Abstract:Summary
Synthetic strategies to activate cytokine receptors so far only address standard dimeric cytokine receptor assemblies. The 19 ligands of the tumor necrosis factor superfamily (TNFSF), however, form noncovalent trimers and receptor trimerization is considered to be essential for receptor activation. Synthetic TNFR1, TNFR2, and Fas/CD95 receptors were activated by synthetic trimeric ligands which induced NF-κB signaling or Caspase-induced apoptosis. Albeit dimeric receptor activation did not i… Show more
“…Recently, we have developed a fully synthetic cytokine/ cytokine receptor system which phenocopied cytokine signaling, exemplified for the pro-inflammatory cytokines Interleukin (IL-)6, IL-23, TNFα, the anti-inflammatory cytokine IL-22 and death ligand Fas (Engelowski et al, 2018;Mossner et al, , 2021. This fully synthetic cytokine receptor system (SyCyR) is based on nanobodies specifically recognizing GFP and mCherry (Rothbauer et al, 2008;Fridy et al, 2014) fused to the transmembrane and intracellular domains of the receptor of interest.…”
Type I interferons (IFNs) are potent inhibitors of viral replication. Here, we reformatted the natural murine and human type I interferon-α/β receptors IFNAR1 and IFNAR2 into fully synthetic biological switches. The transmembrane and intracellular domains of natural IFNAR1 and IFNAR2 were conserved, whereas the extracellular domains were exchanged by nanobodies directed against the fluorescent proteins Green fluorescent protein (GFP) and mCherry. Using this approach, multimeric single-binding GFP-mCherry ligands induced synthetic IFNAR1/IFNAR2 receptor complexes and initiated STAT1/2 mediated signal transduction via Jak1 and Tyk2. Homodimeric GFP and mCherry ligands showed that IFNAR2 but not IFNAR1 homodimers were sufficient to induce STAT1/2 signaling. Transcriptome analysis revealed that synthetic murine type I IFN signaling was highly comparable to IFNα4 signaling. Moreover, replication of vesicular stomatitis virus (VSV) in a cell culture-based viral infection model using MC57 cells was significantly inhibited after stimulation with synthetic ligands. Using intracellular deletion variants and point mutations, Y510 and Y335 in murine IFNAR2 were verified as unique phosphorylation sites for STAT1/2 activation, whereas the other tyrosine residues in IFNAR1 and IFNAR2 were not involved in STAT1/2 phosphorylation. Comparative analysis of synthetic human IFNARs supports this finding. In summary, our data showed that synthetic type I IFN signal transduction is originating from IFNAR2 rather than IFNAR1.
“…Recently, we have developed a fully synthetic cytokine/ cytokine receptor system which phenocopied cytokine signaling, exemplified for the pro-inflammatory cytokines Interleukin (IL-)6, IL-23, TNFα, the anti-inflammatory cytokine IL-22 and death ligand Fas (Engelowski et al, 2018;Mossner et al, , 2021. This fully synthetic cytokine receptor system (SyCyR) is based on nanobodies specifically recognizing GFP and mCherry (Rothbauer et al, 2008;Fridy et al, 2014) fused to the transmembrane and intracellular domains of the receptor of interest.…”
Type I interferons (IFNs) are potent inhibitors of viral replication. Here, we reformatted the natural murine and human type I interferon-α/β receptors IFNAR1 and IFNAR2 into fully synthetic biological switches. The transmembrane and intracellular domains of natural IFNAR1 and IFNAR2 were conserved, whereas the extracellular domains were exchanged by nanobodies directed against the fluorescent proteins Green fluorescent protein (GFP) and mCherry. Using this approach, multimeric single-binding GFP-mCherry ligands induced synthetic IFNAR1/IFNAR2 receptor complexes and initiated STAT1/2 mediated signal transduction via Jak1 and Tyk2. Homodimeric GFP and mCherry ligands showed that IFNAR2 but not IFNAR1 homodimers were sufficient to induce STAT1/2 signaling. Transcriptome analysis revealed that synthetic murine type I IFN signaling was highly comparable to IFNα4 signaling. Moreover, replication of vesicular stomatitis virus (VSV) in a cell culture-based viral infection model using MC57 cells was significantly inhibited after stimulation with synthetic ligands. Using intracellular deletion variants and point mutations, Y510 and Y335 in murine IFNAR2 were verified as unique phosphorylation sites for STAT1/2 activation, whereas the other tyrosine residues in IFNAR1 and IFNAR2 were not involved in STAT1/2 phosphorylation. Comparative analysis of synthetic human IFNARs supports this finding. In summary, our data showed that synthetic type I IFN signal transduction is originating from IFNAR2 rather than IFNAR1.
