Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany.
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Stadt Seeland, Gatersleben, Germany.
PLoS One. 2020 Apr 1;15(4):e0230804. doi: 10.1371/journal.pone.0230804. eCollection 2020.
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 (EC50) 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 EC50 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 EC50 values (GFP3: 0.58 ng/ml; mCherrry3: 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.
细胞因子信号转导是通过细胞表面受体进行的，这些受体作为天然的生物开关，控制着细胞的功能，如免疫反应。最近，我们设计了合成细胞因子受体（SyCyRs），由绿色荧光蛋白（GFP）和 mCherry-纳米体融合到细胞因子受体的跨膜和细胞内结构域组成。用同型和异型 GFP-mCherry 融合蛋白刺激后，所得受体模拟了生理发生的细胞因子诱导的信号。GFP 和 mCherry 融合蛋白在大肠杆菌或 CHO-K1 细胞中产生，但总体产量和稳定性较低。因此，我们应用了两种替代的多聚化策略，实现了免疫球蛋白 Fc 介导的二聚体和卷曲螺旋 GCN4pII 介导的三聚体组装。GFP-和/或 mCherry-Fc 同源二聚体激活了天然响应白细胞介素 6 家族细胞因子的合成 gp130 细胞因子受体。这些合成 gp130 受体的激活导致 STAT3 和 ERK 磷酸化，随后 Ba/F3-gp130 细胞增殖。二聚体 GFP-Fc 和 mCherry-Fc 的半最大有效浓度（EC50）分别为 8.1 ng/ml 和 0.64 ng/ml。这在天然细胞因子的预期 EC50 范围内。此外，我们还产生了四聚体和六聚体 GFP-mCherry-Fc 融合蛋白，它们也具有生物活性。这突出了两个细胞因子受体紧密毗邻对于有效受体激活的重要性。最后，我们使用三聚体 GCN4pII 基序生成同三聚体 GFP 和 mCherry 复合物。这些合成细胞因子显示出改善的 EC50 值（GFP3：0.58 ng/ml；mCherry3：0.37 ng/ml），优于二聚体 Fc 融合变体。总之，我们成功地生成了高效且稳定的多聚化合成细胞因子受体配体，用于合成细胞因子受体的激活。
