RIP2 Caspase-1 Signaling in Macrophages

项目摘要 巨噬细胞释放17 kDa IL-1是一种高度调节的事件， 转录和合成前体31 kDa proIL-1的能力。此事件集中于激活 酶半胱天冬酶-1，并涉及称为炎性体的复杂蛋白质集合。炎症小体 这些基因是存在于植物和动物中的古老先天宿主防御系统的同源物。 目前的建议旨在扩大从IL-1的巨噬细胞生物学中获得的线索， 将这些过程扩展到宿主对脓毒症反应的基础。我们有最新的数据表明， 这种细胞内炎性体复合物的关键成分显著促进了破坏性的 先天性宿主反应的激活定义了感染性休克。更具体地说，caspase-1， 炎性小体的靶点与脓毒症直接相关。半胱天冬酶1基因敲除的动物 保护免于败血症死亡，但是胱天蛋白酶-1炎性体复合物的单个组分（例如， 半胱天冬酶-5，ASC和NALP 1）也与脓毒症结果相关。这些蛋白质相互作用， 其他通过半胱天冬酶募集结构域（CARD）和结构相关的pyrin结构域（PYD）。我们 一种假设是，以这些CARD和PYD结构域为中心的翻译后事件是 脓毒症的发病机制。炎性小体组装不仅调节炎症介质的加工和激活， 炎症细胞因子如IL-1和IL-18，但扩展到NF B和宿主细胞凋亡的调节。 目前的建议旨在将这些令人兴奋的突破应用于巨噬细胞生物学 面对败血症的挑战我们知道炎性小体组装的关键调控事件之一 是CARD/CARD和PYD/PYD相互作用的触发。我们最近证明， 含有CARD的分子（例如，RIP2和ASC）可以指导以caspase-1为中心的炎症反应 或者是NF B或者是IL-1加工。有趣的是，这些事件涉及早期磷酸化， 由于酪氨酸激酶活性的抑制深刻地影响炎性小体， 败血症因此，这项建议试图剖析这些事件是如何调节的分子细节。 中心假设是，决定亲的方向和严重性的主调节开关， 和对脓毒症攻击的抗炎反应在胞质溶胶中由特异性CARD/CARD控制， PYD/PYD相互作用。我们将剖析调节caspase-1和RIP2能力的机制， 相互作用，运输到膜，然后直接宿主炎症。这些事件最初诱导NFkB活化 但随后，通过caspase-1催化活化，可能最终诱导凋亡和NF B下调 事件这些研究将提高我们对脓毒症的基本先天宿主反应的理解， 这样做可以发现治疗感染性休克和其他炎症性疾病的新治疗方法， 由这个以caspase-1为中心的过程调节。

Project Summary Macrophage release of 17 kDa IL-1¿ is a highly regulated event that extends beyond the macrophage's ability to transcribe and synthesize the precursor, 31 kDa proIL-1¿. This event is centered upon activation of the enzyme caspase-1 and involves a complex protein assemblage termed the inflammasome. Inflammasome components are homologues of an ancient innate host defense system that exists in both plants and animals. The present proposal seeks to expand upon the clues derived from the macrophage biology of IL-1¿ and extend these processes to the basics of the host response to sepsis. We have recent data to demonstrate that key components of this intracellular inflammasome complex contribute significantly to the devastating activation of the innate host response that defines septic shock. More specifically, caspase-1, the central target of the inflammasome has been directly linked to sepsis. Not only are caspase-1 knockout animals protected from sepsis mortality but individual components of the caspase-1 inflammasome complex (e.g., caspase-5, ASC and NALP1) have also been linked to sepsis outcomes. These proteins interact with each other via caspase recruitment domains (CARDs) and structurally related pyrin domains (PYDs). Our hypothesis is that posttranslational events centered upon these CARD and PYD domains are central to the pathogenesis of sepsis. Inflammasome assembly not only regulates the processing and activation of inflammatory cytokines like IL-1¿ and IL-18 but extends to the regulation of NF¿B and host cell apoptosis. The current proposal seeks to apply these exciting breakthroughs in the biology of macrophage function to the challenge of sepsis. We know that one of the key regulatory events in inflammasome assembly is the triggering of CARD/CARD and PYD/PYD interactions. We have recently demonstrated that regulating CARD-containing molecules (e.g., RIP2 and ASC) can direct the caspase-1 centered inflammatory response either toward NF¿B or toward IL-1¿ processing. Interestingly, these events involve an early phosphorylation event since inhibition of tyrosine kinase activity profoundly affects the inflammasome and also protects animals from sepsis. Thus, this proposal seeks to dissect the molecular details of how these events are regulated. The central hypothesis is that the master regulatory switch that determines the direction and severity of the pro and anti-inflammatory responses to septic challenge are controlled in the cytosol by specific CARD/CARD and PYD/PYD interactions. We will dissect the mechanisms that regulate the ability of caspase-1 and RIP2 to interact, traffic to membranes and then direct host inflammation. These events initially induce NFkB activation but subsequently, via caspase-1 catalytic activation, may finally induce apoptosis and down regulation of NF¿B events. These studies will improve our understanding of the basic innate host responses to sepsis and in doing so uncover novel therapeutic approaches to septic shock and other inflammatory disorders that are regulated by this caspase-1-centric process.