EP0941319A1 - Peptides capable of inhibiting the endocytosis of the app and corresponding nucleotide sequences - Google Patents

Peptides capable of inhibiting the endocytosis of the app and corresponding nucleotide sequences

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Publication number
EP0941319A1
EP0941319A1 EP96938292A EP96938292A EP0941319A1 EP 0941319 A1 EP0941319 A1 EP 0941319A1 EP 96938292 A EP96938292 A EP 96938292A EP 96938292 A EP96938292 A EP 96938292A EP 0941319 A1 EP0941319 A1 EP 0941319A1
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EP
European Patent Office
Prior art keywords
peptide
app
protein
sequence
interaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96938292A
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German (de)
French (fr)
Inventor
Luc Merken
Alain Fournier
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aventis Pharma SA
Original Assignee
Rhone Poulenc Rorer SA
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Filing date
Publication date
Application filed by Rhone Poulenc Rorer SA filed Critical Rhone Poulenc Rorer SA
Publication of EP0941319A1 publication Critical patent/EP0941319A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein

Definitions

  • the present invention relates to new peptide and nucleotide sequences, and their pharmaceutical use. More particularly, the present invention relates to new peptides capable of at least partially inhibiting the phenomenon of APP endocytosis.
  • amyloid ⁇ peptide the major constituent of the amyloid plaque, is a peptide of about 40 amino acids of 4kDa, resulting from the cleavage of APP (Precursor of
  • Amyloid peptide It accumulates significantly in the brain, not only in Alzheimer's disease but also in Down syndrome, more commonly known as Down's syndrome.
  • APP is a glycosylated protein from 100 to 140kDa, having several domains including a transmembrane region, an extracellular domain and a cytoplasmic domain.
  • the region of the molecule more specifically concerned with the expression of the amyloid ⁇ peptide partly overlaps the transmembrane domain and also extends within the extracellular domain.
  • Three major forms of APP, resulting from alternative splicing, have been characterized.
  • the APP gene is located on chromosome 21 and mutations have been identified there in 3 to 5% of patients with familial forms of Alzheimer's disease.
  • the present invention results from the discovery by the applicant that the protein FE65 does not only constitute a transcriptional activator in the brain but that it also interacts at the level of the cytoplasmic region of APP by intervening in the modulation of the APP endocytosis.
  • the present invention results more particularly from the identification and characterization of particular regions (so-called effector regions) of the FE65 protein, involved in the transduction of signals for the activation of APP endocytosis.
  • regions regions (so-called effector regions) of the FE65 protein, involved in the transduction of signals for the activation of APP endocytosis.
  • the demonstration of the existence of such regions makes it possible to envisage the preparation of new peptides which can be used pharmaceutically.
  • the name protein FE65 covers the protein itself as well as all of its homologous forms.
  • the term “homologous form” is intended to denote any protein equivalent to the protein under consideration, of diverse cellular origin and in particular derived from cells of human origin, or of other organisms, and having an activity of the same type. Such homologous sequences can be obtained by hybridization experiments. Within the meaning of the invention, it is sufficient for a sequence of this type to have a significant percentage of identity to lead to physiological behavior comparable to that of the protein FE 65 as claimed.
  • a first object of the invention therefore relates to peptides capable of interfering at least partially at the level of the interaction of the protein FE65, or one of its homologous forms, with the cytoplasmic region of APP.
  • the claimed peptide can slow, inhibit or at least partially stimulate the interaction between the protein FE65 or one of its homologous forms with the cytoplasmic region of APP.
  • Such peptides are preferably peptides capable of at least partially antagonizing this interaction.
  • the peptides are capable of binding at the level of the interaction domain between the protein FE65 or one of its homologous forms and the cytoplasmic region of APP.
  • the peptides of the invention comprise all or part of the peptide sequence coding for the protein FE65 presented in SEQ ID No. 1, SEQ ID No. 2 or one of its derivatives.
  • the term derivative designates any sequence differing from the sequence considered due to a degeneration of the genetic code, obtained by one or more modifications of a genetic and / or chemical nature, as well as any sequence hybridizing with these sequences or fragments thereof and retaining the ability to interact at the level of the interaction between the protein FE65, or one of its homologs, and the cytoplasmic region of APP.
  • modification of genetic and / or chemical nature one can hear any mutation, substitution, deletion, addition and / or modification of one or more residues.
  • the term derivative also includes sequences homologous to the sequence considered, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type.
  • hybridization experiments can be carried out from nucleic acid libraries, using the native sequence or a fragment thereof as probe, under variable hybridization conditions (Maniatis et al., Cf. general techniques of molecular biology).
  • Such derivatives can be generated for different purposes, such as in particular that of increasing their therapeutic efficacy or reducing their side effects, or that of conferring on them new pharmacokinetic and / or biological properties.
  • a peptide derived from the FE65 protein and homologous forms mention may in particular be made of any peptide capable of interacting with the cytoplasmic region of APP, but carrying an effector region made non-functional. Such peptides can be obtained by deletion, mutation or disruption of this effector region on the protein FE65 and homologous forms. Such modifications can be made, for example, by in vitro mutagenesis, by introduction of additional elements or synthetic sequences, or by deletions or substitutions of the original elements.
  • a derivative as defined above When a derivative as defined above is produced, its activity as a partial inhibitor of the binding of the protein FE65 and of the homologous forms on its APP binding site can be highlighted. Any technique known to those skilled in the art can obviously be used for this purpose.
  • fragments of the sequences indicated above can be generated in different ways.
  • they can be synthesized chemically, on the basis of the sequences given in the present application, using the peptide synthesizers known to those skilled in the art.
  • They can also be synthesized genetically, by expression in a cellular host of a nucleotide sequence coding for the peptide sought.
  • the nucleotide sequence can be prepared chemically using an oligonucleotide synthesizer, on the basis of the peptide sequence given in the present application and the genetic code.
  • the nucleotide sequence can also be prepared from the sequences given in the present application, by enzymatic cleavages, ligation, cloning, etc., according to techniques known to those skilled in the art, or by screening DNA libraries with elaborate probes from these sequences.
  • the peptides of the invention namely capable of slowing down or at least partially inhibiting the interaction between the protein FE65 and homologous forms and the cytoplasmic region of APP can also be peptides having a sequence corresponding to site of interaction of the protein FE65 and homologous forms on the cytoplasmic region of APP.
  • peptides according to the invention are the peptides capable of competing with the peptides defined above for the interaction with their cellular target. Such peptides can be synthesized in particular on the basis of the sequence of the peptide considered, and their ability to compete with the peptides defined above can be determined.
  • Another subject of the invention resides in antibodies or fragments of polyclonal or monoclonal antibodies directed against a peptide as defined above.
  • Such antibodies can be generated by methods known to those skilled in the art.
  • these antibodies can be prepared by immunizing an animal against a peptide of the invention, drawing blood, and isolating the antibodies.
  • These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art.
  • the antibodies or antibody fragments of the invention have the ability to at least partially inhibit the interaction of peptides claimed with the cytoplasmic region of the APP. They can thus be used to modulate the endocytosis of APP.
  • these antibodies can also be used to detect and / or measure the expression or overexpression of APP in biological samples, and therefore, to provide information on its state of activation.
  • the invention provides an equal ment of non-peptidic compounds or non-peptidic pharmaceutically usable exclusively. It is indeed possible, from the active protein motifs described in the present application, to produce molecules which inhibit the signaling pathway dependent on the FE65 protein which are not exclusively peptide and which are compatible with pharmaceutical use.
  • the present invention also relates to any nucleotide sequence coding for a peptide according to the invention. It may in particular be a sequence comprising all or part of the sequence presented in SEQ ID No. 1, SEQ ID No. 2 or one of their derivatives. By derivative sequence is meant within the meaning of the present invention any sequence hybridizing with the sequence presented in SEQ ID No. 1 or in SEQ ID No. 2 or with a fragment thereof and coding for a peptide according to the invention, as well as the sequences resulting from these by degeneration of the genetic code.
  • the different nucleotide sequences of the invention can be of artificial origin or not.
  • D can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained either by screening of DNA libraries (cDNA library, genomic DNA library), or by chemical synthesis, or by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries.
  • nucleotide sequences can be used for the production of the peptides of the invention.
  • the present application thus relates to a process for the preparation of such a peptide according to which a cell containing a nucleotide sequence according to the invention is cultivated, under conditions of expression of said sequence and the peptide produced is recovered.
  • the part coding for said peptide is generally placed under the control of signals allowing its expression in a cellular host.
  • the choice of these signals promoters, terminators, "leader” secretory sequence, etc.
  • the nucleotide sequences of the invention can be part of a vector which can be autonomously replicating or integrative.
  • replication vectors Autonomous can be prepared using autonomous replicating sequences in the chosen host.
  • 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.
  • the cellular hosts which can be used for the production of the peptides of the invention by the recombinant route 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.
  • COS COS
  • CHO CHO
  • C127 cells etc.
  • Aspergillus ssp. or Trichoderma ssp.
  • prokaryotic hosts it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces.
  • the nucleic acid sequences according to the invention can also be used for the production of antisense oligonucleotides or of genetic antisense usable as pharmaceutical agents.
  • the antisense sequences are small Ogonucleotides, complementary to the coding strand of a given gene, and therefore capable of hybridizing specifically with the transcribed mRNA, inhibiting its translation into protein.
  • the subject of the invention is therefore the antisense sequences capable of at least partially inhibiting the interaction of the FE65 proteins on the cytoplasmic region of APP.
  • Such sequences can consist of all or part of the nucleic acid sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides interacting with the cytoplasmic region of APP.
  • Such Ogonucleotides can be obtained by fragmentation, etc., or by chemical synthesis.
  • the claimed sequences can be used in the context of gene therapies, for the transfer and expression in vivo of antisense sequences or of peptides capable of modulating the interaction of the FE65 proteins with the cytoplasmic region of APP.
  • the sequences can be incorporated into viral or non-viral vectors, allowing their administration in vivo (Medicine and Sciences 7 (1991) 705).
  • viral vectors in accordance with the invention, mention may very particularly be made of vectors of the adenovirus, retrovirus, adeno-associated virus or herpes virus type.
  • the present application also relates to defective recombinant viruses comprising a heterologous nucleic sequence coding for a polypeptide according to the invention.
  • the invention also allows the production of nucleotide probes, synthetic or not, capable of hybridizing with the nucleotide sequences defined above, usable in the context of gene therapy.
  • probes can be used in vitro as a diagnostic tool, for the detection of the expression or overexpression of APP, 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 homologous nucleic acid sequences 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 10 bases, and they can for example comprise up to the entirety of one of the abovementioned sequences or of their complementary strand. Preferably, these probes are, prior to their use, marked. For this, different techniques known to those skilled in the art can be used (radioactive, enzymatic labeling, etc.).
  • the invention also relates to any pharmaceutical composition comprising as active principle at least one peptide as defined above.
  • composition comprising as active ingredient at least one antibody and / or an antibody fragment as defined above, as well as any pharmaceutical composition comprising as active ingredient at least one nucleotide sequence as defined above before.
  • compositions in which the peptides, antibodies and nucleotide sequence defined above are associated with each other or with other active ingredients.
  • compositions according to the invention can be used to modulate the activation of APP proteins and therefore to modulate its endocytosis and the production of amyloid ⁇ peptides. More particularly, these pharmaceutical compositions are intended to modulate the interaction between the FE65 proteins and the cytoplasmic region of APP. More preferably, these compositions are intended to slow down or at least partially inhibit the interaction of the FE65 proteins with the cytoplasmic region of APP. More preferably, these are pharmaceutical compositions intended for the treatment of neurodegenerative diseases such as, for example, Alzheimer's disease and trisomy 21.
  • Another subject of the invention is the use of the molecules described above to modulate the activation of APP endocytosis or the typing of diseases neurodegenerative.
  • the invention relates to the use of these molecules to at least partially inhibit activation of APP endocytosis.
  • Figure 1 Representation of the vector pGAL4DB-CAPP
  • Figure 2 Representation of plasmid DNAs on gels obtained according to Example 3.
  • yeast strains used are:
  • the YCM strain of the genus S.cerevisiae (MATa, ura3-52, his3-200, ade2-101, lys2- 801, trpl-901, leu2-3,112, canr, gal4-542, gal80-538, URA3 :: GALlll0- lacZ,
  • LYS :: GAL1I10-HIS3 was used as a tool to screen the brain fusion bank by the two hybrid system.
  • the L40 strain of the genus S.cerevisiae (Mata, his3D200, trpl-901, leu2-3,112, ade2,
  • LYS2 (lexAop) 4-HIS3, URA3: :( lexAop) 8-LacZ, GAL4) was used to verify protein-protein interactions when one of the protein partners is fused to the LexA protein.
  • the latter is capable of recognizing the LexA response element controlling the expression of the reporter genes LacZ and His3.
  • MiUeu YNB rninimum -Yeast Nitrogen Base (without amino acids) (6.7g l) (Difco)
  • This medium can be made solid by adding 20g / l of agar (Difco).
  • auxotrophic yeasts To allow the growth of auxotrophic yeasts on this medium, it is necessary to add to it amino acids or nitrogen bases, on which they are dependent at 50 mg / ml. 100 ⁇ g / ml of ampicillin are added to the medium in order to avoid bacterial contamination.
  • Ampicillin was used at 100 ⁇ g / ml, this antibiotic is used to select the bacteria which have received the plasmids carrying as marker the gene for resistance to this antibiotic.
  • the vector pGBTIO. (Clontech).
  • a 5.4 kb shuttle plasmid which has an origin of bacterial and yeast replication allowing it to replicate with a high number of copies in these two microorganisms.
  • This plasmid contains a multiple cloning site located downstream of the sequence coding for the DNA binding domain of GAL4 and upstream of a terminator to form a fusion protein. It also contains the TRP1 gene from S. cerevisiae which makes it possible to complement yeasts of the trpl genotype in order to select them on a minimum medium containing no tryptophan.
  • This vector carries the ampiciUine resistance gene which makes it possible to select the bacteria possessing it on a medium containing ampiciUine.
  • the vector pGADIO supplied by Clontech and which allows the expression in yeast of fusion proteins between the GAL4 transactivating domain and a protein coded by the cDNA originating from a brain bank, inserted at an EcoRI site.
  • the vector pLex9 (pBTMll ⁇ ) (Bartel et al DA Hartley Ed, Oxford University press page 153) of 5kb homologous to pGBTIO which contains a multiple cloning site located downstream of the sequence coding for the bacterial repressor LexA and upstream of a terminator to form a fusion protein.
  • the Ogonucleotides which made it possible to obtain the PCR fragment corresponding to CAPP with the EcoRI and Sali sites.
  • the Ogonucleotides are synthesized on the Applied System ABI 394-08. They are detached from the synthesis matrix with ammonia and precipitated twice with 10 volumes of n-butanol and then taken up in water. The quantification is carried out by measuring the optical density (1DO corresponds to 30 ⁇ g ml).
  • DNA Small amounts of DNA are prepared in the following manner: the bacteria containing the plasmid are cultured for at least 4 hours in 2 ml of miUeu LB in a shaker shaker. They are then centrifuged for 2 minutes at 14,000 rpm in Ependorf tubes, then the pellet is resuspended in 100 ⁇ l of solution I (50 mM of glucose, 25 mM of Tris HC1 ⁇ H8 buffer, 10 mM EDTA ⁇ H8), lysed with 200 ⁇ l of the solution II (0.2M NaOH, 1% SDS). The lysis solution is then neutralized with 150 ⁇ l of solution III (3M of potassium acetate, 11.5% (v / v) of glacial acetic acid).
  • solution I 50 mM of glucose, 25 mM of Tris HC1 ⁇ H8 buffer, 10 mM EDTA ⁇ H8
  • the lysis solution is then neutralized with 150 ⁇ l of solution III (3M of potassium acetate, 11.5%
  • RNAse 10 mM Tris-HCl solution and ImM EDTA with 50 ⁇ g / ml of RNAse.
  • PCR reactions are carried out in a final volume of 100 ⁇ l in the presence of the DNA template, dNTP (0.2 mM), PCR buffer (Tris-HCL pH 8.5 10 mM, MgCl 2 ImM, KCl 5 mM , 0.01% gelatin), 0.5 ⁇ g of each of the oGonucleotides and 2.5 IU of AmpU Taq DNA polymerase (Perkin Elmer) with or without formamide (5%).
