Patent Application
20250059558
The invention refers to the regulation of function of proteins through insertion of a peptide into the selected protein and its interaction with a regulatory peptide that interacts with the inserted peptide. The method involves insertion of a non-structured peptide, preferably into the solvent exposed loop of the target protein, wherein the target protein retains its function. When a regulatory peptide is added that binds to the inserted peptide, it forms an elongated defined structure that disrupts the functional site of the target protein by an allosteric effect and disrupts the function of said selected protein. Genetic fusion of an inhibitory peptide to the selected protein, wherein said inhibitory peptide interacts with an inserted peptide within the same polypeptide chain results in inhibition of the function of the selected protein. Said selected protein can be reactivated by the addition of a regulatory peptide that binds with higher affinity to the fused inhibitory peptide or by proteolytic cleavage of the linker peptide between the said protein and an inhibitory peptide. The invention can be used to activate or inactivate the function of different selected proteins and therefore to regulate their properties and processes such as enzymatic activity, recognition and binding to other molecules, signaling and cellular localization, useful for pharmacological, therapeutic, diagnostic, sensing, biotechnological and other industrial applications.
Regulation of the biological activity of proteins through interactions with other molecules is a cornerstone of complex biological systems and is achieved by a variety of mechanisms, from ligand binding to proteolysis and many others. In most cases, the posttranslational regulation of protein activity is uniquely adapted to each protein and is difficult to translate to other proteins. Therefore, it would be desirable to have a widely applicable principle of protein function regulation that could be introduced into diverse natural and engineered proteins that could be used for therapeutic, biotechnological and other purposes. Several engineering principles have already been introduced, such as combinations with degrons (CHOMP, Gao et al., 2018), fusion with coiled-coil and proteolysis domains (SPOCK, Fink et al., 2019)), and engineering of interaction domains modeled to introduce alternative arrangements of the secondary structural elements (LOCKR, Langan et al., 2019). However, these systems have limited applicability and require extensive remodeling of the target protein.
Another method of regulating protein activity is based on split proteins, in which protein activity is reconstituted by the proximity of the two split protein chains, which can be triggered by fusion to form interaction domains. It would be sufficient and even advantageous if the two subdomains would remain on the same polypeptide chain and be only slightly displaced to disrupt the function of the protein.
Allostery is a principle of modulation of protein function through conformational changes that occur when a regulator binds at a site remote from the primary functional site. Allosterically engineered proteins have been designed by introducing folded protein domains or domains that compete with the folding of the target protein (Dagliyan et al., 2013, 2019; Ha & Loh, 2012; Ostermeier, n.d.). There are a couple of designed allosterically regulated proteins, that have been designed individually for each protein, based on the introduction of protein domains that are regulated by binding of a ligand that induces complex formation (Dagliyan et al., 2017) or by alternate frame folding (Mitrea et al., 2010), which however need to be extensively tuned for each target protein separately and have not been used for diverse proteins. A method of regulation of the function of al or at least large majority of proteins through the addition of short peptides or small molecules could have important biomedical and biotechnological use. Further it would be of great biotechnological and biomedical use if the invention could provide a way to convert the inactive into an active functional protein by the addition of a peptide or activation of a protease, which can be activated by a small molecule.
The present invention refers to the regulation of the function of selected target proteins by insertion of a non-structured peptide into an appropriate position that maintains the particular function of the target protein. Upon addition of a regulatory peptide that binds to the inserted peptide, a coiled-coil dimer is formed between the regulatory peptide and an inserted peptide, changing its conformation from a random structure to the helical structure, which increases the distance between the termini of the inserted peptide. This locally disrupts the structure of the target protein and inhibits its function. The invention can be used to activate or inactivate the function, structure and properties of a target protein and can be used to regulate processes such as enzymatic activity, recognition of other molecules, signaling, binding to other molecules, cellular localization, optical and other properties, which are useful for pharmacological, therapeutic, diagnostic, sensing, biotechnological and industrial applications.
In the present invention, the appropriate target protein site is defined by testing the function of the target protein with a peptide inserted at different, typically solvent-exposed, loop sites that do not participate directly in protein function. The position of the insertion within the selected target protein is selected in such way that the target protein with an insert peptide retains the selected function. In the presence of an interaction peptide, the target peptide forms a coiled-coil dimer with a peptide inserted into the target protein loop, which impairs the function of the selected target protein.
The present invention refers to the method of activation of a target protein function, wherein the target protein is initially inhibited through insertion of a peptide as described above and an additional fusion of an inhibitory peptide that interacts weakly with an inserted peptide and forms a coiled coil dimer with a peptide inserted into the loop of a target protein, wherein the inhibitory peptide is genetically fused to the target protein via a flexible peptide linker. Said target protein can be activated by the addition of a regulatory peptide that strongly interacts with the inhibitory peptide or through a protease, that cleaves the linker between the target protein and the inhibitory peptide. The present invention can be further implemented to regulate the function of the target protein by a protease whose activity can be regulated through small molecules or though the biological processes, such as a protease specific for the cell type, cell state or a microbial protease, for example viral protease.
The present invention can be further implemented to combine several input signals to implement logic functions that activate or inhibit the function of the target protein, depending on the combination of coiled-coil forming peptides and proteases.
This invention refers to activation or inhibition of the biological function or biological activity such as catalytic activity or binding or other function of various target proteins such as enzymes, such as luciferase, hydrolases, kinases or proteases, proteins that constitute signaling pathways, such as protein kinases, signaling mediators that recruits other proteins to the signaling complex. Further it refers to proteins, which bind specific nucleic acid sequences or that act as transcriptional regulators, and proteins that can be detected and act as reporters, such as fluorescent proteins or luciferases and further it refers to other molecule-recognizing domains such as antibodies and their domains or to structural proteins.
The disclosed invention refers to functional proteins whose activity can be inhibited or activated by the addition or presence of a chemical or biological signal that affects the formation of the dimer involving inserted peptide in the loop of the target protein.
