CZ20001110A3 - Nucleic acids encoding polypeptides capable of interaction with presenilins , process for preparing such polypeptides as well as pharmaceutical preparations in which these polypeptides are comprised - Google Patents

Abstract

New nucleotide and peptide sequences encoding proteins capable of interacting with presenilins, and their pharmaceutical uses.

Description

Nucleic acids encoding polypeptides capable of interacting with presenilins, a process for preparing said polypeptides, and pharmaceutical compositions comprising said polypeptides

Technical field

The present invention relates to novel nucleotide and peptide sequences and their pharmaceutical use. More particularly, the present invention relates to nucleic acids encoding proteins capable of interacting with presenilins, methods of preparing such proteins, and pharmaceutical compositions comprising the proteins.

BACKGROUND OF THE INVENTION

Alzheimer's disease is the most widespread form of dementia. The frequency increases with increasing age, ranging from 5 to 10% over the age of 65 and over 20% over the age of 80.

To date, there is no specific treatment for this disease and only palliative care can be provided to patients. The presence of this disease can only be confirmed by histological analysis of the brain during autopsy, as there is no accurate and timely diagnosis.

Alzheimer's disease is associated with organic brain lesions that form histological features: senile plaques, neurofibrillary degeneration of the neurons, atrophy and neuronal loss at the cortex level.

The etiology of the disease is complex, many genes are involved, and the various physiopathological mechanisms in which these genes are not yet fully understood.

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Anatomical brain lesions observed in patients under 65 years of age associated with presenile dementia have been reported. Currently, the term “Alzheimer's type dementia covers both presenile dementia (65 years ago) and senile dementia (after 65 years) that exhibit the same characteristics.

There are two forms of disease: sporadic forms that are not associated with heredity, and familial forms.

Sporadic forms of the disease are the most common, affecting 60 to 90% of patients and occur most frequently after 65 years of age. In most cases, the onset of the pathological condition remains unknown. But it has been found that head injuries, inflammations, emotional shocks or high stress situations. increase the likelihood of disease (Seubert et al., 1992).

Familial forms of the disease account for 10 to 40% of cases. They are divided into two groups, late form (after 65 years) and early form (65 years ago). In early cases, the transmission of the disease has an autosomal dominant character.

In 1992, the existence of the AD3 locus on chromosome 14 was demonstrated, and in 1995 the presenilin 1 gene (PS-1) was isolated (the gene was initially called S182) (Sherrington et al., 1995). Presenilin 1 gene mutations result in 70% of very early forms of the disease (55 years ago). Very rare cases of early forms are associated with mutations in the presenilin 2 gene located on chromosome 1 (Levy-Lehad et al., 1995). This gene shows 67% identity to the presenilin 1 gene.

The gene consists of 12 exons, exons 3 to 12 encode a protein. It is ubiquitously expressed, giving rise to a 2.7 kb major transcript and a minor sized transcript

7.5 kb (Kovacs, 1996). The protein contains 467 amino acids and exhibits a membrane protein structure. It exhibits at least 8 possible transmembrane domains. Amino acids

The 3 corresponding codons affected by the recorded mutations are distributed throughout the protein, located in both hydrophobic regions (TM) and hydrophilic loops (BL), with two preferred regions, TM2 and BL6.

Currently, more than 35 point mutations have been published and mutations not yet identified are mostly all types of codon sense mutations (the so-called "missense" that lead to a point change in the amino acid of the protein sequence). But recently, another type of splicing mutation has been published (Perez-Tur et. Al., 1995), in which case the gene is altered by the loss of exon 9.

The role of presenilins in normal cells is still poorly understood. Numerous hypotheses have been proposed. One of these hypotheses states that presenilin 1 (PS-1) could play a role in cellular transport. This hypothesis is confirmed by an immunocytochemical outcome that made it possible to localize PS-1 at the level of the endoplasmic reticulum and the 'Golgi apparatus' in human neuroglial cells (Kovacs et al., 1996). In addition, PS-1 could play a role in cellular communication and the mechanism of endocytosis and proteolysis of APP (Dewji and Singer, 1996). The third hypothesis involves PS-1 in interactions with the cytoskeleton, and in particular with Tau-associated microtubules (Lew and Wang, 1996). Finally, considering its structure, PS-1 could play the role of a receptor associated with G proteins involved in signal transduction, or alternatively, a transporter or ion channel (Van Broeckhoven, 1995).

Finding partners that interact with presenilins allows you to understand the true role of presenilins.

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SUMMARY OF THE INVENTION

The present invention is based on the inventor having identified nucleic acids encoding proteins capable of interacting with presenilins. The identification of these nucleic acids makes it possible to consider a preparation based on novel polypeptides which can be used pharmaceutically.

Accordingly, a first aspect of the invention relates to nucleotide sequences encoding proteins capable of interacting with presenilins.

According to a specific embodiment, the nucleotide sequences of the invention encode proteins or polypeptides capable of interacting with all or part of the large loop of presenilin PS-1.

More preferably, the nucleotide sequences of the invention comprise all or part of sequence id. No. 1 or derivatives thereof, or all or part of SEQ ID NO: 1; No. 2 or derivatives thereof. .

For the purposes of the present invention, the term derived nucleotide sequence refers to any sequence that differs from the sequence of interest by degeneracy of the genetic code obtained by one or more modifications of genetic and / or chemical nature, as well as each sequence hybridizing to these sequences or fragments thereof and encoding the polypeptide of invention. It is understood that modifications of a genetic and / or chemical nature mean any mutation, substitution, deletion, addition and / or modification of one or more residues. The term derivative also includes sequences homologous to the sequence of interest that originate from other cellular sources and in particular from cells of human origin or other organisms. Such homologous sequences can be obtained by hybridization experiments. Hybridizations are performed using a nucleic acid library using a native sequence or fragment thereof as a probe under varying hybridization conditions (Maniatis et al., 1989).

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The nucleotide sequences of the invention may be of artificial or other origin. These may be genomic, cDNA or RNA sequences, hybrid sequences, or synthetic or semi-synthetic sequences. These sequences can be obtained, for example, by screening DNA libraries (cDNA library, genomic DNA library) with probes generated based on the sequences of the invention. These libraries are prepared from cells of different origin ^one conventional molecular biological techniques, which are known in the art. Nucleotide sequences of the invention may also be prepared by chemical synthesis or alternatively by mixed methods including chemical or enzymatic modification of sequences obtained by screening libraries. In general, the nucleic acids of the invention may 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 of the invention are capable of interacting with a large loop of presenilin PS-1 and / or a large loop of presenilin PS-2. More preferably, they are polypeptides capable of interacting with the amino acid sequence 310-463 of the large loop of presenilin

PS-1.

For the purposes of the present invention, the name presenilin covers the PS-1 and PS-2 presenilins per se, as well as their homologous forms particularly corresponding to the mutated forms of these proteins.

The present invention also relates to polypeptides comprising all or part of a peptide sequence selected from SEQ ID NOs. 1 or id. 2 or a derivative thereof.

For purposes of the present invention, the term "derived polypeptide sequence" refers to any polypeptide sequence differing from the sequences set forth in SEQ ID NO: 1 feefef feefefef feefefefefef feefefefef · • fe ·· or sequence id. No. 2, which are obtained by one or more modifications of a genetic and / or chemical nature and which have the capacity to interact with presenilins. It is understood that modifications of a genetic and / or chemical nature mean any mutation, substitution, deletion, addition and / or modification of one or more residues.

These derivatives may arise with the intent, in particular, of increasing their therapeutic efficacy or reducing their side effects, or of conferring on them new pharmaceutical and / or biological properties.

The term derivative also includes sequences homologous to the sequences of interest that originate from other cellular sources, and in particular from cells of human origin or other organisms, and which have activity of the same type.

This may also be fragments of the peptide sequences listed above. These fragments can be formed in various ways. In particular, they can be synthesized chemically, based on the sequences given in the present application, using peptide synthesizers known to those skilled in the art. They may also be synthesized by genetic route, expression, nucleotide sequences encoding the desired peptide in a cell host. In this case, the nucleotide sequence may be prepared. chemically using oligonucleotide synthesizers based on the peptide sequence of the present invention and based on the genetic code. Nucleotide sequences can also be prepared from the sequences disclosed in the present application by enzymatic cleavage, ligation, cloning, and the like, using techniques known to those skilled in the art, or by screening libraries with probes prepared according to these sequences.

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It is also an object of the present invention to use the polypeptides of the invention to slow, inhibit, stimulate or modulate the activity of presenilins.

It is really possible to count on the regulation of the function. presenilins by the proteins of the invention and in particular by inhibiting or slowing the activity of the mutated presenilins.

A further object of the present invention relates to a method for preparing the polypeptides of the invention, wherein the cell comprising the nucleotide sequence of the invention is grown under conditions suitable for expression of said sequence and the polypeptide being produced is recovered. In this case, in general, the portion encoding said polypeptide is placed under the control of signals allowing its expression in a cellular host. The choice of these signals (promoters, terminators, secretory leader sequences, etc.) may vary according to the cellular host used. In addition, the nucleotide sequences of the invention may be part of a vector, which may be a replicating or integrating vector autonomously. More specifically, autonomously replicating vectors can be prepared using autonomously replicating sequences in a selected host. For integrating vectors, for example, they can be prepared using sequences homologous to certain regions of the host genome, allowing the vector to be integrated by homologous recombination.

The present invention also provides a host cell transformed with a nucleic acid comprising the nucleotide sequence of the invention. Cell hosts that can be used to produce the peptides of the invention by recombinant means are both eukaryotic and prokaryotic hosts. Suitable eukaryotic hosts include cells of animal origin, yeast or fungi. In particular, in the case of yeast, the yeasts of the genera Saccharomyces, Kluyveromyces, Pichia, Schwanniom.yces or fefefefe • fefefe • fefefe • fefefefe fefefefe fefe fefe fefe

Hansenula. In the case of cells of animal origin, COS, CHO, C127 or human neuroblastoma cells, etc. may be mentioned, and fungi may more specifically include Aspergillus ssp. Or Trichoderma ssp. Streptomyces.

According to a preferred embodiment, the host cells are preferably represented by recombinant yeast strains for expression of the nucleic acids of the invention. as well as for the production of proteins derived therefrom.

Preferably, the host cell comprises at least one sequence or one fragment of a sequence selected from sequence id. 1 or SEQ ID NO. No. 2 for the production of proteins according to the invention.

Another use of the nucleic acid sequences of the invention is the production of antisense oligonucleotides or antisense genes, which can be used as pharmaceutical preparations. Antisense sequences are small-sized oligonucleotides that are complementary to the coding strand of a given gene, and as such are capable of specific hybridization with the transcribed mRNA and thus inhibit its translation into a protein. proteins capable of interacting with presenilins. Such sequences may consist of all or part of the nucleotide sequences defined above and may be obtained by fragmentation and the like, or by chemical synthesis.