  • the mixture is covered with 2 drops of paraffin oil to limit the evaporation of the sample.
  • the device used is the "Crocodile II" from Appligene.
  • All the ligation reactions are carried out at -f-14 ° C overnight in a final volume of 10 ⁇ l in the presence of 100 to 200 ng of vector, 0.5 to 2 ⁇ g of insert, 40 IU of T4 DNA Ugase enzyme (Biolabs) and a ligation buffer (50 mM Tris-HCl pH 7.8; 10 mM MgCl 2 ; 10 mM DTT; 1 mM ATP).
  • the negative control consists of the ugation of the vector in the absence of an insert.
  • the transformation of the bacteria by a plasmid is carried out according to the following protocol :.
  • the entire volume of Ugation (10 ⁇ l) is used to transform the TGl bacteria made competent by the method of Chung et al, (PNAS. 1988 86, 2172-2175).
  • the TGl bacteria are cultured in a liquid LU medium for a few hours in a shaking oven at 37 ° C., until an OD of 0.6 to 600 nm is obtained. The medium is then centrifuged at 6000 rpm for 10 min.
  • the bacteria are made competent by taking up the bacterial pellet with a volume of TSB (miUeu LB + 100 g / 1 of PEG 4000, 5% of DMSO, 10 mM of MgCl 2) 10 mM of MgSO 4 ) corresponding to 1/10 of the volume of the medium of the initial culture. After incubation at 4 ° C for 30 to 60 minutes, 200 ⁇ l of bacteria are brought into contact with the Ugation products for 15 minutes on ice. After adding 200 ⁇ l of LB, the bacteria are incubated for 30 min at 37 ° C. and then spread on an LB + ampicillin medium.
  • TSB miUeu LB + 100 g / 1 of PEG 4000, 5% of DMSO, 10 mM of MgCl 2
  • 10 mM of MgSO 4 10 mM of MgSO 4
  • the DNA is separated according to their size by electrophoresis. To do this; different gels are used depending on the size of the fragments to be separated:
  • the extraction of DNA from the band of an agarose gel is carried out by electroelution as follows:
  • the piece of gel containing the DNA fragment is cut with a scalpel and placed in a dialysis rod closed by two forceps and containing 100 to 500 ⁇ l of TBE.
  • the whole is put in an electrophoresis tank where it undergoes an electric field of 100 Volts.
  • the DNA after being removed from the gel, is then purified by two phenol / chloroform extractions followed by two chloroform extractions, then precipitated in the presence of 0.3M sodium acetate and 2.5 volumes of absolute ethanol. After centrifugation (5 min at 14,000 rpm) the DNA pellet is dried and then taken up in 20 ⁇ l of water.
  • the sequencing is done according to the Sanger method using 4 dideoxyribonucleotides having a different fluorescent marker.
  • the incorporation of one of these dideoxyribonucleotides produces a stop in the replication by Taq polymerase of the DNA to be sequenced. This reaction will give DNA fragments of different sizes, all terminated in 3 'with one of the 4 dideoxyribonucleotides.
  • One ⁇ g of a plasmid and 4 picomoles of a primer are added to 9.5 ⁇ l of a "premix" supplied by AppUed Biosystems under the name of Prism.
  • the final volume must be 20 ⁇ l to carry out a PCR for 25 cycles, decomposing into a denaturation step at 96 ° C for 30 seconds.
  • the DNA fragments, obtained after amplification, are purified on an exclusion column (Chromaspin-30 from Clontech); and are then dried with Speed Vac. The whole is taken up in 5 ⁇ l of a mixture formed of 24 ⁇ l of EDTA (50 mM) and 120 ⁇ l of deionized formamide. After denaturation at 96 ° C for 3 minutes, 3 to 5 ⁇ l are deposited on an electrophoresis gel.
  • the different DNA fragments are separated according to their size and will pass successively in front of a laser reader of the Apparatus 370 DNA sequencer (Applied Biosystems) where the different fluorescences will be detected.
  • the brain cDNA fusion bank is sold as bacteria.
  • the latter contain a plasmid pGADIO containing an insert corresponding to a human brain cDNA.
  • the cDNAs of this bank are formed using the oUgodT technique and the degenerate oUgonucleotide technique which allows to have the 5 'parts of the mRNAs which are difficult to obtain by the first technique. These cDNAs are cloned into the vector pGADIO at the EcoRI site.
  • the yeasts previously cultivated in 100 ml of liquid medium are harvested after centrifugation at 3000 rpm for 3 minutes and suspended in 1 ml of sterile water. After centrifugation at 3000 m for 3 minutes, the cell pellet is resuspended in 1 ml of sterile water and then centrifuged again. This operation is repeated again in order to eliminate all traces of the culture medium.
  • the yeasts are then taken up in 1 ml of the transformation solution I (0.1A LiAc, Tris-HCl pH 7.5 lOmM, EDTA ImM). then centrifuged at 3000 ⁇ m for 3 minutes. The cell pellet is taken up again in 1 ml of the transformation solution I.
  • a transformation solution II LiAc 0.1M, Tris-HCl pH 7.5 lOmM, EDTA ImM in PEG 4000 40%
  • a thermal shock is then applied to the transformation mixture in a water bath at 40 ° C for 15 minutes and then the whole is centrifuged at 15000 ⁇ m for 1 minute in order to collect the cell pellet.
  • This pellet is taken up in 200 ⁇ l of water and then spread over a minimum agar medium which does not contain the amino acids corresponding to the markers provided by the transforming plasmid.
  • the yeasts are then placed in culture for 72 hours at 28 ° C.
  • the yeast used contains the plasmid pGAL4DB-CAPP coding for the C-terminal part of the APP fused to the DNA binding domain of GAL4. It is cultivated in 250 ml of medium minimum YNB-r-His + Lys + Ad + Leu at 28 ° C.
  • Centrifugation (000 ⁇ m for 5 min) is repeated 3 times in succession, each time taking up the pellet with 10 ml of sterile water. The third time the pellet is taken up with 2.5 ml of PBS. Thus PEG toxic for cells has been eliminated.
  • 2.4 ml of this suspension are used to inoculate 250 ml of minimum medium containing the amino acids His, Lys, Ad and cultured overnight in a shaker at 28 ° C. The remaining 100 ⁇ l of this suspension is used to verify the efficiency of the transformation; for this, dilutions of 10 " , 10 " and 10 of this suspension were made and spread on a minimum medium containing the amino acids His, Lys, Ad. After culture at 28 ° C.
  • the overnight culture is centrifuged (3000 ⁇ m for 5 min) and washed with sterile water twice in succession. The pellet is then taken up in 2.5 ml of water. 2.4 ml, the volume of which is brought to 10 ml in sterile water, are used to inoculate 10 dishes of 435 cm containing YNB + Lys + Ad medium and incubated for 3 days. The remaining 100 ⁇ l are used to carry out the same operations as when determining the transformation rate, in order to determine the rate of amplification of the number of colonies during a culture night.
  • DNA (genomic and plasmid) is extracted from yeasts as follows :.
  • the value of an average loop of a yeast clone is put in 200 ⁇ l of a TELT solution (Triton XI 00 2%, SDS 1%, NaCl lOOmM, Tris pH8 lOmM, EDTA ImM), in the presence of 3g of biUes of glass 450 ⁇ m in diameter and 200 ⁇ l of phenol / chloroform. This mixture is vortexed for 15 minutes, then centrifuged for 2 minutes at 14000 ⁇ m. The supernatant is collected without removing the protein cake and the DNA contained in this phase is precipitated with 2.5 volumes of absolute ethanol.
  • a TELT solution Triton XI 00 2%, SDS 1%, NaCl lOOmM, Tris pH8 lOmM, EDTA ImM
  • the DNA pellet is dried and taken up in 20 ⁇ l of TERNAse.
  • This DNA solution which corresponds to a mixture of genomic and plasmid DNA, is used directly to transform bacteria. Only plasmid DNA is capable of replicating in bacteria and can be analyzed by the miniprep technique.
  • a nitrocellulose sheet is previously deposited on the Petri dish containing the individualized yeast clones. Thanks to the phenomenon of addition, we will obtain a faithful image of the location of the clones. This sheet is then immersed in Uquide nitrogen for 30 seconds in order to burst the yeasts and thus to tiberate the ⁇ galactosidase activity.
  • the nitroceUulose sheet is deposited, colonies upwards, in another Petri dish containing Whatman paper previously soaked with 1.5 ml of PBS solution (Na 2 HPO4 60mM, NaH 2 PO 4 40mM, KCl 10MM, MgSO 4 ImM, pH7) and from 10 to 30 ⁇ l of X-Gal (5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside) at 50 mg / ml of N, N-dimethylformamide.
  • PBS solution Na 2 HPO4 60mM, NaH 2 PO 4 40mM, KCl 10MM, MgSO 4 ImM, pH7
  • X-Gal 5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside
  • the box is then placed in an oven at 37 ° C with the lid closed to prevent drying.
  • the appearance time of the blue color can be very variable, from a few minutes to several hours. This test should always
  • EXAMPLE 1 Construction of a vector allowing the expression of a fusion protein between the C-terminal part of the precursor of the amyloid peptide (CAPP) and the DNA binding domain of GAL4
  • the 138 bpd DNA fragment corresponding to the last 46 amino acids of the APP was obtained by PCR from the Ogonucleotides (SEQ ID N ° 3 and N ° 4) which also allowed us to introduce the EcoRI and Dirty at the ends of the sequence.
  • the PCR fragment was introduced between the EcoRI and SalI sites of the multisite for cloning the plasmid pGBTIO downstream of the sequence corresponding to GAL4DB to give the vector pGAL4DB-CAPP (FIG. 2).
  • the construction was verified by DNA sequencing. This verification allowed us to show that this fragment did not present mutations generated during the PCR reaction and that it was fused in the same open reading phase as that of the fragment corresponding to GAL4DB.
  • EXAMPLE 2 SCREENING OF THE BRAIN FUSION BANK.
  • the screening of a fusion bank makes it possible to identify clones producing proteins fused to the transactivating domain of GAL4, which can interact with our protein of interest. This interaction makes it possible to reconstitute a transactivator which will then be capable of inducing the expression of the reporter genes His3 and LacZ in the YCM strain.
  • a fusion library made from cDNA from the human brain As this library was supplied to us in the form of bacteria, the plasmid DNA of the library was first purified.
  • the plasmid DNA from the brain cDNA library was extracted according to the Clontech protocol (see materials and methods, ⁇ 11). During this preparation, it was important to preserve the representativeness of the library, that is to say, to keep the number of independent plasmids which constitute it and which are 1.2.10 6 plasmids. In order to protect us from the loss of plasmids from the library during this preparation, the batch of plasmid DNA that we have assembled was obtained from a number of isolated bacterial colonies corresponding to slightly more than twice the representativeness of the bank, ie 4.10 6 colonies.
  • each plasmid independent of the fusion library is present in at least one yeast at the same time as the plasmid GAL4DB-CAPP.
  • a yeast transformation protocol giving an efficiency of 10 5 ceUules transformed per ⁇ g of DNA.
  • This YCM-CAPP strain of His-, Lys-, Leu- phenotype was transformed with lOO ⁇ g of plasmid DNA from the fusion bank.
  • EXAMPLE 3 ISOLATION OF BANK PLASMIDS
  • the plasmid DNAs of the bacterial colonies obtained after transformation with yeast DNA extracts were analyzed by digestion with restriction enzymes and separation of the DNA fragments on agarose gel.
  • the DNA of clone 3E is obtained from strains of the His + and ⁇ GAL + phenotype.
  • EXAMPLE 4 DETERMINATION OF THE SEQUENCE OF THE INSERTS OF THE IDENTIFIED PLASMIDS.
  • the sequencing was carried out using the oligonucleotide (SEQ ID No. 5) complementary to the GAL4TA region near the insertion site of the brain cDNA library, 52 bpd from the EcoRI site.
  • the comparison of the 3 sequences with the sequences contained in the GENBank and EMBL (European Molecular Biology Lab) databases showed that the sequences of the cDNAs which were in the plasmids derived from strains 9A and 3H show 87% homology at the nucleic level with the mutant gene coding for the protein FE65, they are represented in SEQ ID No. 2, and that the sequence of the plasmid derived from the strain 7D has 60% homology with this same gene (see FIG. 3). Analysis of the sequence of the plasmids from strains 9A and 3H indicates that these contain two overlapping regions corresponding to the same mRNA. LIST OF SEQUENCES
  • NAME RHONE POULENC RORER S.A.

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Abstract

The invention concerns novel peptide and nucleotide sequences, and their pharmaceutical use. More particularly, it concerns novel peptides capable of inhibiting at least partially the phenomenon of APP endocytosis by intervening at the level of the interaction of the FE65 protein with the cytoplasmic region of the APP.

Description

PEPTIDES CAPABLES D'INHIBER L'ENDOCYTOSE DE L'APP ET SEQUENCES NUCLEOTIDIOUES CORRESPONDANTES PEPTIDES CAPABLE OF INHIBITING APP ENDOCYTOSIS AND CORRESPONDING NUCLEOTIDIOUE SEQUENCES
La présente invention concerne de nouvelles séquences peptidiques et nucléotidiques, et leur utilisation pharmaceutique. Plus particulièrement, la présente invention concerne de nouveaux peptides capables d'inhiber au moins partiellement le phénomène d'endocytose de l'APP.The present invention relates to new peptide and nucleotide sequences, and their pharmaceutical use. More particularly, the present invention relates to new peptides capable of at least partially inhibiting the phenomenon of APP endocytosis.
Le peptide β amyloïde, le constituant majeur de la plaque amyloïde, est un peptide d'environ 40 acides aminés de 4kDa, issu du clivage de l'APP (Précurseur duThe amyloid β peptide, the major constituent of the amyloid plaque, is a peptide of about 40 amino acids of 4kDa, resulting from the cleavage of APP (Precursor of
Peptide Amyloïde). Il s'accumule de façon importante dans le cerveau, non seulement dans la maladie d'Alzheimer mais aussi dans le syndrome de Down plus communément appelé trisomie 21.Amyloid peptide). It accumulates significantly in the brain, not only in Alzheimer's disease but also in Down syndrome, more commonly known as Down's syndrome.
L'APP est une protéine glycosylée de 100 à 140kDa, possédant plusieurs domaines dont une région transmembranaire, un domaine extracellulaire et un domaine cytoplasmique. La région de la molécule plus spécifiquement concernée par l'expression du peptide β amyloïde chevauche en partie le domaine transmembranaire et s'étend d'autre part au sein du domaine extracellulaire. Trois formes majoritaires de 1' APP, résultant d'un épissage alternatif, ont été caractérisées. Le gène de l'APP est localisé sur le chromosome 21 et des mutations y ont été identifiées dans 3 à 5% des patients atteints des formes familiales de la maladie d'Alzheimer.APP is a glycosylated protein from 100 to 140kDa, having several domains including a transmembrane region, an extracellular domain and a cytoplasmic domain. The region of the molecule more specifically concerned with the expression of the amyloid β peptide partly overlaps the transmembrane domain and also extends within the extracellular domain. Three major forms of APP, resulting from alternative splicing, have been characterized. The APP gene is located on chromosome 21 and mutations have been identified there in 3 to 5% of patients with familial forms of Alzheimer's disease.
Le rôle physiologique de l'APP n'est, à ce jour, pas complètement élucidé ; toutefois, on pense qu'il est impliqué dans le contact synaptique, agit à titre de facteur de régulation de croissance, et est neuroprotectif .To date, the physiological role of APP has not been fully elucidated; however, it is believed to be involved in synaptic contact, acts as a growth regulator, and is neuroprotective.
