WO2000043524A1 - Polypeptides derives de jnk3 - Google Patents
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- WO2000043524A1 WO2000043524A1 PCT/FR2000/000104 FR0000104W WO0043524A1 WO 2000043524 A1 WO2000043524 A1 WO 2000043524A1 FR 0000104 W FR0000104 W FR 0000104W WO 0043524 A1 WO0043524 A1 WO 0043524A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/10—Antioedematous agents; Diuretics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the field of biology and the regulation of signal transduction pathways in response to extracellular stimuli. More particularly, the present invention relates to new polypeptides derived from the human JNK3 protein, their variants, the corresponding nucleotide sequences, and their uses.
- the protein JNK (c-jun N-terminal kinase), also called SAPK (Stress-Activated Protein Kinase), belongs to the family of MAP (Mitogen-Activated Protein) kinases. It is involved in signal transduction pathways in response to extracellular stimuli (eg proinflammatory cytokines or environmental stress). Its activation requires the phosphorylation of Threonine 221 and Tyrosine 223 within a very conserved T-P-Y tripeptide motif located in the kinase domain (Dérijard et al. 1994).
- the JNK substrates are transcription factors such as c-Jun (phosphorylated on Serine 63 and Serine 73), ATF-2 (phosphorylated on Threonine 69 and Threonine 71) and Elk-1.
- JNKs have many isoforms, and ten isoforms have been identified to date. They derive from three different genes, named JNK1, JNK2 and JNK3.
- the JNK1 and JNK2 genes code for two isoforms ⁇ and ⁇ by alternative splicing (Gupta et al. 1996). For each ⁇ and ⁇ isoform, there is a short version and a long version in C-terminal. The short version is linked to the insertion, during alternative splicing, of five nucleotides which provide a termination codon.
- JNK3 ⁇ l and J K3 ⁇ 2 are the only two isoforms of J K3 listed so far. They differ from JNK1 or JNK2 among other things by an N-terminal extension of 38 amino acids whose function has not yet been established.
- JNK3 The tissue distribution of isoforms is widely dispersed with varying levels of expression.
- JNK3 isoforms are more selectively expressed in the brain (for example in the hippocampus or in the cerebellum (Mohit et al. 1995)), but also in the heart and the testes.
- a growing number of results underlines the importance of JNK3 in the phenomena of neurodegeneration and neuronal death by apoptosis, however, the mode of action of JNK3 is not known to date.
- JNK3 immunoreactivity is diffuse and only cytoplasmic (Zhang et al. 1998).
- JNK3 immunoreactivity is diffuse and only cytoplasmic (Zhang et al. 1998).
- the deletion of the JNK3 gene in mice allows resistance to kainic acid, a glutamate receptor agonist participating in the phenomena of excitotoxicity.
- the authors describe in detail the reduction of epileptic effects and the prevention of neuronal death by apoptosis in the hippocampus, after injection of kainic acid in these mice deleted for JNK3.
- one of the preferred substrates of JNK3 is the transcription factor c-Jun, one of the components of the API complex which is also widely implicated in functions of survival and neuronal degeneration.
- the factor c-Jun seems to carry a double function both in cell death and in neuronal protection (Herdegen et al. 1997). Suppression of c-Jun expression or its functional inhibition protects hippocampal and sympathetic neurons from cell death in culture (Estus et al. 1994, Ham et al. 1995).
- c-Jun is increased in degenerative apoptotic neurons after ischemia, section of nerves, irradiation but also in biopsies of patients suffering from neurodegenerative pathologies such as multiple sclerosis, amyotrophic lateral sclerosis, diseases of 'Alzheimer, Parkinson and Huntington (Anderson et al. 1994, Herdegen et al. 1998, Martin et al. 1996).
- JNK3 protein kinase JNK3 appears today as one of the key elements involved in neuronal degeneration, however the precise nature of its mode of action is not known. In this regard, the identification of new natural isoforms of JNK3 constitutes a major challenge for the understanding of the mechanism of action of this protein in the phenomena of neuronal degeneration and for the identification of new targets for therapeutic purposes.
- the present invention describes the detection, cloning and characterization of new polypeptides derived from JNK3.
- the present invention results from the characterization of three new isoforms of human JNK3 named hJNK3 ⁇ l39, JNK3 ⁇ N ⁇ l and JNK3 ⁇ N ⁇ 2. It follows more particularly from the demonstration that two of these isoforms JNK3 ⁇ N ⁇ l and JNK3 ⁇ N ⁇ 2, lack the N-terminal extension which characterizes the known isoforms of JNK3 and from the demonstration that these isoforms JNK3 ⁇ N ⁇ l and JNK3 ⁇ N ⁇ 2 have different properties from the isoforms of JNK3 already described. It also stems from the discovery that these new isoforms devoid of the N-terminal extension unexpectedly exhibit properties common with the isoforms JNKl ⁇ l and JNK2 ⁇ l.
- JNK3 The identification of these new isoforms of JNK3 makes it possible to envisage many applications. These applications notably cover the identification of new neuroprotective compounds capable of interacting specifically with these isoforms. These compounds can be used for the prevention and treatment of various pathologies caused by neuronal degeneration, among which there may be mentioned Alzheimer's disease, Huntington's disease and Parkinson's disease, senile dementias and those due to AIDS, head trauma, anoxia, hypoxia and cerebral edema. These new isoforms can also be used in molecular modeling to allow a better understanding of the structure and function of these enzymes as well as their involvement in pathologies involving one or more isoforms of JNK3. Finally, these JNK3 isoforms are also useful for demonstrating new proteins involved in intracellular signaling pathways specific to each of them. It is thus possible to identify new relevant targets involved in degenerative neuropathologies.
