FR2769021A1 - Nucleic acid encoding peptides that interact with presenilin - Google Patents

Abstract

Nucleic acid (I) encoding a peptide (II) that interacts with presenilins (III). Independent claims are also included for the following: (1) peptide (II); (2) recombinant production of (II); (3) cells transformed with (I) that produce (II); (4) oligonucleotides antisense to (I); (5) probes that hybridize with (I) or the corresponding mRNA; (6) antibodies, or their fragments, directed against (II); (7) method for detection or isolation of compounds (A) that modulate or inhibit interaction between (II) and (III); (8) ligand for (II) identified by this method; (9) recombinant, defective virus containing (I); and (10) vector containing (I).

Description

The present invention relates to new nucleotide and peptide sequences, and their pharmaceutical use. More particularly, the present invention relates to nucleic acids coding for proteins capable of interacting with presenilins, the processes for producing said proteins as well as compositions containing them.

 Alzheimer's disease is the most common form of dementia. Its overall frequency increases with age, it is 5 to 10% after 65 years and 20% beyond 80 years.

 Currently, there is no specific treatment and only palliative care can be offered to patients. This disease is only really confirmed after histological analysis of the brain post-mortem for lack of an accurate and early diagnosis.

 Alzheimer's disease is linked to organic brain lesions constituting histological signs: senile plaques, neuronal neurofibrillary degeneration, loss and atrophy of neurons in the cortex.

 The etiology of the disease is complex, many genes are involved, and the different pathophysiological mechanisms in which they participate are not fully understood.

 Anatomical brain changes observed in patients under the age of 65 have been described, linked to presenile dementia. Currently, the term "dementia of the Alzheimer type" includes presenile dementia (before 65 years) as well as senile dementia (after 65 years) having the same characteristics.

 There are two forms of the disease: sporadic forms, that is to say not linked to heredity and familial forms.

 The sporadic forms of the disease are the most frequent, they concern 60 to 90% of the patients and appear most often after 65 years. The origins of the pathology remain unknown in most cases. However, it has been established that head trauma, inflammation, emotional shock or situations of great stress increase the probability of developing the disease (Seubert et al., 1992).

 Familial forms of the disease represent 10 to 40% of cases. They are classified into two groups, late forms (after 65 years) and early forms (before 65 years). In early cases, the disease is transmitted in a dominant and autosomal character.

 In 1992, the existence of an AD3 locus was demonstrated on chromosome 14 and in 1995 the presenilin 1 gene (PS-I) was isolated (the gene was originally called S182) (Sherrington et al ., 1995). Mutations in the presenilin 1 gene are implicated in 70% of very early forms of the disease (before 55 years of age). Very rare cases of early forms are linked to mutations in the presenilin 2 gene located on chromosome 1 (Levy-Lahad et al. 1995). This gene has 67% identity with the presenilin 1 gene.

 The gene is made up of 12 exons, exons 3 to 12 code for the protein. It is expressed ubiquitously and generates a majority transcript of 2.7 kb and a minority transcript of 7.5 kb (Kovacs, 1996). The protein comprises 467 amino acids and has the structure of a membrane protein. It presents at least 8 potential transmembrane domains. The amino acids corresponding to the codons affected by the listed mutations are distributed throughout the protein, they are located in both the hydrophobic regions (TM) and in the hydrophilic loops (BL), with, however, two preferred regions, TM2 and BL6.

 Currently, more than 35 point mutations have been reported and the mutations identified so far are almost all of the sense type "(ie they result in the point change of an amino acid in the protein sequence However, another type of mutation affecting splicing has recently been reported (Perez-Tûr et ai., 1995), in which case the gene is altered by the loss of exon 9.

 The role of presenilins in the normal cell is still poorly understood. Many hypotheses have been put forward. Among these hypotheses, presenilin 1 (PS-I) could have a role in cell traffic. This hypothesis is supported by an immunocytochemical result which made it possible to localize PS-1 at the level of the endoplasmic reticulum and the Golgi apparatus in human neuroglioma cells (Kovacs et al., 1996). In addition, PS-I plays a role in cellular communication and the endocytosis mechanisms and proteolysis of APP (Demi and Singer, 1996). A third hypothesis involves PS-I in interactions with the cytoskeleton and in particular the microtubules associated with the Tau protein (Lew and Wang, 1996). Finally, given its structure, PS-I could play the role of a receptor coupled to G proteins participating in signal transduction or that of a transporter or ion channel (Van Broeckhoven, 1995).

 The search for partners interacting with presenilins would make it possible to understand the exact role of presenilins.

The present invention results from the discovery by the applicant of nucleic acids coding for proteins capable of interacting with presenilins. The demonstration of such nucleic acids makes it possible to envisage the preparation of new polypeptides which can be used pharmaceutically.

A first object of the invention therefore relates to nucleotide sequences coding for proteins capable of interacting with presenilins.

According to a particular mode, the nucleotide sequences according to the invention encode proteins or polypeptides capable of interacting with all or part of the large loop of presenilin PS-i.

More preferably, the nucleotide sequences according to the invention comprise all or part of SEQ ID N "1 or its derivatives or all or part of SEQ ID N" 2 or its derivatives.

 Within the meaning of the present invention, the term derived nucleotide sequence designates any sequence different from the sequence considered due to the 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 coding for a polypeptide according to the invention. 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. Such homologous sequences can be obtained by hybridization experiments. Hybridizations can be carried out from a nucleic acid library, using as probe, the native sequence or a fragment thereof, under variable hybridization conditions (Maniatis et al. 1989).

The nucleotide sequences according to the invention can be of artificial origin or not. They can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained for example by screening DNA libraries (cDNA library, genomic DNA library) by means of probes produced on the basis of sequences presented above. Such libraries can be prepared from cells of different origins by standard molecular biology techniques known to those skilled in the art. The nucleotide sequences of the invention can also be prepared by chemical synthesis or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries. In general, the nucleic acids of the invention can be prepared according to any technique known to those skilled in the art.

 The present invention also relates to proteins or polypeptides capable of interacting with presenilins. Preferably, the polypeptides according to the invention are capable of interacting with the large loop of presenilin PS-I and / or the large loop of presenilin PS-2. More preferably, they are polypeptides capable of interacting with the amino acid sequence 310463 of the large loop of presenilin PS-1.

 Within the meaning of the present invention, the name presenilin covers the presenilins PS-I and PS-2 in themselves as well as their homologous forms corresponding in particular to mutated forms of these proteins.

The present invention also relates to polypeptides comprising all or part of a peptide sequence chosen from the sequences SEQ ID N0 1 or SEQ
ID N02 or a derivative thereof.

Within the meaning of the present invention, the term derived polypeptide sequence designates any polypeptide sequence differing from the sequences presented in sequence
SEQ ID N0 1 or SEQ ID N "2, obtained by one or more modifications of genetic and / or chemical nature, and possessing the capacity to interact with the presenilins. By modification of genetic and / or chemical nature, one can hear any mutation, substitution, deletion, addition and / or modification of one or more residues.

 Such derivatives can be generated for different purposes, such as in particular that of increasing their therapeutic efficacy or reducing their side effects, or that of conferring on them new pharmacokinetic and / or biological properties.

 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.

 They can also be fragments of the peptide 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.

A subject of the present invention is also the use of the polypeptides according to the invention, for slowing down, inhibiting, stimulating or modulating the activity of Presenilins.

Indeed, it is possible to regulate the functioning of the Presenilins by means of the proteins according to the invention and in particular to inhibit or slow down the activity of
Mutated presenilins.

Another object of the present invention relates to a process for the preparation of the polypeptides according to the invention according to which a cell containing a nucleotide sequence according to the invention is cultivated, under conditions of expression of said sequence and the polypeptide produced is recovered. In this case, the part coding for said polypeptide is generally placed under the control of signals allowing its expression in a cellular host. The choice of these signals (promoters, terminators, secretory leader 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, autonomously replicating vectors can be prepared using autonomous replicating sequences in the chosen host. As integrative vectors, these can be prepared, for example, by using sequences homologous to certain regions of the genome of the host, allowing, by homologous recombination, the integration of the vector.

The present invention also relates to host cells transformed with a nucleic acid comprising a nucleotide sequence according to the invention. 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, Kiuyvernmyces, Pichia, Schwanniomyces, or Han.semwla. As regards animal cells, mention may be made of COS, CHO, C127, human neuroblastoma 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, ssacillus, or Streptomyces.

 According to a preferred mode, the host cells are advantageously represented by recombinant yeast strains for the expression of the nucleic acids of the invention as well as the production of proteins derived from these.

 Preferably, the host cells comprise at least one sequence or a sequence fragment chosen from the sequences SEQ ID N "1 or SEQ ID NO 2, for the production of the proteins according to the invention.

 Another application of the nucleic acid sequences according to the invention is the production of antisense oligonucleotides or genetic antisenses usable as pharmaceutical agents. The antisense sequences are small oligonucleotides, complementary to the strand coding for a given gene, and therefore capable of specifically hybridizing with the transcribed mRNA, inhibiting its translation into protein.

The subject of the invention is therefore the ante sense sequences capable of inhibiting, at least partially, the expression of proteins capable of interacting with presenilins. Such sequences can consist of all or part of the nucleotic sequences defined above and 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 in vivo production of antisense sequences or the expression of proteins or polypeptides capable of modulating the activity of presenilins. In this regard, the sequences can be incorporated into viral or non-viral vectors, allowing their administration in vivo. The vector can be a plasmid, the production of which is known to a person skilled in the art. 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 or of the corresponding mRNAs, which can be used in the context of gene therapy. Such probes can be used in vitro as a diagnostic tool, for the detection of presenilins, 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.).

 Another object of the invention resides in antibodies or fragment of polyclonal or monoclonal antibodies directed against a polypeptide as defined above. Such antibodies can be generated by methods known to those skilled in the art. In particular, these antibodies can be prepared by immunization of an animal against a polypeptide whose sequence is chosen from the sequences SEQ ID N "1 or SEQ ID N" 2 or any fragment or derivative thereof, followed by removal of the blood and isolation of antibodies. These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art. The antibodies or antibody fragment according to the invention can in particular be used to inhibit and / or reveal the interaction between the presenilins and the polypeptides as defined above.

 Another object of the present invention relates to a method of identifying compounds capable of modulating or totally or partially inhibiting the interaction between the presenilins and the polypeptides according to the invention.

The demonstration and / or isolation of modulators or ligands of the polypeptides according to the invention is carried out according to the following steps
- a molecule or a mixture containing different molecules, possibly unidentified, is brought into contact with a recombinant cell as described above expressing a polypeptide of the invention under conditions allowing the interaction between said polypeptide and said molecule in the case where it has an affinity for said polypeptide, and,
- Molecules linked to said polypeptide of the invention are detected and / or isolated.

 In a particular embodiment, this method of the invention is suitable for the detection and / or isolation of agonists and antagonists of the interaction between the presenilins and the polypeptides of the invention.

 Another object of the invention relates to the use of a ligand or of a modulator identified and / or obtained according to the process described above as a medicament.

Such ligands or modulators can indeed make it possible to treat certain neurological conditions.

