The physiological role of RIPK3-dependent necroptosis

RIPK3依赖性坏死性凋亡的生理作用

• 批准号: 9193610
• 负责人: Andrew Atwell Oberst
• 金额: $ 44.29万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9193610
• 关键词: Ablation; Address; Alpha Cell; Antigen-Presenting Cells; Antigens; Apoptosis; Apoptotic; Autoimmune Diseases; Automobile Driving; Bacterial Infections; Bacterial Model; Bacterial Toxins; Binding; Bioavailable; CASP8 gene; Caspase Inhibitor; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Chemicals; Complex; Emergency Situation; Event; Evolution; Genes; Genetic; Genetic Transcription; Growth; Immune; Immune response; Immune signaling; Immune system; Infection; Inflammatory; Lead; Ligation; Malignant Neoplasms; Mammalian Cell; Manipulative Therapies; Mediating; Messenger RNA; Modeling; NF-kappa B; Necrosis; Outcome; Pathologic; Pathway interactions; Peptide Hydrolases; Phagocytes; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Protein Inhibition; Protein Synthesis Inhibition; Proteins; RIPK3 gene; Receptor Signaling; Role; Signal Transduction; Signaling Molecule; Stress; System; T cell response; TNF gene; TNFRSF1A gene; Tertiary Protein Structure; Testing; Tissues; Toll-like receptors; Transcriptional Activation; Translations; Up-Regulation; Virus; Virus Diseases; Virus Inhibitors; Work; adaptive immune response; cell type; cellular sensitization; cytokine; in vivo; inhibitor/antagonist; insight; interest; novel; paralogous gene; prevent; programs; public health relevance; response; therapy design; tool; tumor progression

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.

描述（由适用提供）：肿瘤坏死因子 - （TNF）和Toll样受体（TLR）信号传导通过驱动促炎基因的转录激活，在配位免疫调查中起关键作用。但是，长期以来，人们已经认识到它们也可以引发凋亡细胞死亡。最近，已经表明，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”。虽然发现坏死的发现引起了人们的考虑，但这种替代细胞死亡程序的身体作用仍然难以捉摸。特别是，坏死性被凋亡蛋白酶caspase-8阻塞，因此大多数研究依赖于遗传消融或caspase-8的化学抑制来触发坏死。这提出了一个问题：在生理条件下，TNF或TLR介导的坏死症何时发生？我们已经表明，caspase-8必须与其旁系同源物翻转一起起作用以阻断坏死作用，并且FLIP可能会被TNF和TLR转录信号传导上调。许多类型的感染和压力会导致抑制炎症信号传导或对蛋白质合成的一般抑制作用。因此，我们建议缺乏翻转 - 抑制caspase-8提供了一种对坏死性敏感性敏感的一般机制。我们进一步假设坏死作用本身是炎症性的，因为坏死性释放损伤与激活免疫细胞的信号分子死亡的细胞。为了解决这种可能性，我们将重点关注三个特定问题：1）如何激活促氧化激酶RIPK3，以及如何被caspase-8/flip抑制这种激活？我们创建了一个系统，可以控制RIPK3的多个步骤。我们将使用该系统来测试RIPK3激活需要磷酸化依赖性组装和RIPK3低聚物的传播的假设，而caspase-8/Flip直接阻止了此过程。 2）caspase-8/flip的抑制是如何在物理条件下允许坏死的抑制作用？我们假设NF-KB信号传导的抑制剂或通用蛋白质翻译，通过防止翻转表达来对坏死的敏感细胞对坏死。我们将使用细菌和病毒感染的病理相关模型以及ER应力在多种细胞类型中测试该模型。我们还将考虑如何在mRNA和蛋白质水平上控制翻转水平。 3）免疫系统如何应对核凋亡与凋亡细胞死亡？我们假设细胞死亡的机制很重要，因为坏死释放了凋亡过程中包含或消除的炎症分子。为了测试这个想法，我们创建了一个系统，使我们能够使用无毒药物触发凋亡或坏死。我们将使用该系统来分析先天和适应性免疫回避细胞死亡。这里提出的工作共同旨在了解体内坏死性的原因和后果，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疗法。