EP1409675A1 - Gene involved in apoptosis regulation - Google Patents

Abstract

The invention concerns a nucleic acid sequence involved in apoptosis regulation and tumoral reversion of the p53 path, and the protein encoded by said nucleic acid sequence, as well as methods for detecting, identifying and/or screening compounds for use in treating cancers and certain neurodegenerative diseases, related to dysfunction in tumour suppression regulation and/or apoptosis in the biological chain of p53, said methods being based on interaction between TSIP4 and TSIP2.

Description

 GENE INVOLVED IN THE REGULATION OF APOPTOSIS

The present invention relates to a nucleic acid sequence involved in the regulation of apoptosis and tumor reversion of the p53 pathway, as well as the protein encoded by this nucleic acid sequence.

Apoptosis, or cell death, is a complex phenomenon that is regulated by many proteins, including the p53 protein. this protein interacts with many other proteins, and its expression, which induces the phenomena of cell death and tumor reversion, can be correlated with the induction or repression of the expression of other cellular genes.

The inventors of the present invention have thus demonstrated genes induced and activated during the cascade leading to tumor repression and / or apoptosis (TSAP for "Tumor Suppressor Activated Pathway"), or repressed genes (TSIP for " Tumor Suppressor Inhibited Pathway ”). These genes have in particular been the subject of patent applications WO 97/22695 or WO 00/08147.

It is important to be able to understand precisely the mechanisms of the p53 cascade, in order to be able to generate new compounds having an anti-tumor activity (which can in particular induce apoptosis or tumor suppression), or which can be used for the treatment of neurological diseases. degenerative. Indeed, the inventors of the present application have demonstrated that presenilin 1 (PSI), whose role had been suggested in Alzheimer's disease, was identical to the TSIP 2 protein described in application WO 97/22695. Thus, it is legitimate to look for drugs that can interfere in apoptosis, in order to reduce this phenomenon, and which could be used in neurodegenerative diseases.

The inventors of the present application have demonstrated the interaction of the protein TSIP 2 (presenilin 1, PSI) with a protein, which has been shown to be itself repressed in a model of cell death. This protein (SEQ ID No. 2) has thus been called TSIP4, and it is an object of the present invention, as well as the nucleic sequences which code for said protein. The TSIP4 gene will in particular be preferred, it being understood that by gene, it may be understood either the cDNA sequence or the genomic DNA sequence, with or without the regulatory elements. Thus, the subject of the present invention is a purified or isolated nucleic acid, characterized in that it comprises a nucleic sequence chosen from the following group of sequences: a) SEQ ID N ⁰ 1; b) the sequence of a fragment of at least 15 consecutive nucleotides of a sequence chosen from SEQ ID No. 1; c) a nucleic sequence having an identity percentage of at least 80%, after optimal alignment with a sequence defined in a) or b); d) a nucleic sequence hybridizing under conditions of high stringency with a nucleic sequence defined in a) or b); e) the complementary sequence or the TARN sequence corresponding to a sequence as defined in a), b), c) or d). The nucleic acid sequence according to the invention defined in c) has a percentage identity of at least 80% after optimal alignment with a sequence as defined in a) or b) above, preferably 90%, more preferably 95%, most preferably 98%, or 99%.

The term “nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNAs. Thus, the nucleic acid sequences according to the invention also include PNA (Peptid Nucleic Acid), or the like. It should be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. These are sequences which have been isolated and / or purified, that is to say that they have been taken directly or indirectly, for example by copy, their environment having been at least partially modified. This also means the nucleic acids obtained by chemical synthesis.

By “percentage of identity” between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. The term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage of identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window" to identify and compare the regions. sequence similarity locale. The optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970 ), using the similarity search method of Pearson and Lipman (1988), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI). In order to obtain optimal alignment, the BLAST program is preferably used with the BLOSUM 62 matrix. The PAM or PAM250 matrices can also be used.

The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences, the nucleic acid or amino acid sequence to be compared can include additions or deletions by compared to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences. By nucleic acid sequences having a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with a reference sequence, is meant the nucleic acid sequences having, with respect to the sequence reference nucleic acid, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion, and / or a substitution, in particular point, and whose nucleic sequence has at least 80%, preferably 90%, more preferred 98%, of identity after optimal alignment with the reference nucleic sequence. They are preferably sequences whose complementary sequences are capable of hybridizing specifically with the sequences SEQ ID No. 1 of the invention. Preferably, the specific hybridization conditions or high stringency will be such that they ensure at least 80%, preferably 90%, more preferably 98% identity after optimal alignment between one of the two sequences and the sequence complementary to each other. Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two complementary DNA fragments. By way of illustration, high stringency conditions of the hybridization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.

DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS. The last washing is carried out in 0.1 × SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides. The high stringency hybridization conditions described above for a polynucleotide of defined size can be adapted by a person skilled in the art for larger or smaller oligonucleotides, according to the teaching of Sambrook et al., 1989.

Among the nucleic sequences having a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with the sequence according to the invention, the nucleic sequences variant of SEQ ID are also preferred. N ° 1, or its fragments, that is to say all of the nucleic sequences corresponding to allelic variants, that is to say individual variations of the sequence SEQ ID N ° 1. These mutated sequences natural correspond to polymorphisms present in mammals, in particular in humans and, in particular, to polymorphisms which can lead to the onset of a pathology, such as for example cell degeneration. Preferably, the present invention relates to the variant nucleic acid sequences in which the mutations lead to a modification of the amino acid sequence of the polypeptide, or of its fragments, encoded by the normal sequence of SEQ ID No. 1.

It is also intended to denote by variant nucleic acid sequence any RNA or cDNA resulting from a mutation and / or variation of a splicing site of the genomic nucleic sequence of which the cDNA has the sequence SEQ ID No. 1.

The invention preferably relates to a purified or isolated nucleic acid according to the present invention, characterized in that it comprises or consists of the sequence SEQ ID TST ° 1, its complementary sequence or the sequence of the RNA corresponding to SEQ ID N ° 1.

The invention also relates to a purified or isolated nucleic acid characterized in that it codes for a polypeptide having a continuous fragment of at least 100, more preferably 150, most preferably 200 amino acids of the protein SEQ ID # 2.

The primers or probes, characterized in that they comprise a sequence of a nucleic acid according to the invention, also form part of the invention. Thus, the present invention also relates to the primers or the probes according to the invention which can make it possible in particular to demonstrate or discriminate the variant nucleic sequences, or to identify the genomic sequence of the genes whose cDNA is represented by SEQ ID # 1, using in particular an amplification method such as the PCR method, or a related method.

The invention also relates to the use of a nucleic acid sequence according to the invention as probe or primer, for the detection, identification, assay or amplification of nucleic acid sequence.

According to the invention, the polynucleotides which can be used as a probe or as a primer in methods of detection, identification, assay or amplification of nucleic sequence, have a minimum size of 15 bases, preferably 20 bases, or better from 25 to 30 bases. The probes and primers according to the invention can be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal.

The unlabeled polynucleotide sequences according to the invention can be used directly as a probe or primer.

The sequences are generally marked to obtain sequences which can be used for numerous applications. The labeling of the primers or probes according to the invention is carried out with radioactive elements or with non-radioactive molecules. Among the radioactive isotopes used, mention may be made of P, P, S, H or ¹²⁵ I. Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens , dyes, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents. The polynucleotides according to the invention can thus be used as a primer and / or probe in methods using in particular the PCR technique (polymerase chain reaction) (Rolfs et al., 1991). This technique requires the choice of pairs of oligonucleotide primers framing the fragment which must be amplified. One can, for example, refer to the technique described in US Pat. No. 4,683,202. The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced. The specificity of the amplification can be controlled using as primers the nucleotide sequences of polynucleotides of the invention and as matrices, plasmids obtaining these sequences or even the amplification products derived therefrom. The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.