 The subject of the invention is also any pharmaceutical composition comprising as active principle at least one polypeptide as defined above.

 It also relates to any pharmaceutical composition comprising as active principle at least one antibody and / or an antibody fragment as defined above, and / or an anti sense oligonucleotide, as well as any pharmaceutical composition comprising as active principle at least one sequence nucleotide as defined above.

 Furthermore, it also relates to pharmaceutical compositions in which the peptides, antibodies and nucleotide sequences defined above or fragments thereof are associated with each other or with other active ingredients.

 It also relates to the compositions in which the nucleotide sequences according to the invention are incorporated into a viral or non-viral recombinant vector.

 The pharmaceutical compositions according to the invention can be used to modulate the activity of presenilins. More preferably, these compositions are intended to slow down or at least partially inhibit the activity of presenilins and in particular the mutated forms. More preferably, these are pharmaceutical compositions intended for the treatment of neurodegenerative diseases such as, for example, Alzheimer's disease and trisomy 21.

List of Figures
Figure 1. Diagram of the presenilin I and of the part used for the double hybrid test.

Figure 2. Isolation of yBM-C and yBM-D strains by yeast auxotrophy test carrying different plasmids containing in particular clone C (corresponding to SEQ
ID N "1) and clone D (corresponding to SEQ ID N 2)
Figure 3. Expression of mRNAs corresponding to peptides C and D in human tissue
Figure 4. Diagram of the mammalian expression vector used.

Figure 5. Expression of C and D fusion proteins in mammalian cells.

Figure 6 (a, b, c). Main vector maps
MATERIALS AND METHODS
1) The yeast strains used are the following: S. cerewsîae YCM 79 (MA Ta, ade2, type, leu2, Iys2, ura3, his3, gal4, gal80, met16 :: GALl / 1O-URA3 lacZ, HlS3 :: GAL7 -phleoR).

S.cerevisiae PCY 2 (MA Ta, Dgal4, Dgal80, ade2, trp1, leu2, lys2, hîs3 URA3 ::
GAL1 / 10-lacZ.

S.cerevisiae HF7 C (MATa, ura3, his2, ade2, trp1, leu2, lys2, gal4, gal80, canR,
URA3 :: GAL4 hinding site- CYCI-lacZ, LYS2 :: GALI- HIS3).

S.cerevisae L 40 (MATa, his3, ade2, trpl, leu2, lys2, URA3 :: LexA-lacZ, LYS2
LexA-HIS3).

2) The strains of bacteria used are the following
E.coli TGI (Fl traD 36, lacl q, lacZ D M15, proA + B + J, supE, thi-1, D (lac-pro AB), D (hsdM-mcrB) 5 rK-mK-McrB-) .

E.coli BL21 (DE3) (F-, ompT, hsdSB (rB-mB-), gal, dcm, (DE3).

3) The culture media used for the various strains are the following: complete YPD medium for yeasts:
-Bacto-yeast extract (10 g / l) (Difco)
-Bacto-peptone (20 g / l) (Difco)
-Glucose (20 g / l) (Merk)
This medium can be solidified by adding agar (20 g / l) and made sterile at 110 ° C. for 20 min in an autoclave. minimum YNB medium for yeasts:
-Bacto-yeast nitrogen base without amino acids (6.7 g / l) (Difco)
-Glucose (20g / l) (Merk)
This medium can be solidified by adding agar (20g / l) and sterilized by autoclaving.

The amino acids or nitrogen bases necessary for the growth of auxotrophic yeasts are then added at 50 mg / l. Ampicillin is also added at 10011g / ml to avoid bacterial contamination. LBcomplete medium for bacteria:
-Bacto-yeast extract (5g / l) (Difco)
-Bacto-tryptone (10 g / l) (Difco)
-NaCl (5 g / l) (Difco)
This medium can be solidified by adding agar (12 g / l) and sterilized by autoclave.

Ampicillin or kanamycin are added to 100 μg / ml to select the bacteria which have been transformed by the plasmids carrying the corresponding resistance markers.

4) The plasmids used are the following
The vector pGBT9 (fig. 6 (a)) is a 5.5 kb shuttle plasmid which can be propagated and selected in E. coli bacteria as well as in yeasts. It has an origin of ColE I replication and an ampicillin resistance gene (propagation and selection in E. coli) as well as a 2mm origin of replication and a TRP1 gene (selection in trpl-deficient yeasts for synthesis tryptophan). This plasmid is used to express in yeast fusion proteins between the DNA binding domain (DLA) of GAL4 and peptides of interest. It has a multiple cloning site downstream of the DLA region of GAL4, the whole being located between the promoter and the terminator of the ADH1 gene coding for alcohol dehydrogenase 1. The
DLA of GAL4 contains a nuclear localization signal sequence.

The sequences corresponding to different parts of PS-I were inserted at the EcoRI / SalI cloning sites.

The vector pGAD10 (fig. 6 (a)), is a 6.6 kb shuttle plasmid. It has an origin of Colt1 replication and an ampicillin resistance gene which allow its propagation and selection in E. coli. It also contains an origin of yeast replication (2mm) and a LEU2 selection gene to isolate processed yeasts. It has a multiple cloning site downstream of the sequence coding for the activation domain (DA) of GAL4 between the promoter and the ADH1 terminator. The DA of
GAL4 contains a nuclear localization signal sequence.

This plasmid was used to make a commercial human brain cDNA library (Clontech). CDNA sequences were inserted at the cloning site
EcoRI.

The vector pLex 9 (fig. 6 (b)) is a 5 kb shuttle plasmid. Like pGBT9, it has the ColEl origin, the AmpR gene, as well as the 2mm replication origin and the TRP1 gene. However, the sequence coding for the DNA binding domain is that of the bacterial repressor Lex A. The promoter and the terminator used for the expression of the fusion protein are also those of the ADH1 gene. The DLA of
Lex A contains the SV40 nuclear localization signal sequence.

The sequences corresponding to the large loop of PS-I were inserted at the EcoRI / SalI cloning sites.

The vector pGEX-4T1 (fig. 6 (b)), is a bacterial plasmid of 4.9 kb. It has an origin of replication pBR322, an ampicillin resistance gene, a lacIq gene to repress the hela-galactosidase system. This plasmid is used to express in the bacteria fission proteins between the protein glutathione S-transferase (GST) and peptides of interest. It has a multiple cloning site downstream of the GST gene to insert the sequences encoding the peptides. The whole is controlled by the tac promoter with a high level of expression inducible by IPTG. After production of the protein, the peptide of interest can be separated from the GST by cleavage with thrombin, since the sequence recognized and cleaved by this enzyme has been introduced between these two regions.

The vector pET-29a (fig. 6 (c)), is a 5.4 kb bacterial plasmid. It has an origin of replication pBR322, an origin of replication of phage f1, a kanamycin resistance gene, a lacIq gene. This plasmid is used to express in the bacterium peptides of interest fused to an S-Tag sequence (catalytic part of the protein S ribonuclease) and a His-Tag sequence (peptide of 10 histidines) which facilitate the detection and the purification of fusion protein. The T7 transcription promoter allows strong expression after induction with IPTG. A sequence recognized and cleaved by thrombin has been introduced between the S-Tag and His sequences
Tag.

5) The synthetic oligonucleotides used:
From the PS-I sequence (Sherrington et al., 1995), different pairs of oligonucleotides were defined in order to obtain the DNA fragments corresponding to the hydrophilic regions of PS-I. These oligonucleotides allow the introduction of a site
EcoRI in 5 'and a SalI site in 3' of the sequence after amplification by PCR.
oligoA CAA GAA TTC TGT CCG AAA GGT CCA CTT (SEQ ID NO 3)
oligoB CAA GTC GAC TCA GGT TGT GTT CCA GTC TCC (SEQ ID N "4)
Oligonucleotides A and B were used to obtain the BL6 and BM fragments from the large loop.
oligoC CAA GAA TTC TCA ACA ATG GTG TGG TTG (SEQ ID N "5)
oligoD CAA GTC GAC TCA TTC CTC TGG GTC TTC ACC (SEQ ID N "6)
Oligonucleotides C and D were used to obtain the BR fragment from a shorter region of the large loop.
oligoE CAA GAA TTC ACA TTA TTA CTC CTT GCC (SEQ ID NO 7)
oligoF CAA GTC GAC TCA GAT ATA AAA TTG ATG CAA (SEQ ID N "8)
The oligonucleotides E and F were used to obtain the Ct fragment, derived from the Cminminal part of PSI.
oligoG CAA GAA TTC ATG ACA GAG TTA CCT GCA (SEQ ID N "9)
oligoH AA GTC GAC TCA ATG CTT GGC GCC ATA TTT (SEQ ID N "10)
The oligonucleotides G and H were used to obtain the Nt fragment, originating from the Nterminal part of PSI.

 Oligonucleotides I, J and K were used to amplify and sequence the inserts of the plasmids pGBT9 and pGAD10. They are, respectively, complementary to the sequences of the DLA of GAL4, of the DA of GAL4 and of the ADH1 terminator. <Mixture is covered with 2 drops of paraffin oil to limit the evaporation of the sample.

The program used has 32 cycles: 1 cycle of denaturation of the matrix (5 min at 95 "C), 30 cycles (1 min of denaturation at 95" C, imin of hybridization of the oligonucleotides on the DNA matrix at 50 " C, 1 min elongation with Taq polymerase at 72 "C) finally 1 final elongation cycle (5 min at 72" C).

PCR can be performed directly on bacteria or yeast cultures in the exponential growth phase.

 Fluorescence sequence.

 The sequencing technique used is derived from the method of Sanger et al. (1977) and adapted for fluorescence sequencing developed by Applied Biosystems. The protocol used is that described by the designers of the system (Perkin Elmer, 1995).

Insertion of a DNA fragment into a plasmid by ligation.

The insert coming from a plasmid DNA or from a PCR reaction and the vector plasmid are digested for 1 hour by restriction enzymes with 1 U / llg of DNA in their respective buffer (Biolabs). The enzymes are chosen according to the cloning sites to introduce the insert in phase with the other domains of the vector.

The fragments from the digestion are separated according to their size by electrophoresis on a 0.8% "preparative" agarose gel made in TBE (Tris 90 mM, 90 mM borate, 2 mM EDTA, pH 8) with 0.5 Rg / ml BET. Deposition blue (1/10 volume) is added to the samples before they are loaded onto the gel next to a molecular weight marker (1 Kb ladder, Gibco BRL). Migration takes place at constant 90 volts / cm in TBE
The DNA is revealed under UV by the fluorescence of the BET which is interposed between the bases. The gel pieces containing the DNA fragments of desired sizes (vector and insert) are cut with a scalpel, introduced into a dialysis rod with TBE and subjected to electrophoresis for 15 min at 100 volts. The DNA extracted from the gel is then recovered, treated with one volume of phenol / chloroform / isoamylate (25/24/1), precipitated with 3 volumes of absolute ethanol in the presence of 0.25 M NaCl, washed once with 1 ethanol 70%, dried and finally taken up in 20 μl of H2O.

