Patent Application
20210179682
The invention pertains to the field of molecular means capable of binding calcium, in particular peptides which are calcium chelators, appropriate for use in vitro or in vivo and preferably capable of targeting specific cellular compartments.
Calcium (Ca2+) is a second messenger central in multiple cellular responses ranging from metabolism and survival to vesicle release and motility. The blockade of Ca2+ signals currently relies on pharmacological strategies to block Ca2+ influx into the cell or chelate the extracellular or intracellular Ca2+. Since Ca2+ is crucial for many signaling pathways and for the function of a wide range of components of the extracellular matrix, those strategies lack cellular specificity and are plagued with a variety of side effects when applied to patients. Since their first appearance, the rise of optogenetics has provided a wide variety of tools to increase intracellular Ca2+. This has been widely used to control electrical activity in excitable cells and provided a vast knowledge focused on neuronal interactions and even opened new promising fields in therapeutics. Nevertheless, this strategy relies on imposing additional Ca2+ influx from the extracellular medium, with no possibility of manipulating endogenous Ca2+ variations, reduce Ca2+ concentration or to target Ca2+ release from internal stores. In addition, the use of optogenetics is handicapped in vivo, when light stimulation are technically challenging (e.g. during early stages of development).
There is thus a need for improved means to manipulate calcium signaling, and in particular intracellular calcium concentration.
The helix-loop-helix calcium-binding motif (EF-hand motif) is the most prevalent Ca2+ binding site in proteins. The canonical EF-hand motif is of about 29 amino acids in length and is structured by two alpha-helices bridged by a flexible calcium-binding loop composed of 12 highly conserved residues.
The inventors have set up polypeptides comprising calcium-binding domains derived from EF hand motifs. As a result, the polypeptides of the invention interact directly with calcium, rather than modifying the activity of calcium channels. They can therefore be utilized as chelators of intracellular Ca2+.
Said polypeptides are formed of two calcium-binding domains having different binding affinity for Ca2+, and which are linked through a peptide linker. The inventors have in particular found that by associating calcium-binding domains derived from different EF-hand motif, and therefore having distinct affinities for Ca2+, the range of calcium-dependent cellular pathways susceptible to be affected is enlarged.
The polypeptides of the invention can be used in vitro and in vivo, for instance to prevent the activation of downstream effectors and calcium-dependent cellular processes. The polypeptides of the invention can easily be further functionalized using simple molecular tools, for instance by addition of fluorescent peptides or signal peptides, so as to target specific compartments within the cells, and can thus be employed in a wide variety of applications.
The invention pertains to a polypeptide comprising a first calcium-binding domain, a peptide linker and a second calcium binding domain, wherein said first and second binding domains are linked through said peptide linker, and wherein:
The polypeptide of the invention is preferably an isolated, recombinant or synthetic polypeptide.
In the context of the invention, the terms ‘differ in at least one calcium-binding site_ indicates that at least one of the calcium binding sites of one calcium-binding domain differs from any of the calcium binding sites of the other calcium-binding domain.
In the context of the invention, the terms ‘calcium-binding site derived from a EF-hand motif_ refer to peptide which sequence derives from the sequence of said EF hand motif by substitution, deletion or addition of amino-acid residues, and which has retained the ability to bind calcium.
The ability to bind calcium can easily be measured using well known methods, for instance using fluorescence methods such as thoroughly detailed by Johnson and Tikunova (Methods in Molecular Biology; 173: 89-102, 2002).
Preferably, each of the calcium-binding sites according to the invention derives from a canonical EF-hand motif.
In the context of the invention, the terms ‘canonical EF-hand motif_ should be construed as generally considered in the field, that is to say as a peptide sequence comprising about 29 amino-acid residues containing a first a-helix designated as E (residues 1-10), a calcium-binding loop (residues 10-21), and a second a-helix designated F (residues 19-29). This motif has been found in a large number of calcium-binding proteins.
Preferably, in the polypeptide of the invention, the calcium-binding sites are derived from canonical EF-hand motif from proteins chosen in the list consisting of Parvalbumin, Calmodulin, Calretinin, D28K, Calbindin, Recoverin, Hippocalcin, NCS-1, MELC, Cacineurin B, MRLC, Caltractin, TnC, Squidulin, Grancalcin, Sorcin, ALG2, Calpain, CaPK, Spectin, sCaBP, and BM-40. Advantageously, the calcium-binding sites are derived from canonical EF-hand motif from proteins chosen in the list consisting of Parvalbumin and Calmodulin.
It has been documented in the field that the calcium-binding function of the canonical EF-hand motif relies mostly on the 12 amino-acid calcium-binding loop. Preferably, each of the calcium-binding sites according to the invention comprises or consists of a calcium-binding loop of a canonical EF-hand motif.
Unless otherwise indicated, the terms ‘calcium-binding loop of a canonical EF-hand motif_ or ‘calcium binding loop_ refer the sequence of 12 amino-acid residues corresponding to the calcium-binding loop of a canonical EF-hand. Canonical EF-hands, their sequence and in particular the 12-amino-acid sequence corresponding to their respective calcium-binding loop are well documented. Preferably, the terms ‘calcium-binding loop of a canonical EF-hand motif_ refer to a peptide sequence corresponding to the consensus sequence SEQ ID NO: 1:
D-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11, in which:
or a functional variant thereof.
