Novel method for transducing protein-protein interactions

Abstract

To provide methods and means for assessing or responding to a protein-protein interaction and transducing this interaction to the expression of a gene of interest.SOLUTION: The present invention relates to a cell comprising a first nucleic acid sequence encoding a first polypeptide fused to N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain, (wherein the first variant does not comprise a transmembrane domain), a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of the histidine kinase comprising a DHp domain and a CA domain, (wherein the second variant does not comprise a transmembrane domain), and a third nucleic acid sequence encoding a response regulatory protein specifically phophorylatable by the DHp domain of the first or the second variant. The present invention further relates to uses of the cell of the invention.SELECTED DRAWING: Figure 4A

Description

[Technical field]

[0001] This application claims the benefit of priority to European Patent Application No. 18200357.4, filed October 15, 2018, which is incorporated herein by reference.

[0002] The present invention relates to methods and means for assessing or responding to protein-protein interactions and for transducing such interactions into the expression of genes of interest. [Background technology]

[0003] Molecular interactions, such as protein-protein interactions, are involved in almost all cellular processes in living cells. Characterization of protein-protein interactions is a key step to better understand and control biological systems. Transduction of protein-protein interactions into detectable signals or expression of genes of interest is important for the development of cells with new functions to be used as cell-based biosensors or cell therapy and for the discovery of new drugs.

[0004] Various intracellular pathways are regulated by the presence or absence of compounds that promote or inhibit protein-protein interactions. Identifying molecules that regulate protein-protein interactions is one of the major tools used in drug discovery and development. Furthermore, transduction of protein-protein interactions into novel responses is a major tool in cell engineering for therapeutic purposes.

[0005] An example of a protein interaction modulated by a compound is the interaction between a G protein-coupled receptor (GPCR) and a downstream pathway protein such as β-arrestin. GPCRs are membrane receptors of mammalian cells that can detect various ligands (endogenous hormones, growth factors, natural or synthetic small molecules). Following the interaction of the GPCR with its ligand, different cascades are induced within the cell that regulate the cell activity. Due to the central function of GPCRs within the cell, many drugs act as targets on GPCRs. To identify and characterize GPCR agonists and antagonists, various assays have been developed. Some of these assays transduce the interaction of the GPCR with one of its protein partners into the expression of a reporter gene.

[0006] Furthermore, protein-protein interactions have been used to design chimeric sensors that can sense various signals and transduce these signals into specific responses. This ability to direct information from a given input to a specific output can be used for many applications, such as generating cell-based biosensors to create new in vitro diagnostic tools or to generate new cell therapies with better safety and efficacy. Although there have been various developments in the field of biosensors, many of them simply generate detectable signals that require manual reading and interpretation by humans. Little progress has been made so far in biosensors that transduce signals into downstream biological activities such as gene expression. The artificial signaling systems described so far mainly utilize the cleavage action of fusion proteins to release transcriptional activators, which then regulate the expression of the gene of interest.

[0007] Two-component system (TCS) signaling cascades are initiated by ligand-induced autophosphorylation of histidine kinase (HK) receptor proteins at histidine residues, followed by phosphorylation to aspartate residues of response regulator (RR) proteins. With some exceptions, HK sensors form homodimers at the plasma membrane, and the structural basis for HK autophosphorylation is the existence of two distinct HK dimer conformations. In the unstimulated state, the catalytic ATP-binding (CA) domain undergoes dimerization and histidine-containing phosphorylation (DHp). Autophosphorylation does not occur because HK is in a conformation that is away from the histidine residues of the DHp (Figure 1a). Upon ligand binding, the CA domain and bound ATP are brought into close proximity to the histidine of DHp, allowing phosphoryl transfer. A cognate response regulator (RR) then binds to the phosphorylated DHp domain, transferring the phosphate from the histidine to one of the aspartates in the receiver domain of the RR, and the phosphorylated RR binds to its target promoters and regulates gene expression. Furthermore, when the phosphorylated RR binds to the unphosphorylated DHp domain, this DHp domain catalyzes the dephosphorylation of the aspartate residue, thus actively shutting down signaling. Thus, HK is a bifunctional enzyme with kinase and phosphatase activities, and the balance between the two determines the strength and dynamics of signaling.

[0008] Given the importance of GPCR signaling in human diseases, various assays have been developed to detect and identify molecules that interact with GPCRs. Among the various methods developed, some take advantage of the interaction of β-arrestins with ligand-activated GPCRs. Examples include the Bioluminescence Resonance Energy Transfer (BRET) assay, the TANGO assay (Invitrogen) (Figure 5), and the ChaCha system. The first system transduces protein-protein interactions into a detectable fluorescent signal. The latter two assays transduce protein-protein interactions into the expression of a gene of interest, such as a reporter gene.

[0009] The TANGO assay is realized by fusing a proteolytically cleavable artificial transcription factor (GAL4-VP16) to the intracellular domain of a GPCR and fusing TEV protease to β-arrestin. Activation of the GPCR by ligand induces recruitment of β-arrestin to the GPCR, binding the TEV protease to the vicinity of the cleavable linker of the GPCR and allowing release of GAL4-VP16. The artificial transcription factor will induce expression of a reporter gene (β-lactamase) driven by a chimeric promoter targeted by GAL4-VP16.

[0010] The ChaCha system was recently developed as a derivative of the TANGO assay. In this system, dCas9 linked to a tripartite transcription activator composed of VP64, p65 activation domain, and Rta (dCas9-VPR) (which cannot cleave DNA but can still bind to it) is fused to β-arrestin, while the intracellular domain of a GPCR is fused to TEV protease. This system also requires the expression of a guide RNA (gRNA), which allows dCas9 to be recruited to a promoter that drives the expression of the gene of interest. Interaction between the GPCR and the β-arrestin-dCas9-VRP fusion releases the dCas9-VRP. The dCas9-VRP regulates the expression of the gene of interest targeted by the co-expressed gRNA in the cell. The promoter can be an endogenous or chimeric promoter.

[0011] Different methods have been developed as well for the probing of general protein-protein interactions, for example in the cytoplasm. One of the most common is the yeast two hybrid approach. In this method, two possible interacting proteins, usually called bait and prey, are fused to split subunits of a protein with a specific detectable biological activity. Each of the split subunits alone does not show the biological activity of interest. The interaction between bait and prey allows the appropriate reconstitution of the domains fused to the bait and prey, respectively. Depending on the arrangement of the bait and prey, different reporter systems have been developed: - To probe nuclear localization, the split proteins reconstitute transcriptional activation of a reporter gene; - For protein-protein interactions occurring in the cytoplasm or membrane, reporter systems are based on yeast growth by activating Ras signaling, uracil requirement, or antibiotic resistance.

[0012] One drawback of the yeast two-hybrid approach is the fact that interactions are quantified in yeast cells, which may not faithfully recapitulate interactions in the native mammalian cellular environment. Summary of the Invention [Problem to be solved by the invention]

[0013] It is therefore an object of the present invention to provide means and methods for responding to and / or assessing protein-protein interactions. [Means for solving the problem]

[0014] Description of the invention This object is solved by a cell and a method thereof as specified in the independent claims. Advantageous embodiments are set out in the dependent claims and in the following description. [Brief description of the drawings]

