Abstract
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 RelAassociated 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 NF?B transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocar-ditis-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.
Original language | English |
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Pages (from-to) | 2604-2617 |
Number of pages | 14 |
Journal | Molecular & Cellular Proteomics |
Volume | 13 |
Issue number | 10 |
DOIs | |
Publication status | Published - Oct 2014 |
Keywords
- affinity proteomics
- Cytokines
- Host-pathogen interaction
- infectious disease
- iTRAQ
- Phosphoproteome
- Signal transduction
- siRNA
- Viruses