EP0941319A1 - Peptide die im stande sind die endozytose von app zu hemmen und entsprechende nuckleotid-sequenzen - Google Patents

Peptide die im stande sind die endozytose von app zu hemmen und entsprechende nuckleotid-sequenzen

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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.)
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Application number
EP96938292A
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English (en)
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/de
Withdrawn legal-status Critical Current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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EP96938292A 1996-11-08 1996-11-08 Peptide die im stande sind die endozytose von app zu hemmen und entsprechende nuckleotid-sequenzen Withdrawn EP0941319A1 (de)

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