Synthetic cytokine receptors transmit biological signals using artificial ligands

Engelowski, Erika; Schneider, Artur; Franke, Manuel; Xu, Haifeng; Clemen, Ramona; Lang, Alexander; Baran, Paul; Binsch, Christian; Knebel, Birgit; Al-Hasani, Hadi; Moll, Jens M.; Floß, Doreen M.; Lang, Philipp A.; Scheller, Jürgen

doi:10.1038/s41467-018-04454-8

Cited by 47 publications

(44 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Importantly, proliferation of Ba/F3-gp130-(GFP VHH -gp130) cells was stimulated by GFP-Fc and Hyper-IL-6, whereas proliferation of Ba/F-gp130 was only induced by Hyper-IL-6 but not by GFP-Fc ( Fig 1D). The half-maximal effective concentration of GFP-Fc to induce cellular proliferation was determined to be 8.1 ng/ml (Fig 1E), which is in good agreement with previous findings using dimeric GFP-GFP-fusion proteins from CHO-K1 cell culture supernatants [2]. The EC 50 of purified Hyper-IL-6-Fc or Hyper-IL-6 cell culture supernatant were 0.7 ng/ml and 0.16 ng/ml, respectively (S2B Fig).…”

Section: Dimeric Gfp-fc and Mcherry-fc Fusion Proteins Are Effective supporting

confidence: 91%

“…Ba/F3-gp130 cells were consecutively retrovirally transduced with two SyCyRs using two different pMOWS plasmids with either puromycin (for GFP VHH -SyCyRs) or hygromycin resistance (for mCherry VHH -SyCyRs) for selection of stably transduced clones. Synthetic ligands were expressed in CHO-K1 cells as previously described [2].…”

Section: Composition Of Sycyrs and Synthetic Fluorescent Ligandsmentioning

confidence: 99%

“…Ba/F3-gp130 cells were retrovirally transduced using pMOWS plasmids coding for different SyCyRs as described previously [2]. The packaging cell line was Phoenix-Eco.…”

Section: Transfection and Selection Of Cellsmentioning

confidence: 99%

“…The on-state might be interrupted by negative feedback mechanisms or depletion of the cytokine and cytokine receptor. Recently, we have designed synthetic cytokine receptors (SyCyRs) which phenocopy IL-6 and IL-23 signaling via homodimeric gp130 and heterodimeric IL-23R/IL-12Rbeta1 receptors [2]. SyCyRs incorporate nanobodies specifically recognizing GFP or mCherry [3,4] fused to transmembrane and intracellular receptor domains.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands

et al. 2020

Self Cite

View full text Add to dashboard Cite

Cytokine signaling is transmitted by cell surface receptors which act as natural biological switches to control cellular functions such as immune reactions. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of green fluorescent protein (GFP)-and mCherry-nanobodies fused to the transmembrane and intracellular domains of cytokine receptors. Following stimulation with homo-and heterodimeric GFP-mCherry fusion proteins, the resulting receptors phenocopied signaling induced by physiologically occurring cytokines. GFP and mCherry fusion proteins were produced in E. coli or CHO-K1 cells, but the overall yield and stability was low. Therefore, we applied two alternative multimerization strategies and achieved immunoglobulin Fc-mediated dimeric and coiled-coil GCN4pII-mediated trimeric assemblies. GFP-and/or mCherry-Fc homodimers activated synthetic gp130 cytokine receptors, which naturally respond to Interleukin 6 family cytokines. Activation of these synthetic gp130 receptors resulted in STAT3 and ERK phosphorylation and subsequent proliferation of Ba/F3-gp130 cells. Half-maximal effective concentrations (EC 50) of 8.1 ng/ml and 0.64 ng/ml were determined for dimeric GFP-Fc and mCherry-Fc, respectively. This is well within the expected EC 50 range of the native cytokines. Moreover, we generated tetrameric and hexameric GFP-mCherry-Fc fusion proteins, which were also biologically active. This highlighted the importance of close juxtaposition of two cytokine receptors for efficient receptor activation. Finally, we used a trimeric GCN4pII motif to generate homo-trimeric GFP and mCherry complexes. These synthetic cytokines showed improved EC 50 values (GFP 3 : 0.58 ng/ml; mCherrry 3 : 0.37 ng/ml), over dimeric Fc fused variants. In conclusion, we successfully generated highly effective and stable multimeric synthetic cytokine receptor ligands for activation of synthetic cytokine receptors.

show abstract

Section: Dimeric Gfp-fc and Mcherry-fc Fusion Proteins Are Effective supporting

confidence: 91%

Section: Composition Of Sycyrs and Synthetic Fluorescent Ligandsmentioning

confidence: 99%

“…Ba/F3-gp130 cells were retrovirally transduced using pMOWS plasmids coding for different SyCyRs as described previously [2]. The packaging cell line was Phoenix-Eco.…”

Section: Transfection and Selection Of Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…[11][12][13] Many natural proteins, in contrast, exhibit exquisite sensitivity to highly related molecules with important biological consequences, as is the case for hormone receptors 14 or cyclic nucleotide binding proteins 15,16 . Unraveling the principles underlying the selectivity of ligand binding is crucial not only for our understanding of native signaling processes, but also for drug design 17 and synthetic biology 18 .…”

Section: Introductionmentioning

confidence: 99%

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Tebo

Moeyaert

Thauvin

et al. 2020

Preprint

View full text Add to dashboard Cite

Fluorescence microscopy is an indispensable tool in biological research, allowing subsecond and sub-micrometer mapping of molecules or processes inside living cells.Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one-and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy. 3

show abstract

Synthetic Biology and Tissue Engineering: Toward Fabrication of Complex and Smart Cellular Constructs

Hoffman

Antovski

Tebon

et al. 2020

Adv Funct Materials

View full text Add to dashboard Cite

Tissue engineering approaches, with the goals of replacing or recovering damaged or diseased tissues, or of reconstituting tissues in vitro for disease modeling and drug development, have the potential to make significant contributions to medicine. Advances in stem cell biology, biomaterial synthesis and characterization, and microscale technologies have made engineered tissues a reality. However, the classic tools used to build tissues in the lab do not allow for complete control of cell behaviors. More recently, synthetic biology principles have developed robust and versatile approaches to program cells with artificial genetic circuits, where cell behavior and function can be manipulated. At the interface between synthetic biology and tissue engineering, there is space for a new area of investigation where material engineering and cellular engineering complement and sustain each other. In this progress report, synthetic biology principles and how they have been used to engineer cells with potential to dictate cell behavior and function in tissue constructs of the future are briefly described. It is our belief that this research area still needs further exploration to fully exploit synthetic biology to make smart and functional cellular constructs for therapeutic and in vitro applications.Received: ((will be filled in by the editorial staff))Revised: ((will be filled in by the editorial staff))

show abstract

Synthetic cytokine receptors transmit biological signals using artificial ligands

Cited by 47 publications

References 47 publications

Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands

Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Synthetic Biology and Tissue Engineering: Toward Fabrication of Complex and Smart Cellular Constructs

Contact Info

Product

Resources

About