title: Phosphoproteomics combined with quantitative 14-3-3-affinity capture identifies SIRT1 and RAI as novel regulators of cytosolic double-stranded RNA recognition pathway.
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 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.
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland;
authors.forename: Tiina
authors.initials: T
authors.lastname: Öhman
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland; §Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland;
authors.forename: Sandra
authors.initials: S
authors.lastname: Söderholm
authors.affiliation: ¶Institute for Molecular Medicine Finland (FIMM), FI-00014 University of Helsinki, Helsinki, Finland;
authors.forename: Petteri
authors.initials: P
authors.lastname: Hintsanen
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland; §Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland;
authors.forename: Elina
authors.initials: E
authors.lastname: Välimäki
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland;
authors.forename: Niina
authors.initials: N
authors.lastname: Lietzén
authors.affiliation: **University of Dundee, Dundee, Scotland DD1 5EH, United Kingdom.
authors.forename: Carol
authors.initials: C
authors.lastname: MacKintosh
authors.affiliation: ¶Institute for Molecular Medicine Finland (FIMM), FI-00014 University of Helsinki, Helsinki, Finland;
authors.forename: Tero
authors.initials: T
authors.lastname: Aittokallio
authors.affiliation: §Finnish Institute of Occupational Health, FI-00250 Helsinki, Finland;
authors.forename: Sampsa
authors.initials: S
authors.lastname: Matikainen
authors.affiliation: From the ‡Institute of Biotechnology, FI-00014 University of Helsinki, Helsinki, Finland; tuula.nyman@helsinki.fi.
authors.forename: Tuula A
authors.initials: TA
authors.lastname: Nyman
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D018188:Cardiovirus Infections / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004686:Encephalomyocarditis virus / Q000235:genetics / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012330:RNA, Double-Stranded / Q000276:immunology / Q000378:metabolism*; D012367:RNA, Viral / Q000276:immunology / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D015398:Signal Transduction; D056564:Sirtuin 1 / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D047908:Intracellular Signaling Peptides and Proteins; C119612:PPP1R13L protein, human; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012097:Repressor Proteins; C447939:SIRT1 protein, human; D056564:Sirtuin 1