[0015] [Figure 1A] Figure 1 is a schematic diagram of native and transplanted two-component signaling pathways. (A) The native pathway consists of a receptor histidine kinase (HK) protein that, in the presence of a signal, autophosphorylates at the level of a single asparagine present in the receiver domain and phosphorylates a cognate response regulator (RR). The phosphorylated RR binds to a specific response element present in the promoter controlled by the RR. [Figure 1B]Figure 1 shows a schematic of the native and transplanted two-component signaling pathways. (B) The TCS transplanted into the mammalian host is expressed from a gene with a human-optimized codon sequence. The histidine kinase transplanted into the mammalian cell is constitutively active, autophosphorylating and phosphorylating the RR. The transplanted RR is enhanced with the VP48 transactivation domain. The phosphorylated RR binds to an RE present in the artificial promoter that drives expression of the gene of interest (in this case, the fluorescent reporter Cerulean). DNB, DNA binding domain; VP48, VP48 transactivation domain; Pmin, minimal mammalian promoter. [Figure 2A-1] Figure 2 shows cis and trans autophosphorylation of HK. (A) Schematic diagram of cis-autophosphorylation (top) and trans-autophosphorylation (bottom) in mammalian cells expressing WT (wild-type) HK (first row), DHp mutant (second row), CA mutant (third row), and DHp and CA mutants (fourth row). [Figure 2A-2] Figure 2 shows cis versus trans autophosphorylation of HK. (A) Schematic diagram of cis-autophosphorylation (top) and trans-autophosphorylation (bottom) in mammalian cells expressing WT (wild-type) HK (first row), DHp mutant (second row), CA mutant (third row), and DHp and CA mutants (fourth row). [Figure 2B] Figure 2 shows cis vs. trans autophosphorylation of HK. (B) Quantitative data from mammalian cells expressing reporter genes alone or with wild-type HK, DHp mutants (EnvZ H243V, NarX H399Q, and DcuS H350L), CA mutants (EnvZ N347A, NarX N509A, and DcuS N445A), or combinations of DHp and CA mutants. Bar graphs show cerulean levels normalized to expression of transfection controls in norm.u. as the mean ± SD of independent biological triplicates. [Figure 3A-1]Figure 3 shows the activity of truncated HKs. (A) Schematic diagram of phosphotransfers occurring between the EnvZ / OmpR (top row) and NarX / NarL (bottom row) elements in mammalian cells expressing WT HK (first row), truncated mutants of the sensor domain (second row), or truncated mutants of the sensor and transduction domains (third row). [Figure 3A-2] Figure 3 shows the activity of truncated HKs. (A) Schematic diagram of phosphotransfers occurring between the EnvZ / OmpR (top row) and NarX / NarL (bottom row) elements in mammalian cells expressing WT HK (first row), truncated mutants of the sensor domain (second row), or truncated mutants of the sensor and transduction domains (third row). [Figure 3B] Figure 3 shows activity of truncated HK. (B) Quantitative data from mammalian cells expressing the reporter gene alone or in combination with wild-type HK, sensor domain truncated mutants (EnvZ180-450 and NarX176-598), or sensor and transduction domain truncated mutants (EnvZ223-450 and NarX379-598). Bar graphs show cerulean levels normalized to expression of transfection controls in units of reference as the mean ± SD of independent biological triplicates. [Figure 4A] Figure 4 shows the design of protein-protein interaction assay. (A) Schematic diagram of PPI assay monitoring the interaction between two proteins P1 and P2. The interaction between P1 and P2, spontaneously or induced by a compound, allows dimerization of a short cytoplasmic domain of HK of the transphosphorylation family mutated at the level of the CA domain fused to P1 with a short cytoplasmic domain of HK of the transphosphorylation family mutated at the level of the DHp domain fused to P2. Dimerization causes phosphorylation of RR, which will bind to RE and induce expression of a reporter gene. [Figure 4B] Figure 4 shows the design of the protein-protein interaction assay. (B) Quantitative data from mammalian cells expressing HK mutants fused to the SZ1 or SZ2 domains. [Figure 4C]Figure 4 shows the design of the protein-protein interaction assay. (C) Quantitative data from mammalian cells expressing HK mutants fused to FKBP or FRB domains in the absence (white bars) or presence (black bars) of A / C heterodimers that induce dimerization of the FKBP and FRB domains. Bar graphs show cerulean levels normalized to expression of transfection control in units of reference as the mean ± SD of independent biological triplicates. [Figure 5A] Figure 5 shows the design of a protein-protein interaction assay (PPI) for GPCR. (A) A schematic diagram of the TANGO assay for monitoring the interaction between GPCR and β-arrestin is shown. Agonist-induced interaction between GPCR and β-arrestin allows the β-arrestin-TEV protease fusion to localize at the level of GPCR-tTA and trigger the release of the transcription factor tTA. [Figure 5B] Figure 5 shows the design of protein-protein interaction assay (PPI) of GPCR. (B) Schematic diagram of PPI assay for monitoring interaction between GPCR and β-arrestin. Agonist-induced interaction between GPCR and β-arrestin allows dimerization of a short cytoplasmic domain of HK of transphosphorylation family mutated at the level of CA domain fused to GPCR and a short cytoplasmic domain of HK of transphosphorylation family mutated at the level of DHp domain fused to β-arrestin. Dimerization causes phosphorylation of RR, which will bind to RE and induce expression of reporter gene. [Figure 5C-D] Figure 5 shows the design of the protein-protein interaction assay (PPI) for GPCR. (C) Quantitative data for mammalian cells expressing TANGO. (D) Quantitative data for mammalian cells expressing HK mutants fused to GPCR or β-arrestin in the absence (white bars) or presence (black bars) of procaterol. Bar graphs show cerulean levels normalized to expression of transfection control in units of reference as the mean ± SD of independent biological triplicates. [Figure 6] Figure 6 shows restoration of two-component signaling by forced dimerization of protein moieties fused at the C- or N-terminus of NarX. Signaling levels are shown in mammalian cells expressing the response regulator NarL and the NarL-regulated mCerulean fluorescent protein reporter alone or in various combinations of SynZip1 and SynZip2 fused at the C- or N-terminus of NarX, as indicated. Bar graphs show Cerulean levels normalized to expression of transfection controls with reference units being the mean ± SD of independent biological triplicates. [Figure 7] FIG. 7 shows a comparison of the CMV and EF1α promoters. (a) iRFP fluorescence of HEK cells transfected with plasmids expressing iRFP from the CMV promoter (white bars) or the EF1α promoter (black bars). Reporter expression in DMEM without any ligand, or in the presence of 1 μM procaterol or 2 μM epinephrine, as indicated. Bar graphs show iRFP levels normalized to the frequency of transfection marker citrine-positive cells (rel.u.) as the mean ± SD of independent biological triplicates. (b) Activity of NarX / NarL expressed from the CMV or EF1α promoters. All transfections contain a NarL-controlled mCerulean fluorescent protein reporter and plasmids expressing NarL and NarX from the CMV, EF1α or EF1α-V1 promoters (as indicated). Bar graphs show Cerulean levels normalized to expression of the transfection control in standard units as the mean ± SD of independent biological triplicates. [Figure 8]Figure 8 shows restoration of two-component signaling via forced dimerization fused to wild-type NarX or various NarX mutants. Signaling levels are shown in mammalian cells expressing the response regulator NarL and the NarL-regulated mCerulean fluorescent protein reporter alone or in different combinations of SynZip1 and SynZip2 fused to wild-type NarX or various NarX mutants, as indicated. Bar graphs show Cerulean levels normalized to expression of transfection controls with reference units being the mean ± SD of independent biological triplicates. [Figure 9] Figure 9 shows restoration of two-component signaling via forced dimerization. Signaling levels in mammalian cells expressing the response regulator NarL and the NarL-regulated mCerulean fluorescent protein reporter alone or with one different variant of the NarX mutant fused to SynZip1 or SynZip2, as indicated in the table. Bar graphs show Cerulean levels normalized to expression of transfection control with reference units as the mean ± SD of independent biological triplicates. [Figure 10A] Figure 10 shows the transduction of cytoplasmic ligand concentration on gene expression. (a) Signaling levels in mammalian cells expressing the response regulator NarL and the NarL-regulated mCerulean fluorescent protein reporter alone or with one of the different variants of NarX mutants fused to FKBP and FRB as indicated. For each pair of bars, the left bar (white) shows the expression of the reporter without A / C ligand and the right bar (black) shows the expression of the reporter with ligand (100 nM). Bars display the levels of Cerulean normalized to the expression of the transfection control in base units. [Figure 10B]Figure 10 shows the transduction of the concentration of ligand in the cytoplasm relative to gene expression. (b) Representative microscopy images of the same transfections as those shown in Figure 3b, including the transfection control channel (red). The top and bottom images of all panels show the expression of the mCherry transfection reporter (red pseudocolor) and the output of the pathway-inducible mCerulean protein (cyan pseudocolor) in the same transfections with and without the ligand, respectively. Bar graphs show Cerulean levels normalized to the expression of the transfection control in reference units taken as the mean ± SD of independent biological triplicates. [Figure 11A] Figure 11 shows rewiring of GPCR activity upon reporter gene expression: (a) Signaling levels in mammalian cells expressing the response regulator NarL and the NarL-regulated mCerulean fluorescent protein reporter, as well as various combinations of the indicated protein domains and their fusions. [Figure 11B] Figure 11 shows the rewiring of GPCR activity to reporter gene expression. (b) Signaling levels in mammalian cells expressing components of the TANGO assay from the CMV or EF1α-V1 promoters. For each pair of bars in panels a and b, the left bar (white) represents reporter expression without procaterol and the right bar (black) represents reporter expression with procaterol (2 μM). Bar graphs show cerulean levels normalized to expression of the transfection control in units of reference as the mean ± SD of independent biological triplicates. [Figure 11C] Figure 11 shows the rewiring of GPCR activity to reporter gene expression. (c) Representative microscopy images of the same selected transfections for which images are shown in Figure 4, also showing expression of the mCherry transfection control. The top and bottom images in all panels show expression of the mCherry transfection reporter (red pseudocolor) and output of the pathway-inducible mCerulean protein (cyan pseudocolor) in the same transfections with or without ligand, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] A first aspect of the invention relates to a recombinant cell, which facilitates the analysis of the interaction of two pairs of polypeptides or proteins with each other. These interaction partners are referred to below as "first polypeptide" and "second polypeptide". Each of these polypeptides is encoded by a nucleic acid sequence, and each of these polypeptides is part of a fusion protein comprising the polypeptide portion that is the subject of the analysis of its interaction with the other polypeptide, and a fragment of a histidine kinase mutant that retains DHp and CA activity.

[0017] The cells according to the invention include: - a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase (EC 2.7.13.3), which comprises a DHp (dimerization and histidine-containing phosphotransfer) domain and a CA (catalytic ATP-binding) domain. The cell further comprises: - a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of a histidine kinase comprising the DHp domain and the CA domain; and - a third nucleic acid sequence encoding a response regulator protein that is specifically phosphorylatable by the DHp domain of the mutant form of the first or said second histidine kinase.

[0018] In other words, this aspect of the invention relates to a cell comprising: a first nucleic acid sequence, wherein the first nucleic acid sequence is o encoding a first polypeptide fused in an N-to-C orientation to the N-terminus of a first variant of a histidine kinase, the interaction of which with a second polypeptide is to be analyzed, wherein the histidine kinase comprises a DHp domain and a CA domain, and both the DHp domain and the CA domain are retained in the first variant; - a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising a DHp domain and a CA domain, wherein both the DHp domain and the CA domain are retained in the second variant; and - a third nucleic acid sequence encoding a response regulator protein that is specifically phosphorylatable by said DHp domain of said first or said second variant.

[0019] In certain embodiments, the first and second polypeptides do not comprise any portion of the histidine kinases described above, in particular they do not comprise the transmembrane domain, the sensor domain or the transduction domain.

[0020] In certain embodiments of the cells of the invention, the first mutant and / or the second mutant does not comprise the transmembrane domain of a histidine kinase.

[0021] In certain embodiments of the cells of the invention, - said first variant does not comprise a functional transduction domain and / or a functional sensor domain of a histidine kinase, and / or - said second variant does not contain the functional transducer and / or sensor domain of a histidine kinase.

[0022] In certain embodiments, the naturally occurring sensor and transduction domains of histidine kinase are replaced by two proteins of interest and their interaction needs to be evaluated. If there is a specific interaction between these proteins, the binding between them promotes the dimerization of the truncated mutant form of histidine kinase, thereby achieving spatial proximity of the CA domain and the DHp domain with ATP. This leads to phosphorylation of the DHp domain, which can then phosphorylate its cognate ligand, the response regulator domain, particularly the receiver domain of the response regulator protein.

[0023] Alternatively, two proteins of interest can form an artificial signal transduction pathway, where binding of the two proteins of interest is triggered by a stimulus, such as a ligand, that is specifically recognizable by one or both of the two proteins of interest. Recognition of the ligand can lead to a desired response mediated by the activity of a response control protein. Such a response can be the expression of a microRNA that affects a cellular process, or a protein, such as a cytokine or an antibody. In certain embodiments, such cells can be used for medical applications, where a beneficial or therapeutic response can be specifically triggered by a disease-related compound, such as a disease-related antigen.

[0024] Alternatively, the effect of a compound on a known interacting protein can be assessed using the cells of the invention, and the effect of the compound can be determined by the activity of the response regulator protein.

[0025] In particular, the response control protein comprises an effector function, which can be determined to assess the interaction between a first polypeptide and a second polypeptide or can be used to elicit a desired response in response to the above-mentioned stimuli. Non-limiting examples of such effector functions include binding to DNA, RNA, or enzymes, such as enzymes that catalyze the formation of cAMP.

[0026] In certain embodiments, the effector function comprises specific binding to a promoter sequence and inducing expression of a gene of interest.

[0027] In a particular embodiment of the cell of the invention, the response control protein comprises a receiver domain fused to an effector domain, the receiver domain being capable of being phosphorylated by the DHp domain of a first or second histidine kinase mutant, and the effector domain being capable of being regulated, in particular activated or inhibited, by the phosphorylated receiver domain. In particular, the activity of the effector domain changes depending on the phosphorylation state of the receiver domain, and thus the activity of the effector domain can be increased, or switched on, or decreased or inhibited by phosphorylation of the receiver domain.

[0028] In certain embodiments of the cells of the invention, - the effector domain is a transcription activation domain, and - the cell comprises a fourth nucleic acid sequence comprising a gene of interest under the control of an inducible promoter recognizable by a transcription activation domain; Activation of the transcription activation domain induces expression of the gene of interest.

[0029] In particular embodiments of the cell of the invention, the gene of interest encodes a protein of interest, in particular a fluorescent or luminescent protein, or an RNA of interest.

[0030] Such a protein of interest may be a fluorescent or luminescent protein, whereby the success of the interaction between the first and second polypeptides may be determined or observed via the fluorescence or luminescence of the protein of interest.

[0031] Alternatively, the protein of interest or RNA of interest may be or may cause a desired response in response to the above-mentioned stimuli, for example a desired therapeutic response (production of cytokines, antibodies, reactive oxygen species, etc.).

[0032] In certain embodiments of the cells of the invention, - the first variant and the second variant are variants of EnvZ kinase (UniProt number P0AEJ4) and the response regulator protein comprises or is the OmpR response regulator (RR) protein (Uniprot number P0AA16), or - the first variant and the second variant are variants of NarX kinase (Uniprot number P0AFA2), the receiver domain comprises or is the NarL response regulator (RR) protein (Uniprot number P0AF28) and the effector domain comprises or is the VP16 transcription activation domain (Vp48, SEQ ID NO: 7).

[0033] As mentioned above, the effector domain can be a part of NarL that is fused to VP16.

[0034] In a particular embodiment of the cell of the invention, the transcription activation domain is, consists of or comprises the amino acid sequence characterized by SEQ ID NO:7.

[0035] In certain embodiments of the cells of the invention, the first mutant or the second mutant is EnvZ 180~450 (SEQ ID NO: 8), EnvZ 223~450 (SEQ ID NO: 9), NarX 176~598 (SEQ ID NO: 10) and NarX 379~598 (SEQ ID NO: 11), or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 8 to 11, or comprising the same.

[0036] In certain embodiments of the cell of the invention, the inducible promoter is selected from the OmpR promoter (SEQ ID NO: 1) and the NarL-RE promoter (SEQ ID NO: 2).