- a first object of the invention relates to polypeptides derived from the JNK3 ⁇ 1 or JNK3 ⁇ 2 isoforms comprising an N-terminal or C-terminal deletion.
- these are derivatives comprising an N-terminal deletion corresponding to the first 38 amino acids of these isoforms.
- these are derivatives comprising a deletion of the C-terminal amino acids from amino acid 139.
- the derivatives according to the invention are polypeptides chosen from the sequences SEQ ID No 23, SEQ ID No 25, SEQ ID No 27 or a variant thereof.
- variant within the meaning of the invention designates any polypeptide whose structure is distinguished from a polypeptide chosen from the sequences SEQ ID No 23 or SEQ ID No 25 or SEQ ID No 27 by one or more modifications of a genetic nature , biochemical and / or chemical and retaining at least one of the biological properties of said polypeptide. It may in particular be any mutation, substitution, deletion, addition and / or modification of one or more residues.
- Such derivatives can be generated for different purposes, such as in particular that of improving their level of production, that of increasing their resistance to proteases or of improving their passage through cell membranes, that of increasing their efficiency therapeutic or to reduce their side effects, that of increasing the affinity of the polypeptides for their sites of interaction, or that of conferring on it new pharmacokinetic and / or biological properties.
- the variants include deletions or mutations relating to amino acids the presence of which is not decisive for the activity of the derivative. Such amino acids can be identified, for example, by cell activity tests as described in the examples.
- a subject of the invention is also a polypeptide as defined according to the sequence SEQ ID No. 29 corresponding to fragment 1-38 of the forms JNK3 ⁇ 1 or JNK3 ⁇ 2.
- Another object of the present invention relates to any nucleic acid coding for a polypeptide derived from JNK3 as defined above.
- the nucleic acid according to the invention can be a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In addition, it may be genomic DNA (gDNA) or complementary DNA (cDNA). It can be of human, animal, viral, synthetic or semi-synthetic origin. It can be obtained in different ways and in particular by chemical synthesis using the sequences presented in the application and for example a nucleic acid synthesizer. It can also be obtained by screening banks using specific probes, in particular as described in the request. It can also be obtained by mixed techniques including chemical modification (elongation, deletion, substitution, etc.) of sequences screened from libraries. In general, the nucleic acids of the invention can be prepared according to any technique known to those skilled in the art.
- the nucleic acid according to the invention is a cDNA or a
- the nucleic acid according to the invention is advantageously chosen from:
- the nucleic acid sequences according to the invention can also be used for the production of antisense oligonucleotides which can be used to control the expression of the various isoforms of JNK3.
- the invention also relates to the antisense sequences, the expression of which in a target cell makes it possible to specifically control the translation of cellular mRNA coding for JNK3 ⁇ N ⁇ l or JNK3 ⁇ N 2 or hJNK3 ⁇ l 39 or also JNK3 ⁇ l or JNK3 ⁇ 2, by hybridization of the oligonucleotides antisense on the sequences specific to the corresponding mRNAs.
- sequences can for example be transcribed in the target cell into RNA complementary to the cellular mRNAs of the different isoforms of JNK3 and thus block their translation into protein according to the technique described in patent EP 140 308.
- sequences can be constituted by all or part of the nucleotide sequences SEQ ID No. 22, 24 or 26 transcribed in the reverse orientation.
- the invention also allows the production of nucleotide probes, synthetic or not, capable of hybridizing with the nucleotide sequences defined above.
- probes can be used in vitro as a diagnostic tool, for the detection of the expression or overexpression of the different isoforms of JNK3, or even for the detection of genetic anomalies (poor splicing, polymorphism, point mutations, etc.) .
- These probes can also be used for the detection and isolation of nucleic acid sequences homologs coding for peptides as defined above, from other cellular sources and preferably from cells of human origin.
- the probes of the invention generally comprise at least 17 bases and advantageously less than 300 bases, but they can also comprise up to the entirety of one of the above-mentioned sequences or of their complementary strand. Preferably, these probes are marked prior to their use. For this, different techniques known to those skilled in the art can be used (radioactive, enzymatic labeling, etc.).
- the nucleotide probes can be used, in particular by PCR, as a diagnostic tool to identify:
- the diagnosis can also be performed by Northern (Maniatis, T. et al. 1989) to identify:
- the present invention also relates to any expression cassette comprising a nucleic acid as defined above, a promoter allowing its expression and a transcription termination signal.
- Another subject of the invention also relates to any vector comprising a nucleotide sequence according to the invention or an expression cassette as defined above.
- the vector of the invention can be for example a plasmid, a cosmid or any DNA not encapsulated by a virus, a phage, an artificial chromosome, a recombinant virus etc. It is preferably a plasmid or a recombinant virus.
- viral vectors in accordance with the invention, mention may very particularly be made of vectors of adenovirus, retrovirus type, virus associated with adenovirus (AAV), herpes virus or vaccinia virus.
- AAV adenovirus
- the present application also relates to defective recombinant viruses comprising a heterologous nucleic sequence coding for a polypeptide according to the invention.
