Patent Application
20210340526
The present invention relates to methods and means for assessing or responding to a protein-protein interaction and transducing this interaction to the expression of a gene of interest.
Molecular interactions, such as protein/protein interactions, are involved in almost every cellular process in living cells. The characterization of a protein/protein interaction is an important step to better understand and control biological systems. The transduction of a protein/protein interaction to a detectable signal or to expression of a gene of interest is important for the discovery of new drugs and the development of cells with new functions used as cell-based biosensors or cell therapies.
Different cellular pathways are modulated by the presence/absence of compounds that promote or inhibit a protein/protein interaction. The identification of molecules modulating protein/protein interaction is one of the main tools used in drug discovery and development. Further, transduction of a protein/protein interaction into a novel response is a major tool for cell engineering for therapeutic purposes.
An example of a protein interaction modulated by a compound is the interaction between G-protein coupled receptors (GPCRs) and downstream pathway proteins, such as beta-arrestin. GPCRs are membrane receptor in mammalian cells that can detect various ligands (endogenous hormones, growth factors, and natural or synthetic small molecules). Following the interaction of the GPCR with its ligand, different cascades in the cells are induced, modulating cellular activity. Due to the central function of the GPCR in the cells, many drugs act on GPCRs as their targets. Different assays have been developed to identify and characterize GPCR agonists and antagonists. Some of these assays transduce the interaction of the GPCR with one of its protein partners into the expression of a reporter gene.
In addition, protein/protein interactions have been used to design chimeric sensors which can sense different signals and transduce these signals to a specific response. This ability to redirect information from defined input to specific output can be used for numerous applications such as the generation of cell-based biosensors to produce new in vitro diagnostic tools or to generate new cell therapies with better safety and efficacy. Despite numerous developments in the area of biosensors, many of them merely generate a detectable signal that requires manual readout and interpretation by a human. Significantly less progress has been made on biosensors that transduce a signal into a downstream biological activity, such as gene expression. Artificial signal transduction systems described so far mainly utilize an act of cleavage of a fusion protein to release a transcriptional activator, which then modulates the expression of a gene of interest.
The two-component system (TCS) signalling cascade is initiated upon ligand-triggered autophosphorylation of the histidine kinase (HK) receptor protein at the histidine residue, followed by phosphoryl transfer to the aspartate residue of a Response Regulator (RR) protein. With a few exceptions, a HK sensor forms a homodimer in the cell membrane, and the structural basis of HK autophosphorylation is the existence of two distinct HK dimer conformations. In the unstimulated state, the conformation is such that the catalytic ATP-binding (CA) domain is distant from the histidine residue in the dimerization and histidine-containing phosphotransfer (DHp) domain (
Due to the importance of the GPCR signaling in human disease, different assays have been developed to detect and identify molecules that interact with GPCRs. Among the different methods developed, some take advantage of the fact that beta-arrestin interacts with ligand-activated GPCR. The bioluminescence resonance energy transfer (BRET) assay, the TANGO assay (Invitrogen) (
The TANGO assay is implemented by fusing to the intracellular domains of GPCR a proteo-lytically cleavable artificial transcription factor (GAL4-VP16) and by fusing a TEV protease to beta-arrestin. The activation of the GPCR by a ligand induces the recruitment of the beta-arrestin to the GPCR, bringing the TEV protease in close proximity of the cleavable linker on the GPCR, and allowing the release of GAL4-VP16. The artificial transcription factor will induce the expression of the reporter gene (beta-lactamase) driven by a chimeric promoter targeted by GAL4-VP16.
The ChaCha system has been recently developed as a derivative of the TANGO assay. In this system dCas9 (unable to cut DNA but still able to bind it) linked to a tripartite transcriptional activator composed of VP64, p65 activation domain, and Rta (dCas9-VPR) is fused to beta-arrestin while the intracellular domains of GPCR is fused to the TEV protease. This system also requires the expression of a guide RNA (gRNA), which allows dCas9 to be recruited to the promoter driving the expression of the gene of interest. The interaction between the GPCR and the beta-arrestin-dCas9-VRP fusion releases the dCas9-VRP. The dCas9-VRP modulates the expression of gene of interest targeted by the gRNA co-expressed in the cell. The promoter can be an endogenous or a chimeric one.
With respect to probing generic protein-protein interactions e.g., in the cytoplasm, different methods have been developed as well. One of the most popular is the yeast two hybrid approach. In this method, two potential interacting proteins, usually called bait and prey, are fused to split subunits of a protein with a particular detectable biological activity. Each of the split subunits alone do not show the biological activity in question. The interaction between the bait and the prey allows proper reconstitution of the domains fused to the bait and the prey, respectively. Different reporter systems have been developed depending of the localization of the bait and the prey:
One drawback of the yeast-two-hybrid approach is the fact that the interaction is quantified in the yeast cells and they may not faithfully recapitulate the interaction in the native mammalian cell milieu.
Accordingly, it is the objective of the invention to provide means and methods for responding to and/or assessing protein-protein interactions.
This objective is solved by a cell and the methods specified in the independent claims. Advantageous embodiments are stated in the dependent claims and the following description.
A first aspect of the invention relates to a recombinant cell. The cell facilitates analysis of the interaction of a pair two polypeptides or proteins with one another. These interaction partners are termed in the following “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 part subject to analysis of its interaction with the other polypeptide, and a fragment of a histidine kinase variant that retains DHp and CA activity.
The cell according to the invention comprises
In other words, this aspect of the invention relates to a cell that comprises
In particular embodiments, the first and the second polypeptide do not comprise any part of the above mentioned histidine kinase, particularly not the transmembrane domain, the sensor domain or the transmitter domain.
In certain embodiments of the cell of the invention, the first variant and/or the second variant does not comprise a transmembrane domain of the histidine kinase.
In certain embodiments of the cell of the invention,
In particular embodiments, the naturally occurring sensor and transmitter domain of the histidine kinase are replaced by two proteins of interest, the interaction of which shall be assessed. If there is a specific interaction between these proteins, binding between them facilitates the dimerization of the truncated variants of the histidine kinase, by which a spatial proximity of the CA domain having an ATP and the DHp domain is achieved. This yields a phosphorylation of the DHp domain, which then is able to phosphorylate a cognate ligand, the response regulatory domain, particularly a receiver domain of the response regulatory protein.
Alternatively, the two proteins of interest may form an artificial signal transduction pathway, wherein binding of the two proteins of interest is triggered by a stimulus, such as a ligand being specifically recognizable by the one of or both of the two proteins of interest. Recognition of the ligand may be connected to a desired response mediated by the activity of the regulator response protein. Such response may be the expression of a microRNA affecting cellular processes of the cell, or a protein, such as a cytokine or an antibody. In particular embodiments, such cells may be used for medical applications, wherein beneficial or therapeutic responses may be specifically triggered by, for example, disease-related compounds such as disease related antigens.