The invention also relates to the nucleic acids capable of being obtained, by amplification using primers according to the invention. Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention. By PCR-like is meant to denote all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified, these techniques are of course known. In general it is the amplification of DNA by a polymerase; when the original sample is an RNA, a reverse transcription should be carried out beforehand. There are currently many methods for this amplification, such as the SDA technique (Strand Displacement Amplification) or strand displacement amplification technique (Walker et al., 1992), the TAS technique (Transcription-based Amplification System) described by Kwoh et al. (1989), the 3SR (Self-Sustained Sequence Replication) technique described by Guatelli et al. (1990), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by Kievitis et al. (1991), the TMA technique (Transcription Mediated Amplification), the LCR technique (Ligase Chain Reaction) described by Landegren et al. (1988), the RCR (Repair Chain Reaction) technique described by Segev (1992), the CPR (Cycling Probe Reaction) technique described by Duck et al. (1990), the Q-beta-replicase amplification technique described by Miele et al. (1983). Some of these techniques have since been perfected. In the case where the target polynucleotide to be detected is an mRNA, it is advantageous to use, prior to the implementation of an amplification reaction using the primers according to the invention or to the implementation of a method detection using the probes of the invention, a transcriptase type enzyme reverse in order to obtain a cDNA from the mRNA contained in the biological sample. The cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection method according to the invention. The probe hybridization technique can be performed in various ways (Matthews et al., 1988). The most general method consists in immobilizing the nucleic acid extracted from cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe).

According to another embodiment of the nucleic acid probes according to the invention, the latter can be used as capture probes. In this case, a probe, called a “capture probe”, is immobilized on a support and is used to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected. thanks to a second probe, called a “detection probe”, marked by an easily detectable element.

Among the nucleic acid fragments of interest, it is thus necessary to cite in particular the antisense oligonucleotides, that is to say those whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product. Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition or an activation of this expression. Such oligonucleotides can be used as therapeutic products and medicaments to regulate the phenomena of apoptosis and tumor repression. The use of its sense or antisense sequences can also be implemented in vitro, to define new means of model and study of apoptosis. The present invention also relates to an isolated polypeptide characterized in that it comprises a polypeptide chosen from: a) a polypeptide of sequence SEQ ID No. 2; b) a polypeptide varying from a polypeptide of sequence defined in a); c) a polypeptide homologous to a polypeptide defined in a) or b), comprising at least 80% identity with said polypeptide of a); d) a fragment of at least 15 consecutive amino acids of a polypeptide defined in a), b) or c); e) a biologically active fragment of a polypeptide defined in a), b) or c). By “polypeptide” is meant, within the meaning of the present invention, denote proteins or peptides.

By “biologically active fragment” is meant a fragment having the same biological activity as the peptide fragment from which it is deduced, preferably in the same order of magnitude (to within a factor of 10). Thus, the examples show that the protein TSIP4 (SEQ ID No. 2) has a potential role in the phenomena of apoptosis. A biologically active fragment of the TSIP4 protein therefore consists of a polypeptide derived from SEQ ID No. 2 also having a role in apoptosis.

Preferably, a polypeptide according to the invention is a polypeptide consisting of the sequence SEQ ID No. 2 (corresponding to the protein encoded by the TSIP4 gene) or of a sequence having at least 80% identity with SEQ ID No. 2 after optimal alignment.

The polypeptide sequence has a percentage identity of at least 80% after optimal alignment with the sequences SEQ ID No. 2, preferably 90 or 95%, more preferably 98%, or 99%.

By polypeptide whose amino acid sequence having a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with a reference sequence, is intended to denote the polypeptides having certain modifications with respect to the reference polypeptide, such as in particular one or more deletions, truncations, an elongation, a chimeric fusion, and / or one or more substitutions.

Among the polypeptides whose amino acid sequence having a percentage identity of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with the sequence SEQ ID No. 2 or with one of its fragments according to the invention, the variant polypeptides encoded by the variant nucleic sequences as defined above are preferred, in particular the polypeptides whose sequence of amino acids has at least one mutation corresponding in particular to a truncation, deletion, substitution and / or addition of at least one amino acid residue with respect to the sequence SEQ DD N ° 2 or with one of its fragments , more preferably variant polypeptides having a mutation linked to a pathology, such as cancer or a neurodegenerative disease. The present invention also relates to the cloning and / or expression vectors comprising a nucleic acid or coding for a polypeptide according to the invention. Such a vector can also contain the elements necessary for the expression and optionally for the secretion of the polypeptide in a host cell. Such a host cell is also an object of the invention. The vectors characterized in that they comprise a promoter and / or regulator sequence according to the invention, also form part of the invention.

Said vectors preferably comprise a promoter, translation initiation and termination signals, as well as suitable regions for regulating transcription. They must be able to be maintained stably in the cell and may possibly have specific signals specifying the secretion of the translated protein.

These different control signals are chosen according to the cellular host used. For this purpose, the nucleic acid sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or vectors integrative of the chosen host.

Among the autonomous replication systems, plasmid or viral type systems are preferably used depending on the host cell, the viral vectors possibly being in particular adenoviruses (Perricaudet et al., 1992), retroviruses, lentiviruses, poxvirus or herpesvirus (Epstein et al., 1992). Those skilled in the art know the technologies that can be used for each of these systems. When it is desired to integrate the sequence into the chromosomes of the host cell, it is possible to use, for example, systems of the plasmid or viral type; such viruses are, for example, retroviruses (Temin, 1986), or AANs (Carter, 1993). Among the non-viral vectors, naked polynucleotides such as naked ADΝ or naked ΓARΝ are preferred according to the technique developed by the company NICAL, artificial bacteria chromosomes (BAC, bacterial artificial chromosome), artificial yeast chromosomes (YAC, yeast artificial chromosome) for expression in yeast, mouse artificial chromosomes (MAC) for expression in murine cells and preferably human artificial chromosomes (HAC, human artificial chromosome) for expression in human cells.

Such vectors are prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.

The invention furthermore includes host cells, in particular eukaryotic and prokaryotic cells, transformed by the vectors according to the invention as well as transgenic animals, preferably mammals, except humans, comprising one of said cells transformed according to the invention. . These animals can be used as models, for the study of the etiology of inflammatory and / or immune diseases, and in particular of inflammatory diseases of the digestive tract, or for the study of cancers. Among the cells which can be used within the meaning of the present invention, mention may be made of bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular cell cultures. mammals (Edwards and Aruffo, 1993), and in particular Chinese hamster ovary (CHO) cells. Mention may also be made of insect cells in which methods can be used, for example using baculoviruses (Luckow, 1993). A preferred cellular host for the expression of the proteins of the invention consists of COS cells. Among the mammals according to the invention, animals such as rodents, in particular mice, rats or rabbits, which express a polypeptide according to the invention, are preferred.

Among the mammals according to the invention, animals such as mice, rats or rabbits are also preferred, characterized in that the gene coding for the protein with sequence SEQ ID No. 2, or whose sequence is coded by homologous gene in these animals, is not functional, is invalidated or has at least one mutation.

These transgenic animals are obtained for example by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the affected chimeras at the level of the reproductive lines, and growth of said chimeras.

The transgenic animals according to the invention can thus overexpress the gene coding for the protein according to the invention, or their homologous gene, or express said gene into which a mutation is introduced. These transgenic animals, in particular mice, are obtained for example by transfection of a copy of this gene under the control of a strong promoter of ubiquitous nature, or selective for a type of tissue, or after viral transcription.

Alternatively, the transgenic animals according to the invention can be made deficient for the gene coding for the polypeptide of the sequence SEQ ID

N ° 2, or its homologous gene, by inactivation using the LOXP / CRE recombinase system (Rohlmann et al., 1996) or any other system of inactivation of the expression of this gene.

The cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and can also be used as an analysis model.

The transformed cells or mammals as described above can also be used as models in order to study the interactions between the polypeptides according to the invention, and the chemical or protein compounds, involved directly or indirectly in the activities of the polypeptides according to the invention, this in order to study the different mechanisms and interactions involved.

They can in particular be used for the selection of products interacting with the polypeptides according to the invention, in particular the protein of sequence SEQ DN No. 2 or its variants according to the invention, as cofactor, or inhibitor, in particular competitive, or else having an agonist or antagonist activity of the activity of the polypeptides according to the invention. Preferably, said transformed cells or transgenic animals are used as a model, in particular for the selection of products making it possible to combat pathologies linked to an abnormal expression of this gene.

The invention also relates to the use of a cell, a mammal or a polypeptide according to the invention for the screening of chemical or biochemical compounds. capable of interacting directly or indirectly with the polypeptides according to the invention, and / or capable of modulating the expression or the activity of these polypeptides.

Similarly, the invention also relates to a method for screening for compounds capable of interacting in vitro or in vivo with a nucleic acid according to the invention, using a nucleic acid, a cell or a mammal according to the invention, and by detecting the formation of a complex between the candidate compounds and the nucleic acid according to the invention.

The compounds thus selected are also objects of the invention.

Such a compound according to the invention can be a compound having a chemical structure, a lipid, a sugar, a protein, a peptide, a hybrid protein-lipid compound, protein-sugar, peptide-lipid, or peptide-sugar, a protein. or a peptide to which chemical ramifications have been added.

