Phosphoproteomics combined with quantitative 14-3-3-affinity capture identifies SIRT1 and RAI as novel regulators of cytosolic double-stranded RNA recognition pathway.
Phosphoproteomics combined with quantitative 14-3-3-affinity capture identifies SIRT1 and RAI as novel regulators of cytosolic double-stranded RNA recognition pathway.
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磷酸化蛋白质组学与定量 14-3-3-亲和力捕获相结合，确定 SIRT1 和 RAI 是胞质双链 RNA 识别途径的新型调节剂。
DOI：
10.1074/mcp.m114.038968
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Öhman T
中科院分区：
文献类型：
--
作者：
Öhman T
病毒双链RNA（dsRNA）是由先天免疫系统的胞质模式识别受体识别的最重要的病毒结构，并且其识别导致刺激抗病毒细胞因子的产生和感染细胞的凋亡的信号级联的激活。14-3-3蛋白是参与多种细胞过程的普遍表达的调节分子，并且14-3-3蛋白介导的信号传导途径由人角质形成细胞中的细胞质dsRNA激活。然而，14-3-3蛋白介导的相互作用在病毒dsRNA刺激过程中的功能作用仍然没有得到表征。在这里，我们使用功能蛋白质组学来鉴定其磷酸化和与14-3-3的相互作用由dsRNA调节的蛋白质，并表征在人HaCaT角质形成细胞中胞质dsRNA诱导的先天免疫应答期间激活的信号通路。磷酸化蛋白质组分析表明，一些MAPK和免疫反应相关的信号通路被激活后，dsRNA刺激。相互作用组分析鉴定了RelA相关抑制剂、高迁移率族蛋白和与宿主对病毒感染的反应相关的几种蛋白作为新的14-3-3靶蛋白。功能研究表明，RelA相关抑制剂调节dsRNA诱导的细胞凋亡和TNF的产生。蛋白质组数据的综合网络分析表明，sirtuin 1是在双链RNA刺激期间受14-3- 3 s调节的中心分子。进一步的实验表明，sirtuin 1负调控dsRNA诱导的NFκB转录活性，抑制抗病毒细胞因子的表达，并保护细胞免于dsRNA刺激和脑心肌炎病毒感染的角质形成细胞的凋亡。总之，我们的数据突出了14-3-3蛋白在抗病毒反应中的重要性，并将RelA相关抑制剂和sirtuin 1鉴定为抗病毒先天免疫反应的新型调节剂。
Viral double-stranded RNA (dsRNA) is the most important viral structure recognized by cytosolic pattern-recognition receptors of the innate immune system, and its recognition results in the activation of signaling cascades that stimulate the production of antiviral cytokines and apoptosis of infected cells. 14-3-3 proteins are ubiquitously expressed regulatory molecules that participate in a variety of cellular processes, and 14-3-3 protein-mediated signaling pathways are activated by cytoplasmic dsRNA in human keratinocytes. However, the functional role of 14-3-3 protein-mediated interactions during viral dsRNA stimulation has remained uncharacterized. Here, we used functional proteomics to identify proteins whose phosphorylation and interaction with 14-3-3 is modulated by dsRNA and to characterize the signaling pathways activated during cytosolic dsRNA-induced innate immune response in human HaCaT keratinocytes. Phosphoproteome analysis showed that several MAPK- and immune-response-related signaling pathways were activated after dsRNA stimulation. Interactome analysis identified RelA-associated inhibitor, high-mobility group proteins, and several proteins associated with host responses to viral infection as novel 14-3-3 target proteins. Functional studies showed that RelA-associated inhibitor regulated dsRNA-induced apoptosis and TNF production. Integrated network analyses of proteomic data revealed that sirtuin1 was a central molecule regulated by 14-3-3s during dsRNA stimulation. Further experiments showed that sirtuin 1 negatively regulated dsRNA-induced NFκB transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocarditis-virus-infected keratinocytes. In conclusion, our data highlight the importance of 14-3-3 proteins in antiviral responses and identify RelA-associated inhibitor and sirtuin 1 as novel regulators of antiviral innate immune responses.