The disclosed invention refers to the method of regulating the activity of proteins and said proteins and nucleic acids encoding them and cells producing said proteins.
In the particular embodiment, the disclosed invention refers to the activation or inhibition of the biological and biochemical function of proteins luciferase, beta-galactosidase, IRAK-1 kinase, MyD88, Lyn kinase, transcription activator-like effector and its implementation as DNA binding domains, Cas9 gRNA-dependent DNA nuclease or specific DNA binding domain, fluorescent proteins, antibody variable domain (Fv), whose activity can be regulated through interactions with a peptide that forms a coiled-coil heterodimer with a peptide inserted into the loop of the target proteins.
In the particular embodiments, the cells such as the mammalian or eukaryotic or bacterial cells, exhibit the function of target proteins depending on the applied signal, which triggers formation or reverses formation of a coiled-coil dimer within the target protein loop, that results in the modification of the biological properties, or cells that produce the modified target proteins, which can be used for medical, biotechnological or other application of cell function.
Moreover, the present invention refers to nucleotide sequences comprising coding sequences for polypeptides described above according to the invention, optionally incorporated in a delivery vector such as a plasmid, a linear or circular nucleic acid or a virus or inserted into the genome of cells. In a further aspect, the present invention refers to a protein comprising the polypeptide according to the invention.
Scheme of the principle of the invention; where insertion of an unstructured inserted peptide (INS) into the loop of the target protein maintains the protein function, while the addition of a coiled-coil regulatory peptide (REG) triggers formation of a coiled-coil dimer, which extends the inserted peptide and inactivates the target protein function.
Peptide N8 was genetically inserted at amino acid K493 of firefly luciferase with different lengths of linkers (fLuc_N8, fLuc_5gs:N8:5gs or fLuc_10gs:N8:10gs) at each side of the inserted peptide sequence. White bar presents the activity of luciferase by insertion of an inserted peptide and shaded show the remaining luciferase activity by coexpression of the N7 peptide by transfection of HEK293T cells by different amount of the plasmid coding for N7 regulatory peptide. Linker regions allow for the additional flexibility around the peptide which maintains the firefly luciferase activity as well as the level of inhibition with the addition of peptide pair. 5gs linker flanking inserted peptide N8 demonstrated the best ratio between ON and OFF state.
Demonstration of inhibition level of fLuc_5gs: N8: 5gs with REG peptides with different affinity. We used REG peptides which had complementary electrostatic motives to N8 (INS), but different helical propensities, to demonstrate how different CC stability facilitates different levels of regulation. N7 with highest affinity to N8, inhibits luciferase activity most efficiently.
Inserted peptide N8 was genetically fused into the firefly luciferarase. It can bind several different N7 variants designed in a range of binding affinities with P7SN weakest affinity with N8 and N7 having strongest affinity for N8. Co-transfection in HEK293T cells demonstrates that the degree of inhibition of luciferase with inserted N8 peptide depends on the binding affinity of the co-expressed N7 peptide variant.
Scheme of the design of inverted INSRTR (ON switch). ON switch was designed based on affinity of CC dimer-forming peptides. CC-peptide P7 (INS) with flanking 5gs linkers was genetically inserted into firefly luciferase. Lower affinity CC-forming peptide (INH) was genetically fused on the C-terminal via linker peptide (inhibitory peptide) and into the loop (inserted peptide) of target protein. Due to high local concentration, the inhibitory and inserted peptide form a coiled coil, which results in inactive target protein (OFF state). The addition of a regulatory peptide (REG), with high affinity for CC dimer formation with inhibitory peptide, removes the INH peptide which results in formation of inhibitory and regulatory peptide CC heterodimer and releases the inserted peptide (INS) and target protein regains its activity (ON state).
N7 peptide and release of inhibition can be delivered through several methods in the inverted INSRTR system; A) Delivery of REG N7 peptide via coexpression, regulated by transcription,; B) Delivery of synthetic REG N7 peptide via liposomal agent DOTAP, externally added peptide with transfection reagent. In addition to different delivery systems, we demonstrated how the addition of a binding N7 REG peptide, regardless of type of delivery regulates luciferase activity. Orthogonal P4 REG peptide that does not bind to the inserted peptide has no effect. C) Scheme of chemically activated protease regulation of INSRTR; protease PPVp that cleaves the linker peptide between the inhibitory peptide and target protein is activated by a chemical signal, in this case Rapa (rapamycin) that leads to the interaction between FKBP and FRB that are fused to the split PPVp protease.
Scheme of the design principle of building modules for allosteric logic circuits based on allosteric regulation. Target protein with inserted peptide (INS) was genetically fused to the inhibitory peptide (INH) by flexible linker (link) with inserted protease cleavage site. Addition of protease results in proteolytic cleavage at the protease cleavage site, releasing the inhibitory peptide from target protein.
HEK293T cells were transfected with plasmids coding for allosterically regulated luciferase constructs as schematically represented in
Peptide N8 was inserted into β-galactosidase on various positions. A) β-galactosidase an exoglycosidase, which hydrolyzes the β-glycosidic bond formed between a galactose and its organic moiety. Activity of all designed β-galactosidase INSRTR versions in the absence or presence of 100 ng of plasmid encoding for regulatory peptide mCherry_N7. B) Insertion position of INS peptide N8 shown here is A239. Inhibition of β-galactosidase_A239 INSRTR variant by titration of co-transfected plasmid encoding regulatory peptide N7.
Peptide P7 was inserted into TEVp at various positions. A) Activity of all designed TEVp INSRTR versions in the absence or presence of 50 ng of plasmid coding for a regulatory peptide mCherry_N8, TEV (Tobacco-etch virus) protease is a highly sequence-specific cysteine protease. B) Insertion position of INS peptide P7 with flanking 5gs linkers shown here is 177. Inhibition of TEVp_177 INSRTR variant by titration of co-transfected plasmid encoding peptide N8, C) Activation of TEVp_177 INSRTR variant (ON switch) with inserted peptide P7, fused to inhibitory peptide by titration of co-transfected plasmid encoding regulatory peptide N7. Autoinhibited (TEVp) with INS peptide P7, INH peptide N8.