Among the chemical compounds envisaged, they may contain one or more rings, aromatic or not, as well as several residues of all kinds

(in particular lower alkyl, that is to say having between 1 to 6 carbon atoms). The invention also relates to the use of a nucleic acid sequence according to the invention for the synthesis of recombinant polypeptides.

The method for producing a polypeptide of the invention in recombinant form, itself included in the present invention, is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultivated in conditions allowing the expression of a recombinant polypeptide encoded by a nucleic acid sequence according to the invention, and that said recombinant polypeptide is recovered. Recombinant polypeptides, characterized in that they are capable of being obtained by said production method, also form part of the invention.

The recombinant polypeptides obtained as indicated above can be both in glycosylated and non-glycosylated form and may or may not have the natural tertiary structure.

The sequences of the recombinant polypeptides can also be modified in order to improve their solubility, in particular in aqueous solvents.

Such modifications are known to those skilled in the art, such as the deletion of hydrophobic domains or the substitution of hydrophobic amino acids with hydrophilic amino acids.

These polypeptides can be produced from the nucleic acid sequences defined above, according to the techniques for producing recombinant polypeptides known to those skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.

An efficient system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.

These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence.

The methods used for the purification of a recombinant polypeptide are known to those skilled in the art. The recombinant polypeptide can be purified from lysates and cell extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoafinity techniques using '' specific monoclonal or polyclonal antibodies, etc.

The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the many known peptide syntheses, for example the techniques using solid phases (see in particular

Stewart et al., 1984) or techniques using partial solid phases, by condensation of fragments or by synthesis in conventional solution. The polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.

Mono- or polyclonal antibodies or their fragments, chimeric or immunoconjugate antibodies, characterized in that they are capable of specifically recognizing a polypeptide according to the invention, form part of the invention.

Specific polyclonal antibodies can be obtained from a serum of an animal immunized against the polypeptides according to the invention, in particular produced by genetic recombination or by peptide synthesis, according to the usual procedures.

We note in particular the interest of antibodies specifically recognizing certain polypeptides, variants, or their immunogenic fragments, according to the invention.

Mono- or polyclonal antibodies or their fragments, chimeric or immunoconjugate antibodies, characterized in that they are capable of specifically recognizing the polypeptide of sequence SEQ ID No. 2 are particularly preferred.

The specific monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas described by Kôhler and Milstein (1975).

The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') ₂ fragments. They can also be in the form of immunoconjugates or labeled antibodies in order to obtain a detectable and / or quantifiable signal.

The invention also relates to methods for the detection and / or purification of a polypeptide according to the invention, characterized in that they use an antibody according to the invention.

The invention further comprises purified polypeptides, characterized in that they are obtained by a method according to the invention.

Furthermore, in addition to their use for the purification of polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, can also be used for the detection of these polypeptides in a biological sample.

They thus constitute a means of immunocytochemical or immunohistochemical analysis of the expression of the polypeptides according to the invention, in particular the polypeptide of sequence SEQ ID No. 2 or one of its variants, on sections of specific tissues, for example by immunofluorescence, gold labeling, enzyme immunoconjugates.

They can in particular make it possible to demonstrate an abnormal expression of these polypeptides in tissues or biological samples.

More generally, the antibodies of the invention can be advantageously used in any situation where the expression of a polypeptide according to the invention, normal or mutated, must be observed.

Thus, a method of detecting a polypeptide according to the invention in a biological sample, comprising the steps of bringing the biological sample into contact with an antibody according to the invention and of demonstrating the antigen-antibody complex formed is also an object of the invention, as well as a kit for implementing such a method. Such a kit contains in particular: a) a monoclonal or polyclonal antibody according to the invention; b) optionally reagents for the constitution of a medium suitable for the immunological reaction; c) reagents for the detection of the antigen-antibody complex produced during the immunological reaction. The antibodies according to the invention can also be used in the treatment of a neurodegenerative disease, or of cancer, in humans, when an abnormal expression of the TSIP4, PSI, p53 or a gene is observed. gene described by the inventors of the present application, in application WO 97/22695 or WO 00/08147. Abnormal expression means over-expression, under-expression or expression of a mutated protein.

These antibodies can be obtained directly from human serum, or from animals immunized with polypeptides according to the invention, then “humanized”, and can be used as such or in the preparation of a medicament intended for the treatment of aforementioned diseases. Also forming part of the invention are the methods for determining an allelic variability, a mutation, a deletion, a loss of heterozygosity or any genetic anomaly of the gene according to the invention, characterized in that they use a nucleic acid sequence, a polypeptide or an antibody according to the invention.

Mutations in the sequence of the TSIP4 gene repressed during apoptosis induced by p53 can be detected directly by analysis of the nucleic acid and of the sequences according to the invention (genomic DNA, RNA or cDNA), but also by l intermediary of the polypeptides according to the invention. In particular, the use of an antibody according to the invention which recognizes an epitope carrying a mutation makes it possible to discriminate between a "healthy" protein and a protein "associated with a pathology". The precise description of the mutations observable in the TSIP4 gene can thus make it possible to lay the foundations for a molecular diagnosis of neurodegenerative diseases and cancers, or any disease involving apoptosis. Such an approach, based on the search for mutations in the gene, will contribute to the diagnosis of these diseases and possibly reduce the importance of certain invasive or costly additional examinations. The invention lays the foundations for such a molecular diagnosis based on the search for mutations in TSIP4.

In particular, a method of diagnosis and / or prognostic evaluation of a neurodegenerative disease or of a cancer is preferred, characterized in that the presence of at least one mutation and / or alteration of expression of the gene corresponding to SEQ ID No. 1 by the analysis of all or part of a nucleic sequence corresponding to said gene.

This method of diagnosis and / or prognostic evaluation can be used preventively (study of a predisposition to a neurodegenerative disease or cancer), or in order to serve for the establishment and / or confirmation of a clinical condition in a patient.

Preferably, the neurodegenerative disease is Alzheimer's disease.

The analysis can be carried out by sequence of all or part of the gene, or by other methods known to those skilled in the art. In particular, methods based on PCR can be used, for example PCR-SSCP which makes it possible to detect point mutations.

It is also possible to carry out the analysis by fixing a probe according to the invention corresponding to one of the sequences SEQ ID No. 1 on a DNA chip and hybridization on these microplates. A DNA chip containing a sequence according to the invention is also one of the objects of the invention.

Likewise, a protein chip containing an amino acid sequence according to the invention is also an object of the invention. Such a protein chip allows the study of interactions between the polypeptides according to the invention and other proteins or chemical compounds, and can thus be useful for the screening of compounds interacting with the polypeptides according to the invention. It is also possible to use the protein chips according to the invention to detect the presence of antibodies directed against the polypetides according to the invention in the serum of patients. It is also possible to use a protein chip containing an antibody according to the invention.

Those skilled in the art also know how to implement techniques allowing the study of the alteration of the expression of a gene, for example by studying the mRNA (in particular by Northern Blot or by experiments of RT-PCR, with probes or primers according to the invention), or of the protein expressed, in particular by Western Blot, using antibodies according to the invention.

The invention also relates to methods for obtaining an allele of the TSIP4 gene, associated with a detectable phenotype, comprising the following steps: a) obtaining a nucleic acid sample from an individual expressing said detectable phenotype; b) bringing said nucleic acid sample into contact with an agent capable of specifically detecting a nucleic acid coding for the TSIP4 protein; c) isolating said nucleic acid coding for the TSIP4 protein. Such a method can be followed by a sequence step of all or part of the nucleic acid coding for the protein TSIP4, which makes it possible to predict the susceptibility to a neurodegenerative disease or to cancer.

The agent capable of specifically detecting a nucleic acid coding for the TSIP4 protein is advantageously an oligonucleotide probe according to the invention, which can be formed from DNA, RNA, PNA, modified or not. The modifications may include radioactive or fluorescent labeling, or be due to modifications in the bonds between the bases (phosphorothioates, or methylphosphonates for example). Those skilled in the art know the protocols for isolating a specific DNA sequence. Step b) of the above process above described can also be an amplification step as described above.

The invention also relates to a method for detecting and / or assaying a nucleic acid according to the invention in a biological sample, comprising the following steps of bringing a probe according to the invention into contact with a biological sample and detecting and / or assaying the hybrid formed between said polynucleotide and the nucleic acid of the biological sample.