- Such vectors are capable of being used in gene therapy for the treatment of peripheral traumas such as in particular traumas of the spinal cord or retinal degenerations.
- the present invention also relates to host cells transformed with a nucleic acid comprising a nucleotide sequence or a vector according to the invention.
- the cellular hosts which can be used for the production of the polypeptides according to the invention are both eukaryotic and prokaryotic hosts.
- suitable eukaryotic hosts there may be mentioned animal cells, yeasts, or fungi.
- yeasts mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula.
- yeasts mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula.
- Sf9 insect cells COS, CHO, C127 cells, human neuroblastoma cells, etc.
- the mushrooms there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
- prokaryotic hosts it is
- the host cells are advantageously represented by recombinant yeast strains.
- the host cells comprise at least one sequence or a sequence fragment chosen from the nucleotide sequences SEQ ID No 22 or SEQ ID No 24 or SEQ ID No 26 for the production of the polypeptides according to the invention.
- Another object of the present invention relates to a process for the preparation of the polypeptides according to the invention according to which a cell containing a nucleotide sequence according to the invention is cultivated, under conditions of expression of said sequence and the polypeptide produced is recovered.
- the part coding for said polypeptide is generally placed under the control of signals allowing its expression in a cellular host.
- the choice of these signals can vary depending on the cell host used.
- nucleotide sequences of the invention can be part of a vector which can be autonomously replicating or integrative. More particularly, autonomously replicating vectors can be prepared using autonomously replicating sequences in the chosen host. As integrative vectors, these can be prepared, for example, by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector.
- Another object of the invention resides in antibodies or fragments of polyclonal or monoclonal antibodies directed against a polypeptide as defined above.
- Such antibodies can be generated by methods known to those skilled in the art.
- these antibodies can be prepared by immunization of an animal against a polypeptide whose sequence is chosen from the sequences SEQ ID No 23 or SEQ ID No 25 or SEQ ID No 27 or any fragment or derivative thereof , then blood collection and isolation of antibodies.
- These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art.
- the antico ⁇ s or fragments antico ⁇ s according to the invention may especially be used in diagnosis for the realization of Western blot (Maniatis, T.
- the invention relates to specific antico ⁇ s of the JNK3 ⁇ l and JNK3 ⁇ 2 isoforms. It may especially be monoclonal antibodies capable of recognizing an epitope located in the N-terminal part of the forms JNK3 ⁇ l and JNK3 ⁇ 2.
- it is an epitope included in the fragment delimited between the amino acids located at positions 1 and 38 of the forms JNK3 ⁇ 1 and JNK3 ⁇ 2; this fragment is represented in the sequence SEQ ID No. 29.
- Such antico ⁇ s are likely to induce a neuroprotective effect by specifically neutralizing the forms JNK3 ⁇ l and JNK3 ⁇ 2.
- These antico ⁇ s are also likely to confer on the protein particular properties which can modify its rate of expression, its stability, its catalytic activity, its specificity of substrates, and its affinity for proteins which intervene in the modulation of these properties.
- the invention also includes the antico ⁇ s derived from the monoclonal antico ⁇ s defined above.
- the term “antico ⁇ s derivatives” means any molecule which comprises the idiotype of the monoclonal antico ⁇ s according to the invention and in particular the chimeric antico ⁇ s, the single chain antico ⁇ s and the Fab fragments.
- Such chimeric antico ⁇ s can be obtained according to the techniques described by Morrison et al., J. Bacteriol. 159: 870 (1984); Neberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985), incorporated herein by reference.
- the Fab fragments which contain the idiotype of the antico ⁇ s according to the invention can be generated by any technique known to those skilled in the art.
- a subject of the invention is also single chain ScFv antico ⁇ s derived from the monoclonal antico ⁇ s defined above. Such single chain antico ⁇ s can be obtained according to the techniques described in US Patents 4,946,778, US 5,132,405 and US 5,476,786.
- the present invention also relates to a method of identifying compounds capable of binding to the polypeptides according to the invention.
- the detection and / or isolation of these compounds can be carried out according to the following steps: - A molecule or a mixture containing different molecules, possibly unidentified, is brought into contact with a polypeptide of the invention under conditions allowing interaction between said polypeptide and said molecule in the event that the latter has an affinity for said polypeptide, and,
- such a method makes it possible to identify molecules capable of specifically blocking the JNK3 ⁇ N ⁇ l and JNK3 ⁇ N ⁇ 2 isoforms and thus of modulating the processes of neuronal degeneration. These molecules are likely to have neuroprotective activity due to the specific inhibition of these isoforms. According to another mode, such a method makes it possible to identify specific inhibitors of the JNK3 ⁇ 1 and JNK3 ⁇ 2 isoforms.
- the present invention further relates to methods of identifying compounds capable of inhibiting the activity of the JNK3 derivatives according to the invention by measuring the growth of microorganisms transformed with a nucleic acid encoding a polypeptide according to the invention.
- the detection and / or isolation of these compounds perhaps carried out by a negative drug selection screen (the drug inhibits the growth of yeast) and or by a positive screen (the drug restores the growth of yeast).
- the latter avoids the selection of antifungals which distort the results.
- These two screens use a Hogl mutant expressing a JNK3 isoform. The first is used in hyperosmotic conditions. The second contains in addition the form PBS2dd which hyperstimulates the Hogl pathway causing the death of yeast in normal condition. PBS2dd is only toxic if Hogl is functional or replaced by JNK3.