Alternatively, the effect of compounds on known interacting proteins may be assessed by the cell of the invention, wherein the effect of the compounds may be determined by the activity of the regulatory response protein.
Particularly, the response regulatory protein comprises an effector function, which can be determined for assessing the interaction between the first polypeptide and the second polypeptide, or used to elicit a desired response in response to the above mentioned stimulus. Non-limiting examples of such effector function include binding to DNA, RNA or enzymes, for example enzymes catalyzing e.g. the formation of cAMP.
In particular embodiments, the effector function comprises the specific binding to a promoter sequence and induction of the expression of a gene of interest.
In certain embodiments of the cell of the invention, the response regulatory protein comprises a receiver domain fused to an effector domain, wherein the receiver domain is capable of being phosphorylated by the DHp domain of the first or the second histidine kinase variant, and the effector domain is capable of being modulated, particularly activated or inhibited, by the phosphorylated receiver domain. Particularly, the activity of the effector domain changes with respect to the phosphorylation state of the receiver domain, thus the activity of the effector domain can increase or can be switched on or decrease or be inhibited by phosphorylation of the receiver domain.
In certain embodiments of the cell of the invention,
In certain embodiments of the cell of the invention, the gene of interest encodes a protein of interest, particularly a fluorescent or a luminescent protein, or an RNA of interest.
Such protein of interest may be a fluorescent or a luminescent protein, whereby successful interaction between the first and second polypeptide may be determined or observed via the fluorescence or luminescence of the protein of interest.
Alternatively, the protein of interest or the RNA of interest may be or trigger the desired response in response to the above-mentioned stimulus, such as a desired therapeutic response (cytokine, antibody, production of reactive oxygene species, etc.).
In certain embodiments of the cell of the invention,
As indicated previously, the effector domain can be part of NarL fused with the VP16.
In certain embodiments of the cell of the invention, the transcriptional activating domain is, consists of or comprises an amino acid characterized by SEQ ID NO 7.
In certain embodiments of the cell of the invention, the first or the second variant is or comprises a variant selected from EnvZ180to450(SEQ ID 8), EnvZ223to450 (SEQ ID 9), NarX176to598 (SEQ ID 10) and NarX379to598 (SEQ ID 11) or a functional equivalent polypeptide having a sequence identity of at least 70%, 80%, 85%, 90%, 95%, 98% or 99% to any one of SEQ ID 8 to 11.
In certain embodiments of the cell of the invention, the inducible promoter is selected from the OmpR promoter (SEQ ID 1) and the NarL-RE promoter (SEQ ID 2).
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 is optimized towards the codon usage of the cell.
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 is under transcriptional control of a constitutive promoter.
In certain embodiments of the cell of the invention, the constitutive promoter is selected from CMV (SEQ ID 3), EF1α (SEQ ID 4), and EF1α-V1 (SEQ ID 5).
In certain embodiments of the cell of the invention, the first variant and the second variant are identical.
In certain embodiments of the cell of the invention, the histidine kinase belongs to the transphosphorylation family.
In certain embodiments of the cell of the invention,
In certain embodiments of the cell of the invention,
In certain embodiments of the cell of the invention, specific binding of the first polypeptide and the second polypeptide is triggerable by a ligand specifically recognizable by the first and/or the second polypeptide.
In certain embodiments of the cell of the invention, the first polypeptide is or comprises a receptor and the second polypeptide is or comprises a binding partner of the receptor, wherein binding of the receptor and the binding partner is triggerable by the ligand recognizable by the receptor.
In certain embodiments of the cell of the invention, the receptor is a transmembrane receptor, and the binding partner is a cytosolic protein. In this case, the ligand recognizable by the receptor and the cytosolic protein as binding partner are particularly separated by a membrane.
In certain embodiments of the cell of the invention,
In certain embodiments of the cell of the invention, the cell is a mammalian cell, particularly a human cell.
Another aspect of the invention relates to a method for assessing a protein-protein interaction. The method comprises:
A further aspect of the invention relates to a method for assessing the effect of a compound on a protein-protein interaction. The method comprises the steps of:
Advantageously, the above method may be used as a screening assay to assess the effect of any compound on any interesting protein-protein interaction.
Yet another aspect of the invention provides a method for for eliciting a desired response in response to a stimulus. The method according to this aspect of the invention comprises the steps of:
Preferably, the first or second polypeptide is a receptor recognizing the stimulus, for example a T cell receptor recognizing a disease related antigen, or a G-coupled receptor, wherein the other polypeptide may be a ligand of this receptor, such as beta-arrestin or ZAP-70, respectively.
The term “specific binding of the first and second polypeptide” particularly refers to a binding with a Kd of less than 10−5 M 10−6 M, 10−7M, 10−8 M, or 10−9 M.
In certain embodiments of the methods of the invention, the response regulatory protein comprises a receiver domain fused to an effector domain, wherein the receiver domain is phosphorylatable by the DHp domain of the first or the second variant, and the effector domain is activatable by the phosphorylated receiver domain.
In certain embodiments of the method of the invention,
In certain embodiments of the methods of the invention, the presence of the expression product of the gene of interest is determined as the activity of the response regulatory protein.
In certain embodiments of the method of the invention, the expression product of the gene of interest has an optical quality, e.g. comprises a luminescent moiety, like in the case of the green fluorescent protein (GFP).
In certain embodiments of the methods of the invention, the expression product of the gene of interest is Cerulean.
In certain embodiments of the methods of the invention, the expression product of the gene of interest is or mediates the desired response. For example, the expression product may be a cytokine intended to elicit an immune response by the cell. The expression product may also be a component of a signal cascade eliciting the desired response further downstream of the cascade. The expression product may also be an RNA able to elicit the desired response, such as such as a microRNA or a guide RNA that itself regulate endogenous genes.
Furthermore, the invention provides a vector that is particularly suitable for transfecting or transducing a mammalian cell, particularly a human cell. The vector comprises:
According to a further aspect of the invention, a fusion protein is provided. The variant comprises a polypeptide fused to a variant of a histidine kinase (E.C. 2.7.13.3) comprising a DHp (dimerization and histidine-containing phosphotransfer) domain and a CA (catalytic ATP-binding) domain.
Particularly, the polypeptide does not comprise any part of the above-mentioned histidine kinase, particularly not the transmembrane domain, the sensor domain or the transmitter domain.
In certain embodiments of the fusion protein of the invention, the variant does not comprise a transmembrane domain of the histidine kinase.
In certain embodiments of the fusion protein of the invention, the variant does not comprise a functional transmitter domain and/or a functional sensor domain of the histidine kinase.