Peptide P7 was inserted into Lck at various positions. A) Activity of designed Lck INSRTR variants in the absence or presence of 50 ng plasmid encoding for the regulatory peptide mCherry_N8, Lck kinase, a member of the tyrosine kinases in lymphocytes. B) The insertion position of INS peptide P7 with flanking 5gs linkers shown here is N266. Inhibition of INSRTR variant Lck_N266 by titration of a co-transfected plasmid encoding regulatory peptide N8.
Peptide N8 was inserted into mIRAK-1 at various positions. A) Activity of designed mIRAK-1 INSRTR variants in the absence or presence of 90 ng of plasmid encoding for regulatory peptide mCherry_N7; the interleukin-1 receptor (IL-1R) associated kinase 1 (mIRAK1) is a member of serine/threonine-protein kinase. B) Insertion position of INS peptide N8 with flanking 5gs linkers shown here is F212. Inhibition of mIRAK-1_F212 INSRTR variant by titration of co-transfected plasmid encoding regulatory peptide N7.
Peptide N8 was inserted into mMyD88 at various positions. A) Activity of designed mMyD88 INSRTR variants in the absence or presence of 90 ng of plasmid coding for the regulatory peptide mCherry_N7. MyD88 is involved in the innate immune response as adaptor protein in Toll-like receptor and IL-1 receptor signaling pathway. B) Insertion position of INS peptide N8 with flanking 5gs linkers shown here is N170. Inhibition of mMyD88_N170 INSRTR variant by titration of co-transfected plasmid encoding regulatory peptide N7
Transcription activator-like effectors (TALEs) is a DNA binding protein that in fusion with VP16 activator domain acts as a transcription activator. Peptide N6 was inserted into TALE-A at T496 position. Peptide N8 was fused at the C-terminal of TALE-A protein. Activation domain VP16 was brought to the DNA binding site by N8-N7 CC dimer formation. A) Insertion position of INS peptide N6 shown here is T496. Inhibition of TALE-A_T496 INSRTR variant by titration of co-transfected plasmid encoding regulatory peptide N5 with NLS signal. B) Activation of TALE-A_T496 INSRTR variant with inserted peptide N6, fused inhibitory peptide N5 by titration of co-transfected plasmid encoding a regulatory peptide N6 with NLS signal. Autoinhibited TALE with INS peptide N6, INH peptide N5.
Peptide N8 was inserted into dCas9 at various positions. A) Activity of designed dCas9 INSRTR versions in the absence or presence of 90 ng of plasmid encoding for regulatory peptide mCherry_N7. Nuclease-dead Cas9 (dCas9) is a catalytically inactive mutant of Cas9 protein. B) Inhibition of dCas9_R535 INSRTR variant by titration of co-transfected plasmid encoding peptide N7 with NLS signal. Insertion position of INS peptide N8 with flanking 5gs linkers shown here is R535. C) Activation of dCas9_R535 INSRTR variant with inserted peptide P7, fused inhibitory peptide N8 by titration of co-transfected plasmid encoding peptide N7 with NLS signal. Autoinhibited dCas with INS peptide P7, INH peptide N8.
CAR-T receptor molecule; Single-chain variable fragment of CD19 antibody (scFv-CD19). Peptide P7 was inserted into scFV CD19 CAR at S196 position.
HEK293T cells were transfected with plasmids coding for fluorescent protein Neon green (ngGFP) with INSRTR strategy and Regulatory peptide (REG) fused to fluorescent protein mCherry. A) Peptide P7 was inserted at position C143. Inhibition of ngGFP_C143 green fluorescent protein by co-transfection with mCherry fused N8. Neon green is a green fluorescent protein. Insertion position of INS peptide P7 with flanking 5gs linkers shown here is C143. B) Activation of ngGFP_C143 INSRTR variant with inserted peptide P7, fused inhibitory peptide N8 by co-transfection with mCherry fused to N7. Autoinhibited ngGFP with INS peptide P7, INH peptide N8.
The term “target protein” refers to any selected protein whose function is modified by insertion of a (coiled-coil dimer forming) peptide into one of its solvent exposed loops.
The term “inserted peptide” refers to a peptide that is inserted into the loop of the target protein, wherein the peptide by itself is preferentially not folded but has tendency to form a complex, preferentially a coiled-coil dimer upon interaction with a regulatory or inhibitory peptide.
The term “regulatory peptide” refers to a peptide that is added and interacts with an inserted peptide in the loop of the target protein forming a structured complex such as the coiled-coil dimer or interacts strongly with an inhibitory peptide.
The term “inhibitory peptide” refers to a peptide that interacts, preferentially forming a coiled-coil dimer, with an inserted peptide wherein the inhibitory peptide is genetically fused to the target protein at its N-or C-terminal ends or both ends and is connected to a target protein through a flexible peptide linker, preferably consisting of flexible small hydrophilic amino acid residues of sufficient length, typically 10-30 amino acid residues, long enough to enable the inhibitory peptide to bind to the inserted peptide within the same molecule.
The term “coiled-coil dimer” refers to the polypeptide motif composed of two peptides, either as individual molecules or as segments within the protein, that have the propensity to interact specifically with each other to form a dimer of helices that are typically wound each around other to form an elongated superhelix. The term “coiled-coil dimer” as used herein, unless explicitly specified, refers to two different peptides forming a heterodimer.
The term “peptide”, as used herein, refers to the polymeric form of amino acids, shorter than protein, typically shorter than 60 amino acid residues, which is used to interact with another peptide or to connect functional segments of proteins or peptides or to have some other biological or chemical function.
The term “protein”, as used herein, refers to the polymeric form of amino acids of any length, which expresses any function, for instance localizing to a specific location, localizing to specific DNA sequence, facilitating and triggering chemical reactions, transcription regulation, structural function, and biological recognition.
