Regulation and Function of ISG15 in Innate Immunity

描述（由申请人提供）：Toll样受体（TLR）和干扰素（IFN）诱导介导先天免疫的基因，并形成适应性免疫应答。TLR激动剂双链RNA（dsRNA）和脂多糖（LPS）可诱导IFN刺激基因（ISG）的亚类，表明它们在宿主防御中起作用。ISG 15在这些ISG中脱颖而出，作为具有免疫调节特性的蛋白质，其是对这些刺激的最早和最强的反应之一; ISG 15在先天免疫中的特定功能尚不清楚。ISG 15是一种泛蛋白样蛋白，在细胞内作用于翻译后修饰细胞蛋白质。游离的、未缀合的ISG 15从细胞中释放，在细胞中它表现出类精氨酸活性。病毒、IFN和TLR激动剂诱导相当水平的ISG 15。然而，在病毒和TLR激动剂刺激的细胞中观察到主要游离的ISG 15，导致其分泌增强;而IFN处理导致ISG 15与细胞蛋白质的稳健缀合，有利于其保留在细胞中。我们假设ISG 15在先天免疫中起双功能作用：i）作为由病毒、细菌和TLR激动剂诱导的细胞外细胞因子;和ii）通过其与细胞蛋白缀合作为IFN作用的细胞内介体。因此，不同诱导剂对ISG 15缀合和解缀合酶的差异调节决定了ISG 15缀合/分泌的程度，因此决定了其主要的生物学功能。为了理解ISG 15缀合的功能意义，鉴定ISG 15缀合物是必要的。蛋白酶体抑制显著增加特异性ISG 15缀合物的水平;我们将采用该条件纯化ISG 15缀合物（目的1）。ISG 15缀合位点的定位和诱变将确定缀合如何影响蛋白质稳定性和亚细胞定位（目的2）。为了剖析不同刺激差异调节ISG 15缀合和分泌的机制，将确定病毒、IFN、dsRNA和LPS对ISG 15缀合和解缀合酶的诱导（目的3）。为了将ISG 15诱导置于先天免疫应答的背景下，我们将测量小鼠中响应微生物刺激的细胞内和细胞外ISG 15的表达（目的4）。了解ISG 15在细胞因子网络中的功能可能会揭示操纵免疫功能用于治疗应用的策略。

DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) and interferons (IFNs) induce genes that mediate innate immunity, and shape the adaptive immune response. A subset of IFN-stimulated genes (ISGs) is induced by the TLR agonists doublestranded RNA (dsRNA) and lipopolysaccharide (LPS), suggesting that they function in host defense. ISG15 stands out among these ISGs as a protein with immunomodulatory properties that is one of the earliest and strongest responses to these stimuli; the specific function(s) of ISG15 in innate immunity are not known. ISG15 is an ubiquitin-like protein that acts within cells to posttranslationally modify cellular proteins. Free, unconjugated ISG15 is released from cells where it exhibits cytokine-like activities. Virus, IFN, and TLR agonists induce comparable levels of ISG15. However, predominantly free ISG15 is observed in virus and TLR agonist-stimulated cells, leading to its enhanced secretion; whereas, IFN treatment results in a robust conjugation of ISG15 to cellular proteins, favoring its retention in cells. We hypothesize that ISG15 serves a bifunctional role in innate immunity: i) as an extracellular cytokine that is induced by virus, bacteria, and TLR agonists; and ii) as an intracellular mediator of IFN action through its conjugation to cellular proteins. Thus, the differential regulation of ISG15 conjugating and deconjugating enzymes by distinct inducers dictates the extent of ISG15 conjugation/secretion, and hence its predominant biologic function. To understand the functional significance of ISG15 conjugation, it is essential to identify ISG15 conjugates. Proteasome inhibition dramatically increases the levels of specific ISG15 conjugates; we will employ this condition to purify ISG15 conjugates (aim 1). Mapping and mutagenesis of ISG15 conjugation sites will determine how conjugation influences protein stability and subcellular location (aim 2). To dissect the mechanism by which distinct stimuli differentially regulate ISG15 conjugation and secretion, the induction of ISG15 conjugating and deconjugating enzymes by virus, IFN, dsRNA and LPS will be determined (aim 3). To place ISG15 induction in the context of the innate immune response, we will measure the expression of intracellular and extracellular ISG15 in response to microbial stimuli in mice (aim 4). An understanding of how ISG15 functions in the cytokine network may reveal strategies to manipulate immune function for therapeutic applications.