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

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Tina Fink
Tina Fink
Tina Fink
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Jan Lonzarić
Jan Lonzarić
Jan Lonzarić
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Arne Praznik at National Institute of Chemistry
Arne Praznik
Tjasa Plaper at National Institute of Chemistry
Tjasa Plaper
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Abstract and Figures

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. © 2018, The Author(s), under exclusive licence to Springer Nature America, Inc.
Design of the proteolysis-based signaling pathways and orthogonal proteases a, Scheme of components of the proteolysis-based signaling pathways. b, Heat map showing the orthogonality of the four potyviral protease homologs tested in HEK293T cells, detected by the cycLuc reporter with a matching protease-cleavage site. c,d, Chemically inducible reconstitution of the split proteases (schematic in c) 24 h after induction with rapamycin (d) (three-dimensional homology models of orthogonal split proteases are shown in Supplementary Fig. 1e). Values in d are the means from four cell cultures ±s.d. and are representative of two independent experiments. Transfection plasmid mixtures are listed in Supplementary Table 1.
Design of the proteolysis-based signaling pathways and orthogonal proteases a, Scheme of components of the proteolysis-based signaling pathways. b, Heat map showing the orthogonality of the four potyviral protease homologs tested in HEK293T cells, detected by the cycLuc reporter with a matching protease-cleavage site. c,d, Chemically inducible reconstitution of the split proteases (schematic in c) 24 h after induction with rapamycin (d) (three-dimensional homology models of orthogonal split proteases are shown in Supplementary Fig. 1e). Values in d are the means from four cell cultures ±s.d. and are representative of two independent experiments. Transfection plasmid mixtures are listed in Supplementary Table 1.
… 
Design of proteolytic-cleavage-responsive CC interaction modules a, Design of the proteolytic-cleavage-responsive CC rearrangement for reconstitution of the functional split protein. After linker cleavage, an autoinhibitory coil is replaced by a displacer segment with higher binding affinity to reconstitute the split effector/reporter. b, ABA- and rapamycin-inducible activation, demonstrated in HEK293T cells measured 30 min after induction with the indicated concentration of ABA or rapamycin. fLuc, firefly luciferase; nRLU, normalized relative light units; n and c prefixes, N and C fragments. c, Design of a proteolytic-cleavage-inactivated module (logical negation). A protease-cleavage site is introduced between the CC-forming segments and split effector/reporter domain. After cleavage of the linker, split luciferase dissociates. d, Decrease in luciferase activity after cotransfection of logical negation functions with plasmids encoding specific proteases. Values in b and d are the means from four cell cultures ±s.d. and are representative of two independent experiments. Transfection plasmid mixtures are listed in Supplementary Table 1.
Design of proteolytic-cleavage-responsive CC interaction modules a, Design of the proteolytic-cleavage-responsive CC rearrangement for reconstitution of the functional split protein. After linker cleavage, an autoinhibitory coil is replaced by a displacer segment with higher binding affinity to reconstitute the split effector/reporter. b, ABA- and rapamycin-inducible activation, demonstrated in HEK293T cells measured 30 min after induction with the indicated concentration of ABA or rapamycin. fLuc, firefly luciferase; nRLU, normalized relative light units; n and c prefixes, N and C fragments. c, Design of a proteolytic-cleavage-inactivated module (logical negation). A protease-cleavage site is introduced between the CC-forming segments and split effector/reporter domain. After cleavage of the linker, split luciferase dissociates. d, Decrease in luciferase activity after cotransfection of logical negation functions with plasmids encoding specific proteases. Values in b and d are the means from four cell cultures ±s.d. and are representative of two independent experiments. Transfection plasmid mixtures are listed in Supplementary Table 1.
… 
Design of Boolean logic functions implemented by SPOC logic a–h, Experimental analysis of the indicated SPOC logic function designs in HEK293T cells with introduced genetic circuits. The design and expected response to all input combinations is schematically shown below the graphs with experimental luciferase-based results. Input signals are combinations of two orthogonal proteases, TEVp and PPVp, and the output signal is split luciferase activity. The remaining Boolean SPOC logic circuits (B, NOT B, NOT A, A nimply B, A imply B and XNOR) are shown in Supplementary Fig. 9. Transfection plasmid mixtures are listed in Supplementary Table 2. Values are the means from three (d–h) and four (a–c,e) cell cultures ±s.d. and are representative of two independent experiments. Significance was tested by one-way analysis of variance (ANOVA) with Tukey’s comparison (values of confidence intervals, degrees of freedom, F and P are indicated).
