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Artificial signaling in mammalian cells enabled by prokaryotic two-component system

Abstract

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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Fig. 1: Identification of HK domains with reduced intrinsic signaling.
Fig. 2: Restoration of two-component signaling via forced dimerization.
Fig. 3: Transduction of cytoplasmic ligand to gene expression.
Fig. 4: Rewiring the GPCR activity to the expression of a reporter gene.
Fig. 5: Multiple GPCRs are rewired to induce gene expression.
Fig. 6: Dynamic characterization of the various signaling approaches.

Data availability

All plasmid sequences and data supporting the findings are available from the corresponding author upon request.

Code availability

The code used to process the time-lapse imaging data is available from the corresponding author upon request.

References

  1. 1.

    Park, S. H., Zarrinpar, A. & Lim, W. A. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 299, 1061–1064 (2003).

    CAS  PubMed  Google Scholar 

  2. 2.

    Jackson, H. J., Rafiq, S. & Brentjens, R. J. Driving CAR T-cells forward. Nat. Rev. Clin. Oncol. 13, 370–383 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Benenson, Y. Biomolecular computing systems: principles, progress and potential. Nat. Rev. Genet. 13, 455–468 (2012).

    CAS  PubMed  Google Scholar 

  4. 4.

    Hansen, J. & Benenson, Y. Synthetic biology of cell signaling. Nat. Comput. 15, 5–13 (2016).

    CAS  Google Scholar 

  5. 5.

    Cho, J. H., Collins, J. J. & Wong, W. W. Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell 173, 1426–1438 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Barnea, G. et al. The genetic design of signaling cascades to record receptor activation. Proc. Natl Acad. Sci. USA 105, 64–69 (2008).

    CAS  PubMed  Google Scholar 

  7. 7.

    Schwarz, K. A., Daringer, N. M., Dolberg, T. B. & Leonard, J. N. Rewiring human cellular input-output using modular extracellular sensors. Nat. Chem. Biol. 13, 202–209 (2017).

    CAS  PubMed  Google Scholar 

  8. 8.

    Kipniss, N. H. et al. Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system. Nat. Commun. 8, 2212 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Baeumler, T. A., Ahmed, A. A. & Fulga, T. A. Engineering synthetic signaling pathways with programmable dCas9-Based chimeric receptors. Cell Rep. 20, 2639–2653 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Xie, M. et al. Beta-cell-mimetic designer cells provide closed-loop glycemic control. Science 354, 1296–1301 (2016).

    CAS  PubMed  Google Scholar 

  11. 11.

    Porter, D. L., Levine, B. L., Kalos, M., Bagg, A. & June, C. H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365, 725–733 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Mootz, H. D. & Muir, T. W. Protein splicing triggered by a small molecule. J. Am. Chem. Soc. 124, 9044–9045 (2002).

    CAS  PubMed  Google Scholar 

  13. 13.

    Slomovic, S. & Collins, J. J. DNA sense-and-respond protein modules for mammalian cells. Nat. Methods 12, 1085–1090 (2015).

    CAS  PubMed  Google Scholar 

  14. 14.

    Morsut, L. et al. Engineering customized cell sensing and response behaviors using synthetic Notch receptors. Cell 164, 780–791 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Angelici, B., Mailand, E., Haefliger, B. & Benenson, Y. Synthetic biology platform for sensing and integrating endogenous transcriptional inputs in mammalian cells. Cell Rep. 16, 2525–2537 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Schreiber, J., Arter, M., Lapique, N., Haefliger, B. & Benenson, Y. Model‐guided combinatorial optimization of complex synthetic gene networks. Mol. Syst. Biol. 12, 899 (2016).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Hansen, J. et al. Transplantation of prokaryotic two-component signaling pathways into mammalian cells. Proc. Natl Acad. Sci. USA 111, 15705–15710 (2014).

    CAS  PubMed  Google Scholar 

  18. 18.

    Rivera-Cancel, G., Ko, W. H., Tomchick, D. R., Correa, F. & Gardner, K. H. Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation. Proc. Natl Acad. Sci. USA 111, 17839–17844 (2014).

    CAS  PubMed  Google Scholar 

  19. 19.

    Dago, A. E. et al. Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis. Proc. Natl Acad. Sci. USA 109, E1733–E1742 (2012).

    CAS  PubMed  Google Scholar 

  20. 20.

    Casino, P., Rubio, V. & Marina, A. Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction. Cell 139, 325–336 (2009).

    CAS  PubMed  Google Scholar 

  21. 21.

    T.N, H., Noriega, C. E. & Stewart, V. Conserved mechanism for sensor phosphatase control of two-component signaling revealed in the nitrate sensor NarX. Proc. Natl Acad. Sci. Usa. 107, 21140–21145 (2010).

