WO1999060117A2

WO1999060117A2 - Prax-1 polypeptide

Info

Publication number: WO1999060117A2
Application number: PCT/FR1999/001070
Authority: WO
Inventors: Pierre Casellas; Sylvaine Galiegue; Omar Jbilo; Gérard Le Fur
Original assignee: Sanofi-Synthelabo
Priority date: 1998-05-15
Filing date: 1999-05-06
Publication date: 1999-11-25
Also published as: WO1999060117A3; AU3528599A; FR2778665B1; FR2778665A1

Abstract

The invention concerns a purified polypeptide, comprising an amino acid sequence selected among: a) sequence SEQ ID n°2; and b) a biologically active sequence derived from SEQ ID n°2; and c) a fragment of the sequence SEQ ID n°2 having a biological activity of the same type as that of the sequence SEQ ID n°2 polypeptide, or immunologically related to the sequence SEQ ID n°2 polypeptide.

Description

"PRAX-1 polypeptide" The invention relates to a new nucleic acid sequence and the corresponding protein sequence.

The invention also relates to the prophylactic, therapeutic and diagnostic applications thereof.

The authors of the present invention have identified the transcript of a new gene as well as the corresponding protein. This PRAX-1 gene was isolated by the double hybrid technique as coding for a protein interacting specifically with the peripheral benzodiazepine receptor (PBR). The present invention therefore relates to a purified protein PRAX-1, or biologically active fragments thereof.

The invention also relates to isolated nucleic acid sequences encoding said protein or its biologically active fragments and to specific oligonucleotides obtained from this sequence. It further relates to the cloning and / or expression vectors containing at least one of the nucleotide sequences defined above, and the host cells transfected with these cloning and / or expression vectors under conditions permitting repiication. and / or the expression of one of said nucleotide sequences.

Methods of producing recombinant PRAX-1 proteins or its biologically active fragments by transfected host cells are also part of the invention.

The invention also includes antibodies or antibody derivatives specific for the proteins defined above.

The invention also relates to any inhibitor or activator of the specific activity of PRAX-1, but also of any protein-protein interaction involving PRAX-1.

It also relates to antisense oligonucleotide sequences, specific for the above nucleic acid sequences, which can modulate the expression of the PRAX-1 gene in vivo. It relates to oligonucleotide sequences defined from the nucleic acid sequences above, probe or primer usable in the diagnosis of pathologies where PRAX-1 is involved.

The invention finally relates to the use in a method of gene therapy of a nucleotide sequence according to the invention, in which vectors such as for example inactivated viral vectors capable of transferring coding sequences for a protein according to the invention are injected into cells deficient for this protein. The subject of the present invention is a purified polypeptide whose amino acid sequence is the sequence SEQ ID No. 2 or any biologically active sequence derived from SEQ ID No. 2.

In the description of the invention, the following definitions are used: - PRAX-1 protein: a polypeptide comprising an amino acid sequence chosen from the sequence SEQ ID No. 2 or any fragment or derivative thereof biologically active.

- derivative: any polypeptide varying from the polypeptide of sequence SEQ ID No. 2 or any molecule resulting from a genetic and / or chemical modification of the sequence SEQ ID No. 2, that is to say obtained by mutation, deletion , addition, substitution and / or chemical modification of a single or a limited number of amino acids, as well as any isoform sequence, that is to say a sequence identical to the sequence SEQ ID No. 2 to 1 'one of its modified fragments or sequences, containing one or more amino acids in the form of the D enantiomer, said variant, modified or isoform sequences having retained at least one of the properties making them biologically active and / or said sequences being immunologically related to the sequence SEQ ID No. 2.

- biologically active: capable of interacting with the peripheral benzodiazepine receptor. - immunologically related: capable of being recognized by the antibodies specific for the polypeptide of sequence SEQ ID No. 2 and / or capable of inducing antibodies which recognize this polypeptide.

The manufacture of derivatives can have different objectives, including in particular that of improving its production rates, of increasing its resistance to proteases, of modifying its biological activities or of imparting to it new pharmaceutical and / or biological properties.

Advantageously, the invention relates to a polypeptide comprising the interaction domain with PBR of one of the preceding polypeptides.

This domain corresponds to the sequence between residue 1564 and residue 1857 of the sequence SEQ ID No. 2.