Design of Boolean logic functions implemented by SPOC logic a–h, Experimental analysis of the indicated SPOC logic function designs in HEK293T cells with introduced genetic circuits. The design and expected response to all input combinations is schematically shown below the graphs with experimental luciferase-based results. Input signals are combinations of two orthogonal proteases, TEVp and PPVp, and the output signal is split luciferase activity. The remaining Boolean SPOC logic circuits (B, NOT B, NOT A, A nimply B, A imply B and XNOR) are shown in Supplementary Fig. 9. Transfection plasmid mixtures are listed in Supplementary Table 2. Values are the means from three (d–h) and four (a–c,e) cell cultures ±s.d. and are representative of two independent experiments. Significance was tested by one-way analysis of variance (ANOVA) with Tukey’s comparison (values of confidence intervals, degrees of freedom, F and P are indicated).
… 
Multilayer design of proteolysis-based signaling pathways a, Two-layer protease-cascade function with a catalytically inactive split tobacco etch virus protease (TEVp*) domain fused to the autoinhibitory CC, showing low leakage and high fold activation (additional data in Supplementary Fig. 11). b, Double inverter consisting of a split TEVp regulated by split PPVp; PPVp is regulated by the rapamycin-induced split SbMVp. c, B nimply A logic function combining human immunodeficiency virus-1 (HIV-1p) and PPVp as input signals. The SQVSQNYPIVQNLQ recognition sequence for HIV-1 protease was used. Transfection plasmid mixtures are listed in Supplementary Tables 1 and 4. Values are the means from three (c) and four (a,b) cell cultures ±s.d. and are representative of at least two independent experiments; significance was tested by one-way ANOVA with Tukey’s comparison between the indicated ON and OFF states (confidence interval, 95%, degrees of freedom, 11; F = 72 (c)).
Multilayer design of proteolysis-based signaling pathways a, Two-layer protease-cascade function with a catalytically inactive split tobacco etch virus protease (TEVp*) domain fused to the autoinhibitory CC, showing low leakage and high fold activation (additional data in Supplementary Fig. 11). b, Double inverter consisting of a split TEVp regulated by split PPVp; PPVp is regulated by the rapamycin-induced split SbMVp. c, B nimply A logic function combining human immunodeficiency virus-1 (HIV-1p) and PPVp as input signals. The SQVSQNYPIVQNLQ recognition sequence for HIV-1 protease was used. Transfection plasmid mixtures are listed in Supplementary Tables 1 and 4. Values are the means from three (c) and four (a,b) cell cultures ±s.d. and are representative of at least two independent experiments; significance was tested by one-way ANOVA with Tukey’s comparison between the indicated ON and OFF states (confidence interval, 95%, degrees of freedom, 11; F = 72 (c)).
… 
Fast kinetics of the proteolysis-mediated signaling pathway a, Scheme of the reconstitution of the split TEVp by ABA and split PPVp by rapamycin, measured with a cycLuc reporter in HEK293 cells. A CRISPR–dCas9-based activator was used as a comparison for the kinetics of the transcriptional control of luciferase activation. b,c, Continuous online monitoring of chemically induced luciferase activity in HEK293T cells (b) and comparison with the kinetics of transcriptional activation (c). Rapa, rapamycin. d, Schematic presentation of building blocks for inducible SPOC logic functions. e, Kinetics of the chemically regulated AND function, in which both segments of the split luciferase are activated by proteolytic cleavage. A substantial response was detected less than 15 min after the addition of chemical inducers. f–h, SPOC logic functions AND (f), OR (g) and B (h), regulated by rapamycin and ABA 15 min after induction. Transfection mixtures are listed in Supplementary Tables 5 and 6. Values are the means from three (f) and four (b,c,e–g) cell cultures ±s.d. and are representative of two independent experiments. Significance was tested with one-way ANOVA with Tukey’s comparison between the indicated ON and OFF states (confidence intervals, degrees of freedom, F and P are indicated (f–h)).
