Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4747—Apoptosis related proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• 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.
• TSAP Tumor Suppressor Activated Pathway
• TSIP Tumor Suppressor Inhibited Pathway
• PSI presenilin 1
• TSIP 2 presenilin 1, PSI
• SEQ ID No. 2 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.
• 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).
• 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
• 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%.
• 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.
• the nucleic acid sequences according to the invention also include PNA (Peptid Nucleic Acid), or the like.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the target polynucleotide to be detected is an mRNA
• 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).
• the latter can be used as capture probes.
• a probe called a “capture probe”
• a 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.
• a second probe called a “detection probe”, marked by an easily detectable element.
• 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.
• 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).
• polypeptide is meant, within the meaning of the present invention, denote proteins or peptides.
• 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).
• 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.
• 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%.
• 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.
• 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.
• a vector can also contain the elements necessary for the expression and optionally for the secretion of the polypeptide in a host cell.
• a host cell is also an object 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.
• control signals are chosen according to the cellular host used.
• 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.
• 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).
• adenoviruses Perricaudet et al., 1992
• retroviruses retroviruses
• lentiviruses lentiviruses
• poxvirus or herpesvirus Epstein et al., 1992.
• herpesvirus Epstein et al., 1992.
• viruses are, for example, retroviruses (Temin, 1986), or AANs (Carter, 1993).
• naked polynucleotides such as naked AD ⁇ or naked ⁇ AR ⁇ 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.
• BAC bacteria chromosome
• YAC yeast artificial chromosome
• MAC mouse artificial chromosomes
• HAC human artificial chromosome
• 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. .
• 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.
• bacterial cells Olins and Lee, 1993
• yeast cells Buckholz, 1993
• animal cells in particular cell cultures.
• mammals Edwards and Aruffo, 1993
• Chinese hamster ovary (CHO) cells in particular Chinese hamster ovary
• a preferred cellular host for the expression of the proteins of the invention consists of COS cells.
• animals such as rodents, in particular mice, rats or rabbits, which express a polypeptide according to the invention, are preferred.
• 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.
• 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.
• 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.
• 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.
• transgenic animals according to the invention can be made deficient for the gene coding for the polypeptide of the sequence SEQ ID NO: 1
• 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.
• polypeptides according to the invention 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.
• 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.
• 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.
• 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.
• 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.
• sequences of the recombinant polypeptides can also be modified in order to improve their solubility, in particular in aqueous solvents.
• 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.
• 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.
• 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
• 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.
• 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.
• the antibodies of the invention in particular the monoclonal antibodies, can also be used for the detection of these polypeptides in a biological sample.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the invention lays the foundations for such a molecular diagnosis based on the search for mutations in TSIP4.
• 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.
• 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.
• methods based on PCR can be used, for example PCR-SSCP which makes it possible to detect point mutations.
• a DNA chip containing a sequence according to the invention is also one of the objects of the invention.
• a protein chip containing an amino acid sequence according to the invention is also an object of the invention.
• 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.
• 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 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.
• kit can also contain positive or negative controls to ensure the quality of the results obtained.
• positive or negative controls to ensure the quality of the results obtained.
• those skilled in the art can also carry out an amplification step using primers chosen from the sequences according to the invention.
• 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.
• 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.
• 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.
• apoptosis 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.
• step b) 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.
• 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.
• 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;
• 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.
• 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.
• 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.
• 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.
• 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
• 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.
• 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 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.
• 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.
• 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.
• 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.
• 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.
• 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 ..
• proteins 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.
• a usable protocol can be the following:
• 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).
• prokaryotic E. coli, B. subtiis
• eukaryotic yeasts such as Saccharomyces, Kluyveromyces ..., mammalian cells (HeLa, Cos, Hep- 2)
• insects using a Baculovirus system
• a Histidine label is chosen, or GST
• the proteins expressed are linked to sepharose beads exhibiting Gluthathione.
• preparation of proteins by in vitro translation 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.
• 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.
• peptide sequence or nucleic or nucleotide sequence 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)
• 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.
• 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 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.
• Figure 1 distribution of TSIP4 mRNA in different tissues.
• Figure 2 differential expression of TSIP 4 in the K562 / KS system.
• TSIP2 as bait
• TSIP4 as prey
• 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,
• 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,
• 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
• 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 (MAT ⁇ 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.
• TSIP 4 protein has two alternative forms, one corresponding to SEQ ID ⁇ ° 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.
• 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.
• 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).
• NP40 NP40 1%; Tris pH 7.4 10 mM; NaCl 150 mM; EDTA ImM; Glycerol 10%; DTT ImM; Aprotinin 2 ⁇ g / ml; Leupeptin 2 ⁇ g / ml; Pepstatin 2 ⁇ g / ml; AEBSF ImM).
• Glutathione-Sepharose 4B beads are available from Amersham Pharmacia Biotech AB under number 17.0756.01.
• 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.
• the gel 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
• Example 3 50 mM TrisHCl, NaC1150 mM, leupeptin 2 ⁇ l / ml, aprotinin 1%, ABESF 1 mM). Then added 5 to 10 ⁇ l of the in vitro translation product as obtained in Example 3, depending on the amount of the product observed by autoradiography.
• the beads are rinsed 10 times with buffer A NP 40, without antiproteases.

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