A publication referring to the invention appeared on January 29, 1999 in the journal Journal of Biological Chemistry, volume 274, no. 5, 2938-2952. It is entitled: Cloning and Characterization of PRAX-1 - A new protein that specifically interacts with the peripheral benzodiazepine receptor by S. Galliegue et al. The nucleotide sequence corresponding to the invention is accessible dens

Genban with access number AF039571. The present invention also relates to the nucleic acid sequences coding for a PRAX-1 protein or biologically active fragments or derivatives thereof.

More preferably, the subject of the invention is isolated nucleic acid sequences chosen from: a) the sequence SEQ ID No. 1, b) the sequence complementary to the sequence SEQ ID No. 1, c) the acid sequences nucleic acid capable of specifically hybridizing to the sequence SEQ ID No. 1 or their complementary sequences, or of hybridizing specifically to their proximal sequences, d) the sequences derived from the sequences a), b) and c), owing to the fact degeneration of the genetic code.

According to a preferred embodiment, the subject of the invention is the nucleotide sequence SEQ ID No. 1 corresponding to the cDNA of the human protein of sequence SEQ ID No. 2.

The different nucleotide sequences of the invention can be of artificial origin or not. They may be DNA or RNA sequences, obtained by screening of sequence banks using probes prepared on the basis of the sequence SEQ ID No. 1. Such libraries can be prepared by conventional techniques. molecular biology, known to those skilled in the art.

The nucleotide sequences according to the invention can also be prepared by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening libraries. These nucleotide sequences allow the production of nucleotide probes, capable of hybridizing strongly and specifically with a nucleic acid sequence, of a genomic DNA or of a messenger RNA, coding for the polypeptide according to the invention or a biologically fragment active of it. Such probes are also part of the invention. They can be used as an in vitro diagnostic tool for the detection, by hybridization experiments, of specific transcripts of the polypeptides of the invention in biological samples or for the detection of aberrant syntheses or genetic anomalies at the level nucleic acid sequences encoding a polypeptide as defined above such as loss of heterozygosity or genetic rearrangement, resulting from polymorphism, mutations or different splicing.

To this end, a nucleotide probe according to the invention is brought into contact with a biological sample under conditions allowing the formation of a hybridization complex between the probe and the nucleotide sequence of interest, possibly after a preliminary step of amplification of said nucleotide sequence; the hybridization complex possibly formed is detected and the nucleotide sequence forming the hybridization complex with the probe of the invention is optionally sequenced. The nucleotide sequences of the invention can also be used for the manufacture of a medicament useful in gene therapy.

The probes of the invention comprise at least 10, preferably at least 16 nuciototides, and at most comprise the entire sequence of the PRAX-1 gene or of its cDNA contained for example in a cosmid. Among the shortest probes, that is to say about 10 to 20 nuciototides, the appropriate hybridization conditions correspond to the stringent conditions usually used by those skilled in the art, in particular of high stringency as defined by Sambrook et al. in Molecular Cloning, a laboratory manual, 2 edition,

ColdSpring Harbor Laboratory Press, 1989. The temperature used is preferably between T _m -5 ° C to T _m -30 ° C, more preferably between Tm -5 ° C and T _m -10 ° C, T being the temperature melting point, the temperature at which 50% of the paired DNA strands separate.

Advantageously, the hybridization conditions used are as follows - temperature: 65 ° C., - hybridization buffer: BSA 1%, SDS 7% NaH2 θ4 0.5M, EDTA 1 mM, as described in the example VII.

Advantageously, these probes are represented by the following oligonucleotides as well as the corresponding complementary sequence nucleotides:

SEQ ID n ° 3: GCT CTG TTT GAC TAT GAC CC SEQ ID n ° 4: AAC TTG AAT CCA ATG AGC AA

SEQ ID n ° 5: TCT GCT GAC CTG AAA GCT CC

SEQ ID n ° 6: CCA TCA GGG GGC ACA GGC TC

SEQ ID No.7: TGG GCC TTG TAC CCC CAG CT

SEQ ID n ° 8: TCC TCC TGA GGA GCT GCT TC SEQ ID n ° 9: CCC CTG CCC GGA AGG GCA GC

SEQ ID n ° 10: TTG ACC AAG GTC ACT GAG CC

SEQ ID n ° 1 1: GGG CCT GCT GGC CCC TGT AA

SEQ ID # 12: GCC ATC GGC ATC CTT GTC CC

SEQ ID # 13: CCT CTC CGG TCT CTG TTG CCC G SEQ ID # 14: AGG CCC AGG GGG CCT CCG AAG GC

SEQ ID n ° 15: CGC TGC CCG CCT CCT GCT CAT CCT C

SEQ ID # 16: TCC TCC AGG GCT GGA GAC TGC CT

SEQ ID n ° 17: GGT TCC AGG ACT TGC TCC TTC CAG AAG SEQ ID No. 18: CAG CTC CCT CAC TGG CCA AG

SEQ ID n ° 19: TGG CTC CGG CCA GCC TGC C

SEQ ID No. 20: TCC GGG TCA CAG GCT ATG CCA T

SEQ ID n ° 21: CC CAG TAC CTA CTG GGC CAC CT SEQ ID n ° 22: GCT TCC ATA TCT GTG TGA ATG GG

SEQ ID n ° 23: GGT AAG GCA CGG CAG GAG GCT C

SEQ ID n ° 24: GCT GGC TCC TTC TCA CAC TG

SEQ ID # 25: GGA GAG CTC ATG GAT GGC CGA AG

SEQ ID No. 26: CCA GTG AGG TAG AGG AGC TGG SEQ ID No. 27: TCC ATT CCC CAG CCT TGC CGG GG

SEQ ID No. 28: GCT CGA AGA ACA GTG CCG CAG CC

SEQ ID # 29: TCG CAA GAG CCA GGG CCT GCA GA

SEQ ID # 30: GCT GGA ATC GGA GCT CAG CAA G

SEQ ID # 31: CCC GTG ATT CTA GGG GAG CCA GAG SEQ ID # 32: GAG TGC AGG GAG CTG CAG GCC A

SEQ ID No. 33: GGC TGA AGC GGA AGA ACG CCG AG

SEQ ID # 34: CAA TCT GGA GCT GCT GAG GG

SEQ ID # 35: TGA GGT CTG AGG AGA GTT CCA AG

SEQ ID No. 36: ATG GAG CCA TGG GCA CTG CCC Preferably, the probes of the invention are marked, prior to their use. For this, several techniques are within the reach of those skilled in the art

(fluorescent, radioactive, chemiluminescent, enzymatic labeling, etc.).

The in vitro diagnostic methods in which these nucleotide probes are used are included in the subject of the present invention. These methods relate, for example, to the detection of abnormal syntheses (eg accumulation of transcripts) or genetic abnormalities, such as loss of heterozygosity and genetic rearrangement, and point mutations in the nucleotide sequences of nucleic acids. coding for a protein

PRAX-1, as defined above. The nucleotide sequences of the invention are also useful for the manufacture and use of oligonucleotide primers for specific sequencing or amplification reactions according to the so-called PCR technique or any variant thereof (Ligase Chain Reaction (LCR) ), ...).

Preferred pairs of primers are constituted by primers advantageously of at least 16 nucleotides, chosen from the nucleotide sequence:

SEQ ID No. 1: human sequence of 7033 nuciotides.

Advantageously, these primers are represented by the following pairs: - couple n ° 1: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n ° 3) antisense primer: AAC TTG AAT CCA ATG AGC AA (SEQ ID n ° 4)

- couple n ° 2: sense primer: TCT GCT GAC CTG AAA GCT CC (SEQ ID n ° 5) antisense primer: TCC TCC TGA GGA GCT GCT TC (SEQ ID n ° 8)

- couple n ° 3: sense primer: TTG ACC AAG GTC ACT GAG CC (SEQ ID n ° 10) antisense primer: AAC TTG AAT CCA ATG AGC AA (SEQ ID n ° 4)

- couple n ° 4: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n ° 3) antisense primer: GCC ATC GGC ATC CTT GTC CC (SEQ ID n ° 12)

- pair n ° 5: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n ° 3) antisense primer: TCC TCC TGA GGA GCT GCT TC (SEQ ID n ° 8) "sense" primers ranging from 5 'to 3 'and the 3' to 5 '"anti-sense" primers.

