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Chemically inducible split protein regulators for mammalian cells

Nature Chemical Biology volume 19, pages 64–71 (2023)Cite this article

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Abstract

Chemically inducible systems represent valuable synthetic biology tools that enable the external control of biological processes. However, their translation to therapeutic applications has been limited because of unfavorable ligand characteristics or the immunogenicity of xenogeneic protein domains. To address these issues, we present a strategy for engineering inducible split protein regulators (INSPIRE) in which ligand-binding proteins of human origin are split into two fragments that reassemble in the presence of a cognate physiological ligand or clinically approved drug. We show that the INSPIRE platform can be used for dynamic, orthogonal and multiplex control of gene expression in mammalian cells. Furthermore, we demonstrate the functionality of a glucocorticoid-responsive INSPIRE platform in vivo and apply it for perturbing an endogenous regulatory network. INSPIRE presents a generalizable approach toward designing small-molecule responsive systems that can be implemented for the construction of new sensors, regulatory networks and therapeutic applications.

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Fig. 1: Design and characterization of INSPIRE.
Fig. 2: Design of NR-INSPIRE.
Fig. 3: Application of designed INSPIRE for inducible regulation of gene expression.
Fig. 4: Orthogonal INSPIRE-mediated gene activation and design of logic circuits.
Fig. 5: Application of designed INSPIRE for inducible regulation of gene expression.

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Data availability

All data supporting the findings of this study are available within the article or Supplementary Information. Plasmid maps, including complete and annotated GenBank files, are provided in Supplementary Data. A subset of key plasmids used in this study will be made available on Addgene. Source data are provided with this paper. Any other relevant data or reagents are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was funded by grants from the Slovenian research agency (P4-0176 (R.J.), N4-0080 (R.J.), J1-9173 (R.J.), J1-2471 (U.B.) and P2-0046 (U.B.).).

Author information

Author notes

  1. These authors contributed equally: Erik Rihtar, Tina Lebar.

Authors and Affiliations

  1. Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia

    Erik Rihtar, Tina Lebar, Duško Lainšček & Roman Jerala

  2. Graduate School of Biomedicine, University of Ljubljana, Ljubljana, Slovenia

    Erik Rihtar

  3. Laboratory of Physical Chemistry and Chemical Thermodynamics, University of Maribor, Maribor, Slovenia

    Katarina Kores, Samo Lešnik & Urban Bren

  4. University of Primorska, Faculty of Mathematics, Natural Sciences and Information Technologies, Glagoljaška ulica 8, 6000 Koper, Slovenia

    Urban Bren

  5. EN-FIST, Centre of Excellence, Ljubljana, Slovenia

    Roman Jerala

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Contributions

E.R. and T.L. performed the experiments on cell culture. D.L. performed the experiments on mice. K.K., S.L. and U.B. performed the bioinformatics analysis. E.R. and T.L. designed and analyzed the experiments. E.R., T.L. and R.J. wrote the manuscript. R.J. conceived of the study.

Corresponding author

Correspondence to Roman Jerala.

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Competing interests

The authors E.R., T.L., and R.J. are inventors of the EPO Patent application LU102654 ‘Engineering Chemically Inducible Split Protein Actuators (INSPIRE)’, submitted by the National Institute of Chemistry. All other authors declare that they have no competing interests.

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Nature Chemical Biology thanks Srivatsan Raman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Design of LYN-based INSPIRE system.

a, Left: crystal structure of Lyn kinase domain (LYN) in complex with dasatinib (DAS) (PDB ID 2zva). Splits are chosen in surface-exposed loops (cyan); α-helixes and β-strands are colored in dark and light gray, respectively. Right: aa sequence of LYN with additional annotations indicating split site locations and secondary structures. b, Schematic illustration showing the process of cloning split LYN fragments into reporter luciferase vectors (nLuc and cLuc). Split fragments were fused to split luciferase segments via a flexible gs10 linker. Additionally, constructs were tagged with either Myc- or HA-tag to confirm the expression of fusions via Western blot. c, Expression of N- (top) and C-terminal (bottom) split LYN fragments in HEK293T cells was verified with Western blot. Constitutively expressed mCitrine fluorescent protein was used as transfection control, and an empty plasmid vector (pcDNA3) was used as a negative control. The mCitrine transfection control was run on a separate gel. The transfection mixtures are provided in Supplementary Table 4. Data are representative of two independent experiments (n = 1). Original uncropped images are provided in the Source Data.