 After having estimated the respective amounts of vector and insert on the gel, the latter are mixed in a ratio of 1/1 in the presence of 40 IU of T4 DNA ligase, of the final 1X ligation buffer (50 mM TrisHCI, pH 7, 5, 10 mM MgCl 2, 10 mM DTT, 1 mM ATP) in a total volume of 20 1. The ligation is carried out at 20 ° C. for at least one hour.

Transformation of bacteria with a plasmid.

1) The so-called "TSB" method (after Chung and Miller, N.A.R. vol 16, 1988) consists in weakening the wall of bacteria by treatment with DMSO to allow the entry of DNA into cells. Its efficiency is 106 transformants / g of DNA.

The bacteria are cultivated in a liquid LB medium for approximately 3 hours with stirring (up to Aoo = 0.6). The culture is centrifuged for 10 min at 3000 rpm, the pellet is taken up in 1/10 of the initial volume with TSB (LB medium, PEG4000 10%, DMSO 5%,
10 mM MgC12, 10 mM MgSO4), and incubated for 15 min at 4 C. About 50 ng of DNA (10, 1 for the ligation product) are added to 100, 1 of competent bacteria, then the mixture is incubated for 30 min. at 4 C. After addition of TSBglu (TSB, 20 mM glucose) and incubation at 37 C for 45 min, the bacteria are spread on LB media with the antibiotic corresponding to the resistance gene carried by the plasmid.

2) Electroporation is a technique 1000 times more efficient than the TSB method, it is used when very small quantities of DNA are available (as after the extraction of the plasmids contained in yeasts). It consists in weakening the bacterial wall by an electric shock to introduce the DNA into the cells.

The bacteria are cultivated in a liquid LB medium with stirring up to A600 = 0.6.

The culture, incubated beforehand 15 min at 4 ° C., is centrifuged for 10 min at 3000 rpm and the pellet is washed successively with I volume of ice-cold H2O, 1/2 volume of ice-cold H2O, and 1/5 volume of 10% glycerol. ice. Between each wash, the bacteria are centrifuged 10 min at 3000 rpm to finally be taken up in 1/1000 volume of ice-cold 10% glycerol. 40 μl of this suspension of bacteria is mixed with 1 l of DNA in an electroporation tank. The tanks are placed in the Gene Pulser
BioRad for bacteria to undergo an electric shock (25 mF, 2.5kV, 200 W)
After incubation at 37 ° C. for 45 min, the bacteria are spread on LB medium containing the antibiotic used for selection.

Hybridization of mRNAs after transfer to a membrane (or Northern Blot).

The membranes used are Clontech commercial nitrocellulose membranes where the mRNAs from different human tissues have already been transferred and fixed.

The radioactive DNA probe is prepared according to the Rediprime DNA protocol
Amersham Labeling System. This protocol makes it possible to obtain an incorporation rate of [32p] dCTP (50 mCi) greater than 55% in the DNA fragment after an incubation of 10 min at 37 ° C. and to detect an RNA band equivalent to 0 , 5 μg after a 2 hour hybridization and one night of autoradiography.

The membranes are prehybridized with 8 ml of hybridization solution (NaCl IM,
NaH2PO4 10 mM pH 7.4, Ficoll 10 mg / ml, polyvinylpyrrolidone 10 mg / ml, BSA 10 mg / ml, salmon sperm DNA 100 mg / ml, SDS 2%) approximately 2 hours at 42 OC with stirring, then they are hybridized with the probe at 2.106 cpm / ml mixed with 8 ml of hybridization solution for 24 hours at 42 OC with stirring, they are then rinsed several times with washing solution 1 (0.3 M NaCl, sodium citrate 30 mM pH 7, SD5 0.05%), washed for 40 min in this same solution, incubated 40 min at 50 ° C. in washing solution 2 (15 mM NaCl, 1.5 mM sodium citrate pH 7, SDS 0, 1%) and finally they are subjected to autoradiography.

 7) Methods for the double hybrid system.

Principle of the system.

The double hybrid system, developed by Song and Fields since 1989, is a technique of cloning by interaction in vivo, in the yeast Saccharomyces cerevisiae.

It is based on the fact that certain eukaryotic transactivators require only two domains to function: a DNA binding domain (DLA) which binds to a specific sequence and an activation domain (DA) which recruits the transcription machinery. (RNA polII). When the two domains are separated, the DA cannot be positioned correctly and transcription is not induced.

In this system, DLA is fused to protein X and DA to protein
Y. If proteins X and Y interact, a functional transactivating complex is formed and induces the expression of a reporter gene placed downstream of the sequence recognized by the DLA. Expression of the gene indirectly reveals the interaction between the two proteins.

Transformation of yeast with one or two different plasmids.

The yeasts are made competent by treatment with LiAc / PEG according to the method described by Gietz (Gietz et al., 1995).

The yeasts are cultured on YPD solid medium at 28 ° C. overnight. They are then resuspended in 1 ml of sterile solution I (0.1 M LiAc, 10 mM TrisHCl,
EDTA ImM pH7.5), centrifuged for 1 min at 3000 rpm and taken up again in 1 ml of solution I. 50 lul of the suspension are then mixed with 5 g of DNA from sperm of salmon (trainer DNA), 1 to 5 g. of plasmid DNA and 300 Cll of sterile solution II (0.1 M LiAc, 10 mM TrisHCl, 1 mM EDTA pH 7.5, PEG0 50%). The mixture is incubated for 30 min at 28 ° C. and then undergoes a thermal shock for 20 min at 42 ° C. After centrifugation for 1 min at 3000 rpm, the cells are washed once with sterile water and taken up in 100 μl of sterile water. Then, the yeasts are spread on YNB agar medium supplemented with amino acids and nitrogen bases necessary for their growth. To allow the selection of transformed yeast strains, the amino acids and nitrogen bases corresponding to the selection markers carried by the plasmids are not not added. The boxes are placed in the oven at 28 ° C. for 3 days.

Transformation of yeast by a cDNA library.

The cDNA bank used is a commercial bank (Clontech) constituted from mRNA of a normal human brain (37-year-old man who died following a brain trauma). The cDNAs were cloned into the plasmid pGAD10 downstream of the DA region of GAL4 at the EcoRI site. The average size of the inserts is 1.4 kb with a standard deviation of 1 kb. The library was amplified once in E.coli DH5a to obtain 1.2x106 independent clones, 80% of which contain an insert.

To preserve a good representativeness of the bank, it is necessary to have a number of transformants two to ten times greater than that of the independent clones obtained during the construction of the bank. We therefore use a protocol which makes it possible to obtain good transformation efficiency (approximately 107 transformants / 100 g of DNA).

The yeasts YCM79 previously transformed with the plasmid pGBT9 containing a sequence corresponding to a region of PS-I are cultivated overnight in 50 ml of YNB + His + Lys + Ade + Met + Leu + Ura medium at 28 OC with stirring. The optical density of this preculture is measured to evaluate the number of cells per ml and allow the calculation of the volume to be inoculated in 250 ml of YPD medium in order to obtain a culture of 107 cells / ml (approximately A600 = 0.8) after one night at 28 OC with stirring.

The cells are then harvested by centrifugation at 3000 rpm for 10 min, washed twice with 100 ml of sterile H2O, taken up in 10 ml of transformation solution I. The suspension is centrifuged for 10 min at 3000 rpm and the cells resuspended in 2.5 ml of this same solution. Part of the suspension is taken (200 l): half will serve as an untransformed negative control, the other half will be transformed by a control plasmid whose transformation efficiency is known. The remaining suspension is mixed with 100 μl of Img / ml cDNA plasmid bank and 20 ml of solution II. The mixture is incubated for 1 hour at 28 ° C. with shaking and then 20 min at 42 ° C. before being centrifuged for 15 min at 3000 rpm. The cell pellet is washed once with water and twice with PBS in order to remove the PEG which has a toxic effect on yeasts, then is taken up in 2.5 ml of sterile PBS (50 mM Na2HPO4,
10 mM KCI, 1 mM MgSO4, pH 7).

250 ml of YNB + His + Lys + Ade + Met + Ura medium are seeded with this suspension and incubated for 4 hours at 28 ° C. with shaking to allow the expression of the hybrid proteins, their possible interaction and the expression of the reporter gene URZ43. However, this step has the effect of increasing the number of positive clones. To evaluate the rate of amplification of the number of colonies, 1 ml of culture is taken before and after this incubation and different dilutions are spread on agar medium YNB + His + Lys + Ade + Met + Ura. After growth, the number of Leu + Trp + clones is determined to evaluate the transformation frequency before and after the incubation period and the amplification rate is deduced therefrom.

 The cells are then centrifuged for 10 min at 3000 rpm, washed twice with sterile water, taken up in 10 ml of NYNB and spread on 10 boxes of 435 cm 2 each containing 200 ml of selective medium YNB + His + Lys + Ade + Met. The boxes are put in the oven at 28 "C for 3 days.

Extraction of plasmid DNA from yeasts.

This technique extracts both plasmids and genomic DNA contained in yeasts. To isolate the plasmids, it suffices to transform bacteria where only the plasmid DNA can replicate and to analyze each clone after mini-preparation
.The yeasts containing the plasmids are cultured on selective YNB solid medium at 28 "C overnight. They are then resuspended in 200 ml of breaking solution (Triton X100 2%, SDS 1%, NaCl 100 mM, Tris 10 mM pH 8, 1 mM EDTA) in the presence of glass beads (450 μm in diameter) and 200 ml of phenol / chloroform, the mixture is stirred for 15 min on a vortex and then centrifuged for 2 min at 14,000 rpm. The aqueous phase is dialyzed for 1 hour. This solution can be used for the transformation of bacteria by electroporation.

Revelation of beta-galactosidase activity.

This test makes it possible to verify the expression of the LacZ reporter gene coding for ss-galactosidase in transformed yeasts.

A nitrocellulose sheet is deposited on the individualized yeast clones. The replica is immersed 3 times for 30 seconds in liquid nitrogen to break the yeasts and allow access of the substrate to beta-galactosidase. The sheet is then placed, colonies upwards, on Whatman paper soaked in a solution of PBSlXgal prepared extemporaneously (100, u1 of a solution of Xgal substrate at 40 mg / ml in dimethylformamide in 5 ml of PBS) and then placed at 37 OC, optimal temperature for the enzymatic activity tested.

The time of appearance of the blue color which reveals the 13-gay activity is very variable, from a few minutes to several hours. The test is carried out in the presence of a positive interaction control which quickly turns blue and a negative control which remains white.

Drop test.

This test makes it possible to analyze the phenotype of a yeast strain. A drop (approximately 5 ml) of a slightly cloudy yeast suspension is deposited on various selective and non-selective agar media, and the growth of the yeasts is observed after 2 days of incubation at 28 "C.

Plasmid segregation.

This involves testing by plasmid segregation whether the phenotype of the transformed yeasts is indeed linked to the presence of the plasmids.

The yeasts are cultivated overnight on complete YPD medium at 28 "C. This non-selective medium allows the growth of yeasts conserving or not conserving the plasmids after cell division. Different dilutions of this culture are spread out to obtain between 50 and 100 clones per box of YPD medium after overnight incubation at 28 "C. The phenotype of the clones is then tested by replicas on velvet on different selective media and the frequency of cells not containing plasmid can be estimated after 2 days of incubation at 28 "C.