Fast kinetics of the proteolysis-mediated signaling pathway a, Scheme of the reconstitution of the split TEVp by ABA and split PPVp by rapamycin, measured with a cycLuc reporter in HEK293 cells. A CRISPR–dCas9-based activator was used as a comparison for the kinetics of the transcriptional control of luciferase activation. b,c, Continuous online monitoring of chemically induced luciferase activity in HEK293T cells (b) and comparison with the kinetics of transcriptional activation (c). Rapa, rapamycin. d, Schematic presentation of building blocks for inducible SPOC logic functions. e, Kinetics of the chemically regulated AND function, in which both segments of the split luciferase are activated by proteolytic cleavage. A substantial response was detected less than 15 min after the addition of chemical inducers. f–h, SPOC logic functions AND (f), OR (g) and B (h), regulated by rapamycin and ABA 15 min after induction. Transfection mixtures are listed in Supplementary Tables 5 and 6. Values are the means from three (f) and four (b,c,e–g) cell cultures ±s.d. and are representative of two independent experiments. Significance was tested with one-way ANOVA with Tukey’s comparison between the indicated ON and OFF states (confidence intervals, degrees of freedom, F and P are indicated (f–h)).
… 
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Articles
https://doi.org/10.1038/s41589-018-0181-6
1Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia. 2Graduate School of Biomedicine, University of
Ljubljana, Ljubljana, Slovenia. 3ENFIST Centre of Excellence, Ljubljana, Slovenia. 4These authors contributed equally: Tina Fink, Jan Lonzarić.
*e-mail: roman.jerala@ki.si
Responsiveness to external and internal signals is a key fea-
ture of living cells that allows for an appropriate response to
environmental conditions, intracellular communication and
many other functions. Physicochemical signals are typically sensed
by diverse protein receptors that relay and transduce signals and
consequently trigger an appropriate cellular response. Signaling
in both prokaryotes and eukaryotes is predominantly achieved
through protein–protein interactions and their post-translational
modifications, such as phosphorylation or proteolytic cleavage and
degradation. Although many proteins in signaling pathways are
composed of related modular domains1, protein–protein interac-
tions have been optimized during evolution for the high specificity
and orthogonality of pathways operating in parallel. Several physi-
ological responses—such as secretion of insulin after increases in
glucose concentration, blood-vessel dilation or response to noxious
agents—must occur rapidly, within minutes. Because de novo bio-
synthesis of signal mediators through transcription and translation
is time consuming, fast signaling responses are often accomplished
through rapid processing of premade mediators. The design and
introduction of new signaling pathways based on protein modifica-
tion rather than transcription regulation may therefore enable ther-
apeutic or biotechnological benefits and contribute to elucidation
of the principles of signaling through naturally evolved pathways.
Signaling pathways have already been rewired and transferred
between organisms, for example, in yeast and mammalian cells2–6.
However, to introduce specific, adjustable and scalable regulation,
the information-processing pathways should preferably be designed
de novo, to minimize unwanted interactions with the cellular chas-
sis and to make the pathways highly programmable for implement-
ing designed cellular logic. To date, most designed cell circuits
have been based on transcriptional regulation, drawing on the
modular DNA-recognition and transcriptional-effector domains7–9.
Transcriptional-regulation-based cell logic is, however, inherently
slower than systems based on protein interaction and modification.
In contrast to responses based on transcriptional regulation,
responses based on protein interaction or modification occur in
cells within minutes and typically combine specific protein–protein
interactions and catalytic steps arranged in several interconnected
layers, thereby enabling multiple input signals, mediators, modi-
fiers and information-processing steps to be combined (Fig. 1a).
Several natural pathways use proteolysis, either through prote-
asome-mediated degradation of selected proteins that generate
or expose a degradation-targeting motif (for example, Iκ B in the
inflammatory signaling cascade) or through cleavage at defined
sites (for example, Notch signaling, apoptosis or the coagulation
cascade)10–12. Proteolytic regulation has already been engineered
into mammalian cells, for example, through degrons4,13 and cleavage
of transcription factors14 and their translocation triggered by pro-
teolytic cleavage15,16. In addition, in vitro systems for the detection
of proteolytic cleavage have been designed for a limited number of
logic functions17,18. However, the design of a fast, modular, scalable
protein-modification-based signaling platform for the construction
of logic functions in mammalian cells has remained a challenge,
especially in terms of achieving the cellular response at the subhour
time scale. The use of proteolysis for logic-circuit design imposes
several prerequisites, specifically (i) the availability of a sufficient
number of orthogonal proteases, each specific to its own substrate
without interfering with other components or processes within the
circuit or the cellular chassis; (ii) a mechanism to activate proteases
by selected internal or external signals; (iii) a mechanism to convert
the proteolytic processing into an output activity in a functionally
complete way, thereby allowing for the design of diverse logic func-
tions; and (iv) a mechanism to render further information process-
ing (protease cleavage) dependent on the output of the upstream
logic function, thus enabling coupling of functional layers in a mod-
ular way (scalability).