In certain embodiments of the fusion protein of the invention, the variant is a variant of the EnvZ kinase (UniProt No POAEJ4) or a variant of of the NarX kinase (UniProt No POAFA2).
In certain embodiments of the fusion protein of the invention, the variant is or comprises a variant selected from EnvZ180to450 (SEQ ID 8), EnvZ223to450 (SEQ ID 9), NarX176to598 (SEQ ID 10) and NarX379to598 (SEQ ID 11) or a functional equivalent polypeptide having a sequence identity of at least 70%, 80%, 85%, 90%, 95%, 98% or 99% to any one of SEQ ID 8 to 11.
In certain embodiments of the fusion protein of the invention, the histidine kinase belongs to the transphosphorylation family.
In certain embodiments of the fusion protein of the invention, the variant comprises a DHp domain that does not comprise a histidine residue accessible by the CA domain of the variant or another variant of the histidine kinase or comprises a CA domain that is not able to bind ATP.
In certain embodiments of the fusion protein of the invention, the variant is or comprises variant NarX379-598 (H399Q) (SEQ ID 12), variant NarX379-598 (N509A) (SEQ ID 13) or a functional equivalent polypeptide having a sequence identity of at least 70%, 80%, 85%, 90%, 95%, 98% or 99% to SEQ ID 12 or 13.
In certain embodiments of the fusion protein of the invention, the polypeptide consists of or comprises a G-protein coupled receptor or a cytosolic ligand of the G-protein coupled receptor, particularly beta-arrestin.
In certain embodiments of the fusion protein of the invention, the polypeptide comprises a T cell receptor or one of its components or a cytosolic ligand of the T cell receptor or its component, particularly ZAP-70 (UniProt No P43403).
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
The present invention provides a novel approach to transduce protein/protein interactions into gene expression in mammalian cells using components derived from the two-component system present in bacteria. The invention can be used to develop new screening assay for protein-protein interactions in general, and for GPCR signaling modulation in particular. The herein described system is characterized by superior dynamic range compared to the state of the art (e.g., the TANGO system from ThermoFischer), and it has great potential for high-throughput multiplexing thanks to an almost unlimited supply of the building blocks.
The present invention may provide the basis of a synthetic signal transduction module in cell-based biosensors and engineered therapeutic cells, with such properties as low background levels, high dynamic range, and reversibility. As the approach can be multiplexed, it will allow the creation of complex logic-based circuits which could provide novel capabilities to the modified cell.
Particularly, the present invention comprises 3 different features:
The HK domains fused to the 2 interacting proteins are mutated to increase the dynamic range. Wild-type domains can also be used, but so far this resulted in lower dynamic range. Nevertheless, the use of the wild type domain can confer some advantages when the system is used to detect homodimerization. In addition, in case of homodimerization of a protein of interest, HK belonging to the cis family can be used. The advantage of this approach is the reduction in the number of genetic constructs. Nevertheless, in all cases the important feature of the invention is the fusion of the protein of interest to a size-reduced domain of the HK that does not dimerize on its own and therefore does not transduce transcriptional activity on its own, unless forced to dimerize with the help of the fused components.
The RR (response regulator protein) used in this experiment is fused with VP48 as transcriptional activating domain functional in mammalian cells. Other transcriptional activating domains could be fused to the RR, for example p65 (RelA) domain or Rta. In the present examples, the inventors used RR that binds directly to DNA, but other types of RR can be used depending of the desired readout. For example, some RR can bind to RNA and others are enzymes, catalyzing the production of a compound feeding into a secondary signal transduction chain, e.g., cyclic di-GMP.
The gene construct expressing the gene of interest (GOI) comprises 2 elements. The first part is a chimeric promoter. The inventors used a minimal promoter linked to an upstream sequence with a number of binding sites for the RR. The distance between the minimal promoter and the number of RE can be modified to tune the expression of the GOI, either up- or down.
The GOI used in the experiment is a fluorescent reporter Cerulean but this can be replaced with any other protein- or RNA-coding gene. For example, GOI could be a miRNA or guide RNA that itself can regulate endogenous genes.
The present invention differs from the aforementioned systems TANGO and ChaCha in that these systems release the transcription factor previously fused, respectively, to the GPCR or beta-arrestin, and this transcription factor accumulates over time. On the contrary, in the present invention, all the elements are still functional after one act of protein-protein interaction and therefore they exhibit multiple turnover. The gene of interest modulated by both the TANGO and the system of the invention is under the regulation of a chimeric promoter. In the ChaCha system, endogenous genes can also be modulated using appropriately designed gRNA. Another difference between this invention and the TANGO assay is that in this invention, one needs an additional component, namely, the RR in addition to the chimeric GPCR fusion and beta-arrestin fusion.
The size of the HK domains fused to the GPCR and to the beta-arrestin are only 223 aa. In the TANGO the size of the fused proteins is 240 aa and 341 aa, respectively. For the ChaCha they are 240 aa and 1900 aa, respectively, in addition to the requirement for gRNA expression. Due to this smaller size the system is easier to construct and it places less burden on the cells.
The system of the invention contains two signal amplification steps. The first step, unique to the invention, results from the reconstituted HK which phosphorylates multiple copies of the RR. The second level of amplification, also present in the TANGO and ChaCha system, is the catalytic nature of gene induction by the RR. Two levels of amplification in the present invention result in a 10-fold improvement in the dynamic range compared to the TANGO system.
The system of the invention is not desensitized in the course of time; the same protein can be re-activated after several cycles of presence/absence of ligand. On the contrary, the elements of the TANGO and ChaCha system can only be used once and therefore their system activity relies on the protein degradation and de novo protein synthesis, an inherently slow process. This characteristic lets the system of the invention switch more rapidly from On state to Off and vice versa.
Due to the fact that the present invention contains one extra level of amplification compared to the other approaches, it can detect low amounts of protein and weak protein-protein interactions. The advantage of this feature is that the expression of the components needed for the assay of the invention can be modulated in the cells from low to high. In this way, the level of expression of the system components can be set at a level that enables an appropriate ligand-inducible dynamic range. Therefore, the present approach reduces ligand-independent signalling that can occur due to protein overexpression in the earlier approaches.
Specifically, the assay of the invention shows higher dynamic range than the TANGO assay. This parameter would facilitate the automated analysis of the result generated by the present invention compared to the TANGO.
Another advantage of the system of the invention is that it can be multiplexed by employing different HK-RR pairs simultaneously. Due to the very large number of natural two-component systems, the potential to multiplex is very large. Each chimeric pair would be independent and induce a different output. Therefore, in the same experiment one will be able to test the effect of a compound on multiple protein-protein interactions or multiple GPCRs at once. Multiplexing is more difficult with other approaches because the number of well characterized TEV proteases is limited.