Parvalbumin has been described in the field as containing two different EF hand motifs: Parv EF-hand1 and 2, and thus comprises the 2 corresponding calcium-binding loops:
and;
Of note, a high affinity variant of the calcium-binding loop of the rat Parv EF-hand1 having the sequence DKDKDGFIDEDE (SEQ ID NO: 5), has also been disclosed.
Calmodulin has been described in the field as containing four different EF hand motifs: CaM EF-hand1, 2, 3 and 4, and thus comprises the 4 corresponding calcium-binding loops:
Yet preferably, in the polypeptide of the invention, each of the calcium-binding sites comprises or consists of a calcium-binding loop chosen in the list consisting of SEQ ID NO: 2 to 9, and functional variants thereof.
In order to increase the capacity of the polypeptide of the invention to bind calcium ions, the person skilled in the art may consider using calcium-binding sites derived from whole canonical EF-hand motifs, that is to say comprising the two a-helix as well as the calcium-binding loop typical of the canonical EF-hand motif.
Advantageously, in the polypeptide of the invention, each of the calcium-binding sites comprises or consists of a canonical EF-hand motif.
Parvalbumin has been described in the field as containing two different EF hand motifs: Parv EF-hand1 and 2:
Calmodulin has been described in the field as containing four different EF hand motifs: CaM EF-hand1, 2, 3 and 4:
Yet preferably, in the polypeptide of the invention, each of the calcium-binding sites comprises or consists of a canonical EF-hand motif chosen in the list consisting of SEQ ID NO: 10 to 15 and functional variants thereof.
Advantageously, the first calcium-binding domain and the second calcium binding domain each comprises at least two calcium-binding sites derived from a EF-hand motif. Preferably, the calcium-binding sites within a calcium-binding domain are derived from EF hand motifs from the same protein, preferably chosen in the list disclosed above.
In a preferred embodiment, in the polypeptide of the invention, the first calcium-binding domain comprises calcium-binding sites derived from EF-hand motifs from Parvalbumin, and the second calcium-binding domain comprises calcium-binding sites derived from EF-hand motifs from Calmodulin.
Preferably, in said embodiment, the first calcium-binding domain comprises two calcium-binding sites derived from EF-hand motifs from Parvalbumin, and the second calcium-binding domain comprises two calcium-binding sites derived from EF-hand motifs from Calmodulin.
More preferably, in said embodiment, the first calcium-binding domain comprises at least one, preferably at least two, calcium-binding site comprising or consisting of calcium-binding loop from Parvalbumin chosen in the list consisting of SEQ ID NO: 2 to 5 and functional variants therefrom, yet preferably in the list consisting of SEQ ID NO: 4, SEQ ID NO: 5 and functional variants therefrom, and the second calcium-binding domain comprises at least one, preferably at least two, calcium-binding site comprising or consisting of calcium-binding loop from Calmodulin chosen in the list consisting of SEQ ID NO: 6 to 9 and functional variants therefrom, yet preferably in the list consisting of SEQ ID NO: 6 or 7 and functional variants thereof.
Yet preferably, in said embodiment, the first calcium-binding domain comprises or consists of at least one, preferably at least two, calcium-binding site comprising or consisting of EF-hand motifs from Parvalbumin chosen in the list consisting of SEQ ID NO: 10 and 11, and the second calcium-binding domain comprises or consists of at least one, preferably at least two, calcium-binding site comprising or consisting of EF-hand motifs from Calmodulin chosen in the list consisting of SEQ ID NO: 12 to 15 and functional variants therefrom, preferably in the list consisting of SEQ ID NO: 12 or 13 and functional variants thereof.
In a yet preferred embodiment, the first calcium-binding domain has a sequence comprising or consisting of the sequence SEQ ID NO: 16 or a functional variant thereof, and the second calcium-binding domain has a sequence comprising or consisting of the sequence SEQ ID NO: 17 or a functional variant thereof.
In the context of the invention, the functional variants of a sequence of reference chosen from SEQ ID NO: 1 to SEQ ID NO: 17 encompass peptides which sequence derives from said sequence of reference by insertions, substitutions, and/or deletions of amino-acid residues at one or more positions of the sequence of reference, and which have retained the ability to allow the localization of a peptide bound thereto or comprising thereof to lipid rafts. Of particular interest are the functional variants which sequence contains conservative substitutions, that is to say wherein amino acids residues of the sequence of reference are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. Preferably, a functional variant of a sequence of reference chosen from SEQ ID NO: 1 to SEQ ID NO: 17 is a peptide which sequence derives from the sequence of reference by one or more conservative substitutions. Yet preferably, a functional variant of a sequence of reference chosen from SEQ ID NO: 1 to SEQ ID NO: 17 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence of reference. In a preferred embodiment, a functional variant of sequence of reference chosen from SEQ ID NO: 1 to SEQ ID NO: 17 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence of reference and is a peptide which sequence derives from the sequence of reference by conservative substitutions.