Des données récentes de la littérature ont montré le rôle important de l'endocytose de l'APP pour la production du peptide β amyloïde, et ont permis d'identifier des éléments de séquence, au sein de la région cytoplasmique, utilisés comme signaux d'endocytose. C'est ainsi que des cellules transfectées avec une construction d'ADNc délétée du domaine C terminal cytosolique de l'APP, permettent uniquement la production de la forme soluble de l'APP et ne produisent pas le peptide β amyloïde. Toutefois, in vivo, on ne connait pas encore la nature précise des événements responsables de l'activation de l'endocytose de l'APP ni de la transduction des signaux qui lui sont associés. L'élucidation du rôle exact de ces signaux d'endocytose dans les cellules, de leur mode de fonctionnement et de leurs caractéristiques constitue donc un enjeu majeur pour la compréhension et l'approche thérapeutique de la maladie d'Alzheimer et plus généralement des maladies neurodégénératives.Recent data from the literature have shown the important role of APP endocytosis for the production of the amyloid β peptide, and have made it possible to identify elements of sequence, within the cytoplasmic region, used as signal signals. endocytosis. Thus cells transfected with a cDNA construct deleted from the cytosolic terminal C domain of APP, only allow the production of the soluble form of APP and do not produce the amyloid β peptide. However, in vivo, we do not yet know the precise nature of the events responsible for the activation of APP endocytosis or the transduction of the signals associated with it. The elucidation of the exact role of these endocytosis signals in cells, their mode of operation and their characteristics therefore constitutes a major challenge for the understanding and the therapeutic approach of Alzheimer's disease and more generally of neurodegenerative diseases .
La présente invention résulte de la mise en évidence par la demanderesse que la protéine FE65 ne constitue pas uniquement un activateur transcriptionnel dans le cerveau mais qu'elle interagit également au niveau de la région cytoplasmique de l'APP en intervenant dans la modulation de l'endocytose de l'APP.The present invention results from the discovery by the applicant that the protein FE65 does not only constitute a transcriptional activator in the brain but that it also interacts at the level of the cytoplasmic region of APP by intervening in the modulation of the APP endocytosis.
La présente invention résulte plus particulièrement de l'identification et de la caractérisation de régions particulières (dites régions effectrices) de la protéine FE65, impliquées dans la transduction des signaux d'activation de l'endocytose de l'APP. La mise en évidence de l'existence de telles régions permet d'envisager la préparation de nouveaux peptides utilisables pharmaceutiquement.The present invention results more particularly from the identification and characterization of particular regions (so-called effector regions) of the FE65 protein, involved in the transduction of signals for the activation of APP endocytosis. The demonstration of the existence of such regions makes it possible to envisage the preparation of new peptides which can be used pharmaceutically.
Au sens de la présente invention, la dénomination protéine FE65 couvre la protéine en soit ainsi que toutes ses formes homologues. Par forme homologue on entend désigner toute protéine équivalente à la protéine considérée, d'origine cellulaire diverse et notamment issue de cellules d'origine humaine, ou d'autres organismes, et possédant une activité de même type. De telles séquences homologues peuvent être obtenues par des expériences d'hybridation. Au sens de l'invention, il suffit qu'une séquence de ce type présente un pourcentage d'identité significatif pour conduire à un comportement physiologique assimilable à celui de la protéine FE 65 tel que revendiqué.Within the meaning of the present invention, the name protein FE65 covers the protein itself as well as all of its homologous forms. The term “homologous form” is intended to denote any protein equivalent to the protein under consideration, of diverse cellular origin and in particular derived from cells of human origin, or of other organisms, and having an activity of the same type. Such homologous sequences can be obtained by hybridization experiments. Within the meaning of the invention, it is sufficient for a sequence of this type to have a significant percentage of identity to lead to physiological behavior comparable to that of the protein FE 65 as claimed.
Un premier objet de l'invention concerne donc des peptides capables d'interférer au moins partiellement au niveau de l'interaction de la protéine FE65, ou de l'une de ses formes homologues, avec la région cytoplasmique de l'APP.A first object of the invention therefore relates to peptides capable of interfering at least partially at the level of the interaction of the protein FE65, or one of its homologous forms, with the cytoplasmic region of APP.
Cette interférence d'un peptide selon l'invention peut se manifester sous différents aspects. Le peptide revendiqué peut ralentir, inhiber ou stimuler au moins partiellement l'interaction entre la protéine FE65 ou l'une de ses formes homologues avec la région cytoplasmique de l'APP. De tels peptides sont préférentiellement des peptides capables ^'antagoniser au moins partiellement cette interaction. Selon un mode particulier de l'invention, les peptides sont capables de se lier au niveau du domaine d'interaction entre la protéine FE65 ou l'une de ses formes homologues et la région cytoplasmique de l'APP.This interference of a peptide according to the invention can manifest itself in different aspects. The claimed peptide can slow, inhibit or at least partially stimulate the interaction between the protein FE65 or one of its homologous forms with the cytoplasmic region of APP. Such peptides are preferably peptides capable of at least partially antagonizing this interaction. According to a particular embodiment of the invention, the peptides are capable of binding at the level of the interaction domain between the protein FE65 or one of its homologous forms and the cytoplasmic region of APP.
Plus préférentiellement, les peptides de l'invention comprennent tout ou partie de la séquence peptidique codant pour la protéine FE65 présentée en SEQ ID N°l, SEQ ID N°2 ou un de ses dérivés.More preferably, the peptides of the invention comprise all or part of the peptide sequence coding for the protein FE65 presented in SEQ ID No. 1, SEQ ID No. 2 or one of its derivatives.
Au sens de la présente invention, le terme dérivé désigne toute séquence différant de la séquence considérée en raison d'une dégénérescence du code génétique, obtenue par une ou plusieurs modifications de nature génétique et/ou chimique, ainsi que toute séquence hybridant avec ces séquences ou des fragments de celles-ci et conservant la capacité d'interagir au niveau de l'interaction entre la protéine FE65, ou l'un de ses homologues, et la région cytoplasmique de l'APP. Par modification de nature génétique et/ou chimique, on peut entendre toute mutation, substitution, délétion, addition et/ou modification d'un ou plusieurs résidus. Le terme dérivé comprend également les séquences homologues à la séquence considérée, issues d'autres sources cellulaires et notamment de cellules d'origine humaine, ou d'autres organismes, et possédant une activité de même type. De telles séquences homologues peuvent être obtenues par des expériences d'hybridation. Les hybridations peuvent être réalisées à partir de banques d'acides nucléiques, en utilisant comme sonde la séquence native ou un fragment de celle-ci, dans des conditions variables d'hybridation (Maniatis et al., Cf techniques générales de biologie moléculaire).Within the meaning of the present invention, the term derivative designates any sequence differing from the sequence considered due to a degeneration of the genetic code, obtained by one or more modifications of a genetic and / or chemical nature, as well as any sequence hybridizing with these sequences or fragments thereof and retaining the ability to interact at the level of the interaction between the protein FE65, or one of its homologs, and the cytoplasmic region of APP. By modification of genetic and / or chemical nature, one can hear any mutation, substitution, deletion, addition and / or modification of one or more residues. The term derivative also includes sequences homologous to the sequence considered, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type. Such homologous sequences can be obtained by hybridization experiments. Hybridizations can be carried out from nucleic acid libraries, using the native sequence or a fragment thereof as probe, under variable hybridization conditions (Maniatis et al., Cf. general techniques of molecular biology).
De tels dérivés peuvent être générés dans des buts différents, tels que notamment celui d'augmenter leur efficacité thérapeutique ou de réduire leurs effets secondaires, ou celui de leur conférer de nouvelles propriétés pharmacocinétiques et/ou biologiques.Such derivatives can be generated for different purposes, such as in particular that of increasing their therapeutic efficacy or reducing their side effects, or that of conferring on them new pharmacokinetic and / or biological properties.
En tant que peptide dérivé de la protéine FE65 et des formes homologues, on peut citer notamment tout peptide capable d'interagir avec la région cytoplasmique de l'APP, mais portant une région effectrice rendue non fonctionnelle. De tels peptides peuvent être obtenus par délétion, mutation ou disruption de cette région effectrice sur la protéine FE65 et des formes homologues. De telles modifications peuvent être effectuées par exemple par mutagénèse in vitro, par introduction d'éléments additionnels ou de séquences synthétiques, ou par des délétions ou des substitutions des éléments originels. Lorsqu'un dérivé tel que défini ci-dessus est réalisé, son activité d'inhibiteur partiel de la fixation de la protéine FE65 et des formes homologues sur son site de fixation sur l'APP peut être mise en évidence. Toute technique connue de l'homme de l'art peut bien évidemment être utilisée à cet effetAs a peptide derived from the FE65 protein and homologous forms, mention may in particular be made of any peptide capable of interacting with the cytoplasmic region of APP, but carrying an effector region made non-functional. Such peptides can be obtained by deletion, mutation or disruption of this effector region on the protein FE65 and homologous forms. Such modifications can be made, for example, by in vitro mutagenesis, by introduction of additional elements or synthetic sequences, or by deletions or substitutions of the original elements. When a derivative as defined above is produced, its activity as a partial inhibitor of the binding of the protein FE65 and of the homologous forms on its APP binding site can be highlighted. Any technique known to those skilled in the art can obviously be used for this purpose.
Il peut également s'agir de fragments des séquences indiquées ci-dessus. De tels fragments peuvent être générés de différentes façons. En particulier, ils peuvent être synthétisés par voie chimique, sur la base des séquences données dans la présente demande, en utilisant les synthétiseurs peptidiques connus de l'homme du métier. Ds peuvent également être synthétisés par voie génétique, par expression dans un hôte cellulaire d'une séquence nucléotidique codant pour le peptide recherché. Dans ce cas, la séquence nucléotidique peut être préparée chimiquement en utilisant un synthétiseur d'oligonucléotides, sur la base de la séquence peptidique donnée dans la présente demande et du code génétique. La séquence nucléotidique peut également être préparée à partir des séquences données dans la présente demande, par coupures enzymatiques, ligature, clonage, etc, selon les techniques connues de l'homme du métier, ou par criblage de banques d'ADN avec des sondes élaborées à partir de ces séquences.They can also be fragments of the sequences indicated above. Such fragments can be generated in different ways. In particular, they can be synthesized chemically, on the basis of the sequences given in the present application, using the peptide synthesizers known to those skilled in the art. They can also be synthesized genetically, by expression in a cellular host of a nucleotide sequence coding for the peptide sought. In this case, the nucleotide sequence can be prepared chemically using an oligonucleotide synthesizer, on the basis of the peptide sequence given in the present application and the genetic code. The nucleotide sequence can also be prepared from the sequences given in the present application, by enzymatic cleavages, ligation, cloning, etc., according to techniques known to those skilled in the art, or by screening DNA libraries with elaborate probes from these sequences.
Par ailleurs, les peptides de l'invention à savoir capables de ralentir ou d'inhiber au moins partiellement l'interaction entre la protéine FE65 et des formes homologues et la région cytoplasmique de l'APP peuvent également être des peptides ayant une séquence correspondant au site d'interaction de la protéine FE65 et des formes homologues sur la région cytoplasmique de l'APP.Furthermore, the peptides of the invention, namely capable of slowing down or at least partially inhibiting the interaction between the protein FE65 and homologous forms and the cytoplasmic region of APP can also be peptides having a sequence corresponding to site of interaction of the protein FE65 and homologous forms on the cytoplasmic region of APP.
D'autres peptides selon l'invention sont les peptides capables d'entrer en compétition avec les peptides définis ci-dessus pour l'interaction avec leur cible cellulaire. De tels peptides peuvent être synthétisés notamment sur la base de la séquence du peptide considéré, et leur capacité à entrer en compétition avec les peptides définis ci-dessus peut être déterminée.Other peptides according to the invention are the peptides capable of competing with the peptides defined above for the interaction with their cellular target. Such peptides can be synthesized in particular on the basis of the sequence of the peptide considered, and their ability to compete with the peptides defined above can be determined.
Un autre objet de l'invention réside dans des anticorps ou fragments d'anticorps polyclonaux ou monoclonaux dirigés contre un peptide tel que défini ci-avant. De tels anticorps peuvent être générés par des méthodes connues de l'homme du métier. En particulier, ces anticorps peuvent être préparés par immunisation d'un animal contre un peptide de l'invention, prélèvement du sang, et isolement des anticorps. Ces anticorps peuvent également être générés par préparation d'hybridomes selon les techniques connues de l'homme de l'art.Another subject of the invention resides in antibodies or fragments of polyclonal or monoclonal antibodies directed against a peptide as defined above. Such antibodies can be generated by methods known to those skilled in the art. In particular, these antibodies can be prepared by immunizing an animal against a peptide of the invention, drawing blood, and isolating the antibodies. These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art.
Plus préférentiellement, les anticorps ou fragments d'anticorps de l'invention présentent la capacité d'inhiber au moins partiellement l'interaction des peptides revendiqués avec la région cytoplasmique de l'APP. Ils peuvent ainsi être utilisés pour moduler l'endocytose de l'APP.More preferably, the antibodies or antibody fragments of the invention have the ability to at least partially inhibit the interaction of peptides claimed with the cytoplasmic region of the APP. They can thus be used to modulate the endocytosis of APP.
Par ailleurs, ces anticorps peuvent également être utilisés pour détecter et/ou doser l'expression ou la surexpression de l'APP dans des échantillons biologiques, et de ce fait, pour renseigner sur son état d'activation.Furthermore, these antibodies can also be used to detect and / or measure the expression or overexpression of APP in biological samples, and therefore, to provide information on its state of activation.
L'invention fournit également des composés non peptidiques ou non exclusivement peptidiques utilisables pharmaceutiquement. Il est en effet possible, à partir des motifs protéiques actifs décrits dans la présente demande, de réaliser des molécules inhibitrices de la voie de signalisation dépendante de la protéine FE65 non exclusivement peptidiques et compatibles avec une utilisation pharmaceutique.The invention provides an equal ment of non-peptidic compounds or non-peptidic pharmaceutically usable exclusively. It is indeed possible, from the active protein motifs described in the present application, to produce molecules which inhibit the signaling pathway dependent on the FE65 protein which are not exclusively peptide and which are compatible with pharmaceutical use.
La présente invention a également pour objet toute séquence nucléotidique codant pour un peptide selon l'invention. Il peut s'agir en particulier d'une séquence comprenant tout ou partie de la séquence présentée en SEQ ID N°l, SEQ ID N°2 ou une de leurs dérivés. Par séquence dérivée, on entend au sens de la présente invention toute séquence hybridant avec la séquence présentée en SEQ ID N°l ou en SEQ ID N°2 ou avec un fragment de celles-ci et codant pour un peptide selon l'invention, ainsi que les séquences résultant de ces dernières par dégénérescence du code génétique. Les différentes séquences nucléotidiques de l'invention peuvent être d'origine artificielle ou non. D peut s'agir de séquences génomiques, d'ADNc, d'ARN, de séquences hybrides ou de séquences synthétiques ou semi-synthétiques. Ces séquences peuvent être obtenues soit par criblage de banques d'ADN (banque d'ADNc, banque d'ADN génomique), soit par synthèse chimique, soit par des méthodes mixtes incluant la modification chimique ou enzymatique de séquences obtenues par criblage de banques.The present invention also relates to any nucleotide sequence coding for a peptide according to the invention. It may in particular be a sequence comprising all or part of the sequence presented in SEQ ID No. 1, SEQ ID No. 2 or one of their derivatives. By derivative sequence is meant within the meaning of the present invention any sequence hybridizing with the sequence presented in SEQ ID No. 1 or in SEQ ID No. 2 or with a fragment thereof and coding for a peptide according to the invention, as well as the sequences resulting from these by degeneration of the genetic code. The different nucleotide sequences of the invention can be of artificial origin or not. D can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained either by screening of DNA libraries (cDNA library, genomic DNA library), or by chemical synthesis, or by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries.
