The physiological role of RIPK3-dependent necroptosis

描述（由申请人提供）：肿瘤坏死因子-β（TNF）和Toll样受体（TLR）信号传导通过驱动促炎基因的转录激活在协调免疫反应中发挥关键作用。然而，人们早就认识到它们也可以引发凋亡性细胞死亡。最近的研究表明，这些信号还可以诱导另一种形式的程序性细胞死亡，称为“坏死性凋亡”。“虽然坏死性凋亡的发现引起了相当大的兴趣，但这种交替细胞死亡程序的生理作用仍然难以捉摸。特别是，坏死性凋亡会被促细胞凋亡蛋白酶caspase-8所阻断，因此大多数研究都依赖于caspase-8的基因消融或化学抑制来触发坏死性凋亡。这就提出了一个问题：在生理条件下，TNF或TLR介导的坏死性凋亡何时发生？我们已经证明，caspase-8必须与其旁叶FLIP协同作用以阻断坏死性凋亡，并且FLIP被TNF和TLR转录信号有力地上调。许多类型的感染和应激导致炎症信号传导的抑制或蛋白质合成的一般抑制。因此，我们提出，FLIP的缺乏，而不是抑制半胱天冬酶-8-提供了一个细胞致敏坏死性凋亡的一般机制。我们进一步假设坏死性凋亡本身是炎症性的，因为坏死性凋亡导致的细胞死亡释放出损伤相关的信号分子，激活免疫细胞。为了解决这种可能性，我们将集中在三个具体的问题：1）促坏死蛋白激酶RIPK 3是如何被激活的，这种激活是如何被caspase-8/FLIP抑制的？我们已经创建了一个系统，其中可以控制RIPK 3的多个步骤。我们将使用该系统来检验RIPK 3激活需要磷酸化依赖的RIPK 3寡聚体的组装和增殖，以及胱天蛋白酶-8/FLIP直接阻断该过程的假设。2)在生理条件下，caspase-8/FLIP的抑制是如何被解除以允许坏死性凋亡的？我们假设NF-kB信号或一般蛋白翻译的抑制剂通过阻止FLIP表达使细胞对坏死性凋亡敏感。我们将使用细菌和病毒感染的病理相关模型以及ER应激在多种细胞类型中测试该模型。我们还将考虑FLIP水平如何在mRNA和蛋白质水平上控制。3)免疫系统如何对坏死性细胞死亡和凋亡性细胞死亡作出反应？我们假设细胞死亡的机制是重要的，因为坏死性凋亡释放出在凋亡过程中被抑制或消除的炎性分子。为了测试这个想法，我们创建了一个系统，允许我们使用无毒药物触发细胞凋亡或坏死性凋亡。我们将使用这个系统来分析细胞死亡的先天性和适应性免疫反应。总之，这里提出的工作旨在了解体内坏死性凋亡的原因和后果，从而允许合理设计治疗方法，在感染，自身免疫性疾病和癌症中操纵这一过程。

DESCRIPTION (provided by applicant): Tumor Necrosis Factor-� (TNF) and Toll-like receptor (TLR) signaling play key roles in coordinating immune responses, by driving the transcriptional activation of pro-inflammatory genes. However, it has long been recognized that they can also trigger apoptotic cell death. More recently, it has been shown that these signals can also induce another form of programmed cell death, called "necroptosis." While the discovery of necroptosis has generated considerable interest, the physiological role of this alternate cell death program remains elusive. In particular, necroptosis is blocked by the pro-apoptotic protease caspase-8, so most studies rely on genetic ablation or chemical inhibition of caspase-8 to trigger necroptosis. This raises a question: when does TNF or TLR-mediated necroptosis occur under physiological conditions? We have shown that caspase-8 must act in concert with its paralog FLIP to block necroptosis, and FLIP is potently up-regulated by TNF and TLR transcriptional signaling. Many types of infection and stress lead to inhibition of inflammatory signaling or general inhibition of protein synthesis. We therefore propose that the absence of FLIP-rather than inhibition of caspase-8-provides a general mechanism for cellular sensitization to necroptosis. We further hypothesize that necroptosis is itself inflammatory, because cells dying by necroptosis release damage- associated signaling molecules that activate immune cells. To address this possibility, we will focus on three specific questions: 1) How is the pro-necroptotic kinase RIPK3 activated, and how is this activation suppressed by caspase-8/FLIP? We have created a system in which multiple steps of RIPK3 can be controlled. We will use this system to test the hypothesis that RIPK3 activation requires phosphorylation- dependent assembly and propagation of a RIPK3 oligomer, and that caspase-8/FLIP directly blocks this process. 2) How is suppression of caspase-8/FLIP relieved to allow necroptosis under physiological conditions? We hypothesize that inhibitors of NF-kB signaling, or of general protein translation, sensitize cells to necroptosis by preventing FLIP expression. We will test this model in multiple cell types using pathologically relevant models of bacterial and viral infection, as well as ER stress. We will also consider how FLIP levels are controlled at both mRNA and protein levels. 3) How does the immune system respond to necroptotic vs. apoptotic cell death? We hypothesize that the mechanism by which a cell dies is important, because necroptosis releases inflammatory molecules that are contained or eliminated during apoptosis. To test this idea, we have created a system that allows us to trigger apoptosis or necroptosis using a non-toxic drug. We will use this system to analyze innate and adaptive immune responses to cell death. Together, the work proposed here seeks to understand the causes and consequences of necroptosis in vivo, and to thereby allow rational design of therapies that manipulate this process in infection, autoimmune disease, and cancer.