In the sense of the present invention, the “percentage identity” or “% identity” between two sequences of nucleic acids or amino acids means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length. The comparison of two nucleic acid or amino acid sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an “alignment window”. Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman, by means of the similarity search method of Pearson and Lipman (1988) or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis., or by the comparison software BLAST NR or BLAST P). The percentage identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally-aligned sequences in which the nucleic acid or amino acid sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the amino acid, nucleotide or residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.
The polypeptide of the invention further comprises a peptide linker
Preferably, the peptide linker according to the invention has a length of at least 5, preferably at least 10, and up to 40, preferably up to 30, and still more preferably up to 20 amino-acids. Preferably the peptide linker has the sequence SEQ ID NO: 18.
In a preferred embodiment, the polypeptide of the invention has the sequence SEQ ID NO: 19.
The polypeptide of the invention may further be functionalized with peptide sequences having properties of interest, such as peptide signals, which are localization sequences that will enable targeting the polypeptide of the invention to specific subcellular compartment, or fluorescent peptides, that will be useful for in vitro detection of the polypeptide.
Peptides signals capable of targeting a polypeptide to specific subcellular compartment are well known in the art. Of particular interest are the sequences capable of targeting peptides to the plasma membrane, and more particularly to lipid rafts. Lipid rafts are sphingolipid- and cholesterol-rich plasma membrane microdomains, which have been identified and proposed to function as platforms where signal transduction molecules may interact with receptors. Peptide signals capable of targeting a peptide to the plasma membrane are well known in the art. For instance, the N terminus domains of proteins from the Src family, and in particular the peptide of sequence SEQ ID NO: 20, which corresponds to the palmitoylation and myristoylation motifs of the N terminus part of the Lyn Kinase, have been shown to enable the localization of said proteins to lipid rafts. In contrast, the peptide of sequence SEQ ID NO: 21 which corresponds to the CaaX-polylysine motif derived from the protein K-Ras, is known to target proteins to the plasma membrane while excluding the localization to lipid rafts. Advantageously, the polypeptide of the invention further comprises a peptide signal, preferably a peptide signal capable of targeting the polypeptide of the invention to the plasma membrane, yet preferably to lipid rafts of the plasma membrane. Preferably, the polypeptide of the invention further comprises a peptide signal of sequence SEQ ID NO: 20 or 21, or functional variants thereof.
In the context of the invention, the functional variants of SEQ ID NO: 20 encompass peptides which sequence derives from the sequence SEQ ID NO: 20 by insertions, substitutions, and/or deletions of amino-acid residues at one or more positions of the sequence SEQ ID NO: 20, and which have retained the ability to allow the localization of a peptide bound thereto or comprising thereof to lipid rafts. Of particular interest are the functional variants which sequence contains conservative substitutions, that is to say wherein amino acids residues of the sequence SEQ ID NO: 20 are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. Preferably, a functional variant of SEQ ID NO: 20 is a peptide which sequence derives from the sequence SEQ ID NO: 20 by one or more conservative substitutions. Yet preferably, a functional variant of SEQ ID NO: 20 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 20. In a preferred embodiment, a functional variant of SEQ ID NO: 20 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 20 and is a peptide which sequence derives from the sequence SEQ ID NO: 20 by conservative substitutions.
In the context of the invention, the functional variants of SEQ ID NO: 21 encompass peptides which sequence derives from the sequence SEQ ID NO: 21 by insertions, substitutions, and/or deletion, of amino-acid residues at one or more positions of the sequence of reference, and which have retained the ability to allow the localization of a peptide bound thereto or comprising thereof to the cellular membrane, at the exclusion of lipid rafts. Of particular interest are the functional variants which sequence contains conservative substitutions, that is to say wherein amino acids residues of the sequence SEQ ID NO: 21 are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. Preferably, a functional variant of SEQ ID NO: 21 is a peptide which sequence derives from the sequence SEQ ID NO: 21 by one or more conservative substitutions. Yet preferably, a functional variant of SEQ ID NO: 21 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 21. In a preferred embodiment, a functional variant of SEQ ID NO: 21 has a sequence which has at least 80, 85, 90 or 95% identity with the sequence SEQ ID NO: 21 and is a peptide which sequence derives from the sequence SEQ ID NO: 21 by conservative substitutions.
The capacity of a peptide signal to allow the localization of a peptide bound thereto or comprising thereof to the cellular membrane, and in particular to lipid rafts or at the exclusion of lipid rafts may easily be determined using standard methods, in particular based on fluorescence microscopy and/or density gradients, such as detailed in the experimental part of the present application
The peptide signal may be in the C terminus or the N terminus part of the polypeptide.
Fluorescent peptides are well known in the art. Examples of fluorescent peptides are the green fluorescent protein (GFP), or GFP variants such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP) and their variants such as the optimized CFP Turquoise or the YFP variant Venus. Advantageously, the polypeptide of the invention further comprises a fluorescent peptide, preferably chosen in the list consisting of green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP), and their variants such as the optimized CFP Turquoise or the YFP variant Venus. The fluorescent protein may be in the C terminus or the N terminus part of the polypeptide.