Source data

Extended Data Fig. 2 Analysis of split protein–ligand complex structural parameters.

a, Selected LBP is computationally split, and the contact number is calculated between nSplit–ligand (P1-L), nSplit–cSplit (P1-P2), and cSplit–ligand (P2-L). b–d, Calculated contact numbers for LYN in complex with DAS (PDB ID 2zva) (b), DHFR in complex with MTX (PDB ID 1u72) (c) and GR2 in complex with MOF (PDB ID 4e2j) (d). nSplit–ligand contacts (P1-L), cSplit–ligand (P2-L), and nSplit–cSplit (P1-P2) are represented by blue, yellow, and red lines, respectively. Arrows indicate the split site positions, and light shaded regions show the split sites showing the highest efficacy (split 267 for LYN, 173 for DHFR, and 175 for GR2). e, The results page of the ProteinSplit web application (http://elixir.fkkt.um.si/ProteinSplitIndex.html). f, Percentage of database proteins with the minimum number of α-helices or β-sheets located in the shorter segment up to a possible cut indicated on the x-axis. The proteins, where the shorter segment contains 1, 2, or 3 α-helixes, β-sheets, or combinations of both, represent 80% of proteins from various organisms and 84% of human proteins. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 3 Design of DHFR-based INSPIRE system.

a, Left: crystal structure of dihydrofolate reductase (DHFR) in complex with methotrexate (MTX) (PDB ID 1u72). Splits are chosen in surface-exposed loops (orange); α-helixes and β-strands are colored in dark and light gray, respectively. Right: aa sequence of DHFR with additional annotations indicating split site locations and secondary structures. b, Expression of N- (left) and C-terminal (right) split DHFR fragments in HEK293T cells verified with Western blot. Constitutively expressed mCitrine fluorescent protein was used as transfection control, and an empty plasmid vector (pcDNA3) was used as a negative control. The mCitrine transfection control was run on a separate gel. The transfection mixtures are provided in Supplementary Table 4. Data are representative of two independent experiments (n = 1). Original uncropped images are provided in the Source Data file.

Source data

Extended Data Fig. 4 Design of GR2-based INSPIRE system.

a, Left: crystal structure of ligand-binding domain (LBD) of glucocorticoid receptor 2 (GR2) in complex with mometasone furoate (MOF) (PDB ID 4e2j). Splits are chosen in surface-exposed loops (magenta); α-helixes and β-strands are colored in dark and light gray, respectively. Right: aa sequence of GR2, with additional annotations indicating split site locations and secondary structures. b, Expression of N- (left) and C-terminal (right) split GR2 fragments in HEK293T cells verified with Western blot. Constitutively expressed mCitrine fluorescent protein was used as transfection control, and an empty plasmid vector (pcDNA3) was used as a negative control. The mCitrine transfection control was run on a separate gel. Transfection mixtures are provided in Supplementary Table 4. Data are representative of two independent experiments (n = 1). Original uncropped images are provided in the Source Data file.

Source data

Extended Data Fig. 5 INSPIRE-mediated activation of a reporter gene.

a, Schematic presentation of the transcriptional activator INSPIRE systems (TA-INSPIRE). Ligand-induced dimerization of split protein fragments results in the expression of firefly luciferase gene (fLuc) on a reporter plasmid with 10 binding sites for gRNA upstream of the minimal promoter (Pmin). b–g, Transcriptional activation of the fLuc reporter gene by employing LYN267- (b), DHFR173- (c), GR2175- (d), TRβ412- €, ERβ446- (f), and PPARγ429- (g) based TA-INSPIRE systems after stimulation of HEK293T cells with the indicated ligands. Results are shown as fold activation in reporter gene expression compared to non-stimulated mock cells transfected with reporter plasmid only. Detailed descriptions of the genetic components and transfection mixtures are provided in Supplementary Table 4. Plots shows the means ± s.d. of n = 8 biological replicates combined from two independent experiments. See Supplementary Table 8 for sample sizes (n) and P values. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 6 INSPIRE-mediated activation of individual endogenous genes.

a,b, Activation of individual endogenous genes by employing GR2- (a) or ERβ- (b) based TA-INSPIRE systems. HEK293T cells were transfected with individual gRNAs targeting TTN, ASCL1, NEUROD1, and TUNAR endogenous genes and stimulated with indicated ligands. Results are shown as fold activation in reporter gene expression compared to non-stimulated mock cells transfected with an empty plasmid (pcDNA3.1). Detailed descriptions of the genetic components and transfection mixtures are provided in Supplementary Table 4. Plots shows the means ± s.d. of n ≤ 6 biological replicates combined from two independent experiments. Conditions were compared using a two-sided unpaired t-test with Welch correction (****P < 0.0001; ***P < 0.001; **P < 0.01; ns > 0.05). See Supplementary Table 8 for sample sizes (n) and P values. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 7 Characterization of reversibility of TA-INSPIRE systems.