The claimed sequences can be used in the context of gene therapy for the transfer and production of in vivo antisense sequences or expression of proteins or polypeptides capable of modulating the activity of presenilins. In this regard, the sequences may be incorporated into viral or non-viral vectors,

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0 0 0 0 · »· allowing them to be administered in vivo. The vector may be a plasmid known in the art. Examples of viral vectors according to the invention include, in particular, vectors of the type adenovirus, retrovirus, adeno-associated virus or herpes virus. The present invention also provides a defective recombinant virus comprising a nucleotide sequence encoding a polypeptide of the invention.

The invention also allows for the production of synthetic or non-synthetic nucleotide probes capable of hybridizing as defined above - or with the corresponding mRNA, which can be used in the context of gene therapy. These probes can be used in vitro as presenilins or abnormalities (bad, etc.). These probes isolating homologous diagnostic aids for detection alternatively for the detection of genetic splicing, polymorphism, point mutations can also be used to detect α nucleic acid sequences encoding peptides as defined above from other cell sources and preferably from cells of human origin. The probes of the invention generally contain at least 10 bases, and may, for example, contain up to one or more of the above sequences or their complementary strand. Preferably, these probes are labeled prior to use. Various techniques known to the person skilled in the art (radioactive or enzyme labeling, etc.) can be used for this.

Another object of the invention is to provide polyclonal or monoclonal antibodies or antibody fragments directed against polypeptides as defined above. These antibodies may be generated by methods which are within the skill of the art to prepare these antibodies for a polypeptide having the sequence of SEQ ID NO: 1 or SEQ ID NO. No. 2 or known. In particular, immunizing the animal against can be selected from sequence id.

of any fragment or derivative thereof, and then collecting blood · ♦ · · ·

- 10 isolation of antibodies. These antibodies may be. also produced by the preparation of hybridomas according to techniques known to those skilled in the art. The antibodies or antibody fragments of the invention; in particular, they may be used to inhibit and / or detect the interaction between presenilins and polypeptides as defined above.

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

The identification and / or isolation of modulators or ligands of polypeptides of the invention is carried out according to the following steps:

a molecule or mixture comprising various molecules, optionally unidentified, is contacted with a recombinant cell as described above, expressing the polypeptide of the invention, under conditions allowing interaction between. said polypeptide and said molecule, which should have an affinity for said polypeptide, and, molecules bound to said polypeptides of the invention are detected and / or isolated.

In a specific embodiment, the method of the invention is adapted to identify and / or isolate agonists and antagonists of the interaction between presenilins and polypeptides of the invention.

Another object of the invention relates to the use of a ligand or modulator identified and / or obtained according to the method described above as a medicament. Indeed, these ligands or modulators may allow the treatment of certain neurological conditions.

The present invention also provides any pharmaceutical composition comprising as an active ingredient at least one polypeptide as defined above.

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The present invention also provides any pharmaceutical composition comprising as active ingredient at least one antibody and / or antibody fragment as defined above and / or one antisense oligonucleotide, as well as any pharmaceutical composition comprising as active ingredient at least one nucleotide sequence as defined above .

In addition, the invention also relates to pharmaceutical compositions in which the peptides, antibodies and nucleotide sequences defined above or fragments thereof are combined with each other or with other active ingredients.

The invention also provides compositions wherein the nucleotide sequences of the invention are incorporated into viral or non-viral recombinant vectors.

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

Description of the picture

Figure 1. Schematic representation of presenilin 1 and the part used for the double hybrid assay.

Figure 2. Isolation of yBM-C and yBM-D strains carrying various plasmids containing mainly clone C (corresponding to SEQ ID NO: 1) and clone D (corresponding to SEQ ID NO: 2) by a yeast auxotrophy assay.

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Figure 3. Expression of mRNA corresponding in human tissues.

Picture. 4. Schematic representation of the mammalian expression vector used.

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

Figure 6 a, b, c). Maps of major vectors used.

-Materials and methods used in the invention

1) The following yeast strains were used:

S. cerevisiae YCM 79 (MATa, ade2, suffer, leu2, lys2, ura3, his3, gal4, gal180, metal 6 :: GAL1 10-URA3 LacZ, HIS3 :: GAL7- phleo ^R ). S. cerevisiae PCY 2 (MATa, Dgal4 DgalSO , ade2, suffer, leu2, lys2, his3, URA3 :: GAL1 10-lacZ). S. cerevisiae HF7. C (MATa, ura3, his3, ade2, suffer, leu2, lys2, gal4, gall80, canR, URA3 :: GAL 4 Coupling place - CY Tar- lacZ, LYS2 :: GAL1-HIS3). S. cerevisiae L 40 (MATa, his3) ade2, suffer, leu2, ly s2,

URA3:: LexA-lacZ, LYS2 :: LexA-HIS3).

2) The following bacterial strains were used:

E. coli TG1 (F '[traD 36, lacF, lacZ D M15, for A ⁺ B ⁺ ], supE, thi-1, D (lac-for AB), D (hsdM-mcrB) 5, (r _K- m _K) _McrB ~).

E. coli BL21 (DE3) (F-, ompT, HsdS _B (r _B m _B ~) gal dcm (DE3).

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3) Culture media used for different strains:

YPD Complete Yeast Medium:

- yeast extract Bacto (10 g / l) (Difco)

Bacto-peptone (20 g / l) (Difco).

- glucose (20 g / l) (Merck)

This medium can be made solid by adding agar (20 g / l) and autoclaved at 110 ° C for 20 minutes.

YNB minimal medium for yeast:

yeast nitrogen base. Bacto. without amino acids (6.7 g / l) (Difco)

- glucose (20 g / l) (Merck)

This medium can be made solid by adding agar (20 g / L) and autoclaved. The amino acids or nitrogen bases necessary for the growth of the auxotrophic yeast are then added in an amount of 50 mg / l. Ampicillin is then added at 100 pg / ml to prevent bacterial contamination.

Complete LB medium for bacteria:

- yeast extract Bacto (5 g / l) (Difco)

- Bacto-tryptone (10 g / l) (Difco).

- NaCl (5 g / l) (Difco)

This medium can be made solid by the addition of agar (12 g / L) and autoclaved. To select bacteria that have been transformed with plasmids carrying the corresponding resistance marker, ampicillin or kanamycin is added in an amount of 100 pg / ml fefe.

- 14 fefe ···· fe fe fe · fefe fefe · • fefe fe fefe fefe fefe fefe fefe · • fefe · fefe · • fefe «• fe fefe

4) The following plasmids were used:.

The vector pGBT9 (Fig. 6 (a)) is a 5.5 kb shuttle plasmid that can be multiplied and selected in E. coli as well as yeast. It has a ColE1 origin of replication and an ampicillin resistance gene (reproduction and selection in E. coli) as well as a replication. origin 2mm. and the TRP1 gene (selection in yeast suffer - defective in tryptophan synthesis). This plasmid is used to express fusion proteins. between the GAL4 DNA-binding domain (DBD) and the desired peptides in yeast. It has a multiple cloning site downstream of the DBD GAL4 region, the whole is situated between the promoter and terminator of the ADH1 gene encoding alcohol dehydrogenase 1. The DBD GAL4 comprises a nuclear localization signal sequence.

Sequences corresponding to different portions of PS-1 were inserted into EcoRI / SalI cloning sites.

The vector pGAD10 (FIG. 6 (a)) is a 6.6 kb shuttle plasmid. It has a ColE1 origin of replication and an ampicillin resistance gene, which allows its reproduction and selection in E. coji. It also contains a yeast replication origin (2mm) and a LEU2 selection gene for the isolation of transformed yeast. It has a multiple cloning site downstream of the sequence encoding the GAL4 activating domain (AD) between the promoter and the terminator of the ADH1 gene. AD GAL4 contains a nuclear localization signal sequence.

This plasmid was used to produce a commercial human brain cDNA library (Clontech). The cDNA sequences were inserted into the EcoRI cloning site.

The vector pLex9 (FIG. 6 (b)) is a 5 kb shuttle plasmid. As pGBT9, it has a ColE1 origin and an Amp ^R gene, as well as a 2mm replication origin and a TRP1 gene. But the sequence ·· · to · tototo ·

This coding DNA binding domain is the sequence of the bacterial repressor Lex A. The promoter and terminator used to express the fusion protein are also from the ADH1 gene. The Lex A DBD contains the SV40 nuclear localization signal sequence.

Sequences corresponding to the large PS-1 loop were inserted into EcoRI / SalI cloning sites.

The vector pGEX-4T1 (FIG. 6 (b)) is a 4.9 kb bacterial plasmid. It has a replication origin of pBR322, a gene for resistance to. ampicillin ,. laclq gene for repression of the beta-galactosidase system. This plasmid is used to express fusion proteins between glutathione S-transferase (GST) and the desired peptides in bacteria. It has a multiple cloning site downstream of the GST gene for insertion of sequences encoding peptides. The whole is under the control of the tac promoter, which has a high level of expression inducible by IPTG. After protein production, the desired peptides can be separated from GST by thrombin cleavage because the sequence recognized and cleaved by this enzyme has been inserted between the two regions.

The vector 'pET-29a (Fig. 6 (c)) is a 5.4 kb bacterial plasmid. It has a pBR322 origin of replication, a phage fl origin of replication, a kanamycin resistance gene, a laclq gene. This plasmid is used for expression in bacteria of the desired peptides fused to the S-Tag sequence (catalytic portion of the S ribonuclease protein) and His-Tag sequence (peptide of 10 histidines), which allows the fusion protein to be detected and purified. The T7 transcriptional promoter allows high expression upon induction with IPTG. The sequence recognized and cleaved by thrombin was inserted between the S-Tag and His-Tag sequences.

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5) Synthesis of used oligonucleotides:

Different oligonucleotide pairs were determined from the sequence 'PS-1 (Sherrington et al., 1995) to obtain DNA fragments corresponding to the hydrophilic regions of PS-1. These oligonucleotides made it possible to introduce the EcoRI site at the 5 'end and the SalI site at the 3' end of the sequence after PCR amplification (polymerase chain reaction).

Oligo A CAA GAA TTC TGT CCG AAA GGT CCA CTT (SEQ ID NO: 3)

OligoB CAA GTC GAC TCA GGT TGT GTT CCAGTC TCC (SEQ ID NO: 4)

Oligonucleotides A and B were used to obtain large-loop derived BL6 and BM fragments.