The system of the invention also enables reversibility due to the fact that the RR spontaneously dephosphorylates. In the absence of the interaction between protein pairs, the kinase is not active anymore and does not phosphorylate the RR. The unphosphorylated RR cannot induce the expression of the GOI. In the case of the TANGO and ChaCha, the reversibility of the system is difficult because it requires time-consuming degradation of the transcriptional activator released after the interaction between the GPCR and beta-arrestin.
Lastly, the assay of the invention is highly modular. The same pair of complementing HK fragments can be utilized to detect a protein-protein interaction in the cytoplasm and membrane-localized protein-protein interaction, as in the case of GPCR induction assay. Other assays require big adjustments for probing different types of protein-protein interaction, and assays such as TANGO are specific to GPCR pathways and have not been used to probe generic protein-protein interaction.
The present invention can have multiple applications.
1) It can be used for the development of a new screening assay for the identification of compounds interacting with a GPCR. The resulting assay is expected to have better specificity and a higher dynamic range than the existing ones. It will also be easier to multiplex. The commercialization of the assays can be done by selling stable cell lines containing the GPCR fused to the HK, similar to what is currently done by DiscoverX and ThermoFischer (PathHunter and the TANGO).
2) It can be used to screen for compounds modulating protein-protein interactions and therefore for drug discovery. As opposed to the yeast 2 hybrid, the screening assays could be done in mammalian cells, which is a more relevant system. In addition, with the present invention, the same assay can be used for proteins localized to different cell compartments (nucleus, cytoplasm or at the membrane).
3) Existing therapeutic cell-based agents often use signaling pathways that involve protein-protein interactions at the cytoplasmic side of the membrane. This includes CAR-T cells where the binding of the antigen to the extracellular antibody fragment recruits protein interaction partners; these interactions can then be rewired to result in therapeutic effects using the inventors' approach.
It is known that intracellular cytoplasmic domains of HK are capable of dimerizing and autophosphorylating.
The present invention is based on the question whether partial cytoplasmic domains have a reduced inherent capacity to signal. To this end, the inventors undertook stepwise truncation mutagenesis of the HKs to identify domains that fail to dimerize on their own (
The inventors used a known interaction pair SynZip1 and SynZip2, and fused them to the N- and the C-termini of the short NarX domain NarX379to598. It was observed (
For any synthetic signalling system, it is important to avoid non-specific changes to the signalling readout. In the system of the invention, the components are expressed from 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 via unrelated endogenous pathways. This would result in the change of constitutive expression, and apparent change in the signalling readout that is unrelated to the studied effect and is artefactual. To eliminate these confounding factors, the inventors examined a number of constitutive promoters for their robustness under various stimulation conditions and compared the expression of iRFP from the CMV and EF1α promoters in the presence of different compounds often used to induce cell signalling (Epinephrine and procaterol). In the medium without any compound, the activity of both promoters is similar. However, in presence of epinephrine and procaterol the activity of the CMV promoter is induced by a factor of two, while the activity of the EF1α promoter is not affected (
The activity of the NarX/NarL system expressed from CMV, EF1α, and EF1α-V1 promoters was quantified to determine whether a too-high expression of the truncated HK cytoplasmic domain would increase the background level and respond to non-specific interactions. The EF1α-V1 promoter is around 5 times weaker than EF1α. The results demonstrate that comparable expression of the reporter gene is obtained with NarX expressed from any of the tested promoter (
The inventors further explored possibilities to optimize the effect. It is known that the HKs can be divided into two families with respect to the autophosphorylation mechanism. In the “cis”-family, the phosphoryl group is transferred to the histidine from an ATP molecule bound to a CA domain of the same monomer. In the “trans”-family, the phosphoryl group is transferred from an ATP bound to one monomer to the phosphorylatable histidine in the other monomer (
The inventors hypothesized that dimerization between complementing mutants could result in a more efficient transduction due to reduced phosphatase activity of the mutant HK towards its cognate response regulator. Using protein alignment, the inventors identified the putative residues important for ATP binding in the CA domains of NarX.
Mutational analysis of the aa present in the CA domain of EnvZ had allowed to identify the asparagine in position 347 to be primordial for kinase activity of EnvZ. The CA domains of the histidine kinase belongs to the large family of the ATPase domain of HSP90 chaperone/DNA topoisomerase II/histidine kinase protein (Superfamily 55874, http://supfam.org/SUPERFAMILY/cgi-bin/scop.cgi?sunid=55874). To identify if the N347 of EnvZ is conserved in NarX, the inventors aligned EnvZ and NarX with proteins containing the ATPase domain. The alignment identified the Asn509 for NarX as a conserved residue that is potentially important for ATP binding.
Because the full-length HKs were shown to signal constitutively in the mammalian cells, the inventors set up the complementation assay in HEK293 cells by cotransfecting different combinations of codon-optimized mutants of the NarX together with the cognate downstream RR NarL and the reporter gene driven by NarL-responsive promoter. In this assay (
Next, the same mutations were introduced in the short cytoplasmic domain of NarX (
In summary, these experiments suggest that NarX-enabled signalling can be restored upon forced dimerization of the complementing, truncated mutant domains, and that this restoration is dose- and interaction strength dependent. Consistently with the current knowledge of two component signalling stoichiometry with about 1:30 HK:RR ratio in E. coli, NarX expression of about 10% compared to NarL fully activates the reporter output. This is because the promoter driving NarL, EF1α-V1 (see Methods), is about 5 times weaker than wild-type EF1α promoter driving NarL, and in addition plasmid dosage ratio of 1:2 (i.e., 50 ng of NarX-derived plasmid vs 100 ng of the RR-encoding plasmid) already saturates the response.
To enable bona fide signalling, the dimerization of the NarX domains should be preferably controlled by an external stimulus. Inducible protein-protein interaction is a common signalling mechanism both in the cytoplasm and across the membrane. A well characterized ligand-induced heterodimerization takes place between the proteins FK506-binding protein 12 (FKBP) and FKBP12-rapamycin binding domain (FRB) mutant FRBT2098L in the presence of the small molecule A/C heterodimerize (a rapamycin analog Cl 6-(S)-7-methylindolerapamycin, known also as AP21967).
