Regulation and Function of ISG15 in Innate Immunity

描述（由申请人提供）：toll样受体（TLRs）和干扰素（ifn）诱导介导先天免疫的基因，并形成适应性免疫反应。ifn刺激基因（ISGs）的一个子集被TLR激动剂双链RNA （dsRNA）和脂多糖（LPS）诱导，表明它们在宿主防御中起作用。ISG15作为一种具有免疫调节特性的蛋白质在这些isg中脱颖而出，是对这些刺激最早和最强烈的反应之一；ISG15在先天免疫中的具体功能尚不清楚。ISG15是一种泛素样蛋白，在细胞内起作用，翻译后修饰细胞蛋白。游离的、未偶联的ISG15从细胞中释放出来，并表现出细胞因子样活性。病毒、IFN和TLR激动剂诱导的ISG15水平相当。然而，ISG15在病毒和TLR激动剂刺激的细胞中主要游离，导致其分泌增强；然而，IFN处理导致ISG15与细胞蛋白的强大结合，有利于其在细胞中保留。我们假设ISG15在先天免疫中具有双重功能：i)作为一种由病毒、细菌和TLR激动剂诱导的细胞外细胞因子；ii)通过与细胞蛋白的结合，作为IFN作用的细胞内介质。因此，不同的诱导剂对ISG15偶联酶和解偶联酶的不同调控决定了ISG15偶联/分泌的程度，从而决定了其主要的生物学功能。为了了解ISG15偶联的功能意义，有必要对ISG15偶联物进行鉴定。蛋白酶体抑制显著增加特异性ISG15偶联物的水平；我们将利用这个条件纯化ISG15偶联物（目的1）。ISG15偶联位点的定位和诱变将决定偶联如何影响蛋白质稳定性和亚细胞定位（目的2）。为了剖析不同刺激对ISG15偶联和分泌的不同调节机制，将确定病毒、IFN、dsRNA和LPS对ISG15偶联和解偶联酶的诱导作用（目的3）。为了将ISG15诱导置于先天免疫反应的背景下，我们将测量小鼠在微生物刺激下细胞内和细胞外ISG15的表达（目的4）。了解ISG15如何在细胞因子网络中发挥作用，可能会揭示治疗应用中操纵免疫功能的策略。

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.