 8) Methods for the in vitro interaction test.

Principle of the test.

This involves verifying the interaction between two proteins using a biochemical technique of affinity chromatography. A cellular extract containing one of the supposed partners of the protein is added to a support where the bait protein has been previously fixed. If the interaction exists between the protein and its possible partner, this one too, will be retained on the support.

 Expression of fusion proteins in Ecoli.

The fusion proteins are produced in E. coli BL21 (DE3) via the expression vectors pET and pGEX inducible by IPTG (Studier, 1991).

The BL2 1 bacteria (DE3), previously transformed by the recombinant plasmids, are grown overnight with stirring in LB medium with the antibiotic used for selection (ampicillin for pGEX or kanamycin for pET at 100 μg / ml). 5 ml of this preculture are inoculated in 45 ml of LB + antibiotic medium in order to obtain a culture at Ao = 0.6 after 1 hour of agitation at 37 ° C. Protein production is then induced by the addition of 0.1 mM IPTG and the bacteria are returned to 37 ° C. with stirring between 1 hour and 4 hours. The cells expressing the proteins are harvested by centrifugation at 6000 rpm for 5 min. The pellets can be kept at -20 C.

Extraction and analysis of proteins produced in the bacteria.

The bacterial pellets containing the fusion proteins are resuspended in 5 ml of
TE (50 mM TrisHCI pH 8, 2 mM EDTA). The cells are lysed with lysozyme 0.1 mg / ml final and 1% Triton X-100. They are incubated for 15 min at 30 ° C. before being placed in ice to be subjected to ultrasound until the bacterial suspension becomes fluid. The bacterial lysate is centrifuged for 15 min at 10,000 rpm at 4 C. The supernatant containing the solubilized fusion protein can be stored at -20 C.

At this stage, the various fractions obtained are analyzed by denaturing gel electrophoresis (SDS PAGE). 60 μl of the bacterial suspension before centrifugation (total fraction), 60 μl of supernatant (soluble fraction) as well as 60 μl of the residue resuspended in 5 ml of TE (insoluble fraction) are removed. The proteins contained in the various fractions are denatured for 10 min at 95 ° C. in the presence of 1/4 volume of 4xL deposition blue (0.2 M TrisHCI pH 6.8, 5% SDS, 5% glycerol, 0.5 mercaptoethanol %, bromophenol blue) and loaded onto a 12% SDS polyacrylamide gel where they are separated according to their molecular mass by electrophoresis. After migration at 125 volts / cm, the proteins are detected by staining the gel with Coomassie blue.

Solubilization of proteins.

When an exogenous gene is expressed in E. coli, aggregates of the exogenous protein may appear, forming inclusion bodies. The recombinant protein is then easy to purify by centrifugation but it is often difficult to dissolve.

The proteins found in the insoluble fractions are denatured by urea 6
M and 10 mM DTT for 10 min at 37 ° C. and then renatured by dialysis in three successive buffers for at least 6 hours each (5 mM TrisHCI pH 8/2 M urea;
5 mM TrisHCl, pH 8/1 M urea, 5 mM TrisHCI, pH 8). After 15 min of centrifugation at 10,000 rpm, approximately 5 to 10% of the protein is in the supernatant which can be stored at -20 "C.

Affinity chromatography.

For protein retention to occur under good conditions, the protein concentration of the samples must be greater than 40 mg / ml. This concentration can be estimated by a colorimetric method (Bradford reagent, for example).

200 μl of soluble fraction containing the non-fused GST or GST-BM (mutated loop of presenilin 1) are incubated for 2 min at 20 ° C. in the presence of 300 μl of glutathione resin. The fraction not retained by the beads is taken. The resin is washed 4 times with washing buffer (TrisHCl pH 8, 20 mM, 150 mM NaCl, 0.1% Triton X100) in order to remove the fixed proteins by non-specific adsorption. 150 μl of resin having bound the GST or the GST-BM are incubated for 1 hour at 4 "C. with shaking with 600 μl of soluble fraction containing STag or STag-Pepl26. The agarose beads are then centrifuged I min at 3000 rpm and washed 4 times with washing buffer after removal of the non-retained fraction.

An aliquot of each fraction (soluble, not retained and retained by the beads) is deposited on SDS PAGE gel after denaturation at 95 "C in the presence of deposition blue and separated by electrophoresis. The proteins are then transferred to the nitrocellulose membrane. then the membrane is stained with Ponceau red (20 CLg / ml) to verify that the transfer of proteins has been carried out.

In order to be able to specifically detect the proteins fused with the S-Tag, the nitrocellulose membrane is saturated with 1% of gelatin in TBST (10 mM TrisHCl pH 8, 150 mM NaCl, 0.1% Tween20) for 15 min and incubated 15 min with protein S linked to alkaline phosphatase. After 4 washes with TBST, the activity of alkaline phosphatase is revealed by the addition of a PA 1X buffer containing NBT and BCIP according to the Novagen method.

 9) Methods of expressing recombinant proteins in mammalian cells.

Construction of an expression vector in mammalian cells
For expression in mammalian cells, the sequences ID N "1 and ID NO 2 were subcloned by conventional methods in a vector under the control of a strong viral promoter (CMV).

Cell transfection
The method established for COS 1 cells or CHO cells consists in using a lipofectant in a ratio of I for 8 (weight / weight) relative to DNA and a synthetic peptide (H1) in the same ratio in order to optimize DNA compaction and transfection efficiency. This method is based in particular on the neutralization of the charges of DNA phosphates by the positive charges of the lipofectant.

COS i cells are cultured in an incubator at 37 OC, 95% humidity and 5% CO2 in DMEM medium (Dulbecco's Modified Eagle's Medium) containing 4.5 g / l glucose (Gibco-BRL) supplemented with 3% L- Glutamine, 1% penicillinestreptomycin and 10% Fetal Calf Serum.

The day before transfection, the cells are seeded at a density of 2.5 106 cells per 100 mm dish. On the day of the transfection, the cells are rinsed twice with PBS (Phosphate Buffer Saline) and once with OptiMEM (patented composition;
Gibco-BRL) for habituation of at least 15 minutes in an incubator.

Per 100 mm dish equivalent, a total of 8 Fg of plasmid DNA is added to 300 µl of OptiMEM and 64 µg of synthetic peptide (H1). After having vigorously vortexed for 10 seconds, the lipofectamine (32 μl, ie 64 μg) diluted in 300 Zl of OptiMEM is added to the preceding mixture, after 5 minutes of waiting.

The whole is again vigorously vortexed and then left to stand for 30 minutes. Five milliliters of OptiMEM are added per tube and the vortexed mixture is placed on the cells (from which the medium has previously been aspirated). The cells are then placed in an incubator for 4 hours, at the end of which the mixture is replaced by complete medium.

Cell lysis and protein assay
The cells are most often lyzed 48 hours after transfection (at the usual maximum expression). The lysis buffer contains 10 mM Tris pH 7.5, 1 mM EDTA, 1% Triton X100, 1% NP40 and a cocktail of protease inhibitors (Complete, Boehringer-Mannheim). For each plate, after rinsing with PBS, 800 μl of cold buffer is added. The lysates then undergo sonication followed by stirring with a magnetic bar at 4 "C. overnight. A centrifugation for 30 minutes at 15,000 × g separates the pellet from the supernatant.

The soluble proteins are then dosed according to the BCA kit (Pierce) in order to be able to standardize the following experiments.

Immunoblot
The samples (cell lysates) are denatured in an equal volume of deposition buffer (125 mM Tris pH 6.8, 4% m / v SDS, 20% glycerol, 0.02% Bromophenol Blue, 50 mM Dithiothreitol) at 95 "C for 5 For the analysis of the expression of the presenilins, the samples are denatured in the presence of 8 M urea and at 37 "C in order to avoid the aggregation specific to the presenilins at 95" C.

The samples are deposited on Tris-Glycine gels (Novex), with a different percentage of acrylamide depending on the molecular weight to be discriminated. A molecular weight marker is also deposited (Broad Range, BioRad). The migration takes place for approximately 2 hours at 100 constant volts in final SDS 1X buffer (Novex). The gel is then transferred to a nitrocellulose or PVDF membrane (1 X final transfer buffer (Novex) with 10% methanol) for 2 hours at 150 mA constant.

After transfer, the membrane is blocked for 2 hours at room temperature in 50 ml of PBS-T (PBS with 0.5% Tween) containing 2% skimmed milk (Merck).

The primary antibody (diluted to the optimal concentration of the order of 1 / 1000th to 1 / 5000th, in PBS-T with or without 2% skim milk) is left overnight at 4 "C. After a brief rinsing in PBS-T, the membrane is incubated for 45 minutes in the presence of the second antibody (anti-mouse or anti-rabbit Ig G, as the case may be, coupled with horseradish peroxidase) diluted with l / SOOOC in a buffer called ECL (Tris 20 mM,
150 mM NaCI, Tween 0.1%).

The membrane is then rinsed 4 times 15 minutes in the ECL buffer. It can be revealed by the ECL reagent (Amersham) consisting of 2 buffers to be mixed extemporaneously in equal volume. Various exhibitions of a photographic film (Hyperfilm ECL, Amersham) are carried out, followed by development.

EXAMPLES
Example 1: Isolation of Specific Partners of PS I by Screening of a Brain cDNA Fusion Library
In order to isolate specific partners of the PS 1 protein, a human brain cDNA library was screened by the double-hybrid technique. A cDNA library fused with the DA of GAL4 was used and different bait proteins were constructed by fusing the hydrophilic regions of PS-I with the DLA of GAL4.

1.1 Construction of the vectors allowing the expression of fusion proteins between the
DLA of GAL4 and the hydrophilic regions of PS 1.

Five fusion proteins were produced using the vector pGBT9 into which the sequences corresponding to different hydrophilic regions of PS 1 were introduced in the same reading frame as the DLA sequence of GAL4. It was chosen to identify proteins interacting with the N-terminal region (Nt, codons 1 to 81), the large loop (BL6, codons 263 to 407), the large loop with the L286V mutation (BM), a region shorter of the large loop (BR, codons 290 to 376) and finally the Cterminal part of the protein (Ct, codons 421 to 467).

The DNA fragments corresponding to these different regions were obtained by
PCR from the entire PS 1 cDNA sequence. The various oligonucleotides used allowed the introduction of the EcoRI and SalI sites at the ends of each amplified sequence. So the fr DNA sequencing. This verification showed that the fragments did not present any mutation generated by the PCR and that they were indeed in the same open reading frame as that of the DLA sequence of GAL4.

1.2 Obtaining yeast strains expressing the fusion proteins between the DLA of GAL4 and the hydrophilic regions of PS 1.

As the cotransformation of yeast by two different plasmids is a less frequent event than its transformation by a single plasmid, a yeast strain previously transformed by the vector coding for the bait protein was used, in order to have the best possible efficiency during cDNA library screening.