To find out if NarX domains are capable of transducing this interaction (
The above results illustrate the ability of TCS-based components to mediate signal transduction in the cytoplasm. However, a bulk of signalling takes place across the membrane. Many transmembrane signalling pathways involve protein-protein interactions at the cytoplasmic surface of the lipid bilayer, including an important class of signalling pathways initiated by G-protein coupled receptors (GPCRs), a family of a few hundred proteins. A key step of GPCR signal transduction is the formation of a complex between the GPCR itself and the protein beta-arrestin, followed by various processes that include GPCR internalization, recycling, and signaling. This interaction was previously shown to be sufficient for rewiring GPCR signalling by specific proteolytic cleavage of a fused transcriptional activator. The inventors hypothesized that this interaction could also enable catalytic transmembrane signalling via the two-component pathway. To this end, the inventors fused truncated histidine mutant of NarX, NarX379to598H399Q, to a GPCR ADRB2-AVPR2: the procaterol-activated chimera of Adrenoceptor Beta 2 (ADRB2) and the cytoplasmic fragment of Arginine Vasopressin Receptor 2 (AVPR2). The inventors also fused the truncated asparagine mutant of NarX, NarX379to598N509A, to beta-arrestin 2 (
First, it was confirmed that procaterol did not affect the signalling via NarX/NarL system. HEK293 cells co-transfected with NarL and NarL-activated reporter with either NarX or NarX379to598, in the absence or presence of 100 nM procaterol, showed respectively fully induced and background reporter expression independent of the procaterol (
To determine whether the synthetic signalling cascade is able to recapitulate the effects of different known GPCR ligands, the inventors characterized the dose response of a system comprising the pair ADRB2-AVPR2::Hmut and beta-arrestin::Nmut in the presence of two agonists (procaterol, isoproterenol) and one partial agonist (clenbuterol). It was observed that the two full agonists, procaterol (
Implementing non-native signalling modalities in cells, in particular mammalian cells, is highly desirable for rational control of cell behaviour and ultimately, engineering novel cellular functions for basic research, biotechnology and medicine. Two-component signalling is evolutionary extremely divergent from vertebrate signalling, and to the best of the inventors' knowledge, not a single instance of histidine to aspartate phosphoryl transfer has been described in vertebrate cells. The native mechanism of TCS signal transduction in prokaryotes relies on ligand-induced conformation change of the HK dimer in the membrane, but direct implementation of this mechanism in mammalian cells has been elusive. Instead, here the inventors pursued a different strategy to achieve essentially the same end result by controlling the signalling via switching between dissociated and associated states of the HK cytoplasmic domains. In the cases the inventors show here, the switching was accomplished by ligand-induced dimerization of proteins fused, respectively, to histidine and asparagine mutant of a truncated cytoplasmic domain of an HK NarX. A similar qualitative effect is observed when wild-type truncated domain is used instead of the mutants. However, the quantitative behaviour is inferior and more importantly, the resulting effect does not distinguish between ligand-induced dimerization of the two interaction partners and homo-dimerization of one of the partners, as was the case with 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.
The approach retains many of the features of the original prokaryotic signalling. It is an amplifying, multiple turnover process with a single NarX dimer capable of phosphorylating multiple copies of the response regulator NarL, which in turn can induce multiple transcription initiation events. The inventors speculate that the two-step amplification resulted in a greatly improved dynamic range compared to the proteolytic-cleavage based approach. Further, upon stimulus withdrawal, the signalling will cease due to spontaneous dephosphorylation of the RR; this can be facilitated by judicious employment of the wild-type HK domains that retain their full phosphatase activity if quick signalling quiescence is required. Given the rich variety of TCS pathways, multiplexing of the synthetic signalling pathways is feasible by using the methods described above. Together, the result point toward a novel modality for sensing and signal transduction in mammalian cells, both in the cytoplasm and across the membrane.
Standard molecular cloning techniques were employed as available to the skilled person.
Plasmids were constructed using standard cloning techniques. All restriction enzymes used in this work were purchased from New England Biolabs (NEB). Q5 High-Fidelity DNA Polymerase (NEB) was used for fragment amplification. Single-strand oligonucleotides were synthesized by Sigma-Aldrich. Digestion products or PCR fragments were purified using GenElute Gel Extraction Kit or Gen Elute PCR Clean Up Kit (Sigma-Aldrich). Ligations were performed using T4 DNA Ligase (NEB) by temperature cycle ligation with 140 cycles between 30 s at 10° C. and 30 s at 30° C. Gibson assembly was done as described below. 5 μl of the ligation product or the Gibson assembly product were transformed into chemically competent E. Coli DH5a or E. coli TOP10 that were plated on LB Agar with Ampicillin at 100 μg/ml. The resulting clones where screened directly by colony-PCR (Dream Taq Green PCR Master Mix, Thermo Scientific). The inventors expanded single clones in LB Broth Miller Difco (BD) supplemented with ampicillin and purified their plasmid DNA using GenElute Plasmid Miniprep Kit (Sigma-Aldrich). All the resulting plasmids were sequence-verified by Microsynth using Sanger sequencing method. The DNA for mammalian transfection was obtained from 100 ml of liquid culture using the Promega PureYield™ Plasmid Midiprep System (A2495). The recovered DNA was further purified using the Norgen Endotoxin Removal Kit Mini (Cat. #27700) or Midi (Cat. #52200). A short cloning procedure for each construct used within this work is described below.
The Gibson assembly was performed in 10 μl final volume by mixing vectors (0.018 pmol) and inserts (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 assemblies included vectors alone. The Gibson assembly was realized at 50° C. for 1 h.
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 CMV-envZN347A (pMZ37): The 5′ and the 3′ fragments of envZ was PCR amplified with PR3687/PR3708 and PR3707/PR3709 from the plasmid CMV-envZ (pJH001). The primers were designed to introduce a mutation exchanging the codon encoding for the asparagine (N) at the 347th position to codon encoding for an alanine (A). Both PCR products and the plasmid CMV-envZ (pJH00114) digested with XhoI and PvuII were assembled using Gibson mix.
CMV-envZ223to450 (pMZ123): The 3′ fragments of envZ was PCR amplified with PR4345/PR4346 from the plasmid CMV-envZ (pJH1). The primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding for the phosphorylable histidine at the position 243 till the end of the gene, and to insert ATG sequence in front of this amplified sequence. The PCR products and the plasmid CMV-envZ (pJH001) digested with XhoI and AgeI were assembled using Gibson mix.
CMV-narX N509A (pMZ160): The 5′ and the 3′ fragments of narX were PCR amplified with PR4122/PR4541 and PR4346/PR4542 from the plasmid CMV-narX (pJH002). The primers were designed to introduce a mutation exchanging the codon encoding for the asparagine (N) at the 509th position to codon encoding for an alanine (A). Both PCR products and the plasmid CMV-envZ (pJH001) digested with XhoI and AgeI were assembled using Gibson mix.
CMV-narX379to598 (pMZ163): The 3′ fragment of narX was PCR amplified with PR4345/PR4546 from the plasmid CMV-narX (pJH00214). The primers were designed to amplify the sequence from the 20th codon upstream the codon encoding for the phosphorylable histidine at the position 399 till the end of the gene, and to insert ATG sequence in front of this amplified sequence. The PCR products and the plasmid CMV-envZ (pJH001) digested with XhoI and AgeI were assembled using Gibson mix.