Oligo CAA GAA TTC TCA. ACA ATG GTG TGG TTG (SEQ ID NO: 5)

OligoD CAA GTC GAC TCA TTC TTC CTC TGG GTC TTC ACC (SEQ ID NO: 6)

Oligonucleotides C and D were used to obtain BR derived from the shorter region of the large loop.

fragment

OligoE CAA GAA TTC ACA TTA CTA CTTC GCC (SEQ ID NO: 7)

OligoF CAA GTC GAC TCA GAT ATA AAA TTG ATG CAA (SEQ ID NO: 8)

Oligonucleotides E and F were used to obtain a Ct fragment derived from the C-terminal portion of PS-1.

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OligoG CAA GAA TTC ATG ACA GAG TTA CCT GCA (SEQ ID NO: 9)

OligoH AA GTC GAC TCA ATG CTT GGC GCC ΑΤΑ TTT (SEQ ID NO: 10)

Oligonucleotides G and H were used to obtain an Nt fragment derived from the N-terminal portion of PS-1.

Oligonucleotides 1, J and K were used for amplification and sequencing of the plasmid inserts pGBT9 and pGAD10. They are complementary to the DBD GAL4, AD GAL4, and ADH1 terminator sequences.

Oligol GCG ACA TCA TCA TCG GAA GAG AG (sequence id. C. 11 OligoJ CTA TTC GAT. GAT GAA GAT ACC CC (sequence id. C. 12 OligoK GGC GAA GAA GTC CAA AGC (sequ c e i d .. c. 13)

6) the biology used in the invention.

Molecular methods

Preparation of plasmid DNA

Both small and large amounts of plasmid DNA were isolated according to the protocols described by Maniatis et al. (1989).

Polymerase Chain Amplification by Thermostable Taq Polymerase (PCR)

PCR allows the amplification of the DNA sequence between two known regions where oligonucleotides (about 20 bp) that can be used as primers can bind. The reaction is performed in a final volume of 100 μΐ in the presence of DNA template (50 ng), dNTP (0.2 mM), PCR buffer (10 mM TrisHCl pH 8.5, 1 mM MgCl ₂ , 5 mM KCl, 0.01% gelatin), two

- 18 • 0 ···· oligonucleotides (each 500 ng) and 2.5 IU AmpliTaq DNA polymerase. Cover the mixture with two drops of paraffin oil to limit evaporation of the sample.

The program used contains 32 cycles, 1 cycle for template denaturation (5 minutes at 95 ° C, 30 cycles (1 minute denaturation at 95 ° C), 1 minute oligonucleotide hybridization to

DNA template at 50 ° C, 1 minute extension by Taq polymerase at 72 ° C), final 1 extension cycle (5 minutes at 72 ° C).

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

Fluorescent sequencing

The sequencing technique used is based on the method of Sanger et al. (1977) and is adapted for fluorescent sequencing developed by Applied Biosystems. The protocol used is the one described by system designers (Perkin Elmer, 1995).

Insertion of DNA fragment into plasmids by ligation

The insert obtained from the plasmid DNA or from the PCR reaction and the plasmid vector are digested with 1 U of restriction enzymes per 1 &lt; 1 &gt; Enzymes are selected by cloning sites to insert the insert in accordance with other vector domains.

The fragments resulting from the cleavage are separated according to their size by electrophoresis on a 0.8% preparative agarose gel prepared from TBE (90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8) with 0.5 µg / ml BET. Addition blue (1/10 volume) is added to the samples before being applied to the gel along with a molecular weight standard (1 kb ladder, Gibco BRL). Migration is performed at a constant voltage of 90 V / cm in TBE.

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DNA appeared under UV due to BET fluorescence, which is intercalated between bases. The gel pieces containing DNA fragments of the desired size (vector and insert) are cut with a scalpel, inserted into dialysis tubing with TBE and electrophoresed for 15 minutes at 10Ό V. The DNA extracted from the gel is then removed, treated with one volume of phenol / chloroform / isoamyl alcohol. 24/1), precipitated with 3 volumes of absolute ethanol in the presence of 0.25M NaCl, washed once with 70% ethanol, dried and finally dissolved in 20 μΐ H;

After the appropriate amounts of vector and insert have been determined on the gel, they are mixed at a ratio of 1/1 in the presence. 40 IU T4 DNA ligase, 1 x final ligation buffer (50 mM TrisHCl, pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP) · in a total volume of 20 µ objemu. the ligation is performed at 20 ° C for at least one hour.

Plasmid transformation of bacteria

1) So-called; the TSB method (according to Chung and Miller, NAR, 16, 1988) consists in making the bacterial wall fragile after treatment with DMSO, thereby allowing DNA to enter cells. Its efficiency is 10 ⁶ transformants per 1 pg of DNA. Bacteria are grown in LB liquid medium for approximately 3 hours with agitation (up to A ₆₀ o = 0/6). • The culture is centrifuged for 10 minutes at 3000 rpm, the pellet is resuspended in 1/10 of the original volume of TSB (LB medium. % PEG ₄ (1.5% DMSO, 10mM MgCl ₂ , 10mM MgSO ₄ ), and incubated for 15 minutes at 4 ° C.

Approximately 50 ng of DNA (10 μΐ of ligation product). is added to 100 μΐ of competent bacteria and then incubated for 30 minutes at 4 ° C. After addition of TSBglu (TSB, 20 mM glucose) and incubation at 37 ° C for 45 minutes, the bacteria are plated on LB medium with an antibiotic corresponding to the resistance gene carrying the plasmid.

- 20 2) Electroporation is a technique 1000 times more efficient than the TSB method, when very small amounts of DNA are available (as after the extraction of plasmids contained in yeast). It is that the bacterial wall becomes fragile by electric shock, so that DNA can enter the cells.

The bacteria are grown in LB liquid medium with agitation up to A = 0.6). The culture, pre-incubated for 15 minutes at 4 ° C, is centrifuged for 10 minutes at 3000 rpm and the pellet is washed successively with 1 volume of ice cold H ₂ O, 1/2 volume of ice cold H ₂ O and 1/5 volume of 10% ice cold. glycerol. Between each wash, the bacteria are centrifuged for 10 minutes at 3000 rpm and then finally resuspended in 1/1000 volume of 10% ice-cold glycerol. 40 μΐ of this bacterial suspension is mixed with 1 μΐ of DNA in an electroporation cuvette. The cuvettes are placed in the BioRad Gene Pulser and the bacteria are subjected to electric shock (25 mF, 2.5 kV, 200 W). After incubation at 37 ° C for 45 minutes, the bacteria are plated on LB medium containing the antibiotic used for selection.

Hybridization of mRNA after transfer to 'membrane (or Northern blot)'

The membranes used are Clontech's commercially available nitrocellulose membranes to which mRNA originating from various human tissues has been transferred and fixed.

The radioactive DNA probe is prepared according to the Amersham Rediprime DNA Labeling System protocol. This protocol makes it possible to obtain (32 P) dCTP (50 mCi) with a rate of incorporation into the DNA fragment greater than 55% after incubation for minutes at 37 ° C and to detect an RNA band corresponding to 0.5 µg after 2 hours hybridization and film exposure overnight.

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The membranes are prehybridized with 8 ml hybridization solution (1M NaCl, 10 mM NaH-PCh, pH 7.4, 10 mg / ml Ficoll, mg / ml polyvinylpyrrolidone, 10 mg / ml BSA, 100 mg / ml salmon sperm DNA, 2% SDS) for approximately 2 hours at 42 ° C with shaking, and then hybridized with the probe at 2 x 10 ® cpm / ml mixed with 8 ml of hybridization solution for 24 hours at 42 ° C with shaking, then rinsed several times wash solution 1 (0.3M NaCl, 30mM sodium citrate, pH 7, 0.05% SDS), washed for 40 minutes in the same solution, incubated for 4.0 minutes at 50 ° C in wash solution 2 (15M NaCl / 1.5mM sodium citrate pH 7, 0.1% SDS) and finally exposed to film.

7) Methods for double hybrid system

System principle

The double hybrid system developed by Song and Fields in 1989 is a technique of cloning by in vivo interaction in yeast Saccharomyces cerevisiae. It is based on the fact that some eukaryotic transactivators require only two domains to function: a DNA binding domain (DBD) that binds to a specific sequence, and an activation domain (AD) that triggers a transcriptional mechanism (RNA polil). When these two domains are separated, AD cannot be in the correct position and transcription is not induced.

In this system, DBD is fused to protein X and AD to protein Y. When X and Y proteins interact, a functional transactivation complex forms and induces expression of a reporter gene located downstream of the sequence recognized by the DBD. Gene expression makes it possible to indirectly detect the interaction between the two proteins.

- 22 Yeast transformation with one or two different plasmids

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

The yeast is grown 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, 1 mM EDTA, pH 7.5), centrifuged for 1 minute at 3000 rpm and resuspended in 1 ml of solution I. The 50 μ 50 suspension is then mixed with 5 µg salmon sperm DNA (DNA carrier), 1 to 5 µg plasmid DNA and 300 µΐ sterile solution II (0.1M LiAc, 10mM TrisHCl, 1mM EDTA, pH 7.5, 50%

PEG 400). The mixture is incubated for 30 minutes at 28 ° C and then subjected to heat shock for 20 minutes at 42 ° C. After centrifugation for 1 minute at 3000 rpm, the cells are washed once with sterile water and resuspended in 100 μΐ sterile water, the yeast is sown on YNB agar medium supplemented with the amino acids and nitrogen bases necessary for their growth. In order to allow selection of transformed yeast strains, amino acids and nitrogen bases corresponding to the selectable markers carrying the plasmids are not added. Plates are placed in an incubator at 28 ° C for 3 days. ,

Transformation of yeast cDNA library

The cDNA library used is a commercial library (Clontech) containing mRNA from a normal human brain (a 37-year-old man who died after a head injury). The cDNAs were cloned into plasmid pGAD10 downstream of the AD GAL4 region at the EcoRI site. The average insert size is 1.4 kb with a standard deviation of 1 kb. The library was amplified once in E. coli DH5α to obtain 1.2 x 10® independent clones, 80% of which contain an insert.

In order to maintain good representativity of the library, it is necessary to have a number of transformants that is twice to

- 23 times greater than the number of independent clones obtained during library construction. Therefore, a protocol that allows good transformation efficiency (approximately 10 'transformants per 100 µg DNA) is used.

YCM79 yeast, pre-transformed with plasmid pGBT9 containing the sequence corresponding to the PS-1 region, are grown overnight in 50 ml of YNB + His + Lys + Ade + Met + Leu + Ura at 28 ° C with shaking. The optical density of this pre-culture is measured to evaluate the number of cells per ml and to calculate the volume to be seeded into 250 ml of YPD medium to obtain a culture of 10 ⁷ cells / ml (approximately A ₆₀ o = 0). 8) After one night for shaking.

