RIP2 caspase-1 signaling in macrophages

描述(申请人提供)：巨噬细胞释放17 kDa IL-12是一个高度受调控的事件，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，涉及一种称为炎症体的复杂蛋白质组合。炎症体成分是一种古老的天然宿主防御系统的同系物，这种防御系统存在于植物和动物中。本提案试图扩展IL-12巨噬细胞生物学的线索，并将这些过程扩展到宿主对脓毒症的基本反应。我们最近的数据表明，这种细胞内炎性小体复合体的关键成分对定义感染性休克的固有宿主反应的破坏性激活起着重要作用。更具体地说，炎症小体的中心靶点caspase-1与脓毒症直接相关。不仅caspase-1基因敲除的动物免受脓毒症死亡的影响，而且caspase-1炎症体复合体的个别成分(例如，caspase-5、ASC和NALP1)也与脓毒症的结局有关。这些蛋白质通过半胱氨酸酶募集结构域(CADS)和结构相关的吡咯结构域(PYD)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脓毒症发病机制的核心。炎性小体组装不仅调节IL-12和IL-18等炎性细胞因子的处理和激活，而且延伸到对核因子-β和宿主细胞凋亡的调节。目前的提案寻求将巨噬细胞功能生物学上的这些令人兴奋的突破应用于脓毒症的挑战。我们知道，炎症小体组装中的关键调控事件之一是触发卡/卡和PYD/PYD相互作用。我们最近证明，调控含卡分子(如RIP2和ASC)可以将以caspase-1为中心的炎症反应导向NF-β或IL-12的处理。有趣的是，这些事件涉及早期的磷酸化事件，因为抑制酪氨酸激酶活性会深刻地影响炎症体，并保护动物免受败血症的影响。因此，这项提议试图剖析这些事件如何受到调控的分子细节。中心假说是，决定败血症激发的促炎和抗炎反应的方向和严重程度的主调控开关在细胞质中由特定的卡片/卡片和PYD/PYD相互作用控制。我们将剖析调控caspase-1和RIP2相互作用、运输到膜然后引导宿主炎症的机制。这些事件最初诱导核因子？B的激活，但随后通过caspase-1的催化激活，可能最终诱导细胞凋亡和下调核因子-？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，并在这样做的过程中发现治疗败血症休克和其他炎症性疾病的新方法，这些疾病由caspase-1为中心的过程调节。与公共卫生相关：感染性休克是一种由压倒性感染导致的全身损伤，是一种常见的疾病，在美国每年导致20多万人死亡。在这种情况下，身体对感染性物质的反应涉及到新描述的感应蛋白，我们认为这些蛋白对防御损伤至关重要。这项建议试图了解这些防御蛋白如何与感染因子反应来调节细胞死亡和炎症反应，特别是在脓毒症的背景下。

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.