The term “target protein” as used herein refers to the protein whose function is modified by modifications such as the insertion of a peptide, genetic fusion with the inhibitory peptide ad addition of a regulatory peptide or addition of a small molecule that affects the activity of a protease or other proteins.
The term “function” of a target protein refers to the property of the protein that is characteristic for each protein and includes the catalytic activity, tertiary structure, dynamics, stability, interactions with other molecules, physicochemical properties and biological activity either in vitro or within cells or organisms.
The term “protein domain”, as used herein, refers to a folding functional unit of a protein. For example, a part of a protein that can be folded and expressed independently of the entire protein and is typically composed of one or more secondary structural elements, such as alpha helices or beta strands.
The term “antibody” as used herein refers to the protein that is able to bind to other molecules due to the amino acid sequence and conformation of the loops of the proteins that contribute to interactions whereas this protein belongs to the immunoglobulin family and refers also to the antibody fragments comprising variable antibody domain in two chains or connected into a single chain or nanobodies and its molecule binding fragments, which are composed of a single heavy chain.
The term “enzyme” as used herein refers to the protein whose function is to catalyze a reaction so that it occurs faster, with higher efficiency under the physiological conditions.
The term “allostery” or “allosteric” refers to the effect that interaction of an effector molecule with a target protein has on the properties of the protein, such as its activity or binding, while the effector allosteric binding to site is not the same site, which is involved in protein function.
The term “INSRTR” used herein refers to the technique of affecting the function or properties of the target protein through the addition of a regulatory peptide that interacts with either inserted or inhibitory peptide genetically incorporated into the target protein.
The term “ligand”, used herein, refers to any small molecule with low molecular weight (<5000 Da and preferably <900 Daltons). The ligands include but are not limited to for example lipids, monosaccharide, second messengers, hormones, inhibitors, other natural products and metabolites, as well as drugs and other synthetic small molecules.
The term “sensor”, used herein refers to a molecule or molecular complex where the presence of a selected ligand triggers the generation of measurable output signal as e.g. emitted light, fluorescence, electric current, or other chemical or physical signal or change of physicochemical property.
The term “genetic fusion”, as used herein refers to the insertion of DNA coding for the inserted peptide into the DNA coding region of the target protein, wherein the resulting DNA codes for the protein with an inserted peptide, which can be fused to the amino-or carboxy terminus of said protein or inside the protein polypeptide sequence. The single chain polypeptide comprises polypeptide of two or more constituents that are consecutive or between them are short linker polypeptides that prevent steric overlap, typically comprising 1-10 small polar flexible amino acid residues, typically glycine or serine or similar small hydrophilic amino acid residues.
The term “cell”, used herein, refers to a eukaryotic or prokaryotic cell, a cellular or multicellular organism (cell line) cultured as a single cell entity that has been used as a recipient of nucleic acids and includes the daughter cells of the original cell that has been genetically modified by the inclusion of nucleic acids. The term refers primarily to cells of higher developed eukaryotic organisms, preferably vertebrates, preferably mammals. This invention relies also on non-vertebrate cells, preferably plant cells.
The term “cells” also refers to human or animal primary cells or cell lines. Naturally, the descendants of one cell are not necessarily completely identical to the parents in morphological form and its DNA complement, due to the consequences of natural, random or planned mutations. A “genetically modified host cell” (also “recombinant host cell”) is a host cell into which the nucleic acid has been introduced. The eukaryotic genetically modified host cell is formed in such a way that a suitable nucleic acid or recombinant nucleic acid is introduced into the appropriate eukaryotic host cell. The invention hereafter includes host cells and organisms that contain a nucleic acid according to the invention (transient or stable) bearing the operon record according to the invention. Suitable host cells are known in the field and include eukaryotic cells. It is known that proteins can be expressed in cells of the following organisms: human, rodent, cattle, pork, poultry, rabbits and the like. Host cells may include cultured cell lines of primary or immortalized cell lines.
The term “recombinant”, as used herein, means that a particular nucleic acid (DNA or RNA) is a product of various combinations of cloning, restriction and/or ligation or chemical synthesis leading to a construct having structurally coding or non-coding sequences different from endogenous nucleic acids in a natural host system.
The term “nucleic acid”, used herein, refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length and is not limited to single, double or higher chains of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers with a phosphorothioate polymer backbone made from purine and pyrimidine bases or other natural, chemical or biochemically modified, synthetic or derived nucleotide bases.
The term “DNA-binding domain”, used herein, refers to any protein domain with the ability to bind a DNA molecule. The DNA-binding protein could be of natural origin or artificially designed whole protein or only a segment with characteristic to bind to nucleic acid in sequence specific manner.
The term “effector domain”, in the description refers to any protein domain with a specific function, for example, but not limited to nuclease domains, transcriptional activation domains, and chromatin silencing domains.
The insertion of the vectors into the host cells is carried out by conventional methods known from the field of science, and the methods relate to transformation or transfection and include e.g.: chemically induced insertion, electroporation, micro-injection, DNA lipofection, cellular sonication, gene bombardment, viral DNA input, as well as other methods. The entry of DNA may be of transient or stable. Transient refers to the insertion of a DNA with a vector that does not incorporate the DNA of the invention into the cell genome. A stable insertion is achieved by incorporating DNA of the invention into the host genome. The insertion of the DNA of the invention, in particular for the preparation of a host organism having stably incorporated a nucleic acid, e.g. a DNA, of the invention, can be screened by the presence of markers. The DNA sequence for markers refers to resistance to antibiotics or chemicals and may be included on a DNA vector of the invention or on a separate vector.
The present invention refers to the regulation of the function of selected target proteins by insertion of a peptide, preferably in the range between 20 and 50 amino acid residues (e.g. 30-40 amino acid residues), into an appropriate position that maintains the particular function of the target protein. Upon addition of a regulatory peptide that binds to the inserted peptide, a coiled-coil dimer is formed between the regulatory peptide and the inserted peptide, changing its conformation from a random structure to a helical structure, which increases the distance between the termini of the inserted peptide. This locally disrupts the structure of the target protein and inhibits its function. The invention can be used to activate or inactivate function, structure and other properties of a target protein and can be used to regulate processes such as enzymatic activity, recognition of other molecules, signaling, binding to other molecules, cellular localization, optical and other properties, which are useful for pharmacological, therapeutic, diagnostic, sensing, biotechnological and industrial applications.
