Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.

• MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• exprese genu MeSH
• genový knockout MeSH
• hepatocyty imunologie   metabolismus   MeSH
• interferonové regulační faktory genetika   metabolismus   MeSH
• interferony nedostatek   genetika   metabolismus   MeSH
• interleukiny nedostatek   genetika   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• receptor interleukinu-10 - beta-podjednotka nedostatek   genetika   metabolismus   MeSH
• receptory interferonů nedostatek   genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• signální transdukce MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Interferon gamma (IFN-γ) is one of the key players in the immune system of vertebrates. The evolution and properties of IFN-γ and its receptors in fish species are of special interest as they point to the origin of innate immunity in vertebrates. We studied the phylogeny, biophysical and structural properties of IFN-γ and its receptors. Our phylogeny analysis suggests the existence of two groups of IFN-γ related proteins, one specific for Acanthomorpha, the other for Cypriniformes, Characiformes and Siluriformes. The analysis further shows an ancient duplication of the gene for IFN-γ receptor 1 (IFN- γR1) and the parallel existence of the duplicated genes in all current teleost fish species. In contrast, only one gene can be found for receptor 2, IFN- γR2. The specificity of the interaction between IFN- γ and both types of IFN- γR1 was determined by microscale thermophoresis measurements of the equilibrium dissociation constants for the proteins from three fish species. The measured preference of IFN- γ for one of the two forms of receptor 1agrees with the bioinformatic analysis of the coevolution between IFN- γ and receptor 1. To elucidate structural relationships between IFN-γ of fish and other vertebrate species, we determined the crystal structure of IFN-γ from olive flounder (Paralichthys olivaceus, PoliIFN-γ) at crystallographic resolution of 2.3 Å and the low-resolution structures of Takifugu rubripes, Oreochromis niloticus, and Larimichthys crocea IFN-γ by small angle X-ray diffraction. The overall PoliIFN-γ fold is the same as the fold of the other known IFN- γ structures but there are some significant structural differences, namely the additional C-terminal helix G and a different angle between helices C and D in PoliIFN-γ.

• MeSH
• fylogeneze MeSH
• interferon gama genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• receptory interferonů genetika   metabolismus   MeSH
• ryby genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity.

• MeSH
• interferon gama chemie   metabolismus   MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely * MeSH
• receptory interferonů chemie   genetika   metabolismus   MeSH
• sbalování proteinů * MeSH
• substituce aminokyselin * MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Type I interferon (IFN), mainly produced by dendritic cells (DCs), is critical in the host defence against tick-transmitted pathogens. Here, we report that salivary cysteine protease inhibitor from the hard tick Ixodes scapularis, sialostatin L2, affects IFN-β mediated immune reactions in mouse dendritic cells. Following IFN receptor ligation, the Janus activated kinases/signal transducer and activator of transcription (JAK/STAT) pathway is activated. We show that sialostatin L2 attenuates phosphorylation of STATs in spleen dendritic cells upon addition of recombinant IFN-β. LPS-stimulated dendritic cells release IFN-β which in turn leads to the induction of IFN-stimulated genes (ISG) through JAK/STAT pathway activation. The induction of two ISG, interferon regulatory factor 7 (IRF-7) and IP-10, was suppressed by sialostatin L2 in LPS-stimulated dendritic cells. Finally, the interference of sialostatin L2 with IFN action led to the enhanced replication of tick-borne encephalitis virus in DC. In summary, we present here that tick salivary cystatin negatively affects IFN-β responses which may consequently increase the pathogen load after transmission via tick saliva.

• MeSH
• Borrelia burgdorferi fyziologie   MeSH
• cystatiny imunologie   MeSH
• dendritické buňky imunologie   MeSH
• fosforylace MeSH
• interferon beta imunologie   MeSH
• interferonový regulační faktor 7 imunologie   MeSH
• klíště imunologie   mikrobiologie   MeSH
• lipopolysacharidy imunologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• receptory cytokinové imunologie   MeSH
• receptory interferonů metabolismus   MeSH
• slinné cystatiny imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor.

• MeSH
• interferon gama chemie   genetika   metabolismus   MeSH
• lidé MeSH
• povrchová plasmonová rezonance MeSH
• receptory interferonů chemie   genetika   metabolismus   MeSH
• sbalování proteinů * MeSH
• simulace molekulární dynamiky * MeSH
• substituce aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Aims: To follow the IFNγ receptor expression on monocytes and granulocytes of cardiac surgical patients with respect to the type of cardiopulmonary bypass (CPB). Methods: Expression of IFNγ receptor on monocytes and granulocytes of 26 cardiac surgical patients operated with the use of either "standard" or "miniaturised" CPB was determined by flow cytometry. Results: The significant increase in IFNγ receptor expression on monocytes on the 1(st) and on the 3(rd) postoperative days was revealed in both groups of patients (p<0.001) irrespective of the type of CPB used, being non-significantly different between groups. In contrast, the expression of IFNγ on granulocytes displayed significant differences in terms of the CPB used. Whereas, in "standard" CPB patients, granulocyte INFγ receptor expression reached its maximum immediately after surgery (p<0.01), in "miniivasive" CPB patients, the peak in INFγ receptor expression was postponed to the 1(st) postoperative day (p<0.05). Statistically significantly higher IFNγ receptor expression on granulocytes was found in "standard" CPB patients (p<0.05). Conclusion: Compared to "miniaturised" CPB patients, the significantly higher IFNγ receptor expression on granulocytes was found in "standard" CPB patients (p<0.05) on the 1(st) postoperative day.

• MeSH
• granulocyty metabolismus   MeSH
• kardiochirurgické výkony MeSH
• kardiopulmonální bypass klasifikace   metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• miniaturizace MeSH
• monocyty metabolismus   MeSH
• pooperační období MeSH
• průtoková cytometrie MeSH
• receptory interferonů metabolismus   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

• MeSH
• antivirové látky antagonisté a inhibitory   MeSH
• buněčné linie MeSH
• interferon alfa antagonisté a inhibitory   fyziologie   metabolismus   MeSH
• lidé MeSH
• neutralizační testy MeSH
• receptory interferonů genetika   metabolismus   MeSH
• virové proteiny metabolismus   MeSH
• virus vakcinie genetika   MeSH
• Check Tag
• lidé MeSH