The cells are then harvested by centrifugation at 3000 rpm for 10 minutes, washed twice with 100 ml sterile H ₂ O, and resuspended in 10 ml transformation solution I. The suspension is centrifuged (10 minutes at 3000 rpm) and the cells are resuspended in 2.5 ml of the same solution. Part of the suspension is removed (200 μΐ): one half will serve as a untransformed negative control and the other half will be transformed with the control plasmid of which. transformation efficiency is known. The remaining suspension is mixed with 100 μΐ plasmid library cDNA at 1 mg / ml and 20 ml of solution II. The mixture is incubated for 1 hour at 28 ° C with shaking and then for 20 minutes at 42 ° C before being centrifuged for 15 minutes at 3000 rpm. The cell pellet is washed once with water and twice with PBS to thoroughly remove PEG, which has a yeast toxic effect, and then resuspended in 2.5 ml of sterile PBS (50 mM Na ₂ HPO ₄ , 10 mM · KCl, 1 mM MgSO ₄₎ . pH 7).

250 ml of YNB + His + Lys + Ade + Met + Leu + Ura medium are inoculated with this suspension and incubated. 4 hours at 28 ° C with shaking to allow expression of hybrid proteins, their possible interactions and expression of the URA 3 reporter gene.

- 24 fc fc • fc the result of this step is also an increase in the number of positive clones. In order to evaluate the rate of colony amplification, 1 ml of culture is taken before and after this incubation and different dilutions are seeded on YNB + His + Lys + Ade + Met + Leu + Ura plates. After cultivation, the number of Leu + Trp + clones is determined in order to evaluate the transformation frequency before and after the incubation period and to derive the amplification rate therefrom. '

The cells are then centrifuged for 10 minutes at 3000 rpm, washed twice with sterile water, resuspended in 10 ml YNB and plated in 10 435 cm ² dishes each containing 200 ml of selective YNB + His + Lys + Ade + Met medium. . Plates are placed in an incubator at 28 ° C for 3 days.

Extraction of plasmid DNA from yeast

This technique extracts both plasmid and genomic DNA contained in yeast. Tibas have been isolated with plasmids, it is sufficient to transform bacteria in which only plasmid DNA can replicate and analyze each clone after miniprep. Yeast containing plasmids are grown on selective solid YNB at 28 ° C overnight. They are then resuspended in 200 ml of disruption solution (2% Triton X100, 1% SDS, 100mM NaCl, 10mM Tris, pH 8, 1mM EDTA) in the presence of glass beads (450 μη on average) and 200ml phenol / chloroform. The mixture was shaken for 15 minutes on a vortex, and then centrifuged for 2 minutes at 14,000 rpm. The aqueous phase was dialyzed for 1 hour on the membrane. This solution can be used to transform bacteria by electroporation.

Detection of beta-galactosidase activity

This assay makes it possible to verify the expression of the LacZ reporter gene encoding β-galactosidase in transformed yeast.

- 25. The nitrocellulose membrane is placed on individualized yeast clones. The impression is immersed 3 times in liquid nitrogen for 30 seconds to disrupt the yeast and allow access to the beta-galactosidase substrate. The membrane is then. -plated with colonies on top of Whatman paper soaked with freshly prepared PBS / Xgal solution (100 μΐ of substrate Xgal at 40 mg / ml in dime tyl formamide in 5 ml PBS) and then placed at 37 ° C, optimal temperature for the enzyme assayed activity.

The time when the blue color that detects β-gal activity appears is highly variable, from a few minutes to several hours. The assay is performed in the presence of a positive control for an interaction that changes rapidly to blue and in the presence of a negative control that remains white.

Drip 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 yeast growth is observed after 2 days incubation at 28 ° C.

Segregation of plasmids

This involves testing whether the transformed yeast phenotype is indeed associated with the appearance of plasmids, using plasmid segregation.

The yeast is grown overnight on complete YPD medium at 28 ° C. This non-selective medium allows the growth of yeasts that retain plasmids after cell division and those that do not retain the plasmids. Various dilutions of this culture are plated in plates so that 50-100 clones are obtained on a YPD medium dish after incubation overnight at 28 ° C. Then

- 26 tests the phenotype of clones by imprinting the plates on the surface (replica)

plating on vel vet) different selective media and po 2 days incubation in 28 ° C se can evaluate the frequency of cells without no plasmid. 8) Methods for testing in vitro interaction Test principle It includes verification interaction between two proteins

using the biochemical affinity chromatography technique. The cell extract containing one of the putative protein partners is added to a carrier to which the test (bait) protein has previously been attached. If there is an interaction between the protein and its possible partner, the second added protein will also be retained on the carrier.

Expression of fusion proteins in E. coli

Fusion proteins are produced in E. coli BL21 (DE3) by pET and pGEX expression vectors that are inducible by IPTG (Studier, 1991).

BL21 (DE3) bacteria pre-transformed with recombinant plasmids are grown overnight, with shaking, in LB medium with the antibiotic used for this selection (ampicillin for pGEX or kanamycin for pET at a concentration of 100 pg / ml). 5 ml of this pre-culture is inoculated into 45 ml of LB + antibiotic medium to obtain a culture of _eO 0 = _0.6 after shaking for 1 hour at 37 ° C. Protein production is then induced by the addition of 0.1 mM IPTG and the bacteria are reintroduced to 37 ° C with shaking for 1 hour to 4 hours. Protein expressing cells are harvested by centrifugation at 6000 rpm for 5 minutes. The pellets can be stored at -20 ° C.

• 9,999

- 27 99 9999

99 9 9

9 9 9 9 9

9 9 9 9 9 9 9 9 9 9

9 9 9 9 9 9

Extraction and analysis of proteins produced in bacteria

The bacterial pellets containing the fusion proteins are resuspended in 5 ml TE (50 mM TrisHCl, pH 8.2, 2 mM EDTA). The cells are then lysed with lysozyme at a final concentration of 0.1 mg / ml and 1% Triton X-100. Cells are incubated for 15 minutes at 30 ° C before being placed on ice to be sonicated until the bacterial suspension becomes liquid. The bacterial lysate is centrifuged for 15 minutes at 10,000 rpm at 4 ° C. The supernatant containing the solubilized fusion protein may. stored at -20 ° C.

At this stage, the various fractions obtained are analyzed by denaturing gel electrophoresis (SDS-PAGE). 60 μΐ of the bacterial suspension prior to centrifugation (complete fraction), 60 μΐ of the supernatant (soluble fraction) and 60 μΐ of the pellet resuspended in 5 ml TE (insoluble fraction) are collected. Proteins contained in different fractions are denatured for 10 minutes at 3.5 ° C in the presence of 1/4 volume of 4HL loading blue (0.2M TrisHCl, pH 6.8, 5% SDS, 5% glycerol, 0.5% 2-mercaptoethanol, bromophenol) blue) and are loaded onto a 12% SDS polyacrylamide gel on which they are separated by electrophoresis according to their molecular weight. After migration at 125 V / cm, proteins are detected by staining with Coomassie Blue.

Solubilization of proteins

Exogenous gene expression in E. coli may be accompanied by the appearance of exogenous protein aggregates that form inclusion bodies. The recombinant protein is then easy to purify by centrifugation, although it is often difficult to solubilize.

Proteins present in the insoluble fractions are denatured with 6M urea and 10mM DTT for 10 minutes at 37 ° C and then renatured by dialysis in three sequential buffers, each for at least 6 hours (5mM TrisHCl, pH 8 ·· ····

28 / 2M urea, 5 mM TrisHCl, pH 8 / 1M urea, 5 mM TrisHCl, pH 8). After 15 minutes of centrifugation at 10,000 rpm, approximately 5-10% of the protein is present in the supernatant, which can be stored at -20 ° C.

Affinity chromatography

For protein retention to occur under good conditions, the protein concentration in the samples should be greater than 40 mg / ml. This concentration can be determined by a colorimetric method (e.g., Bradford reagent).

200 μΐ of the soluble fraction containing unfused GST or GST-ML (mutated presenilin 1 loop) is incubated for 2 minutes at 20 ° C in the presence of 300 μΐ of glutathione resin. The fraction that is not retained by the beads is collected. The resin is washed 4 times with wash buffer (20 mM TrisHCl, pH 8, 150 mM NaCl, 0.1% Triton X100). so as to remove proteins bound by non-specific adsorption. 150 μΐ of GST or GST-ML bound resin is incubated for 1 hour at 4 ° C with shaking, with 600 μΐ of the soluble fraction containing STag or STag-Pep126. The agarose beads are then centrifuged for 1 minute at 3000 rpm and washed 4 times with Wash Buffer buffer after collection of the non-retained fraction.

An aliquot of each fraction (soluble, non-retained and retained by the beads) is loaded onto SDS PAGE gel after denaturation at 95 ° C in the presence of loading blue and aliquots are separated by electrophoresis. The proteins are then transferred to a nitrocellulose membrane and the membrane is stained with Ponceau red (20 µg / ml) to verify that the proteins have indeed been transferred.

In order to specifically detect S-Tag fusion proteins, the nitrocellulose membrane is saturated with 1% gelatin in TBST (10 mM TrisHCl, pH 8, 150 mM NaCl, 0.1% Tween 20) after this.

- 29 this to it to it to it to it to it 15 minutes and incubated for 15 minutes with protein S bound to alkaline phosphatase. After 4 washes with TBST, alkaline phosphatase activity is revealed by adding 1X AP buffer containing NBT and BCIP according to the Novagen method.

9) Methods of expression of recombinant proteins in mammalian cells

Construction of a vector for expression in mammalian cells

For expression in mammalian cells, the sequences were SEQ ID NOs. 1 and SEQ ID NO. No. 2 subcloned by conventional methods into a vector under the control of the strong viral promoter (CMV).

Transfection of cells

The method introduced for COSI or CHO cells is to use a lipofectant in a ratio of 1 to 8 (w / w) relative to DNA and synthetic peptide (H1) in the same ratio to optimize DNA condensation and transfection efficiency. This method is based in particular on neutralizing charges of DNA phosphates by positive charges of the lipofectant.

COSI cells are grown in an incubator at 37 ° C, 95% humidity and 5% CO ₂ in DMEM (Dulbecco's Modified Eagle's Medium) containing 4.5 g / L glucose (Gibco BRL) supplemented with 3% L-glutamine, 1% penicillin-streptomycin and 10% fetal calf serum.

The day before transfection, cells are seeded at density

2.5 χ 10 ⁶ cells per 100-mm dish. On the day of transfection, cells are washed twice with PBS (phosphate buffered saline) and once with OptiMEM (patented formulation, Gibco BRL) to adapt for at least 15 minutes in the incubator.

pg of plasmid DNA in total is added to 300 μΐ OptiMEM and 64 pg of synthetic peptide (H1), corresponding to • 0 ·· · 0

000 · ··

0 0 0 0

0 0 0 · 0 0 0 0

0 0 0 · · 100mm bowl. After vigorous shaking for 10 seconds on the vortex, lipofectamine (32 μΐ, 64 pg) undiluted in 300 μΐ OptiMEM is added to the previous mixture after waiting for 5 minutes. The whole is again vortexed and allowed to stand for 30 minutes. 5 ml of OptiMEM is added to each tube and the shaken mixture is poured over the cells (from which the media was previously aspirated). The cells are then placed in an incubator for 4 hours, after which time the mixture is replaced with complete medium.

