| 1. |
- Grönholm, Juha, et al.
(author)
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Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells
- 2012
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In: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:3, s. 1239-1250
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Journal article (peer-reviewed)abstract
- The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-gamma- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.
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| 2. |
- Valanne, Susanna, et al.
(author)
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Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-kappa B Signaling
- 2010
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In: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 184:11, s. 6188-6198
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Journal article (peer-reviewed)abstract
- Because NF-kappa B signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-kappa B pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappa B regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk) 2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila I kappa B homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappa B regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-kappa B signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappa B reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll(10b)-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-kappa B signaling. The Journal of Immunology, 2010, 184: 6188-6198.
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| 3. |
- Vanha-Aho, Leena-Maija, et al.
(author)
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Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster
- 2012
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:5, s. e37153-
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Journal article (peer-reviewed)abstract
- Drosophila is a well-established model organism for studying innate immunity because of its high resistance against microbial infections and lack of adaptive immunity. In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved. Upon infection, activation of the immune signaling pathways, Toll and Imd, leads to the expression of multiple immune response genes, such as the antimicrobial peptides (AMPs). Previously, we identified an uncharacterized gene edin among the genes, which were strongly induced upon stimulation with Escherichia coli in Drosophila S2 cells. Edin has been associated with resistance against Listeria monocytogenes, but its role in Drosophila immunity remains elusive. In this study, we examined the role of Edin in the immune response of Drosophila both in vitro and in vivo. We report that edin expression is dependent on the Imd-pathway NF-κB transcription factor Relish and that it is expressed upon infection both in vitro and in vivo. Edin encodes a pro-protein, which is further processed in S2 cells. In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo. Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo. However, edin RNAi flies showed modestly impaired resistance to E. faecalis infection. We conclude that Edin has no potent antimicrobial properties but it appears to be important for E. faecalis infection via an uncharacterized mechanism. Further studies are still required to elucidate the exact role of Edin in the Drosophila immune response.
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