A person skilled in the art knows how to implement such a method, and can in particular use a reagent kit comprising: a) a polynucleotide according to the invention, used as a probe; b) the reagents necessary for the implementation of a hybridization reaction between said probe and the nucleic acid of the biological sample; c) the reagents necessary for the detection and / or the assay of the hybrid formed between said probe and the nucleic acid of the biological sample; which is also an object of the invention.

Such a kit can also contain positive or negative controls to ensure the quality of the results obtained. However, in order to detect and / or measure a nucleic acid according to the invention, those skilled in the art can also carry out an amplification step using primers chosen from the sequences according to the invention.

Finally, the invention also relates to the compounds chosen from a nucleic acid, a polypeptide, a vector, a cell, or an antibody according to the invention, or the compounds obtained by the screening methods according to the invention, as a medicament. , in particular for the prevention and / or treatment of a neurodegenerative disease or of cancer, associated with the presence of at least one mutation of the gene corresponding to SEQ ID No. 1, and / or with an expression abnormal protein corresponding to SEQ ID NO: 2. The present invention also relates to methods of screening and identifying products which may interfere in the p53 cascade, and thus induce tumor reversion and / or apoptosis or conversely, reduce the phenomena of apoptosis. This aspect of the present invention is based on the fact that the TSIP4 protein, object of the present application, binds to the TSIP2 protein described in patent application WO 97/22695, whose role in Alzheimer's disease has been reported.

In a first embodiment, the invention is directed to a method of screening, selection or identification of a compound interfering with, reducing or inhibiting the binding of TSIP4 to TSIP2, having the steps of: a) bringing said compound into contact with a system allowing the in vitro determination of the link between TSIP4 and TSIP2; b) identification of the decrease and / or inhibition of the link between TSIP4 and TSIP2.

A subject of the present invention is also in particular a method for screening, selecting or identifying a compound for the reduction and / or inhibition of tumor repression and / or cell death (apoptosis), presenting the steps of : a) bringing compounds into contact with a system allowing the in vitro determination of the bond between TSIP4 and TSIP2; b) identification of the compounds inducing the reduction and / or inhibition of the link between TSEP4 and TSIP2; c) study of the compounds identified in step b) in a model making it possible to measure the phenomena of apoptosis and / or tumor repression; d) identification of the reduction and / or inhibition of tumor repression and / or cell death in said model compared to a control model with which said compound has not been brought into contact.

Certain models making it possible to study / measure apoptosis and / or tumor repression have already been described, and consist in particular of the in vitro models described by Tellerman et al. (1993, Proc. Natl. Acad. Sci. USA, 90, 8702-6), Amson et al.

(1996, Proc. Natl. Acad. SCI; USA, 93, 3953-7), or Nemani et al. (1996, Proc. Natl. Acad. Sci. USA, 93, 9039-42).

In order to determine compounds allowing the increase in tumor repression and / or cell death (apoptosis), it is also possible to combine different methods according to the invention, it is in particular possible to thus obtain a method for screening, selecting or identifying compounds having the function of an increase in tumor repression and / or cell death (apoptosis), comprising the steps of: a) bringing said compound into contact with a system allowing the determination in in vitro of the link between TSIP4 and TSIP2; b) identification of the compounds inducing the reduction and / or inhibition of the link between TSIP4 and TSIP2. c) bringing the compounds selected in step b) into contact in a model making it possible to measure the phenomena of apoptosis and / or tumor repression; d) identification of the increase in tumor repression and / or cell death in said model compared to a control model with which said compound has not been brought into contact.

Such a method is therefore also an object of the present invention. The present invention therefore makes use of the fact that the proteins TSIP4 and TSIP2 can bind to each other. It is therefore interesting to identify the domains of each protein which are actually in contact with the other protein. Indeed, this should make it possible to be able to use the peptides thus identified as decoys or agonists of complete proteins. This can thus make it possible to define compounds which will interfere in the TSIP4 - TSIP2 link, and which may possibly induce tumor suppression and / or apoptosis, or conversely reduce these phenomena.

More generally, the present invention allows the identification of TSIP4 regions involved in the binding with the TSIP2 protein, by a method comprising the steps of: a) contacting peptides derived from the TSIP4 protein in a system allowing the in vitro determination of the link between TSIP4 and TSIP2; b) identification of the peptides resulting in the reduction of the link between TSIP4 and TSIP2 in said system;

It is obvious that the present invention also makes it possible to determine the regions of TSIP2 involved in the binding with TSIP4, according to methods similar to the methods described above that these regions can in particular be used as decoys when it is desired to reduce tumor repression and / or apoptosis.

By “region” of a protein is meant in particular peptides of primary sequence originating from the primary sequence of said protein. These regions thus identified can be tested like the products described above for their properties in the regulation of tumor repression and / or apoptosis.

The present invention therefore makes it possible to identify products making it possible to interact with the TSIP4 - TSIP2 link, and therefore which may have an interest in the regulation of apoptosis and / or tumor repression. However, it is possible that these products, to be able to be used for a therapeutic treatment, in particular cancer or neurodegenerative diseases, need to be optimized, in order to have a higher activity, and / or a lower toxicity.

Indeed, the development of a drug is often carried out on the following principle:

- screening of compounds having a desired activity by an appropriate method,

- selection of compounds meeting the "specifications",

- determination of the structure (in particular the sequence (possibly tertiary) in the case of peptides, formula and backbone in the case of chemical compounds) of the selected compounds,

- optimization of the selected compounds, by modification of the structure (for example by the change of the steroeochemical conformation (for example passage of L in D of amino acids in a peptide), addition of substituents on the peptide or chemical skeletons, in particular by grafting residues on the skeleton, modification of peptides (see in particular Gante ("Peptidomimetics", in Angewandte Chemie-International Editio Engl. 1994, 33. 1699-1720)

- passage and screening of the compounds thus obtained on appropriate models which are often models closer to the pathology studied. At this stage, we often use animal models, generally in rodents (mice, rats ...) or in dogs, even primates.

The animal models which can be used are, for example, for cancer, models based on immunocompromised mice (for example scid / scid), to which tumor cells are injected (in particular subcutaneously), which will lead to the development of tumors. The efficacy of potentially anti-tumor compounds is studied, for example by measuring the size of the tumors formed.

One can, for the study of neurodegenerative diseases, use the model described by Amson et al. (2000, Proc. Natl. Acad. Sci. USA, 97, 5346-50), which consists of mice deficient in p53, or the model described in Chen et al, Janus et al, and Morgan et al. (2000, Nature, 408 pp. 975-985)

Thus, the object of the present invention is in particular to allow the identification of compounds which could be used for the treatment of cancer, in that they have an activity of increasing tumor repression and / or apoptosis. One of the objects of the present invention is therefore a method comprising the steps of: a) implementing a method according to the invention, making it possible to identify compounds having a certain activity of increasing tumor repression and / or apoptosis, b) modification of the product selected in step a), c) test of the product modified in step b) in in vitro and / or in vivo methods, on relevant models of tumor repression and / or apoptosis, d) identification of the product making it possible to obtain a tumor repression and / or apoptosis activity greater than the activity obtained for the product selected in step a). Step d) can be replaced by a step d '), which would be: d') identification of the product making it possible to obtain the biological effect sought with less toxicity in an animal model

(when one of the models used in step c) is in vivo). When testing on apoptosis or tumor suppression models in vitro, one can for example use the K256 / KS model described by Tellerman et al. (1993, Proc. Natl. Acad. Sci. USA, 90, 8702-6). It is also possible to use the Ml-LTR cells, described by Amson et al. (1996, Proc. Natl. Acad. SCI; USA, 93, 3953-7), or the U937 / US3-US4 cells, described by Nemani et al. (1996, Proc. Natl. Acad. Sci. USA, 93, 9039-42). In vivo tests can be carried out by injecting these cells from animals, in particular immunocompromised mice and by studying the effects of the various compounds tested.

Those skilled in the art will know how to define the conditions and thresholds necessary for the identification of a product which can be used as a medicine, according to regulatory requirements (in particular toxicology), in relation to the benefit provided by the product thus identified.

Likewise, the invention also relates to the methods for optimizing products repressing tumor repression and / or apoptosis, identified by the methods described above, and allowing the identification of products which can be used as medicaments.

Thus the invention also relates to a method of identifying a product having an activity of reduction and / or inhibition of tumor repression and / or apoptosis, characterized in that it comprises the steps of: a ) implementation of a method according to the invention, making it possible to identify compounds having a certain activity of decreasing tumor repression and / or apoptosis, b) modification of the product selected in step a) in particular by grafting residues onto the chemical skeleton, c) testing of the modified product in step b) in in vitro and / or in vivo methods, on relevant models of tumor suppression and / or apoptosis, d) identification of the product making it possible to obtain a tumor repression and / or apoptosis activity reduced compared to the activity obtained for the product selected in step a). Step d) can also be replaced by a step d '), which would be: d') identification of the product making it possible to obtain the biological effect sought with less toxicity in an animal model (when one of the models used in the step c) is in vivo). It is indeed still a question of being able to obtain the product which has the best ratio (biological activity and clinical effect) / (potential risks of use).

