Protein Chemistry Research Group, Institute of Biotechnology, University of Helsinki, Finland.
J Proteome Res. 2010 Mar 5;9(3):1549-64. doi: 10.1021/pr901040u.
The skin is the primary boundary between the body and the environment. In addition to its properties as a physical barrier, skin keratinocytes actively participate in many defense mechanisms. Viral double-stranded RNA (dsRNA) is the most important viral structure involved in activation of immune response. Intracellular detection of dsRNA by cytoplasmic receptors activates well-characterized antiviral response, as well as pro-inflammatory response and apoptosis of virus-infected cells. Here, we have used quantitative subcellular proteomics to characterize the signaling pathways activated by cytosolic dsRNA recognition pathway in human keratinocytes. Cytoplasmic and mitochondrial proteomes were analyzed using 2-DE in combination with MS, immunoblotting and confocal microscopy. We have identified 239 reproducibly differentially expressed proteins upon dsRNA stimulation. The identified proteins include several key proteins involved in cytoskeletal dynamics, cell signaling, cell death, and stress response. Our analysis provides novel information how the cytokeratin network is disrupted in a caspase-dependent manner upon dsRNA stimulation as well as Encephalomyocarditis virus or Vesicular stomatitis virus infection. We show that this caspase-dependent disruption of cytokeratin is activated by cytoplasmic RNA recognition pathway. In addition, we show that viral infection activates 14-3-3 protein mediated signaling pathways in human keratinocytes which suggest an important role of 14-3-3 proteins in antiviral innate immune response.
皮肤是机体与环境之间的主要边界。除了作为物理屏障的特性外，皮肤角质形成细胞还积极参与许多防御机制。病毒双链 RNA（dsRNA）是参与激活免疫反应的最重要的病毒结构。细胞质受体对 dsRNA 的细胞内检测激活了特征明确的抗病毒反应以及病毒感染细胞的炎症反应和细胞凋亡。在这里，我们使用定量亚细胞蛋白质组学来表征人角质形成细胞中细胞质 dsRNA 识别途径激活的信号通路。使用 2-DE 结合 MS、免疫印迹和共聚焦显微镜分析细胞质和线粒体蛋白质组。在 dsRNA 刺激后，我们鉴定了 239 个可重复差异表达的蛋白质。鉴定的蛋白质包括参与细胞骨架动力学、细胞信号转导、细胞死亡和应激反应的几个关键蛋白。我们的分析提供了新的信息，即 dsRNA 刺激以及脑炎心肌炎病毒或水疱性口炎病毒感染如何以 Caspase 依赖性方式破坏细胞角蛋白网络。我们表明，这种 Caspase 依赖性细胞角蛋白破坏是由细胞质 RNA 识别途径激活的。此外，我们表明病毒感染激活了人角质形成细胞中 14-3-3 蛋白介导的信号通路，这表明 14-3-3 蛋白在抗病毒先天免疫反应中起重要作用。
