RIP2 caspase-1 signaling in macrophages

描述（由申请人提供）：巨噬细胞释放17 kDa IL-12是一个高度调控的事件，超出了巨噬细胞转录和合成前体31 kDa proIL-12的能力。这一事件以caspase-1酶的激活为中心，涉及一种称为炎性体的复杂蛋白质组合。炎性小体成分是存在于植物和动物中的一种古老的先天宿主防御系统的同源物。本研究旨在扩展巨噬细胞IL-12生物学的线索，并将这些过程扩展到宿主对败血症反应的基础。我们最近的数据表明，这种细胞内炎性体复合物的关键成分对定义感染性休克的先天宿主反应的破坏性激活起着重要作用。更具体地说，炎性体的中心靶点caspase-1与败血症直接相关。不仅caspase-1基因敲除动物免于败血症死亡，而且caspase-1炎性体复合体的个别成分（如caspase-5、ASC和NALP1）也与败血症的结果有关。这些蛋白通过caspase募集结构域（CARDs）和结构相关的pyrin结构域（PYDs）相互作用。我们的假设是，以这些CARD和PYD结构域为中心的翻译后事件是败血症发病机制的核心。炎性小体组装不仅调节炎性细胞因子如IL-12和IL-18的加工和激活，而且延伸到调节NF-？B和宿主细胞凋亡。目前的提案旨在将巨噬细胞功能生物学方面的这些令人兴奋的突破应用于败血症的挑战。我们知道炎症小体组装的关键调控事件之一是触发CARD/CARD和PYD/PYD相互作用。我们最近已经证明，调节含有card的分子（如RIP2和ASC）可以将caspase-1中心的炎症反应导向NF-？B或向IL-12加工方向发展。有趣的是，这些事件涉及早期磷酸化事件，因为酪氨酸激酶活性的抑制深刻影响炎性体，也保护动物免受败血症。因此，本提案试图剖析这些事件是如何调控的分子细节。核心假设是，在细胞质中，由特定的CARD/CARD和PYD/PYD相互作用控制的主要调控开关决定了对脓毒性挑战的促炎性和抗炎反应的方向和严重程度。我们将剖析调节caspase-1和RIP2相互作用，运输到膜然后指导宿主炎症的能力的机制。这些事件最初诱导NF-？但随后，通过caspase-1催化活化，最终可能诱导细胞凋亡和NF-？B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，并由此发现新的治疗脓毒症休克和其他炎症性疾病的方法，这些方法是由caspase-1中心过程调节的。公共卫生相关性：感染性休克是一种全身损伤，可因严重感染而发生，是常见的，是美国每年超过20万人死亡的原因。在这种情况下，身体对感染因子的反应涉及到新描述的传感蛋白，我们认为这些蛋白对防御损伤至关重要。该建议旨在了解这些防御蛋白如何与感染因子反应，以调节细胞死亡和炎症反应，特别是在败血症的背景下。

DESCRIPTION (provided by applicant): Macrophage release of 17 kDa IL-12 is a highly regulated event that extends beyond the macrophage's ability to transcribe and synthesize the precursor, 31 kDa proIL-12. 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-12 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-12 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-12 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 NF-?B 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. PUBLIC HEALTH RELEVANCE: Septic shock, the whole body injury that can occur as a result of overwhelming infections, is common and represents the cause of over 200,000 deaths annually in the United States. In this context, the body's response to infectious agents involves newly described sensing proteins that we believe are critical to the defense against the injuries. This proposal seeks to understand how these defense proteins react with infectious agents to regulate cell death and inflammatory responses, particularly in the context of sepsis.