De telles séquences nucléotidiques peuvent être utilisées pour la production des peptides de l'invention. La présente demande concerne ainsi un procédé de préparation d'un tel peptide selon lequel on cultive une cellule contenant une séquence nucléotidique selon l'invention, dans des conditions d'expression de ladite séquence et on récupère le peptide produit. Dans ce cas, la partie codant pour ledit peptide est généralement placée sous le contrôle de signaux permettant son expression dans un hôte cellulaire. Le choix de ces signaux (promoteurs, terminateurs, séquence "leader" de sécrétion, etc) peut varier en fonction de l'hôte cellulaire utilisé. Par ailleurs, les séquences nucléotidiques de l'invention peuvent faire partie d'un vecteur qui peut être à réplication autonome ou intégratif. Plus particulièrement, des vecteurs à réplication autonome peuvent être préparés en utilisant des séquences à réplication autonome chez l'hôte choisi. S'agissant des vecteurs intégratifs, ceux-ci peuvent être préparés par exemple en utilisant des séquences homologues à certaines régions du génome de l'hôte, permettant, par recombinaison homologue, l'intégration du vecteur. Les hôtes cellulaires utilisables pour la production des peptides de l'invention par voie recombinante sont aussi bien des hôtes eucaryotes que procaryotes. Parmi les hôtes eucaryotes qui conviennent, on peut citer les cellules animales, les levures, ou les champignons. En particulier, s'agissant de levures, on peut citer les levures du genre Saccharomyces , Kluyveromyces , Pichia, Schwanniomyces , ou Hansenula. S'agissant de cellules animales, on peut citer les cellules COS, CHO, C127, etc. Parmi les champignons, on peut citer plus particulièrement Aspergillus ssp. ou Trichoderma ssp. Comme hôtes procaryotes, on préfère utiliser les bactéries suivantes E.coli, Bacillus, ou Streptomyces .Such nucleotide sequences can be used for the production of the peptides of the invention. The present application thus relates to a process for the preparation of such a peptide according to which a cell containing a nucleotide sequence according to the invention is cultivated, under conditions of expression of said sequence and the peptide produced is recovered. In this case, the part coding for said peptide is generally placed under the control of signals allowing its expression in a cellular host. The choice of these signals (promoters, terminators, "leader" secretory sequence, etc.) can vary depending on the cell host used. Furthermore, the nucleotide sequences of the invention can be part of a vector which can be autonomously replicating or integrative. More particularly, replication vectors Autonomous can be prepared using autonomous replicating sequences in the chosen host. As regards 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. The cellular hosts which can be used for the production of the peptides of the invention by the recombinant route are both eukaryotic and prokaryotic hosts. Among suitable eukaryotic hosts, there may be mentioned animal cells, yeasts, or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127 cells, etc. Among the mushrooms, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces.
Les séquences d'acides nucléiques selon l'invention peuvent également servir à la réalisation d'oligonucléotides antisens ou d' antisens génétiques utilisables comme agents pharmaceutiques. Les séquences antisens sont des oUgonucléotides de petite taille, complémentaire du brin codant d'un gène donné, et de ce fait capables d'hybrider spécifiquement avec l'ARNm transcrit, inhibant sa traduction en protéine. L'invention a ainsi pour objet les séquences antisens capables d'inhiber au moins partiellement l'interaction des protéines FE65 sur la région cytoplasmique de l'APP. De telles séquences peuvent être constituées par tout ou partie des séquences nucléiques définies ci- avant. Il s'agit généralement de séquences ou de fragments de séquences complémentaires de séquences codant pour des peptides interagissant avec la région cytoplasmique de l'APP. De tels oUgonucléotides peuvent être obtenus par fragmentation, etc, ou par synthèse chimique.The nucleic acid sequences according to the invention can also be used for the production of antisense oligonucleotides or of genetic antisense usable as pharmaceutical agents. The antisense sequences are small Ogonucleotides, complementary to the coding strand of a given gene, and therefore capable of hybridizing specifically with the transcribed mRNA, inhibiting its translation into protein. The subject of the invention is therefore the antisense sequences capable of at least partially inhibiting the interaction of the FE65 proteins on the cytoplasmic region of APP. Such sequences can consist of all or part of the nucleic acid sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides interacting with the cytoplasmic region of APP. Such Ogonucleotides can be obtained by fragmentation, etc., or by chemical synthesis.
Les séquences revendiquées peuvent être utiUsées dans le cadre de thérapies géniques, pour le transfert et l'expression in vivo de séquences antisens ou de peptides capables de moduler l'interaction des protéines FE65 avec la région cytoplasmique de l'APP. A cet égard, les séquences peuvent être incorporées dans des vecteurs viraux ou non viraux , permettant leur administration in vivo (Médecine et Sciences 7 (1991) 705). A titre de vecteurs viraux conformes à l'invention on peut tout particulièrement citer les vecteurs de type adénovirus, rétrovirus, virus adéno-associé ou virus de l'herpès. La présente demande a également pour objet des virus recombinants défectifs comprenant une séquence nucléique hétérologue codant pour un polypeptide selon l'invention. L'invention permet également la réalisation de sondes nucléotidiques, synthétiques ou non, capables de s'hybrider avec les séquences nucléotidiques définies ci-avant, utilisables dans le cadre d'une thérapie génique. De telles sondes peuvent être utiUsées in vitro comme outil de diagnostic, pour la détection de l'expression ou surexpression de l'APP, ou encore pour la mise en évidence d'anomalies génétiques (mauvais épissage, polymorphisme, mutations ponctuelles, etc). Ces sondes peuvent également être utiUsées pour la mise en évidence et l'isolement de séquences d'acides nucléiques homologues codant pour des peptides tels que définis précédemment, à partir d'autres sources cellulaires et préférentieUement de ceUules d'origines humaines. Les sondes de l'invention comportent généralement au moins 10 bases, et elles peuvent par exemple comporter jusqu'à l'intégralité d'une des séquences précitées ou de leur brin complémentaire. Préférentiellement, ces sondes sont, préalablement à leur utilisation, marquées. Pour cela, différentes techniques connues de l'homme du métier peuvent être employées (marquage radioactif, enzymatique, etc).The claimed sequences can be used in the context of gene therapies, for the transfer and expression in vivo of antisense sequences or of peptides capable of modulating the interaction of the FE65 proteins with the cytoplasmic region of APP. In this regard, the sequences can be incorporated into viral or non-viral vectors, allowing their administration in vivo (Medicine and Sciences 7 (1991) 705). By way of viral vectors in accordance with the invention, mention may very particularly be made of vectors of the adenovirus, retrovirus, adeno-associated virus or herpes virus type. The present application also relates to defective recombinant viruses comprising a heterologous nucleic sequence coding for a polypeptide according to the invention. The invention also allows the production of nucleotide probes, synthetic or not, capable of hybridizing with the nucleotide sequences defined above, usable in the context of gene therapy. Such probes can be used in vitro as a diagnostic tool, for the detection of the expression or overexpression of APP, 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 homologous nucleic acid sequences 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 10 bases, and they can for example comprise up to the entirety of one of the abovementioned sequences or of their complementary strand. Preferably, these probes are, prior to their use, marked. For this, different techniques known to those skilled in the art can be used (radioactive, enzymatic labeling, etc.).
L'invention a encore pour objet toute composition pharmaceutique comprenant comme principe actif au moins un peptide tel que défini ci-avant.The invention also relates to any pharmaceutical composition comprising as active principle at least one peptide as defined above.
Elle a aussi pour objet toute composition pharmaceutique comprenant comme principe actif au moins un anticorps et/ou un fragment d'anticorps tel que défini ci- avant, ainsi que toute composition pharmaceutique comprenant comme principe actif au moins une séquence nucléotidique telle que définie ci-avant.It also relates to any pharmaceutical composition comprising as active ingredient at least one antibody and / or an antibody fragment as defined above, as well as any pharmaceutical composition comprising as active ingredient at least one nucleotide sequence as defined above before.
Par ailleurs, elle a aussi pour objet les compositions pharmaceutiques dans lesquelles les peptides, anticorps et séquence nucléotidique définis ci- avant sont associés entre-eux ou avec d'autres principes actifs.Furthermore, it also relates to pharmaceutical compositions in which the peptides, antibodies and nucleotide sequence defined above are associated with each other or with other active ingredients.
Les compositions pharmaceutiques selon l'invention peuvent être utiUsées pour moduler l'activation des protéines APP et de ce fait pour moduler son endocytose et la production de peptides β amyloïde. Plus particulièrement, ces compositions pharmaceutiques sont destinées à moduler l'interaction entre les protéines FE65 et la région cytoplasmique de l'APP. Plus préférentiellement, ces compositions sont destinées à ralentir ou inhiber au moins partieUement l'interaction des protéines FE65 avec la région cytoplasmique de l'APP. Il s'agit plus préférentiellement de compositions pharmaceutiques destinées au traitement de maladies neurodégénératives comme par exemple la maladie d'Alzheimer et la trisomie 21.The pharmaceutical compositions according to the invention can be used to modulate the activation of APP proteins and therefore to modulate its endocytosis and the production of amyloid β peptides. More particularly, these pharmaceutical compositions are intended to modulate the interaction between the FE65 proteins and the cytoplasmic region of APP. More preferably, these compositions are intended to slow down or at least partially inhibit the interaction of the FE65 proteins with the cytoplasmic region of APP. More preferably, these are pharmaceutical compositions intended for the treatment of neurodegenerative diseases such as, for example, Alzheimer's disease and trisomy 21.
L'invention a encore pour objet l'utilisation des molécules décrites ci-avant pour moduler l'activation de l'endocytose de l'APP ou le typage de maladies neurodégénératives. En particuUer, l'invention concerne l'utiUsation de ces molécules pour inhiber au moins partieUement l'activation de l'endocytose de l'APP.Another subject of the invention is the use of the molecules described above to modulate the activation of APP endocytosis or the typing of diseases neurodegenerative. In particular, the invention relates to the use of these molecules to at least partially inhibit activation of APP endocytosis.
D'autres avantages de la présente invention apparaîtront à la lecture des exemples et figures qui suivent, qui doivent être considérés comme illustratifs et non limitatifs.Other advantages of the present invention will appear on reading the examples and figures which follow, which should be considered as illustrative and not limiting.
Légendes des figures :Legends of the figures:
Figure 1: Représentation du vecteur pGAL4DB-CAPPFigure 1: Representation of the vector pGAL4DB-CAPP
Figure 2: Représentation d'ADNs plasmidiques sur gels obtenus selon l'exemple 3.Figure 2: Representation of plasmid DNAs on gels obtained according to Example 3.
Figure 3: Comparaison de fragments nucléotidiques de FE65 d'origines diversesFigure 3: Comparison of FE65 nucleotide fragments of various origins
MATERIELS ET TECHNIQUES MIS EN OEUVREMATERIALS AND TECHNIQUES IMPLEMENTED
1) Les souches de levures.utilisées sont:1) The yeast strains used are:
La souche YCM du genre S.cerevisiae (MATa, ura3-52, his3-200, ade2-101, lys2- 801, trpl-901, leu2-3,112, canr, gal4-542, gal80-538, URA3::GALlll0-lacZ,The YCM strain of the genus S.cerevisiae (MATa, ura3-52, his3-200, ade2-101, lys2- 801, trpl-901, leu2-3,112, canr, gal4-542, gal80-538, URA3 :: GALlll0- lacZ,
LYS::GAL1I10-HIS3) a été utilisée comme outil de criblage de la banque de fusion de cerveau par le système deux hybrides.LYS :: GAL1I10-HIS3) was used as a tool to screen the brain fusion bank by the two hybrid system.
La souche L40 du genre S.cerevisiae (Mata, his3D200, trpl-901, leu2-3,112, ade2,The L40 strain of the genus S.cerevisiae (Mata, his3D200, trpl-901, leu2-3,112, ade2,
LYS2:: (lexAop)4-HIS3, URA3::(lexAop)8-LacZ, GAL4) a été utilisée pour vérifier les interactions protéine-protéine quand l'un des partenaires protéiques est fusionné à la protéine LexA. Cette dernière est capable de reconnaître l'élément de réponse LexA contrôlant l'expression des gènes rapporteurs LacZ et His3.LYS2 :: (lexAop) 4-HIS3, URA3: :( lexAop) 8-LacZ, GAL4) was used to verify protein-protein interactions when one of the protein partners is fused to the LexA protein. The latter is capable of recognizing the LexA response element controlling the expression of the reporter genes LacZ and His3.
Elles ont été cultivées sur les milieux de culture suivants:.They were cultivated on the following culture media :.
Milieu YPD complet: -Extrait de levures (lOg 1) (Difco)Complete YPD medium: -Yeast extract (lOg 1) (Difco)
-Bactopeptone (20g/l) (Difco) -Glucose (20g/l) (Merck) Ce miUeu a été rendu solide par addition de 20g/ d'agar (Difco).-Bactopeptone (20g / l) (Difco) -Glucose (20g / l) (Merck) This medium was made solid by the addition of 20 g / agar (Difco).
MiUeu YNB rninimum:-Yeast Nitrogen Base (sans acides aminés) (6,7g l) (Difco)MiUeu YNB rninimum: -Yeast Nitrogen Base (without amino acids) (6.7g l) (Difco)
- Glucose (20g/l) (Merck) Ce milieu peut être rendu solide par addition de 20g/l d'agar (Difco).- Glucose (20g / l) (Merck) This medium can be made solid by adding 20g / l of agar (Difco).
Pour permettre la croissance des levures auxotrophes sur ce miUeu, il est nécessaire d'y ajouter les acides aminés ou les bases azotées, pour lesquelles eUes sont dépendantes à 50mg/ml. 100 μg/ml d'ampicilline sont ajoutés au miUeu afin d'éviter les contaminations bactériennes.To allow the growth of auxotrophic yeasts on this medium, it is necessary to add to it amino acids or nitrogen bases, on which they are dependent at 50 mg / ml. 100 μg / ml of ampicillin are added to the medium in order to avoid bacterial contamination.
2) La souche de bactérie utilisée est :.2) The strain of bacteria used is:.
La souche TGl d'Escherichia coU de génotype supE, hsdΔ5, thi, Δ(lac-proAB), F'[tra D36 pro A+B+ lacIqlacZΔM15]. EUe a été employée comme moyen d'ampUfication et d'isolement de plasmides recombinants utilisés.The Escherichia coU TGl strain of genotype supE, hsdΔ5, thi, Δ (lac-proAB), F '[tra D36 pro A + B + lacI q lacZΔM15]. It has been used as a means of ampUfication and isolation of the recombinant plasmids used.
Elle a été cultivée sur Milieu LB: -NaCl (5g/l) (Difco) -Bactotryptone (lOg/l) (Difco)It was cultivated on LB medium: -NaCl (5g / l) (Difco) -Bactotryptone (lOg / l) (Difco)
-Extrait de levure (5g l) (Difco) Ce milieu peut être rendu solide par addition de 20g/l d'agar (Difco).-Yeast extract (5g l) (Difco) This medium can be made solid by adding 20g / l of agar (Difco).
L'ampicilline a été utilisée à lOOμg/ml, cet antibiotique sert à sélectionner les bactéries ayant reçu les plasmides portant comme marqueur le gène de résistance à cet antibiotique.Ampicillin was used at 100 μg / ml, this antibiotic is used to select the bacteria which have received the plasmids carrying as marker the gene for resistance to this antibiotic.
3) Les plasmides mis en oeuyre sont:.3) The plasmids used are :.
Le vecteur pGBTIO. (Clontech).un plasmide navette de 5,4kb qui possède une origine réplication bactérienne et de levure lui permettant de se répliquer à haut nombre de copies chez ces deux micro-organismes. Ce plasmide contient un site multiple de clonage situé en aval de la séquence codant pour le domaine de liaison à l'ADN de GAL4 et en amont d'un terminateur pour former une protéine de fusion. Il contient également le gène TRP1 de S. cerevisiae qui permet de complémenter les levures de génotype trpl afin de les sélectionner sur un milieu minimum ne contenant pas de tryptophane. Ce vecteur porte le gène de résistance à l'ampiciUine qui permet de sélectionner les bactéries le possédant sur un miUeu contenant de l'ampiciUine.The vector pGBTIO. (Clontech). A 5.4 kb shuttle plasmid which has an origin of bacterial and yeast replication allowing it to replicate with a high number of copies in these two microorganisms. This plasmid contains a multiple cloning site located downstream of the sequence coding for the DNA binding domain of GAL4 and upstream of a terminator to form a fusion protein. It also contains the TRP1 gene from S. cerevisiae which makes it possible to complement yeasts of the trpl genotype in order to select them on a minimum medium containing no tryptophan. This vector carries the ampiciUine resistance gene which makes it possible to select the bacteria possessing it on a medium containing ampiciUine.
Le vecteur pGADIO. fourni par Clontech et qui permet l'expression chez la levure de protéines de fusion entre le domaine transactivateur de GAL4 et une protéine codée par l'ADNc provenant d'une banque de cerveau, inséré au niveau d'un site EcoRI.The vector pGADIO. supplied by Clontech and which allows the expression in yeast of fusion proteins between the GAL4 transactivating domain and a protein coded by the cDNA originating from a brain bank, inserted at an EcoRI site.
Le vecteur pLex9 (pBTMllό) (Bartel et al D.A Hartley Ed, Oxford University press page 153) de 5kb homologue au pGBTIO qui contient un site multiple de clonage situé en aval de la séquence codant pour le répresseur bactérien LexA et en amont d'un terminateur pour former une protéine de fusion.The vector pLex9 (pBTMllό) (Bartel et al DA Hartley Ed, Oxford University press page 153) of 5kb homologous to pGBTIO which contains a multiple cloning site located downstream of the sequence coding for the bacterial repressor LexA and upstream of a terminator to form a fusion protein.