EF1α-V1-envZ-mCherry (pMZ194): The EF1α-V1, as shortened version of EF1α, was PCR amplified with PR4733/PR4734 from the plasmid pRA114 (Altamura et al, manuscript in preparation). The promoter and the plasmid EnvZ-GGGGS-mCherry (pEM01714) digested with PspOMI and AgeI were assembled using Gibson mix.
CMV-SynZip1::narX379to598 (pMZ200): the inventors performed de novo synthesis of gBlock sequence encoding for SynZip1 and G4S linker (gBlock264) via IDT. The coding sequence of NarX379to598 was PCR amplified with PR4346/PR4747 from the plasmid CMV-narX176to598 (JH01014). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.
CMV-narX379to598::SynZip1 (pMZ202): The inventors performed a de novo synthesis of gBlock sequence encoding for G4S linker and SynZip1 (gBlock265) via IDT. The coding sequence of NarX379to598 was PCR amplified with PR4122/PR4747 from the plasmid CMV-narX379to598 (pMZ163). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.
CMV-SynZip2::narX379to598 (pMZ206): The inventors performed a de novo synthesis of gBlock sequence encoding for SynZip2 and G4S linker (gBlock269) via IDT. The coding sequence of NarX379to598 was PCR amplified with PR4346/PR4747 from the plasmid CMV-narX176to598 (JH010). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.
CMV-narX379to598::SynZip1 (pMZ208): The inventors performed a de novo synthesis of gBlock sequence encoding for G4S linker and SynZip1 (gBlock270) via IDT. The coding sequence of NarX379to598 was PCR amplified with PR4122/PR4748 from the plasmid CMV-narX379to598 (pMZ163). The gBlock, the PCR product and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.
CMV-FRB T2098L::CBRC (pMZ211): The 5′ of FRB and the 3′ fragments of FRB with CBRC were PCR amplified with PR4122/PR4541 and PR4346/PR4542 from the plasmid FRB::CBRC27. The primers were designed to introduce a mutation exchanging the codon encoding for the threonine (T) at Both098th position (relative to the full protein Serine/Threonine-protein kinase TOR1) to codon encoding for a leucine (L). Both PCR products and the plasmid FRB::CBRC digested with BamHI and AgeI were assembled using Gibson mix.
CMV-FKBP::narX379to598 (pMZ214): The sequence encoding for FKBP was PCR amplified with PR4766/PR4767 from the plasmid CBRN::FKBP27. The coding sequence of NarX379to598 was PCR amplified with PR4346/PR4771 from the plasmid CMV-narX176to598 (pJH010). The primers were designed to insert (G4S)2 linker between the amplified fragment. Both PCR products and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.
CMV-FRB T2098L::narX379to598 (pMZ215): The sequence encoding for FRB T2098L was PCR amplified with PR4769/PR4770 from the plasmid CMV-FRB::CBRC (pMZ211). The coding sequence of NarX379to598 was PCR amplified with PR4346/PR4771 from the plasmid CMV-narX176to598 (pJH010). The primers were designed to insert (G4S)2 linker between the amplified fragment. Both PCR products and the plasmid CMV-envZ (pJH001) digested with AgeI and XhoI were assembled using Gibson mix.
NarL_RE-cerulean (pMZ219): The minimal response element NarL_RE was formed out by annealing the primers PR4892 and PR4893. The annealed product and the plasmid OmpR_RE-cerulean (pMZ1) digested with AscI and NdeI were assembled using Gibson mix.
EF1α-V1-SynZip1::narX379to598 (pMZ221): The sequence encoding for SynZip1, G4S linker and NarX379to383 was PCR amplified with PR3687/PR4971 from the plasmid CMV-SynZip1:: NarX379to598 (pMZ200). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-SynZip2::narX379to598 (pMZ222): The sequence encoding for SynZip2, G4S linker and NarX379to383 was PCR amplified with PR3687/PR4971 from the plasmid CMV-SynZip2::narX379to598 (pMZ206). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-SynZip1::narX379to598 H399Q (pMZ223): The sequence encoding for SynZip1 and G4S linker was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip1::narX379to598 (pMZ200). The sequence encoding for NarX379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-SynZip2::narX3′ H399Q (pMZ224): The sequence encoding for SynZip2 and G4S linker was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip2::narX379to598 (pMZ206). The sequence encoding for NarX379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-SynZip1::narX379to598 N509A (pMZ225): The sequence encoding for SynZip1 was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip1::narX379to598 (pMZ200). The sequence encoding for NarX379to598 N509A was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-SynZip2::narX379to598 N509A (pMZ226): The sequence encoding for SynZip2 was PCR amplified with PR4971/PR4973 from the plasmid CMV-SynZip2::narX379to598 (pMZ206). The sequence encoding for NarX379to598 N509A was PCR amplified with PR3687/PR4972 from CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-FKBP::narX379to598 H399Q (pMZ229): The sequence encoding for FKBP and (G4S)2 linker was PCR amplified with PR4974/PR4973 from the plasmid CMV-FKBP:: NarX379to598 (pMZ214). The sequence encoding for NarX379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-FRB T2098L::narX379to598 H399Q (pMZ230): The sequence encoding for FRB T2098L and (G4S)2 linker was PCR amplified with PR4975/PR4973 from the plasmid CMV-FRB T2098L::narX379to598 (pMZ215). The sequence encoding for NarX379to598 H399Q was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX H399Q (pEM014). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-FKBP::narX379to598 N509A (pMZ231): The sequence encoding for FKBP and (G4S)2 linker was PCR amplified with PR4974/PR4973 from the plasmid CMV-FKBP::narX379to598 (pMZ214). The sequence encoding for NarX379to598 N509A was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-FRB T2098L::narX379to598 N509A (pMZ232): The sequence encoding for FRB T2098L and (G4S)2 linker was PCR amplified with PR4975/PR4973 from the plasmid CMV-FRB T2098L::narX379to598 (pMZ215). The sequence encoding for NarX379to598 N509A was PCR amplified with PR3687/PR4972 from the plasmid CMV-narX N509A (pMZ160). Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-narX (pMZ239): The sequence encoding for NarX was PCR amplified with PR3687/PR4979 from the plasmid CMV-narX (pJH00214). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix
EF1α-V1-narX379to598 (pMZ241): The 3′ fragment of narX was PCR amplified with PR4977/PR3687 from the plasmid CMV-narX (pJH002). The primers were designed to amplify the sequence from the 20th codon upstream the codon encoding for the phosphorylable histidine at the position 399 till the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-narX H399Q (pMZ242): The sequence encoding for NarX was PCR amplified with PR3687/PR4979 from the plasmid CMV-narX H399Q (pEM01414). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-narX379to598 H399Q (pMZ244): The 3′ fragments of narX was PCR amplified with PR4977/PR3687 from the plasmid CMV-narX H399Q (pEM014). The primers were designed to amplify the sequence from the 20th codon upstream of the codon encoding for the phosphorylable histidine at the position 399 till the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-narX N509A (pMZ245): The sequence encoding for NarX was PCR amplified with PR3687/PR4979 from the plasmid CMV-narX N509A (pMZ160). The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-narX379to598 N509A (pMZ247): The 3′ fragment of narX was PCR amplified with PR4977/PR3687 from the plasmid CMV-narX N509A (pMZ160). The primers were designed to amplify the sequence from 20 codon upstream the codon encoding for the phosphorylable histidine at the position 399 to the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR product and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-narL (pMZ248): The EF1α promoter was PCR amplified with PR4732/PR4978 from the plasmid pRA58 (Altamura et al, manuscript in preparation). The PCR product and the plasmid the plasmid CMV-narL (pJH004) digested with PspOMI and AgeI were assembled using Gibson mix
EF1α-V1-narL (pMZ249): The EF1α-V1 promoter was PCR amplified with PR4734/PR4978 from the plasmid pRA114 (Altamura et al, manuscript in preparation). The PCR product and the plasmid the plasmid CMV-narL (pJH004) digested with PspOMI and AgeI were assembled using Gibson mix.
