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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, Phosphoproteomics Combined with Quantitative 14-3-3-Affinity Capture Identifies SIRT1 and RAI as Novel Regulators of Cytosolic Double-Stranded RNA Recognition Pathway. Molecular and Cellular Proteomics 13(10), 2604–17, 2014.

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.

BibTeX entry:

@ARTICLE{jOSxHiVxLiMaAiMaNy14a,
  title = {Phosphoproteomics Combined with Quantitative 14-3-3-Affinity Capture Identifies SIRT1 and RAI as Novel Regulators of Cytosolic Double-Stranded RNA Recognition Pathway},
  author = {Öhman, Tiina and Söderholm, Sandra and Hintsanen, Petteri and Välimäki, Elina and Lietzén, Niina and MacKintosh, Carol and Aittokallio, Tero and Matikainen, Sampsa and Nyman, Tuula A.},
  journal = {Molecular and Cellular Proteomics},
  volume = {13},
  number = {10},
  pages = {2604–17},
  year = {2014},
}

Belongs to TUCS Research Unit(s): Biomathematics Research Unit (BIOMATH)

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