The parameters to be used to obtain these results are all known and within the reach of those skilled in the art, wishing to develop new drugs, and can be found, for example, in the directives of organizations such as the Medicines Agency, the Commission European, or the Federal Drug Agency.

The implementation of the methods according to the present invention requires models making it possible to determine the link between TSIP4 and TSIP2, as well as easy measurement of increases or decreases in the amount of protein TSIP2 or p53 in a eukaryotic cell.

The amount of TSIP2 or p53 protein can be studied by Western Blot, the revelation being effected by the use of an antibody directed against said protein. Such antibodies, directed to p53, can in particular be found in Calbiochem, under the reference OP09L. In order to implement the methods according to the invention requiring study and screening on mammalian cells, it may be advantageous to overexpress one or the other of the proteins TSIP4, TSIP2 and p53 in said cells, two of between them, or all three together.

Thus, it is easier to be able to study the variations in the quantity of protein TSIP2 or of p53, in particular by comparison of the cells on which the compounds of interest are tested with the same cells, to which the compounds are not added than the 'we want to screen.

It is understood that the expression of the two proteins TSIP4, TSIP2, and p53 can be increased by introduction of the genes (in particular the cDNAs) coding for these proteins in the cells, either by being placed on vectors, or by introduction into the chromosome.

When an episomal expression is chosen, said mammalian cell is transfected with at least one vector chosen from a vector carrying a DNA fragment coding for TSIP4, a vector carrying a DNA fragment coding for TSIP2, a vector carrying a fragment of DNA encoding p53, a vector carrying a DNA fragment encoding more than one of these proteins. Thus, the same vector can express all the proteins, alternatively it is possible to introduce several vectors. It is possible to use vectors allowing the expression and the easy purification of the proteins TSIP4 and TSIP2, for example in prokaryotic (E. coli, B. subtiis ...) or eukaryotic (yeasts such as Saccharomyces, Kluyveromyces .. ., mammalian cells (HeLa, Cos, Hep-2 ...) or insects (using a Baculovirus system). Thus, it may be advantageous for proteins to have a label at their N- or C-terminal end. , in order to facilitate the purification. A Histidine or GST label is chosen in particular. These methods are well known to those skilled in the art, who find the appropriate plasmids in the catalogs of companies such as Stratagene. Methods according to the invention on in vitro models, to study the link between TSIP2 and TSIP4, there are several ways of operating.

A usable protocol can be the following:

- expression and purification of the TSIP4 and TSIP2 proteins, for example in prokaryotic (E. coli, B. subtiis ...) or eukaryotic (yeasts such as Saccharomyces, Kluyveromyces ..., mammalian cells (HeLa, Cos, Hep- 2) ...) or insects (using a Baculovirus system). It may be advantageous for the proteins to have a label at their N- or C-terminal end, in order to facilitate purification. In particular, a Histidine label is chosen, or GST These methods are well known to those skilled in the art, who find the appropriate plasmids in the catalogs of companies such as Stratagene.

- binding of proteins to appropriate beads. When using a GST label, the proteins expressed are linked to sepharose beads exhibiting Gluthathione. - preparation of proteins by in vitro translation. This can easily be done using commercial vectors (for example available from Promega), which make it possible to clone the cDNAs under the control of well known promoters (T7 or T3), and to use the appropriate RNA polymerases to produce the RNAs, then perform protein expression in vitro, using the available kits, and following the manufacturer's directions.

- protein co-precipitation, by adding the proteins obtained by in viti-o translation to the sepharose - glutathione beads on which the fusion proteins with GST are fixed. After sufficient contact time, wash the beads and perform an SDS-PAGE electrophoresis gel analysis and autoradiography. The appearance of the bands corresponding to the two proteins clearly shows the bond between them.

The use of appropriate controls thus makes it possible to define the reduction and / or the inhibition of the bond between TSIP4 and TSIP2, by comparison of the quantities of proteins released after addition of the compound tested during the coprecipitation step with the quantities of proteins. released in controls.

We can also study protein binding using the FRET system

(Fluorescence Resonance Energy Transfer) which consists in labeling each of the proteins with an appropriate residue, the binding of the two proteins inducing a reaction between each of the two residues and the emission of an easily detectable fluorescence.

A subject of the present invention is also the compounds which can be obtained by a process according to the invention, in particular the compounds having an activity of increasing tumor repression and / or apoptosis, those having an activity of inhibiting the TSIP4 - TSIP2 linkage, as well as those having an activity of reduction and / or inhibition of tumor repression and / or apoptosis.

The present invention also relates to the peptide sequences corresponding to a TSIP4 region interacting with the TSIP2 protein, which can in particular be identified by a method according to the invention.

The invention also relates to the peptide sequences corresponding to a TSIP2 region interacting with the TSIP4 protein, which can in particular be identified by a method according to the invention, the method making it possible to identify the peptide sequences of TSIP4 interacting with TSIP2 able to be adapted. to determine the peptide sequences of TSIP2 interacting with TSIP4, in particular by adapting the in vitro protocol developed above.

The invention also relates to the nucleotide sequences coding for the peptide sequences thus identified. It is clear that the term peptide sequence or nucleic or nucleotide sequence (the latter two terms being used interchangeably) represent isolated sequences, that is to say out of their natural state, and can in particular be modified by replacement of their base units by non-natural units, or by modifying the links between base units (for example phosphorothioates (nucleic acid) or Peptid Nucleic Acids)

It is an object of the invention to allow the identification of compounds interfering with the binding TSIP4 and TSIP2, some of these compounds possibly being able to induce effects on the cascade of p53. Thus, the compounds according to the invention, the peptide sequences according to the invention, or the nucleotide sequences according to the invention, as medicaments, are also objects of the invention.

A compound identified by a method according to the invention can be a compound having a chemical structure, a lipid, a sugar, a protein, a peptide, a hybrid protein-lipid compound, protein-sugar, peptide-lipid, or peptide-sugar. , a protein or peptide to which chemical ramifications have been added.

Among the chemical compounds envisaged, they may contain one or more (in particular 2 or 3) rings, aromatic or not, having from 3 to 8 carbon atoms, as well as several residues of all kinds (in particular lower alkyl, c ' that is to say having between 1 to 6 carbon atoms).

These compounds, nucleic sequences, peptide sequences, can thus be used for the preparation of a medicament, intended in particular for the treatment of cancer, or of a neurodegenerative disease, according to the pro or anti-apoptosis / tumor reversion effect. .

The inventors of the present application have, for the first time, demonstrated the fact that the TSIP4 protein binds to the TSIP2 protein. Thus, the present invention also relates to a complex consisting of a TSIP4 protein and a TSIP2 protein.

The present invention also relates to a method of inhibiting the binding of TSIP4 to TSIP2 in a cell, comprising the step of: a) bringing said cell into contact with a compound identified by a method according to the invention, inhibiting binding TSIP4-TSIP2. The compound thus envisaged can also be a “decoy” peptide derived from the TSIP4 protein or from the TSIP2 protein. The method can be carried out in vitro or in vivo. The present invention is also directed to a method for the treatment of cancer, characterized in that a compound identified according to the present invention which increases apoptosis and / or tumor reversion is administered to a patient. A method for the treatment of a neurodegenerative disease, consisting in administering, to a patient, a compound according to the present invention and decreasing or inhibiting apoptosis, is also an object of the present invention.

The following examples provide a better understanding of the advantages of the invention and should not be considered as limiting the scope of the invention.

DESCRIPTION OF THE FIGURES

Figure 1: distribution of TSIP4 mRNA in different tissues. Figure 2: differential expression of TSIP 4 in the K562 / KS system.

EXAMPLES

Example 1: Linking TSIP4 and TSIP2

The link between TSIP 4 and TSIP 2 was observed in a double-hybrid system, derived from the system developed by Finley and Brent (Interaction trap cloning with yeast, 169-203, in DNA Cloning, Expression Systems: a practical

Approach, 1995, Oxford Universal Press, Oxford), using presenilin 1

(TSIP2) as bait, and TSIP4 as prey.