EF1α-V1-ARRB2::narX379to598 (pMZ250): The sequence encoding for ARRB2 was PCR amplified with PR4980/PR4981 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence encoding for NarX379to598 was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX (pJH2). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and EF1α-V1-envZ-mCherry the plasmid (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-ARRB2::narX379to598 H399Q (pMZ251): The sequence encoding for ARRB2 was PCR amplified with PR4980/PR4981 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence encoding for NarX379to598 H399Q was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX H399Q (pEM014). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-ARRB2::narX3′N509A (pMZ252): The sequence encoding for ARRB2 was PCR amplified with PR4980/PR4981 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence encoding for NarX379to598 N509A was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX N509A (pMZ160). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-ADRB21to371::AVPR2343to371::narX379to598 H399Q (pMZ257): The sequence encoding for ADRB21to341::AVPR2343to371 was PCR amplified with PR4983/PR4985 from the plasmid CMV-ADRB21to341::AVPR2343to371::tTA (pBH312). The sequence encoding for NarX379to598 H399Q was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX H399Q (pEM014). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
EF1α-V1-ADRB21to341::AVPR2343to371::narX379to598 N509A (pMZ258): The sequence encoding for ADRB21to341::AVPR2343to371 was PCR amplified with PR4983/PR4985 from the plasmid CMV-ADRB21to341::AVPR2343to371::tTA (pBH312). The sequence encoding for NarX379to598 N509A was PCR amplified with PR3687/PR4982 from the plasmid CMV-narX N509A (pMZ160). The primers were designed to insert G4S linker between the amplified fragment. Both PCR products and the plasmid EF1α-V1-envZ-mCherry (pMZ194) digested with AgeI and XhoI were assembled using Gibson mix.
tTA_RE-cerulean (pMZ290): The promoter regulated by tTA was PCR amplified with PR5226/PR5227 from the plasmid tTA_RE-mCherry (pIM00312). The PCR product and the plasmid DcuR_RE-cerulean (pMZ259) digested with AscI and AgeI were assembled using Gibson mix.
EF1α-V1-ARRB2::TEV protease (pMZ291): The sequence encoding for ARRB2283to409 and for the TEV protease was PCR amplified with PR5228/PR5229 from the plasmid CMV-ARRB2::TEV protease (pBH302). The sequence of the bGH poly(A) signal was PCR amplified with PR5230/PR5231 from the plasmid EF1α-V1-ARRB2::narX379to598 (pMZ250). Both PCR products and the plasmid EF1α-V1-ARRB2::narX379to598 (pMZ250) digested with BsaI and AvrII were assembled using Gibson mix.
EF1α::iRFP (pCS184): The iRFP coding sequence from CMV-iRFP (pCS12) was PCR amplified with PR2258/PR2259. The PCR product and the plasmid EF1a::citrine (pRA001, Altamura et al, manuscript in preparation) digested with BmtI and XbaI were assembled using ligation mix.
EF1α-V1-ADRB21to341::AVPR2343to371::tTA (pBH292): The sequence encoding for ADRB2254to341 AVPR2343to371 and tTA was PCR amplified with PR5232/PR5233 from the plasmid CMV-ADRB21to341::AVPR2343to371::tTA (pBH312). The PCR products and the the plasmid EF1α-V1-ADRB21to341::AVPR2343to371::narX379to598 H399Q (pMZ257) digested with BglII and XhoI were assembled using Gibson mix.
CMV-ARRB2::TEV protease (pBH302): the inventors performed de novo synthesis of gBlock sequence encoding for Beta-arrestin-2 fused with the TEV protease with 2 gBlock (gBlock112 and gBlock 113) via IDT. The gBlock and the plasmid pZsYellow1-N1 (Clontech 632445) digested with NotI and EcoRI were assembled using Gibson mix.
CMV-OPRK11to345::AVPR2343to371::tTA (pBH309): Via IDT, the inventors performed de novo synthesis of gBlock (gBlock114) sequence encoding for KOR-1 and of gBlock (gBlock115) sequence encoding for V2R fused to tTA. The sequence encoding for KOR-11to345 was PCR amplified with PR2442/PR2443 from gBlock114. The PCR product, gBlock115 and the plasmid pZsYellow1-N1 (Clontech 632445) digested with XhoI and MfeI were assembled using Gibson mix.
CMV-ADRB21to341::AVPR2343to371::tTA (pBH312): Via IDT, the inventors performed de novo synthesis of gBlock (gBlock118) sequence encoding for Beta-2 adrenergic receptor. The sequence encoding for ADRB21to341 was PCR amplified with PR2442/PR2444 from gBlock118. The PCR product and the plasmid CMV-OPRK11to345::AVPR2343to371::tTA (pBH309) digested with XhoI and BasHII were assembled using Gibson mix.
CMV-narX176to598 (pJH010): The 3′ fragment of narX was PCR amplified with PR1021/PR1023 from CMV-narX (pJH002). The primers were designed to amplify the sequence of NarX from the codon encoding the alanine at the position 176 to the end of the gene and to insert the ATG sequence in front of this amplified sequence. The PCR products and CMV-narX (pJH002) were digested with XhoI and AgeI. The two digested products are then ligated together.