Cell lysis and protein assay

Cells are most often lysed 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 protease inhibitor cocktail (Complete8, Boehringer Mannheim). After rinsing with PBS, it is added to each dish. 800 μΐ cold buffer. Lysates are then sonicated followed by magnetic rod stirring at 4 ° C overnight. Cehtrifugation for 30 minutes at 15,000 x g separates the pellet from the supernatant.

Soluble proteins are then tested on a BCA kit (Pierce) to normalize future experiments. '

Immunotransmission

Samples (cell lysates) are denatured in an equal volume of loading buffer (125 mM Tris, pH 6.8, 4% (w / v) SDS, 20% glycerol, 0.02% bromophenol blue, 50 mM dithiothreitol) at 95 ° C. 5 minutes. For analysis of presenilin expression, samples are denatured in the presence of 8M urea and at 37 ° C to prevent aggregation at 95 ° C that is specific to presenilins.

• · · ·

- 31 • fe fefefefe

The samples are run on Tris-glycine gels (Novex), with different percentages of acrylamide depending on the molecular weight to be differentiated. A molecular weight standard (Broad Range, Biorad) is also applied. Migration occurs after approximately 2 hours at a constant voltage of 100 V in 1x final SDS buffer (Novex). The gel is then transferred to a nitrocellulose membrane or a PVDF membrane (1x final transfer buffer (Novex) with 10% methanol) for 2 hours at a constant current of 150 mA.

After transfer, the membrane is blocked for 2 hours at room temperature in 50 ml PBS-T (PBS with 0.5% Tween) containing 2% skim milk (Merck). The primary antibody (diluted to optimal; concentrations of the order of 1/1000 to 1/5000 PBS-T with or without 2% skim milk) is left overnight at 4 ° C. After briefly rinsing with PBS-T, the membrane is incubated for 45 minutes in the presence of a secondary antibody (anti-mouse or anti-rabbit IgG depending on the case, conjugated with horseradish peroxidase) diluted 1/5000 with so-called ECL buffer (20mM Tris, 150mM NaCl, 0.1% Tween).

The membrane is then rinsed 4 times for 15 minutes in ECL buffer. It may be necessary to prepare the ECL reagent fresh by mixing an equal volume of the two buffers it consists of. Various exposures of the photographic film (Hyperfilm ECL, Amersham) are performed followed by developing.

- 32 • to totototo • to tototo • to this to * · · • ·· to • ··>

• ·· · ·· ··

DETAILED DESCRIPTION OF THE INVENTION

Example 1 '

Isolation of PS-1 specific partners by screening the fusion cDNA library from the brain

In order to isolate PS-1 specific partners, a cDNA library was screened from the human brain using a double hybrid technique. A cDNA library fused to AD GAL4 was used and various bait proteins were constructed by fusing the hydrophilic regions of PS-1 with DBD GAL4.

1.1 Construction of vectors allowing expression of proteins from fusion between DBD GAL4 and hydrophilic regions of PS-1

Five fusion proteins were generated using the pGBT9 vector into which they were introduced, sequences corresponding to different hydrophilic regions of PS-1 in the same reading frame as the DBD GAL4 sequence. Proteins interacting with the N-terminal region (Nt, codons 1-81), the large loop (BL, codons 263- 407), the large loop with the L286V mutation (BM), and the shorter large loop region (BR, codons) were selected for identification. 290-376) and finally the C-terminal portion of the protein (Ct, codons 421-467).

DNA fragments corresponding to these different regions were obtained by PCR from the entire cDNA sequence of PS-1. The various oligonucleotides used allowed the introduction of EcoRI α-SalI sites at the ends of each amplified sequence. Thus, it was possible that the PCR fragments were inserted between the EcoRI and SalI sites of the multiple cloning site of pGBT9 downstream and in accordance with the DBD GAL4 sequence. The various constructs obtained, pGBT-Nt, pGBTBL6, pGBT-BM, pGBT-BR and pGBT-Ct, were verified by sequencing> fcfc fc fcfc fcfcfc fcfcfcfcfc fcfcfc

fcfcfc • ·

DNA. This verification showed that the fragments did not show any mutation resulting from PCR and that. they were indeed in the same open reading frame as the DBD GAL4 sequence.

1.2 Production of yeast strains expressing proteins from fusion between DBD GAL4 and hydrophilic regions of PS-1

Since yeast co-transformation (co-transformation) with two different plasmids is less frequent than transformation with a single plasmid, a yeast strain pre-transformed with a vector encoding the bait protein was used to achieve the best possible efficiency during screening of the cDNA library.

To this end, the yeast strain YCM 79 was transformed with each of the plasmids pGBT-Nt, pGBT-BL6, pGBT-BM, pGBT-BR and pGBTCt. The yeast containing these plasmids was selected according to their Trp + phenotype on a medium that did not contain tryptophan and named respectively yNt, yBL6, yBM, yBR and yCt. The presence of these different plasmids in each strain was verified by performing PCR directly on yeast, using oligonucleotides complementary to the DBD and tADH1 sequences located at the 5 'and 3' ends of the insert. The size of the PCR fragments obtained makes it possible to distinguish different strains except for the yBL6 and yBM strains.

1.3. Tests of fusion proteins containing hydrophilic regions of PS-1 for transactivation activity and for interaction with AD GAL4

If one of the fusion proteins has transactivating activity or interacts with AD GAL4, it cannot be used as bait in the double hybrid system, since it systematically gives a protein reaction in the absence of each interaction.

The first test, therefore, consists in checking that the hydrophilic regions of PS-1 do not have transactivating activity when they are fefe · fe ····

fefe are fused to DBD GAL4, i.e. they are capable of inducing the transcription of reporter genes by themselves. This type of activity may be associated, for example, with the presence of an acidic domain in the fusion. protein.

For this assay, expression of the LacZ and URA3 reporter genes was directly directed in the yNt, yBL6, yBM, yBR and yCt strains.

For the second assay, which is to control the lack of interaction between fusion proteins and AD GAL4, strains of yNt, yBL6, yBM, yBR and yCt of the Trp + Leu phenotype were transformed with plasmid pGAD424 (identical to pGAD10), which encodes AD GAL4. The yeast was picked on medium without tryptophan or leucine and expression of the LacZ and URA3 reporter genes allowing interaction control was tested on the obtained clones.

In all cases, the results of the tests for β-galactosidase activity as well as the drip tests on selective media without uracil were negative.

Thus, fusion proteins comprising the hydrophilic portions of PS-1 do not have a substantial transactivating property and do not interact with the GAL4 activation domain. They can therefore be used as bait proteins for screening libraries.

1.4 Transformation of various yeast cDNA strains by fusion library

Screening of the fusion library makes it possible to identify clones expressing peptides fused to AD GAL4 that interact with the bait protein. If this interaction exists, it should allow for the reconstitution of a functional transactivator capable of inducing the expression of the LacZ and URA3 reporter genes in the bait protein-containing strain.

For screening to be representative, each independent plasmid forming the library after transformation must be present in at least one yeast clone. For this purpose • · 999 9 ·

9

A protocol was used that theoretically allowed to obtain a number of .transformants greater than the number of independent plasmids in the library (cf. Materials and Methods).

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

At the end of the first selection, 32 clones were obtained which had a Ura + phenotype in the yNt strain, 450 Ura + clones in yBL6, 128 in yBM, 250 in yBR and 67 in yCt. To verify the expression of the second LacZ reporter gene, a β-galactosidase activity assay was performed on these.

transformants

A total of 6 clones had a Ura +, P-gal + double phenotype: 1 clone for the yNt strain, 1 clone for the yBL6, 2 clones for each of the yBM and yBR strains, and no clone for yCt. The clones were respectively named yNt.A, yBL6.B, yBM.C, yBM.D.

Example 2

Isolation of plasmids from library

Tibs were identified for proteins capable of interacting with the mutated loop or reduced loop PS-1, and the plasmids contained in yBM.C and yBM.D. were extracted. The plasmids present in the DNA preparations were used to transform bacteria by electroporation.

Plasmids extracted from. bacterial clones were digested with various enzymes. This analysis identified 5 different restriction profiles.

Cleavage of plasmids extracted from yBM.C and yBM.D showed a general profile corresponding to plasmids pGBT-BM and two

36 different profiles corresponding to plasmid pGAD10 which contained an insert of 2900 bp corresponding to SEQ ID NO. No. 1 (pGAD-C derived from the yBM.C strain) and a 1400 bp insert corresponding to SEQ ID NO: 1. No. 2 (pGAD-D derived from yBM. D strain).

2.1. Control of plasmids isolated by transformation of various yeast strains

During transformation of yeast with plasmids from the brain library, it is possible that several different plasmids are introduced into the same cell. In order to verify that the results of the interaction are indeed due to the presence of separate and non-surplus plasmids, fusion proteins derived from the screening library were again tested against various hydrophilic regions of PS-1 in YCM 79, but also in HF7 C and PCY 2 strains.

HF7 C has two reporter genes, HIS3 and LacZ. The LacZ gene has a different promoter environment; from that which exists in the YCM 79 strain, which makes it possible to verify that the production of β-galH- clones is not associated with a specific promoter environment, but with an interaction between the proteins tested. PCY 2 has the advantage of having only one LacZ reporter gene that is more strongly expressed than in the other two strains where the GAL4 transactivator is functional.

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

Taking into account the variation in the sensitivity of the tests, the interaction is confirmed when the transformed yeast YCM 79 and the YCM 79 are transformed. · · ·· · »

37 HF7 C develop on selective media without uracil or without histidine, i.e. that they confidently express at least one of their reporter genes.

It is believed that the intensity of the reaction (colonies that are blue to a greater or lesser degree, or that evolved to a greater or lesser degree) is in proportion to the degree of expression of the reporter genes. Variations in this step can be explained in various ways. The low degree may result from poor interaction between proteins that reconstitute the GAL4 transactivator. It may also be due to the intervention of a third protein that could destabilize complex formation, or alternatively, to the instability of the fusion proteins in yeast, or possibly to the state of the yeast. Yeast may be in a state of low transcriptional activity associated, for example, with the problem of toxicity of exogenous proteins.

Fusion proteins from the brain library that have been isolated do not interact with the N-terminus and the C-terminus of PS-1. On the other hand, they all appear to interact with the hydrophilic region of BL6, the mutated hydrophilic region of BL6 (BM), as well as the reduced form of this region (BR).

