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

脂多糖（LPS）诱导的toll样受体4 （TLR4）信号的激活可引起巨噬细胞的促炎反应。这种炎症反应被自噬终止。我们发现Pellino3a是一种e3泛素连接酶和tlr4信号传导的支架蛋白，在lps刺激后巨噬细胞受自噬调节。Pellino3a蛋白在lps处理后6 h稳定，在lps刺激后24 h降解。这种损耗是由于与自噬接头蛋白p62/SQSTM1结合，启动其降解而引起的。目前尚不清楚Pellino3a短期蛋白稳定是如何实现的，以及它的阻断是否可以改善败血症的预后。由于Pellino3a在巨噬细胞中对lps - tlr4介导的促炎细胞因子如il -1 β、TNFalpha、IL-6和IFNgamma的表达至关重要，我们假设抑制Pellino3a表达的早期增加可以阻断高炎症反应。由于没有关于炎症中Pellino3a表达调控的机制见解，我们首先通过质谱（MS）和荧光偏振分析来表征Pellino3a和/或p62/SQSTM1的修饰导致它们相互作用。接下来，我们检查是否可以通过p62/SQSTM1的过表达或Torin2的药理学处理来增强诱导自噬和Pellino3a降解，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 - fret分析确定这种相互作用是否阻断Pellino3的结合，从而稳定Pellino3a。我们通过质谱澄清是否pellino3a依赖的泛素化其他蛋白质有助于其降解。此外，我们分析了脓毒症期间Pellino3a蛋白的表达是否受到转录调节，以及这是否可以用于抑制Pellino3a的表达。这些实验将在J774A.1和RAW264.7巨噬细胞中进行，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Pellino3a蛋白的早期稳定，并探索将这一机制直接转化为小鼠内毒素模型的体内设置。我们假设抑制巨噬细胞中Pellino3a或p62/SQSTM1的表达可以减少体内的高炎症反应。

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.