a–g, Reversibility of FKBP-FRB-based CID system (a) and LYN267- (b), DHFR173- (c), GR2175- (d), TRβ412- (e), ERβ446- (f), and PPARγ429- (g) based TA-INSPIRE systems after ligand-mediated induction of reporter gene expression. Cells were continuously stimulated with the indicated ligands for 72 h (ON conditions) or stimulated for 24 h and then washed and cultured without the ligand (ON–OFF conditions). Results are shown as fold activation in reporter gene expression compared to non-stimulated mock cells transfected with only reporter plasmid. Detailed descriptions of the genetic components and transfection mixtures are provided in Supplementary Table 4. Plots shows the means ± s.d. of n = 8 biological replicates combined from two independent experiments. See Supplementary Table 8 for sample sizes (n) and P values. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 8 Orthogonality of inducible GR2- and ERβ-based INSPIRE components and construction of logic gates by orthogonal TA-INSPIRE.

a, Orthogonality of GR2- and ERβ-based INSPIRE components (nSplit and cSplit fragments) was tested in HEK293T cells by co-transfected individual split fragments fused to split luciferase. Orthogonality was assessed by measuring luciferase activity after cell stimulation with indicated synthetic ligands (MOF and 4-OHT) or natural ligands (COR and EST). b, Schematic presentation of the transcriptional activation system used in (c) to simultaneously modulate expression of two reporter genes (BFP and mCit) using orthogonal GR2- and ERβ-based TA-INSPIRE. pMin represents a minimal promoter, and mCit and BFP represent the mCitrine and TagBFP fluorescent protein genes, respectively. c, Conditional activation of reporter genes mCit and BFP after stimulation of the cells with either synthetic ligands MOF (1 µM) and 4-OHT (10 µM) (left) or natural ligands COR (100 µM) and EST (10 µM) (right), detected with fluorescent confocal microscopy. Scale bars represent 100 µm. Microscopic images are representative of two independent experiments (n = 1 biologically independent sample) and four separate observations within the same experiment. d, Construction of functional OR gate, AND gate, and switching between transcriptional activation and repression (ACT/REP) utilizing GR2- and ERβ-based TA-INSPIRE in combination with TALE DNA-binding domain (see schematics in Fig. 4b–d). Cells were stimulated with natural ligands COR (100 µM) and EST (10 µM) as indicated. Results are shown as fold activation in reporter gene expression compared to non-stimulated mock cells transfected with only reporter plasmid. Detailed descriptions of the genetic components and transfection mixtures are provided in Supplementary Table 4. Plots shows the means ± s.d. of n = 8 biological replicates combined from two independent experiments. See Supplementary Table 8 for sample sizes (n) and P values. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 9 Application of GR2-INSPIRE system for ligand-mediated reconstitution of split TEV protease.

a, Schematic presentation of the split-tobacco etch virus protease (TEVp) reconstitution utilizing INSPIRE (nSplit and cSplit). Ligand-mediated reconstitution of split TEVp leads to the cleavage and activation of cyclic luciferase reporter (cycLuc). b, Ligand-mediated induction of split TEVp reconstitution utilizing INSPIRE systems after stimulation of HEK293T cells with COR (left) or MOF (right). Results are shown as fold activation in reporter gene expression compared to non-stimulated mock cells transfected with only reporter plasmid. Detailed descriptions of the genetic components and transfection mixtures are provided in Supplementary Table 4. Plots shows the means ± s.d. of n = 8 biological replicates combined from two independent experiments. See Supplementary Table 8 for sample sizes (n) and P values. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 10 Evaluation of GR2-INSPIRE responsiveness to corticosterone (CORT) and prescreen of the effective gRNAs targeting the mouse 11β-HSD2 gene.

a, The dose-response curve of GR2175-based INSPIRE system for corticosterone (CORT), utilizing dual luciferase assay in HEK293T cells. b, Activation of the mouse 11β-HSD2 gene in Neuro2a cells transfected with dCas9-VPR and indicated gRNAs. The light shaded region indicates the gRNA with the highest fold activation. Results are shown as fold activation in endogenous gene expression compared to mock cells transfected with an empty plasmid (pcDNA3). Detailed descriptions of the genetic components and transfection mixtures are provided in Supplementary Table 4. Plots shows the means ± s.d. of n = 6 biological replicates combined from two independent experiments. Conditions were compared using a two-sided unpaired t-test with Welch correction (****P < 0.0001; **P < 0.01; ns > 0.05). See Supplementary Table 8 for sample sizes (n) and P values. Source data are provided as a Source Data file.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–3, Tables 1–8 and Note.

Reporting Summary

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Rihtar, E., Lebar, T., Lainšček, D. et al. Chemically inducible split protein regulators for mammalian cells. Nat Chem Biol 19, 64–71 (2023). https://doi.org/10.1038/s41589-022-01136-x

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