Design of fast proteolysis-based signaling and logic circuits in mammalian cells


Cellular signal transduction is predominantly based on protein interactions and their post-translational modifications, which enable a fast response to input signals. Owing to difficulties in designing new unique protein–protein interactions, designed cellular logic has focused on transcriptional regulation; however, that process has a substantially slower response, because it requires transcription and translation. Here, we present de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled coils (CC) and implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains with tunable affinity for competitive displacement after proteolytic cleavage. This framework enabled the implementation of Boolean logic functions and signaling cascades in mammalian cells. The designed split-protease-cleavable orthogonal-CC-based (SPOC) logic circuits enable response to chemical or biological signals within minutes rather than hours and should be useful for diverse medical and nonmedical applications.

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Fig. 1: Design of the proteolysis-based signaling pathways and orthogonal proteases.
Fig. 2: Design of proteolytic-cleavage-responsive CC interaction modules.
Fig. 3: Design of Boolean logic functions implemented by SPOC logic.
Fig. 4: Multilayer design of proteolysis-based signaling pathways.
Fig. 5: Fast kinetics of the proteolysis-mediated signaling pathway.

Data availability

The authors declare that the data supporting the findings of this study are available in the paper and its supplementary information files. A set of plasmids for SPOC logic, comprising the split orthogonal proteases, cycLuc reporters and CC-building modules, have been deposited with Addgene under Addgene IDs 118966, 118967, 118968, 118969, 118970, 119182, 119207, 119208, 119209, 119210, 119211, 119212, 119213, 119214, 119299, 119300, 119302 and 119303. The raw data are available from the corresponding author upon reasonable request.


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The idea and proof of principle for this work were conceived as part of the Slovenian iGEM 2016 project, and members of the team who are not among the authors—M. Meško, M. Mraz, M. Moškon, D. Križaj, R. Krese, N. Franko, L. Magdevska, M. Gradišek, Ž. Pušnik, S. Roškar and K. Cerović—are acknowledged for their contribution. The project was funded by the Slovenian Research Agency (P4-0176 and J3-7034) and an ERC project MaCChines (to R.J.). We thank N. Landau (Division of AIDS, NIAID)) for providing materials.

Author information

T.F., J.L., A.P., T.P., K.L., N.J. and E.M. performed the experiments and analyzed the results; T.F., J.L. and T.L. designed SPOC logic gates; Ž.S. and F.L. designed antiparallel CCs; M.B., T.F., J.L. and R.J. wrote and edited the manuscript; M.B. supervised the experimental work; and R.J. conceptualized the study and acquired funding.

Correspondence to Roman Jerala.

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Fink, T., Lonzarić, J., Praznik, A. et al. Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nat Chem Biol 15, 115–122 (2019).

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