- the present invention therefore relates to a method for identifying compounds which inhibit the growth of microorganisms transformed with a nucleic acid coding for a polypeptide according to the invention, comprising the following steps: a molecule or a mixture containing different molecules, possibly unidentified, is brought into contact with at least one microorganism transformed with a nucleic acid coding for a polypeptide according to the invention, under conditions allowing the growth of said microorganism, and
- a molecule or a mixture containing different molecules, possibly unidentified is brought into contact with at least one microorganism transformed with a nucleic acid coding for a polypeptide according to the invention, under conditions inhibiting the growth of said microorganism, and
- the molecules allowing the growth of said microorganism are detected and / or isolated.
- This microorganism can be a yeast containing an inactive Hogl gene and expressing one of the new isoforms of JNK3 according to the invention, such as that described in the examples which follow, or any other microorganism having equivalent properties.
- This yeast grows in hyperosmotic condition. Inhibitors of these new JNK3 isoforms are likely to inhibit or slow the growth of this strain under hyperosmotic conditions.
- a mutated form of PBS2 is expressed in the strain indicated above: PBS2 DD (Wurgler-Mu ⁇ hy et al. 1997) which has a toxic effect on the growth of yeast.
- PBS2 DD Wood-Mu ⁇ hy et al.
- This toxicity depends on the activity of the downstream protein kinase, which according to the present invention is a new isoform of JNK3.
- Inhibitors of these new isoform of JNK3 are likely in this case to inhibit the toxic effect of mutated PBS2 and thus allow the restoration of the growth of this strain.
- Another subject of the invention relates to compounds or ligands capable of inhibiting the activity of the polypeptides according to the invention and capable of being obtained using one of these microorganisms as a screening tool
- Another subject of the invention relates to compounds or ligands capable of binding to the polypeptides according to the invention and capable of being obtained according to one of the methods defined above.
- Another subject of the invention relates to the use of a compound or ligand identified and / or obtained according to one of the methods described above as a medicament.
- Such compounds are in fact capable of being used for the prevention, improvement or treatment of various pathologies caused by neuronal degeneration, among which may be mentioned Alzheimer's disease, Huntington's disease and Parkinson's disease , senile dementia and those due to AIDS, head trauma, anoxia, hypoxia and cerebral edema.
- the subject of the invention is also any pharmaceutical composition comprising as active principle at least one compound or ligand as defined above.
- polypeptides of the invention are also useful for the identification of other partners involved in the mechanisms of neuronal degeneration or in the field of cardiac ischemia by research and identification of molecules interacting in vivo with these polypeptides.
- another object of the present invention relates to a method for identifying specific partner polypeptides of the various natural isoforms of JNK3. They may be specific partner polypeptides of the forms JNK3 ⁇ l ⁇ N or JNK3 ⁇ 2 ⁇ N or hJNK3 ⁇ l 39 or specific partner polypeptides of the forms JNK3 ⁇ 1 and JNK3 ⁇ 2.
- the identification of the specific partner polypeptides of the different isoforms of JNK3 can be carried out by the technique of cloning in double hybrid according to the techniques described by Fields et al. 1994; the bait protein can in particular be the whole protein JNK3 ⁇ N ⁇ l or JNK3 ⁇ N ⁇ 2, or the N-terminal extension of 38 amino acids of JNK3 which is absent in the JNK3 ⁇ N isoforms.
- the bait protein can in particular be the whole protein JNK3 ⁇ N ⁇ l or JNK3 ⁇ N ⁇ 2, or the N-terminal extension of 38 amino acids of JNK3 which is absent in the JNK3 ⁇ N isoforms.
- Figure 1 comparison of nucleotide sequences between the different isoforms of human JNK3.
- the nucleotide sequences of the 5 'region of the different JNK3 isoforms have been aligned with each other.
- the sequence differences compared to the published sequences of JNK3 ⁇ l and 3 ⁇ 2 are noted in bold and the termination codons provided are underlined.
- Figure 2 comparison between the nucleotide sequences of the 5 'region of human JNK3 ⁇ N and of rat SAPK ⁇ .
- the nucleotide sequences of the 5 'region of the human isoforms of JNK3 ⁇ N ⁇ l and ⁇ N ⁇ 2 were aligned with respect to that of rat SAPK ⁇ . The differences between the sequences are indicated by asterisks.
- Figure 3 comparison between the polypeptide sequences of the various human JNK3s.
- the polypeptide sequences of the different JNK3 isoforms were aligned with one another showing a difference in the N-terminal region.
- S.cerevisiae W303a MATa, ade2, trpl, leu2, ura3, his3.
- E.coli TG1 F'f traD 36, lacf>, A (lacZ) Ml 5, proA + B + J supEA (hsdm-mcrb) 5 (r ⁇ m ⁇ ⁇ McrB), thi ⁇ (lac pro AB)
- This medium can be solidified by adding agar (20 g / l) and sterilized by autoclaving at 110 ° C for 20 minutes. 0.5M or 0.9M NaCl yes M of Sorbitol is added to this medium if necessary, to be in hyperosmotic condition.
- YNB medium minimum medium for yeasts: Bacto-yeast nitrogen base w / o amino acids (Difco) 6.7 g / 1, Glucose (Merk) 20 g / 1.