2.2 Assays for Interaction of Library Fusion Proteins with Non-PS-1 Loop Proteins

This assay makes it possible to determine whether library-derived fusion proteins interact non-specifically through their structure, their charge, their hydrophobicity with bait proteins (false positivity type III) or DBD GAL4 (false positivity type II).

Strains YCM 79, HF7 C and PCY 2 were transformed with plasmid pairs: pGBT9 / pGAD-Y (Y corresponding to C or D), pGBTLAM / pGAD-Y, pGBT9 / pGAD424 (negative control) and pGBTCtAPP / pGAD-Fe65 (positive control) . pGBT9 only encodes DBD ····

000 · ··· ·

0 0 0 · · ·

0 0 0 0 0 t 0 0 000 · 0 · 00 00 0 0 · 00 00 '<0 · ··

- 38 GAL4, while pGBT-LAM provided by Clontech encodes a protein from a fusion between DBD GAL4 and a lamin that interacts non-specifically with the hydrophobic regions of other proteins.

As a result, none of the yeasts thus transformed were capable of expressing their reporter genes. Thus, yeast YCM 79 exhibited the phenotype of Ura-, β-gal-,. yeast HF7 C were all His-, β-gal- and yeast PCY2 β-gal-.

These results suggest that library fusion proteins that are thus isolated interact specifically with the hydrophilic region of BL6 of PS-1 and do not bind lamin or DBD GAL4.

2.3. Interaction assay of yeast transformed with pLex 9 vector containing the PS-1 loop coding region.

This assay makes sure that protein interactions are not due to the structure taken up by different fragments from BL6 only when fused to DBD GAL4.

To verify this, the BL6 and BM fragments are fused to another DBD that is from the LexA bacterial repressor. Two additional plasmid constructs (pLex-BS and pLexBM) were generated where the BL6 hydrophilic loop sequence and the mutated forms were fused to the LexA gene at the EcoRI / SalI site of plasmids pLex9.

Further, a yeast strain L40 was transformed in which expression of the HIS3 and LacZ reporter genes is induced by the attachment of the DBD-LaxA / AD-GAL4 complex to the LexA operator site. The interaction with plasmid pairs 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, pLexBM / pGAD-RAF (negative interaction control).

• Φ φ · · · • • • • • • • • • • •

- 39 φ φ

φ •

φ φ φ φ

The results of the drip assays for selective media without histidine and for β-gal activity were positive for strains containing plasmid pLex-BL6 regardless of the plasmid pGAD-Y tested. The same applies to strains containing pLex-BM regardless of the plasmid pGAD-Y tested. This assay showed that the interaction obtained is not associated with the particular structure that the BL6 region of PS-1 assumes when fused to the GAL4 DNA-binding domain.

Example 3

Identification of plasmid inserts derived from screening

3.1. Sequencing of plasmids pGAD-C and pGAD-D.

The inserts of plasmids pGA.D-C and .pGAD-D were completely sequenced. Sequencing was initiated using oligonucleotides complementary to the AD sequence of GAL4 and tADH1 to identify the sequence at the beginning and at the end of each insert. Synthetic oligonucleotides were then prepared at the end of each sequence to allow sequencing on both strands. In this way, the sequencing of the 1400 bp insert D corresponding to SEQ ID NO: 2 was completed. On the other hand, for the 2900 bp C insert (SEQ ID NO: 1), it was necessary to first perform restriction mapping and then to subclone the various fragments to obtain the entire sequence.

The entire insert D sequence was an open reading frame (SEQ ID NO: 2). In contrast, insert C contains a stop codon at the end of the sequence (SEQ ID NO: 1).

to · ····

• to · to · to · to ··· · ·

Their peptide sequences exhibit a somewhat hydrophilic profile, which is in accordance with the selected screening method that is more suitable for hydrophilic proteins.

3.2. Sequence comparison

Sequence comparison will make it possible to determine whether the inserts encode peptide domains whose function is known.

Nucleic and peptide sequences of the inserts were compared to sequences from the Gen Bank and EMBL (European Laboratory of Molecular Biology) databases. No similarity was found between the inserts and the genes (or proteins) recorded in these databases.

The sequences of the inserts were also compared to each other. They show no resemblance.

Furthermore, the occurrence of peptide motifs capable of providing some information about the function of the peptides encoded by the inserts was evaluated. Several potential sites for glycosylation, phosphorylation and myristilation have been identified, but this was not enough to formulate a hypothesis about the possible function of polypeptides.

3.3., MRNA expression corresponding to peptides selected from various human tissues

To confirm that the interaction between the two peptides and PS-1 actually exists in vivo, it was verified that the expression of mRNA peptides isolated from the library was localized to the same tissues as the expression of PS-1 mRNA

The expression of genes containing isolated sequences in specific tissues was evaluated and the level of mRNA expression of the corresponding inserts C and D in various human tissues was analyzed by northern blotting.

The radioactive probe prepared from insert C (SEQ ID NO: 1) allowed hybridization of 9.5 kb mRNA expressed specifically in the brain and most often in the cerebral • 0 ♦

0 0 0 ·

- 41 0 0 0 0 0 0 0 0 0 0 * 0 0 0 0 0 0 0 00 0 000 0 · · 0 00 0 «0 · 0 00 0» * · ** cortex, frontal lobe and temporal lobe (cf. 3). The size of these mRNAs suggests that the translated proteins are of considerable size.

Example 4

In vivo interaction test

This test made it possible to determine whether the interactions were dependent on the yeast context. Reconstitution of a functional GAL4 transactivator is not only due to the direct interaction between two proteins, but may also result from the formation of a protein complex between the fusion proteins and one or more endogenous yeast proteins. Thus, interactions can only exist in the presence of yeast-mediated intermediate proteins, and outside this context there are no such interactions.

Therefore, a cell-free system was selected to confirm interactions between the hydrophilic stretch of BL6 from PSI and isolated peptides called PepC and PepD.

Proteins were produced in E. coli BL21 (DE3) to obtain large quantities. Both isolated peptides were fused to the marker peptides to allow their detection and binding to the carrier. For this purpose, GST protein, which binds to glutathione, to various forms of the BL6 region and to the S-Tag peptide specifically recognized by protein S, was fused to the isolated peptides (see Materials and Methods for the principle of this method).

- 42 4.1. Production of fusion protein between S-Tag and peptides isolated in library screening

In order to construct fusion proteins between S-Tag and peptides PepC and PepD, various EcoRI / EcoRI fragments obtained from plasmid pGAD-C and pGAD-D were inserted into the pET-29a vector in the same reading frame as the S-Tag sequence.

At this stage, a plasmid (pET-D) containing an insert corresponding to PepD was obtained. Partial sequencing of this plasmid showed that fragment insertion was performed in accordance with the desired reading frame.

This plasmid allowed the production of a 52 kDa protein corresponding to the STag-PepD fusion protein. The STag-PepD protein is. produced in BL21 (DE3) after induction of IPTG.

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

For fusion of GST with one of the three forms of the BL6 hydrophilic region of PS-1, the vector pGEX-4T1 was used, into which EcoRI / SalI fragments obtained from plasmids pGBT-BL6, pGBT-BM and pGBT-BR were introduced.

The plasmid pGEX-BM corresponding to the mutated PS-1 loop was obtained. Sequencing showed that the insertion was indeed performed in the reading frame of the GST sequence.

This plasmid allowed the production of a 42 kD GST-ML fusion protein in BL21 (DE3) after induction of IPTG.

4.3 Solubilization of the GST-ML fusion protein

After lysis of the bacteria, the GST-ML protein remained exclusively in the insoluble fraction, while the STag peptide, as well as the STag-PepD protein and GST, were partially found soluble in the fraction.

Solubilization of fusion proteins is absolutely necessary in order to perform affinity chromatography. First, it was protein

4 · · · · ·

- 43> · 4 4

4 4

GST-ML specifically linked to glutathione. resin covalently bound to agarose beads to sequentially test the interaction of GST-ML with PS-1 peptide partners fused to STag (cf. Material and Methods). If proteins are not solubilized, they sediment with pearls to which they do not specifically bind.

Following solubilization of the GST-BM fusion protein, the assay was performed under the conditions described in Section 8, Materials and Methods.

Example 5

Expression of PS-1 specific partners in mammalian cells

This assay makes it possible to verify that protein interactions revealed in yeast are also detectable in connection with mammalian cells and with the complete PS-1 protein inserted into their membrane environment. SEQ ID NO. 1 and SEQ ID NO. No. 2 of the partners were subcloned into the mammalian cell expression vector pCDNA3 carrying at the 5 'end another sequence corresponding to the myc epitope and an EcoRI restriction site (Figure 4) allowing subcloning according to the sequences of the partners.

A restriction fragment was isolated. EcoRI of 1400 bp and was subcloned in accordance with the EcoRI site of the expression vector. Constructs carrying CDNAs in the correct orientation were selected by enzyme restriction analysis.

For clone C, the EcoRI (1) -HindIII (391j) fragment from pGAD-C was first isolated, and a second HindIII (392) -MscI (2892) fragment was isolated by partial digestion (the second blunt-ended site is present in the pGAD10 vector). downstream of the coding region of clone C. EcoRI (1) -HindIII (391) fragments feefefe ·

- 44 «fefefefe fe

fefe fefe-fefe-fefe-fefe-fefe-fefe-fefe-fefe and HindIII (392) -MscI (2892) were ligated together with an EcoRI-and EcoRV digested expression vector, the second site being blunt-ended. The correct constructs carrying the entire coding sequence C were identified by restriction analysis.

These constructs allow the generation of myc epitope-bearing fusion proteins and peptides corresponding respectively to SEQ ID NOs. 1 and id. No. 2.

5.2 Expression in mammalian cells

These constructs were transfected into GOS1 cells using standard methods. After cell lysis, samples were analyzed by immunotransmission and antibody testing. anti-myc. Partner expression was detected by immunotransfer using anti-Myc 9E10 antibody (Figure 5). It was possible to detect a very strong band of 120 kD, as expected with PepC, demonstrating high expression (lane C). In D, the intensity of the band detected was not always constant. On the other hand, it was possible to detect expression in CHO cells. In this cell type, D may be slightly more stable and appears to migrate at approximately 80 kD (lane D).

It was therefore possible to express and detect these two proteins in mammalian cells.