Peptides that serve as the inserted peptide, inhibitory peptide or regulatory peptide are selected from the set of designed coiled-coil dimers that typically comprise 20-50 amino acid residues. The peptides are in isolation weakly structured or nonstructured and only form a coiled-coil dimer in the presence of the partner peptide from the set of designed coiled-coil heterodimers (Drobnak et al., 2017; Gradišar & Jerala, 2011; Plaper et al., 2021) (
The insertion site is selected in a manner that insertion of a peptide maintains the function of the protein that we are interested in, such as the catalysis, dynamics or binding. According to the invention, typically several insertion sites can be functional for each protein.
In the present invention the appropriate target protein site is identified by testing the function of the target protein with an inserted peptide inserted at different, typically solvent-exposed, loop sites that do not participate directly in protein function. The position of the insertion within the selected protein is selected in such way that the target protein with an inserted peptide retains the function. In the presence of an interaction peptide, the target peptide forms a coiled-coil dimer with a peptide inserted into the target protein loop, which impairs the function of the selected protein.
The present invention also refers to the method of activation of a target protein function (ON switch), where the target protein is initially inhibited through a combination of an inserted peptide as described above with an additional C- or N-terminal fusion to the target protein of an inhibitory peptide that interacts weakly with an inserted peptide and forms a coiled-coil dimer with a inserted peptide within a target protein, wherein the inhibitory peptide is genetically fused to the target protein via a flexible peptide linker. Weaker affinity between the inserted peptide and an inhibitory peptide in this context means affinity in the micro-to millimolar concentration range, that is sufficient to form intramolecular coiled-coil dimer yet can be displaced by a stronger, binding regulatory peptide, with affinity in the nanomolar or lower range. Weaker affinity between the inserted peptide and inhibitory peptide also ensures that the inhibitory peptide dissociates if it is no longer covalently connected to the target protein, which can be accomplished by the proteolytic cleavage of the linker between the protein and inhibitory peptide.
Said target protein can be activated by the addition of a regulatory peptide that strongly interacts with an inhibitory peptide or through a protease that cleaves the linker between the target protein and the inhibitory peptide. If a regulatory peptide with high affinity for the inhibitory peptide is added the inserted peptide is outcompeted from the inhibitory peptide and the protein regains the activity.
The present invention can be further implemented to regulate the function of the target protein by a protease whose activity can be regulated through small molecules that can trigger reconstitution of a split protease or though proteases characteristic for biological processes, such as e.g. proteases specific for the cell type, physiological cell state or a microbial protease, for example viral protease present due to the infection.
For precise recognition or response more than one input signal may be required, that can more precisely specify the context in which a selected biological process should be activated. The presence of the first signal and the absence of the second signal or presence of both signals or presence of at least one of the two signals or other combinations of input signals define logic functions, which could be beneficial to precisely specify activation of the selected protein that may have a role in a physiological, therapeutic or biotechnological process. The invention describes construction of those logic functions that are achieved by combinations of insertions and fusions of interacting peptides and protease cleavage sites that can be combined to generate logic functions that combine two or more input signals that define under which conditions the selected protein is in active or inactive form. The proteins can also be proteases that are able to represent an input signal to the logic gates at higher level and generate more complex logic circuits.
This invention refers to activation or inhibition of the diverse biological function or biological activity such as catalytic activity or binding or other function of various target proteins such as, but not exclusively, enzymes, such as luciferase, hydrolases, kinases or proteases, proteins that constitute signaling pathways, such as protein kinases, signaling mediators that recruits other proteins to the signaling complex, further it refers to proteins which bind specific nucleic acid sequences or that act as transcriptional regulators, and proteins that can be detected and act as reporters, such as fluorescent proteins or luciferases and further it refers to other molecule-recognizing domains such as antibodies and their domains or to structural proteins.
The disclosed invention refers to functional proteins whose activity can be inhibited or activated by the addition or presence of a chemical or biological signal that affects the formation of the dimer involving the inserted peptide in the loop of the target protein.
The disclosed invention refers to the method of regulating the activity of proteins and said proteins and nucleic acids encoding them and cells producing said proteins.
In the particular embodiment, the disclosed invention refers to the activation or inhibition of the biological and biochemical function of proteins luciferase, beta-galactosidase, IRAK-1 kinase, MyD88, Lyn kinase, transcription activator-like effector and its implementation as DNA binding domains, Cas9 gRNA-dependent DNA nuclease or specific DNA binding domain, fluorescent proteins, antibody variable domain (Fv), whose activity can be regulated through interactions with a peptide that forms a coiled-coil heterodimer with a peptide inserted into the loop of the target proteins.
In the particular embodiments, the cells such as the mammalian or eukaryotic or bacterial cells exhibit the function of target proteins depending on the applied signal, which triggers formation or reverses formation of a coiled-coil dimer within the target protein loop that results in the modification of the biological properties or cells that produce the modified target proteins, which can be used for medical, biotechnological or other application of cell function.
Moreover, the present invention refers to nucleotide sequences comprising coding sequences for polypeptides described above according to the invention, optionally incorporated in a delivery vector such as a plasmid, a linear or circular nucleic acid or a virus or inserted into the genome of cells. In a further aspect, the present invention refers to a protein comprising the polypeptide according to the invention.
The invention refers to a method of regulating a function of a target protein by:
The invention refers to the method to allosterically regulate function of the target protein wherein the peptide is inserted into said target protein where the regulatory peptide pair to regulate function of the said protein are selected from the designed heterodimeric pairs comprising or consisting of the amino acid sequences as shown in SEQ ID NO: 121 to SEQ ID NO: 146.