Preferably, the invention also pertains to modified polypeptides, which are derived from the polypeptide of the invention or its functional variants by introduction of chemical modification into one or more amino acid residues, peptide bonds, N- and/or C-terminal ends of the protein, aimed at increasing the stability, bioavailability or bioactivity of the protein, as long as the modified polypeptide remains functional.
Another aspect of the invention relates to a polynucleotide encoding the polypeptide of the invention, and a recombinant vector encoding said polynucleotide.
In the context of the invention, the term ‘polynucleotide_ refers to a polydeoxyribonucleotides or polyribonucleotides, in the form of a separate fragment or a larger construct, and includes DNA, cDNA and RNA sequences which encode the polypeptide of the invention. Preferably, the polynucleotide of the invention is an isolated polynucleotide. The term ‘isolated_ as used herein includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated.
Preferably, said recombinant vector is an expression vector capable of expressing said polynucleotide when transfected or transformed into a host cell such as a eukaryotic or prokaryotic cell like a mammalian, bacterial or fungal cell. ‘Expression vectors_ are DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. Suitable selectable markers may for instance be chosen among the genes encoding neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRP1 for S. cerevisiae. Some expression vectors do not contain an origin of replication for autonomous replication in host cells but rather depend on the ability of the vector to stably integrate (either randomly or by a homologous integration event) using a marker to select for integration/maintenance. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. The polynucleotide is inserted into the expression vector in proper orientation and correct reading frame for expression. Preferably, the polynucleotide is operably linked to at least one transcriptional regulatory sequence and, optionally to at least one translational regulatory sequence. Such vectors comprise YAC (yeast artificial chromosome), BAC (bacterial artificial), baculovirus vectors, phage, phagemid, cosmid, viral vector, plasmid, RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA. Suitable vectors according to the present invention comprise, but are not limited to, YAC (yeast artificial chromosome), BAC (bacterial artificial), baculovirus vectors, phage, phagemid, cosmid, viral vector, plasmid, RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA. Suitable plasmids to be used as mammalian expression vectors include but are not limited to, pcDNA3, pcDNA3.1, pcDNAI, pcDNAIamp (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and ≅ZD35 (ATCC 37565). Suitable plasmids to be used as bacterial expression vectors include but are not limited to pCR2.1 (Invitrogen), pET11a (Novagen), lambda gt11 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia), and pQE vectors (Qiagen) including pQE7O, pQE6O, pQE-9. Suitable plasmids to be used as expression vectors in fungal cells include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen). Suitable plasmids to be used as expression vectors in insect cell include but are not limited to pBlueBacIII, pBlueBacHis2 (Invitrogen) and pFastBac1, pFastBacHT (Life Technologies). Viral vectors include retrovirus, adenovirus, parvovirus (e. g., adenoassociated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, Dtype viruses, HTLV-BLV group, lentivirus, spumavirus. Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.
Another aspect of the invention provides a host cell or a non-human organism transformed with said polynucleotide or recombinant vector. The non-human transgenic organism is obtained from a unicellular or pluricellular microorganism or a higher eukaryotic organism. The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to infection, transformation, transfection, lipofection, protoplast fusion, and electroporation. Modified host cells containing the expression vector may be clonally propagated and individually analyzed to determine whether they produce the polypeptide of the invention.
In a preferred embodiment said modified host cell is a eukaryotic cell such as a human cell. In another embodiment, said non-human transgenic organism is a transgenic plant, nematode, zebrafish or algae.
The polynucleotide, vector, host cell, and non-human transgenic organism of the invention are useful for the production of the polypeptide/chimeric protein of the invention using well-known recombinant DNA techniques. Another aspect of the invention relates to the non-therapeutic use of the polypeptide, polynucleotide, or vector comprising thereof, in vitro or in vivo, as calcium chelating agent. Preferably the invention relates to the non-therapeutic use of the polypeptide, polynucleotide, or vector comprising thereof for stabilizing and/or lowering calcium concentration, and/or for inhibiting calcium signalization in vivo and/or in vitro. Yet preferably, the non-therapeutic use, when performed in vivo, is performed in a healthy subject.
The terms ‘stabilizing calcium concentration_ should be construed as generally understood in the art, that is to say as the action of minimizing the variations of the concentration of calcium in a composition upon addition of calcium or induction of calcium concentration variations in said composition. The variation, and in particular the increase, of calcium concentration may easily be measured using for instance the biosensor Twitch2B, by monitoring the FRET/CFP ratio, as disclosed in the experimental part. In the context of the invention, calcium concentration can be considered as stabilized in a composition when the FRET/CFP ratio of the biosensor Twitch2B varies, preferably increases, of less than 10%, preferably less than 5%, of the variation detected without the polypeptide of the invention upon of addition of calcium or induction of calcium concentration variations in said composition.
The terms ‘inhibiting calcium signal_, ‘calcium signal inhibition_ should be construed as generally understood in the art, that is to say as the action of inhibiting the downstream effectors known to be activated by calcium as a second messenger, for instance calcineurin. Methods and kits for assessing the calcineurin cellular activity are known and in the art, such as for instance the calcineurin cellular activity assay kit commercialized by the company Enzo (under reference BML-AK816-0001).