[0037] In certain embodiments of the cells of the invention, the first nucleic acid sequence and / or the second nucleic acid sequence and / or the third nucleic acid sequence are optimized for the codon usage of the cell.

[0038] In certain embodiments of the cell of the invention, the first nucleic acid sequence and / or the second nucleic acid sequence and / or the third nucleic acid sequence are under the transcriptional control of a constitutive promoter.

[0039] In certain embodiments of the cell of the invention, the constitutive promoter is selected from CMV (SEQ ID NO: 3), EF1α (SEQ ID NO: 4), and EF1α-V1 (SEQ ID NO: 5).

[0040] In certain embodiments of the cells of the invention, the first variant and the second variant are identical.

[0041] In a particular embodiment of the cell of the invention, the histidine kinase belongs to the transphosphorylation family.

[0042] In certain embodiments of the cells of the invention, - the first variant comprises a DHp domain that does not contain a histidine residue accessible to the CA domain of the first variant or the second variant of the histidine kinase, and / or - The second variant contains a CA domain that is unable to bind ATP.

[0043] In certain embodiments of the cells of the invention, - The first mutation is the mutant NarX 379~598 (H399Q) (SEQ ID NO: 12), or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to (SEQ ID NO: 12), and / or - The second mutant is mutant NarX 379~598 (N509A) (SEQ ID NO:13), or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to (SEQ ID NO:13), or comprising the same.

[0044] In certain embodiments of the cells of the invention, specific binding of the first and second polypeptides is inducible by a ligand that can be specifically recognized by the first and / or second polypeptide.

[0045] In certain embodiments of the cells of the invention, the first polypeptide is or comprises a receptor and the second polypeptide is or comprises a binding partner of the receptor, and binding between the receptor and the binding partner is inducible by a ligand recognized by the receptor.

[0046] In a particular embodiment of the cell of the invention, the receptor is a transmembrane receptor and the binding partner is a cytoplasmic protein, in which case the ligand recognizable by the receptor and the cytoplasmic protein that is the binding partner are separated, in particular, by a membrane.

[0047] In certain embodiments of the cells of the invention, - the first polypeptide consists of or comprises a G protein-coupled receptor and the second polypeptide consists of or comprises a cytoplasmic ligand of the G protein-coupled receptor, in particular a β-arrestin, or - said first polypeptide consists of or comprises a T cell receptor or one of its components and said second polypeptide is or comprises a cytoplasmic ligand of the T cell receptor, or a component thereof, in particular ZAP-70 (UniProt number P43403).

[0048] In certain embodiments of the cells of the invention, the cells are mammalian cells, in particular human cells.

[0049] Another aspect of the present invention relates to a method for assessing protein-protein interactions, the method comprising: - providing a cell according to the invention, said cell comprising: a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of histidine kinase (EC 2.7.13.3) comprising a DHp domain and a CA domain; a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second mutant form of histidine kinase comprising the DHp domain and the CA domain; and a third nucleic acid sequence encoding a response regulator protein that is specifically phosphorylatable by the first or second variant DHp domain; and - determining the activity of a response regulator (RR) protein; Here, when the first and second polypeptides specifically bind, the CA domain of the first or second mutant phosphorylates the DHp domain of the first or second mutant, and the first and second mutant dimerize such that the activity of the response control protein is regulated, in particular activated or inhibited, by phosphorylation by the DHp domain of the first or second mutant.

[0050] A further aspect of the present invention relates to a method for assessing the effect of a compound on a protein-protein interaction, the method comprising the steps of: - providing a cell according to the invention, said cell comprising: a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain; a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said histidine kinase comprising the DHp domain and the CA domain; and a third nucleic acid sequence encoding a response regulator protein that can be specifically phosphorylated by the DHp domain of the first or second variant, and - contacting the cell with a compound; and - determining the activity of said response regulator protein; wherein, upon specific binding of the first and second polypeptides, the CA domain of the first variant or the second variant phosphorylates the DHp domain of the first variant or the second variant, and the first variant and the second variant dimerize such that the activity of a response regulator protein is modulated, in particular activated or inhibited, by phosphorylation by the DHp domain of said first variant or the second variant; and The effect of the compound on the specific binding between the first and second polypeptide is determined by the activity of the response regulator protein.

[0051] Advantageously, the above method can be used as a screening assay to evaluate the effect of any compound on any protein-protein interaction of interest.

[0052] Yet another aspect of the present invention provides a method for eliciting a desired response in response to a stimulus. The method according to this aspect of the present invention comprises the steps of: - providing a cell according to the invention, said cell comprising: a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a first variant of a histidine kinase comprising a DHp domain and a CA domain; a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second mutant form of histidine kinase comprising the DHp domain and the CA domain; wherein the specific binding between the first polypeptide and the second polypeptide is induced by a stimulus; and a third nucleic acid sequence encoding a response regulator protein that can be specifically phosphorylated by the first or second variant DHp domain; wherein upon specific binding of the first and second polypeptides, the CA domain of the first variant or the second variant phosphorylates the DHp domain of the first variant or the second variant, and the first variant and the second variant dimerize such that the activity of the response regulator protein is regulated, in particular activated or inhibited, by phosphorylation by the DHp domain of the first variant or the second variant; and - exposing the cell to a stimulus, where the desired response is mediated by or is the activity of a response regulator protein.

[0053] Preferably, the first or second polypeptide is a receptor that recognizes a stimulus, such as a T cell receptor that recognizes a disease-associated antigen, or a G-coupled receptor, and the other polypeptide may be a ligand of this receptor, such as β-arrestin or ZAP-70, respectively.

[0054] The term "specific binding between a first polypeptide and a second polypeptide" particularly refers to a polypeptide having a specific binding site. -5 M, 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 M means binding with a Kd of less than M.

[0055] In certain embodiments of the methods of the invention, the response control protein comprises a receiver domain fused to an effector domain, the receiver domain being phosphorylatable by a first or second mutant DHp domain, and the effector domain being activateable by the phosphorylated receiver domain.

[0056] In certain embodiments of the method of the present invention, - the response control protein comprises a receiver domain fused to an effector domain, the receiver domain being phosphorylatable by said first or second mutant DHp domain, and the effector domain being activateable by the phosphorylated receiver domain; - the effector domain is a transcription activation domain, - the cell further comprises a fourth nucleic acid sequence encoding a gene of interest under the control of an inducible promoter recognizable by said transcription activation domain; - Activation of the transcription activation domain induces expression of the gene of interest.

[0057] In certain embodiments of the methods of the present invention, the presence of the expression product of the gene of interest is determined as the activity of a response regulator protein.

[0058] In certain embodiments of the methods of the invention, the expression product of the gene of interest is of optical quality and includes a light-emitting moiety, as is the case for example with green fluorescent protein (GFP).

[0059] In a particular embodiment of the method of the present invention, the expression product of the gene of interest is cerulean.

[0060] In certain embodiments of the method of the present invention, the expression product of the gene of interest is a desired response or mediates a desired response. For example, the expression product can be a cytokine intended to elicit an immune response by a cell. The expression product can also be a component of a signal cascade that elicits a desired response further downstream in the signal cascade. The expression product can also be an RNA that can elicit a desired response, for example, a microRNA or guide RNA that itself controls an endogenous gene.

[0061] Furthermore, the present invention provides a vector that is particularly suitable for transfecting or transducing mammalian cells, in particular human cells, said vector comprising: - a first nucleic acid sequence as contained in a cell of the invention, - a second nucleic acid sequence as contained in the cell of the invention, - a third nucleic acid sequence as contained in the cell of the invention, and Optionally, a fourth nucleic acid sequence as comprised in the cell of the invention.

[0062] According to a further aspect of the invention there is provided a fusion protein comprising a polypeptide fused to a mutant of histidine kinase (EC 2.7.13.3) comprising a DHp (dimerization and histidine-containing phosphotransfer) domain and a CA (catalytic ATP-binding) domain.

[0063] In particular, the polypeptide does not contain any part of the above-mentioned histidine kinases, in particular the transmembrane domain, the sensor domain, the transduction domain.

[0064] In certain embodiments of the fusion protein of the invention, the mutant does not include the transmembrane domain of a histidine kinase.

[0065] In a particular embodiment of the fusion protein of the invention, the mutant does not contain a functional transduction domain and / or a functional sensor domain of a histidine kinase.

[0066] In a particular embodiment of the fusion protein of the invention, the mutant is a mutant of EnvZ kinase (UniProt number P0AEJ4) or a mutant of NarX kinase (UniProt number P0AFA2).

[0067] In certain embodiments of the fusion proteins of the invention, the mutant form is EnvZ 180~450 (SEQ ID NO: 8), EnvZ 223~450 (SEQ ID NO: 9), NarX 176~598 (SEQ ID NO: 10) and NarX 379~598 (SEQ ID NO: 11), or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 8 to 11.

[0068] In a particular embodiment of the fusion protein of the invention, the histidine kinase belongs to the transphosphorylation family.

[0069] In certain embodiments of the fusion proteins of the invention, the mutant comprises a DHp domain that does not contain a histidine residue that is accessible by the mutant CA domain or another mutant form of a histidine kinase, or comprises a CA domain that is unable to bind ATP.

[0070] In certain embodiments of the fusion protein of the invention, the mutant is a mutant NarX. 379~598 (H399Q) (SEQ ID NO: 12), mutant NarX 379~598 (N509A) (SEQ ID NO: 13), or a functionally equivalent polypeptide having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12 or 13.

[0071] In a particular embodiment of the fusion protein of the invention, the polypeptide consists of or comprises a G protein-coupled receptor or a cytoplasmic ligand of a G protein-coupled receptor, in particular a β-arrestin.

[0072] In a particular embodiment of the fusion protein of the invention, the polypeptide comprises a T cell receptor or one of its components, or a cytoplasmic ligand of the T cell receptor or a component thereof, in particular ZAP-70 (UniProt No. P43403).

[0073] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived, which are intended to illustrate the invention and are not intended to limit the scope of the invention.

[0074] Detailed Description of Certain Embodiments of the Invention The present invention provides a novel approach to transduce protein-protein interactions into gene expression in mammalian cells using components derived from a two-component system present in bacteria. The present invention can be used to develop new screening assays for protein-protein interactions in general, and for the modulation of GPCR signaling in particular. The system described herein is characterized by a superior dynamic range compared to previous methods (e.g., ThermoFischer's TANGO system) and has great potential for high-throughput multiplexing due to an almost unlimited supply of building blocks.

[0075] The present invention can provide the basis for cell-based biosensors and synthetic signal transduction modules in artificial therapeutic cells with properties such as low background levels, high dynamic range, and reversibility. Because this approach is multiplexable, complex logic-based circuits can be created, which can provide novel functions to engineered cells.

[0076] In particular, the present invention includes three distinct features: - HK (histidine kinase) component (split into two different variants) - RR component - A gene construct containing a chimeric promoter allowing the expression of the gene of interest.

[0077] The HK domains fused to the two interacting proteins are mutated to increase the dynamic range. Wild-type domains can also be used, but so far this results in a reduced dynamic range. Nevertheless, the use of wild-type domains offers some advantages when the system is used to detect homodimerization. Furthermore, in the case of homodimerization of the protein of interest, HKs belonging to the cis family can be used. The advantage of this approach is that it reduces the number of gene constructs. Nevertheless, in all cases, the key feature of the invention is the fusion of the protein of interest to a domain of HK with a reduced size (which does not dimerize by itself unless forced to do so with the aid of a fused component, and therefore does not transduce transcriptional activity by itself).

[0078] The RR (response regulator protein) used in this experiment is fused to VP48, a transcription activation domain that functions in mammalian cells. Other transcription activation domains can also be fused to the RR, such as the p65 (RelA) domain or Rta. In this example, we used an RR that directly binds to DNA, but other types of RRs can be used depending on the desired readout. For example, some RRs can bind to RNA, and others are enzymes that catalyze the production of compounds that feed into the secondary signal transduction chain, such as cyclic di-GMP.