- This medium can be solidified by adding agar (20g / l) and sterilized by autoclaving. The amino acids or nitrogen bases necessary for the growth of the auxotrophic yeasts, previously sterilized by filtration, are then added to 50 mg / 1. Ampicillin (100 ⁇ g / ml) can be added to this medium to avoid bacterial contamination.
- LB complete medium for bacteria: Bacto-yeast extract (Difco) 5 g / 1, Bactos tryptone (Difco) 10g / l, NaCl (Difco) 5 g / 1.
- This medium can be solidified by adding agar (12 g / 1) and sterilized by autoclaving. Ampicillin is added to 100 ⁇ g / ml to select the bacteria which have been transformed by the plasmids carrying the corresponding resistance marker.
- the plasmid pIC20R is a 2.7 kb bacterial plasmid derived from pUC19. It has an origin of ColEl replication and an ampicillin resistance gene. This plasmid is used to clone the protein kinase STE1 1. It has a multiple cloning site into which the sequences coding for the kinases are inserted.
- the plasmid pYX 232 is a shuttle plasmid of 7.4 kb and 9.5 kb respectively, which can be propagated and selected in E. coli bacteria as well as in yeasts. It has an origin of ColEl replication and an ampicillin resistance gene (propagation and selection in E. coli) as well as a 2 ⁇ m origin of replication and a selection gene TRP1 (selection in trpl deficient yeasts for the synthesis of tryptophan).
- TRP1 selection in trpl deficient yeasts for the synthesis of tryptophan.
- a multiple cloning site placed downstream of the TPI (Triose Phosphate Isomerase) promoter makes it possible to insert the sequences of the genes to be expressed.
- Plasmid pYX232 is used to express in yeast the proteins JNKl ⁇ l, JNK2 ⁇ l and the various isoforms of human JNK3. The sequences coding for these proteins were inserted at the EcoRI cloning site previously made freeboard by digestion with the Klenow fragment of the DNA polymerase of E. coli.
- the plasmid pFA6a-kan MX4 is a 2.5 kb plasmid derived from the plasmid pSP72 (Promega).
- This plasmid was used to carry out the deletion of the gene coding for Hogl in the strain W303a.
- the selection of transformants is carried out on medium supplemented with geneticin (G418).
- the sequences corresponding to the various JNKs were amplified with synthetic oligo nucleotides and from double hybrid libraries of yeast genomic DNA or from plasmids.
- oligonucleotides were chosen so as to amplify only the coding phase corresponding to JNK3 ⁇ l and 3 ⁇ 2. In bold, the initiation and termination codons are indicated and in italics the restriction sites used for cloning into the appropriate vectors.
- Oligonucleotide corresponding to the 5 ′ region of JNK3 ⁇ l and 3 ⁇ 2 (SEQ ID No. 1)
- Oligonucleotide corresponding to the 3 'region of JNK3 ⁇ 2 (SEQ ID No. 3)
- Oligonucleotide corresponding to the 5 ′ region of JNK3 ⁇ l and 3 ⁇ 2 (SEQ ID N ° 4)
- Oligonucleotide corresponding to the 3 'region of JNK3 ⁇ l (SEQ ID No. 5)
- Oligonucleotide corresponding to the 3 'region of JNK3 ⁇ 2 (SEQ ID No. 6)
- Oligonucleotide corresponding to the 5 'region of JNK3 ⁇ N ⁇ l and ⁇ N ⁇ 2 (SEQ ID N ° 7)
- Oligonucleotide corresponding to the 3 'region of JNK3 ⁇ N ⁇ l (SEQ ID No. 8)
- Oligonucleotides used to amplify JNKl ⁇ 1 and human JNK2 ⁇ l by PCR The oligonucleotides were chosen so as to amplify only the coding phase corresponding to JNKl ⁇ l and JNK2 ⁇ l. In bold, the initiation and termination codons are indicated and in italics the restriction sites used for cloning in the yeast expression vector.
- Oligonucleotide corresponding to the 5 ′ region of JNKl ⁇ 1 (SEQ ID No. 10)
- the first pair of oligonucleotides hybridizes to the G418 resistance gene and to the yeast Hogl gene upstream of the initiation codon (SEQ ID No. 14) or downstream of the termination codon (SEQ ID No. 15):
- the second pair of oligonucleotides hybridizes to the yeast gene Hogl and to the part corresponding to the Hogl gene sequence of the first pair of oligonucleotides (SEQ ID N 0 14 or 15):
- Oligonucleotides to amplify part of the deleted Hogl gene by insertion of the G418 resistance gene are:
- thermostable Taq polymerase or PCR
- PCR makes it possible to amplify a DNA sequence between two known regions where oligonucleotides (of around 20 nucleotides) can bind, which will serve as primers for a thermostable polymerase.
- the reactions are carried out in a final volume of 100 ml in the presence of the DNA template (50 ng), dNTP (0.2 M), PCR buffer (TrisHCl pH8.5 l OmM; MgC l, 5mM; KC1 5mM; 0.01% gelatin), two oligonucleotides (500 ng each) and 2.5 IU of Taq DNA Ampli polymerase.
- the mixture is covered with 2 drops of paraffin oil to limit the evaporation of the sample.
- the program used comprises 32 cycles: 1 cycle of denaturation of the matrix (5 min at 95 ° C), 30 cycles (1 min of denaturation at 94 ° C, 1 min of hybridization of the oligonucleotides on the DNA matrix at 50 ° C, 1 min elongation by Taq polymerase at 72 ° C), finally 1 final elongation cycle (5 min at 72 ° C).