“…Apoptosis of Ba/F3-gp130-C VHH Fas cells was induced upon addition of trimeric mCherry (3C) fused to an Fc part of an IgG1 antibody ( 6 ). The IC 50 of the synthetic FasL ligand was determined to be in the range of 0.5 and 3.2 ng/ml, demonstrating that low quantitative doses were sufficient to efficiently prevent Hyper-IL-6 (HIL-6)–induced proliferation of Ba/F3-gp130 cells expressing synthetic Fas as control ( Fig.…”
Section: Resultsmentioning
confidence: 99%
“…3 , A and B ). HIL-6 is a fusion protein of IL-6 and the soluble IL-6R, which specifically activates gp130 receptor signal transduction and proliferation of Ba/F3-gp130 cells ( 6 ). Figure 3 LOF Fas variants failed to inhibit HIL-6–induced cellular proliferation.…”
Section: Resultsmentioning
confidence: 99%
“…Synthetic biology has become an alternative option to analyze cytokine signal transduction as well as for the development of personalized therapies ( 5 ). Recently, we generated fully synthetic cytokine receptors to phenocopy prototypical trimeric receptors for TNF and FasL ( 6 ). In our synthetic cytokine receptors for Fas (Fas-SyCyR), we use nanobodies as extracellular ligand-binding domains ( 7 , 8 ), fused to the transmembrane and intracellular domain of the receptor of interest.…”
“…Homo- and heterodimerization of such synthetic receptors can be flexibly controlled by corresponding homo- or heteromeric mEGFP/mCherry variants that bind to the nanobodies ( 38 ). The capabilities of this framework has been validated for diverse receptor systems ( 39 , 40 ) including murine IFNAR ( 41 ) . Here, we deployed this approach to design an artificial IFN-I receptor (AIR) to study the role of JAKs in human IFN-I signaling.…”
Promiscuous binding of different Janus kinases (JAKs) to class I/II cytokine receptors has been reported, yet its role in signaling is unclear. To systematically explore JAK pairing in type I interferon (IFN-I) signaling, we generated an artificial IFN-I receptor (AIR) by replacing the extracellular domains of IFNAR1 and IFNAR2 with anti mEGFP and mCherry nanobodies. The heterodimeric AIR restored near-native IFN-I activity, while the homomeric variant of IFNAR2 (AIR-dR2) initiated much weaker signaling despite harboring docking sites for signal transducer and activator of transcription (STAT) proteins. AIR-dR1 was signaling inactive, yet, pulldown uncovered its ICD to bind both TYK2 and JAK1. To further investigate the roles of JAKs on the receptors, knockout (KO) JAK1, JAK2, TYK2, and JAK2/TYK2 were generated. JAK1 KO led to complete loss of IFN-I signaling, which was partially restored by TYK2 overexpression. TYK2 KO cells retained partial activity, which was elevated by JAK1 overexpression, suggesting both JAKs to partially substitute each other. Conversely, JAK2 KO only moderately impacted the biological activity of IFN-Is, even in JAK2/TYK2 KO cells. Live cell micropatterning confirmed promiscuous binding of JAK1, JAK2 and TYK2 to IFNAR1 and IFNAR2, in line with an AlphaFold model that shows JAKs interchangeability on IFNAR ICDs. Similar promiscuity of JAK binding was observed for TPOR and GHR but not EPOR, accompanied by different downstream signaling activity. The competitive binding of JAKs to cytokine receptors together with the highly diverse absolute and relative JAK expression levels can account for cell type-dependent signaling pleiotropy observed for cytokine receptors.One Sentence SummaryPromiscuous and interchangeable binding of JAKs to cytokine receptors enables cell type-specific pleiotropic signaling.
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