新型泛素连接酶E3调控TLR信号通路和炎症反应的研究

Ubiquitin ligase E3 plays an important role in the regulation of TLR signaling and inflammatory response in macrophages.Our project aims to screen and identify the new E3 molecule that regulates TLR signaling and inflammatory response in macrophages. More than 280 E3 siRNAs were screened and a new E3 named HD, which locates in the endoplasmic reticulum, was found to enhance the production of IL-6. Moreover, HD’s regulation in inflammatory response was dependent on its E3 ubiquitin ligase activity. More importantly, the mice specifically KO of HD in macrophages increased the survival rates in response to LPS-induced endotoxin shock. Considering that USP15 was identified to interact with HD, we will look for the ubiquitination sites in USP15 and study the function and molecular mechanism of TLR signaling regulation by HD with wild-type USP15 and its ubiquitination site mutant. Bacterial infection and other disease models will also be used to verify the function of HD in regulation of inflammatory response in vivo. This project will provide innovative information to further understanding of the role of E3 in inflammatory response, which will help us to identify new targets to block endotoxin shock.

泛素连接酶E3在调控巨噬细胞的TLR信号通路和炎症反应中发挥着重要作用。本申请项目期望能筛选和鉴定巨噬细胞中调控TLR信号和炎症反应的新型E3分子。申请人前期利用包含280多个E3的siRNA文库，筛选到定位于内质网的新型E3 HD能正向调控IL-6的产生，并且HD调控炎症反应的功能依赖其E3泛素连接酶活性。更重要的是，通过构建在巨噬细胞中条件性敲除HD的小鼠，本项目已证实LPS诱导内毒素休克的HD KO小鼠存活率显著升高。鉴于初步筛选发现USP15能与HD结合，申请者拟进一步探索HD对USP15进行泛素化修饰的位点以及野生型USP15与其泛素化位点突变体调控TLR信号的功能和分子机制；并进一步利用细菌感染等模型验证HD调控炎症反应的功能。这将为深入认识E3调控炎症反应以及寻找新靶点阻断内毒素休克提供创新性的信息。

特殊细胞器定位的信号分子MAVS、STING和TLR3在清除病毒感染中发挥着重要的作用。通过TLR4激活的NF-κB信号通路可以产生促炎细胞因子帮助细菌的清除。虽然关于TLR4通路已经有了深入地研究，但TLR4通路中具有特殊细胞器定位的效应分子还有待发现。本课题筛选了280多个E3泛素连接酶，发现内质网定位的E3分子Hrd1在细菌感染过程中可以正向调节TLR4诱导的炎症反应。机制上，Hrd1可以与去泛素化酶Usp15直接结合，进而泛素化Usp15。与以往报道的Hrd1泛素化底物后使其降解的功能不同，Usp15并没有降解，而是失去了活性，导致IκB降解速率减缓，从而使NF-κB过度激活。利用LPS和盲肠结扎穿刺诱导的脓毒性休克模型，我们揭示了巨噬细胞中缺失Hrd1可以保护小鼠免受脓毒性休克，并且在Hrd1敲除巨噬细胞中敲低Usp15可回复IL-6炎症因子的水平。我们的研究揭示了ER定位的E3分子Hrd1与细胞膜定位的信号分子TLR4协同作用，共同调控炎症反应的机制，为研究细胞器膜与细胞膜信号传递提供了新思路。