Augmenting live cells with new signal transduction capabilities is a key objective in genetic engineering and synthetic biology. We showed earlier that two-component signaling pathways could function in mammalian cells, albeit while losing their ligand sensitivity. Here, we show how to transduce small-molecule ligands in a dose-dependent fashion into gene expression in mammalian cells using two-component signaling machinery. First, we engineer mutually complementing truncated mutants of a histidine kinase unable to dimerize and phosphorylate the response regulator. Next, we fuse these mutants to protein domains capable of ligand-induced dimerization, which restores the phosphoryl transfer in a ligand-dependent manner. Cytoplasmic ligands are transduced by facilitating mutant dimerization in the cytoplasm, while extracellular ligands trigger dimerization at the inner side of a plasma membrane. These findings point to the potential of two-component regulatory systems as enabling tools for orthogonal signaling pathways in mammalian cells.
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All plasmid sequences and data supporting the findings are available from the corresponding author upon request.
The code used to process the time-lapse imaging data is available from the corresponding author upon request.
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The study was funded by ETH Zurich, NCCR Molecular Systems Engineering (grant no. 51NF40-182895) and Swiss National Science foundation (grant no. 31003A_149802). We thank B. Haefliger, R. Altamura, J. Hansen, T. Littmann, G. Bernhardt and C. Stelzer for plasmids, Benenson lab members for discussions, H.M. Kaltenbach for help with statistical analysis and the members of the Single Cell Unit for their help with imaging and flow cytometry.
A patent application has been filed describing the result in this study, with A.M. and Y.B. listed as coinventors. Y.B. is a coinventor of a background patent to this filing.
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Mazé, A., Benenson, Y. Artificial signaling in mammalian cells enabled by prokaryotic two-component system. Nat Chem Biol 16, 179–187 (2020). https://doi.org/10.1038/s41589-019-0429-9
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