The TSIP2 gene (cDNA) was cloned into the plasmid pEG202 known to those skilled in the art for such an application (promoter 67-1511, lexA 1538-2227,

ADH Ter 2209-2522, pBR remnants 2540-2889, 2μ ori 2890-4785, YSCNFLP

4923-5729, HIS3 7190-5699, TYIB 7243-7707, RAFjart 7635-7976, skeleton pBR 7995-10166, bla 8131-8988).

The TSIP4 gene (cDNA) was cloned into the plasmid pJG4-5, also well known to those skilled in the art (promoter GAL 1-528, fusion cassette 528-849,

ADH Ter 867-1315, 2μ ori 1371-3365, TRP1 3365-4250, skeleton pUC 4264-

6422, Ap 4412-5274). The reporter plasmid pSH18-34, also known to those skilled in the art, is also used. This plasmid is in particular available from Invitrogen, under the reference number N611-20, and also already transformed in the strain EGY48

(also called RFY 231), from the same supplier (only strain reference: C835-00, transformed by pSHl 8-34: C836-00)

The binding has been demonstrated in the yeast strain RFY 231 (described in Finley Jr, et al, 1998, Proc atl Acad Sci USA, 95, 14266-71). This yeast strain has the genotype (MATα trplAv.hisG his3 ura3-l leu2 :: 3Lexop-LEU2), and is derived from EGY48 (Guris et al., 1993, Cell, 75, 791-803). The reporter gene was the LacZ gene.

The study is carried out on a medium containing galactose, not containing leucine, and the presence of colored colonies on these dishes is studied.

It has thus been possible to show that the protein TSEP21 binds to the protein TSIP4, and that the binding is on the 204 C-terminal amino acids of the protein TSIP2.

EXAMPLE 2 Analysis of the TSIP4 Protein

It appears that the TSIP 4 protein has two alternative forms, one corresponding to SEQ ID Ν ° 2, coded by SEQ ID No 1, the other corresponding to amino acids 198-400 of SEQ ID No 2, coded by bases 592 to 1200 of SEQ ID No. 1. a putative signal peptide corresponds to amino acids 198-243 of SEQ ID

No. 2 (bases 592 to 729 of SEQ ID No. 1).

The sequences described above are also objects of the invention.

The TSIP4 protein (complete) has 4 predicted trans-membrane domains (amino acids 224-244, 260-280, 320-340, 350-370), the N- and C-terminal ends being located in the cytoplasm. These transmembrane domains, as well as the intra- or extra-cellular domains are also objects of the invention, as well as the sequences which encode them.

Example 3: Expression of the TSIP 4 Protein Hybridization of a Northern Blot from different human tissues with a probe corresponding to a perturbation sequence of TSIP 4 shows the existence of two transcripts (approximately 1.8 and 6 kb). Most mRNA is found in the heart, skeletal muscle, and the brain. It is also less present important in the placenta, pancreas and kidney. Its expression is very weak in the lung and almost nonexistent in the liver (Figure 1).

Example 4 Differential Expression of TSIP 4 in the K562 / KS System Hybridization of a Northern Blot with the TSIP4 probe showed an attenuated signal compared to the K562 line, whereas an equivalent signal is obtained with the control probe GAPDH (Figure 2). The 562 / KS model has been described by Telerman et al (1993, Proc. Natl. Acad. Sci. USA, 90, 8702-6).

The TSIP4 protein is therefore repressed in a tumor repression model.

EXAMPLE 5 Expression of the GST Fusion Proteins

To obtain GST fusion proteins, the following protocol can be followed: Preparation of a preculture from a colony isolated from B121 (DE3), transformed with the plasmid pGEX-Pl-TSIP4 or pGEX-Pl-TSIP2, in an SB medium with 100 μg / ml of ampicillin, at 37 ° C.

Plasmids are available from Amersham Pharmacia Biotech AB.

Proteins are human proteins, encoded by complementary cDNAs corresponding to sequences SEQ ID No. 1 (TSIP4) and the complete sequence of TSIP2 (GenBank U50957).

The next day, inoculate 250 ml of SB + Amp with 5 ml of the preculture.

Growth at 28 ° C or 37 ° C depending on the toxicity of the proteins to the host bacteria, up to an optical density between 0.5 and 0.7. Addition of 0.1 mM IPTG to induce protein synthesis.

Growth at 28 ° C or 37 ° C for 1 hour or 1 hour 30 minutes.

Centrifugation 3000 rpm for 10 min (1800g, 4 ° C)

Resuspension of the precipitate in 10 ml of buffer A NP40 (NP40 1%; Tris pH 7.4 10 mM; NaCl 150 mM; EDTA ImM; Glycerol 10%; DTT ImM; Aprotinin 2μg / ml; Leupeptin 2μg / ml; Pepstatin 2μg / ml; AEBSF ImM).

Sonication 3 times for 15 s at 50 power, on ice.

Centrifugation at 12,000 rpm for 10 min (18000g, 4 ° C)

Keep the supernatant at -80 ° C. EXAMPLE 6 Protocol to be followed for the binding of fusion proteins to sepharose-glutathione beads

Addition of 2 ml of supernatant to 200 μl of beads (prepared after 3 rinses in PBS, 1 rinse in buffer A NP40, resuspension to 50% (weight by volume) in buffer A NP40, centrifugation at 3000 rpm each time ).

Glutathione-Sepharose 4B beads are available from Amersham Pharmacia Biotech AB under number 17.0756.01.

Stir gently for at least 1 hour at 4 ° C. Rinse the beads 3 times in buffer A NP40 without protease inhibitor.

Resuspend the beads in 1 ml of NP40 buffer A with protease inhibitor.

For SDS-PAGE electrophoresis analysis, take 20 to 40 μl of the resuspended beads, centrifuge for 5 min, discard the supernatant, resuspend the beads in 10 μl of X loading buffer, heat to 97 ° C for 7 min.

Load onto gel and analyze after revelation by Coomassie blue, to normalize the quantity of fusion proteins to be used.

EXAMPLE 7 Protocol for Translating Proteins In Vitro The Promega TNT Coupled Reticulocyte Lysate System kit is used with T7 or T3 RNA polymerase depending on the vector used to translate and express proteins (T7 for AIPI and TSIP4 1, T3 for TSIP2). The kit is used according to the manufacturer's instructions (Reference L4610)

The proteins incorporate methionine S ³⁵ (Amersham Pharmacia). The products obtained in vitro are analyzed by SDS-PAGE electrophoresis.

After electrophoresis, the gel is placed in fixing buffer (5% methanol, 15% acetic acid, 80% water) for 1/2 hour and the signal is amplified by immersion of the gel in the Amplify product. from Amersham Pharmacia

(Ref: NAMP100). A Kodak Biomax MR film is then exposed on the dried gel for a period varying from one hour to one week, then developed. EXAMPLE 8 Protocol for the Co-Precipitation of Proteins

30 μl of sepharose-glutathione beads coupled to fusion proteins

GST, after normalization of the quantities, are rinsed in buffer B (NP40 1%,

50 mM TrisHCl, NaC1150 mM, leupeptin 2 μl / ml, aprotinin 1%, ABESF 1 mM). Then added 5 to 10 μl of the in vitro translation product as obtained in Example 3, depending on the amount of the product observed by autoradiography.

After a night of contact, the beads are rinsed 10 times with buffer A NP 40, without antiproteases.

Analysis by SDS-PAGE and autoradiography. We can thus show the link between the proteins TSIP4 and TSIP2.

EXAMPLE 9 Protocol for the Screening of Compounds Interfering with the TSIP4-TSIP2 Link

The tests described in Examples 5 to 8 are carried out, adding the compounds which it is desired to study in the step of Example 8, and comparison is made with the results obtained when the compounds are not added.

