Phosphoproteomics combined with quantitative 14-3-3-affinity capture identifies SIRT1 and RAI as novel regulators of cytosolic double-stranded RNA recognition pathway

Tiina Öhman, Sandra Söderholm, Petteri Hintsanen, Elina Välimäki, Niina Lietzén, Carol MacKintosh, Tero Aittokallio, Sampsa Matikainen, Tuula A. Nyman (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    9 Citations (Scopus)

    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 languageEnglish
    Pages (from-to)2604-2617
    Number of pages14
    JournalMolecular & Cellular Proteomics
    Volume13
    Issue number10
    DOIs
    Publication statusPublished - Oct 2014

    Keywords

    • affinity proteomics
    • Cytokines
    • Host-pathogen interaction
    • infectious disease
    • iTRAQ
    • Phosphoproteome
    • Signal transduction
    • siRNA
    • Viruses

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