These primers correspond to the sequences respectively between:

- nucleotide n ° 5091 and nucleotide n ° 5110 on SEQ ID n ° 1 for SEQ ID n ° 3

- nucleotide n ° 6700 and nucleotide n ° 6719 on SEQ ID n ° 1 for SEQ ID n ° 4.

- nucleotide n ° 5475 and nucleotide n ° 5494 on SEQ ID n ° 1 for SEQ ID n ° 5 - nucleotide n ° 5939 and nucleotide n ° 5958 on SEQ ID n ° 1 for SEQ ID n ° 8

- nucleotide n ° 6226 and nucleotide n ° 6245 on SEQ ID n ° 1 for SEQ ID n ° 10

- nucleotide n ° 5188 and nucleotide n ° 5207 on SEQ ID n ° 1 for SEQ ID n ° 12

Thus, PRAX-1 as a new protein can be considered according to the invention as a new element integrating gene therapy. The nucleotide sequences encoding the PRAX-1 protein can be transferred to target cells via inactivated viral vectors. The prospect of using this protein in a gene therapy treatment comes first from the chromosomal localization of the human gene coding for this protein. Located on chromosome 17 in the q22-q23 region, this gene is associated with markers of serious pathologies affecting the central nervous or immune system (neuroblastomas or leukemias). An application of this protein in gene therapy is also entirely possible according to the invention if we consider the particular expression of this protein associated with three major systems in the body: - the central nervous system, the expression of this new protein is predominant in the brain. Its expression is associated with certain well-defined brain regions.

- the immune system, the expression profile of the protein in the thymus is quite remarkable.

- the endocrine system with an expression in the adrenal glands, the pituitary gland, the kidney, the skin.

The nucleotide sequences according to the invention can moreover be used for the production of PRAX-1 recombinant proteins, according to the definition which has been given to this term.

These proteins can be produced from the nucleotide sequences defined above, according to techniques for the production of recombinant products known to those skilled in the art. In this case, the nucleotide sequence used is placed under the control of signals allowing its transcription and expression in a cellular host.

An efficient system for producing a recombinant protein requires a vector, for example of plasmid or viral origin, and a compatible host cell.

The cell host can be chosen from prokaryotic systems, such as bacteria, or eukaryotic systems, such as, for example, yeasts, insect or mammalian cells, in particular CHO (Chinese hamster ovary cells) or any other system advantageously available. . A preferred cellular host for the expression of the proteins of the invention consists of the bacterium E. coli, in particular the strain MC 1061 (Clontech). The vector must include a promoter, translation initiation and termination signals, as well as the appropriate regions for transcription regulation. It must be able to be maintained stably in the cell and may possibly have particular signals specifying the secretion of the translated protein. These different control signals are chosen according to the cellular host used. To this end, the nucleotide sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or integrative vectors of the chosen host. Such vectors will be 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, electroporation. Cloning and / or expression vectors containing at least one of the nucleotide sequences defined above also form part of the present invention.

A cloning and expression vector used is the plasmid pZeoSV2 (+) (Invitrogen, V850-01) which contains both the elements necessary for its use as a cloning vector in E. coli (origin of replication in E. coli and Zeocin resistance gene), and as an expression vector in animal cells (promoter, polyadenylation signal, origin of replication of the SV40 virus), as well as the elements allowing its single-strand copying for the purpose of sequencing ( origin of replication of phage f1).

The integration of cDNAs originating from the nucleic acid sequences of the invention is further described in the examples below.

According to a preferred embodiment, the proteins of the invention are in the form of fusion proteins, in particular in the form of protein fused with glutathione S-transferase (GST). An expression vector designated in this case is represented by the plasmid vector pGEX-4T-2 (Pharmacia ref-27.4581 -01).

The invention further relates to host cells transfected with these preceding vectors. 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.

These cells can be used in a method for producing a recombinant polypeptide of sequence SEQ ID No. 2 or any fragment or biologically active derivative thereof. The method for producing a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transfected cells are cultured under conditions allowing the expression of a recombinant polypeptide of sequence SEQ ID No. 2 or any biologically active fragment or derivative thereof, and that said recombinant polypeptide is recovered. The purification methods used are known to those skilled in the art. The recombinant polypeptide can be purified from cell lysates and extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using '' specific mono or polyclonal antibodies, etc. A preferred variant consists in producing a recombinant polypeptide fused to a "carrier" protein (chimeric protein). The advantage of this system is that it allows stabilization and a decrease in the proteolysis of the recombinant product, an increase in solubility during renaturation in vitro and / or a simplification of the purification when the fusion partner has an affinity for a specific ligand.