- PCR can be performed directly on bacteria or yeast cultures in the exponential growth phase in place of the DNA template.
- the sequencing technique used is derived from the method of Sanger et al. (1977) and adapted for fluorescence sequencing developed by Applied Biosystems.
- the protocol used is that described by the designers of the system (Perkin Elmer, 1995).
- the insert originating from a plasmid DNA or from a PCR reaction is digested for 1 h by restriction enzymes appropriate to 1U / mg of DNA in their respective buffer (Biolabs).
- the fragments resulting from the digestion are separated according to their size by electrophoresis on a gel "0.8% agarose preparation prepared in a TBE buffer (Tris 90mM, Borate 90mM, EDTA 2mM PH8) with 0.5 ⁇ g / ml of Bet. Blue deposit (1/10 volume) is added to the samples before loading onto the gel next to a molecular weight marker (1KB Ladder, Gibco BRL).
- Migration takes place at 90 volts / cm 2 constant in buffer DNA is revealed under UV by the fluorescence of Bet which is inserted between the bases.
- the pieces of gel containing the DNA fragments of desired sizes are cut with a scalpel, introduced into a rod. dialysis with TBE and subjected to electrophoresis for 30 min at 90 volts.
- the DNA extracted from the gel is then recovered, treated with one volume of phenol / chloroform / isoamylate (25/24/1), precipitated with 3 volumes of absolute ethanol in the presence of 0.25M NaCl, washed once with 70% ethanol o , dried, and finally taken up in 20 ⁇ l H 2 0.
- ligation IX final Tris, HC1 50mM PH7.5, MgCl 2 lOmM, ATP ImM
- Ligation takes place at 20 ° C for at least 1 hour.
- TLB Temporal et a, 1988
- This method consists of weakening the wall of bacteria by treatment with DMSO to allow the entry of DNA into cells. Its efficiency is 10 6 transformants / ⁇ g of DNA.
- the culture is centrifuged for 10 min at 3000 ⁇ m, the pellet is taken up in 1/10 of the initial volume with TSB (LB medium, PEG 4000 10%, DMSO 5%, MgCl 2 10 mM, MgSO 4 10 mM), and incubated 15 min at 4 ° C.
- DNA 10 ⁇ l for the ligation product
- TSBglu 20 mM glucose
- the yeasts are made competent by a LiAc / PEG treatment according to the method described by Gietz (Gietz et al, 1995).
- the yeasts are cultured on YPD solid medium at 28 ° C. overnight. They are then resuspended in 1 ml of sterile solution I (LiAc 0.1 M, Tris.HCL lOmM, EDTA ImM pH7.5), centrifuged for 1 min at 3000 ⁇ m and taken up again in 1 ml of solution I. 50 ⁇ L of the suspension are then mixed with 5 ⁇ g of salmon sperm DNA (trainer DNA), 1 to 5 ⁇ g of plasmid DNA and 300 ⁇ l of sterile solution II (LiAc 0.1M, Tris, HCl lOmM, EDTA ImM PH7.5, PEG 400 or 50%) .
- sterile solution I LiAc 0.1 M, Tris.HCL lOmM, EDTA ImM pH7.5
- sterile solution II LiAc 0.1M, Tris, HCl lOmM, EDTA ImM PH7.5, PEG 400 or 50%
- the mixture is incubated for 30 min at 28 ° C and then undergoes thermal shock for 20 min at 42 ° C. After centrifugation for 1 min at 3000 ⁇ m, the cells are washed once with sterile water and taken up in 100 ⁇ l of sterile water. Then, the yeasts are spread on YB agar medium supplemented with the amino acids necessary for their growth. To allow the selection of the transformed yeast strains, the amino acids and the nitrogen bases corresponding to the selection markers carried by the plasmids are not added. After spreading the yeast cells, the dishes incubated at 28 ° C for 3 days. 1 1) Drop test:
- This test makes it possible to analyze the phenotype of a yeast strain.
- a drop (approximately 5 ⁇ l) of a slightly cloudy yeast suspension (approximately 10 5 cells / ml) is deposited on various hyperosmotic and non-hyperosmotic agar media, and the growth is observed after 2 days of incubation at 28 ° C.
- Example 1 - cloning of cDNA coding for new isoforms of human J K3: JNK3 ⁇ N ⁇ l, JNK3 ⁇ N ⁇ 2 and JNK3 ⁇ l39.
- JNK3 Molecular cloning of human JNK3 was carried out by PCR from a human cerebellum cDNA expression library (Clontech®) or a double hybrid human brain cDNA library (Clontech®).
- the nucleotide primers were chosen so as to amplify by PCR the coding phase corresponding to JNK3 ⁇ l and 3 ⁇ 2 (Genbank U34820 and U34819) (see Materials and Methods).
- a first series of oligonucleotides used to amplify JNK3 from the cerebellum library made it possible to provide the BamHI cloning sites in 5 ′ and Kpnl in 3 ′ so as to be able to introduce the amplified fragments of DNA into cloning or appropriate expressions (SEQ ID N ° 1, SEQ ID N ° 2 and SEQ ID N ° 3).
- the second series used on the bank of two brain cDNA hybrids, made it possible to introduce the Smal and BamHI sites in 5 'and Smal and Xhol in 3' (SEQ ID N ° 4, SEQ ID N ° 5 and SEQ ID No. 6).