The invention refers to the method of regulating a protein function, wherein:
An inserted peptide that is by itself not structured is inserted into the selected target protein at such position of the target protein, preferably into a solvent exposed loop, that the function of the protein is maintained.
Inhibitory peptides that can interact with the inserted peptide in the target protein according to a) are fused to the target protein via a nonstructured linker peptide, preferentially comprising 3 to 30 amino acid residues, more preferably 5 to 20 amino acid residues or 10 to 15 amino acid residues, wherein the inhibitory peptides can bind to the inserted peptide within the same molecule thereby inhibiting the function of said protein in the constitutively expressed state, wherein the function of the target protein can be regained by the addition of a regulatory peptide which binds tightly to the inhibitory peptide or by cleaving the linker between the inhibitory peptide and the target protein by a protease that has a recognition sequence within the nonstructured linker.
The invention refers to a method of allosteric regulation of a protein function based on insertion of a peptide into said target protein and fusion of an inhibitory protein to the target protein, wherein the activity of the target protein is regulated by the proteolytic cleavage by a protease that cleaves between the target protein and the inhibitory peptide, wherein the protease can be regulated by a small molecule or wherein a protease is used that is characteristic for a desired physiological process or is derived from pathogen, such as a virus, bacteria, fungi or a parasite.
The invention refers to a method of allosteric regulation of protein function by insertion of a peptide, where the function of the target protein is regulated by combination of input signals that affect the structure of the insert peptide which comprise logic functions to combine several chemical or biological signals.
The invention refers to proteins allosterically regulated by insertion of a peptide where the function of the target proteins can be regulated by the addition or release of a peptide that regulates the function of the target protein wherein the target protein is an enzyme or a DNA binding protein domain or a signaling protein.
The invention refers to proteins allosterically regulated by the peptide that interacts with the peptide inserted or fused to the target protein where the target protein is a firefly luciferase or MyD88 or IRAK1 or Lck kinase or beta-galactosidase or tobacco-etch virus protease or a Transcription-activator-like effector or Cas9 or antibody or its single chain variable domain (scFv) comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, or 120.
The invention refers to an allosterically regulated protein based on insertion of a peptide into a target protein wherein the target protein is an antibody or nanobody or its single chain variable domain (scFv) of SEQ ID NO: 116.
The invention refers to nucleic acids coding for proteins with sequences as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, or 145.
The invention refers to cells containing nucleic acids coding for allosterically regulated proteins based on insertion of a peptide into the target proteins where the properties of the cell are regulated by the proteins encoded by the nucleic acids and peptides interacting with them.
The inventions refer to a method of regulating the response of immune cells, preferentially T cells expressing chimeric antigen receptor (CAR), wherein the recognition of target cells is achieved by an allosterically regulated antibody domain, wherein the recognition of proteins on target cells can be regulated by the addition of a peptide that binds to the insert in the variable fragment of the antibody and affects its recognition of target cells through the allosteric effect.
The invention refers to proteins comprising or consisting of an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 144, or 146.
The examples described in further detail have no intention of limiting the scope of the invention and its applicability, but are merely intended to provide a better understanding of the invention and its applicability.
DNA constructs were prepared using methods of molecular biology that are described in any molecular biology handbook and are known to experts. In order to prepare DNA constructs, the inventors used experimental techniques and methods such as: chemical synthesis of DNA with a defined polynucleotide sequence, DNA fragmentation with restriction enzymes, DNA amplification using polymerase chain reaction-PCR, PCR ligation, DNA concentration determination, agarose gel electrophoresis, purification of DNA fragments from agarose gels, ligation of DNA fragments into a vector, the Gibson assembly method, transformation of chemically competent cells E. coli DH5α, isolation of plasmid DNA with commercially available kits, screening and selection. DNA segments were characterized by restriction analysis and sequencing.
All plasmids, completed constructs and partial constructs were transformed into bacterium E. coli DH5alfa by chemical transformation. Plasmids for transfection into the cell line HEK293T were isolated using GeneJet Plasmid DNA Isolation Kit (In Vitrogen).
All protein coding constructs have a Kozak sequence (GCCACC) before the coding region and were cloned into the pcDNA3 backbone vector for high-level expression in mammalian cells.
In the described examples, the target proteins are modified by an inserted peptide, introduced into the positions determined from the tertiary structures or molecular models of proteins which are located in solvent exposed loops, that are close to the active or functional site of the target protein, typically within 2-3 nm.
The DNA coding for the modified target proteins were in-frame inserted into vectors. All protein-coding DNA construct demonstrated as examples are listed in Table 1.
5gs linker of 5 aminoacids in length consisting of serine and glycine 10gs linker of 10 aminoacids in length consisting of serine and glycine
To demonstrate the design of inhibition of protein function by regulatory peptides, the inventors inserted the insertion peptide into the target protein into the position exposed to the solvent in the vicinity but not directly in the active or binding site of the target protein. The inserted peptide is selected from the set of designed pairs of coiled-coil heterodimers (Drobnak et al., 2017; Gradišar & Jerala, 2011; Plaper et al., 2021), where each individual peptide is in isolation intrinsically unstructured, however in the presence of the regulatory peptide, which is its designed binding partner, it adopts a helical secondary structure, and the inserted and regulatory peptides form a coiled-coil dimer (
Plasmids encoding for the split firefly luciferase, were transiently transfected into the HEK293T cell line. A Renilla constitutively expressed luciferase (phRL-TK http://www.promega.com/vectors/prltk.txf) was used as control of transfection efficiency to quantitatively determine the effect on the firefly activity.