It is well documented that a number of pathologies are associated with calcium signaling dysfunction. The polypeptide, polynucleotide, vector, and host cell of the invention, may thus be particularly useful in preventing or treating such pathologies. In this context, the polypeptide, polynucleotide, vector, or host cell of the invention, may be formulated into a composition suitable for pharmaceutical use.
Another aspect of the invention relates to a pharmaceutical composition, comprising at least one polypeptide, polynucleotide, vector, and/or host cell of the invention, and, preferably, a pharmaceutically acceptable carrier. In the context of the invention, the terms “pharmaceutically acceptable” refer to carriers which can be used in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable, and which is acceptable for veterinary use as well as for human pharmaceutical use. Suitable vehicles or carriers include any pharmaceutically acceptable vehicle such as buffering agents, stabilizing agents, diluents, salts, preservatives, and emulsifying agents. Examples of suitable buffering agents include buffered solutes, such as phosphate buffered solution, chloride solutions or Ringer{hacek over ( )}s solution. Examples of suitable stabilizing agents include human serum albumin (HSA), polyvinylpyrrolidone or hyaluronic acid.
The composition of the invention may be formulated according to the intended administration route. For instance, formulations suitable for oral administration include liquid or solid formulations such as tablets, pills, powders (hard or soft gelatine capsules) or granules. Typically, in such formulations, the polypeptide, polynucleotide, vector, and/or host cell of the invention is mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica, in a stream of argon. These compositions may also comprise substances other than diluents, for example lubricants such as magnesium stearate or talc, coloring agent, coating (coated tablets) or varnish. Formulations suitable for injection, such as used in parenteral administration, are preferably sterile and fluid, and may preferably be aqueous or non-aqueous solutions, suspensions or emulsions.
Another aspect of the invention relates to the polypeptide, the polynucleotide, vector comprising thereof, host cell of the invention, or a composition comprising thereof for use as a medicine, preferably for use in the prevention and/or treatment of pathologies associated with intracellular calcium signaling dysfunction.
In the context of the invention, the terms ‘pathologies associated with intracellular calcium signaling dysfunction_ should be construed according to their general meaning in the art. Preferably, said pathologies are associated with intracellular calcium signaling dysfunction due to an abnormally high intracellular concentration of calcium. In the context of the invention, ‘an abnormally high intracellular concentration of calcium_ refer to an intracellular concentration of calcium which is significantly higher than in cells from a healthy donor. Yet preferably, said pathologies are chosen in the list consisting of retinitis pigmentosa, neurodegenerative diseases, preferably Alzheimer disease, epilepsy, stroke, cardiac dysrhythmia, heart failure, hypertension, diabetes, and cancer.
In other terms, the invention pertains to a method for the prevention and/or treatment of pathologies associated with intracellular calcium signaling dysfunction, wherein said pathologies are chosen in the list consisting of retinitis pigmentosa, neurodegenerative diseases, preferably Alzheimer disease, epilepsy, stroke, cardiac dysrhythmia, heart failure, hypertension, diabetes, and cancer, comprising the step of administering the polypeptide, polynucleotide, vector, host cell of the invention, or a composition comprising thereof to a subject in need thereof, preferably in an effective amount.
In the context of the invention, the terms ‘effective amount_ means that the amount of polypeptide, polynucleotide, vector, host cell of the invention, or of a composition comprising thereof is of sufficient quantity to obtain appropriate prevention and or regression of the symptoms of the above-recited pathologies. The effective amount and dosage regimen may be determined by the attending physician based on usual clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
The polypeptide, polynucleotide, vector, host cell of the invention, or composition comprising thereof referred to herein can administered once or more than one time, in order to prevent or to treat a pathology as recited herein. Efficacy of the prevention and/or treatment can be monitored by periodic assessment.
The polypeptide, polynucleotide, vector, host cell of the invention, or composition comprising thereof referred to herein can be administered topically or systemically, through the known routes of administration. Preferably, the polypeptide, polynucleotide, vector, host cell of the invention, or composition comprising thereof referred to herein is administered through the enteric route, in particular by oral, sublingual, or rectal administration, or through the parenteral route, in particular by intracerebral, intramuscular, intradermal, transdermal, intraperitoneal or nasal administration.
In addition to the preceding features, the invention further comprises other features which will emerge from the following description, which refers to examples illustrating the present invention, as well as to the appended figures.
0.05; ***P
0.001; (A,C) Two-way ANOVA and Bonferroni post hoc tests.
0.001, Kruskal-Wallis test followed by Dunn{hacek over ( )}s multiple comparison test.
0.001, Mann-Whitney test.
0.001, Mann-Whitney test.
subunit of cholera toxin (CtB, enriched in fractions 3 and 4, peaks in fraction 3) and (b) differs from the expression of Caveolin (enriched 2+sp in fraction 4). Ca2+Sp/SpiCee-Kras is mostly enriched in fraction 4, with a profile resembling (d) Caveolin, and (c) distinct from CtB. Data are mean±s.e.m.; *P
0.05, ***P
0.01, ***P
0.001, Two-way ANOVA and Bonferroni post hoc tests.