«· ··» ·

- 45 LIST OF SEQUENCES (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 1: '(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2892 bp (B) TYPE: nucleotide (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) REVERSE ORIENTATION: no (ix) FEATURES:

(A) NAME / MARKING: ODS (B) POSITION: 1..2736 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 1:

CGG ATG Arg Met 1 ATT CCC ATC TTT CAT GAC ATG Met ATG GAC TGG GAG AGA AAA 48 Ile For Ile Phe His Asp Met 10 Asp Trp Glu Gin. Arg 15 Dec Lys AAT GGC AAC TTC AAA CAG GTG GAG GCC GAG TTG ATT GAC AAG CTG GAC 96 Asn Gly Asn Phe Lys Gin Wall Glu Ala Glu Leu Ile Asp Lys Leu Asp 20 May 25 30 AGC ATG GTG TCA GAA GGG AAA GGT GAC GAG AGC TRAY AGG GAG CTC TTC 144 Ser Met Wall 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 TRAY CCC AGC CTG CTG GAG AAG 192 Ser Leu Leu Thr Gin Leu Phe Gly For Tyr For Ser Leu Leu Glu Lys 50 55 60 GTT GAA CAA GAA ACA TGG CGC GAG ACC. GGC ATT TCC TTT GTG ACC TCA 24 0 Wall Glu Gin Glu Thr Trp Arg Glu Thr Gly Ile Ser Phe Wall Thr Ser 65 70 75 80 GTC ACC CGC CTC ATG GAA CGT CTT CTT GAC TRAY AGG GAC TGC ATG AAA 288 Wall 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 Wall Asn Leu Met

100 105 110

0 ··

0000 «0

- 47 - 4 0 00 « 0 * ) 0 0 '0' · 0 0 * • « 0 0 0 0 0 < 0 0 0 0 * 0 «» · • · · • · · . · · CTG CAG ATG GAT AGG GTA CCA GAT CGA GTC AAG AGC TTC MELT CGC GTC 1008 Leu Gin Met Asp Arg Wall For Asp Arg Wall Lys Ser Phe Tyr Arg Wall 325 330 335 AAC AAT GTG AGG AAG TTC CGG MELT GAC AGG CCT TTT CAC AAA GGC CCC 1056 Asn Asn Wall Arg Lys Phe Arg Tyr Asp Arg For Phe His Lys Gly For 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 AGČ TTG CCT GGC ATC TCT CGG TGG TTT GAA GTG 1152 Leu Thr Leu Thr His Ser Leu For Gly Ile Ser Arg Trp Phe Glu Wall 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 Wall Glu Wall Ser For Leu Glu Asn Ala Ile Gin 385 390 395 400 GTG GTT GAG AAT AAG AAC CAG GAG CTA CGC TCC CTG ATC AGC CAG MELT 1248 Wall Wall Glu Asn Lys Asn Gin Glu Leu Arg S'er Leu Ile Ser Gin Tyr 405 410 415 CAA CAC AAG CAG GTG CAT GGC AAC ATT AAC CTG CTA AGC ATG TGC CTG 1296 Gin His Lys Gin Wall 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 MELT CAG 344

Asn Gly Wall 435 'Ile Asp Ala Ala Wall 440 Asn Gly Gly Ile Ala 445 Arg Tyr Gin GAG GCC TTC TTT GAT AAA GAT TRAY ATC AAC AAG CAC CCA GGA GAT GCT 1392 Glu Ala 450 Phe Phe Asp Lys Asp 45.5 Tyr Ile Asn Lys His 460 For Gly Asp Ala GAG AAG ATC ACC CAG CTC AAG GAG CTT ATG CAG GAG CAG GTT CAT GTC 1440 Glu 465 Lys Ile Thr Gin Leu 470 Lys Glu Leu Met Gin 475 Glu Gin Wall His Wall 480 CTT GGA GTT GGG CTA GCA GTT CAT GAG AAG TTT GTG CAC CCA GAA ATG 488 Leu Gly Wall Gly Leu 485 Ala Wall His Glu Lys 490 Phe Wall His For Glu 495 Met CGG CCT CTG CAT AAG AAG CTA ATT GAT CAG TTC CAG ATG ATG CGG GCC 1536 Arg For Leu His 500 Lys Lys Leu Ile Asp 505 Gin Phe Gin Met Met 510 Arg Ala AGT CTC TRAY CAT GAG TTT CCA GGT TTG GAT AAG CTA AGT CCT GCA TGT 1584 Ser Leu Tyr 515 His Glu Phe For Gly 520 Leu Asp Lys Leu Ser 525 For Ala Cys TCA GGC ACC AGC ACC CCA CGG GGA AAT GTT CTG GCA TCC CAT AGC CCC 1632 Ser Gly Thr Ser Thr For Arg Gly Asn Wall Leu Ala Ser His Ser For

530 535 540

·· · «·· ·« this ··

ATG AGT CCG GAG AGC ATC AAG ATG ACC Met 545 Ser For Glu Ser Ile 550 Lys Met Thr TTG ATG GGC ACA GGC CGC CAT TCA TCA Leu Met Gly Thr Gly Arg His Ser Ser TCT AGT GAA GCA GGA AAC ATG GTG ATG Ser Ser Glu Ala 5S0 Gly Asn Met Wall Met 585 GAT GCT CCT GAG GAC CTG TRAY CAC CAC Asp Ala For 595 Glu Asp Leu Tyr His 600 His CCC AGG TRAY CAA GGC TCA GTC ACC AAC For Arg 610 Tyr Gin Gly Ser Wall 615 Thr Asn CAG GCA AGC CCT TCT TCC TCC AGC CTG Gin 625 Ala Ser For Ser Ser. 630 Ser Ser Leu TCC CAG ATG ATT ACC TCT GCC CCT TCC Ser Gin Met Ile Thr 645 Ser Ala For Ser CAT GCC CGT GÁA ATG ATG TTG TTG CTG His Ala Arg Glu 660 Met Met Leu Leu Leu 665 AGC AGT GCC ATG MELT CCA GCA GCC ATC Ser Ser Ala 675 Met Tyr For Ala Ala 680 Ile AAT TTC CAG CGA GCC CTG TTC CAG CAA Asn Phe 690 Gin Arg Ala Leu Phe 695 Gin Gin TGC AGT GAT CCC AAT CTG TCT GTG GCT Cys · 705 Ser Asp For Asn Leu 710 Ser Wall Ala CAC TTT GAC GCC TTC CAC CAC CCT CTG His Phe Asp Ala Phe 725 His His For Leu CCT GCC CGG ACC CTG CGC AAG TCT CCT For Ala Arg Thr 740 Leu Arg Lys Ser For 745 CCC ACA AGC CCC CAG TCA GGT CTG GAC For Thr Ser 755 For Gin Ser Gly Leu 760 Asp

• to 9 to • to •.

Toto to to to • toto toto toto toto toto toto toto toto toto toto toto toto toto toto

CAC CGG CAC £ GC CCC ATG AAC 1680 His Arg 555 His Ser For Met Asn 560 TCC TCT CTC TCC TCA CAT GCG 1728 Ser 570 Ser Leu Ser Ser His 575 Ala CTG GGT GAC GGC TCC ATG GGT 1776 Leu Gly Asp Gly Ser 590 Met Gly ATG CAG CTC GCG MELT CCC AAC 1824 Met Gin Leu Ala 605 Tyr For Asn GTC TCT GTT CTG TCC TCG TCC 1872 Wall Ser Wall 620 Leu Ser Ser Ser AGT TCC ACT CAC TCA GCA CCA 1920's Ser Ser 635 Thr His Ser Ala For 64 0 AGT GCC CGA GAT AAG TRAY CGC 1968 Ser 650 Ala. Arg Asp Lys Tyr 655 Arg CCC ACA TRAY CGG GAC CGC CCA 17. Nov. 2016 For Thr Tyr Arg Asp 670 Arg For CTG GAG AAC GGA CAG CCG CCG 2064 Leu Glu Asn Gly 685 Gin For For GTG GTC GGA GCC TGC AAA CCC 2112 Wall Wall Gly 700 Ala Cys Lys For GAA AAA GGT CAT TRAY TCC CTA 2160 Glu Lys 715 Gly His Tyr Ser Leu 720 GGT GAT ACC CCC CCA GCC CTC 2208 Gly 730 Asp Thr For For Ala 735 Leu CTC CAC CCT ATC CCA GCC TCC 225S Leu His For Ile For 750 Ala Ser GGC AGC AAC TCT ACG CTG TCC 2304 Gly Ser Asn Ser Thr Leu Ser

765

this this »·

- 49 • to to to to to to to 49 49 49 49 49 49 to

GGC AGT GCC AGC TCC Ser TCC TTG AGT GAG 2352 Gly Ser Ala Ser Ser Gly Val Ser Leu Ser Glu Ser Asn 780 Phe Gly CAC TCC TCG GAG GCC CCA CCT CGC ACT GAC ACC ATG GAC TCC ATG CCA 2400 His Ser Ser Glu Ala For For Arg Thr Asp Thr Met Asp Ser Met For 785 790 795 800 AGT CAG GCC TGG AAT GCT GAC GAA GAT CTT GAG CCA CCC TRAY CTC CCT 2448 Ser Gin Ala Trp Asn Ala Asp Glu Asp Leu Glu For For Tyr Leu For 805 810 815 GTC CAC TRAY AGC CTC TCT GAG TCT GCC GTC CTG GAC TCC ATC AAG GCC 2496 Wall His Tyr ' Ser Leu Ser Glu Ser Ala Wall 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 Gin For Cys Arg Ser His Ser Ala For Gly Cys Wall Ile For Gin Asp 835 840 845 CCC ATG GAC CCG CCT GCG CTG CCG CCC AAG CCC TRAY CAC CCC CGC CTG 2592 For Met Asp For For Ala Leu For For Lys' For Tyr His For Arg Leu 850 ♦ 855 860

CCG GCC CTG GAG CAC GAT GAG GGG 2640 For Al Leu 865 Glu His Asp 870 Glu Gly Wall Leu Leu Arg Glu Glu Thr Glu 880 AGG CCT CGA GGC CTG CAC CGC AAG GCT CCA TTG CCT CCT GGG AGC GCT 2688 Arg For Arg Gly Leu His Arg Lys Ala For Leu For  For 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 Gin Ala Arg Met Ala Trp Glu His Gly Arg Gly Glu Gin 900 905 910

TGAGGGGCAA CGAGGCGGCT GGGATGCCGC CCTCAGTAAG CAGCTTGCCA ATCACTCCAG 2796

GTCTGAAAAG CAAGTCCCCC AGCCCCACCC CAGGGAGCCA GAGAGGCTTG CACTCAGGAG 2856

AGAACCACCC

CCAAGTCTCC GTTCTACTGC CGCCCG

2892 fe · ···· · fefefefe fefe fefe • fe fefefe · fefefefe • · fe ·· fe fe · · · · fefefe fefe fefe · • · fe ··· fefe ·· · fefefe · fefe fefe · ·

- 50 (2) INFORMATION FOR THE SEQUENCE WITH IDENTIFICATION NUMBER 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1363 bp (B) TYPE: nucleotide (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) REVERSE ORIENTATION: no (ix) FEATURES:

