The physiological role of RIPK3-dependent necroptosis

项目摘要/摘要-依赖RIPK3的坏死性下垂的生理作用 肿瘤坏死因子和Toll样受体信号在协调中起关键作用 免疫反应，通过驱动促炎基因的转录激活。然而，它已经有很长时间了 人们已经认识到，它们也可以引发细胞凋亡。最近，有研究表明，这些 信号还可以引起另一种形式的程序性细胞死亡，称为“坏死性下垂”。虽然发现了 坏死性下垂引起了相当大的兴趣，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水解酶caspase-8阻断，所以大多数研究 依赖于基因消融或caspase-8的化学抑制来触发坏死性下垂。这就提出了一个问题：什么时候 在生理条件下是否会发生肿瘤坏死因子或肿瘤坏死因子受体介导的坏死性下垂？我们已经证明了caspase-8 必须与其Paralog Flip协同作用以阻止坏死性下垂，而Flip可被肿瘤坏死因子和 TLR转录信号。许多类型的感染和应激导致炎症信号或 对蛋白质合成的全面抑制。因此，我们建议没有翻转--而不是 抑制caspase-8-为细胞对坏死性下垂的敏化提供了一般机制。我们 进一步假设，坏死性下垂本身就是炎症性的，因为死于坏死性下垂的细胞会释放损伤- 激活免疫细胞的相关信号分子。为了应对这种可能性，我们将重点关注三个方面 具体问题：1)促坏死因子激酶RIPK3是如何被激活的，这种激活是如何被激活的 被caspase-8/flip抑制？我们已经创建了一个系统，其中可以使用RIPK3的多个步骤 控制住了。我们将使用这个系统来测试RIPK3激活需要磷酸化的假设- 依赖于RIPK3寡聚体的组装和增殖，且caspase-8/Flip直接阻断这一过程 进程。2)生理性下垂如何解除对caspase-8/flip的抑制以允许坏死性下垂 条件呢？我们假设，核因子-kB信号或一般蛋白质翻译的抑制物可使 通过阻止翻转表达使细胞发生坏死性下垂。我们将在多种类型的细胞中测试此模型 细菌和病毒感染的病理相关模型，以及内质网应激。我们还将考虑如何 FliP水平在mRNA和蛋白质水平上都受到控制。3)免疫系统如何应对 坏死性细胞死亡与凋亡性细胞死亡？我们假设细胞死亡的机制很重要， 因为坏死性下垂会释放炎性分子，而这些分子在细胞凋亡过程中会被抑制或消除。至 测试这一想法，我们已经创造了一个系统，允许我们使用无毒的 毒品。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。团结在一起， 这里提出的工作旨在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。

Project Summary/Abstract- The physiological role of RIPK3-dependent necroptosis 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.