For this, the yeast strain YCM 79 was transformed with each of the plasmids pGBT-Nt, pGBT-BL6, pGBT-BM, pGBT-BR and pGBT-Ct. The yeasts containing these plasmids were selected for their Trp + phenotype on medium not containing tryptophan and respectively called yNt, yBL6, yBM, yBR and yCt. The presence of these different plasmids in each strain was verified by carrying out a PCR directly on the yeasts, using oligonucleotides complementary to the sequences
DLA and tADH1 located 5 'and 3' from the insert. The size of the PCR fragments obtained makes it possible to distinguish the different strains with the exception of the yBL6 and yBM strains.

1.3 Tests for transactivating activity and interaction with the DA of GAL4 of the fusion proteins containing the hydrophilic regions of PS 1.

If one of the fusion proteins has transactivating activity or if it interacts with the DA of GAL4, it cannot be used as bait in the double hybrid system because it systematically gives a positive response outside any protein-protein interaction.

The first test therefore consists in verifying that the hydrophilic regions of PS-I do not have transactivating activity when they are fused to the DLA of GAL4, that is to say capable of inducing, on their own, transcription reporter genes. This type of activity can, for example, be linked to the presence of an acid domain in the fusion protein.

For this test, the expression of the reporter genes LacZ and URA3 were directly checked on the strains yNt, yBL6, yBM, yBR and yCt.

For the second test which consists in checking the absence of interaction between the fusion proteins and the DA of GAL4, the strains yNt, yBL6, yBM, yBR and yCt of phenotype Trp + Leu- were transformed by the plasmid pGAD424 (equivalent pGAD10) which codes for the DA of GAL4. The yeasts were selected on a medium without tryptophan or leucine and the expression of the reporter genes LacZ and URA3 making it possible to control the interaction was tested on the clones obtained.

In all cases, the results of the ss-galactosidase activity tests as well as those of the drop tests on selective medium without uracil were negative.

The fusion proteins containing the hydrophilic parts of PS-I therefore do not have an intrinsic transactivating property and do not interact with the activation domain of GAL4. They can therefore be used as bait proteins for screening the bank.

1.4 Transformation of the different yeast strains by the cDNA fusion bank.

The screening of the fusion library makes it possible to identify clones expressing peptides fused to the DA of GAL4 interacting with the bait protein. If this interaction exists, it must allow the reconstitution of a functional transactivator capable of inducing the expression of the reporter genes URA3 and LacZ in the strain containing the bait protein.

For the screening to be representative, it is necessary that each independent plasmid constituting the library is present in, at least, a yeast clone after transformation. For this, a protocol theoretically making it possible to obtain a number of transformants greater than the number of plasmids independent of the bank was used (cf. materials and methods).

The 5 strains yNt, yBL6, yBM, yBR and yCt of phenotype His-, Lys-, Ade-, Met-,
Ura-, Leu-, were successively transformed with 100 μg of plasmid DNA from the fusion library and the transformed cells were selected on YNB + His + Lys + Ade + Met medium.

After the first selection, we obtained 32 clones with a phenotype
Ura + with the yNt strain, 450 Ura + clones with yBL6, 128 with yBM, 250 with yBR and 67 with yCt. An ss-galactosidase activity test was carried out on these transformants in order to verify the expression of the second LacZ reporter gene. A total of 6 clones exhibited the double phenotype Ura +, equal +: I clone for the strain yNt, 1 clone for yBL6, 2 clones for each of the strains yBM and yBR, no clone for yCt.

The clones are respectively named yNt.A, yBL6.B, yBM.C, yBM.D.

EXAMPLE 2 Isolation of the plasmids from the library.

In order to identify the proteins which can interact with the mutated loop or the reduced loop of PS 1, the plasmids contained in the yeasts yBM.C and yBM.D were extracted. The plasmids present in DNA preparations have been used to transform bacteria by electroporation.

The plasmids, extracted from the bacterial clones obtained, were digested with different enzymes. This analysis revealed 5 different restriction profiles.

The digests of the plasmids extracted from the yBM.C and yBM.D yeasts have a common profile corresponding to the plasmid pGBT-BM and two different profiles corresponding to the plasmid pGAD10 containing an insert of 2900bp corresponding to the sequence ID N "1 (pGAD-C being from the strain yBM.C) and an insert of 1400 bp corresponding to the sequence ID N "2 (pGAD-D being from the strain yBM.D).

2.1 Control of the plasmids isolated by transformation of different strains of yeast.

During the transformation of yeasts by plasmids from the brain bank, it is possible that several different plasmids are introduced into the same cell.

In order to verify that the interaction results are due to the presence of the isolated plasmids and not to supernumerary plasmids, we again tested the fusion proteins resulting from the screening of the library against the various hydrophilic regions of
PS-I in the YCM 79 strain but also in the HF7 C and PCY 2 strains.

HF7 C has two reporter genes HIS3 and LacZ The LacZ gene has a different promoter environment than that existing in the YCM 79 strain, which makes it possible to control that obtaining equal + clones is not linked to a specific promoter environment but rather to an interaction between the proteins tested. PCY 2 has the advantage of having only one LacZ reporter gene which is expressed more strongly than in the other two strains when the GAL4 transactivator is functional.

We transformed the three yeast strains YCM 79, HF7 C and PCY 2 with the different pairs of plasmids pGBT-X / pGAD-Y (X corresponding to Nt, BL6, BM,
BR or Ct; Y corresponding to C or D), pGBT9 / pGAD424 (negative interaction control) and pGBT-CtAPP / pGAD-Fe65 (positive interaction control) (Russo et al., 1995). We tested auxotrophy and equal activity on 3 to 5 clones from each transformation. The results of the boxes are presented in Figure 2.

Given the difference in sensitivity of the tests, the interaction is confirmed when the transformed YCM 79 and HF7 C yeasts develop on selective media without uracil or histidine, that is to say that they express, in a safe manner, at least one of their reporter gene.

The intensity of the response (more or less blue, more or less developed colonies) is assumed to be proportional to the rate of expression of the reporter genes. The variations in this rate can be explained in different ways. A low level can result from a weak interaction between the proteins reconstituting the transactivator
GAL4. It may also be due to the intervention of a third protein which would destabilize the formation of the complex, or else to an instability of the fusion proteins in the yeast, or else to the state of the yeasts. The yeasts may be in a state of low transcriptional activity linked, for example, to a problem of toxicity of exogenous proteins.

The brain fusion proteins that have been isolated do not interact with the N-terminal part and with the C-terminal of PS 1. On the other hand, they all seem to interact with the hydrophilic region BL6, the hydrophilic region BL6 mutated (BM), as well as the reduced form of this region (BR).

2.2 Interaction tests of the fusion proteins of the library with proteins not containing the PS 1 loop.

This test makes it possible to determine whether the fusion proteins originating from the library interact in a non-specific manner by their structure, their charge, their hydrophobicity with the bait proteins (false positives of type III) or with the DLA of
GAL4 (false positive type II).

We transformed the YCM 79, HF7 C and PCY 2 strains with the plasmid pairs: pGBT9 / pGAD-Y (Y corresponding to C or D), pGBT-LAM / pGAD-Y, pGBT9 / pGAD424 (negative control) and pGBT -CtAPP / pGAD-Fe65 (positive control). The pGBT9 codes only for the DLA of GAL4 while the pGBT-LAM provided by
Clontech codes for a fusion protein between the DLA of GAL4 and the lamin which interacts in a non-specific manner with the hydrophobic regions of other proteins.

 As a result, none of the yeasts thus transformed were capable of expressing their reporter genes. Thus, the YCM 79 yeasts had the Ura-, ss-gal- phenotype, the HF7 C yeasts were all His-, ss-gal- and the PCY 2 yeasts, ss-gal-.

These results suggest that the library fusion proteins thus isolated, interact specifically with the hydrophilic region BL6 of PS-I and do not bind with the lamin or the DLA of GAL4.

2.3 Interaction test on yeasts transformed by the vector pLex 9 containing the region coding for the loop of PS 1.

This test makes it possible to ensure that the interaction of the proteins is not due to a structure adopted by the different fragments of BL6 only when they are fused with the DLA of GAL4.

To verify this, the BL6 and BM fragments were fused to another DLA, that of the bacterial repressor LexA. Two additional plasmid constructs (pLex-BS and pLex-BM) were performed, where the sequences of the hydrophilic loop BL6 and the mutated form were fused to the LexA gene at the site level
EcoRI / SalI of the plasmid pLex9.

Then the yeast strain L40 in which the expression of the reporter genes HIS3 and LacZ is induced by the fixation of the DLA-LexA / DA-GAL4 complex on the operator site of LexA has been transformed. The interaction with the pairs of plasmids was tested pLex-BL6 / pGAD-Y (Y for C or D), pLex-BM / pGAD-Y, pLex
RAS / pGAD-RAF (positive interaction control) (Vojtek et al., 1993) and pLex
RAS / pGAD424, pLex-BL6 / pGAD424, pLex-BL6 / pGAD-RAF, pLex-BM / pGAD424, pLex-BM / pGAD-RAF (negative interaction controls).

The results of the drop tests on selective media without histidine and of the tests of equal activity were positive for the strains containing the plasmid pLex.
BL6 whatever the plasmid pGAD-Y tested. The same is true for strains containing pLex-BM whatever the plasmid pGAD-Y tested. This test shows that the interaction obtained is not linked to a particular conformation that the BL6 region of PS-I would adopt when it is fused with the DNA binding domain of GAL4.

EXAMPLE 3 Identification of the inserts of the plasmids resulting from the screening.

3.1 Sequence of the inserts of the plasmids pGAD-C and pGAD-D.

The inserts of the plasmids pGAD-C and pGAD-D were completely sequenced. We started sequencing using the complementary oligonucleotides of
DA of GAL4 and tADHl in order to know the sequence of the beginning and the end of each insert. Then, starting from the end of each sequence, we constituted synthetic oligonucleotides which allowed us to advance in the sequences on the two strands. In this way, we completely sequenced the 1400 bp of the insert
D corresponding to the sequence SEQ ID N "2. On the other hand, for insert C of 2900 bp (sequence SEQ ID N" 1), it was necessary to establish a restriction map and then to carry out a subcloning of different fragments in order to obtain the entire sequence.

Insert D has an open reading phase over its entire sequence (Seq ID N "2). On the other hand, insert C contains a stop codon at the end of sequence (Seq ID
N01).

Their peptide sequence has a rather hydrophilic profile which is in accordance with the choice of the screening method which is more applicable to hydrophilic proteins.

3.2 Comparison of sequences
This comparison should make it possible to know whether the inserts encode a peptide domain whose function is known.

The nucleic acid and peptide sequences of the inserts were compared with sequences listed in the Zen Bank and EMBL (European Molecular Biology Lab) databases. No significant similarity was found between the inserts and the genes (or proteins) stored in these libraries.

The sequences of the inserts were also compared with each other. They have no similarity.

In addition, the existence of peptide motifs capable of providing information on the function of the peptides encoded by the inserts has been sought. Some potential sites for glycosylation, phosphorylation and myristylation have been found but which do not allow hypothesis as to a possible function of the peptides.