[0079] The gene construct expressing the gene of interest (GOI) contains two elements. The first part is a chimeric promoter. We used a minimal promoter linked to an upstream sequence with several binding sites for RRs. The distance between the minimal promoter and the number of REs can be adjusted to adjust up or down for the expression of the GOI.

[0080] The GOI used in the experiments is the fluorescent reporter Cerulean, but this can be replaced by genes encoding other proteins or RNAs, for example the GOI could be a miRNA or guide RNA that can itself regulate an endogenous gene.

[0081] The present invention differs from the aforementioned systems TANGO and ChaCha in that they release transcription factors previously fused to GPCR or β-arrestin, respectively, which accumulate over time. In contrast, in the present invention, all elements are still functional after one action of protein-protein interaction, and therefore they show multiple turnover. The gene of interest regulated by both TANGO and the present system is under the control of a chimeric promoter. In the ChaCha system, endogenous genes can also be regulated using appropriately designed gRNAs. Another difference between the present invention and the TANGO assay is that the present invention requires an additional component, RR, in addition to the chimeric GPCR fusion and β-arrestin fusion.

[0082] The size of the HK domain fused to GPCR and β-arrestin is only 223 amino acid residues (aa). For TANGO, the size of the fused protein is 240aa and 341aa, respectively. For ChaCha, in addition to the expression requirements of gRNA, they are 240aa and 1900aa, respectively. Due to this smaller size, the system is easier to construct, which reduces the burden on the cell.

[0083] The system of the present invention contains two signal amplification steps. The first step, unique to the present invention, results in the reconstitution of HK, which phosphorylates multiple copies of the RR. The second level of amplification, also present in the TANGO and ChaCha systems, is that of the catalytic nature of gene induction by the RR. The two levels of amplification of the present invention result in a 10-fold improvement in the dynamic range compared to the TANGO system.

[0084] The systems of the present invention do not desensitize over time, and the same protein can be reactivated after several cycles in the presence / absence of ligand. In contrast, the elements of the TANGO and ChaCha systems can only be used once, and therefore their system activity depends on protein degradation and de novo protein synthesis, which is an inherently slow process. This property allows the systems of the present invention to switch from the on state to the off state (and vice versa) more quickly.

[0085] The present invention contains one extra amplification level compared to other approaches, so that the present invention can detect low-abundance proteins and weak protein-protein interactions. The advantage of this feature is that the expression of the components required for the assay of the present invention can be adjusted from low to high in cells. In this way, the expression level of the components of the system can be set at a level that allows a suitable ligand-induced dynamic range. Therefore, the present approach reduces ligand-independent signaling that can occur due to overexpression of proteins in previous approaches.

[0086] In particular, the assay of the present invention exhibits a higher dynamic range than the TANGO assay, a parameter that will facilitate automated analysis of the results generated by the present invention as compared to TANGO.

[0087] Another advantage of the present system is that it can be multiplexed by using different HK-RR pairs simultaneously. The number of natural two-component systems is very large, so the multiplexing possibilities are very large. Each chimeric pair is independent and induces a different output. Therefore, in the same experiment, multiple protein-protein interactions and the effects of compounds on multiple GPCRs can be tested at once. In other approaches, multiplexing is more difficult due to the limited number of well-characterized TEV proteases.

[0088] In addition, the reversibility of the system of the present invention is achieved by spontaneous dephosphorylation of the RR. In the absence of interaction between the protein pair, the kinase is no longer activated and does not phosphorylate the RR. The unphosphorylated RR cannot induce the expression of the GOI. In the case of TANGO and ChaCha, the reversibility of the system is difficult because the transcription activator released after the interaction between the GPCR and β-arrestin needs to be degraded over time.

[0089] Finally, the assay of the present invention is highly modular: as in the GPCR induction assay, the same pair of complementary HK fragments can be utilized to detect cytoplasmic and membrane-localized protein-protein interactions. Other assays require significant adjustments to probe different types of protein-protein interactions, and assays such as TANGO are specific to the GPCR pathway and have not been used to probe general protein-protein interactions.

[0090] The present invention can have multiple applications. 1) It can be used to develop new screening assays to identify compounds that interact with GPCRs. The resulting assay is expected to have better specificity and a higher dynamic range than existing assays. It will also be easier to multiplex. Commercialization of the assay can be done by selling stable cell lines containing GPCRs fused to HK, as is currently done by DiscoverX and ThermoFischer (PathHunter and TANGO).

[0091] 2) It can be used to screen compounds that modulate protein-protein interactions, and therefore can be used in drug discovery. In contrast to yeast two-hybrid, the screening assay can be performed in mammalian cells, which is a more important system. Moreover, the present invention allows the same assay to be used for proteins that are localized in different cellular compartments (nucleus, cytoplasm, or membrane).

[0092] 3) Existing therapeutic cell-based drugs often use signaling pathways that involve protein-protein interactions on the cytoplasmic side of the membrane. This includes CAR-T cells, where binding of antigen to an extracellular antibody fragment recruits protein interaction partners; these interactions can then be rewired to produce a therapeutic effect using the inventors' approach. EXAMPLES

[0093] The cytoplasmic domain of HK is known to be capable of dimerization and autophosphorylation.

[0094] The present invention is based on the question whether partial cytoplasmic domains have a reduced ability to send intrinsic signals. To this end, we performed mutagenesis of stepwise truncations of HK to identify domains that cannot dimerize by themselves (FIG. 1b). The first set of truncation mutants consisted of the entire cytoplasmic domains of EnvZ and NarX (EnvZ and NarX, respectively). 180~450 and NarX 176~598 ), and the second set featured a deeper truncation with approximately 20 amino acids (aa) N-terminal upstream of the histidine (EnvZ, respectively). 223~450 and NarX 379~598 ). We found that truncated mutants of EnvZ coexpressed with the cognate response regulator OmpR in HEK293 cells were able to signal constitutively and induce expression of the OmpR-regulated reporter m-Cerulean at levels comparable to wild-type EnvZ (Fig. 1c). This result is consistent with a small histidine-containing EnvZ domain ("domain A", aa 223-289) being responsible for homodimerization. On the other hand, truncated mutants of NarX showed a size-dependent reduction in basal signaling in the presence of the cognate RR NarL and a NarL-inducible reporter, with the shortest mutant, NarX, showing a size-dependent reduction in basal signaling in the presence of the cognate RR NarL and a NarL-inducible reporter. 379~598Background levels were reached at 100 ng / mL (Fig. 1d). There are several possible explanations for this result, including: (1) decreased protein stability, (2) decreased kinase activity and / or increased phosphatase activity of the truncated mutants, and (3) the inability of the mutants to dimerize with themselves. Of these explanations, only the last one supports the eventual establishment of synthetic signaling. To see if forced dimerization would restore signaling, we attempted to fuse the truncated NarX domain to a pair of proteins that form strong heterodimers in mammalian cells. The reasoning was that these same mutants bound to proteins with strong affinity for each other would dimerize and transduce signals downstream if only dimerization was impaired.

[0095] SynZip1 / SynZip2 We used the known interacting pair, SynZip1 and SynZip2, and coupled them to a short NarX domain, NarX. 379~598 The short NarX domains were fused to the N- and C-termini of HK. We observed that co-expression of SynZip1 and SynZip2 fusions to the N-terminus of the short NarX domains resulted in increased reporter expression, albeit much lower than the signaling of full-length NarX (Figure 6). On the other hand, co-expression of SynZip fusions to the C-terminus of NarX did not result in an increase in reporter expression induced by dimerization. Overall, the results suggested the possibility of forced dimerization as a signaling mechanism via fusion to the N-terminus of the short cytoplasmic NarX domain, which is consistent with the location of the sensor domain relative to the cytoplasmic domain of full-length HK. However, the quantitative behavior was poor. In parallel with this study, we optimized the promoter driving the constructs to ensure that they were not responsive to external stimuli and that expression levels were balanced (Figure 7).

[0096] For synthetic signaling systems, it is important to avoid non-specific changes in the signaling readout. In the present system, the components are expressed by constitutive promoters. However, these promoters are in fact controlled by highly expressed transcription factors such as Sp1, and there is always a risk that these factors are directly affected by external stimuli through unrelated endogenous pathways. This would result in changes in constitutive expression and apparent changes in the signaling readout that are unrelated to the studied action and are artificial. To eliminate these confounding factors, we investigated several constitutive promoters for their robustness under various stimulation conditions and compared the expression of iRFP by the CMV promoter and the EF1α promoter in the presence of different compounds (epinephrine and procaterol) that are often used to induce cell signaling. In medium without any compound, the activity of both promoters is similar. However, in the presence of epinephrine and procaterol, the activity of the CMV promoter is induced two-fold, while the activity of the EF1α promoter is not affected (Figure 7a).

[0097] To determine whether too high expression of the truncated HK cytoplasmic domain increases background levels and is responsible for nonspecific interactions, we quantified the activity of the NarX / NarL system expressed from the CMV, EF1α, and EF1α-V1 promoters. The EF1α-V1 promoter is about 5-fold weaker than EF1α. The results show that comparable expression of the reporter gene is obtained with NarX expressed from either tested promoter (Fig. 7b, compare lanes 2 and 3, and lanes 5 and 6). However, a decrease in reporter gene expression was observed when NarL was expressed from a weaker promoter (Supplementary Fig. 2b). In light of these results, we used EF1α-V1 to drive the NarX-derived construct and EF1α to drive NarL expression.

[0098] We further investigated the possibility of optimizing the action. It is known that HKs are divided into two families with respect to the autophosphorylation mechanism. In the "cis" family, the phosphoryl group is transferred from an ATP molecule bound to the CA domain of the same monomer to a histidine. In the "trans" family, the phosphoryl group is transferred from ATP bound to one monomer to a phosphorylatable histidine of the other monomer (Figure 2a). For all HKs, the phosphoryl group transfer can be stopped by mutating either the ATP binding site or the histidine. The heterodimer formed between the ATP binding site mutant and the histidine mutant cannot signal in the case of the "cis" family HKs, but in the case of the "trans" family HKs, it is actually still able to signal via a unidirectional phosphoryl transfer from the histidine mutant monomer to the ATP binding site mutant monomer (Figure 2b). Therefore, these mutants complement each other for the "trans" family HKs.

[0099] We hypothesized that dimerization between complementary mutants could result in more efficient transduction due to reduced phosphatase activity of mutant HK toward its cognate response regulator.Using protein alignments, we identified putative residues in the CA domain of NarX that are important for ATP binding.

[0100] Mutational analysis of the amino acids (aa) present in the CA domain of EnvZ allowed us to identify the asparagine at position 347 as essential for the kinase activity of EnvZ. The CA domain of histidine kinases belongs to a large family of ATPase domains of HSP90 chaperone / DNA topoisomerase II / histidine kinase proteins (superfamily 55874, http: / / supfam.org / SUPERFAMILY / cgi-bin / scop.cgi?sunid=55874). To identify whether N347 of EnvZ is conserved in NarX, we aligned EnvZ and NarX with proteins containing ATPase domains. The alignment identified Asn509 of NarX as a conserved residue potentially important for ATP binding.

[0101] Since full-length HK was shown to constitutively signal in mammalian cells, we set up a complementation assay in HEK293 cells by cotransfecting different combinations of codon-optimized mutants of NarX with the cognate downstream RR NarL and a reporter gene driven by a NarL-responsive promoter. This assay (Fig. 2c) shows that (i) full-length wild-type NarX is able to signal as shown previously, (ii) mutants of either the aspartic acid in the CA domain, which is important for ATP binding, or the histidine in the DHp domain, are unable to signal by themselves, as expected, and (iii) coexpression of the complemented mutants of NarX partially restores signaling to the level obtained with the wild type.