Here, we present a platform for the design of a proteolysis-based
signaling pathway, called SPOC logic. We demonstrate the design of
Design of fast proteolysis-based signaling and
logic circuits in mammalian cells
TinaFink 1,2,4, JanLonzarić1,4, ArnePraznik1,2, TjašaPlaper 1,2, EsteraMerljak 1,2, KatjaLeben 1,2,
NinaJerala1, TinaLebar1, ŽigaStrmšek1,2, FabioLapenta1,2, MojcaBenčina1,3 and RomanJerala 1,3*
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 prote-
olysis and designed coiled coils (CC) and implemented in mammalian cells. A set of split proteases with highly specific orthogo-
nal 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.
NATURE CHEMICAL BIOLOGY | VOL 15 | FEBRUARY 2019 | 115–122 | www.nature.com/naturechemicalbiology 115
The Nature trademark is a registered trademark of Springer Nature Limited.
... Recent advancements in artificial intelligence-assisted protein structure prediction and de novo protein design have fueled significant interest in engineering proteinlevel switches for ON/OFF control of protein functions in a signal-dependent manner (Figure 1c). Engineered protein-protein interactions were proven effective in regulating TEV protease activity through trigger-inducible reconstitution of split-TEV protease [34]. By directly cleaving the deactivating or destabilizing domains of target proteins, this approach enables a markedly faster response to input signals when directly compared to traditional transcription-and translationlevel gene switches that also use inducible protein-protein interactions for ON/OFF switching [34,35] ( Figure 1a,b). ...
... Engineered protein-protein interactions were proven effective in regulating TEV protease activity through trigger-inducible reconstitution of split-TEV protease [34]. By directly cleaving the deactivating or destabilizing domains of target proteins, this approach enables a markedly faster response to input signals when directly compared to traditional transcription-and translationlevel gene switches that also use inducible protein-protein interactions for ON/OFF switching [34,35] ( Figure 1a,b). The trigger-inducible protease system has been widely adopted for modulation of transcriptional activation and protein localization, offering precise and versatile multidimensional control over complex biological processes. ...
... Currently, there are two major classes of architectures how gene switches can be assembled to form higher order computational networks [43] (Figure 2). In singlelayered network topologies [8,34,44,[47][48][49], all computation tasks are completed within a same gene regulation level and using a same type of gene switches for each individual part (Figure 2a). In contrast, multi-layered biocomputers involve hierarchical topologies that comprise multiple interconnected gene switches across same or different regulation levels [13,14,17,50,51] (Figure 2b). ...
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... Synthetic biological regulations design can also be conducted at the translation level, which has a shorter time scale than the tr anscription le v el (Fink et al. 2019 ). In this section, we pr ovide an ov ervie w of differ ent orthogonal tr anslation r egulations, including ribosomes , ribos witches , and aminoac yl-tRN A synthetase (aaRS)-tRNA pairs based on non-canonical amino acids (ncAAs). ...
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Article
  • Sep 2018
Building smarter synthetic biological circuits Synthetic genetic and biological regulatory circuits can enable logic functions to form the basis of biological computing; synthetic biology can also be used to control cell behaviors (see the Perspective by Glass and Alon). Andrews et al. used mathematical models and computer algorithms to combine standardized components and build programmable genetic sequential logic circuits. Such circuits can perform regulatory functions much like the biological checkpoint circuits of living cells. Circuits composed of interacting proteins could be used to bypass gene regulation, interfacing directly with cellular pathways without genome modification. Gao et al. engineered proteases that regulate one another, respond to diverse inputs that include oncogene activation, process signals, and conditionally activate responses such as those leading to cell death. This platform should facilitate development of “smart” therapeutic circuits for future biomedical applications. Science , this issue p. eaap8987 , p. 1252 ; see also p. 1199
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Programming gene and engineered-cell therapies with synthetic biology
Article
  • Feb 2018
Toward programmed therapeutics Advances in synthetic biology are enabling the development of new gene and cell therapies. Kitada et al. review recent successes in areas such as cancer immunotherapy and stem cell therapy, point out the limitations of current approaches, and describe prospects for using synthetic biology to overcome these challenges. Broader adoption of these therapies requires precise, context-specific control over cellular behavior. Gene circuits can be built to give sophisticated control over cellular behaviors so that therapeutic functions can, for example, be programmed to activate in response to disease biomarkers. Science , this issue p. eaad1067
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Synthetic biology – Engineering cell-based biomedical devices
Article
  • Oct 2017
Synthetic biology applies rational bottom-up engineering principles to create cell-based biological systems with novel and enhanced functionality to address currently unmet clinical needs. In this review, we provide a brief overview of the state-of-the-art in cell-based therapeutic solutions, focussing on how these integrated biological devices can enhance and complement the natural functionality of cells in order to provide novel treatments. We also highlight some blueprints for synthetic biology-inspired approaches to developing cell-based cancer therapies, and briefly discuss their future clinical potential.
