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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要干扰素诱导型 GTP 酶是细胞自主免疫中最有效的效应物之一。他们是由促炎细胞因子，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 酶通过多种方式消除或限制细胞内细菌和原生动物寄生虫策略。最突出的策略是破裂含有病原体的液泡（PCV）以暴露病原体进入细胞质模式识别受体（PRR），并直接攻击和裂解细菌膜。另一方面，干扰素诱导型 GTPases 受到严格控制，以避免对宿主的无差别攻击细胞内膜。干扰素诱导型 GTP 酶的关键作用最近受到关注和赞赏，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作用细胞和微生物因素的调节仍然难以捉摸。在这个提案中，我们研究了激活和使用鸟苷酸结合蛋白 2 (GBP2) 作为干扰素诱导 GTP 酶的调节机制原型。在我们的初步研究中，我们纯化了GBP2，确定了其晶体结构，并获得了初步的结果深入了解其结构和功能。在本研究中，我们将利用这些初步数据确定 GBP 在溶液中和目标膜上活化的结构和机制基础，以及细胞和微生物因素的调节机制。我们将首先阐明 GBP2 寡聚状态和溶液中的 GTP 酶活性。然后我们将确定 GBP2 在各种核苷酸结合中的原子结构使用 X 射线晶体学和低温电子显微镜 (cryo-EM) 进行状态。接下来，我们修改了GBP2 与法呢基团结合，并表征其与脂质和脂质体的相互作用。然后我们将确定使用荧光脂质体法尼基化 GBP2 对其靶膜的重塑/裂解作用泄漏测定和 EM 技术。我们还计划表征膜的高阶组装模式附2英镑。最后，我们将定义 GBP2 如何自我抑制并受到其他细胞和病原体的调节 - 衍生蛋白质。这项研究的成功完成将填补我们在这方面知识的一个重大空白细胞内病原体检测和限制的分子机制。这项工作也将拓展我们的对大 GTP 酶的激活和组装的总体了解。最终，这些发现将有助于微生物感染和自身炎症性疾病新治疗策略的开发。