0.05; ***P
0.001; Wilcoxon paired test
encoding a polynucleotide according to the invention, was designed in silico in frame with a tandem repeat of the nucleotide sequence encoding the Lyn Kinase N-terminus domain of sequence SEQ ID NO: 23:
,
and the desired sequences obtained as oligonucleotides from Sigma and Invitrogen, respectively. The oligos were annealed and cloned into pcDNA3-mRFP in frame with the reporter sequence. Lipid-raft-excluded and cytosolic forms were obtained by subcloning the above polynucleotide into pcDNA3 with or without the sequence encoding the CaaX-polylysine motif of Kras of sequence SEQ ID NO: 24:
respectively.
For expression on retinal ganglion cell (RGCs), the constructs were subcloned into pcX. Twitch2B was targeted to the membrane microdomains using the In-Fusion HD cloning kit (Clontech) and subcloned into pcDNA3 or pcX.
Cell culture: HEK293T cells were kept in a 37° C., 5% CO2 incubator and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer{hacek over ( )}s protocol and imaged the day following transfection or fixed and processed for immunocytofluorescence.
Production of purified Ca2+Sp/SpiCeeF134W and Ca2+Sp/SpiCeeF103W: The full-length CaSponge cDNA was subcloned into a pGST-Parallel2 vector (derived from pGEX-4T-1; Amersham) in frame with the N-terminal glutathione S-transferase (GST) tag. Ca2+Sp/SpiCeeF134W and Ca2+Sp/SpiCeeF103W clones were prepared from Ca2+Sp/SpiCee using the QuickChange II Site-directed mutagenesis kit (Agilent Technologies). Proteins were produced in BL21 (DE3) pLysS E. coli cells. The GST-tagged proteins were purified using a glutathione Sepharose 4B column. The GST tag was cleaved using recombinant tobacco etch virus NIa proteinase. The recombinant proteins were eluted in a buffer containing 30 mM MOPS (3-Morpholinopropanesulfonic acid), pH 7.2 and 100 mM KCl. Protein concentrations were deduced from A280 measurements using a computed extinction coefficient of 5500 M−1 cm−1 for both Ca2+Sp/SpiCeeF134W and Ca2+Sp/SpiCeeF103W.
Measurement of Ca2+ affinity for the labeled site of Ca2+Sp/SpiCeeF103W (parvalbumin-derived): Ca2+ binding to the labeled site of Ca2+Sp/SpiCeeF103W was monitored by measuring tryptophan fluorescence (285 nm excitation, 330 nm emission, 5 nm bandpass) at 20° C. in a 4 mm×10 mm quartz cuvette (Jasco FP-8300 spectrofluorimeter). The protein was diluted to 2.25 μM in standard buffer (30 mM MOPS-KOH pH7.2; 0.1 M KCl) and 254 μL samples were loaded in the cuvette. It was first observed that addition of CaCl2 did not increase fluorescence, whereas addition of millimolar amounts of EGTA led to large decreases (up to 27% at a final EGTA concentration of 7.5 mM). This showed that the labeled site of Ca2+Sp/SpiCeeF103W was saturated with Ca2+. Dissociation constant of Ca2+ was deduced from a competition experiment in which the protein solution was titrated with EGTA. 2 μl samples of solutions containing increasing concentrations of EGTA (1.95 to 500 mM) were added to the cuvette. Fluorescence was measured after each addition. Fluorescence values were corrected for dilution and plotted as a function of the concentration of EGTA. Data were fitted to the quadratic equation 1 as described previously by Weeks, K. M. & Crothers (D. M. Biochemistry 31, 10281-10287 (1992)), assuming that the two parvalbumin-derived sites were equivalent for Ca2+ binding.
Measurement of Ca2+ affinity for the labeled site of Ca2+Sp/SpiCeeF134W (calmodulin-derived): Ca2+ binding to the first calmodulin site of Ca2+Sp/SpiCeeF134W was monitored by measuring tryptophan fluorescence (285 nm excitation, 340 nm emission, 5 nm bandpass) at 20° C. Ca2+Sp/SpiCeeF134W was diluted to 1.6 μM in standard buffer and 258 μL samples were loaded in the cuvette. Addition of CaCl2 increased fluorescence, whereas addition of millimolar amounts of EGTA led to fluorescence decrease, showing that the calmodulin-derived sites of Ca2+Sp/SpiCee were partially filled with Ca2+ in the sample. The value of the fluorescence of the protein with Ca2+-free calmodulin sites was measured in the presence of 2 mM EGTA, a concentration found to be saturating. Total concentration of Ca2+ in the sample (excluding Ca2+ bound to the parvalbumin derived sites of Ca2+Sp/SpiCee) was measured as described above using Quin-2 and found to be 2.5 μM. Dissociation constant of Ca2+ from the labeled site of Ca2+Sp/SpiCeeF134W was deduced from an experiment where the protein solution was titrated with increasing amounts of CaCl2 (2 μL additions of solutions from 0.05 to 6.25 mM). Fluorescence values were corrected for dilution, and plotted as a function of total Ca2+ concentration (added Ca2+ plus 2.5 μM). Data were fitted to a binding curve assuming that the two calmodulin sites were equivalent for Ca2+ binding.