[0102] We then introduced the same mutations into the short cytoplasmic domain of NarX (Fig. 2d) and fused the resulting mutants at their N-terminus to peptides from SynZip1 and SynZip2, as was done previously with the wild-type domain, resulting in the constructs SynZip1::NarX, respectively. 379~598 H399Q (hereafter, for simplicity, SynZip1::H mut ), and SynZip2::NarX 379~598 N509A(SynZip2::Nmut We also inverted the fusion pair to create SynZip2::NarX 379~598 H399Q(SynZip2::H mut ) and SynZip1::NarX 379~598 N509A (SynZip1 ::Nmut ) were generated. When these pairs of constructs were co-expressed in HEK293 cells with the NarL RR in the presence of a NarL-responsive reporter, NarL-mediated signaling was fully restored to the levels obtained with full-length wild-type NarX and was found to generate a much stronger signal compared to the wild-type short NarX fusion (Fig. 8). The restoration was the same for both pairs (Fig. 2f, bars 11 and 12). When one or both of the fused SynZip domains were deleted, signaling was abolished (Fig. 2f, bars 4-8, Fig. 9), indicating that dimerization of the fusion domains is necessary and sufficient to restore signaling. Interestingly, a pair of complementary NarX mutants, both fused to SynZip1 (SynZip1::H mut and SynZip1::N mut ) also resulted in elevated signaling activity (Fig. 2f, bar 9), consistent with the (weak) effect observed under conditions similar to those of the wild-type domain (Fig. 6, bar 4). This leads to the hypothesis that the SynZip1 domain can homodimerize, albeit with reduced affinity compared to the SynZip1-SynZip2 interaction. Indeed, examination of the original literature suggests that SynZip1 exists as both a monomer and a dimer in size-exclusion chromatography experiments, and as such to a much broader extent than SynZip2 alone. To assess the dose-response behavior of signaling strength, we characterized output levels for varying plasmid doses of NarX-derived constructs (Fig. 2g).

[0103] Activity increases linearly with plasmid dosage, with full-length wild-type NarX showing the highest dosage sensitivity and perhaps the strongest dimerization constant, the SynZip1-SynZip2 pair showing slightly reduced but comparable dimerization behavior, and SynZip1 clearly showing inferior dimerization. For example, reducing the amount of plasmid approximately 16-fold compared to the conditions initially used (6.25 ng instead of 100 ng) reduces SynZip1-SynZip1 signaling to background levels, but on the other hand, as expected, results in strong SynZip1-SynZip2 dimerization.

[0104] In summary, these experiments suggest that possible signaling of NarX can be restored by forced dimerization of the complementary truncated mutant domains, and that this restoration depends on the dose and strength of the interaction. In agreement with the current knowledge of the two-component signaling stoichiometry with a HK:RR ratio of about 1:30 in E. coli, about 10% NarX expression compared to NarL fully activates the reporter output. This is because the promoter EF1α-V1 (see Methods) driving NarL is about 5-fold weaker than the wild-type EF1α promoter driving NarL, and furthermore, a plasmid dosage ratio of 1:2 (i.e., 50 ng of NarX-derived plasmid to 100 ng of RR-encoding plasmid) already saturates the response.

[0105] FK506 / FKBP To allow for genuine signal transduction, dimerization of the NarX domains should preferably be controlled by external stimuli. Inducible protein-protein interactions are a common signal transduction mechanism both in the cytoplasm and across membranes. A well-characterized ligand-induced heterodimerization has been demonstrated in the presence of the small molecule A / C heterodimerizer (rapamycin analog C16-(S)-7-methylindolerapamycin, also known as AP21967) with the proteins FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding domain (FRB) mutant FRB12. T2098L occurs between

[0106] To test whether the NarX domain could transduce this interaction (Fig. 3a), we cloned the NarX mutants complementing the histidine and asparagine residues described above at their N-termini with FKBP (FK) proteins and FRB1, respectively. T2098L (FR) protein and FK::NarX 379~598 H399Q(FK::H mut ) and FR::NarX 379~598 N509A(FR::N mut ) and the reverse FR::H fusion mut and Free Kick :: N mut First, NarX or NarX were isolated in the absence or presence of 100 nM A / C. 379~598 into HEK cells to confirm that A / C does not affect the wild-type NarX / NarL system (Fig. 3b, bars 2 and 3). We then expressed the different fusion mutants and control constructs in HEK293 cells in the presence of the response regulator NarL and a NarL activation reporter construct, this time with and without ligand, to probe ligand-induced signaling in a manner similar to that used for the SynZip1-SynZip2 experiments (Fig. 3b, Fig. 8). As expected, full-length wild-type NarX was constitutively active. In all cases except for wild-type NarX, no signaling occurred in the absence of ligand. High levels of ligand could induce strong signaling only when (i) the NarX-derived domains contained complementary histidine and asparagine mutations and (ii) they were fused to FKBP and FRB interaction partners, respectively (Fig. 3b, bars 11 and 12). Next, the inventors mut and FK::N mutWe proceeded to characterize the dose-response behavior of this modified signal transduction pathway using the pair (Figure 3c). The dose-response showed the expected Hill function dependence, from which the EC50 in the assay was determined to be 1.3 nM, compared to published values ​​of 10 nM and 36 nM.

[0107] The above results indicate the ability of TCS-based components to mediate signal transduction in the cytoplasm. However, the majority of signal transduction takes place across membranes. Many transmembrane signaling pathways involve protein-protein interactions at the cytoplasmic surface of lipid bilayers, including an important class of signaling pathways initiated by G protein-coupled receptors (GPCRs), a family of hundreds of proteins. A key step in GPCR signal transduction is the formation of a complex between the GPCR itself and the protein β-arrestin, followed by various processes such as GPCR internalization, recycling, and signal transduction. This interaction was previously shown to be sufficient for rewiring GPCR signaling by specific proteolytic cleavage of the fused transcriptional activator. We hypothesized that this interaction may also enable catalytic transmembrane signaling via a two-component pathway. To this end, we have developed a truncated histidine mutant of NarX, NarX. 379~598 H399Q was fused to a procaterol-activated chimera of the GPCR ADRB2-AVPR2:adrenergic receptor beta 2 (ADRB2) and a cytoplasmic fragment of the arginine vasopressin receptor 2 (AVPR2). We also used a truncated asparagine mutant of NarX, NarX 379~598 N509A was fused to β-arrestin 2 (FIG. 4a). Finally, we reversed these fusions to see if the effects were symmetric.

[0108] First, we confirmed that procaterol does not affect signal transduction via the NarX / NarL system. NarL and the NarL activation reporter were stimulated with either NarX or NarX in the absence or presence of 100 nM procaterol. 379~598 HEK293 cells co-transfected with either NarX or GPCR showed fully induced and background reporter expression, respectively, independent of Procaterol (Fig. 4b, bars 2 and 3). In experiments combining complementary NarX mutants fused to GPCR receptors, β-arrestins, or both, respectively (Fig. 4b, Fig. 11a), a certain amount of Procaterol-independent signaling was found to occur with β-arrestins and GPCR receptors alone (Fig. 4b, bars 9 and 10). This effect was more pronounced with GPCRs, suggesting that the receptors dimerize in a ligand-independent manner. Importantly, when the complementary NarX mutants were fused to GPCR receptors and β-arrestins, respectively, very strong Procaterol-induced signaling occurred, with an induction dynamic range exceeding three orders of magnitude (Fig. 4b, bars 11 and 12). The dynamic range obtained with the two-component-based system was higher than that obtained with the TANGO assay, a proteolysis-based assay of GPCR activation (Figure 11b).

[0109] To determine whether the synthetic signaling cascade could recapitulate the actions of different known GPCR ligands, we investigated the ADRB2-AVPR2::H signaling cascade in the presence of two agonists (procaterol, isoproterenol) and a partial agonist (clenbuterol). mut and β-arrestin::N mut.We characterized the dose response of the system containing the pair of agonists, procaterol (Figure 4c, blue line) and isoproterenol (Figure 4c, red line). We observed that the two full agonists, procaterol (Figure 4c, blue line) and isoproterenol (Figure 4c, red line), induce potent downstream gene expression in a dose-dependent manner, reaching the same maximum response at saturating doses. In the presence of the partial agonist, clenbuterol, reporter gene expression is 3.5-fold lower than in the presence of the full agonist (Figure 4c, compare green line with blue and red lines). Furthermore, single-cell flow cytometry data suggest a unimodal rather than bimodal induction (Figure 4d, TCS data). We determined the EC50 values ​​of procaterol, isoproterenol, and clenbuterol to be 5 nM, 30 nM, and 14 nM, respectively, from dose-response curves. These values ​​are similar to those described in the literature and those determined using the TANGO assay (Figure 4c, dashed line, secondary axis). Note that although the TANGO assay results in high absolute reporter expression, leakage is much higher compared to the TCS-based mechanism and the single-cell data are bimodal (Fig. 4d, TANGO data). We also characterized the action of the antagonist propranolol in the presence of procaterol and found that it inhibited the action of procaterol in a dose-dependent manner in our signaling cascade (Fig. 4e, blue line). We found that the IC 50 was determined to be 2 nM, which was similar to the value obtained by using the TANGO assay (FIG. 4e, dashed line). These results demonstrate that the system of the present invention can faithfully transduce the various known effects of agonists and antagonists on GPCR activity and can be used to extract quantitative data of interaction parameters.

[0110] overview Implementing non-native signaling approaches in cells, especially mammalian cells, is highly desirable to rationally control cellular behavior and ultimately engineer novel cellular functions for basic research, biotechnology, and medicine. Two-component signaling has evolved very differently from vertebrate signaling, and to the inventors' knowledge, one example of phosphoryl transfer from histidine to aspartate has not been disclosed in vertebrate cells. The native mechanism of TCS signal transduction in prokaryotes relies on ligand-induced conformational changes of HK dimers in the membrane, but direct implementation of this mechanism in mammalian cells has been elusive. Instead, here we pursued a different strategy to achieve essentially the same end result by controlling signaling through switching between dissociated and bound states of the HK cytoplasmic domain. In the case we present here, we achieved switching by ligand-induced dimerization of proteins fused to histidine and asparagine mutants, respectively, of the truncated cytoplasmic domain of HK NarX. A similar qualitative effect is observed when the wild-type truncated domain is used instead of the mutant. However, the quantitative behavior is poorer and, more importantly, the resulting effect does not distinguish between ligand-induced dimerization of the two interaction partners and homodimerization of one of the partners, as is the case for SynZip1 and GPCR. One reason for the reduced dynamic range could be the stronger phosphatase activity of the wild-type domain compared to the histidine mutant.

[0111] This approach retains many of the features of the original prokaryotic signaling. It is an amplifiable, multiple turnover process with a single NarX dimer that can phosphorylate multiple copies of the response regulator NarL, thus inducing multiple transcription initiation events. We speculate that the two-step amplification results in a significantly improved dynamic range compared to proteolytic-cleavage-based approaches. Furthermore, spontaneous dephosphorylation of the RRs stops signaling when the stimulus is withdrawn, which can be facilitated by the judicious use of wild-type HK domains that retain full phosphatase activity when rapid signaling quiescence is required. Given the diversity of TCS pathways, multiplexing of synthetic signaling pathways is feasible by using the methods described above. Together, the results demonstrate a novel approach for sensing and signaling in mammalian cells, both in the cytoplasm and across membranes.

[0112] method Standard molecular cloning techniques available to those skilled in the art were used.

