, and gained initial insights into its structures and functions. In this research, we will take advantage of these preliminary data to determine the structural and mechanistic basis of GBP activation in solution and on target membrane, and the regulation mechanism by cellular and microbial factors. We will first elucidate GBP2 oligomerization status and GTPase activity in solution. We will then determine the atomic structures of GBP2 in various nucleotide-bound states using X-ray crystallography and cryogenic electron microscopy (cryo-EM). Next, we have modified GBP2 with farnesyl group and will characterize its interaction with lipids and liposomes. We will then determine the remodeling/lysis effect of farnesylated GBP2 on its target membranes using fluorescence-based liposome leakage assay and EM techniques. We also plan to characterize the higher-order assembly mode of membrane- attached GBP2. Finally, we will define how GBP2 is self-inhibited and regulated by other cellular and pathogen- derived proteins. Successful accomplishment of this proposed research will fill a major gap in our knowledge of the molecular mechanisms of intracellular pathogen detection and restriction. This work will also expand our understanding of activation and assembly of large GTPases in general. Ultimately, these findings will facilitate the development of novel therapeutic strategies for microbial infections and autoinflammatory diseases.

项目摘要 干扰素诱导的GTPase是细胞自主免疫中最有效的效应子之一。他们是 促炎细胞因子，尤其是I型和II型干扰素高度诱导的动力蛋白样大GTP酶。 干扰素诱导的GTP酶消除或限制细胞内细菌和原生动物寄生虫通过一种品种 策略。最突出的策略是含病原体液泡（PCV）破裂以暴露 病原体到胞质模式识别受体（PRR），以及细菌膜的直接攻击和裂解。 另一方面，干扰素诱导的GTPases受到严格控制 细胞内膜。最近，IFN诱导GTPases的关键作用最近引起了人们的关注和欣赏， 然而，它们激活的分子机制，其效应子在靶膜上的功能及其 细胞和微生物因子的调节仍然存在。在此提案中，我们研究激活和 使用鸟苷结合蛋白2（GBP2）作为干扰素诱导GTP酶的调节机制作为 原型。在我们的初步研究中，我们纯化了GBP2，确定了其晶体结构并获得了初始 深入了解其结构和功能。在这项研究中，我们将利用这些初步数据 确定溶液和靶膜中GBP激活的结构和机械基础，以及 通过细胞和微生物因子调节机制。我们将首先阐明GBP2低聚状态和 溶液中的GTPase活性。然后，我们将确定各种核苷酸结合的GBP2的原子结构 使用X射线晶体学和低温电子显微镜（Cryo-EM）的状态。接下来，我们修改了GBP2 与Farnesyl基团，将表征其与脂质和脂质体的相互作用。然后，我们将确定 使用基于荧光的脂质组 泄漏测定和EM技术。我们还计划表征膜的高阶组装模式 附着的GBP2。最后，我们将定义GBP2如何自抑制和受到其他细胞和病原体的调节 衍生的蛋白质。这项拟议研究的成功完成将填补我们对 细胞内病原体检测和限制的分子机制。这项工作也将扩大我们的 一般来说，了解大型GTPases的激活和组装。最终，这些发现将有助于 用于微生物感染和自身炎症性疾病的新型治疗策略的发展。