Regulation and Function of ISG15 in Inmate Immunity

超出提供的空间。Toll样受体(TLRs)和干扰素(IFN)诱导基因介导天然免疫，并塑造获得性免疫反应。TLR激动剂双链RNA(DsRNA)和脂多糖(LPS)诱导了干扰素刺激基因(ISGs)的一部分，表明它们在宿主防御中发挥作用。在这些ISG中，ISG15作为一种具有免疫调节特性的蛋白质而脱颖而出，这是对这些刺激最早和最强烈的反应之一；ISG15在天然免疫中的特定功能(S)尚不清楚。ISG15是一种泛素样蛋白，在细胞内作用于翻译后修饰细胞蛋白。游离的、未结合的ISG15是从细胞中释放出来的，在那里它表现出细胞因子样的活性。病毒、干扰素和TLR激动剂可诱导类似水平的ISG15。然而，在病毒和TLR激动剂刺激的细胞中观察到主要是游离的ISG15，导致其分泌增加；而干扰素处理导致ISG15与细胞蛋白的牢固结合，有利于其在细胞内的保留。我们假设ISG15在先天免疫中起着双功能作用：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；以及ii)作为一种通过与细胞蛋白结合的干扰素作用的细胞内介质。因此，不同诱导剂对ISG15结合和去结合酶的不同调节决定了ISG15结合/分泌的程度，从而决定了ISG15的主要生物学功能。为了理解ISG15接合的功能意义，鉴定ISG15接合是必不可少的。蛋白酶体抑制显著增加了特异的ISG15结合物的水平；我们将利用这个条件来纯化ISG15结合物(目标1)。ISG15结合位点的作图和突变将决定结合如何影响蛋白质稳定性和亚细胞定位(目标2)。为了剖析不同刺激对ISG15结合和分泌的不同调控机制，将测定病毒、干扰素、dsRNA和内毒素诱导的ISG15结合和去结合酶(目标3)。为了将ISG15的诱导置于先天免疫反应的背景下，我们将测量小鼠细胞内和细胞外ISG15对微生物刺激的反应(目标4)。了解ISG15如何在细胞因子网络中发挥作用，可能会揭示为治疗应用而操纵免疫功能的策略。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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 double- stranded RNA (dsRNA) and tipopolysaccharide (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. PERFORMANCE SITE ========================================Section End===========================================