[0113] Plasmid construction Plasmids were constructed using standard cloning techniques. All restriction enzymes used were purchased from New England Biolabs (NEB). Q5 High-Fidelity DNA polymerase (NEB) was used for fragment amplification. Single-stranded oligonucleotides were synthesized at Sigma-Aldrich. Digestion products or PCR fragments were purified using the GenElute Gel Extraction Kit or Gen Elute PCR Clean Up Kit (Sigma-Aldrich). Ligation was performed by temperature cycle ligation using T4 DNA Ligase (NEB) for 140 cycles between 10°C for 30 s and 30°C for 30 s. Gibson assembly was performed as described below. Five microliters of the ligation product or Gibson assembly product was transformed into chemically competent E. coli DH5α or E. coli TOP10 plated on LB agar medium supplemented with 100 μg / ml ampicillin. The resulting clones were directly screened by colony PCR (Dream Taq Green PCR Master Mix, Thermo Scientific). We expanded single clones in LB Broth Miller Difco (BD) supplemented with ampicillin and purified their plasmid DNA using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). All the resulting plasmids were sequence verified by Microsynth using Sanger sequencing method. DNA for mammalian transfection was obtained from 100 ml liquid cultures using Promega PureYield™ Plasmid Midiprep System (A2495). Recovered DNA was further purified using Norgen Endotoxin Removal Kit Mini (Cat. No. 27700) or Midi (Cat. No. 52200). Brief cloning procedures for each construct used in this work are described below.

[0114] Gibson Assembly Protocol Gibson assembly was performed in a final volume of 10 μl by mixing vector (0.018 pmol) and insert (0.09 pmol) in 1× Gibson assembly buffer (0.1 M Tris-HCl, pH 7.5, 0.01 M MgCl2, 0.2 mM dGTP, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, 0.01 M DTT, 5% (w / v) PEG-8000, 1 mM NAD), 0.04 units of T5 exonuclease (NEB), 0.25 units of Phusion DNA polymerase (NEB), and 40 units of Taq DNA ligase (NEB). Negative controls for Gibson assembly contained vector only. Gibson assembly was achieved at 50° C. for 1 hour.

[0115] Recombinant DNA Cloning Protocols OmpR_RE-Cerulean (pMZ1): The mCerulean coding sequence from EF1α-Cerulean (pKH24) was digested with NotI and SmaI and cloned into the plasmid OmpR_RE-amCyan (pJH008) digested with AfeI and PspOMI.

[0116] CMV-envZ N347A (pMZ37): The 5' and 3' fragments of envZ were PCR amplified from plasmid CMV-envZ (pJH001) using PR3687 / PR3708 and PR3707 / PR3709. The primers were designed to introduce a mutation that exchanges the codon encoding asparagine (N) at position 347 for a codon encoding alanine (A). Both the XhoI and PvuII digested plasmid CMV-envZ (pJH001) and the PCR product were assembled using Gibson mix.

[0117] CMV-envZ 223~450(pMZ123): The 3' fragment of envZ was PCR amplified from plasmid CMV-envZ (pJH1) using PR4345 / PR4346. Primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding the phosphorylatable histidine at position 243 to the end of the gene and to insert an ATG sequence in front of this amplified sequence. Plasmid CMV-envZ (pJH001) digested with XhoI and AgeI and the PCR product were assembled using Gibson mix.

[0118] CMV-narX N509A (pMZ160): The 5' and 3' fragments of narX were PCR amplified from plasmid CMV-narX (pJH002) using PR4122 / PR4541 and PR4346 / PR4542. The primers were designed to introduce a mutation that exchanges the codon encoding asparagine (N) at position 509 for a codon encoding alanine (A). Both plasmid CMV-envZ (pJH001) digested with XhoI and AgeI and the PCR product were assembled using Gibson mix.

[0119] CMV-narX 379~598 (pMZ163):The 3' fragment of narX was PCR amplified from plasmid CMV-narX (pJH002) using PR4345 / PR4546. Primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding the phosphorylatable histidine at position 399 to the end of the gene and to insert an ATG sequence in front of this amplified sequence. Plasmid CMV-envZ (pJH001) digested with XhoI and AgeI and the PCR product were assembled using Gibson mix.

[0120] EF1α-V1-envZ-mCherry (pMZ194): EF1α-V1, a truncated version of EF1α, was PCR amplified from plasmid pRA114 in PR4733 / PR4734 (Altamura et al., manuscript in preparation). Plasmid EnvZ-GGGGS-mCherry (pEM017) and promoter were digested with PspOMI and AgeI and assembled using Gibson mix.

[0121] CMV-SynZip1::narX 379~598 (pMZ200): We performed de novo synthesis of the gBlock sequence encoding SynZip1 and a G4S linker via IDT (gBlock264). 379~598 The coding sequence of is located in plasmid CMV-narX 176~598 (JH010) was PCR amplified using PR4346 / PR4747. Plasmid CMV-envZ (pJH001), gBlock, and the PCR product digested with AgeI and XhoI were assembled using Gibson mix.

[0122] CMV-narX 379~598 ::SynZip1 (pMZ202): We performed de novo synthesis of the gBlock sequence encoding the G4S linker and SynZip1 via IDT (gBlock265). 379~598 The coding sequence of is located in plasmid CMV-narX 379~598 (pMZ163) was PCR amplified using PR4122 / PR4747. Plasmid CMV-envZ (pJH001), gBlock, and the PCR product digested with AgeI and XhoI were assembled using Gibson mix.

[0123] CMV-SynZip2::narX 379~598 (pMZ206): We performed de novo synthesis of the gBlock sequence encoding SynZip2 and a G4S linker via IDT (gBlock269). 379~598 The coding sequence of is located in plasmid CMV-narX 176~598(JH010) was PCR amplified using PR4346 / PR4747. Plasmid CMV-envZ (pJH001), gBlock, and the PCR product digested with AgeI and XhoI were assembled using Gibson mix.

[0124] CMV-narX 379~598 ::SynZip1 (pMZ208): We performed de novo synthesis of the gBlock sequence encoding the G4S linker and SynZip1 via IDT (gBlock270). 379~598 The coding sequence of is located in plasmid CMV-narX 379~598 (pMZ163) was PCR amplified using PR4122 / PR4748. Plasmid CMV-envZ (pJH001), gBlock, and the PCR product digested with AgeI and XhoI were assembled using Gibson mix.

[0125] CMV-FRB T2098L::CBRC (pMZ211): The 3' fragment of FRB and the 5' fragment of FRB carrying CBRC were PCR amplified from the plasmid FRB::CBRC using PR4122 / PR4541 and PR4346 / PR4542. Primers were designed to introduce a mutation that exchanges both codons encoding threonine (T) at position 98 for codons encoding leucine (L) (compared to the intact protein serine / threonine-protein kinase TOR1). Both the plasmid FRB::CBRC digested with BamHI and AgeI and the PCR product were assembled using Gibson mix.

[0126] CMV-FKBP::narX 379~598 (pMZ214): The sequence encoding FKBP was PCR amplified from plasmid CBRN::FKBP using PR4766 / PR4767. 379~598 The coding sequence of is located in plasmid CMV-narX 176~598(pJH010) was PCR amplified using PR4346 / PR4771. Primers were designed to insert a (G4S)2 linker between the amplified fragments. Both the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0127] CMV-FRB T2098L::narX 379~598 (pMZ215): The sequence encoding FRB T2098L was PCR amplified from plasmid CMV-FRB::CBRC (pMZ211) using PR4769 / PR4770. 379~598 The coding sequence of is located in plasmid CMV-narX 176~598 (pJH010) was PCR amplified using PR4346 / PR4771. Primers were designed to insert a (G4S)2 linker between the amplified fragments. Both the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0128] NarL_RE-Cerulean (pMZ219): The minimal response element NarL_RE was formed by annealing primers PR4892 and PR4893. Plasmid OmpR_RE-Cerulean (pMZ1) was digested with AscI and NdeI and the annealed product was assembled using Gibson mix.

[0129] EF1α-V1-SynZip1::narX 379t~598 (pMZ221): SynZip1, G4S linker and NarX 379~383 The coding sequence is contained in the plasmid CMV-SynZip1::NarX 379~598 (pMZ200) was PCR amplified using PR3687 / PR4971. The plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0130] EF1α-V1-SynZip2::narX 379~598 (pMZ222): SynZip2, G4S linker and NarX 379~383 The coding sequence is located in the plasmid CMV-SynZip2::narX 379~598 (pMZ206) was PCR amplified using PR3687 / PR4971. The plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0131] EF1α-V1-SynZip1::narX 379~598 H399Q (pMZ223): The sequence encoding SynZip1 and the G4S linker is contained in the plasmid CMV-SynZip1::narX 379~598 (pMZ200) was amplified by PCR using PR4971 / PR4973. 379~598 The sequence encoding H399Q was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0132] EF1α-V1-SynZip2::narX 379~598 H399Q (pMZ224): The sequence encoding SynZip2 and the G4S linker is contained in the plasmid CMV-SynZip2::narX 379~598 (pMZ206) was amplified by PCR using PR4971 / PR4973. 379~598 The sequence encoding H399Q was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0133] EF1α-V1-SynZip1::narX 379~598 N509A (pMZ225): The sequence encoding SynZip1 is contained in the plasmid CMV-SynZip1::narX 379~598 (pMZ200) was amplified by PCR using PR4971 / PR4973. 379~598 The sequence encoding N509A was PCR amplified from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0134] EF1α-V1-SynZip2::narX 379~598 N509A (pMZ226): The sequence encoding SynZip2 is contained in the plasmid CMV-SynZip2::narX 379~598 (pMZ206) was amplified by PCR using PR4971 / PR4973. 379~598 The sequence encoding N509A was PCR amplified from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0135] EF1α-V1-FKBP::narX 379~598 H399Q (pMZ229): The sequence encoding FKBP and the (G4S)2 linker is contained in the plasmid CMV-FKBP::narX 379~598 (pMZ214) was amplified by PCR using PR4974 / PR4973. 379~598 The sequence encoding H399Q was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0136] EF1α-V1-FRB T2098L::narX 379~598 H399Q (pMZ230): The sequence encoding FRB T2098L and the (G4S)2 linker is contained in the plasmid CMV-FRB T2098L::narX 379~598 (pMZ215) was amplified by PCR using PR4975 / PR4973. 379~598 The sequence encoding H399Q was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0137] EF1α-V1-FKBP::narX 379~598 N509A (pMZ231): The sequence encoding FKBP and the (G4S)2 linker is contained in the plasmid CMV-FKBP::narX 379~598 (pMZ214) was amplified by PCR using PR4974 / PR4973. 379~598 The sequence encoding N509A was PCR amplified from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0138] EF1α-V1-FRB T2098L::narX 379~598 N509A (pMZ232): The sequence encoding FRB T2098L and the (G4S)2 linker is contained in the plasmid CMV-FRB T2098L::narX 379~598 (pMZ215) was amplified by PCR using PR4975 / PR4973. 379~598The sequence encoding N509A was PCR amplified from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4972. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0139] EF1α-V1-narX (pMZ239): The NarX coding sequence was PCR amplified from plasmid CMV-narX (pJH002) using PR3687 / PR4979. Plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0140] EF1α-V1-narX 379~598 (pMZ241): The 3' fragment of narX was PCR amplified with PR4977 / PR3687 from plasmid CMV-narX (pJH002). Primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding the phosphorylatable histidine at position 399 to the end of the gene and to insert an ATG sequence in front of this amplified sequence. The PCR product and plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.