Measurement of kinetic dissociation constants: Values of kinetic dissociation constants (koff) for the tryptophan-labeled Ca binding sites of Ca2+/Sp/SpiCee (Ca2+Sp/SpiCeeF103W and Ca2+Sp/SpiCeeF134W) were measured by mixing proteins with appropriate amounts of EGTA at 20° C. in an Applied Photophysics stopped flow apparatus. Reacting solutions were excited at 285 nm and fluorescence was measured using a photomultiplier equipped with a 320 nm cutoff filter. For the Ca2+Sp/SpiCeeF103W, the protein (2.3 μM in standard buffer) was mixed 1:1 with 15 mM EGTA in the same buffer. For Ca2+Sp/SpiCeeF134W, the protein (1.7 μM in standard buffer plus 50 μM CaCl2) was mixed 1:1 with 4 mM EDTA in standard buffer plus 50 μM CaCl2. Fluorescence was recorded as a function of time and data were fitted with single exponential curves from which rate constants were derived. Reported values are the mean±standard deviation from three measurements.
Cell death assay: HEK293 Cells were plated on poly-lysine-coated coverslips and transfected the following day with a pCX-mRFP or a pCX-Ca2+Sp/SpiCee vector using Lipofectamine 2000 (Thermo Fisher) following the manufacturer{hacek over ( )}s instructions. Three days after plating, cells were either fixed with 4% paraformaldehyde and processed for immunocytochemistry with the antibodies against Cleaved Caspase 3 (Asp175; Cell Signaling; lot #0043) and -tubulin (Sigma) or treated with the CellEvent Caspase 3/7 Green Detection Reagent (Thermo Fisher) for 30 minutes and then fixed and labeled with an -tubulin antibody.
Membrane fractionation by detergent-free method: Electroporated retinas were pelleted (195 g for 5 min at 4° C.) and resuspended in 1.34 mL of 0.5 M sodium carbonate, pH 11.5, with protease inhibitor cocktail and phosphatase inhibitor cocktail 1, 2 and 3 (Sigma-Aldrich). The homogenate was sheared through a 26-gauge needle and sonicated three times for 20 s bursts. The homogenate was adjusted to 40% sucrose by adding 2.06 mL of 60% sucrose in MBS (25 mM MES, pH 6.4, 150 mM NaCl, and 250 mM sodium carbonate), placed under a 5-30% discontinuous sucrose gradient, and centrifuged at 34,000 rpm for 15-18 h at 4° C. in a Beckman SW 41Ti rotor. Nine fractions (1.24 mL each) were harvested from the top of the tube mixed with 9 volumes of MBS, and centrifuged at 40,000 rpm for 1 h at 4° C. (Beckman SW-41Ti rotor). Supernatants were discarded, and membrane pellets were resuspended in 100 μl of 1% SDS.
For immunoblotting, samples were separated on 4-15% Mini-Protean TGX Tris-Glycine-buffer SDS PAGE (Biorad) and transferred onto 0.2 μm Trans-Blot Turbo nitrocellulose membranes (Biorad). Membranes were blocked for one hour at room temperature in 1×TBS (10 mM Tris pH 8.0, 150 mM NaCl) supplemented with 5% (w/v) dried skim milk powder. Primary antibody incubation was carried out overnight at 4° C., with the following antibodies: rabbit anti-GFP (1/200; A11122; Life Technologies), rabbit anti-DsRed (1/200; 632476; Clontech), rabbit anti--Adaptin (1/200; sc-10762; Santa Cruz) and rabbit anti-Caveolin (1/500; 610060; BD Transduction Laboratories). A goat anti-rabbit-HRP coupled secondary antibody was used for detection (Jackson ImmunoResearch, West Grove, Pa.). After antibody incubations, membranes were extensively washed in TBS T (TBS containing 2.5% Tween-20). Western blots were visualized using the enhanced chemiluminescence method (ECL prime Western Blotting detection reagent, Amersham).
Collapse assay: Retinas of E15 mice were electroporated with mRFP, Lyn-Calcium Sponge, Calcium Sponge or Calcium Sponge-Kras using two poring pulses (square wave, 175V, 5 ms duration, with 50 ms interval) followed by four transfer pulses (40V, 50 ms and 950 ms interpulse). Retinas were dissected and kept 24 hours in culture medium (DMEM-F12 supplemented with glutamine (Sigma Aldrich), penicillin/streptomicin (Sigma Aldrich), BSA (Sigma Aldrich) and glucose), in a humidified incubator at 37° C. and 5% CO2. The day after, they were cut into 200 μm squares with a Tissue-Chopper (McIlwan) and explants were plated on glass coverslips coated with poly-L-lysine and Laminin (Sigma Aldrich). Cells were cultured for 24 hours in culture medium supplemented with 0.4% methyl cellulose and treated with rmSlit-1 (R&D Systems) for 1 hour.
Immunodetection: Retinal explants, or Hek cells coexpressing the targeted versions of Calcium Sponge and GFP or the targeted versions of Twitch2B and mRFP, were fixed with 4% PFA in PB for 30 minutes, permeabilyzed blocked and with 1% Triton and 3% BSA in PBS, then immunized against DsRed (Clontech) followed by a secondary antibody coupled to AlexaFluor 594 (Invitrogen) and GFP (invitrogen) or -Tubulin (Sigma) followed by a secondary antibody coupled to AlexaFluor 488 (Invitrogen).