4) les oUgonucléotides de synthèse employés sont:.4) the synthetic oGonucleotides used are :.
CAA GTC GAC CTA GTT CTG CAT CTG CTC (SEQ ID N°3) C AA GAA TTC AAG AAA CAG TAC ACA TCC (SEQ ID N°4)CAA GTC GAC CTA GTT CTG CAT CTG CTC (SEQ ID N ° 3) C AA GAA TTC AAG AAA CAG TAC ACA TCC (SEQ ID N ° 4)
OUgonucléotides qui ont permis d'obtenir le fragment PCR correspondant au CAPP avec les sites EcoRI et Sali. Les oUgonucléotides sont synthétisés sur l'appareU Applied System ABI 394-08. Us sont décrochés de la matrice de synthèse par de l'ammoniac et précipités deux fois par 10 volumes de n-butanol puis repris dans de l'eau. La quantification est effectuée par mesure de la densité optique (1DO correspond à 30μg ml).OUgonucleotides which made it possible to obtain the PCR fragment corresponding to CAPP with the EcoRI and Sali sites. The Ogonucleotides are synthesized on the Applied System ABI 394-08. They are detached from the synthesis matrix with ammonia and precipitated twice with 10 volumes of n-butanol and then taken up in water. The quantification is carried out by measuring the optical density (1DO corresponds to 30 μg ml).
5) Préparation des ADN plasmidiques.5) Preparation of the plasmid DNAs.
Les grandes quantités d'ADN sont préparées en utiUsant le Kit de préparation rapide d'ADN de Proméga.Large amounts of DNA are prepared using the Proméga DNA Rapid Preparation Kit.
Les petites quantités d'ADN sont préparées de la manière suivante: les bactéries contenant le plasmide sont cultivées pendant au moins 4 heures dans 2ml de miUeu LB dans un shaker à agitation. Elles sont ensuite centrifugées pendant 2 minutes à 14000 rpm dans des tubes Ependorf, puis le culot est remis en suspension dans lOOμl de la solution I (50mM de glucose, 25mM de tampon Tris HC1 ρH8, lOmM EDTA ρH8), lysées par 200μl de la solution II (0.2M de NaOH, 1% de SDS). La solution de lyse est ensuite neutralisée par 150μl de la solution III (3M d'acétate de potassium, 11.5% (v/v) d'acide acétique glacial). Après agitation des tubes jusqu'à obtenir un précipité floconneux, 150μl u un mélange de phénol/Chloroforme (50% de phénol et 50% de chloroforme saturés en eau) sont ajoutés, et l'ensemble est agité 30 secondes. La phase acqueuse contenant l'ADN est récupérée après centrifugation pendant 2 minutes à 14000 rpm. L'ADN est ensuite précipité par addition de 0,5 volume d'isopropanol puis centrifugé 5 minutes à 14000 rpm et séché à l'air pour enfin être repris dans 20μl de TE RNAse (solution de Tris-HCl lOmM et d'EDTA ImM avec 50μg/ml de RNAse).Small amounts of DNA are prepared in the following manner: the bacteria containing the plasmid are cultured for at least 4 hours in 2 ml of miUeu LB in a shaker shaker. They are then centrifuged for 2 minutes at 14,000 rpm in Ependorf tubes, then the pellet is resuspended in 100 μl of solution I (50 mM of glucose, 25 mM of Tris HC1 ρH8 buffer, 10 mM EDTA ρH8), lysed with 200 μl of the solution II (0.2M NaOH, 1% SDS). The lysis solution is then neutralized with 150 μl of solution III (3M of potassium acetate, 11.5% (v / v) of glacial acetic acid). After stirring the tubes until a flaky precipitate is obtained, 150 μl u a mixture of phenol / Chloroform (50% phenol and 50% chloroform saturated in water) are added, and the whole is stirred for 30 seconds. The the aqueous phase containing the DNA is recovered after centrifugation for 2 minutes at 14,000 rpm. The DNA is then precipitated by adding 0.5 volumes of isopropanol, then centrifuged for 5 minutes at 14,000 rpm and air dried to finally be taken up in 20 μl of TE RNAse (10 mM Tris-HCl solution and ImM EDTA with 50μg / ml of RNAse).
6) Amplification enzymatique d'ADN ou PCR (Polymerase Chain Reaction).6) Enzymatic amplification of DNA or PCR (Polymerase Chain Reaction).
Les réactions de PCR sont effectuées dans un volume final de lOOμl en présence de la matrice d'ADN, de dNTP (0,2mM), de tampon PCR (Tris-HCL pH 8,5 10 mM, MgCl2 ImM, KCl 5 mM, gélatine 0,01%), de 0,5 μg de chacun des oUgonucléotides et de 2,5 UI d'AmpU Taq DNA polymerase (Perkin Elmer) avec ou sans formamide (5%). Le mélange est recouvert de 2 gouttes d'huile de paraffine pour limiter l'évaporation de l'échantiUon. L'appareil utiUsé est le "Crocodile II" d'Appligene. Nous avons utiUsé une température de dénaturation de la matrice de 90°C, une température d'hybridation des oUgonucléotides à la matrice inférieure de 5 à 10 degrés à la température de séparation des oUgonucléotides et une température d'élongation par l'enzyme à 72°C.The PCR reactions are carried out in a final volume of 100 μl in the presence of the DNA template, dNTP (0.2 mM), PCR buffer (Tris-HCL pH 8.5 10 mM, MgCl 2 ImM, KCl 5 mM , 0.01% gelatin), 0.5 μg of each of the oGonucleotides and 2.5 IU of AmpU Taq DNA polymerase (Perkin Elmer) with or without formamide (5%). The mixture is covered with 2 drops of paraffin oil to limit the evaporation of the sample. The device used is the "Crocodile II" from Appligene. We used a denaturation temperature of the matrix of 90 ° C, a hybridization temperature of the Ogonucleotides to the matrix 5-10 degrees lower than the separation temperature of the Ogonucleotides and an elongation temperature of 72 ° C.
7) Les ligatures.7) The ligatures.
Toutes les réactions de ligation sont effectuées à -f-14°C pendant une nuit dans un volume final de 10 μl en présence de 100 à 200 ng de vecteur, 0.5 à 2 μg d' insert, 40 UI d'enzyme T4 DNA Ugase (Biolabs) et un tampon de ligation (Tris-HCl 50 mM pH 7,8; MgCl2 10 mM; DTT 10 mM; ATP 1 mM). Le témoin négatif est constitué par la Ugation du vecteur en l'absence d' insert. 8) La tranformation des bactéries par un plasmide est effectuée selon le protocole suivant:.All the ligation reactions are carried out at -f-14 ° C overnight in a final volume of 10 μl in the presence of 100 to 200 ng of vector, 0.5 to 2 μg of insert, 40 IU of T4 DNA Ugase enzyme (Biolabs) and a ligation buffer (50 mM Tris-HCl pH 7.8; 10 mM MgCl 2 ; 10 mM DTT; 1 mM ATP). The negative control consists of the ugation of the vector in the absence of an insert. 8) The transformation of the bacteria by a plasmid is carried out according to the following protocol :.
La totalité du volume de Ugation (lOμl) est utiUsée pour transformer les bactéries TGl rendues compétentes par la méthode de Chung et al, ( PNAS.1988 86, 2172-2175). Les bactéries TGl sont mises en culture dans un miUeu LB Uquide pendant quelques heures dans une étuve à agitation à 37°C, jusqu'à obtenir une DO de 0,6 à 600nm. Le miUeu est ensuite centrifugé à 6000 rpm pendant 10 mn. Les bactéries sont rendues compétentes en reprenant le culot bactérien avec un volume de TSB (miUeu LB + 100 g/1 de PEG 4000, 5% de DMSO, 10 mM de MgCl2) 10 mM de MgSO4) correspondant à 1/10 du volume du miUeu de la culture initiale. Après incubation à 4°C pendant 30 à 60 minutes, 200μl de bactéries sont mises au contact des produits de Ugation pendant 15 minutes sur la glace. Après addition de 200μl de LB, les bactéries sont incubées 30 mn à 37°C puis étalées sur un milieu LB + ampicilline.The entire volume of Ugation (10 μl) is used to transform the TGl bacteria made competent by the method of Chung et al, (PNAS. 1988 86, 2172-2175). The TGl bacteria are cultured in a liquid LU medium for a few hours in a shaking oven at 37 ° C., until an OD of 0.6 to 600 nm is obtained. The medium is then centrifuged at 6000 rpm for 10 min. The bacteria are made competent by taking up the bacterial pellet with a volume of TSB (miUeu LB + 100 g / 1 of PEG 4000, 5% of DMSO, 10 mM of MgCl 2) 10 mM of MgSO 4 ) corresponding to 1/10 of the volume of the medium of the initial culture. After incubation at 4 ° C for 30 to 60 minutes, 200 μl of bacteria are brought into contact with the Ugation products for 15 minutes on ice. After adding 200 μl of LB, the bacteria are incubated for 30 min at 37 ° C. and then spread on an LB + ampicillin medium.
9) Les séparation et extraction des ADN sont effectuées comme suit:9) The DNA separation and extraction are carried out as follows:
La séparation des ADN est réalisée en fonction de leur taille par électrophorèse. Pour ce faire; différents gels sont utilisés en fonction de la taille des fragments à séparer:The DNA is separated according to their size by electrophoresis. To do this; different gels are used depending on the size of the fragments to be separated:
-gels de polyacrylamides précoulés (Novex) à 6%, 10% et 20% pour la séparation des petits fragments d'ADN (75 à 500pb)- 6%, 10% and 20% polyacrylamide precast polyacrylamide gels for the separation of small DNA fragments (75 to 500 bp)
-gel d'agarose à 1% (Gibco BRL) dans un tampon TBE (Tris base 90mM; Borate 90mM; EDTA 2mM) pour la séparation de grands fragments d'ADN (supérieurs à 500pdb)- 1% agarose gel (Gibco BRL) in TBE buffer (Tris base 90mM; Borate 90mM; EDTA 2mM) for the separation of large DNA fragments (greater than 500 bpd)
-gel d'agarose NuSieve à 2% (FMC Bioproducts) dans un tampon TBE pour la séparation de petits fragments (inférieurs à 500 pdb). Toute migration sur gel d'agarose ou sur gel de polyacrylamide est réalisée dans un tampon TBE et en présence d'un marqueur de poids moléculaire (1Kb ladder, Gibco BRL). L'ADN est mélangé avec 1/10 du volume du bleu de dépôt (200g/l de Ficoll, 0,5g/l de bleu de bromophénol, 50mM d'EDTA) avant d'être déposé sur gel. Après migration à 100 Volts et coloration au bromure d'éthydium (concentration 0.5μg/ml de gel), les bandes sont visualisées sous la lampe à UV.- 2% NuSieve agarose gel (FMC Bioproducts) in TBE buffer for the separation of small fragments (less than 500 bpd). Any migration on agarose gel or on polyacrylamide gel is carried out in a TBE buffer and in the presence of a molecular weight marker (1Kb ladder, Gibco BRL). The DNA is mixed with 1/10 of the volume of the deposition blue (200 g / l of Ficoll, 0.5 g / l of bromophenol blue, 50 mM of EDTA) before being deposited on gel. After migration to 100 volts and staining with ethydium bromide (concentration 0.5 μg / ml of gel), the bands are visualized under the UV lamp.
L'extraction de l'ADN de la bande d'un gel d'agarose est réalisée par électroélution comme suit: Le morceau de gel contenant le fragment d'ADN est découpé au scalpel et mis dans un boudin de dialyse fermé par deux pinces et contenant de 100 à 500μl de TBE. L'ensemble est mis dans une cuve à électrophorèse ou il subit un champ électrique de 100 Volts. L'ADN, après être sorti du gel, est ensuite purifié par deux extractions au phénol/chloroforme suivies de deux extractions au chloroforme, puis précipité en présence d'acétate de sodium 0.3M et de 2,5 volume d'éthanol absolu. Après centrifugation (5 mn à 14000 rpm) le culot d'ADN est séché puis repris dans 20μl l'eau.The extraction of DNA from the band of an agarose gel is carried out by electroelution as follows: The piece of gel containing the DNA fragment is cut with a scalpel and placed in a dialysis rod closed by two forceps and containing 100 to 500μl of TBE. The whole is put in an electrophoresis tank where it undergoes an electric field of 100 Volts. The DNA, after being removed from the gel, is then purified by two phenol / chloroform extractions followed by two chloroform extractions, then precipitated in the presence of 0.3M sodium acetate and 2.5 volumes of absolute ethanol. After centrifugation (5 min at 14,000 rpm) the DNA pellet is dried and then taken up in 20 μl of water.
10) Séquençage fluorescent des ADN plasmidiques.10) Fluorescent sequencing of plasmid DNAs.
Le séquençage est fait suivant le méthode de Sanger en utilisant 4 didéoxyribonucléotides possédant un marqueur fluorescent différent. L'incorporation de l'un de ces didéoxyribonucléotides produit un arrêt dans la réplication par la Taq polymerase de l'ADN à sequencer. Cette réaction donnera des fragments d'ADN de tailles différentes, tous terminés en 3' par un des 4 didéoxyribonucléotides. Un μg d'un plasmide et 4 picomoles d'un primer sont ajoutés à 9,5μl d'un "prémix" fourni par AppUed Biosystems sous le nom de Prism . Le volume final doit être de 20μl pour effectuer une PCR pendant 25 cycles se décomposant en une étape de dénaturation à 96°C pendant 30 secondes.une étape d'hybridation à 50°C pendant 15 secondes et une étape d'élongation à 60°C pendant 4 minutes. Les fragments d'ADN, obtenus après amplification, sont purifiés sur colonne d'exclusion (Chromaspin-30 de Clontech); et sont ensuite séchés au Speed Vac. L'ensemble est repris par 5μl d'un mélange formé de 24μl d'EDTA (50mM) et 120μl de formamide désionisée. Après dénaturation à 96°C pendant 3 minutes, 3 à 5μl sont déposés sur un gel d'électrophorèse. Les différents fragments d'ADN sont séparés suivant leur taille et vont passer successivement devant un lecteur laser de l'Appareil 370 DNA sequencer (Applied Biosystems) où les différentes fluorescences vont être détectées.The sequencing is done according to the Sanger method using 4 dideoxyribonucleotides having a different fluorescent marker. The incorporation of one of these dideoxyribonucleotides produces a stop in the replication by Taq polymerase of the DNA to be sequenced. This reaction will give DNA fragments of different sizes, all terminated in 3 'with one of the 4 dideoxyribonucleotides. One μg of a plasmid and 4 picomoles of a primer are added to 9.5 μl of a "premix" supplied by AppUed Biosystems under the name of Prism. The final volume must be 20 μl to carry out a PCR for 25 cycles, decomposing into a denaturation step at 96 ° C for 30 seconds. A hybridization step at 50 ° C for 15 seconds and an elongation step at 60 °. C for 4 minutes. The DNA fragments, obtained after amplification, are purified on an exclusion column (Chromaspin-30 from Clontech); and are then dried with Speed Vac. The whole is taken up in 5 μl of a mixture formed of 24 μl of EDTA (50 mM) and 120 μl of deionized formamide. After denaturation at 96 ° C for 3 minutes, 3 to 5 μl are deposited on an electrophoresis gel. The different DNA fragments are separated according to their size and will pass successively in front of a laser reader of the Apparatus 370 DNA sequencer (Applied Biosystems) where the different fluorescences will be detected.
11) Préparation de plasmides de la banque de cerveau (Clontech®).11) Preparation of plasmids from the brain bank (Clontech®).
La banque de fusion d'ADNc de cerveau est vendue sous forme de bactéries. Ces dernières contiennent un plasmide pGADIO renfermant un insert correspondant à un ADNc de cerveau humain. Les ADNc de cette banque sont constitués grâce à la technique des oUgodT et la technique des oUgonucléotides dégénérés qui permet d'avoir les parties 5' des ARNm qui sont difficiles à obtenir par la première technique. Ces cDNA sont clones dans le vecteur pGADIO au niveau du site EcoRI.The brain cDNA fusion bank is sold as bacteria. The latter contain a plasmid pGADIO containing an insert corresponding to a human brain cDNA. The cDNAs of this bank are formed using the oUgodT technique and the degenerate oUgonucleotide technique which allows to have the 5 'parts of the mRNAs which are difficult to obtain by the first technique. These cDNAs are cloned into the vector pGADIO at the EcoRI site.
