Role of autophagy-dependent Pellino3a downregulation during TLR4 signaling in macrophages

自噬依赖性 Pellino3a 下调在巨噬细胞 TLR4 信号传导过程中的作用

• 批准号: 284401149
• 负责人: Professor Dr. Andreas von Knethen
• 金额: --
• 依托单位: Georg-Speyer-Haus, Institut für Tumorbiologie und experimentelle Therapie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/284401149?language=en
• 关键词: Role; autophagy; dependent; Pellino3a; downregulation

Lipopolysaccharide (LPS)-induced activation of toll-like receptor 4 (TLR4) signaling provokes a proinflammatory response in macrophages. This inflammatory reaction is terminated by autophagy. We found that Pellino3a, which is an E3-ubiquitin-ligase and scaffold protein in TLR4-signaling, is regulated by autophagy in macrophages after LPS-stimulation. Pellino3a protein gets stabilized 6 h after LPS-treatment and subsequently is degraded 24 h after LPS-stimulation. This depletion is caused by binding to the autophagy adaptor protein p62/SQSTM1, initiating its degradation. It remains obscure, how Pellino3a short time protein stabilization is achieved and whether its blockade might improve sepsis outcome. Because Pellino3a is essential in macrophages for LPS-TLR4-mediated expression of proinflammatory cytokines such as IL-1beta, TNFalpha, IL-6, and IFNgamma, we hypothesize that inhibiting the early increase of Pellino3a expression blocks the hyperinflammatory response. Since no mechanistic insights are available for regulation of Pellino3a expression in inflammation, we characterize first by mass spectrometry (MS) and a fluorescence polarization assay which modification(s) of Pellino3a and/or p62/SQSTM1 cause their interaction. Next we check, whether induction of autophagy and Pellino3a degradation can be enhanced by overexpression of p62/SQSTM1 or pharmacologically by Torin2 treatment, thus decreasing proinflammatory cytokine expression. Considering that Keap1 binds to p62/SQSTM1 as well, we determine by FACS-FRET-analysis whether this interaction blocks Pellino3 binding, thereby stabilizing Pellino3a. We clarify by MS whether Pellino3a-dependent ubiquitination of other proteins contributes to its degradation. Moreover, we analyze whether Pellino3a protein expression is transcriptionally modulated during sepsis and whether this can be used to inhibit Pellino3a expression. These experiments will be performed in J774A.1 and RAW264.7 macrophages, following endogenous gene tagging by CRSIPR/Cas9. We aim at identifying, how the early stabilization of Pellino3a protein can be blocked and explore to directly translate this mechanism to an in vivo setup in the mouse, the endotoxin model. We act on the assumption that inhibition of Pellino3a or p62/SQSTM1 expression in macrophages decreases the hyperinflammatory response in vivo.

脂多糖(LPS)激活Toll样受体4(TLR4)信号可引起巨噬细胞的促炎反应。这种炎症反应通过自噬终止。我们发现，Pellino3a是一种E3泛素连接酶，也是TLR4信号转导的支架蛋白，受内毒素刺激后巨噬细胞自噬的调节。Pellino3a蛋白在内毒素处理后6h稳定下来，随后在内毒素刺激后24h降解。这种耗尽是由于与自噬适配器蛋白p62/SQSTM1结合，启动其降解而引起的。目前尚不清楚Pellino3a是如何实现短时间蛋白质稳定的，以及阻断Pellino3a是否可能改善脓毒症的预后。由于Pellino3a在巨噬细胞内毒素-TLR4介导的促炎细胞因子如IL-1β、TNFpha、IL-6和IFNGamma的表达中是必不可少的，我们假设抑制Pellino3a表达的早期增加可以阻止高炎症反应。由于Pellino3a在炎症中的表达调控机制尚不清楚，我们首先通过质谱仪(MS)和荧光偏振分析来表征Pellino3a的修饰(S)和/或p62/SQSTM1导致它们之间的相互作用。接下来，我们检查p62/SQSTM1的过度表达是否可以增强自噬和Pellino3a降解的诱导，或者Torin2治疗是否在药理上可以增强自噬和Pellino3a的降解，从而减少促炎细胞因子的表达。考虑到Keap1也与p62/SQSTM1结合，我们通过FACS-FRET-分析确定这种相互作用是否阻止Pellino3结合，从而稳定Pellino3a。我们通过MS阐明了依赖于Pellino3a的其他蛋白质的泛素化是否有助于其降解。此外，我们还分析了脓毒症期间Pellino3a蛋白的表达是否受到转录调控，以及这是否可以用来抑制Pellino3a的表达。这些实验将在CRSIPR/Cas9内源性基因标记后在J774A.1和RAW264.7巨噬细胞中进行。我们的目标是确定如何阻止Pellino3a蛋白的早期稳定，并探索将这一机制直接转化为小鼠体内的设置，即内毒素模型。我们假设抑制巨噬细胞中Pellino3a或p62/SQSTM1的表达可以降低体内的高炎症反应。