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Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity
Article
  • May 2017
  • J AM CHEM SOC
The coiled-coil dimer is a widespread protein structural motif and due to its designability represents an attractive building block for assembling modular nanostructures. The specificity of coiled-coil dimer pairing is mainly based on the hydro-phobic and electrostatic interactions between residues at positions a and d, and e and g of the heptad repeat, respectively. Binding affinity, on the other hand, can also be affected by surface residues that face away from the dimerization interface. Here we show how design of the local helical propensity of interacting peptides can be used to tune the stabilities of coiled-coil dimers over a wide range. By designing intramolecular charge pairs, regions of high local helical propensity can be engineered to form trigger sequences and dimer stability is adjusted without changing the peptide length or any of the directly interacting residues. This general principle is demonstrated by a change in thermal stability by more than 30 °C as a result of only two mutations outside the binding interface. The same approach was successfully used to modulate the stabilities in an orthogonal set of coiled-coils without affecting their binding preferences. The stability effects of local helical propensity and peptide charge are well described by a simple linear model, which should help improve current coiled-coil stability prediction algorithms. Our findings enable tuning the stabilities of coiled-coil-based building modules match a diverse range of applications in synthetic biology and nanomaterials.
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The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force
Article
  • May 2017
The Notch signaling pathway relies on a proteolytic cascade to release its transcriptionally active intracellular domain, on force to unfold a protective domain and permit proteolysis, on extracellular domain glycosylation to tune the forces exerted by endocytosed ligands, and on a motley crew of nuclear proteins, chromatin modifiers, ubiquitin ligases, and a few kinases to regulate activity and half-life. Herein we provide a review of recent molecular insights into how Notch signals are triggered and how cell shape affects these events, and we use the new insights to illuminate a few perplexing observations.
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Rewiring human cellular input-output using modular extracellular sensors
Article
  • Dec 2016
  • NAT CHEM BIOL
Engineered cell-based therapies comprise a promising emerging strategy for treating diverse diseases. Realizing this promise requires new tools for engineering cells to sense and respond to soluble extracellular factors, which provide information about both physiological state and the local environment. Here, we report such a biosensor engineering strategy, leveraging a self-contained receptor-signal transduction system termed modular extracellular sensor architecture (MESA). We developed MESA receptors that enable cells to sense vascular endothelial growth factor (VEGF) and, in response, secrete interleukin 2 (IL-2). By implementing these receptors in human T cells, we created a customized function not observed in nature-an immune cell that responds to a normally immunosuppressive cue (VEGF) by producing an immunostimulatory factor (IL-2). Because this platform utilizes modular, engineerable domains for ligand binding (antibodies) and output (programmable transcription factors based upon Cas9), this approach may be readily extended to novel inputs and outputs. This generalizable approach for rewiring cellular functions could enable both translational applications and fundamental biological research.
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Engineering dynamical control of cell fate switching using synthetic phospho-regulons
Article
  • Nov 2016
  • P NATL ACAD SCI USA
Significance Many long-term cellular decisions in development, synaptic plasticity, and immunity require cells to recognize input dynamics such as pulse duration or frequency. In dynamically controlled cells, incoming stimuli are often processed and filtered by a rapid-acting signaling layer, and then passed to a downstream slow-acting layer that locks in a longer-term cellular response. Directly testing how such dual-timescale networks control dynamical regulation has been challenging because most tools in synthetic biology allow rewiring of slow gene expression circuits, but not of rapid signaling circuits. In this work, we developed modular peptide tags for engineering synthetic phosphorylation circuits. We used these phospho-regulons to build synthetic dual-timescale networks in which the dynamic responsiveness of a cell fate decision can be selectively tuned.
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