Après vérification du titre de la banque, 2μl de bactéries de la banque de fusion de cerveau, mises au préalablement dans 8 ml de LB, sont étalées de façon non confluente sur un miUeu solide afin de garder la représentativité de cette banque. Nous avons ainsi étalé 16 boites de 770 cm contenant un miUeu LB+ampiciUine. Les colonies apparues sont reprises pour chacune des boites avec 30ml de LB+ampiciUine Uquide. Les suspensions obtenues sont ensuite mises dans un Erlen et mises à incuber dans un shaker à 37°C pendant 3 heures. L'ADN est ensuite extrait de ces souches par la technique de la Maxiprep. La concentration en ADN sera déterminée à 260nm.After checking the title of the bank, 2 μl of bacteria from the brain fusion bank, put beforehand in 8 ml of LB, are spread non-confluently on a solid medium in order to keep the representativeness of this bank. We have thus spread 16 boxes of 770 cm containing a LU + ampicUine miUeu. The colonies that have appeared are taken up for each of the dishes with 30 ml of LB + ampicine Uquide. The suspensions obtained are then placed in an Erlenmeyer flask and incubated in a shaker at 37 ° C for 3 hours. DNA is then extracted from these strains by the Maxiprep technique. The DNA concentration will be determined at 260nm.
12) Transformation de la levure par un plasmide.12) Transformation of the yeast with a plasmid.
Les levures préalablement cultivées dans 100ml de milieu Uquide sont récoltées après centrifugation à 3000 rpm pendant 3 minutes et mises en suspension dans 1ml d'eau stérile. Après centrifugation à 3000 m pendant 3 minutes le culot ceUulaire est remis en suspension dans 1 ml d'eau stérile puis centrifugé à nouveau. Cette opération est répétée une nouvelle fois afin d'éliminer toute trace du milieu de culture. Les levures sont ensuite reprises par 1 ml de la solution I de transformation (LiAc 0.1M, Tris-HCl pH 7,5 lOmM, EDTA ImM). puis centrifugées à 3000 φm pendant 3 minutes. Le culot cellulaire est repris à nouveau dans 1 ml de la solution I de transformation. 50μl de cette suspension de levures sont mis en présence de 50μg d'ADN de sperme de saumon et de 1 à 5 μg d'ADN plasmidique. 300μl d'une solution II de transformation (LiAc 0.1M, Tris-HCl pH 7,5 lOmM, EDTA ImM dans du PEG4000 40%) sont ensuite ajoutés, puis l'ensemble est mis à incuber à 28°C pendant 30 minutes. Un choc thermique est ensuite appUqué sur le mélange de transformation dans un bain-marie à 40°C pendant 15 minutes puis l'ensemble est centrifugé à 15000 φm pendant 1 mn afin de récolter le culot cellulaire. Ce culot est repris dans 200μl d'eau puis étalé sur un milieu minimum gélose ne contenant pas les acides aminés correspondant aux marqueurs apportés par le plasmide transformant. Les levures sont ensuite mises à cultiver pendant 72 heures à 28°C.The yeasts previously cultivated in 100 ml of liquid medium are harvested after centrifugation at 3000 rpm for 3 minutes and suspended in 1 ml of sterile water. After centrifugation at 3000 m for 3 minutes, the cell pellet is resuspended in 1 ml of sterile water and then centrifuged again. This operation is repeated again in order to eliminate all traces of the culture medium. The yeasts are then taken up in 1 ml of the transformation solution I (0.1A LiAc, Tris-HCl pH 7.5 lOmM, EDTA ImM). then centrifuged at 3000 φm for 3 minutes. The cell pellet is taken up again in 1 ml of the transformation solution I. 50 μl of this yeast suspension are placed in the presence of 50 μg of salmon sperm DNA and from 1 to 5 μg of plasmid DNA. 300 μl of a transformation solution II (LiAc 0.1M, Tris-HCl pH 7.5 lOmM, EDTA ImM in PEG 4000 40%) are then added, then the whole is incubated at 28 ° C for 30 minutes . A thermal shock is then applied to the transformation mixture in a water bath at 40 ° C for 15 minutes and then the whole is centrifuged at 15000 φm for 1 minute in order to collect the cell pellet. This pellet is taken up in 200 μl of water and then spread over a minimum agar medium which does not contain the amino acids corresponding to the markers provided by the transforming plasmid. The yeasts are then placed in culture for 72 hours at 28 ° C.
Dans le cas particulier de la tranformation de la levure par la banque d'ADNc de cerveau, on procède comme suit: La levure utiUsée contient le plasmide pGAL4DB-CAPP codant pour la partie C- terminale de l'APP fusionnée au domaine de tiaison à l'ADN de GAL4. EUe est cultivée dans 250ml de miUeu rninimum YNB-r-His+Lys+Ad+Leu à 28°C sousIn the particular case of the transformation of yeast by the brain cDNA library, the procedure is as follows: The yeast used contains the plasmid pGAL4DB-CAPP coding for the C-terminal part of the APP fused to the DNA binding domain of GAL4. It is cultivated in 250 ml of medium minimum YNB-r-His + Lys + Ad + Leu at 28 ° C.
•η agitation jusqu'à une densité de 10 ceUules/ml. Les ceUules sont récoltées par centrifugation à 3000φm pendant 10 minutes et reprises dans 250ml d'eau. Après une nouveUe centrifugation le culot ceUulaire est repris dans 100ml d'eau et à nouveau centrifugé. Le culot est alors repris dans 10ml de la solution I de transformation et incubé pendant 1 heure à 28°C sous agitation. Après centrifugation les ceUules sont à nouveau reprises dans 2,5 ml de la solution I de transformation, de 100 μl de la banque d'ADNc de cerveau et de 20 ml de la solution II de transformation, puis incubées pendant 1 heure à 28°C sous agitation. Un choc thermique est effectué sur ce mélange de transformation à 42°C pendant 20 minutes. Une centrifugation (3000 φm pendant 5mn) est répétée 3 fois de suite, en reprenant à chaque fois le culot avec 10 ml d'eau stérile. La troisième fois le culot est repris avec 2,5 ml de PBS. Ainsi le PEG toxique pour les ceUules a été étiminé. 2,4 ml de cette suspension sont utilisés pour ensemmencer 250 ml de mitieu minimum contenant les acides aminés His, Lys, Ad et cultivés durant une nuit dans un shaker à 28°C. Les 100 μl de cette suspension restants servent à vérifier l'efficacité de la transformation; pour cela des dilutions de 10" , 10" et 10 de cette suspension ont été réalisées et étalées sur un milieu minimum contenant les acides aminés His, Lys, Ad. Après culture à 28°C durant 2 jours les colonies obtenues ont été comptées et le taux de transformation est déterminé en utilisant la formule : "nombre de colonies x facteur de dilution". La culture de nuit est centrifugée (3000 φm pendant 5 mn) et lavée à l'eau stérile deux fois de suite. Le culot est ensuite repris dans 2,5 ml d'eau. 2,4 ml, dont le volume est amené à 10ml dans de l'eau stérile, sont utilisés pour ensemencer 10 boites de 435 cm contenant un milieu YNB+Lys+Ad et mis à incuber pendant 3 jours. Les 100 μl restants sont utilisés pour effectuer les mêmes opérations que lors de la détermination du taux de transformation, afin de déterminer le taux d'amplification du nombre de colonies au cours d'une nuit de culture.• Stirring up to a density of 10 ceUules / ml. The cells are harvested by centrifugation at 3000 àm for 10 minutes and taken up in 250ml of water. After a new centrifugation, the cell pellet is taken up in 100 ml of water and centrifuged again. The pellet is then taken up in 10 ml of the transformation solution I and incubated for 1 hour at 28 ° C. with shaking. After centrifugation, the cells are again taken up in 2.5 ml of the transformation solution I, 100 μl of the brain cDNA bank and 20 ml of the transformation solution II, then incubated for 1 hour at 28 ° C with stirring. A thermal shock is carried out on this transformation mixture at 42 ° C for 20 minutes. Centrifugation (3000 φm for 5 min) is repeated 3 times in succession, each time taking up the pellet with 10 ml of sterile water. The third time the pellet is taken up with 2.5 ml of PBS. Thus PEG toxic for cells has been eliminated. 2.4 ml of this suspension are used to inoculate 250 ml of minimum medium containing the amino acids His, Lys, Ad and cultured overnight in a shaker at 28 ° C. The remaining 100 μl of this suspension is used to verify the efficiency of the transformation; for this, dilutions of 10 " , 10 " and 10 of this suspension were made and spread on a minimum medium containing the amino acids His, Lys, Ad. After culture at 28 ° C. for 2 days, the colonies obtained were counted and the transformation rate is determined using the formula: "number of colonies x dilution factor". The overnight culture is centrifuged (3000 φm for 5 min) and washed with sterile water twice in succession. The pellet is then taken up in 2.5 ml of water. 2.4 ml, the volume of which is brought to 10 ml in sterile water, are used to inoculate 10 dishes of 435 cm containing YNB + Lys + Ad medium and incubated for 3 days. The remaining 100 μl are used to carry out the same operations as when determining the transformation rate, in order to determine the rate of amplification of the number of colonies during a culture night.
L'ADN (génomique et plasmidique) est extrait des levures de la manière suivante:.DNA (genomic and plasmid) is extracted from yeasts as follows :.
La valeur d'une anse moyenne d'un clone de levure est mise dans 200μl d'une solution TELT (Triton XI 00 2%, SDS 1%, NaCl lOOmM, Tris pH8 lOmM, EDTA ImM), en présence de 3g de biUes de verre de 450 μm de diamètre et de 200μl de phénol/chloroforme. Ce mélange est vortexé pendant 15 minutes, puis centrifugé pendant 2 minutes à 14000 φm. Le surnageant est collecté sans prélever la galette protéique et l'ADN contenu dans cette phase est précipité avec 2,5 volumes d'éthanol absolu. Après centrifugation pendant 2 minutes à 14000 φ , le culot d'ADN est séché et repris dans 20μl de TERNAse. Cette solution d'ADN, qui correspond à un mélange d'ADN génomique et plasmidique, sert directement à transformer des bactéries. Seul l'ADN plasmidique est capable de se répliquer dans les bactéries et peut être analysé par la technique de miniprep.The value of an average loop of a yeast clone is put in 200 μl of a TELT solution (Triton XI 00 2%, SDS 1%, NaCl lOOmM, Tris pH8 lOmM, EDTA ImM), in the presence of 3g of biUes of glass 450 μm in diameter and 200 μl of phenol / chloroform. This mixture is vortexed for 15 minutes, then centrifuged for 2 minutes at 14000 φm. The supernatant is collected without removing the protein cake and the DNA contained in this phase is precipitated with 2.5 volumes of absolute ethanol. After centrifugation for 2 minutes at 14,000 φ, the DNA pellet is dried and taken up in 20 μl of TERNAse. This DNA solution, which corresponds to a mixture of genomic and plasmid DNA, is used directly to transform bacteria. Only plasmid DNA is capable of replicating in bacteria and can be analyzed by the miniprep technique.
13) Test d'activité de la βgalactosidase.13) βgalactosidase activity test.
Une feuUle de nitrocellulose est préalablement déposée sur la boite de Pétri contenant les clones de levures individuaUsés. Grâce au phénomène d'adsoφtion on obtiendra une image fidèle de l'emplacement des clones. Cette feuille est ensuite plongée dans de l'azote Uquide pendant 30 secondes afin de faire éclater les levures et de tibérer ainsi l'activité βgalactosidase. Après décongélation, la feuille de nitroceUulose est déposée, colonies vers le haut, dans une autre boite de Pétri contenant un papier Whatman préalablement imbibé de 1,5ml de solution PBS (Na2HPO4 60mM, NaH2PO4 40mM, KCl lOmM, MgSO4 ImM, pH7) et de 10 à 30μl de X-Gal (5-bromo-4-chloro-3- indoyl-β-D-galactoside) à 50mg/ml de N,N-diméthylformamide. La boite est ensuite placée dans une étuve à 37°C couvercle fermé pour éviter le dessèchement . Le temps d'apparition de la couleur bleue peut être très variable, de quelques minutes à plusieurs heures. Ce test doit toujours se faire en présence d'un témoin positif dont l'interaction est connue et vire rapidement au bleu.A nitrocellulose sheet is previously deposited on the Petri dish containing the individualized yeast clones. Thanks to the phenomenon of addition, we will obtain a faithful image of the location of the clones. This sheet is then immersed in Uquide nitrogen for 30 seconds in order to burst the yeasts and thus to tiberate the βgalactosidase activity. After thawing, the nitroceUulose sheet is deposited, colonies upwards, in another Petri dish containing Whatman paper previously soaked with 1.5 ml of PBS solution (Na 2 HPO4 60mM, NaH 2 PO 4 40mM, KCl 10MM, MgSO 4 ImM, pH7) and from 10 to 30 μl of X-Gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside) at 50 mg / ml of N, N-dimethylformamide. The box is then placed in an oven at 37 ° C with the lid closed to prevent drying. The appearance time of the blue color can be very variable, from a few minutes to several hours. This test should always be done in the presence of a positive control whose interaction is known and quickly turns blue.
EXEMPLE 1: Construction d'un vecteur permettant l'expression d'une protéine de fusion entre la partie C-terminale du précurseur du peptide amyloïde (CAPP) et le domaine de liaison à l'ADN de GAL4EXAMPLE 1 Construction of a vector allowing the expression of a fusion protein between the C-terminal part of the precursor of the amyloid peptide (CAPP) and the DNA binding domain of GAL4
Le criblage d'une banque utilisant le système double hybride nécessite que la région C- terminale de l'APP (CAPP) soit fusionnée au domaine de liaison à l'ADN de la protéine transactivatrice GAL4. L'expression de cette protéine de fusion est réaUsée grâce au vecteur pGBTIO (cf matériels et méthodes), dans lequel nous avons introduit, dans le même cadre de lecture que la séquence correspondant au domaine de Uaison à l'ADN de GAL4 (GAL4DB), la séquence codant pour le CAPP figurant dans la séquence présentée en SEQ ID N°l.Screening of a library using the double hybrid system requires that the C-terminal region of the APP (CAPP) be fused to the DNA binding domain of the GAL4 transactivating protein. The expression of this fusion protein is achieved using the vector pGBTIO (see materials and methods), in which we have introduced, in the same reading frame as the sequence corresponding to the Uaison domain to the DNA of GAL4 (GAL4DB), the sequence coding for CAPP appearing in the sequence presented in SEQ ID No. 1.
Le fragment d'ADN de 138 pdb correspondant aux 46 derniers acides aminés de l'APP a été obtenu par PCR à partir des oUgonucléotides (SEQ ID N°3 et N°4) qui nous ont également permis d'introduire les sites EcoRI et Sali aux extrémités de la séquence. Le fragment de PCR a été introduit entre les sites EcoRI et Sali du multisite de clonage du plasmide pGBTIO en aval de la séquence correspondant au GAL4DB pour donner le vecteur pGAL4DB-CAPP (fig.2). La construction a été vérifiée par séquençage de l'ADN. Cette vérification nous a permis de montrer que ce fragment ne présentait pas de mutations générées au cours de la réaction de PCR et qu'il était fusionné dans la même phase ouverte de lecture que celle du fragment correspondant au GAL4DB.The 138 bpd DNA fragment corresponding to the last 46 amino acids of the APP was obtained by PCR from the Ogonucleotides (SEQ ID N ° 3 and N ° 4) which also allowed us to introduce the EcoRI and Dirty at the ends of the sequence. The PCR fragment was introduced between the EcoRI and SalI sites of the multisite for cloning the plasmid pGBTIO downstream of the sequence corresponding to GAL4DB to give the vector pGAL4DB-CAPP (FIG. 2). The construction was verified by DNA sequencing. This verification allowed us to show that this fragment did not present mutations generated during the PCR reaction and that it was fused in the same open reading phase as that of the fragment corresponding to GAL4DB.
EXEMPLE 2: CRIBLAGE DE LA BANQUE DE FUSION DE CERVEAU.EXAMPLE 2: SCREENING OF THE BRAIN FUSION BANK.