[0141] EF1α-V1-narX H399Q (pMZ242): The NarX coding sequence was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR3687 / PR4979. Plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0142] EF1α-V1-narX 379~598The 3' fragment of H399Q (pMZ244):narX was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR4977 / PR3687. Primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding the phosphorylatable histidine at position 399 to the end of the gene and to insert an ATG sequence in front of this amplified sequence. Plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0143] EF1α-V1-narX N509A (pMZ245): The NarX coding sequence was PCR amplified from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4979. Plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0144] EF1α-V1-narX 379~598 The 3' fragment of N509A(pMZ247):narX was PCR amplified from plasmid CMV-narX N509A(pMZ160) using PR4977 / PR3687. Primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding the phosphorylatable histidine at position 399 to the end of the gene and to insert an ATG sequence in front of this amplified sequence. Plasmid EF1α-V1-envZ-mCherry(pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0145] EF1α-narL (pMZ248): The EF1α promoter was PCR amplified from plasmid pRA58 using PR4732 / PR4978 (Altamura et al., manuscript in preparation). Plasmid CMV-narL (pJH004) digested with PspOMI and AgeI and the PCR product were assembled using Gibson mix.

[0146] EF1α-V1-narL (pMZ249): The EF1α promoter was PCR amplified with PR4734 / PR4978 from plasmid pRA114 (Altamura et al., manuscript in preparation). Plasmid CMV-narL (pJH004) digested with PspOMI and AgeI and the PCR product were assembled using Gibson mix.

[0147] EF1α-V1-ARRB2::narX 379~598 (pMZ250): The sequence coding for ARRB2 was PCR amplified from the plasmid CMV-ARRB2::TEV protease (pBH302) using PR4980 / PR4981. 379~598 The coding sequence was PCR amplified from plasmid CMV-narX (pJH2) using PR3687 / PR4982. Primers were designed to insert a G4S linker between the amplified fragments. Both the EF1-V1-envZ-mCherry plasmid (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0148] EF1α-V1-ARRB2::narX 379~598 H399Q (pMZ251): The sequence coding for ARRB2 was PCR amplified from the plasmid CMV-ARRB2::TEV protease (pBH302) using PR4980 / PR4981. 379~598 The sequence coding for H399Q was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR3687 / PR4982. Primers were designed to insert a G4S linker between the amplified fragments. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0149] EF1α-V1-ARRB2::narX 379~598N509A (pMZ252): The sequence coding for ARRB2 was PCR amplified from the plasmid CMV-ARRB2::TEV protease (pBH302) using PR4980 / PR4981. 379~598 The sequence encoding N509A was PCR amplified from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4982. Primers were designed to insert a G4S linker between the amplified fragments. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0150] EF1α-V1-ADRB2 1~341 ::AVPR2 343~371 ::narX 379~598 H399Q(pMZ257):ADRB2 1~341 ::AVPR2 343~371 The coding sequence is plasmid CMV-ADRB2 1~341 ::AVPR2 343~371 ::tTA(pBH312) was amplified by PCR using PR4983 / PR4985. 379~598 The sequence coding for H399Q was PCR amplified from plasmid CMV-narX H399Q (pEM014) using PR3687 / PR4982. Primers were designed to insert a G4S linker between the amplified fragments. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0151] EF1α-V1-ADRB2 1~341 ::AVPR2 343~371 ::narX 379~598 N509A(pMZ258):ADRB2 1~341 ::AVPR2 343~371 The coding sequence is plasmid CMV-ADRB2 1~341 ::AVPR2 343~371::tTA(pBH312) was amplified by PCR using PR4983 / PR4985. 379~598 The sequence encoding N509A was PCR amplified from plasmid CMV-narX N509A (pMZ160) using PR3687 / PR4982. Primers were designed to insert a G4S linker between the amplified fragments. Both plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI and the PCR product were assembled using Gibson mix.

[0152] tTA_RE-Cerulean (pMZ290): The promoter controlled by tTA was PCR amplified from plasmid tTA_RE-mCherry (pIM003) using PR5226 / PR5227. Plasmid DcuR_RE-Cerulean (pMZ259) digested with AscI and AgeI and the PCR product were assembled using Gibson mix.

[0153] EF1α-V1-ARRB2::TEV protease(pMZ291):ARRB2 283~409 The sequence encoding the and TEV protease was PCR amplified from the plasmid CMV-ARRB2::TEV protease (pBH302) using PR5228 / PR5229. The sequence of the bGH poly(A) signal was amplified from the plasmid EF1α-V1-ARRB2::narX 379~598 (pMZ250) was PCR amplified using PR5230 / PR5231. Plasmid EF1-V1-ARRB2::narX digested with BsaI and AvrII 379~598 Both (pMZ250) and the PCR product were assembled using Gibson mix.

[0154] EF1α::iRFP (pCS184): The iRFP coding sequence of CMV-iRFP (pCS12) was PCR amplified using PR2258 / PR2259. Plasmid EF1α::citrine (pRA001, Altamura et al., manuscript in preparation) digested with BmtI and XbaI and the PCR product were assembled using a ligation mix.

[0155] EF1α-V1-ADRB2 1~341 ::AVPR2 343~371 ::tTA(pBH292):ADRB2 254~341 AVPR2 343~371 The sequence encoding tTA was inserted into plasmid CMV-ADRB2 1~341 ::AVPR2 343~371 PCR amplification was performed using PR5232 / PR5233 from ::tTA(pBH312). Plasmid EF1α-V1-ADRB digested with BglII and XhoI. 21~341 ::AVPR 2343~371 ::narX 379~598 H399Q(pMZ257) and the PCR product were assembled using Gibson mix.

[0156] CMV-ARRB2::TEV protease (pBH302): We performed IDT-mediated de novo synthesis of a gBlock sequence encoding β-arrestin-2 fused to TEV protease with two gBlocks (gBlock112 and gBlock113). Plasmid pZsYellow1-N1 (Clontech 632445) digested with NotI and EcoRI and the gBlocks were assembled using Gibson mix.

[0157] CMV-OPRK1 1~345 ::AVPR2 343~371 ::tTA(pBH309): We de novo synthesized, via IDT, the gBlock (gBlock114) sequence encoding KOR-1 and the gBlock (gBlock115) sequence encoding V2R fused to tTA. 1~345The coding sequence was PCR amplified from gBlock114 using PR2442 / PR2443. Plasmid pZsYellow1-N1 (Clontech 632445), digested with XhoI and MfeI, the PCR product and gBlock115 were assembled using Gibson mix.

[0158] CMV-ADRB2 1~341 ::AVPR2 343~371 Via ::tTA(pBH312):IDT, the inventors performed the de novo synthesis of the gBlock (gBlock118) sequence encoding the beta-2 adrenergic receptor. ADRB2 1~341 The coding sequence was PCR amplified from gBlock118 using PR2442 / PR2444. Plasmid CMV-OPRK digested with XhoI and BssHII. 11~345 ::AVPR 2343~371 ::tTA(pBH309) and the PCR product were assembled using Gibson mix.

[0159] CMV-narX 176~598 (pJH010): The 3' fragment of narX was PCR amplified from CMV-narX (pJH002) using PR1021 / PR1023. Primers were designed to amplify the sequence of NarX from the codon encoding alanine 176 to the end of the gene and to insert an ATG sequence before this amplified sequence. The PCR product and CMV-narX (pJH002) were digested with XhoI and AgeI. The two digestion products were then ligated together.

[0160] The following plasmids were previously reported: CMV-envZ (pJH001), CMV-narX (pJH002), CMV-ompR (pJH003), CMV-narL (pJH004), OmpR_RE-AmCyan (pJH008), CMV-envZ_cyt (pJH009), CMV-envZ H243V (pEM013), CMV-narX H399Q (pEM014), and EnvZ-GGGGS-mCherry (pEM017) (Hansen, J. et al., Proc Natl Acad Sci USA 111, 15705-15710 (2014)). CBRN::FKBP, and FRB::CBRC (Schramm, A. et al., Int J Mol Sci 19 (2018)). Ef1α-mCerulean (pKH024), Ef1α-Citrine (pKH025), Ef1α-mCherry (pKH026), and Junk-DNA (pBH265) (Prochazka, et al., Nat Commun 5, 4729 (2014)). pTRE bidirectional mCherry-pA (pIM003) (Angelici, B., et al., Cell Rep 16, 2525-2537 (2016)). Plasmid CMV-iRFP (pCS12) was obtained from Addgene (plasmid 31857 (Filonov, G.S., et al., Angewandte Chemie International Edition 51, 1448-1451 (2012))).

[0161] cell culture The experiments in this study were carried out in HEK293 purchased from Life technologies (catalog number 11631-017). Cells were cultured at 37 °C in 5% CO2 in DMEM (Gibco, Life Technologies; catalog number 41966-052) supplemented with 10% FBS (Sigma-Aldrich; catalog number F9665) and 1% penicillin / streptogamine solution (Sigma-Aldrich, catalog number P4333). Splitting was performed every 3-4 days using 0.25% trypsin-EDTA (Gibco, Life Technologies; catalog number 25200-072). Cultures were grown for up to 2 months and then replaced with fresh cell stocks.

[0162] Transfection All transfections were performed using Lipofectamine2000 transfection reagent (Life Technologies; catalog number 11668027). All transfections were performed in 24-well plates (Thermo Scientific Nunc; NC-142475) and transfected with 400 ng of DNA. Cells were seeded at a density of 50,000 cells per well in 500 μl of DMEM 24 h prior to transfection. Plasmids for each sample were mixed as shown in Supplementary Tables 3–18 and completed with a volume of Opti-MEM I reduced serum (Gibco, Life Technologies catalog number 31985–962) to a final volume of 50 μl. 1.5 μl of Lipofectamine2000 was diluted in 50 μl of Opti-MEM I per sample to a final volume of 3.75:1 μl reagent / μg DNA ratio. After at least 5 minutes of incubation, diluted Lipofectamine was added to the mixed DNA sample. The resulting mixture was mixed briefly by gentle vortexing and incubated for 20 minutes at room temperature before being added to the cells. Four hours after DNA was added to the cells, the medium was removed and replaced with 500 μl of fresh medium. If required, 5 μl of the chemical to be tested was added to the medium. Different stock solutions were prepared at 100 times the desired final concentration, as shown below.

[0163] A / C heterodimer (Clontech; catalog number 635057) stock solutions were prepared in ethanol (Honeywell; catalog number 02860): 250 μM, 50 μM, 20 μM, 8 μM, 3.2 μM, 1.28 μM, 512 nM, 205 nM, 81.9 nM, 32.8 nM, 13.1 nM, 5.24 nM, 1.04 nM.

[0164] Procaterol (Sigma; Catalog No. P9180~10MG) stock solutions were prepared in DMSO (Sigma; Catalog No. D4540, BCBT0803): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 10 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM, and 12.7 nM.

[0165] Isoproterenol (Sigma; Cat. No. I6504) stock solutions were prepared in DMSO (Sigma; Cat. No. D4540): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM, and 12.7 nM.

[0166] Clenbuterol (Sigma; Catalog No. C5423) stock solutions were prepared in DMSO (Sigma; Catalog No. D4540): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM, and 12.7 nM.

[0167] Propranolol (Sigma; Catalog No. P0884) stock solutions were prepared in water (Invitrogen; Catalog No. 10977-035): 1 mM, 286 μM, 81.6 μM, 23.3 μM, 6.6 μM, 1.9 μM, 544 nM, 155 nM, 44.4 nM, and 12.7 nM.

[0168] Microscopy Microscopic images were taken 48 hours after transfection. We used a Nikon Eclipse Ti microscope equipped with a mechanized stage and a temperature-controlled chamber that was kept at 37°C during image acquisition. Excitation light was generated by a Nikon IntensiLight C-HGFI mercury lamp and filtered with a set of optimized Semrock filter cubes. The resulting images were collected by a Hamamatsu ORCA R2 camera using a 10x objective. Each Semrock cube is assembled from an excitation filter, a dichroic mirror, and an emission filter. To minimize crosstalk between the different fluorescent proteins, we used the following settings: Cerulean; CFP HC (HC438 / 24, BS458, HC483 / 32), mCherry; TxRed HC (HC624 / 40, BS593, HC562 / 40). Images were acquired with 40 ms exposures for Cerulean and mCherry. The acquired images were processed by ImageJ software, which performed uniform contrast enhancement to improve visualization.