3.3 Expression of mRNAs corresponding to the peptides selected in different human tissues.

In order to reinforce the idea that the interaction between these peptides and PS 1 does indeed exist in vivo, it has been versified that the expression of the mRNAs of the peptides isolated from the library is located in the same tissues as the expression of the PS 1 mRNA. Expression in specific tissues, genes containing the isolated sequences was sought and the level of expression of mRNAs corresponding to insert C and D in different human tissues was analyzed by Northern blot.

The radioactive probe prepared from insert C (sequence ID N "1) made it possible to hybridize a mRNA of 9.5 kb expressed specifically in the brain and more particularly in the cerebral cortex, the frontal lobe and the temporal lobe ( see Fig3).

The size of this mRNA suggests that the translated protein is large.

EXAMPLE 4 In vitro interaction test.

This test makes it possible to know that the interactions are not dependent on the yeast context. The reconstitution of the functional transactivator GAL4 is not always due to a direct interaction between the two proteins, but can also be the result of the formation of a protein complex between the fusion proteins and one or more endogenous yeast proteins. Thus, interactions would only exist in the presence of intermediate proteins from yeast and would have no reality outside this context.

It was chosen to confirm the interactions between the hydrophilic region BL6 of PS I and the isolated peptides respectively called PepC and PepD in an acellular system.

The proteins are produced in E. coli BL21 (DE3) in order to obtain a large quantity thereof. The 2 isolated peptides have been fused to tag peptides allowing their detection and their fixation on supports. For this, it was chosen to merge the GST protein, which binds to glutathione, to the various forms of the BL6 region and the S-Tag peptide, recognized specifically by the S protein, to the isolated peptides (cf. material and method for the principle of the test).

4.1 Production of a fission protein between the S-Tag and the peptides isolated during the screening of the cDNA library.

* In order to build the fusion proteins between the S-Tag and the two peptides PepC and
PepD. The different EcoR // EcoRI fragments from the plasmids pGAD-C and pGAD
D were inserted in the same reading frame as the S-Tag sequence of the vector pET-29a.

At this stage, a plasmid (pET-D) was obtained containing the insert corresponding to the
PepD. The partial sequencing of this plasmid showed that the insertion was carried out in the desired reading phase.

This plasmid made it possible to obtain a 52kDa protein corresponding to the STag-PepD fusion protein. The STag-PepD protein is produced in BL21 (DE3) bacteria after induction with IPTG.

4.2 Production of a fusion protein between the GST and the hydrophilic loop of PS 1.

To merge GST with one of the three forms of the BL6 hydrophilic region of PS 1, the vector pGEX-4T1 was used in which the EcoRI / SalI fragments originating from the plasmids pGBT-BL6, pGBT-BM and pGBT-BR were used. been introduced.

The plasmid pGEX-BM corresponding to the mutated loop of PS-I was obtained. The sequencing showed that the insertion was indeed carried out in the context of reading the GST sequence.

This plasmid enabled the production of the GST-BM 42kDa fusion protein in BL21 (DE3) bacteria after induction with IPTG.

4.3 Solubilization of the GST-BM fusion protein.

After the lysis of the bacteria, the GST-BM protein remains entirely in the insoluble fraction while the STag peptide as well as the STag-PepD protein and the GST are partly found in the soluble fraction.

The solubilization of the fusion proteins is absolutely necessary for carrying out the affinity chromatography. First, the GST-BM protein was specifically fixed on a glutathione resin covalently coupled to agarose beads in order to then be able to test the interaction of GST-BM with the peptide partners of PS 1 merged with STag (cf. Materials and methods). If the proteins are not dissolved, they sediment with the beads in the absence of any specific bond with them.

After solubilization of the GST-BM fusion protein, the test is carried out according to the conditions described in paragraph 8 of the materials and methods section.

EXAMPLE 5 Expression of the specific partners of PS-I in mammalian cells.

5.1 Construction of appropriate expression vectors.

This test makes it possible to verify whether the protein interactions revealed in yeast are also detectable in the context of mammalian cells and with the complete PS1 protein inserted into its membrane environment. The sequences SEQ ID N 1 and SEQ ID
N02, partners, were subcloned into a pCDNA3 mammalian cell expression vector carrying an additional sequence corresponding to the 5'terminal myc epitope and an EcoRI restriction site (FIG. 4) allowing the phase subcloning of the sequences partners.

The 1400 bp pGAD-D EcoRI restriction fragment was isolated and phase-subcloned into the EcoRI site of the expression vector. The constructs carrying the cDNA in the correct orientation were chosen by enzyme restriction analysis.

With regard to clone C, the EcoRI (I) -HindIII (391) fragment of pGAD-C was first isolated. A second HindIII (392) -MscI (2892) fragment (the latter blunt-ended site located within the vector pGAD10 downstream of the coding region of clone C) was then isolated by partial digestion. EcoRI fragments (1)
HindIII (391) and HindIII (392) -MscI (2892) were ligated together with the expression vector digested with EcoRI and EcoRV, the latter site being blunt-ended.

The correct constructs carrying the entire coding sequence of C were identified by restriction analysis.

These constructions make it possible to generate fusion proteins carrying the myc epitope and the peptides corresponding respectively to the sequences ID N "I and ID N" 2.

5.2 Expression in mammalian cells
These constructs were transfected into COS1 cells by standard methods.

After cell lysis, the samples are analyzed by immunoblot and revelation with the anti-myc antibody. The expression of the partners could be detected by immunoblotting using the anti-Myc antibody 9E10 (FIG. 5). We were able to detect a very intense 120 kDa band as expected for pepC, demonstrating strong expression (lane C). For D, the intensity of the band detected was not always constant. On the other hand, the expression in CHO cells could be detected. In this cell type, D is perhaps a little more stable and appears as a protein migrating towards 80 kDa (lane D).

The two proteins could therefore be expressed and detected in mammalian cells.

LIST OF SEQUENCES
(1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: RHONE-POULENC RORER
(B) STREET: 20 avenue Raymon Aron
(C) CITY: Antony
(E) COUNTRY: France
(F) POSTAL CODE: 92165
(G) TELEPHONE: 01.55.71.69.22
<H) FAX: 01.55.71.72.91
(ii) TITLE OF THE INVENTION: Nucleic acids encoding for
proteins capable of interacting with presenilins.

