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

项目摘要 干扰素诱导型GTP酶是细胞自主免疫中最有效的效应子之一。他们是 由促炎细胞因子，特别是I型和II型干扰素高度诱导的动力蛋白样大GTP酶。 干扰素诱导型GTP酶通过多种途径消除或限制细胞内细菌和原生动物寄生虫。 的战略。最突出的策略是破裂含病原体的空泡（PCV）， 病原体与胞质模式识别受体（PRR）的结合，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，以避免对宿主的不加选择的攻击 细胞内膜IFN-诱导型GTP酶的关键作用最近受到关注和重视， 然而，它们的激活的分子机制，它们对靶膜的效应功能，以及它们的 细胞和微生物因素的调节仍然是模糊的。在这个建议中，我们研究了激活和 以鸟苷酸结合蛋白2（GBP 2）为靶点的干扰素诱导型GTP酶的调控机制 样机在我们的前期研究中，我们对GBP 2进行了纯化，测定了其晶体结构，并初步获得了其结构的初步结果。 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GBP在溶液中和靶膜上活化的结构和机制基础， 细胞和微生物因素的调节机制。我们将首先阐明GBP 2寡聚化状态， 溶液中的GT酶活性。然后，我们将确定GBP 2在各种核苷酸结合的原子结构， 状态使用X射线晶体学和低温电子显微镜（cryo-EM）。接下来，我们修改了GBP 2 与法尼基基团，并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体的法尼基化GBP 2对其靶膜的重构/溶解作用 泄漏分析和EM技术。我们还计划表征膜的高阶组装模式- 附GBP 2。最后，我们将定义GBP 2是如何自我抑制和调节其他细胞和病原体- 衍生蛋白质。这项拟议研究的成功完成将填补我们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。这项工作也将扩大我们的 一般理解大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性疾病的新治疗策略的开发。

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.