泛素化降解IFI16的分子机制及其在宿主抗病毒免疫中的调控作用

胞浆DNA受体IFI16可有效识别病毒DNA，通过接头蛋白STING活化下游I型干扰素(IFN-I)信号触发宿主抗病毒免疫。同时，IFI16亦可被IFN诱导表达，病毒感染威胁解除之后如何清除积累的IFI16，防止过度免疫，其分子机制尚不明确。本研究发现，STING不仅介导IFI16触发的固有免疫，也可以促进IFI16蛋白的泛素化降解从而负反馈调控该信号通路。前期结果显示，高表达STING能显著促进IFI16的泛素化，降低其蛋白水平；而敲减内源性STING后，IFI16蛋白水平上调。将降解IFI16的关键位点突变之后，其IFN-β诱导能力显著增强。通过质谱筛选IFI16的E3泛素连接酶，我们发现TRIM21与IFI16具有较强的相互作用，且STING能促进TRIM21依赖的IFI16泛素化降解。本项目将深入探讨STING负反馈调控IFI16的分子机制及其在宿主抗感染免疫中的生理病理意义。

IFI16, a member of PYHIN family, is one of the most well-known cytosolic DNA sensors. DNA from invaded viruses could be effectively recognized by IFI16, and thus triggers the STING-dependent downstream signaling to produce large amount of type I interferon (IFN-I) including IFN-β and multiple IFN-α, which play important roles in the host antiviral immunity by inducing downstream IFN-stimulated genes (ISGs). However, it is still unclear how to negatively regulate the IFI16-IFN-ISG signaling axis in order to avoid the autoimmunity caused by the excessive IFN-β production. Our recent studies have shown that STING directly binds to IFI16 and recruits the E3 ubiquitin ligase TRIM21, which finally promotes IFI16 degradation via the ubiquitin-proteasome system. In our preliminary results, we found that STING overexpression significantly enhanced the ubiquitination of IFI16, especially when cells were exposed to stimulus like IFN-γ. Overexpression of STING consistently reduced IFI16 protein level, while knockdown of STING by shRNA elevated the basal level and IFN-γ-inducible level of IFI16 protein. Mutations at the STING-dependent ubiquitination site extended the half-life of IFI16, which led to stronger IFN-β production in response to HSV60mer transfection. To identify the IFI16 E3 ubiquitin ligase, IFI16-interacting proteins were efficiently isolated and analyzed by Nano-LC-ESI-MS/MS. Several IFI16-interacting E3 ligases were observed, including the high-confidence identification of TRIM21. TRIM21 effectively bound to IFI16 and promoted its ubiquitin-proteasome degradation. STING enhanced IFI16 and TRIM21 interaction, and promoted TRIM21-mediated IFI16 ubiquitination, suggesting that STING might be the adaptor that bridged these two proteins. Our study will further investigate the molecular mechanism for STING-mediated degradation of IFI16, explore the physiological and pathological significance of this negative feedback regulation during host innate immunity against invaded DNA viruses.