Imaging: Images were acquired with an inverted DMI6000B epifluorescence microscope (Leica) coupled to a 40× oil-immersion objective (N.A. 1.3) and the software Metamorph (Molecular Devices). For live imaging experiments, cells were perfused with HBS buffer with 0.2 or 2 mM CaCl. Thapsigargin was used at 1 mM). Images were acquired simultaneously for the CFP (483/32 nm) and YFP (542/27) channels every 20 seconds. Images were processed in ImageJ, corrected for background and bleedthrough and then the ratio CFP/YFP was calculated. Confocal images were acquired with a 63× oil immersion objective (N.A. 1.45) and a Z-stack containing the whole specimen was sampled at nyquist frequency. Images were rendered in ImageJ and Photoshop.
Statistics: Two-Way ANOVA and Bonferroni post-hoc tests were calculated with GraphPad Prism (GraphPad Software Inc.). **=p<0.001
Results:
An example of a polypeptide according to the invention, hereafter designated Ca2+Sp, Calcium Sponge, SpiCee, or Ca2+Sp/SpiCee those three terms referring to the exact same peptide, was designed as a fusion protein containing the Ca2+ binding domains of both Parvalbumin and Calmodulin.
The fluorescent protein mRFP was fused to this construct for easy identification of sponge-expressing cells in fixed or live cell experiments.
To characterize the Ca2+ binding properties of Ca2+Sp/SpiCee, we used the tryptophan fluorescence of two variants of Ca2+Sp/SpiCee (Ca2+Sp/SpiCeeF134W and Ca2+Sp/SpiCeeF103W). Each of these mutants enables the evaluation of the Ca2+ binding properties of either the calmodulin or the parvalbumin moiety (Ca2+Sp/SpiCeeF134W and Ca2+Sp/SpiCeeF103W respectively). Using Ca2+ titration, we found that Ca2+Sp/SpiCeeF134W has a Kd for Ca2+ of 2.8±0.9 μM and stopped-flow experiments were performed to evaluate its dissociation rate, koff=529±28 s−1 at 20° C. (
The buffering properties of Ca2+Sp/SpiCee were investigated using Twitch2B, an optimized Ca2+ FRET sensor. The FRET over CFP ratio, reflecting intracellular Ca2+ concentration, was monitored in Twitch2B-expressing HEK293 cells. The release of intracellular Ca2+ stores induced by a 20 s pulse of thapsigargin induced an increase in FRET/CFP ratio, that was drastically reduced in cells co-expressing Ca2+Sp/SpiCee (
To assess Ca2+Sp/SpiCee capability to interfere with physiological processes in vivo, Ca2+Sp/SpiCee was expressed in newly generated cortical neurons using in utero electroporation of the lateral ventricle in 14.5 day-old (E14.5) mice embryos. The migration of early born cortical neurons is a Ca2+−dependent process. The position of migrating electroporated neurons was assessed at E18.5. The majority of GFP and mRFP co-electroporated neurons reached the cortical plate, and form a dense layer of neuron near the marginal zone (
Calcium signals in the cell are often restricted to subcellular compartments. Since genetic-encoding confers the ability to restrict the expression of the constructs to a specific organelle, the functionality of Ca2+Sp/SpiCee in this scenario was assessed. A tandem of palmitoylation-myristoylation motifs from Lyn Kinase was fused to the n-terminus of Ca2+Sp/SpiCee to target lipid rafts, a compartment of the plasma membrane (Lyn-Ca2+Sp/SpiCee). Alternatively Ca2+Sp/SpiCee was excluded from lipid rafts but still targeted to the plasma membrane by the c-terminus fusion of the CaaX-polylysine motif derived from K-Ras (Ca2+Sp/SpiCee-Kras). These targeting sequences have been previously used and validated. Using detergent-free plasma membrane fractionation methods, Lyn-Ca2+Sp/SpiCee was found enriched in lipid raft fractions, whereas Ca2+Sp/SpiCee localization was shifted towards the non-raft fractions ( subunit of cholera toxin (CtB) and caveolin respectively (
Using Lyn-Ca2+Sp/SpiCee and Ca2+Sp/SpiCee-Kras, the potential of Ca2+Sp/SpiCee to manipulate specific physiological Ca2+ dependent cellular processes depending on its subcellular localization was investigated. To this aim the response of retinal axons to the repellent guidance molecules slit1 and ephrinA5 was analyzed, a process requiring calcium signaling. In control conditions, including untransfected axons and mRFP-electroporated axons, slit1 and ephrinA5 induced the collapse of non-electroporated or mRFP-expressing growth cones, characterized by the depolymerization of lamellipodial actin and drastic reduction of the growth cone area (
In conclusion, the polypeptide of the invention functions as a Ca2+ scavenger that has the capability to interfere with Ca2+-dependent physiological functions. The polypeptide of the invention has the potential to alter calcium responses in a cell-specific manner and with subcellular resolution, opening new fields for the precise study of signaling cascades and paving the way for therapeutic implementation.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17306514.5 | Nov 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/080032 | 11/2/2018 | WO | 00 |