- the PCR reaction was carried out with 1 ⁇ g of DNA from one of the two expression libraries and 0.5 ⁇ g of each oligonucleotide flanking the region to be amplified.
- the DNA fragments obtained were cloned into suitable cloning vectors to analyze their sequence.
- the complete nucleotide sequence of JNK3 ⁇ N ⁇ l is presented in the sequence SEQ ID No. 22 and the complete nucleotide sequence of JNK3 ⁇ N ⁇ 2 is presented in the sequence SEQ ID No. 24.
- the corresponding polypeptide sequences are presented respectively in the sequence SEQ ID No. 23
- the protein expressed from these sequences has a deletion of 38 amino acids corresponding to the N-terminal extension of JNK3 which distinguishes it from other JNKs (FIG. 3).
- the third sequence corresponding to the isoform JNK3 ⁇ l 39 is characterized by a substitution of the nucleotide C in T, in position 418 of the ATG, in the common part of JNK3 ⁇ l and JNK3 ⁇ 2 (FIG. 1). This mutation allows the creation of a termination codon.
- the protein expressed from this sequence is deleted from a large part of the C-terminal region of JNK3.
- This polypeptide only includes the first 139 amino acids common to JNK3 ⁇ l and 3 ⁇ 2 ( Figure 3) which includes the ATP binding site.
- the nucleotide sequence of JNK3 ⁇ 139 is presented in the sequence SEQ ID No. 26 and the corresponding polypeptide sequence is presented in the sequence SEQ ID No. 27.
- the deletion of the Hogl gene was carried out according to the technique described by Wach (Wach et al. 1994).
- This technique uses a DNA deletion fragment obtained directly by PCR. This corresponds to a part of the sequence of the gene to be destroyed interrupted by a resistance marker.
- a pair of oligonucleotides was used whose sequence makes it possible to amplify by PCR the gene for resistance to G418, while generating at the ends of the amplified fragment the sequences corresponding to the gene to be destroyed.
- This resistance marker is under the control of the promoter and the terminator of a highly expressed gene, the TEF gene from Ashbya gossypii.
- the first pair hybridizes to the terminal ends of the G418 resistance gene carried by the plasmid pFA6a-kanMX4. These oligonucleotides also contain a short floating sequence of 20 base pairs each corresponding respectively to the region just upstream of the ATG and downstream of the stop codon of Hogl.
- the second pair of oligonucleotides makes it possible to extend these floating sequences by 20 base pairs during a second PCR step.
- the purpose of this extension is to increase the frequency of crossing-over in these floating sequences and, therefore, the deletion of the Hogl gene.
- the deletion fragment was carried out in two stages of PCR using in the first the plasmid pFA6a-kan MX4 as template and in the second the product of the first PCR. The fragment was used to transform the W303a strain.
- the transformed strains were first selected for their resistance to
- G418 which is conferred by the deletion fragment, then for the osmosensitivity phenotype corresponding to the total inactivation of the hyperosmotic stress pathway via the deletion of the Hogl gene.
- YPD complete medium
- NaCl sodium chloride
- the pair of oligonucleotides (SEQ ID No. 20 and SEQ ID No. 21) used to amplify the region corresponding to the deleted Hogl gene made it possible to obtain a PCR fragment only on genomic DNA originating from osmo-sensitive strains , thus showing that these strains were indeed deleted in the Hogl gene.
- the other pair of oligonucleotides (SEQ ID No. 18 and SEQ ID No. 19) could only generate a fragment from the genomic DNA of the osmo-resistant strains. This confirms that these strains are not deleted in the Hogl gene.
- the yeast strain deleted in the Hogl gene is called ylMl.
- Example 3 Complementation of the osmo-sensitive yeast strain with the different isoforms of human JNK3.
- This example illustrates the properties of the various human JNK3 isoforms.
- the complementation test of the osmo-sensitive strain mutated in the Hogl gene (y ⁇ Ml) was carried out with the different isoforms of JNK3.
- the kinases JNKl ⁇ 1 (u35004) and JNK2 ⁇ l (u34821) were used as a control.
- kinases having a function homologous to kinase HOG1 are capable of restoring the growth of yeast ylMl under conditions of hyperosmotic stress.
- the expression vector pYX232 which replicates with a high number of copies in the cell was chosen to express the various JNKs in yeast. It has the T ⁇ l gene as a selection marker and an expression cassette constituted by the promoter TPI (Triose phosphate isomerase) and a polyA sequence separated by a cloning multisite.
- TPI Teriose phosphate isomerase
- the various inserts coding for JNKl ⁇ l, JNK2 ⁇ l and the human JNK3 isoforms were introduced at the EcoRI site of this plasmid, previously cleaved by the corresponding restriction enzyme and made blunt by the Klenow fragment of DNA polymerase I .
- the fragments JNKl ⁇ l, JNK2 ⁇ l, JNK3 ⁇ l, JNK3 ⁇ 2, JNK3 ⁇ N ⁇ l and JNK3 ⁇ N ⁇ 2 were obtained by PCR from plasmids containing the corresponding cDNA and oligonucleotides, described in Materials and Methods (SEQ ID No. 10 and No.
- the various constructions allowing the expression of human JNKs as well as the plasmid pYX332 without insert were used to transform the yeast osmo- sensitive Hogl-.
- the clones containing the various plasmids were selected on minimum YNB medium supplemented with amino acids or nitrogen bases necessary for the growth of the strain W303a with the exception of tryptophan.