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Claims

claims 1. Purified or isolated nucleic acid, characterized in that it comprises a nucleic sequence chosen from the following group of sequences: a) SEQ ID N ⁰ 1; b) the sequence of a fragment of at least 15 consecutive nucleotides of SEQ ID N ⁰ l; c) a nucleic sequence having an identity percentage of at least 80%, after optimal alignment with a sequence defined in a) or b); d) a nucleic sequence hybridizing under conditions of high stringency with a nucleic sequence defined in a) or b); e) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a), b), c) or d). 2. Purified or isolated nucleic acid according to claim 1, characterized in that it comprises or consists of a sequence chosen from the group consisting of SEQ ID No. 1, the sequence complementary to SEQ ID No. 1 and the sequence of RNA corresponding to SEQ ID No. 1. 3. Purified or isolated nucleic acid characterized in that it codes for a polypeptide having a continuous fragment of at least 200 amino acids of the protein SEQ ID N ⁰ 2. 4. Isolated polypeptide characterized in that it comprises a polypeptide chosen from: a) a polypeptide corresponding to SEQ ID No. 2; b) a polypeptide varying from a polypeptide of sequence defined in a); c) a polypeptide homologous to a polypeptide defined in a) or b), comprising at least 80% homology with said polypeptide of a); d) a fragment of at least 15 consecutive amino acids of a polypeptide defined in a), b) or c); e) a biologically active fragment of a polypeptide defined in a), b) or c). 5. A cloning and / or expression vector comprising a nucleic acid according to one of claims 1 to 3 or coding for a polypeptide according to claim 4. 6. Host cell characterized in that it is transformed by a vector according to claim 5. 7. Animal, except man, characterized in that it comprises a cell according to claim 6. 8. Use in vitro of a nucleic acid sequence according to one of claims 1 to 3 as a probe or primer, for the detection and / or amplification of nucleic acid sequences. 9. Use in vitro of a nucleic acid according to one of claims 1 to 3 as sense or antisense oligonucleotide. 10. Use of a nucleic acid sequence according to one of claims 1 to 3 for the production of a recombinant polypeptide. 11. A method of obtaining a recombinant polypeptide characterized in that a cell according to claim 6 is cultured under conditions allowing the expression of said polypeptide and that said recombinant polypeptide is recovered. 12. Recombinant polypeptide characterized in that it is obtained by a method according to claim 11. 13. Monoclonal or polyclonal antibody characterized in that it selectively binds a polypeptide according to one of claims 4 or 12. 14. Method for detecting a polypeptide according to one of claims 4, or 12, characterized in that it comprises the following steps: a) bringing a biological sample into contact with an antibody according to claim 13; b) highlighting of the antigen-antibody complex formed. 15. Kit of reagents for the implementation of a method according to claim 14, characterized in that it comprises: a) a monoclonal or polyclonal antibody according to claim 13; b) optionally reagents for the constitution of a medium suitable for the immunological reaction; c) reagents for the detection of the antigen-antibody complex produced during the immunological reaction. 16. Method for diagnosis and / or prognostic evaluation of a neurodegenerative disease or of a cancer characterized in that the presence of at least one mutation is determined from a biological sample from a patient and / or an alteration of expression of the gene corresponding to SEQ ID No. 1 by the analysis of all or part of a nucleic sequence corresponding to said gene. 17. DNA chip characterized in that it contains a nucleic sequence according to one of claims 1 to 3. 18. Protein chip characterized in that it contains a polypeptide according to one of claims 4 or 12, or an antibody according to claim 13. 19. Method for detecting and / or assaying a nucleic acid according to one of claims 1 to 3 in a biological sample, characterized in that it comprises the following steps: a) contacting a labeled polynucleotide according to one of claims 1 to 3; b) detection and / or assay of the hybrid formed between said polynucleotide and the nucleic acid of the biological sample. 20. A method of detecting and / or assaying a nucleic acid according to one of claims 1 to 3 in a biological sample, characterized in that it comprises a step of amplifying the nucleic acids of said biological sample to the using primers chosen from nucleic acids according to one of claims 1 to 2. 21. A method of screening for compounds capable of binding to a polypeptide of sequence SEQ ID No. 2, characterized in that it comprises the steps of bringing a polypeptide into contact according to one of claims 4 or 12, d a cell according to claim 6, or a mammal according to claim 7, with a candidate compound and detecting the formation of a complex between said candidate compound and said polypeptide. 22. A method of screening for compounds capable of interacting in vitro or in vivo with a nucleic acid according to one of claims 1 to 3, characterized in that it comprises the steps of contacting a nucleic acid according to l one of claims 1 to 3, of a cell according to claim 6, or of a mammal according to claim 7, with a candidate compound and for detecting the formation of a complex between said candidate compound and said nucleic acid 23. Use of a compound obtained by a process according to one of claims 21 or 22, for the preparation of a medicament intended for the treatment of a neurodegenerative disease or of cancer. 24. Compound characterized in that it is chosen from a) a nucleic acid according to one of claims 1 to 3; b) a polypeptide according to one of claims 4 or 12; c) a vector according to claim 5; d) a cell according to claim 6; e) an antibody according to claim 13, as a medicament. 25. Compound according to claim 24, for the prevention and / or treatment of a neurodegenerative disease or of a cancer associated with the presence of at least one mutation or of an alteration of expression of the gene corresponding to SEQ ID N ⁰ 1. 26. Method for identifying a compound inhibiting the binding of TSIP4 to TSIP2, presenting the steps of: a) bringing said compound into contact with a system allowing the in vitro determination of the bond between TSII and TSIP2; b) identification of the decrease and / or inhibition of the link between TSIP4 and TSIP2. 27. Method for identifying a compound for the reduction and / or inhibition of tumor repression and / or cell death (apoptosis), comprising the steps of: a) bringing the compounds into contact with a system allowing the in vitro determination of the link between TSIP4 and TSIP2; b) identification of the compounds inducing the reduction and / or inhibition of the link between TSIP4 and TSIP2; c) bringing the compounds identified in step b) into contact in a model making it possible to measure the phenomena of apoptosis and / or tumor repression; d) identification of the reduction and / or inhibition of tumor repression and / or cell death in said model compared to a control model with which said compound has not been brought into contact. 28. Method for identifying a compound for increasing tumor repression and / or cell death (apoptosis), comprising the steps of: a) bringing compounds into contact with a system allowing the in vitro determination of the link between TSIP4 and TSIP2; b) identification of the compounds inducing the reduction and / or inhibition of the link between TSIP4 and TSIP2; c) bringing the compounds identified in step b) into contact in a model making it possible to measure the phenomena of apoptosis and / or tumor repression; d) identification of the increase in tumor repression and / or cell death in said model compared to a control model with which said compound has not been brought into contact. 29. A method of identifying the regions of TSIP4 involved in the binding with the TSIP2 protein, comprising the steps of: a) contacting peptides derived from the TSIP4 protein in a system allowing the in vitro determination of the binding between TSIP4 and TSIP2; b) identification of the peptides resulting in the reduction of the link between TSIP4 and TSIP2 in said system; 30. Method for identifying a product having an activity of increasing tumor repression and / or apoptosis, characterized in that it comprises the steps of: a) implementation of a method according to l one of claims 26 or 28, b) modification of the product selected in step a) in particular by grafting of residues on the chemical skeleton, c) test of the modified product in step b) in in vitro methods and / or in vivo, on relevant models of tumor repression and / or apoptosis, d) identification of the product making it possible to obtain a tumor repression and / or apoptosis activity greater than the activity obtained for the product selected for step a). 31. Method for identifying a product having an activity of reduction and / or inhibition of tumor repression and / or apoptosis, characterized in that it comprises the steps of: a) implementation of '' a method according to one of claims 26 or 27, b) modification of the product selected in step a) in particular by grafting of residues on the chemical skeleton, c) test of the modified product in step b) in methods in vitro and / or in vivo, on relevant models of tumor repression and / or apoptosis, d) identification of the product making it possible to obtain tumor repression and / or apoptosis activity reduced compared to the activity obtained for the product selected in step a). 32. Use of a compound identified by a process according to one of claims 26, 28 or 30, having an activity of increasing tumor repression and / or apoptosis, for the preparation of a medicament intended cancer treatment. 33. Use of a compound identified by a method according to one of claims 26, 27, or 31, having an activity of reduction and / or inhibition of tumor repression and / or apoptosis, for the preparation a drug intended for the treatment of a neurodegenerative disease. 34. Peptide sequence corresponding to a TSIP4 region which can be identified by a method according to claim 29. 35. Nucleotide sequence coding for a peptide sequence according to claim 34. 36. Peptide sequence according to claim 34, or nucleotide sequence according to claim 35 as a medicament. 37. Use of a peptide sequence according to claim 34, or a nucleotide sequence according to claim 35 for the preparation of a medicament. 38. Complex consisting of a TSIP4 protein and a TSEP2 protein. 