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）诱导的Toll样受体4（TLR4）信号传导激活巨噬细胞中的促炎反应。这种炎症反应由自噬终止。我们发现，pellino3a是TLR4信号中的E3-泛素 - 连接酶和支架蛋白，在LPS刺激后的巨噬细胞中受到自噬的调节。 Pellino3a蛋白在LPS处理后6小时稳定，随后在LPS刺激后24小时降解。这种耗竭是由与自噬衔接蛋白p62/sqSTM1结合引起的，从而引发其降解。它仍然晦涩难懂，如何实现短期蛋白质稳定以及其封锁是否可以改善败血症的结果。由于Pellino3a在LPS-TLR4介导的促炎细胞因子（例如IL-1Beta，Tnfalpha，IL-6和Ifngamma）的巨噬细胞中至关重要，因此我们假设这抑制了Pellino3a表达的早期增加，则抑制了高炎性反应。由于没有机械洞察可用于调节炎症中的佩里诺3A表达，因此我们首先以质谱（MS）和荧光偏振测定法进行了特征，这些测定法对Pellino3a的修饰和/或P62/SQSTM1进行了修饰。接下来，我们检查是否可以通过p62/sqSTM1的过表达来增强自噬和pellino3a降解或通过TORIN2处理在药理学上诱导，从而降低促炎细胞因子的表达。考虑到KEAP1也与p62/sqSTM1结合，我们通过FACS-触发分析确定这种相互作用是否阻止了pellino3结合，从而稳定了pellino3a。我们通过MS阐明其他蛋白质的依赖性依赖性泛素化是否有助于其降解。此外，我们分析了脓毒症期间佩里诺3A蛋白表达是否是转录调节的，以及是否可以用来抑制pellino3a的表达。这些实验将在CRSIPR/CAS9标记的内源基因后，在J774A.1和RAW264.7巨噬细胞中进行。我们的目的是确定如何阻止Pellino3a蛋白的早期稳定化并将这种机理直接转化为内毒素模型中的体内设置。我们的作用是假设巨噬细胞中对pellino3a或p62/sqstm1表达的抑制会降低体内的高炎反应。