The following plasmids were reported previously: 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)). The plasmid CMV-iRFP (pCS12) was obtained from Addgene (plasmid 31857 (Filonov, G. S. et al. Angewandte Chemie International Edition 51, 1448-1451 (2012))).
The experiments in this work are performed on HEK293 purchased from Life technology (Cat #11631-017). Cell were cultured at 37° C., 5% CO2 in DMEM (Gibco, Life Technologies; Cat #41966-052), supplemented with 10% FBS (Sigma-Aldrich; Cat #F9665) and with 1% Penicillin/Streptogamine Solution (Sigma-Aldrich, Cat #P4333). Splitting was performed every 3-4 days using 0.25% Trypsin-EDTA (Gibco, Life technologies; Cat #25200-072). Cultures were propagated for at most two months before being replaced by fresh cell stock.
All transfections were performed using Lipofectamine 2000 Transfection Reagent (Life Technologies; Cat #11668027). All transfections were performed in 24-well plates (Thermo Scientific Nunc; NC-142475) and 400 ng of DNA was transfected. The cells were seeded 24 h before transfection at a density per well of 50 000 in 500 μl of DMEM. The plasmids for each sample were mixed as indicated in Supplementary Tables 3-18 and completed with the volume of Opti-MEM I Reduced Serum (Gibco, Life technologies Cat #31985-962) to have final volume of 50 μl. 1.5 μl of lipofectamine 2000 was diluted in 50 μl Opti-MEM I per sample to have a final amount of 3.75:1 μl Reagent/μg DNA ratio. After an incubation at least of 5 minutes the diluted Lipofectamine was added to the mixed DNA sample. The resulting mixture was briefly mixed by gentle vortexing and incubated 20 minutes at room temperature before being added to the cells. 4 hours after the DNA was added to the cells the medium was removed and replaced with 500 μl of fresh medium. When required 5 μl of the chemical tested were added to the medium. The different stock solution at 100× of the desired final concentration were prepared as indicated below:
A/C Heterodimerizer (Clontech; Cat #635057) stock solution was prepared in ethanol (Honeywell; Cat #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.
Procaterol (Sigma; Cat #P9180-10MG) stock solution was prepared in DMSO (Sigma; Cat #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
Isoproterenol (Sigma; Cat #16504) stock solution was prepared in DMSO (Sigma; Cat #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
Clenbuterol (Sigma; Cat #C5423) stock solution was prepared in DMSO (Sigma; Cat #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
Propranolol (Sigma; Cat #P0884) stock solution was prepared in water (Invitrogen; Cat #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
Microscopy images were taken from 48 h after transfection. The inventors used a Nikon Eclipse Ti microscope equipped with a mechanized stage and temperature control chamber held at 37° C. during the image acquisition. The excitation light was generated by a Nikon IntensiLight C-HGFI mercury lamp and filtered through a set of optimized Semrock filter cubes. The resulting images were collected by an Hamamatsu, ORCA R2 camera using a 10× objective. Each Semrock cube is assembled from an excitation filter, a dichroic mirror and an emission filter. In order to minimize the crosstalk between the different fluorescent proteins the inventors used the following setup: Cerulean: CFP HC (HC 438/24, BS 458, HC 483/32), mCherry: TxRed HC (HC 624/40, BS 593, HC 562/40). The images were acquired with an exposure of 40 ms for Cerulean and mCherry.
The acquired images were processed by ImageJ software performing uniform contrast-enhancement to improve visualization.
The cells were prepared for FACS analysis 48 h after transfection by removing the medium and incubating the cells with 200 μl StemPro™ Accutase™ Cell Dissociation Reagent (Gibco, cat #A11105-01) at 37° C. for 5 minutes. After incubation, the plates was transferred on ice. To avoid potential cell damage the samples were prepared in successive batches so that no single sample was kept on ice for more than 1 h. The prepared samples were measured using a BD LSR Fortessa II Cell Analyzer with a combination of excitation and emission that minimizes the crosstalk between different fluorescent reporters. Cerulean was measured with a 445 nm laser and a 473/10 nm emission filter, mCherry with a 561 nm excitation laser coupled to a 600 nm longpass filter and 610/20 emission filter. The Cerulean and the mCherry were measured, respectively, at PMT voltage of 330 and 310 in all the experiments. SPHERO RainBow Calibration particles (Spherotech; Cat #RCP-30-5A, BD) were used to ensure constant device performance.
General flow cytometry data analysis for bar charts was performed using FlowJo software. In this work, the fluorescence values in the bar charts, shown as normalized expression units (Cerulean, norm. u.) are calculated as follows. Live cells are gated based on their forward and side scatters readouts. From this population single cell are gated based on their forward scatters area and forward scatters height. Within this gate, cells positive in a Cerulean are gated based on a negative control such that 99.9% of cells in this control sample fall outside of the selected gate. For each Cerulean positive cell, the mean value of the fluorescent intensity is calculated and multiplied by the frequency of the positive cells. This value is used as a measure for the total reporter signal in a sample and can be defined as Total Intensity (TI). The TI of the Cerulean is normalized by the TI of mCherry-positive cells (constitutive transfection control). The relative formula is therefore: Reporter intensity in norm. u.=[mean(Reporter in Reporter+ cells)*Frequency (Reporter+ cells)]/[mean (Transfection Marker in Transfection Marker+ cells)*Frequency (Transfection Marker+ cells)].
The nucleic and amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file named 95083_315_29_seqlist, created Apr. 15, 2021, about 42 KB, which is incorporated by reference herein. In case that the sequences given below vary from the Sequence Listingsubmitted herewith in text format, the below sequences shall prevail.
The sequences of the synthetic promoters are indicated in the following table (underlinded sequences indicates the RR DNA binding sites, italic letters indicate the TATA Box. Start codon is shown in bold)
ATTTACATTTTGAAACATCTATAGCGCCGGCATTTACATTTTGAAACATC
TATCCATATGCTCTAGAGGGTATATAATGGGGGCCACTAGTCTACTACC
TACCCCTATAGGGGTATAGCGCCGGCTACCCCTATAGGGGTATCCATA
The sequence of the promoter CMV, EF1α, and EF1α-V1
DLRTPLTRIRLATEMMSEQDGYLAESINKDIEECNAIIEQFIDYLRTGQEMPMEMADLN
DLRTPLTRIRLATEMMSEQDGYLAESINKDIEECNAIIEQFI
DSIAQSLSCMKMQVSCLQ
DSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNEL
| Number | Date | Country | Kind |
|---|---|---|---|
| 18200357.4 | Oct 2018 | EP | regional |
The present application is the US National Stage of International Patent Application No. PCT/EP2019/077962 submitted Oct. 15, 2019, which claims benefit of the priority of European patent application EP18200357.4 submitted Oct. 15, 2018, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/077962 | 10/15/2019 | WO | 00 |
