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

脂多糖（LPS）诱导的Toll样受体4（TLR 4）信号转导激活在巨噬细胞中引起促炎反应。这种炎症反应通过自噬终止。我们发现，Pellino 3a，这是一个E3-泛素连接酶和支架蛋白TLR 4信号，是调节自噬在巨噬细胞LPS刺激后。Pellino 3a蛋白在LPS处理后6小时稳定，随后在LPS刺激后24小时降解。这种消耗是由与自噬衔接蛋白p62/SQSTM 1结合引起的，从而引发其降解。Pellino 3a如何实现短时间蛋白稳定以及其阻断是否可能改善脓毒症结局仍不清楚。由于Pellino 3a在巨噬细胞中对于LPS-TLR 4介导的促炎细胞因子如IL-1 β、TNF α、IL-6和IFN γ的表达是必需的，我们假设抑制Pellino 3a表达的早期增加阻断了炎症反应。由于没有机制的见解可用于调节炎症中的Pellino 3a表达，我们首先通过质谱法（MS）和荧光偏振测定表征Pellino 3a和/或p62/SQSTM 1的修饰引起它们的相互作用。接下来，我们检查是否可以通过Torin 2处理过表达p62/SQSTM 1或Pellino 3a来增强自噬和Pellino 3a降解的诱导，从而降低促炎细胞因子的表达。考虑到Keap 1也与p62/SQSTM 1结合，我们通过FACS-FRET分析确定这种相互作用是否阻断Pellino 3结合，从而稳定Pellino 3a。我们澄清MS是否Pellino 3a依赖于其他蛋白质的泛素化有助于其降解。此外，我们分析了Pellino 3a蛋白表达在脓毒症期间是否受到转录调节，以及这是否可用于抑制Pellino 3a表达。这些实验将在J774A.1和RAW264.7巨噬细胞中进行，随后通过CRSIPR/Cas9进行内源性基因标记。我们的目标是确定如何阻断Pellino 3a蛋白的早期稳定化，并探索将这种机制直接转化为小鼠体内的内毒素模型。我们采取行动的假设，抑制Pellino 3a或p62/SQSTM 1表达的巨噬细胞减少体内的炎症反应。

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.