The physiological role of RIPK3-dependent necroptosis

RIPK3依赖性坏死性凋亡的生理作用

• 批准号: 8786057
• 负责人: Andrew Atwell Oberst
• 金额: $ 50.09万
• 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/8786057
• 关键词: Ablation; Accounting; Address; Antigen-Presenting Cells; Antigens; Apoptosis; Apoptotic; Autoimmune Diseases; Automobile Driving; Bacterial Infections; Bacterial Toxins; Binding; Bioavailable; Caspase Inhibitor; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Chemicals; Complex; Decision Making; Emergency Situation; Event; Evolution; Genes; Genetic; Growth; Health; Immune; Immune response; Immune system; Infection; Inflammatory; Lead; Ligation; Malignant Neoplasms; Mammalian Cell; Mediating; Messenger RNA; Modeling; NF-kappa B; Necrosis; Outcome; 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; TNFRSF1A gene; Tertiary Protein Structure; Testing; Tissues; Toll-like receptors; Transcriptional Activation; Translations; Tumor Necrosis Factor-alpha; Up-Regulation; Virus; Virus Diseases; Virus Inhibitors; Work; adaptive immunity; caspase-8; cell type; cellular sensitization; cytokine; in vivo; inhibitor/antagonist; insight; interest; novel; paralogous gene; prevent; programs; 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-¿（TNF）和toll样受体（TLR）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，人们早就认识到它们也可以引发凋亡细胞死亡。最近，研究表明这些信号也可以诱导另一种形式的程序性细胞死亡，称为“坏死性死亡”。虽然坏死性上睑下垂的发现引起了相当大的兴趣，但这种细胞交替死亡程序的生理作用仍然难以捉摸。特别是，坏死坏死被促凋亡蛋白酶caspase-8阻断，因此大多数研究依赖于基因消融或化学抑制caspase-8来触发坏死坏死。这就提出了一个问题：在生理条件下，TNF或tlr介导的坏死性上睑下垂何时发生？我们已经证明caspase-8必须与其平行的FLIP协同作用以阻止坏死下垂，并且FLIP被TNF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，我们提出，flip的缺失，而不是caspase-8的抑制，提供了细胞致敏坏死的一般机制。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损伤相关的信号分子，激活免疫细胞。为了解决这一可能性，我们将重点关注三个具体问题：1)促坏死坏死激酶RIPK3是如何激活的，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RIPK3的多个步骤可以控制。我们将使用该系统来验证RIPK3激活需要磷酸化依赖的RIPK3寡聚物的组装和繁殖，而caspase-8/FLIP直接阻断了这一过程的假设。2)在生理条件下，caspase-8/FLIP的抑制是如何解除的，从而允许坏死下垂？我们假设NF-kB信号或一般蛋白质翻译的抑制剂通过阻止FLIP表达使细胞对坏死坏死敏感。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)免疫系统如何对坏死细胞死亡和凋亡细胞死亡作出反应？我们假设细胞死亡的机制很重要，因为坏死下垂释放的炎症分子在细胞凋亡过程中被包含或消除。为了验证这个想法，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。我们将使用这个系统来分析细胞死亡的先天和适应性免疫反应。总之，本文提出的工作旨在了解体内坏死下垂的原因和后果，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。