Methods and techniques for cell line cultivation are well-known to experts in the field and are explained here only indicatively, with the intention of clarifying the example. Cell cultures of HEK293T cells were grown at 37° C. and 5% CO2. For cultivation, DMEM medium with 10% FBS, containing all the necessary nutrients and growth factors was used. When the cell population reached a sufficient density, the cells were transferred to a new flask and/or diluted. For use in experiments, the number of cells was determined by the Countess automated cell counter (Invitrogen). A 96-well microtiter plate, suitable for growing cell cultures, was inoculated with 2*104 cells per well 24 hours prior to transfection. Inoculated plates were incubated at 37° C. and 5% CO2. At 30-90% confluence, they were transfected with a mixture of DNA and PEI (6 μl/500 ng DNA, stock concentration 0.324 mg/ml, pH 7.5) and further incubated at 37° C. and 5% CO2. Cells were lysed 24-48 hours after transfection with passive lysis buffer (Promega) according to manufacturer's instructions. The inventors first measured firefly activity (Fluc), followed by renilla luciferase activity (Rluc). Renilla luciferase is expressing independently and presents the transfection efficiency; firefly luciferase is a direct output for our designed systems. The Fluc/Rluc ration (RLU; relative luciferase units) is thus the Fluc values normalized to transfection efficiency of each individual well.
Results: The results shown in
In
Besides the inhibition of target protein activity or other function, it is sometimes desirable to have the protein in the inactive state until the signal for activation is applied. The inventors demonstrated the adaptation of inserted peptide for such an occasion. The inventors generated autoinhibited firefly luciferase reporter wherein the peptide was inserted peptide into loop and at the C-terminus of the luciferase the inventors extended the protein by 20 amino acids long flexible linker with a further appended sequence of the inhibitory peptide. Inhibitory peptide was selected to have a weaker affinity to the inserted peptide; however due to high local concentration the inhibitory peptide and inserted peptide as they are in the same polypeptide chain they form an intramolecular coiled-coil dimer. This disrupts the luciferase tertiary structure, maintaining the target protein in the inhibited state. Upon the addition of a regulatory protein, which was selected to have high affinity for the inhibitory peptide, the low affinity inhibitory inserted peptide coiled-coil is disassembled and a high affinity inhibitory peptide-regulatory peptide coiled-coil dimer forms. This leads to release of the inserted peptide, which loses its secondary structure and allows the target protein to attain its original tertiary structure and regain the functionality (
Different ways for delivery of the regulatory peptide are possible. The inventors present three different strategies, although additional types of delivery known in the art are possible: co-expression regulated by the transcriptional regulation; synthetic peptide delivery into cells; externally regulated protease that cleaves the linker between the protein and inhibitory peptide, which leads to the inhibitory peptide release.
The plasmids encoding for INSRTR modified firefly luciferase, were transiently transfected into the HEK293T cell line. A constitutively expressed Renilla luciferase (phRL-TK http://www.promega.com/vectors/prltk.txf) was used as control of transfection efficiency.
The HEK293T cells were cultured and transfected as described above. For the regulatory peptide delivery with transfection reagent, 24 hours after transfection cells were stimulated with the mix of peptide with DOTAP transfection reagent. After 6 hours the media was removed and cells were lysed and measured as described above. External regulation of target protein activity was achieved with rapamycin stimulation 24 hours post transfection. Cells were lysed 24 hours post stimulation and measured as described above.
Results: As seen in
For precise recognition or response more than one input signal may be required, that can more precisely specify the context in which a selected biological process should be activated. Presence of the first signal and the absence of the second signal, presence of both signals, presence of only one signal and other combinations define logic functions, which could be beneficial to specify activation of the selected protein. The invention describes those logic functions.
The HEK293T cells were cultured, transfected and stimulated as described above.
Results: The results shown in
In one embodiment of the invention, logic functions can be designed based on the combination of chemical signals that affect the function of a target protein. To this end, split PPV and SbMV protease were used that are regulated by addition of rapamycin or abscisic acid. We constructed firefly luciferase with P7 coiled-coil-forming segment inserted at position K490 and intramolecular fusion of the N8 CC segment at the C-terminal ends. This allows intramolecular binding of CC segments, forming a dimer, and resulted in constitutive inactivation of firefly luciferase. The cleavage site for the PPV protease was introduced into the flexible linker between the C-termini of the luciferase and the N8 coiled-coil forming segment. Addition of rapamycin restored the function of PPV protease which cleavage of the N8 coiled-coil, resulting in an ON switch. In the same manner, logic functions can be constructed for other proteins that are allosterically regulated by the formation of an extended structure in the loop of the target protein.
β-galactosidase is an exoglycosidase which hydrolyzes the β-glycosidic bond formed between a galactose and its organic moiety. It is a powerhouse of energy production by breaking down lactose to galactose and glucose and is therefore commonly used in food industry for production of lactose-free products. Amino acids chosen for insertion of the inserted peptide were L25, A35, T45, L55, D223, A229, A239, D507, P513, D610, T799 and S1000.
HEK293T cells were cultured and transfected as described in the second example. At the time point of 24-48 hours post transfection, activity of beta-galactosidase was assessed with β-gal reporter gene assay, chemiluminescent (Roche) by manufacturers' instructions. Luminescence produced as a result of a chemical reaction directly correlates with the amount of active beta-galactosidase. Luminescence was recorded with Luminescence plate reader Orion II.
Results: As seen in
Tobacco-etch virus protease (TEVp) is a highly sequence-specific cysteine protease. In synthetic biology, it is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo. Many proteases are inactive in innate state and regain their activity upon specific signal is applied. The inventors therefore present INSRTR method for both downregulation and upregulation of TEVp activity. Amino acids chosen for insertion of the inserted peptide were G27, L72, 177, G79, K147, F172, T175, K184, G212.
HEK293T cells were cultured, transfected and measured as described in the second example. Cyclic firefly luciferase reporter with TEVp cleavage site (cycLuc_tevs) was used to determine the activity of TEVp.
Results: Results in
Lck kinase is a member of the SRC family of non-receptor tyrosine kinases. They act by phosphorylating tyrosine residues, thereby regulating the activity of proteins involved in intracellular signaling of T cells. Lck kinase is the first kinase in the TCR signaling pathway and its activity controls the entire orchestra of kinases/phosphatases. N266, Q256, G278, D374 were chosen as amino acids for the insertion of the peptide. A specific phosphorylation-based luciferase reporter was constructed by introducing the phosphopeptide binding domain (SH2) and Lck specific substrate (GPLDGSLYAKVKKKD) into the luciferase such that the engineered luciferase becomes inactive and regains activity only when Lck kinase is present. HEK293T cells were cultured, transfected and relative luciferase activity was measured as described in the second example.
