Vimentin-mediated regulation of the inflammasome in acute lung injury

 描述（由申请人提供）：炎性小体响应于已知引起急性肺损伤（ALI）的广谱感染因子而被激活，但也必须对其进行严格调控以避免来自广泛的病原体、病原体相关分子模式和病原体相关分子模式的有害作用，已知这些病原体、病原体相关分子模式和病原体相关分子模式触发其激活。炎性小体的激活需要两种不同的促炎刺激。在第一步中，Toll样受体（TLR）信号转导激活NF-κB并上调IL-1β前体。第二步涉及多蛋白复合物的组装，其诱导半胱天冬酶-1的自切割，然后介导前IL-1β切割成其生物活性形式。越来越多的证据表明IL-1β是急性炎症和急性肺损伤的重要介质。我们将分别使用两种建立良好的NLRP 3和NLRC 4炎性小体激活的小鼠模型LPS和铜绿假单胞菌来检验我们的假设。我们将进行实验以证明NLR蛋白NLRP 3和NLRC 4通过其富含亮氨酸的重复序列（LRR）与波形蛋白相互作用，并且这种蛋白质-蛋白质相互作用是pro-IL-1β加工和成熟为生物活性IL-1β所必需的。我们将进一步研究在线粒体外膜的NLR蛋白的定位是否需要在炎性小体激活过程中与波形蛋白的相互作用。我们的数据表明，波形蛋白IF网络作为一个额外的检查点，以控制炎症体的激活，我们将测试使用波形蛋白/NLR蛋白相互作用的药理学抑制剂在体外和体内的假设。这些发现很可能对 由于靶向NLR和波形蛋白之间的相互作用，因此具有生物学和临床意义，可以提供一套治疗ALI/ARDS患者的新疗法。我们制定了三个相互关联的具体目标，研究波形蛋白中间丝在体内和体外急性肺损伤模型中的调节：具体目标1：确定波形蛋白（III型中间丝蛋白）激活炎症和急性肺损伤所需的炎性小体的机制。具体目标二：为了鉴定波形蛋白和NLR（NLRP 3和NLRC 4）之间的蛋白质：蛋白质相互作用，所述蛋白质：蛋白质相互作用是炎性小体激活所需的。具体目标3：确定NLR蛋白易位至线粒体膜是否是波形蛋白介导的炎性小体组装和激活所必需的。

 DESCRIPTION (provided by applicant): The inflammasome is activated in response to a broad spectrum of infectious agents known to cause acute lung injury (ALI), but it must also be tightly regulated to avoid deleterious effects from the wide array of pathogens, pathogen-associated molecular patterns and danger-associated molecular patterns, which are known to trigger its activation. Activation of the inflammasome requires two distinct pro-inflammatory stimuli. In the first step, Toll-like receptor (TLR) signaling activates NF-κB and upregulates pro IL-1β. The second step involves the assembly of the multiprotein complex, which induces self-cleavage of caspase-1, and then mediates the cleavage of pro- IL-1β into its biologically active form. Growing evidence indicates that IL-1β is a critical mediator of acute inflammation and acute lung injury. We will test our hypothesis using two well- established murine models of NLRP3 and NLRC4 inflammasome activation, LPS and Pseudomonas aeruginosa, respectively. We will conduct experiments to demonstrate that NLR proteins, NLRP3 and NLRC4, via their leucine-rich-repeat (LRR) interact with vimentin and that this protein-protein interaction is required for the processing and maturation of pro-IL-1β into biologically active IL-1β. We will further examine whether the localization of the NLR proteins at the outer mitochondrial membrane is required for the interaction with vimentin during inflammasome activation. Our data suggest that the vimentin IF network acts as an additional checkpoint to control the activation of the inflammasome, a hypothesis we will test using a pharmacologic inhibitor of vimentin/NLR protein interactions in vitro and in vivo. These findings are likely to have important biological and clinical significance as targeting the interaction between NLRs and vimentin could provide a suite of novel therapeutics to treat patients with ALI/ARDS. We have formulated three interrelated specific aims to study the regulation of vimentin intermediate filaments in both in vivo and in vitro models of acute lung injury: Specific Aim 1: To determine the mechanism by which vimentin, a type III intermediate filament protein, activates the inflammasome, which is required for inflammation and acute lung injury. Specific Aim 2: To identify the protein:protein interactions between vimentin and NLRs, NLRP3 and NLRC4, required for the activation of the inflammasome. Specific Aim 3: To determine whether translocation of NLR proteins to the mitochondrial membrane is required for the vimentin-mediated assembly and activation of the inflammasome.