39. A method of inhibiting the TSIP4 - TSIP2 binding in a cell in vitro, comprising the step of: a) bringing said cell into contact with a compound identified by a method according to claim 26. AMENDED CLAIMS [received by the International Bureau on May 22, 2002 (22.05.02); original claims 1-39 replaced by amended claims 1-38 (8 pages)] 1. Purified or isolated nucleic acid, characterized in that it comprises a nucleic sequence chosen from the following group of sequences: a) SEQ ID No. 1; b) a nucleic sequence having an identity percentage of at least 80%, after optimal alignment with a sequence defined in a); c) a nucleic sequence hybridizing under conditions of high stringency with a nucleic sequence defined in a); d) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a), b) or c). 2. Purified or isolated nucleic acid according to claim 1, characterized in that it comprises or consists of a sequence chosen from the group consisting of SEQ ID No. 1, the sequence complementary to SEQ ID No. 1 and the sequence of RNA corresponding to SEQ ID No. 1. 3. Isolated polypeptide characterized in that it comprises a polypeptide chosen from: a) a polypeptide corresponding to SEQ ID No. 2; b) a polypeptide varying from a polypeptide of sequence defined in a); c) a polypeptide homologous to a polypeptide defined in a) or b), comprising at least 80% homology with said polypeptide of a); d) a fragment of at least 15 consecutive amino acids of a polypeptide defined in a), b) or c); e) a biologically active fragment of a polypeptide defined in a), b) or c). 4. Cloning and / or expression vector comprising a nucleic acid according to one of claims 1 to 2 or coding for a polypeptide according to claim 3. 5. Host cell characterized in that it is transformed by a vector according to claim 4. 6. Animal, except man, characterized in that it comprises a cell according to claim 5. 7. Use in vitro of a nucleic acid sequence according to one of claims 1 to 2 as a probe or primer, for the detection and / or amplification of nucleic acid sequences. 8. Use in vitro of a nucleic acid according to one of claims 1 to 2 as sense or antisense oligonucleotide. 9. Use of a nucleic acid sequence according to one of claims 1 to 2 for the production of a recombinant polypeptide. 10. A method of obtaining a recombinant polypeptide characterized in that a cell is cultured according to claim 5 under conditions allowing the expression of said polypeptide and that said recombinant polypeptide is recovered. 11. Recombinant polypeptide characterized in that it is obtained by a method according to claim 10. 12. Monoclonal or polyclonal antibody characterized in that it selectively binds a polypeptide according to one of claims 3 or 11. 13. Method for detecting a polypeptide according to one of claims 3, or 11, characterized in that it comprises the following steps: a) bringing a biological sample into contact with an antibody according to claim 12; b) highlighting of the antigen-antibody complex formed. 14. Kit of reagents for the implementation of a method according to claim 13, characterized in that it comprises: a) a monoclonal or polyclonal antibody according to claim 12; b) optionally reagents for the constitution of a medium suitable for the immunological reaction; c) reagents for the detection of the antigen-antibody complex produced during the immunological reaction. 15. Method of diagnosis and / or prognostic evaluation of a neurodegenerative disease or of a cancer characterized in that the presence of at least one mutation is determined from a biological sample from a patient and / or an alteration of expression of the gene corresponding to SEQ ID No. 1 by the analysis of all or part of a nucleic sequence corresponding to said gene. 16. DNA chip characterized in that it contains a nucleic sequence according to one of claims 1 to 2. 17. Protein chip characterized in that it contains a polypeptide according to one of claims 3 or 11, or an antibody according to claim 12. 18. A method of detecting and / or assaying a nucleic acid according to one of claims 1 and 2 in a biological sample, characterized in that it comprises the following steps: a) contacting a polynucleotide according to one of claims 1 and 2, marked; b) detection and / or assay of the hybrid formed between said polynucleotide and the nucleic acid of the biological sample. 19. A method of detecting and / or assaying a nucleic acid according to one of claims 1 and 2 in a biological sample, characterized in that it comprises a step of amplifying the nucleic acids of said biological sample to the using primers chosen from nucleic acids according to either of Claims 1 and 2. 20. A method of screening for compounds capable of binding to a polypeptide of sequence SEQ ID No. 2, characterized in that it comprises the steps of bringing a polypeptide into contact according to one of claims 3 or 11, d a cell according to claim 5, or a mammal according to claim 6, with a candidate compound and detecting the formation of a complex between said candidate compound and said polypeptide. 21. A method of screening for compounds capable of interacting in vitro or in vivo with a nucleic acid according to one of claims 1 and 2, characterized in that it comprises the steps of bringing a nucleic acid according to l one of claims 1 and 2, a cell according to claim 5, or a mammal according to claim 6, with a candidate compound and for detecting the formation of a complex between said candidate compound and said nucleic acid 22. Use of a compound obtained by a process according to one of claims 20 or 21, for the preparation of a medicament intended for the treatment of a neurodegenerative disease or of cancer. 23. Compound characterized in that it is chosen from a) a nucleic acid according to one of claims 1 and 2; b) a polypeptide according to one of claims 3 or 11; c) a vector according to claim 4; d) a cell according to claim 5; e) an antibody according to claim 12, as a medicament. 24. Compound according to claim 23, for the prevention and / or treatment of a neurodegenerative disease or of a cancer associated with the presence of at least one mutation or of an alteration of expression of the gene corresponding to SEQ ID N ° 1. 25. Method for identifying a compound inhibiting the binding of TSIP4 to TSIP2, comprising the steps of: a) bringing said compound into contact with a system allowing the in vitro determination of the bond between TSIP4 and TSIP2; b) identification of the decrease and / or inhibition of the link between TSIP4 and TSIP2. 26. Method for identifying a compound for the reduction and / or inhibition of tumor repression and / or cell death (apoptosis), comprising the steps of: a) bringing the compounds into contact with a system allowing the in vitro determination of the link between TSIP4 and TSIP2; b) identification of the compounds inducing the reduction and / or inhibition of the link between TSIP4 and TSIP2; c) bringing the compounds identified in step b) into contact in a model making it possible to measure the phenomena of apoptosis and / or tumor repression; d) identification of the reduction and or the inhibition of tumor repression and / or cell death in said model compared to a control model with which said compound has not been brought into contact. 27. Method for identifying a compound for increasing tumor repression and / or cell death (apoptosis), comprising the steps of: a) bringing the compounds into contact with a system allowing the in vitro determination of the link between TSIP4 and TSIP2; b) identification of the compounds inducing the reduction and / or inhibition of the link between TSIP4 and TSIP2; c) bringing the compounds identified in step b) into contact in a model making it possible to measure the phenomena of apoptosis and / or tumor repression; d) identification of the increase in tumor repression and / or cell death in said model compared to a control model with which said compound has not been brought into contact. 28. Method for identifying the regions of TSIP4 involved in the binding with the protein TSIP2, comprising the steps of: a) bringing together peptides derived from the protein TSIP4 in a system allowing the in vitro determination of the binding between TSIP4 and TSIP2; b) identification of the peptides resulting in the reduction of the link between TSIP4 and TSIP2 in said system; 29. Method for identifying a product having an activity of increasing tumor repression and / or apoptosis, characterized in that it comprises the steps of: a) implementation of a method according to l one of claims 25 or 27, b) modification of the product selected in step a) in particular by grafting of residues on the chemical skeleton, c) test of the modified product in step b) in in vitro methods and / or in vivo, on relevant models of tumor suppression and / or apoptosis, d) identification of the product making it possible to obtain a tumor suppression and / or apoptosis activity greater than the activity obtained for the product selected for step a). 30. Method for identifying a product having an activity of reduction and / or inhibition of tumor repression and / or apoptosis, characterized in that it comprises the steps of: a) implementation of a method according to one of claims 26 or 26, b) modification of the product selected in step a) in particular by grafting of residues on the chemical skeleton, c) test of the modified product in the step b) in in vitro and / or in vivo methods, on relevant models of tumor suppression and / or apoptosis, d) identification of the product making it possible to obtain tumor suppression and / or apoptosis activity reduced by report on the activity obtained for the product selected in step a). 31. Use of a compound identified by a method according to one of claims 25, 27 or 29, having an activity of increasing tumor repression and / or apoptosis, for the preparation of a medicament intended cancer treatment. 32. Use of a compound identified by a method according to one of claims 25, 26, or 30, having an activity of reduction and / or inhibition of tumor repression and / or apoptosis, for the preparation a drug intended for the treatment of a neurodegenerative disease. 33. Peptide sequence corresponding to a region of TSIP4 which can be identified by a method according to claim 28. 34. Nucleotide sequence coding for a peptide sequence according to claim 33. 35. A peptide sequence according to claim 33, or a nucleotide sequence according to claim 34 as a medicament. 36. Use of a peptide sequence according to claim 33, or a nucleotide sequence according to claim 34 for the preparation of a medicament. 37. Complex consisting of a TSIP4 protein and a TSIP2 protein. 38. A method of inhibiting the TSIP4 - TSIP2 binding in a cell in vitro, comprising the step of: a) bringing said cell into contact with a compound identified by a method according to claim 25.