    Google Scholar 

  22. 22.

    Landry, B. P., Palanki, R., Dyulgyarov, N., Hartsough, L. A. & Tabor, J. J. Phosphatase activity tunes two-component system sensor detection threshold. Nat. Commun. 9, 10 (2018).

    Google Scholar 

  23. 23.

    Ashenberg, O., Rozen-Gagnon, K., Laub, M. T. & Keating, A. E. Determinants of homodimerization specificity in histidine kinases. J. Mol. Biol. 413, 222–235 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Tomomori, C. et al. Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ. Nat. Struct. Biol. 6, 729–734 (1999).

    CAS  PubMed  Google Scholar 

  25. 25.

    Thompson, K. E., Bashor, C. J., Lim, W. A. & Keating, A. E. SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains. ACS Synth. Biol. 1, 118–129 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Cai, S. J. & Inouye, M. Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ). J. Mol. Biol. 329, 495–503 (2003).

    CAS  PubMed  Google Scholar 

  27. 27.

    Casino, P., Miguel-Romero, L. & Marina, A. Visualizing autophosphorylation in histidine kinases. Nat. Commun. 5, 3258 (2014).

    PubMed  Google Scholar 

  28. 28.

    Cai, S. J. & Inouye, M. EnvZ-OmpR interaction and osmoregulation in Escherichia coli. J. Biol. Chem. 277, 24155–24161 (2002).

    CAS  PubMed  Google Scholar 

  29. 29.

    Bayle, J. H. et al. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem. Biol. 13, 99–107 (2006).

    CAS  PubMed  Google Scholar 

  30. 30.

    Schramm, A. et al. Establishing a split luciferase assay for proteinkinase G (PKG) interaction studies. Int. J. Mol. Sci. 19, 20 (2018).

    Google Scholar 

  31. 31.

    Edwards, S. R. & Wandless, T. J. The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain. J. Biol. Chem. 282, 13395–13401 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Kobilka, B. & Schertler, G. F. X. New G-protein-coupled receptor crystal structures: insights and limitations. Trends Pharmacol. Sci. 29, 79–83 (2008).

    CAS  PubMed  Google Scholar 

  33. 33.

    Billerbeck, S. et al. A scalable peptide-GPCR language for engineering multicellular communication. Nat. Commun. 9, 12 (2018).

    Google Scholar 

  34. 34.

    Shukla, A. K. et al. Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature 512, 218–222 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Luttrell, L. M. & Lefkowitz, R. J. The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J. Cell Sci. 115, 455–465 (2002).

    CAS  PubMed  Google Scholar 

  36. 36.

    Cahill, T. J. et al. Distinct conformations of GPCR-beta-arrestin complexes mediate desensitization, signaling, and endocytosis. Proc. Natl Acad. Sci. USA 114, 2562–2567 (2017).

    CAS  PubMed  Google Scholar 

  37. 37.

    Baker, J. G. The selectivity of β‐adrenoceptor agonists at human β1‐, β2‐and β3‐adrenoceptors. Br. J. Pharmacol. 160, 1048–1061 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat. Struct. Mol. Biol. 22, 362–369 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    O’Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J. & Bouvier, M. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor. J. Biol. Chem. 264, 7564–7569 (1989).

    PubMed  Google Scholar 

  40. 40.

    Sadeghi, H. M., Innamorati, G., Dagarag, M. & Birnbaumer, M. Palmitoylation of the V2 vasopressin receptor. Mol. Pharmacol. 52, 21–29 (1997).

    CAS  PubMed  Google Scholar 

  41. 41.

    Klumpp, S. & Krieglstein, J. Reversible phosphorylation of histidine residues in proteins from vertebrates. Sci. Signal. 2, pe13 (2009).

    PubMed  Google Scholar 

  42. 42.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  43. 43.

    Haefliger, B., Prochazka, L., Angelici, B. & Benenson, Y. Precision multidimensional assay for high-throughput microRNA drug discovery. Nat. Commun. 7, 12 (2016).

    Google Scholar 

  44. 44.

    Pandy-Szekeres, G. et al. GPCRdb in 2018: adding GPCR structure models and ligands. Nucleic Acids Res. 46, D440–D446 (2018).

    CAS  PubMed  Google Scholar 

  45. 45.

    Corpet, F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16, 10881–10890 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

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A.M. conceived research, performed the experiments, analyzed data and wrote the paper. Y.B. conceived research, analyzed data and wrote the paper.

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Correspondence to Yaakov Benenson.

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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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Supplementary Tables 1–27, Figs. 1–13 and Notes 1 and 2.

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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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