The physiological role of RIPK3-dependent necroptosis

描述(申请人提供)：肿瘤坏死因子-�(肿瘤坏死因子)和Toll样受体信号在协调免疫反应中发挥关键作用，通过驱动促炎基因的转录激活。然而，人们很早就认识到，它们也可以引发细胞凋亡。最近，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“坏死性下垂”。虽然坏死性下垂的发现引起了相当大的兴趣，但这种交替的细胞死亡程序的生理作用仍然难以捉摸。尤其是，坏死性下垂被促凋亡的蛋白酶caspase-8阻断，因此大多数研究依赖于caspase-8的基因消融或化学抑制来触发坏死性下垂。这就提出了一个问题：在生理条件下，肿瘤坏死因子或肿瘤坏死因子受体介导的坏死性下垂何时发生？我们已经证明caspase-8必须与其Paralog Flio协同作用来阻止坏死性下垂，而Flip被肿瘤坏死因子和TLR转录信号有效上调。许多类型的感染和应激导致炎症信号的抑制或蛋白质合成的普遍抑制。因此，我们认为，缺乏翻转-而不是抑制caspase-8-提供了细胞对坏死性下垂增敏的一般机制。我们进一步假设，坏死性下垂本身是炎症性的，因为死于坏死性下垂的细胞释放与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上：1)促坏死因子激酶RIPK3是如何被激活的，以及这种激活是如何被caspase-8/flip抑制的？我们已经创建了一个可以控制RIPK3的多个步骤的系统。我们将使用这个系统来验证这样的假设，即RIPK3的激活需要依赖于磷酸化的RIPK3寡聚体的组装和增殖，而caspase-8/Flip直接阻止这一过程。2)在生理条件下，如何解除对caspase-8/flip的抑制以允许坏死性下垂？我们假设，核因子-kB信号或一般蛋白翻译的抑制剂通过阻止翻转表达而使细胞对坏死性下垂敏感。我们将使用细菌和病毒感染的病理相关模型以及内质网应激，在多种细胞类型中测试这一模型。我们还将考虑如何在mRNA和蛋白质水平上控制翻转水平。3)免疫系统对坏死性和凋亡性细胞死亡有何反应？我们假设细胞死亡的机制很重要，因为坏死性下垂会释放在细胞凋亡过程中被抑制或消除的炎性分子。为了测试这一想法，我们创造了一个系统，允许我们使用无毒药物触发细胞凋亡或坏死性下垂。我们将使用这个系统来分析对细胞死亡的先天和获得性免疫反应。总之，这里提出的工作试图了解体内坏死性下垂的原因和后果，从而允许合理设计在感染、自身免疫性疾病和癌症中操纵这一过程的治疗方法。

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.