Le criblage d'une banque de fusion permet d'identifier des clones produisant des protéines fusionnées au domaine transactivateur de GAL4, pouvant interagir avec notre protéine d'intérêt. Cette interaction permet de reconstituer un transactivateur qui va alors être capable d'induire l'expression des gènes rapporteurs His3 et LacZ dans la souche YCM.The screening of a fusion bank makes it possible to identify clones producing proteins fused to the transactivating domain of GAL4, which can interact with our protein of interest. This interaction makes it possible to reconstitute a transactivator which will then be capable of inducing the expression of the reporter genes His3 and LacZ in the YCM strain.
Pour effectuer ce criblage nous avons choisi une banque de fusion réalisée à partir d'ADNc provenant de cerveau humain. Comme cette banque nous a été fournie sous forme de bactéries, l'ADN plasmidique de la banque a tout d'abord été purifié.To carry out this screening, we chose a fusion library made from cDNA from the human brain. As this library was supplied to us in the form of bacteria, the plasmid DNA of the library was first purified.
2.1) Préparation de l' ADN plasmidique d'une banque de fusion..2.1) Preparation of the plasmid DNA of a fusion library.
L'ADN plasmidique de la banque d'ADNc de cerveau a été extrait suivant le protocole Clontech (voir matériels et méthodes, § 11). Lors de cette préparation il était important de préserver la représentativité de la banque, c'est à dire, conserver le nombre de plasmides indépendants qui la constitue et qui sont au nombre de 1.2.106 plasmides. Afin de nous préserver de la perte de plasmides de la banque au cours de cette préparation, le lot d'ADN plasmidique que nous avons constitué a été obtenu à partir d'un nombre de colonies bactériennes isolées correspondant à un peu plus de deux fois la représentativité de la banque soit 4.106 colonies.The plasmid DNA from the brain cDNA library was extracted according to the Clontech protocol (see materials and methods, § 11). During this preparation, it was important to preserve the representativeness of the library, that is to say, to keep the number of independent plasmids which constitute it and which are 1.2.10 6 plasmids. In order to protect us from the loss of plasmids from the library during this preparation, the batch of plasmid DNA that we have assembled was obtained from a number of isolated bacterial colonies corresponding to slightly more than twice the representativeness of the bank, ie 4.10 6 colonies.
2.2) Transformation par banque de cerveau et sélection par le test d'activité βgalactosidase.2.2) Transformation by brain bank and selection by the βgalactosidase activity test.
Lors du criblage il est nécessaire de préserver la probabilité que chaque plasmide indépendant de la banque de fusion soit présent dans au moins une levure en même temps que le plasmide GAL4DB-CAPP. Pour préserver cette probabilité il est important d'avoir une bonne efficacité de transformation de la levure. Pour cela nous avons choisi un protocole de transformation de la levure donnant une efficacité de 105 ceUules transformées par μg d'ADN. De plus comme la cotransformation de la levure par deux plasmides différents réduit cette efficacité, nous avons préféré utiliser une levure préalablement transformée par le plasmide pGAL4DB-CAPP. Cette souche YCM-CAPP de phénotype His-, Lys-, Leu- a été transformée par lOOμg d'ADN plasmidique de la banque de fusion. Cette quantité d'ADN nous a permis d'obtenir après estimation (voir matériels et méthodes) 10 cellules transformées, ce qui correspond à un nombre légèrement supérieur au nombre de plasmides indépendants que constitue la banque. D'après ce résultat nous pouvons penser que la quasi totalité des plasmides de la banque a servi à transformer les levures. La sélection des cellules transformées, capables de reconstituer un transactivateur GAL4 fonctionnel, a été faite sur un milieu YNB+Lys+Ad.During screening, it is necessary to preserve the probability that each plasmid independent of the fusion library is present in at least one yeast at the same time as the plasmid GAL4DB-CAPP. To preserve this probability, it is important to have a good yeast transformation efficiency. For this we have chosen a yeast transformation protocol giving an efficiency of 10 5 ceUules transformed per μg of DNA. In addition, since the cotransformation of the yeast by two different plasmids reduces this efficiency, we preferred to use a yeast previously transformed by the plasmid pGAL4DB-CAPP. This YCM-CAPP strain of His-, Lys-, Leu- phenotype was transformed with lOOμg of plasmid DNA from the fusion bank. This quantity of DNA allowed us to obtain after estimation (see materials and methods) 10 transformed cells, which corresponds to a number slightly higher than the number of independent plasmids that constitute the bank. From this result we can think that almost all of the plasmids from the bank were used to transform yeasts. The selection of the transformed cells, capable of reconstituting a functional GAL4 transactivator, was made on a YNB + Lys + Ad medium.
A l'issu de cette sélection 1000 clones avec un phénotype His+ ont été obtenus. Sur ces transformants un test d'activité βgalactosidase a été effectué afin de vaUder par l'expression de l'autre gène rapporteur, LacZ, ce nombre de clones obtenus. 68 des 1000 clones obtenus présentaient le double phénotype His+, βGal+ pouvant correspondre à une interaction protéine-protéine.At the end of this selection 1000 clones with a His + phenotype were obtained. On these transformants, a βgalactosidase activity test was carried out in order to assess the number of clones obtained by the expression of the other reporter gene, LacZ. 68 of the 1000 clones obtained exhibited the double His + phenotype, βGal + which may correspond to a protein-protein interaction.
EXEMPLE 3: ISOLEMENT DES PLASMIDES DE LA BANQUEEXAMPLE 3: ISOLATION OF BANK PLASMIDS
Afin d'identifier les protéines pouvant interagir avec le CAPP, nous avons extrait les plasmides de la banque de fusion contenus dans les levures sélectionnées lors du criblage double hybride. Pour pouvoir en obtenir en grande quantité, cet isolement nécessite au préalable une transformation d'E.coli par un extrait d'ADN des souches de levures positives. Comme le plasmide de la banque contenu dans cet extrait est un plasmide navette levure/E.coli il pourra facilement se répUquer chez la bactérie. Afin d'éviter d'isoler le plasmide GAL4 DB - CAPP qui est lui aussi présent dans la levure nous avons travaillé sur des souches qui en sont dépourvues.In order to identify the proteins that can interact with CAPP, we extracted the plasmids from the fusion bank contained in the yeasts selected during the double hybrid screening. To be able to obtain large quantities, this isolation requires a transformation of E. coli beforehand with a DNA extract of the positive yeast strains. As the bank plasmid contained in this extract is a yeast / E. coli shuttle plasmid, it can easily repute in bacteria. In order to avoid isolating the plasmid GAL4 DB - CAPP which is also present in yeast, we have worked on strains which do not have it.
Les ADN plasmidiques des colonies bactériennes obtenues après transformation par des extraits d'ADN de levures ont été analysées par digestion avec des enzymes de restriction et séparation des fragments d'ADN sur gel d'agarose. Nous avons obtenu 3 profils de restriction différents sur les 5 clones analysés (7D, 3E, 9A, 3H, 3G) (fig 2). Ces résultats ont montré que 2 plasmides identiques sont au moins représentés dans 2 clones de levures différents, les clones 7D, ainsi que les clones 3H et 3G. L'ADN du clone 3E est obtenu à partir de souches de phénotype His+ et βGAL+.The plasmid DNAs of the bacterial colonies obtained after transformation with yeast DNA extracts were analyzed by digestion with restriction enzymes and separation of the DNA fragments on agarose gel. We obtained 3 different restriction profiles on the 5 clones analyzed (7D, 3E, 9A, 3H, 3G) (fig 2). These results showed that 2 identical plasmids are at least represented in 2 different yeast clones, the 7D clones, as well as the 3H and 3G clones. The DNA of clone 3E is obtained from strains of the His + and βGAL + phenotype.
EXEMPLE 4: DETERMINATION DE LA SEQUENCE DES INSERTS DES PLASMIDES IDENTIFIES.EXAMPLE 4: DETERMINATION OF THE SEQUENCE OF THE INSERTS OF THE IDENTIFIED PLASMIDS.
Le séquençage a été réaUsé à partir de l'oligonucléotide (SEQ ID N°5) complémentaire de la région de GAL4TA à proximité du site d'insertion de la banque de cDNA de cerveau, à 52 pdb du site EcoRI. La comparaison des 3 séquences avec les séquences contenues dans les banques de données GENBank et EMBL (European Molecular Biology Lab) a montré que les séquences des ADNc qui étaient dans les plasmides issus des souches 9A et 3H présentent 87% d'homologie au niveau nucléique avec le gène mutin codant pour la protéine FE65, elles sont représentées en SEQ ID N°2, et que la séquence du plasmide issu de la souche 7D présente 60% d'homologie avec ce même gène ( voir figure 3). L'analyse de la séquence des plasmides issus des souches 9A et 3H indique que ceux- ci contiennent deux régions chevauchantes correspondant à un même ARNm. LISTE DE SEQUENCESThe sequencing was carried out using the oligonucleotide (SEQ ID No. 5) complementary to the GAL4TA region near the insertion site of the brain cDNA library, 52 bpd from the EcoRI site. The comparison of the 3 sequences with the sequences contained in the GENBank and EMBL (European Molecular Biology Lab) databases showed that the sequences of the cDNAs which were in the plasmids derived from strains 9A and 3H show 87% homology at the nucleic level with the mutant gene coding for the protein FE65, they are represented in SEQ ID No. 2, and that the sequence of the plasmid derived from the strain 7D has 60% homology with this same gene (see FIG. 3). Analysis of the sequence of the plasmids from strains 9A and 3H indicates that these contain two overlapping regions corresponding to the same mRNA. LIST OF SEQUENCES
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(ni) NOMBRE DE SEQUENCES: 5(ni) NUMBER OF SEQUENCES: 5
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(2) INFORMATIONS POUR LA SEQ ID NO: 1:(2) INFORMATION FOR SEQ ID NO: 1:
(î) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 1500 (B) TYPE: nucléotide(î) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1500 (B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(n) TYPE DE MOLECULE: ADNc(n) TYPE OF MOLECULE: cDNA
(Xl ) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1(Xl) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1
(2) INFORMATIONS POUR LA SEQ ID NO: 2:(2) INFORMATION FOR SEQ ID NO: 2:
(î) CARACTERISTIQUES DE LA SEQUENCE:(î) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 1275(A) LENGTH: 1275
(B) TYPE: nucléotide (C) NOMBRE DE BRINS: Simple(B) TYPE: nucleotide (C) NUMBER OF STRANDS: Single
(D) CONFIGURATION: linéaire (il) TYPE DE MOLECULE: ADNc(D) CONFIGURATION: linear (il) TYPE OF MOLECULE: cDNA
(xi ) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
CGG GAG GAG TCC CAG CTC ACC TGG ACA GGT TTT GCT CAT GGA GAA GGC 48 Arg Glu Glu Ser Gin Leu Thr Trp Thr Gly Phe Ala His Gly Glu Gly 1 5 10 15CGG GAG GAG TCC CAG CTC ACC TGG ACA GGT TTT GCT CAT GGA GAA GGC 48 Arg Glu Glu Ser Gin Leu Thr Trp Thr Gly Phe Ala His Gly Glu Gly 1 5 10 15
TTT GAG GAT GGA GAA TTT TGG AAG GAT GAA CCC AGT GAT GAG GCC CCA 96 Phe Glu Asp Gly Glu Phe Trp Lys Asp Glu Pro Ser Asp Glu Ala Pro 20 25 30TTT GAG GAT GGA GAA TTT TGG AAG GAT GAA CCC AGT GAT GAG GCC CCA 96 Phe Glu Asp Gly Glu Phe Trp Lys Asp Glu Pro Ser Asp Glu Ala Pro 20 25 30
ATG GAG CTG GGA CTG AAG GAA CCT GAG GAG GGG ACG TTG ACC TTC CCA 144 Met Glu Leu Gly Leu Lys Glu Pro Glu Glu Gly Thr Leu Thr Phe Pro 35 40 45ATG GAG CTG GGA CTG AAG GAA CCT GAG GAG GGG ACG TTG ACC TTC CCA 144 Met Glu Leu Gly Leu Lys Glu Pro Glu Glu Gly Thr Leu Thr Phe Pro 35 40 45
GCT CAG AGC CTC AGC CCA GAG CCG TTG CCC CAA GAG GAG GAG AAG CTT 192 Ala Gin Ser Leu Ser Pro Glu Pro Leu Pro Gin Glu Glu Glu Lys Leu 50 55 60GCT CAG AGC CTC AGC CCA GAG CCG TTG CCC CAA GAG GAG GAG AAG CTT 192 Ala Gin Ser Leu Ser Pro Glu Pro Leu Pro Gin Glu Glu Glu Lys Leu 50 55 60
CCC CCA CGG AAT ACC AAC CCA GGG ATC AAG TGT TTC GCC GTG CGC TCC 240CCC CCA CGG AAT ACC AAC CCA GGG ATC AAG TGT TTC GCC GTG CGC TCC 240
Pro Pro Arg Asn Thr Asn Pro Gly Ile Lys Cys Phe Ala Val Arg SerPro Pro Arg Asn Thr Asn Pro Gly Ile Lys Cys Phe Ala Val Arg Ser
65 70 75 8065 70 75 80
CTA GGC TGG GTA GAG ATG ACC GAG GAG GAG CTG GCC CCT GGA CGC AGC 288CTA GGC TGG GTA GAG ATG ACC GAG GAG GAG CTG GCC CCT GGA CGC AGC 288
Leu Gly Trp Val Glu Met Thr Glu Glu Glu Leu Ala Pro Gly Arg SerLeu Gly Trp Val Glu Met Thr Glu Glu Glu Leu Ala Pro Gly Arg Ser
85 90 95 AGT GTG GCA GTC AAC AAT TGC ATC CGT CAG CTC TCT TAC CAC AAA AAC 33685 90 95 AGT GTG GCA GTC AAC AAT TGC ATC CGT CAG CTC TCT TAC CAC AAA AAC 336
Ser Val Ala Val Asn Asn Cys Ile Arg Gin Leu Ser Tyr His Lys AsnSer Val Ala Val Asn Asn Cys Ile Arg Gin Leu Ser Tyr His Lys Asn
100 105 110100 105 110
AAC CTG CAT GAC CCC ATG TCT GGG GGC TGG GGG GAA GGA AAG GAT CTG 384 Asn Leu His Asp Pro Met Ser Gly Gly Trp Gly Glu Gly Lys Asp LeuAAC CTG CAT GAC CCC ATG TCT GGG GGC TGG GGG GAA GGA AAG GAT CTG 384 Asn Leu His Asp Pro Met Ser Gly Gly Trp Gly Glu Gly Lys Asp Leu
115 120 125115 120 125
CTA CTG CAG CTG GAG GAT GAG ACA CTA AAG CTA GTG GAG CCA CAG AGC 432CTA CTG CAG CTG GAG GAT GAG ACA CTA AAG CTA GTG GAG CCA CAG AGC 432
Leu Leu Gin Leu Glu Asp Glu Thr Leu Lys Leu Val Glu Pro Gin Ser 130 135 140Leu Leu Gin Leu Glu Asp Glu Thr Leu Lys Leu Val Glu Pro Gin Ser 130 135 140
CAG GCA CTG CTG CAC GCC CAA CCC ATC ATC AGC ATC CGC GTG TGG GGC 480CAG GCA CTG CTG CAC GCC CAA CCC ATC ATC AGC ATC CGC GTG TGG GGC 480
Gin Ala Leu Leu His Ala Gin Pro Ile Ile Ser Ile Arg Val Trp GlyGin Ala Leu Leu His Ala Gin Pro Ile Ile Ser Ile Arg Val Trp Gly