(A) NAME / MARKING: CDS (B) POSITION: 1..1363 (xi) SEQUENCE DESCRIPTION: SEQUENCE WITH IDENTIFICATION NUMBER 2:

GAA Glu TTC Phe CGG Arg 915 GGT Gly TGT Cys GTG Wall AAG Lys ATG GAG TTT CCC 48 Met 920 Glu Phe For Gly Gly 925 Asn Asp Asn TRAY CTG ACG ATC ACA GGG CCT TCG CAC CCC TTC CTG TCA GGG GCC GAG 96 Tyr Leu Thr Ile Thr Gly For Ser His For 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 For Ser Leu Gly Asp Glu Glu Phe Glu Ile. For For 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 For Ser Leu Ala Wall Ser Asp Wall Wall Gly ? 65 970 975 CAC TTT GAT GAC CTG GCA GAC CCT TCC TCT TCA CAG GAT GGC AGT TTT 240 His Phe Asp Asp Leu Ala Asp For Ser Ser Ser Gin Asp Gly Ser Phe 980 985 990 TCA GCC CAG MELT GGG GTC CAG ACA TTG GAC ATG CCT GTG GGC ATG ACC 288 Ser Ala Gin Tyr Gly Wall Gin Thr Leu Asp Met For Wall 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 Gin Gly Gly Gly Leu Leu Ser Gly Gly Leu Thr 1010 1015 1020 ATG GAC TTG GAC CAC TCT ATA GGA ACT CAG MELT AGT GCC AAC CCA CCT 384 Met Asp Leu Asp His Ser Ile Gly Thr Gin Tyr Ser Ala Asn For For

• · φ · φ ·

-51φ φφφφ φ

φ

Φ 9

Φ

Φ Φ Φ Φ Φ Φ Φ Φ

Φ Φ Φ Φ Φ

ΦΦΦ Φ

ΦΦ ΦΦ

Φ Φ «Φ Φ Φ Φ Φ

Φ Φ

1025 1030 1035 1040 GTT ACA ATT GAT GTA CCA ATG ACA GAC ATG ACA TCT GGC GGG 432 Val Thr Ile Asp Val Pro Met Thr Asp 1045 Met Thr Ser Gly 1050 1055 CAT AGC CAG TTG ACC ACC ATT GAT CAG TCA GAA CTG AGC TCC CAG CTG 480 His Ser Gin Leu Thr Thr Ile Asp Ser Glu Leu Ser Ser Gin 1060 1065 1070 Leu GGT TTG AGC CTA GGG GGT ACC ATC CTG CCA CCT GCC CAG TCA CCT 528 Gly Leu Gly Gly Thr Ile 1075 1080 Leu Pro Pro Ala Gin Ser 1085 For GAA GAT CGT CTT TCA ACC ACC CCT TCA CCT ACT AGT GAA 576 Asp Glu 1090 Leu Ser Thr Thr Pro Ser 1095 Pro Thr Ser Ser Leu His 1100 Glu GAT GGT GTT GAG GAT TTC CTT CCC AGC CAG AAG ACA GTC 624 Asp Gly Val Glu Asp Phe Arg A.rg Gin 1110 Leu Pro Ser Gin Lys Thr 1115 Wall 1120 GTG GTG GAA GCA GGG AAA AAG GCC CCA AAG AGA AAA AAG 672 Val Glu Ala Gly Lys Gin Lys 1125 Ala Lys Lys Arg Lys Lys 1130 1135 AAA GAT CCT AAT GAA CCT CAG GTT TCA GCA TAT GCT TTA TTu 72 Lys Asp Pro Asn Glu For Gin Lys For Val Ser Ala Tyr Ala Leu 1140 1145 1150 Phe TTT CGT GAT ACA CAG GCT ATCC AAG GGA CAG AAT ACT 768 Phe Arg Asp Thr Gin Ala Ala Ile Lys 1155 1160 Gly Gin Asn Pro Asn Ala Thr TTT GGT GAG GTT TCA AAA ATT GTG GCC TCG ATG TGG GAT AGT CTT GGA 816 Phe Gly Glu 1170 Val Ser Lys Ile - Val Ala '1175 Ser Met Trp Asp Ser Leu Gly GAG GAG CAA AAA CAG GAT TAT AAG AGG AAA ACT GAG GCT GCC AAG AAA 864 Glu 1185 Lys Gin Val Tyr Lys Thr Glu Ala Ala Lys Lys 1200 GAG TAT CTG AAG GCA GCT GCT GCT TAC AAA GAC CAG 912 Glu Tyr Leu Lys Ala Leu Lys Asp Asn Gin 1210 1215 GCC ACT GTG GAA ACA GTG CCA CCA CCA CCA TCA CAA ACT 960 Ala Thr Val Glu Thr Val Glu Leu Asp 1220 1225 Pro Ala Pro Pro Ser Gin; 1230 Thr CCT TCT CCA CCT ATG GCT ACT GTT GCT CCA GCA TCT CCA GCA CCA 1008 Pro Ser Pro 1225 Pro Pro Ala Ala Pro Pro Ala 1240 1245 For

- 53 • · • 0 0 ·

9

9999 9

0

0

0 '0

0000

0 0 0

0 0 0

0 0 ·

0 0 9

9 9 9

99

GCT Ala TCA ATA GAG CCC CCT GCC CTG TCC CCA TCC ATT 1056 Ser Ile 1250 Glu For For Leu Ala ¹²⁵⁵ Ser Pro Ser Ile Val Wall Asn Ser ACC CTT TCA TCC MELT GTG GCA AAC CAG GCA TCT TCT GGA GCT GGG GGT 1104 Thr Leu Ser Ser Tyr Wall Ala Asn Gin Al'a 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 Gin Pro Asn Ile Thr Lys Leu Ile Ile Thr Lys Gin Met Leu For 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 Gin Gly Gly Met Val Thr Val Ile For Ala Thr 1300 1305 1310 GTG GTG ACC TCC CGG GGG CTC CAA CTA GGC CAA ACC AGT ACA GCT ACT 1248 Wall Val Thr Ser Arg Gly Leu Gin Leu Gly Gin 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 Gin Pro Ser Gin Gin Ala Gin Ile Val Thr Arg Ser Wall Leu Gin 1330 1335 134 0 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 Gin Leu For For For 1345 1350 1355 1360 CGA GTA CAG CCC GGA ATT C 1363 Arg Val Gin For Gly Ile

1365

0000

0000 0

00 0 · 0 0 0 ·

0 0 0 0 · · 0 0 00 00 0

0 0 0 0 0

00 00 00

PATENTOVÉ

J> l / - Wo

Claims (14)

Claims A nucleotide sequence comprising the entire sequence of id. No. 1, i or a portion thereof that encodes a protein that is. able to interact with presenilins. 2. A nucleotide sequence comprising the sequence of SEQ ID NO. No. 2 or a portion thereof or derivatives thereof that encodes a protein capable of interacting with presenilins. 3. Polypeptides comprising a peptide sequence selected from the group consisting of SEQ ID NO. No. 1 according to claim 1 or SEQ ID NO. No. 2 or derivatives thereof according to claim 2, or a part thereof, wherein the polypeptides are capable of interacting with presenilins. 4. Polypeptides according to claim 3, with a large loop of presenilins which PS-1. j sou capably interaction The polypeptides of claim 3, which j sou capably interaction with an amino acid sequence presenilins PS-1. 310 up to 463 large loops > 0 ' The polypeptides of claim 3, with a large loop of presenilins which PS-2. j sou capably interaction Use of the polypeptides according to any one of claims 3 to 6 for the preparation of a medicament intended to slow, stimulate or modulate the activity of presenilins. • fe ·· ♦ · fe .fefefe · · · · · ♦ ······ · · fe ··· fe fefefe • fefefe · fefe fefe fefe fefe - 54 A method for preparing a polypeptide according to any one of claims 3 to 6, characterized in that the cells comprising the nucleotide sequence according to any one of claims 1 to 2 are cultured under conditions where the sequence is expressed and the generated polypeptide is isolated. A host cell for producing a polypeptide according to any one of claims 3 to 6 which has been transformed with a nucleic acid comprising a nucleotide sequence according to any one of claims 1 to 2. An antisense oligoriucleotide having the sequence of any one of claims 1 to 2. A nucleotide probe that is capable of hybridizing to the nucleotide sequence of any one of claims 2 to 2 or the corresponding mRNA. An antibody which is directed against the polypeptide of any one of claims 3 to 6, or a fragment thereof. The antibody of claim 12, which is directed against a sequence selected from peptide sequences id. No. 1 and Id. No. 2 or derivatives thereof, or a fragment thereof. The antibody of claim 12 or 13, which has the ability to inhibit and / or detect the interaction between presenilins and the polypeptides of any one of claims 3 to 6, or a fragment thereof. - 55 fcfc fcfcfcfc · · fcfcfcfc • · · fcfcfcfc fcfc · fcfcfcfc • · fcfcfc fc · · • fcfc · fc fcfc · fcfc fcfc fcfc fcfc A method for identifying or isolating compounds capable of modulating or inhibiting the interaction between presenilins and polypeptides according to any one of claims 3 to 6, comprising the steps of: a) a molecule or mixture comprising different molecules, optionally unidentified, · contacted with a recombinant cell according to claim 9 expressing the polypeptides of any one of claims 3 to 6 under conditions that allow interaction between the polypeptide and the molecule if the molecule has affinity for the polypeptide and b) the molecule bound to said polypeptide is detected and / or isolated. A polypeptide ligand according to any one of claims 3 to 6, which can be prepared by a method according to claim 15. Use of the ligands according to claim 16 for the preparation of a medicament for the treatment of neurological diseases. Defective recombinant virus, which ot > reach e nucleotide sequence. any of claims 3 to 6; coding 6. polypeptide .according to Vector contains any of claims 1 to nucleotide sequence 2. according to Vector set of claim 19, which is a plasmid vector. Vector set of claim 19, which is viral vector. • 0000 «0 ·· 0000« · 0 0 0 0 0 · 00 00 00 ·· · The vector of claim 21, which is a replication-defective virus. A pharmaceutical composition comprising one or more vectors according to any one of claims 18 to 22. 24. A pharmaceutical composition comprising as active ingredient at least one of the polypeptides of any one of claims 3 to 6. Pharmaceutical preparation, characterized in that it contains at least one antibody according to any one of claims 12 to 14 or a fragment thereof and / or one antisense oligonucleotide according to claim 10 and / or an enin prepared according to Claim 1, as active ingredient. A formulation according to any one of claims 23 to 25, characterized in that it is intended for. modulating, slowing or inhibiting the activity of presenilins or their mutated forms. A composition according to any one of claims 23 to 25 for the treatment of neurodegenerative diseases.