(iii) NUMBER OF SEQUENCES: 2
(iv) COMPUTER-DETACHABLE FORM:
(A) TYPE OF SUPPORT: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release &num; 1.0, Version &num; 1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF SEQUENCE 1:
(A) LENGTH: 2892 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETIC: NO
(iv) ANTI-SENSE: NO
(ix) CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..2736
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
CGG ATG ATT CCC ATC TTT CAT GAC ATG ATG GAC TGG GAG CAG AGA AAA 48
Arg Met Ile Pro Ile Phe His Asp Met Met Asp Trp Glu Gln Arg Lys
15 10 15
AAT GGC AAC TTC AAA CAG GTG GAG GCC GAG TTG ATT GAC AAG CTG GAC 96
Asn Gly Asn Phe Lys Gln Val Glu Ala Glu Leu Ile Asp Lys Leu Asp
20 25 30
AGC ATG GTG TCA GAA GGG AAA GGT GAC GAG AGC TAC AGG GAG CTC TTC 144
Ser Met Val Ser Glu Gly Lys Gly Asp Glu Ser Tyr Arg Glu Leu Phe
35 40 45
AGC CTA CTA ACC CAG CTG TTT GGG CCC TAC CCC AGC CTG CTG GAG AAG 192
Ser Leu Leu Thr Gln Leu Phe Gly Pro Tyr Pro Ser Leu Leu Glu Lys
50 55 60
GTT GAA CAA GAA ACA TGG CGC GAG ACC GGC ATT TCC TTT GTG ACC TCA 240
Val Glu Gln Glu Thr Trp Arg Glu Thr Gly Ile Ser Phe Val Thr Ser
65 70 75 80
GTC ACC CGC CTC ATG GAA CGT CTT CTT GAC TAC AGG GAC TGC ATG AAA 288
Val Thr Arg Leu Met Glu Arg Leu Leu Asp Tyr Arg Asp Cys Met Lys
85 90 95
GGA GAG GAA ACA GAG AAT AAG AAG ATA GGC TGC ACT GTT AAC CTG ATG 336
Gly Glu Glu Thr Glu Asn Lys Lys Ile Gly Cys Thr Val Asn Leu Met
100 105 110
AAT TTT TAC AAA TCT GAG ATT AAC AAG GAA GAA ATG TAT ATC CGC TAC 384
Asn Phe Tyr Lys Ser Glu Ile Asn Lys Glu Glu Met Tyr Ile Arg Tyr
115 120 125
ATC CAT AAG CTT TGT GAC ATG CAC TTG CAG GCC GAA AAC TAC ACA GAG 432
Ile His Lys Leu Cys Asp Met His Leu Gln Ala Glu Asn Tyr Thr Glu
130 135 140
GCC GCA TTT ACC CTG CTC CTT TAC TGT GAG CTG CTG CAG TGG GAG GAG 480
Ala Ala Phe Thr Leu Leu Leu Tyr Cys Glu Leu Leu Gln Trp Glu Asp 145 150 155 160
CGG CCA CTA CGG GAA TTC CTC CAC TAC CCA TCG CAG ACA GAG TGG CAG 528
Arg Pro Leu Arg Glu Phe Leu His Tyr Pro Ser Gln Thr Glu Trp Gln
165 170 175
CGG AAG GAG GGA CTG TGC CGG AAG ATC ATT CAC TAC TTC AAC AAA GGC 576
Arg Lys Glu Gly Leu Cys Arg Lys Ile Ile His Tyr Phe Asn Lys Gly
180 185 190
AAG AGC TGG GAG TTT GGG ATC CCA CTG TGC AGG GAG CTG GCG TGT CAG 624
Lys Ser Trp Glu Phe Gly Ile Pro Leu Cys Arg Glu Leu Ma Cys Gln
195 200 205
TAC GAG AGC CTC TAT GAT TAC CAG AGC CTC AGC TGG ATT CGG AAA ATG 672
Tyr Glu Ser Leu Tyr Asp Tyr Gln Ser Leu Ser Trp Ile Arg Lys Met
210 215 220
GAG GCC AGC TAC TAT GAC AAC ATT ATG GAG CAG CAA CGC CTG GAG CCT 720
Glu Ala Ser Tyr Tyr Asp Asn Ile Met Glu Gln Gln Arg Leu Glu Pro 225 230 235 240
GAG TTC TTT CGG GTC GGC TTC TAT GGC AGG AAG TTT CCT TTC TTT CTT 768
Glu Phe Phe Arg Val Gly Phe Tyr Gly Arg Lys Phe Pro Phe Phe Leu
245 250 255
CGG AAC AAA GAA TAC GTG TGC CGT GGC CAT GAG TAC GAG AGG CTG GAG 816
Arg Asn Lys Glu Tyr Val Cys Arg Gly His Asp Tyr Glu Arg Leu Glu
260 265 270
GCC TTC CAG CAG AGG ATG CTC AGT GAG TTT CCG CAG GCT GTC GCC ATG 864
Ala Phe Gln Gln Arg Met Leu Ser Glu Phe Pro Gln Ala Val Ala Met
275 280 285
CAG CAC CCC AAC CAT CCT GAT GAC GCC ATC CTA CAG TGC GAT GCC CAG 912 Gln His Pro Asn His Pro Asp Asp Ala Ile Leu Gln Cys Asp Ala Gln
290 295 300
TAC TTG CAG ATC TAT GCA GTG ACG CCC ATT CCA GAT TAT GTG GAT GTT 960
Tyr Leu Gln Ile Tyr Ala Val Thr Pro Ile Pro Asp Tyr Val Asp Val 305 310 315 320
CTG CAG ATG GAT AGG GTA CCA GAT CGA GTC AAG AGC TTC TAT CGC GTC 1008
Leu Gln Met Asp Arg Val Pro Asp Arg Val Lys Ser Phe Tyr Arg Val
325 330 335
AAC AAT GTG AGG AAG TTC CGG TAT GAC AGG CCT TTT CAC AAA GGC CCC 1056
Asn Asn Val Arg Lys Phe Arg Tyr Asp Arg Pro Phe His Lys Gly Pro
340 345 350
AAG GAC AAG GAG AAT GAA TTC AAG AGC CTG TGG ATT GAA CGT ACC ACA 1104
Lys Asp Lys Glu Asn Glu Phe Lys Ser Leu Trp Ile Glu Arg Thr Thr
355 360 365
CTG ACC CTG ACC CAC AGC TTG CCT GGC ATC TCT CGG TGG TTT GAA GTG 1152
Leu Thr Leu Thr His Ser Leu Pro Gly Ile Ser Arg Trp Phe Glu Val
370 375 380
GAG AGG AGG GAA CTG GTG GAG GTG AGC CCT CTG GAG AAT GCC ATC CAA 1200
Glu Arg Arg Glu Leu Val Glu Val Ser Pro Leu Glu Asn Ala Ile Gln 385 390 395 400
GTG GTT GAG AAT AAG AAC CAG GAG CTA CGC TCC CTG ATC AGC CAG TAT 1248
Val Val Glu Asn Lys Asn Gln Glu Leu Arg Ser Leu Ile Ser Gln Tyr
405 410 415
CAA CAC AAG CAG GTG CAT GGC AAC ATT AAC CTG CTA AGC ATG TGC CTG 1296 Gln His Lys Gln Val His Gly Asn Ile Asn Leu Leu Ser Met Cys Leu
420 425 430
AAT GGT GTC ATT GAT GCA GCT GTC AAT GGA GGC ATT GCA CGC TAT CAG 344
Asn Gly Val Ile Asp Ala Ala Val Asn Gly Gly Ile Ala Arg Tyr Gln
435 440 445
GAG GCC TTC TTT GAT AAA GAT TAC ATC AAC AAG CAC CCA GGA GAT GCT 1392
Glu Ala Phe Phe Asp Lys Asp Tyr Ile Asn Lys His Pro Gly Asp Ala
450 455 460
GAG AAG ATC ACC CAG CTC AAG GAG CTT ATG CAG GAG CAG GTT CAT GTC 1440
Glu Lys Ile Thr Gln Leu Lys Glu Leu Met Gln Glu Gln Val His Val 465 470 475 480
CTT GGA GTT GGG CTA GCA GTT CAT GAG AAG TTT GTG CAC CCA GAA ATG 488
Leu Gly Val Gly Leu Ala Val His Glu Lys Phe Val His Pro Glu Met
485 490 495
CGG CCT CTG CAT AAG AAG CTA ATT GAT CAG TTC CAG ATG ATG CGG GCC 1536
Arg Pro Leu His Lys Lys Leu Ile Asp Gin Phe Gln Met Met Arg Ala
500 505 510
AGT CTC TAC CAT GAG TTT CCA GGT TTG GAT AAG CTA AGT CCT GCA TGT 1584
Ser Leu Tyr His Glu Phe Pro Gly Leu Asp Lys Leu Ser Pro Ala Cys
515 520 525
TCA GGC ACC AGC ACC CCA CGG GGA AAT GTT CTG GCA TCC CAT AGC CCC 1632
Ser Gly Thr Ser Thr Pro Arg Gly Asn Val Leu Ala Ser His Ser Pro
530 535 540
ATG AGT CCG GAG AGC ATC AAG ATG ACC CAC CGG CAC AGC CCC ATG AAC 1680
Met Ser Pro Glu Ser Ile Lys Met Thr His Arg His Ser Pro Met Asn 545 550 555 560
TTG ATG GGC ACA GGC CGC CAT TCA TCA TCC TCT CTC TCC TCA CAT GCG 1728
Leu Met Gly Thr Gly Arg His Ser Ser Ser Ser Leu Ser Ser His Ala
565,570,575
TCT AGT GAA GCA GGA AAC ATG GTG ATG CTG GGT GAC GGC TCC ATG GGT 1776
Ser Ser Glu Ala Gly Asn Met Val Met Leu Gly Asp Gly Ser Met Gly
580 585 590
GAT GCT CCT GAG GAC CTG TAC CAC CAC ATG CAG CTC GCG TAT CCC AAC 1824
Asp Ala Pro Glu Asp Leu Tyr His His Met Gln Leu Ala Tyr Pro Asn
595 600 605
CCC AGG TAC CAA GGC TCA GTC ACC AAC GTC TCT GTT CTG TCC TCG TCC 1872
Pro Arg Tyr Gln Gly Ser Val Thr Asn Val Ser Val Leu Ser Ser Ser
610 615 620
CAG GCA AGC CCT TCT TCC TCC AGC CTG AGT TCC ACT CAC TCA GCA CCA 1920 Gln Ala Ser Pro Ser Ser Ser Ser Leu Ser Ser Thr His Ser Ala Pro 625 630 635 640
TCC CAG ATG ATT ACC TCT GCC CCT TCC AGT GCC CGA GAT AAG TAC CGC 1968
Ser Gln Met Ile Thr Ser Ala Pro Ser Ser Ala Arg Asp Lys Tyr Arg
645 650 655
CAT GCC CGT GAA ATG ATG TTG TTG CTG CCC ACA TAC CGG GAC CGC CCA 2016
His Ala Arg Glu Met Met Leu Leu Leu Pro Thr Tyr Arg Asp Arg Pro
660 665 670
AGC AGT GCC ATG TAT CCA GCA GCC ATC CTG GAG AAC GGA CAG CCG CCG 2064
Ser Ser Ala Met Tyr Pro Ala Ala Ile Leu Glu Asn Gly Gln Pro Pro
675 680 685
AAT TTC CAG CGA GCC CTG TTC CAG CAA GTG GTC GGA GCC TGC AAA CCC 2112
Asn Phe Gln Arg Ala Leu Phe Gln Gln Val Val Gly Ala Cys Lys Pro
690 695 700
TGC AGT GAT CCC AAT CTG TCT GTG GCT GAA AAA GGT CAT TAC TCC CTA 2160
Cys Ser Asp Pro Asn Leu Ser Val Ala Glu Lys Gly His Tyr Ser Leu 705 710 715 720
CAC TTT GAC GCC TTC CAC CAC CCT CTG GGT GAT ACC CCC CCA GCC CTC 2208
His Phe Asp Ala Phe His His Pro Leu Gly Asp Thr Pro Pro Ala Leu
725 730 735
CCT GCC CGG ACC CTG CGC AAG TCT CCT CTC CAC CCT ATC CCA GCC TCC 2256
Pro Ala Arg Thr Leu Arg Lys Ser Pro Leu His Pro Ile Pro Ala Ser
740 745 750
CCC ACA AGC CCC CAG TCA GGT CTG GAC GGC AGC AAC TCT ACG CTG TCC 2304
Pro Thr Ser Pro Gln Ser Gly Leu Asp Gly Ser Asn Ser Thr Leu Ser
755,760,765
GGC AGT GCC AGC AGC GGC GTG TCC TCC TTG AGT GAG AGT AAC TTT GGG 2352
Gly Ser Ala Ser Ser Gly Val Ser Ser Leu Ser Glu Ser Asn Phe Gly
770,775,780
CAC TCC TCG GAG GCC CCA CCT CGC ACT GAC ACC ATG GAC TCC ATG CCA 2400
His Ser Ser Glu Ala Pro Pro Arg Thr Asp Thr Met Asp Ser Met Pro 785 790 795 800
AGT CAG GCC TGG AAT GCT GAC GAA GAT CTT GAG CCA CCC TAC CTC CCT 2448
Ser Gln Ma Trp Asn Ala Asp Glu Asp Leu Glu Pro Pro Tyr Leu Pro
805 810 815
GTC CAC TAC AGC CTC TCT GAG TCT GCC GTC CTG GAC TCC ATC AAG GCC 2496
Val His Tyr Ser Leu Ser Glu Ser Ala Val Leu Asp Ser Ile Lys Ala
820 825 830
CAG CCA TGC CGA AGC CAC TCA GCC CCA GGG TGC GTC ATC CCT CAG GAC 2544 Gln Pro Cys Arg Ser His Ser Ala Pro Gly Cys Val Ile Pro Gln Asp
835 840 845
CCC ATG GAC CCG CCT GCG CTG CCG CCC AAG CCC TAC CAC CCC CGC CTG 2592
Pro Met Asp Pro Pro Ma Leu Pro Pro Lys Pro Tyr His Pro Arg Leu
850 855 860
CCG GCC CTG GAG CAC GAT GAG GGG GTG CTG CTG CGT GAA GAG ACT GAG 2640
Pro Ala Leu Glu His Asp Glu Gly Val Leu Leu Arg Glu Glu Thr Glu 865 870 875 880
AGG CCT CGA GGC CTG CAC CGC AAG GCT CCA TTG CCT CCT GGG AGC GCT 2688
Arg Pro Arg Gly Leu His Arg Lys Ma Pro Leu Pro Pro Gly Ser Ala
885 890 895
AAG GAG GAG CAG GCC CGC ATG GCC TGG GAG CAC GGC CGA GGG GAG CAG 2736
Lys Glu Glu Gln Ala Arg Met Ala Trp Glu His Gly Arg Gly Glu Gln
900 905 910
TGAGGGGCAA CGAGGCGGCT GGGATGCCGC CCTCAGTAAG CAGCTTGCCA ATCACTCCAG 2796
GTCTGAAAAG CAAGTCCCCC AGCCCCACCC CAGGGAGCCA GAGAGGCTTG CACTCAGGAG 2856
AGAACCACCC CCAAGTCTCC GTTCTACTGC CGCCCG 2892
(2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 1363 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETIC: NO
(iv) ANTI-SENSE: NO
(ix) CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1363
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
GAA TTC CGG GGT TGT GTG AAG ATG GAG TTT CCC GGA GGA AAT GAC AAT 48
Glu Phe Arg Gly Cys Val Lys Met Glu Phe Pro Gly Gly Asn Asp Asn
915 920 925
TAC CTG ACG ATC ACA GGG CCT TCG CAC CCC TTC CTG TCA GGG GCC GAG 96
Tyr Leu Thr Ile Thr Gly Pro Ser His Pro Phe Leu Ser Gly Ala Glu
930 935 940
ACA TTC CAT ACA CCA AGC TTG GGT GAT GAG GAA TTT GAA ATC CCA CCT 144
Thr Phe His Thr Pro Ser Leu Gly Asp Glu Glu Phe Glu Ile Pro Pro 945 950 955 960
ATC TCC TTG GAT TCT GAT CCC TCA TTG GCT GTC TCA GAT GTG GTT GGC 192
Ile Ser Leu Asp Ser Asp Pro Ser Leu Ala Val Ser Asp Val Val Gly
965,970,975
CAC TTT GAT GAC CTG GCA GAC CCT TCC TCT TCA CAG GAT GGC AGT TTT 240
His Phe Asp Asp Leu Ala Asp Pro Ser Ser Ser Gln Asp Gly Ser Phe
980 985 990
TCA GCC CAG TAT GGG GTC CAG ACA TTG GAC ATG CCT GTG GGC ATG ACC 288
Ser Ala Gln Tyr Gly Val Gln Thr Leu Asp Met Pro Val Gly Met Thr
995 1000 1005
CAT GGC TTG ATG GAG CAG GGC GGG GGG CTC CTG AGT GGG GGC TTG ACC 336
His Gly Leu Met Glu Gln Gly Gly Gly Leu Leu Ser Gly Gly Leu Thr
1010 1015 1020
ATG GAC TTG GAC CAC TCT ATA GGA ACT CAG TAT AGT GCC AAC CCA CCT 384
Met Asp Leu Asp His Ser Ile Gly Thr Gln Tyr Ser Ala Asn Pro Pro 1025 1030 1035 1040
GTT ACA ATT GAT GTA CCA ATG ACA GAC ATG ACA TCT GGC TTG ATG GGG 432
Val Thr Ile Asp Val Pro Met Thr Asp Met Thr Ser Gly Leu Met Gly
1045 1050 1055
CAT AGC CAG TTG ACC ACC ATT GAT CAG TCA GAA CTG AGT TCC CAG CTG 480
His Ser Gln Leu Thr Thr Ile Asp Gln Ser Glu Leu Ser Ser Gln Leu
1060 1065 1070
GGT TTG AGC CTA GGG GGT GGC ACC ATC CTG CCA CCT GCC CAG TCA CCT 528
Gly Leu Ser Leu Gly Gly Gly Thr Ile Leu Pro Pro Ala Gln Ser Pro
1075 1080 1085
GAA GAT CGT CTT TCA ACC ACC CCT TCA CCT ACT AGT TCA CTT CAC GAA 576
Glu Asp Arg Leu Ser Thr Thr Pro Ser Pro Thr Ser Ser Leu His Glu
1090 1095 1100
GAT GGT GTT GAG GAT TTC CGG AGG CAA CTT CCC AGC CAG AAG ACA GTC 624
Asp Gly Val Glu Asp Phe Arg Arg Gln Leu Pro Ser Gln Lys Thr Val 1105 1110 1115 1120
GTG GTG GAA GCA GGG AAA AAG CAG AAG GCC CCA AAG AAG AGA AAA AAG 672
Val Val Glu Ala Gly Lys Lys Gln Lys Ala Pro Lys Lys Arg Lys Lys
1125 1130 1135
AAA GAT CCT AAT GAA CCT CAG AAA CCA GTT TCA GCA TAT GCT TTA TTC 720
Lys Asp Pro Asn Glu Pro Gln Lys Pro Val Ser Ala Tyr Ala Leu Phe
1140 1145 1150
TTT CGT GAT ACA CAG GCT GCC ATC AAG GGA CAG AAT CCT AAT GCC ACT 768
Phe Arg Asp Thr Gln Ala Ala Ile Lys Gly Gln Asn Pro Asn Ala Thr
1155 1160 1165
TTT GGT GAG GTT TCA AAA ATT GTG GCC TCC ATG TGG GAT AGT CTT GGA 816
Phe Gly Glu Val Ser Lys Ile Val Ala Ser Met Trp Asp Ser Leu Gly
1170 1175 1180
GAG GAG CAA AAA CAG GTA TAT AAG AGG AAA ACT GAG GCT GCC AAG AAA 864
Glu Glu Gln Lys Gln Val Tyr Lys Arg Lys Thr Glu Ala Ala Lys Lys 1185 1190 1195 1200
GAG TAT CTG AAG GCA CTG GCT GCT TAC AAA GAC AAC CAG GAG TGT CAG 912
Glu Tyr Leu Lys Ala Leu Ala Ala Tyr Lys Asp Asn Gln Glu Cys Gln
1205 1210 1215
GCC ACT GTG GAA ACA GTG GAA TTG GAT CCA GCA CCA CCA TCA CAA ACT 960
Ala Thr Val Glu Thr Val Glu Leu Asp Pro Ala Pro Pro Ser Gln Thr
1220 1225 1230
CCT TCT CCA CCT CCT ATG GCT ACT GTT GAC CCA GCA TCT CCA GCA CCA 1008
Pro Ser Pro Pro Pro Met Ala Thr Val Asp Pro Ala Ser Pro Ala Pro
1235 1240 1245
GCT TCA ATA GAG CCC CCT GCC CTG TCC CCA TCC ATT GTT GTT AAC TCC 1056 Ma Ser Ile Glu Pro Pro Ala Leu Ser Pro Ser Ile Val Val Asn Ser
1250 1255 1260
ACC CTT TCA TCC TAT GTG GCA AAC CAG GCA TCT TCT GGA GCT GGG GGT 1104
Thr Leu Ser Ser Tyr Val Ala Asn Gln Ma Ser Ser Gly Ala Gly Gly 1265 1270 1275 1280
CAG CCC AAT ATC ACC AAG TTG ATT ATT ACC AAA CAA ATG TTG CCC TCT 1152 Gln Pro Asn Ile Thr Lys Leu Ile Thr Lys Gln Met Leu Pro Ser
1285 1290 1295
TCT ATT ACT ATG TCT CAA GGA GGG ATG GTT ACT GTT ATC CCA GCC ACA 1200
Ser Ile Thr Met Ser Gln Gly Gly Met Val Thr Val Ile Pro Ala Thr
1300 1305 1310
GTG GTG ACC TCC CGG GGG CTC CAA CTA GGC CAA ACC AGT ACA GCT ACT 1248
Val Val Thr Ser Arg Gly Leu Gln Leu Gly Gln Thr Ser Thr Ala Thr
1315 1320 1325
ATC CAG CCC AGT CAA CAA GCC CAG ATT GTC ACT CGG TCA GTG TTG CAG 1296
Ile Gln Pro Ser Gln Gln Ala Gln Ile Val Thr Arg Ser Val Leu Gln
1330 1335 1340
GCA GCA GCA GCT GCT GCT GCT GCT GCT TCT ATG CAA CTG CCT CCA CCC 1344
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Met Gln Leu Pro Pro Pro 1345 1350 1355 1360
CGA GTA CAG CCC GGA ATT C 1363
Arg Val Gln Pro Gly Ile
1365

