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: 10393611
• 负责人: Qian Yin
• 金额: $ 37.29万
• 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/10393611
• 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.

项目总结 干扰素诱导的GTP酶是细胞自主免疫中最有效的效应器之一。他们是 促炎症细胞因子，尤其是I型和II型干扰素高度诱导的动力素样大GTP酶。 干扰素诱导的GTP酶通过多种途径清除或限制细胞内的细菌和原生动物寄生虫 战略的问题。最突出的策略是破裂含有病原体的空泡(PCV)以暴露 病原体对胞浆模式识别受体(PRRs)的作用，以及细菌膜的直接攻击和裂解。 另一方面，干扰素诱导的GTP酶受到严格控制，以避免对宿主的不分青红皂白的攻击 细胞内膜。近年来，干扰素诱导的GTP酶的关键作用受到了人们的关注和重视。 然而，它们激活的分子机制、它们在靶膜上的效应器功能以及它们的 细胞和微生物因素的调控仍然是回避的。在这个方案中，我们研究了激活和 以鸟苷酸结合蛋白2(GBP2)为靶点的干扰素诱导GTP酶的调控机制 原型。在我们的初步研究中，我们纯化了gbp2，测定了它的晶体结构，并获得了初步的 对其结构和功能的洞察。在这项研究中，我们将利用这些初步数据来 确定了GBP在溶液中和靶膜上活化的结构和机理基础，以及 细胞和微生物因素的调控机制。我们将首先阐明GBP2齐聚状态和 溶液中GTP酶活性。然后我们将确定不同核苷酸结合的GBP2的原子结构 使用了X射线结晶学和低温电子显微镜(Cryo-EM)。接下来，我们修改了GBP2 并将表征其与脂类和脂质体的相互作用。然后我们将确定 荧光脂质体对法尼化GBP2靶膜的重塑/裂解作用 渗漏试验和EM技术。我们还计划表征膜的高阶组装模式-- 随附的GBP2。最后，我们将定义GBP2是如何被其他细胞和病原体自我抑制和调节的- 衍生蛋白质。这项拟议研究的成功完成将填补我们对 细胞内病原体检测与限制的分子机制。这项工作也将扩大我们的 大体上理解大的GTP酶的激活和组装。最终，这些发现将有助于 微生物感染和自身炎症性疾病的新治疗策略的开发。