- Three clones corresponding to each construct were analyzed by drop test on complete medium containing 0.5 or 0.9 M NaCl or 1 M sorbitol. The strains are incubated for three days at 30 degrees. The results are presented in the table below.
- the different strains indicated in the 1st column are tested in culture on different complete media (YPD) added or not with Sorbitol or NaCl as indicated in columns n ° 2 to n ° 5.
- the sign + indicates the aptitude for growth, the sign - indicates the absence of growth.
- the clones transformed by the plasmids coding for JNKl ⁇ 1, JNK2 ⁇ l and two of the isoforms of JNK3 deleted from the N-terminal region are capable of growing on complete medium containing 0.5 M NaCl or 1M sorbitol.
- the clones expressing JNKl ⁇ l are the only ones which show a slight growth on medium containing 0.9 M NaCl.
- JNKl ⁇ l, JNK2 ⁇ l and two of the JNK3 isoforms deleted from their N-terminal region are functional in yeast and are capable of replacing the HOG kinase
- JNKl a protein kinase stimulated by UV light and Ha- Ras that binds and phosphorylates the c-Jun activation domain.
- Herdegen, T. et al. (1997). The c-Jun protein-transcriptional mediator of neuronal survival, regeneration and death. Trends Neurosci. 20, 227-231. Herdegen, T. et al. (1998). Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury. J. Neurosci. 18, 5124-5135.
- p493F12 kinase a novel MAP kinase expressed in a subset of neurons in the human nervous System. Brain Res Mol Brain Res. 35, 47-57.
- a splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous System. Proc. Natl. Acad. Sci. USA. 95, 2586-2591.
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IL14395000A IL143950A0 (en) | 1999-01-20 | 2000-01-19 | Polypeptides derived from jnk3 |
CA002358914A CA2358914A1 (fr) | 1999-01-20 | 2000-01-19 | Polypeptides derives de jnk3 |
NZ512986A NZ512986A (en) | 1999-01-20 | 2000-01-19 | Polypeptides derived from JNK3 |
JP2000594932A JP2002534982A (ja) | 1999-01-20 | 2000-01-19 | Jnk3から誘導されるポリペプチド |
AU30560/00A AU3056000A (en) | 1999-01-20 | 2000-01-19 | Polypeptides derived from jnk3 |
EP00900608A EP1144651A1 (fr) | 1999-01-20 | 2000-01-19 | Polypeptides derives de jnk3 |
US09/909,650 US6649388B2 (en) | 1999-01-20 | 2001-07-19 | Polypeptides derived from JNK3 |
NO20013568A NO20013568L (no) | 1999-01-20 | 2001-07-19 | Polypeptider avledet fra JNK3 |
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FR9900586A FR2788531B1 (fr) | 1999-01-20 | 1999-01-20 | Nouveaux polypeptides derives de la proteine jnk3 humaine, leurs variants, les sequences nucleotidiques correspondantes, et leurs utilisations |
FR99/00586 | 1999-01-20 | ||
US12217599P | 1999-02-26 | 1999-02-26 | |
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CA2148898A1 (fr) * | 1994-05-09 | 1995-11-10 | John M. Kyriakis | Proteine p54 activee par le stress |
US5804399A (en) * | 1993-07-19 | 1998-09-08 | The Regents Of The University Of California | Oncoprotein protein kinase |
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US6162613A (en) * | 1998-02-18 | 2000-12-19 | Vertex Pharmaceuticals, Inc. | Methods for designing inhibitors of serine/threonine-kinases and tyrosine kinases |
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US5804399A (en) * | 1993-07-19 | 1998-09-08 | The Regents Of The University Of California | Oncoprotein protein kinase |
CA2148898A1 (fr) * | 1994-05-09 | 1995-11-10 | John M. Kyriakis | Proteine p54 activee par le stress |
Non-Patent Citations (2)
Title |
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MICHIHITO ITO ET AL.: "JSPA1, a novel Jun N-terminal protein kinase (JNK)-binding protein that functions as a scaffold factor in the JNK signaling pathway", MOLECULAR AND CELLULAR BIOLOGY, vol. 19, no. 11, November 1999 (1999-11-01), pages 7539 - 7548, XP000901824 * |
SHASHI GUPTA ET AL.: "Selective interaction of JNK protein kinase isoforms with transcription factors", EMBO JOURNAL, vol. 18, no. 11, 1996, EYNSHAM, OXFORD GB, pages 2760 - 2770, XP002915477 * |
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WO2002012338A2 (fr) * | 2000-08-03 | 2002-02-14 | Grünenthal GmbH | Procede de criblage |
WO2002012338A3 (fr) * | 2000-08-03 | 2002-12-19 | Gruenenthal Gmbh | Procede de criblage |
EP1469316A1 (fr) * | 2000-08-03 | 2004-10-20 | Grünenthal GmbH | Procédé de criblage |
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US20020165386A1 (en) | 2002-11-07 |
NO20013568L (no) | 2001-09-06 |
NZ512986A (en) | 2004-01-30 |
JP2002534982A (ja) | 2002-10-22 |
AU3056000A (en) | 2000-08-07 |
IL143950A0 (en) | 2002-04-21 |
NO20013568D0 (no) | 2001-07-19 |
EP1144651A1 (fr) | 2001-10-17 |
CA2358914A1 (fr) | 2000-07-27 |
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