[0169] Flow cytometry 48 hours after transfection, cells were prepared for FACS analysis by removing the medium and incubating the cells with 200 μl of StemPro™ Accutase™ cell dissociation reagent (Gibco, catalog no. A11105-01) for 5 min at 37 °C. After incubation, the plates were transferred onto ice. To avoid possible cell damage, samples were prepared in successive batches and no single sample was kept on ice for more than 1 h. Prepared samples were measured using a BD LSR Fortessa II cell analyzer with excitation and emission combinations that minimized crosstalk between the different fluorescent reporters. Cerulean was measured with a 445 nm laser and a 473 / 10 nm emission filter, while mCherry was measured with a 561 nm excitation laser coupled with a 600 nm long pass filter and a 610 / 20 emission filter. Cerulean and mCherry were measured at PMT voltages of 330 and 310, respectively, in all experiments. SPHERO RainBow calibration particles (Spherotech; catalog no. RCP-30-5A, BD) were used to ensure consistent device performance.

[0170] Data analysis Typical bar graph flow cytometry data analysis was performed using FlowJo software. In this study, bar graph fluorescence values, as shown as normalized expression units (Cerulean, reference units), are calculated as follows: Live cells are gated based on forward and side scatter readings. From this population, single cells are gated based on forward scatter area and forward scatter height. Within this gate, Cerulean-positive cells are gated based on the negative control, such that 99.9% of the cells in this control sample fall outside the selected gate. For each Cerulean-positive cell, the mean value of the fluorescence intensity is calculated and multiplied by the frequency of positive cells. This value is used as a measure of the total reporter signal within the sample and can be defined as the total intensity (TI). The TI of Cerulean is normalized by the TI of mCherry-positive cells (constitutive transfection control). Thus, the relative formula is: Reporter Intensity in Reference Units = [Mean (Reporter+ (number of reporters in cells) × frequency (number of reporters + cells)] / [mean(transfection marker + Transfection marker in cells) x frequency (transfection marker) + cell)].

[0171] array In the event that the sequences below differ from the sequence protocol submitted herewith in text form, the sequences below will control.

[0172] [Table 1] JPEG2025072478000003.jpg216153JPEG2025072478000004.jpg217153JPEG2025072478000005.jpg43153

[0173] [Table 2] JPEG2025072478000007.jpg204153JPEG2025072478000008.jpg62153

[0174] The sequence of the synthetic promoter is shown in the table below (underlined indicates RR DNA binding site, italicized letters indicate TATA box, start codon is shown in bold):

[0175] [Table 3]

[0176] Promoter sequences of CMV, EF1α, and EF1α-V1 >CMV promoter (SEQ ID NO:3) gcgttgacattgattattgactagttataatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggccccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaag tctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaact

[0177] EF1α

[0178] EF1α-V1 ttaagctcgggcccTGGGCGGGATTCGTCTTGGGCGGGATCCTTGTCCACGTGATCGGGGGAGGGACTTTCCCGCTGGAGTGACTCATCTAGCCCACGTGATCTTCCATGCCACGTGATCGATATGGGGACTTTCCTGACTCCCACGTGATCGCACCCCCACGTGATCCCGTAAGGG ACTTTCCCTACTTTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTCCCCGTGCTCAGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGTTAACTAGCACAGAACACAGGTAAGTGCCGTGTGT GGTTCCCGCGGGCGGCGACGGGGCCCGTGCCCACGTGATCAGGAGTTGGGCGGGATGTTATGAGTGACTCACGCCATCCACGTGATCTCAGACGGGACTTTCCATATTAAGTGACTCAGGATAAGGGACTTTCCCTACGGCCACGTGATCTCTTTTTGGGCGGGATGAGATTGGGA CTTTCCTGTCCTGGGACTTTCCTACAGTTCAAACTCGACCACGTGATCTTATGACTGACGGGCGGGTGAGTCACCCACGGTGGCATGGGGGAACTTTCCTTTAGGCGTTCATGTGACTCCACGGACAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAa

[0179] narL's sequence: NarL MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASNGEQGIELAESLDPDLILLLDLNMPGMNGLETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLL KDMEPEDLLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRERDILKLIKLIAQGLPNKMIARRLDITESTVKVHVKHMLKKMKLKSRVEAAVWVHQERIF

[0180] Sequence of VP48 >VP48 (SEQ ID NO: 7) GPADALDDFDLDMLPADALDDFDLDMLPADALDDFLDMLPG

[0181] EnvZ 180~450 , EnvZ 223~450 , NarX 176~598 , and NarX 379~598 The aa sequence of is shown below, with the phosphorylatable histidines underlined and italicized, and the asparagine important for the ATP binding domain underlined and bold.

[0182] [Table 4]

[0183] NarX 379~598 In the mutated version of aa, the mutated aa is shown in bold and underlined.

[0184] [Table 5]

Claims

1. A set of one or more nucleic acids for expression in a mammalian cell, comprising: a first nucleic acid sequence encoding a first polypeptide fused to the N-terminus of a truncated first variant of NarX histidine kinase comprising a histidine-containing phosphotransfer (DHp) domain and a catalytic ATP-binding (CA) domain; and a second nucleic acid sequence encoding a second polypeptide fused to the N-terminus of a second variant of said truncated NarX histidine kinase comprising the DHp domain and the CA domain; the truncated first variant and the truncated second variant do not contain a histidine kinase sensor domain and a histidine kinase transduction domain; the truncated first variant and the truncated second variant are incapable of dimerizing with themselves, and Upon specific binding of the first polypeptide and the second polypeptide, the first variant and the second variant dimerize such that the CA domain of the first variant or the second variant phosphorylates the DHp domain of the first variant or the second variant. The set of nucleic acids.

2. A set of nucleic acids as described in claim 1, wherein the first truncated mutant form and / or the second truncated mutant form do not contain the transmembrane domain of the histidine kinase.

3. A set of nucleic acids described in any one of claims 1 to 2, comprising a third nucleic acid sequence encoding a response control protein that can be specifically phosphorylated at an aspartic acid residue in its receiver domain by the first or second mutant type of the DHp domain, wherein the response control protein has an effector function, and the effector function of the response control protein is controlled by phosphorylation by the first or second mutant type of the DHp domain.

4. A set of nucleic acids as described in claim 3, wherein the response control protein comprises a receiver domain fused to an effector domain, the receiver domain being phosphorylatable by the first or second mutant type of the DHp domain, and the effector domain being activatable or inhibitable by the phosphorylated receiver domain.

5. --the effector domain is a transcription activation domain; - the set of nucleic acids comprises a fourth nucleic acid sequence comprising a gene of interest under the control of an inducible promoter recognizable by the transcription activation domain; Activation of the transcription activation domain induces expression of the gene of interest. A set of nucleic acids according to any one of claims 3 to 4.

6. A set of nucleic acids described in claim 5, wherein the target gene encodes a target protein or a target RNA.

7. The receiver domain comprises or is a NarL response control protein (SEQ ID NO: 6), and the effector domain comprises or is a VP16 transcription activation domain (Vp48, SEQ ID NO: 7). A set of nucleic acids according to any one of claims 4 to 6.

8. A set of nucleic acids described in any one of claims 5 to 7, wherein the inducible promoter is selected from OmpR promoter (sequence number 1) and NarL-RE (sequence number 2).

9. A set of nucleic acids described in any one of claims 1 to 8, wherein the specific binding between the first polypeptide and the second polypeptide can be induced by a ligand that can be specifically recognized by the first and / or second polypeptide.

10. A set of nucleic acids as described in claim 9, wherein the first polypeptide is or comprises a receptor, and the second polypeptide is or comprises a binding partner of the receptor, and binding between the receptor and the binding partner can be induced by the ligand recognizable by the receptor.

11. A set of nucleic acids described in claim 10, wherein the receptor is a transmembrane receptor and the binding partner is a cytoplasmic protein. Claim 12: The first polypeptide consists of or comprises a G protein-coupled receptor and the second polypeptide consists of or comprises a cytoplasmic ligand of the G protein-coupled receptor; or - said first polypeptide consists of or comprises a T cell receptor and said second polypeptide is or comprises ZAP-70; A set of nucleic acids according to any one of claims 1 to 11.

13. The set of nucleic acids described in claim 12, wherein the cytoplasmic ligand of the G protein-coupled receptor is β-arrestin.

14. A set of nucleic acids described in any one of claims 5 to 12, wherein the target gene encodes an immune protein or microRNA that affects the function or internal state of a cell.

15. A set of nucleic acids described in claim 14, wherein the immune protein is a cytokine or an antibody.

16. A set of nucleic acids according to any one of claims 1 to 15, wherein the first truncated variant and the second truncated variant each comprise a polypeptide sequence having at least 90% sequence identity with NarX 379-598 (SEQ ID NO: 11).

17. A set of nucleic acids according to any one of claims 1 to 16, wherein the first variant comprises a polypeptide sequence having at least 90% sequence identity with NarX 379-598 (H399Q) (SEQ ID NO: 12), and / or the second variant comprises a polypeptide sequence having at least 90% sequence identity with NarX 379-598 (N509A) (SEQ ID NO: 13).

18. A vector comprising a set of nucleic acids described in any one of claims 1 to 17.

19. A cell comprising a set of nucleic acids described in any one of claims 1 to 2.

20. A cell comprising a set of nucleic acids described in any one of claims 3 to 17.

21. A method for assessing protein-protein interactions, said method comprising the steps of: - providing a cell according to claim 20, and - determining the activity of said response regulator protein; Including, Upon specific binding between the first polypeptide and the second polypeptide, the CA domain of the truncated first or second variant phosphorylates the DHp domain of the truncated first or second variant, and the truncated first variant and the second variant dimerize such that phosphorylation by the DHp domain of the truncated first variant and / or second variant modulates the activity of the response regulator protein. The method.

22. A method for assessing the effect of a compound on a protein-protein interaction, said method comprising the steps of: - providing a cell according to claim 20, and - contacting said cells with a compound, and - determining the activity of said response regulator protein; Including, the specific binding of the first polypeptide to the second polypeptide causes the CA domain of the truncated first or second variant to phosphorylate the DHp domain of the truncated first or second variant, and the truncated first variant and the second variant dimerize such that phosphorylation by the DHp domain of the truncated first variant and / or second variant modulates the activity of the response regulator protein; and the effect of the compound on the specific binding between the first polypeptide and the second polypeptide is determined by the activity of the response control protein. The method.

23. A method for eliciting a desired response in response to a stimulus, said method comprising the steps of: - providing a cell according to claim 20, Including, wherein specific binding between the first polypeptide and the second polypeptide causes the CA domain of the truncated first or second variant to phosphorylate the DHp domain of the truncated first or second variant, and the truncated first variant and the second variant dimerize such that phosphorylation by the DHp domain of the truncated first variant and / or second variant modulates the activity of the response regulator protein. The process, - exposing said cell to said stimulus, wherein said desired response is mediated by or is the activity of said response regulator protein; The method comprising:

24. The response control protein comprises a receiver domain fused to an effector domain, the receiver domain being phosphorylatable by the truncated first or second variant DHp domain, and the effector domain being activatable by the phosphorylated receiver domain; - said effector domain is a transcription activation domain, - the cell further comprises a fourth nucleic acid sequence encoding a gene of interest under the control of an inducible promoter recognizable by the transcription activation domain; Activation of the transcription activation domain induces expression of the gene of interest. The method according to any one of claims 21 to 23.

25. - the presence of an expression product of the gene of interest is determined as the activity of the response regulator protein; or - the expression product of said gene of interest is or mediates said desired response, 25. The method of claim 24.

26. A set of nucleic acids described in any one of claims 1 to 17, a vector described in claim 18, or a cell described in any one of claims 19 to 20, used for in vitro diagnosis or cell therapy.

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