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（由申请人提供）：肿瘤坏死因子-α (TNF) 和 Toll 样受体 (TLR) 信号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“坏死性凋亡”。虽然坏死性凋亡的发现引起了人们极大的兴趣，但这种替代性细胞死亡程序的生理作用仍然难以捉摸。特别是，坏死性凋亡被促凋亡蛋白酶 caspase-8 阻断，因此大多数研究依赖 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：生理条件下TNF或TLR介导的程序性坏死何时发生？我们已经证明 caspase-8 必须与其旁系同源物 FLIP 协同作用来阻止坏死性凋亡，并且 FLIP 受到 TNF 和 TLR 转录信号传导的有效上调。许多类型的感染和应激会导致炎症信号传导的抑制或蛋白质合成的全面抑制。因此，我们认为 FLIP 的缺失（而不是 caspase-8 的抑制）提供了细胞对坏死性凋亡敏感的一般机制。我们进一步假设坏死性凋亡本身是炎症性的，因为因坏死性凋亡而死亡的细胞会释放与损伤相关的信号分子，从而激活免疫细胞。为了解决这种可能性，我们将重点关注三个具体问题：1）促坏死性激酶 RIPK3 是如何激活的，以及 caspase-8/FLIP 如何抑制这种激活？我们创建了一个可以控制 RIPK3 多个步骤的系统。我们将使用该系统来测试以下假设：RIPK3 激活需要 RIPK3 寡聚体的磷酸化依赖性组装和增殖，并且 caspase-8/FLIP 直接阻断该过程。 2) 如何解除 caspase-8/FLIP 的抑制，从而在生理条件下实现坏死性凋亡？我们假设 NF-kB 信号传导抑制剂或一般蛋白质翻译抑制剂通过阻止 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以及内质网应激的病理相关模型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 FLIP 水平。 3) 免疫系统如何应对坏死性细胞死亡和凋亡性细胞死亡？我们假设细胞死亡的机制很重要，因为坏死性凋亡会释放在细胞凋亡过程中被抑制或消除的炎症分子。为了测试这个想法，我们创建了一个系统，允许我们使用无毒药物触发细胞凋亡或坏死性凋亡。我们将使用该系统来分析对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。