Claims (31)

 CLAIMS 1. Nucleotide sequence coding for a protein capable of interacting with presenilins. 2. Nucleotide sequence according to claim 1 characterized in that it codes for a polypeptide or a protein capable of interacting with all or part of the large loop of presenilin PS-1. 3. Nucleotide sequence according to claim I or 2 characterized in that it comprises all or part of the sequence SEQ ID N0 i or of its derivatives 4. Nucleotide sequence according to claim 1 or 2 characterized in that it comprises all or part of the sequence SEQ ID N "2 or its derivatives 5. Polypeptide characterized in that it is capable of interacting with presenilins. 6. Polypeptide according to claim 5 characterized in that it is capable of interacting with the large loop of presenilin PS- i. 7. Polypeptide according to claim 6 characterized in that it is capable of interacting with the amino acid sequence 310-463 of the large loop of presenilin PS-I. 8. Polypeptide according to claim 5 characterized in that quril is capable of interacting with the large loop of presenilin PS-2 9. Polypeptide according to one of claims 5 to 8 characterized in that it comprises all or part of the peptide sequence SEQ ID N "1 or a sequence derived therefrom. 10. Polypeptide according to one of claims 5 to 8 characterized in that it comprises all or part of the peptide sequence SEQ ID N "2 or a sequence derived therefrom. 11. Use of a polypeptide according to claims 5 to 10 for obtaining a medicament intended to slow, stimulate or modulate the activity of presenilins. 12. Method for preparing a polypeptide according to one of claims 5 to 10 characterized in that a cell containing a nucleotide sequence according to one of claims 1 to 4 is cultured under conditions of expression of said sequence and we recover the polypeptide produced 13. Host cell for the production of polypeptide according to one of claims 5 to 10, characterized in that it has been transformed with a nucleic acid comprising a nucleotide sequence according to one of claims 1 to 4. 14. Antisense oligonucleotide of a sequence according to claim 3 or 4 15. Nucleotide probe capable of hybridizing with a nucleotide sequence according to one of claims 1 to 4 or the corresponding mRNA. 16. Antibody or antibody fragment directed against a polypeptide according to one of claims 5 to 10. 17. Antibody or antibody fragment according to claim 16 characterized in that it is directed against a sequence chosen from the peptide sequences presented in SEQ ID N "I or SEQ ID N" 2 or their derivatives. 18. Antibody or antibody fragment according to claim 16 or 17 characterized in that it has the ability to inhibit and / or reveal the interaction between presenilins and a polypeptide according to one of claims 5 to 10. 19. Method for detecting or isolating compounds capable of modulating or inhibiting the interaction between presenilins and the polypeptides as defined in claims 5 to 10, characterized in that the following steps are carried out  a - a molecule or a mixture containing different molecules, possibly unidentified, is brought into contact with a recombinant cell as described above expressing a polypeptide of the invention under conditions allowing the interaction between said polypeptide and said molecule in the event that it has an affinity for said polypeptide, and,  b - the molecules linked to said polypeptide are detected and / or isolated. 20. Ligand of a polypeptide as defined according to claims 5 to 10, capable of being obtained according to the method of claim 19. 21. Use of a ligand according to claim 20 for the preparation of a medicament intended for the treatment of neurological conditions. 22. A defective recombinant virus comprising a nucleotide sequence coding for a polypeptide according to one of claim 5 to 10. 23. Vector comprising a nucleotide sequence according to one of claims 1 to 4. 24. Vector according to claim 23 characterized in that it is a plasmid vector. 25. Vector according to claim 23 characterized in that it is a viral vector. 26. Vector according to claim 25 characterized in that it is a virus defective for replication. 27. Pharmaceutical composition comprising one or more vectors according to one of claims 23 to 26. 28. Pharmaceutical composition comprising as active ingredient at least one polypeptide according to one of claims 5 to 10. 29. Pharmaceutical composition comprising as active principle at least one antibody or antibody fragment according to one of claims 16 to 18, and / or an antisense oligonucleotide according to claim 14 and / or a compound prepared according to claim 19. 30. Composition according to claim 27 to 29 intended to modulate slow down or inhibit the activity of presenilins or their mutated forms. 31 Composition according to one of claims 27 to 29 intended for the treatment of neurodegenerative diseases.