Results:
The interleukin-1 receptor (IL-1R) associated kinase 1 (mIRAK1) is a member of serine/threonine-protein kinase. It plays a critical role against foreign pathogens by initiating innate immune response. Amino acids chosen for insertion of the inserted peptide were F212, G221, R232, S281.
HEK293T cells were cultured, transfected and measured as described in the second example. Activity of mIRAK1 was assessed by detection of downstream signaling cascade protein NF-KB. Reporter plasmid included NF-KB binding site in front of minimal promotor for firefly luciferase.
Results:
MyD88 is involved in the innate immune response as adaptor protein in Toll-like receptor and IL-1 receptor signaling pathway. Amino acids chosen for insertion of the inserted peptide were located in death-domain T66, G80; and in Toll-Interleukin receptor (TIR) domain N170, S209, S224.
HEK293T cells were cultured, transfected and measured as described in the previous example.
Results: Results in
Transcription activator-like effectors (TALEs) is a DNA binding protein that in fusion with VP16 activator domain acts as a transcription activator. Amino acids chosen for insertion of the inserted peptide were T496.
HEK293T cells were cultured, transfected and measured as described in the second example. Reporter plasmid with ten TALE binding sites in front of the minimal promoter for firefly luciferase expression was used to determine the activity of TALE.
Results: Results in
Nuclease-dead Cas9 (dCas9) is a catalytically inactive mutant of Cas9 protein. In synthetic biology it is mainly used as a DNA binding protein which is guided to a specific DNA site by guide RNA (gRNA). In fusion with activator domain like VP16 or VPR it acts as a transcriptional activator. Amino acids chosen for insertion of the inserted peptide were S55, D147, R535, G1104, K1153.
HEK293T cells were cultured, transfected and measured as described in the second example. Plasmid with gRNA was used to direct dCas9 to the TALE binding sites on the reporter plasmid in front of the minimal promoter for firefly luciferase expression was used to determine the activity of dCas9.
Results: As shown in
The second generation CD19 CAR-T, bearing FMC63 single-chain variable fragment, with 4-1BB and cd35 costimulatory domain recognizes CD19 ligand expressed by normal B cells and B cell leukemias and lymphomas. Chimeric antigen receptor composed of the scFv domain, hinge, transmembrane domain, 4-1 BB and CD35 domain endows T cells with specific B-cell recognition and killing ability. The ability to control the activity of CAR T cells is very important for therapy as the excessive activation of CAR T cells may lead to the adverse therapeutic effects, such as the cytokine storm or on the other hand T cell exhaustion, which may either harm the patient or impair the anticancer ability of CAR T cells. Therefore, introduction of the ability to reduce the affinity of scFv to cancer antigens, such as CD19 but also others could be used to improve the therapeutic efficiency. Amino acid position for insertion of the inserted peptide into the scFv antigen recognition domain was S196.
Jurkat cells were grown at 37° C. and 5% CO2. For cultivation, RPMI 1650 medium with 10% FBS, containing all the necessary nutrients and growth factors was used. When the cell population reached a sufficient density, the cells were diluted. For use in experiments, the number of cells was determined by the Countess automated cell counter (Invitrogen). Jurkat cells were mixed with a plasmid (10 μg), electroporated using Neon transfections system according to manufacturer's instructions and seeded into 2 mL of fresh RPMI 1650 medium. The next day, 24 h post electroporation the cells were counted and seeded into 96 well plate with target Raji cells in the ratio Effector (Jurkat-CAR-T): Target (Raji)=10:1. Stimulation with target cells was terminated after 24 hours by removal of media. Media was used for ELISA detection of interleukin-2 (IL-2), produced as a result of Jurkat CAR-T cell activation. The amount of IL-2 directly corresponds to activity of scFv-CD19.
Elisa test was performed to determine secreted hIL2 from electroporated and stimulated Jurkat cells. High binding half-well plates (Greiner) were used. Human IL-2 was measured using standard ELISA assay according to manufacturer's protocol (hIL-2 ELISA Invitrogen 88-7025-88). In brief, plates were coated with primary antibody and incubated overnight (4° C.) Next day plates were washed with PBS+0,05% Tween20 using ELISA plate washer (Tecan). Next, plates were blocked for 1 h at RT with ELISA diluent (PBS+3% FBS) solution. Afterwards, plates were again washed. Then serial dilution of hIL2 standard and 1:2 diluted samples were added and incubated at RT for 2 h. Next, plates were washed and afterwards detection antibody was added. Plates were incubated 1 h at RT. Next plates were washed and HRP conjugated avidin was added and incubated for 30 min. After the addition of substrate (TMB solution) the reaction was stopped with 0.16 m sulfuric acid. The plates were read on a microplate reader at 450 nm, and again at 630 nm for correction by subtraction of the reading at 630 nm from that at 450 nm.
Results:
Green fluorescent protein ngGFP is frequently used as a reporter. The inventors therefore present INSRTR method for both downregulation and upregulation of ngGFP activity. Amino acid chosen for insertion of the inserted peptide was C143.
HEK293T cells were cultured and transfected as described in the second example. Activity of fluorescent protein ngGFP was measured with confocal microscopy.
Results: Results in
Dagliyan, O., Shirvanyants, D., Karginov, A. V., Ding, F., Fee, L., Chandrasekaran, S. N., Freisinger, C. M., Smolen, G. A., Huttenlocher, A., Hahn, K. M., & Dokholyan, N. V. (2013). Rational design of a ligand-controlled protein conformational switch. Proceedings of the National Academy of Sciences of the United States of America, 110(17), 6800-6804. https://doi.org/10.1073/pnas.1218319110
| Number | Date | Country | Kind |
|---|---|---|---|
| 501018 | Dec 2021 | LU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/085613 | 12/13/2022 | WO |