145 150 155 160145 150 155 160
GTC GGG CGG GAC AGT GGA AGA GAG AGG GAC TTT GCC TAC GTA GCT CGT 528 Val Gly Arg Asp Ser Gly Arg Glu Arg Asp Phe Ala Tyr Val Ala Arg 165 170 175 GAT AAG CTG ACC CAG ATG CTC AAG TGC CAC GTG TTT CGC TGT GAG GCA 576 Asp Lys Leu Thr Gin Met Leu Lys Cys His Val Phe Arg Cys Glu Ala 180 185 190GTC GGG CGG GAC AGT GGA AGA GAG AGG GAC TTT GCC TAC GTA GCT CGT 528 Val Gly Arg Asp Ser Gly Arg Glu Arg Asp Phe Ala Tyr Val Ala Arg 165 170 175 GAT AAG CTG ACC CAG ATG CTC AAG TGC CAC GTG TTT CGC TGT GAG GCA 576 Asp Lys Leu Thr Gin Met Leu Lys Cys His Val Phe Arg Cys Glu Ala 180 185 190
CCT GCC AAG AAC ATC GCC ACC AGC CTG CAT GAG ATC TGC TCT AAG ATC 624 Pro Ala Lys Asn Ile Ala Thr Ser Leu His Glu Ile Cys Ser Lys Ile 195 200 205CCT GCC AAG AAC ATC GCC ACC AGC CTG CAT GAG ATC TGC TCT AAG ATC 624 Pro Ala Lys Asn Ile Ala Thr Ser Leu His Glu Ile Cys Ser Lys Ile 195 200 205
ATG GCC GAA CGG GGT AAT GCC CGC TGC TTG GTA AAT GGA CTC TCC CTG 672 Met Ala Glu Arg Gly Asn Ala Arg Cys Leu Val Asn Gly Leu Ser Leu 210 215 220ATG GCC GAA CGG GGT AAT GCC CGC TGC TTG GTA AAT GGA CTC TCC CTG 672 Met Ala Glu Arg Gly Asn Ala Arg Cys Leu Val Asn Gly Leu Ser Leu 210 215 220
GAC CAC TCT AAA CTT GTG GAT GTC CCT TTC CAA GTG GAA TTC CCA GCG 720 Asp His Ser Lys Leu Val Asp Val Pro Phe Gin Val Glu Phe Pro Ala 225 230 235 240GAC CAC TCT AAA CTT GTG GAT GTC CCT TTC CAA GTG GAA TTC CCA GCG 720 Asp His Ser Lys Leu Val Asp Val Pro Phe Gin Val Glu Phe Pro Ala 225 230 235 240
CCT AAG AAT GAG TTG GTC CAG AAG TTC CAA GTC TAT TAC CTG GGG AAT 768 Pro Lys Asn Glu Leu Val Gin Lys Phe Gin Val Tyr Tyr Leu Gly Asn 245 250 255 GTA CCT GTT GCT AAA CCT GTT GGG GTA GAT GTG ATT AAT GGG GCC CTC 816 Val Pro Val Ala Lys Pro Val Gly Val Asp Val Ile Asn Gly Ala Leu 260 265 270 GAG TCA GTC CTG TCC TCC AGC AGC CGT GAA CAA TGG ACC CCA AGT CAT 864 Glu Ser Val Leu Ser Ser Ser Ser Arg Glu Gin Trp Thr Pro Ser His 275 280 285CCT AAG AAT GAG TTG GTC CAG AAG TTC CAA GTC TAT TAC CTG GGG AAT 768 Pro Lys Asn Glu Leu Val Gin Lys Phe Gin Val Tyr Tyr Leu Gly Asn 245 250 255 GTA CCT GTT GCT AAA CCT GTT GGG GTA GAT GTG ATT AAT GGG GCC CTC 816 Val Pro Val Ala Lys Pro Val Gly Val Asp Val Ile Asn Gly Ala Leu 260 265 270 GAG TCA GTC CTG TCC TCC AGC AGC CGT GAA CAA TGG ACC CCA AGT CAT 864 Glu Ser Val Leu Ser Ser Ser Ser Arg Glu Gin Trp Thr Pro Ser His 275 280 285
GTC AGT GTG GCC CCT GCT ACC CTC ACC ATC TTG CAC CAG CAG ACA GAG 912 Val Ser Val Ala Pro Ala Thr Leu Thr Ile Leu His Gin Gin Thr Glu 290 295 300GTC AGT GTG GCC CCT GCT ACC CTC ACC ATC TTG CAC CAG CAG ACA GAG 912 Val Ser Val Ala Pro Ala Thr Leu Thr Ile Leu His Gin Gin Thr Glu 290 295 300
GCA GTG CTG GGA GAG TGT CGG GTG CGT TTC CTC TCC TTC CTG GCC GTG 960 Ala Val Leu Gly Glu Cys Arg Val Arg Phe Leu Ser Phe Leu Ala Val 305 310 315 320GCA GTG CTG GGA GAG TGT CGG GTG CGT TTC CTC TCC TTC CTG GCC GTG 960 Ala Val Leu Gly Glu Cys Arg Val Arg Phe Leu Ser Phe Leu Ala Val 305 310 315 320
GGC AGA GAT GTC CAC ACG TTT GCA TTC ATC ATG GCT GCC GGC CCA GCC 1008 Gly Arg Asp Val His Thr Phe Ala Phe Ile Met Ala Ala Gly Pro Ala 325 330 335GGC AGA GAT GTC CAC ACG TTT GCA TTC ATC ATG GCT GCC GGC CCA GCC 1008 Gly Arg Asp Val His Thr Phe Ala Phe Ile Met Ala Ala Gly Pro Ala 325 330 335
TCC TTC TGC TGT CAC ATG TTC TGG TGC GAG CCC AAT GCT GCC AGC CTC 1056 Ser Phe Cys Cys His Met Phe Trp Cys Glu Pro Asn Ala Ala Ser Leu 340 345 350 TCA GAG GCT GTG CAG GCT GCG TGC ATG CTT CGC TAC CAG AAG TGT CTG 1104 Ser Glu Ala Val Gin Ala Ala Cys Met Leu Arg Tyr Gin Lys Cys Leu 355 360 365TCC TTC TGC TGT CAC ATG TTC TGG TGC GAG CCC AAT GCT GCC AGC CTC 1056 Ser Phe Cys Cys His Met Phe Trp Cys Glu Pro Asn Ala Ala Ser Leu 340 345 350 TCA GAG GCT GTG CAG GCT GCG TGC ATG CTT CGC TAC CAG AAG TGT CTG 1104 Ser Glu Ala Val Gin Ala Ala Cys Met Leu Arg Tyr Gin Lys Cys Leu 355 360 365
GAT GCC CGT TCC CAG GCC TCC ACC TCC TGC CTC CCA GCA CCC CCT GCT 1152 Asp Ala Arg Ser Gin Ala Ser Thr Ser Cys Leu Pro Ala Pro Pro Ala 370 375 380GAT GCC CGT TCC CAG GCC TCC ACC TCC TGC CTC CCA GCA CCC CCT GCT 1152 Asp Ala Arg Ser Gin Ala Ser Thr Ser Cys Leu Pro Ala Pro Pro Ala 370 375 380
GAG TCT GTG GCA CGG GGT GTA GGG TGG ACT GTC CGC AGG GGT GTT CAG 1200 Glu Ser Val Ala Arg Gly Val Gly Trp Thr Val Arg Arg Gly Val Gin 385 390 395 400GAG TCT GTG GCA CGG GGT GTA GGG TGG ACT GTC CGC AGG GGT GTT CAG 1200 Glu Ser Val Ala Arg Gly Val Gly Trp Thr Val Arg Arg Gly Val Gin 385 390 395 400
TCG CTG TGG GGC TCC CTG AAG CCC AAA CGG GTG GGG GGC CAT ACC CCA 1248 Ser Leu Trp Gly Ser Leu Lys Pro Lys Arg Val Gly Gly His Thr Pro 405 410 415TCG CTG TGG GGC TCC CTG AAG CCC AAA CGG GTG GGG GGC CAT ACC CCA 1248 Ser Leu Trp Gly Ser Leu Lys Pro Lys Arg Val Gly Gly His Thr Pro 405 410 415
TGA AGA AGC CCC ACC TTC CCT CCA CCT 1275 * Arg Ser Pro Thr Phe Pro Pro Pro 420 425TGA AGA AGC CCC ACC TTC CCT CCA CCT 1275 * Arg Ser Pro Thr Phe Pro Pro Pro 420 425
(2) INFORMATIONS POUR LA SEQ ID NO: 3:(2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 27 (B) TYPE: nucléotide(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 (B) TYPE: nucleotide
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3: CAAGTCGACC TAGTTCTGCA TCTGCTC 27(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CAAGTCGACC TAGTTCTGCA TCTGCTC 27
(2) INFORMATIONS POUR LA SEQ ID NO: 4:(2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR(A) LENGTH
(B) TYPE: nucléotide (C) NOMBRE DE BRINS: simple(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
CAAGAATTCA AGAAACAGTA CACATCCCAAGAATTCA AGAAACAGTA CACATCC
(2) INFORMATIONS POUR LA SEQ ID NO : 5:(2) INFORMATION FOR SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 23(A) LENGTH: 23
(B) TYPE: nucléotide (C) NOMBRE DE BRINS: simple(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO : 5:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
CTATTCGATG ATGAAGATAC CCC 23 CTATTCGATG ATGAAGATAC CCC 23

Claims

REVENDICATIONS
1. Peptide caractérisé en ce qu'il est capable d'interférer au niveau de l'interaction de la protéine FE65 ou l'une de ses formes homologues avec la région cytoplasmique de l'APP.1. Peptide characterized in that it is capable of interfering at the level of the interaction of the protein FE65 or one of its homologous forms with the cytoplasmic region of APP.
2. Peptide selon la revendication 1 caractérisé en ce que l'interférence se traduit par un ralentissement, une inhibition ou une stimulation de ladite interaction.2. Peptide according to claim 1 characterized in that the interference results in a slowing down, an inhibition or a stimulation of said interaction.
3. Peptide selon l'une des revendications 1 ou 2 caractérisé en ce qu'il est capable de se lier au niveau du domaine d'interaction entre la protéine FE65 ou l'une de ses formes homologues et la région cytoplasmique de l'APP.3. Peptide according to one of claims 1 or 2 characterized in that it is capable of binding at the level of the interaction domain between the protein FE65 or one of its homologous forms and the cytoplasmic region of APP .
4. Peptide selon la revendication 1, 2 ou 3 caractérisé en ce qu'il comprend tout ou partie de la SEQ ID N°l, SEQ ID N°2 ou d'un dérivé de celles-ci.4. Peptide according to claim 1, 2 or 3 characterized in that it comprises all or part of SEQ ID No. 1, SEQ ID No. 2 or a derivative thereof.
5. Peptide capable d'entrer en compétition avec un peptide selon l'une quelconque des revendications 1 à 4 au niveau du domaine d'interaction entre la protéine FE65 ou l'une de ses formes homologues et la région cytoplasmique de l'APP.5. Peptide capable of competing with a peptide according to any one of claims 1 to 4 at the level of the interaction domain between the protein FE65 or one of its homologous forms and the cytoplasmic region of APP.
6. Composé non peptidique ou non exclusivement peptidique capable de moduler l'interaction entre la protéine FE65 et la région cytoplasmique de l'APP obtenu par reproduction des motifs actifs des peptides selon les revendications 1 à 5 par des structures non peptidiques ou non exclusivement peptidiques.6. A non-peptide or non-exclusively peptide compound capable of modulating the interaction between the FE65 protein and the cytoplasmic region of the APP obtained by reproduction of the active motifs of the peptides according to claims 1 to 5 by non-peptide or non-exclusively peptide structures .
7. Séquence nucléotidique codant pour un peptide tel que défini en revendication 1 à 5.7. Nucleotide sequence coding for a peptide as defined in claims 1 to 5.
8. Séquence nucléotidique selon la revendication 7 caractérisée en ce qu'il s'agit d'une séquence comprenant tout ou partie de la SEQ ID N°l, SEQ ID N°2 ou d'une séquence dérivée de celles-ci.8. Nucleotide sequence according to claim 7 characterized in that it is a sequence comprising all or part of SEQ ID No. 1, SEQ ID No. 2 or a sequence derived therefrom.
9. Séquence selon la revendication 7 ou 8 introduite dans un vecteur viral ou non viral.9. The sequence of claim 7 or 8 introduced into a viral or non-viral vector.
10. Oligonucléotide antisens d'une séquence selon la revendication 7 ou 8. 10. Antisense oligonucleotide of a sequence according to claim 7 or 8.
11. Anticoφs ou fragment d'anticoφs caractérisé en ce qu'il est dirigé contre un peptide selon l'une des revendications 1 à 5.11. Anticoφs or fragment of anticoφs characterized in that it is directed against a peptide according to one of claims 1 to 5.
12. Sonde nucléotidique capable de s'hybrider avec un acide nucléique selon l'une des revendications 1 à 5 ou avec l'ARNm correspondant.12. Nucleotide probe capable of hybridizing with a nucleic acid according to one of claims 1 to 5 or with the corresponding mRNA.
13. Composition pharmaceutique comprenant comme principe actif au moins un peptide selon l'une des revendications 1 à 6 ou un anticoφs ou fragment d'anticoφs selon la revendication 11 ou une séquence nucléotidique selon l'une des revendications 7 à 9.13. Pharmaceutical composition comprising as active principle at least one peptide according to one of claims 1 to 6 or an anticoφs or fragment of anticoφs according to claim 11 or a nucleotide sequence according to one of claims 7 to 9.
14. Composition pharmaceutique selon la revendication 13 caractérisée en ce qu'eUe comprend un vecteur viral ou non viral recombinant dans lequel est incorporée une séquence nucléotidique selon la revendication 7 ou 8.14. Pharmaceutical composition according to claim 13 characterized in that eUe comprises a recombinant viral or non-viral vector in which is incorporated a nucleotide sequence according to claim 7 or 8.
15. Composition pharmaceutique selon la revendication 14 caractérisée en ce qu'U s'agit d'un vecteur viral choisi parmi les rétrovirus et les adénovirus.15. Pharmaceutical composition according to claim 14 characterized in that U is a viral vector chosen from retroviruses and adenoviruses.
16. Composition pharmaceutique selon une des revendications 13 à 15 destinée à moduler l'interaction entre la protéine FE65 et la région cytoplasmique de l'APP.16. Pharmaceutical composition according to one of claims 13 to 15 intended to modulate the interaction between the protein FE65 and the cytoplasmic region of APP.
17. Composition pharmaceutique selon la revendication 16 ou 17 destinée à interférer au niveau de l'interaction entre la protéine FE65 et la région cytoplasmique de l'APP.17. Pharmaceutical composition according to claim 16 or 17 intended to interfere with the interaction between the protein FE65 and the cytoplasmic region of APP.
18. Composition pharmaceutique selon la revendication 16 destinée au traitement des maladies neurodégénératives.18. Pharmaceutical composition according to claim 16 intended for the treatment of neurodegenerative diseases.
19. Utilisation d'un anticoφs ou fragment d'anticoφs selon la revendication 11 et/ou d'une séquence nucléotidique selon l'une des revendications 7 ou 8 pour le typage de maladies neurodégénératives.19. Use of an anticoφs or fragment of anticoφs according to claim 11 and / or a nucleotide sequence according to one of claims 7 or 8 for the typing of neurodegenerative diseases.
20. Procédé de préparation d'un peptide selon l'une quelconque des revendications 1 à 5 caractérisé en ce que l'on cultive une cellule contenant une séquence nucléotidique selon la revendication 7 ou 8 dans des conditions d'expression de ladite séquence et on récupère le peptide produit. 20. A method of preparing a peptide according to any one of claims 1 to 5 characterized in that a cell containing a nucleotide sequence according to claim 7 or 8 is cultured under conditions of expression of said sequence and recover the peptide produced.
21. Virus recombinant defectif comprenant une séquence nucléique hétérologue codant pour un polypeptide selon l'une des revendications 1 à 5. 21. Defective recombinant virus comprising a heterologous nucleic sequence coding for a polypeptide according to one of claims 1 to 5.
EP96938292A 1996-11-08 1996-11-08 Peptides capable of inhibiting the endocytosis of the app and corresponding nucleotide sequences Withdrawn EP0941319A1 (en)

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US7622446B2 (en) 2001-04-18 2009-11-24 The Open University Polypeptides, derivatives and uses thereof
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US7491702B2 (en) 2001-04-18 2009-02-17 The Open University Polypeptides related to amyloid precursor protein, pharmaceutical compositions thereof, and methods of treatment using the same
US6927236B2 (en) 2001-09-22 2005-08-09 Aventis Pharma Deutschland Gmbh. Coniosulfides and their derivatives, processes for preparing them, and their use as pharmaceuticals
DE10146737A1 (en) 2001-09-22 2003-04-10 Aventis Pharma Gmbh Coniosulfides and their derivatives, process for their preparation and use as medicines
JPWO2005023286A1 (en) * 2003-09-05 2007-11-01 久光製薬株式会社 Drug for prevention and / or treatment of Alzheimer's disease

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