Advantageously, the polypeptides of the invention are fused with glutathione S-transferase in the N-terminal position ("GST" Pharmacia system). The fusion product is in this case detected and quantified thanks to the enzymatic activity of GST. The color reagent used is a glutathione acceptor, a substrate for GST. The recombinant product is purified on a chromatography support to which glutathione molecules have previously been coupled.

Mono or polyclonal antibodies specifically directed against a PRAX-1 protein according to the definition given above also form part of the invention. Polyclonal antibodies can be obtained from the serum of an animal immunized against the protein, produced for example by genetic recombination according to the method described above, according to the usual procedures.

The monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas described by Kôhler and Milstein, Nature, 1975, 256, 495-497.

Advantageous antibodies are antibodies directed against epitopes located in the region between residue 1564 and residue 1857 for the sequence SEQ ID No. 2.

The antibodies according to the invention can be in the form of immunoconjugates or labeled antibodies.

Furthermore, in addition to their use for the purification of the recombinant 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 protein PRAX-1 on sections of specific tissues, for example by immunofluorescence, gold labeling, enzyme immunoconjugates.

More generally, the antibodies of the invention can be advantageously used in any situation where the expression of a PRAX-1 protein must be observed. The invention therefore also relates to a method of in vitro diagnosis of pathological conditions correlated with an abnormal expression or accumulation of PRAX-1 proteins, from a biological sample, characterized in that at least one contact is made an antibody of the invention with said biological sample, under conditions allowing the possible formation of specific immunological complexes between a PRAX-1 protein and the said antibody or antibodies and in that the specific immunological complexes possibly formed are quantitatively detected. The invention also relates to a kit for the in vitro diagnosis of an abnormal expression or accumulation of the protein PRAX-1 in a biological sample and / or for the measurement of the level of expression thereof in said sample comprising: - at least one antibody specific for the PRAX-1 protein, optionally attached to a support,

- Means for revealing the formation of specific antigen / antibody complexes between the PRAX-1 protein and said antibody and / or means for quantifying these complexes. The invention also relates to any method of diagnosis by the demonstration in the serum of an individual, of autoantibodies directed against the protein PRAX-1.

Such an early diagnosis method is characterized in that a serum sample taken from an individual is brought into contact with a polypeptide of the invention, optionally attached to a support, under conditions allowing the formation of specific immunological complexes between said polypeptide and the autoantibodies possibly present in the serum sample, and in that the specific immunological complexes possibly formed are detected.

The PRAX-1 protein can indeed be considered as a diagnostic element for pathologies occurring in the systems where it is expressed and finds its functionality, in particular the immune, central nervous and endocrine systems.

The invention also relates to a method for determining an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic abnormality of the PRAX-1 gene. , which can be involved in pathologies, characterized in that it uses at least one nucleotide sequence described above. Among the methods for determining an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic abnormality of the PRAX-1 gene, a method comprising a preferred method is preferred. at least one step of amplification by PCR of the target nucleic sequence of PRAX-1 capable of exhibiting a polymorphism, a mutation, a deletion or an insertion using pairs of primers of nucleotide sequences defined above, a step during which the amplified products are treated with appropriate restriction enzymes and a step during which at least one of the products of the enzymatic reaction is detected or assayed . The PRAX-1 protein can finally be envisaged according to the invention as a new pharmacological target by its function involving the peripheral benzodiazepine receptor (PBR). It can in fact be the target of molecules defined to interfere, to modulate the ability of this protein to interact with PBR and thus modify its function. It can also be the target of molecules capable of modulating the expression levels of this protein. Elements for regulating the expression of the PRAX-1 protein are recognized on the promoter of the human gene coding for PRAX-1. By regulating, either by induction or inhibition, the transcription of the gene into mRNA, these elements indirectly modulate the expression of the protein PRAX-1.

The invention also includes pharmaceutical compositions comprising as active ingredient a polypeptide corresponding to the preceding definitions, preferably in soluble form, associated with a pharmaceutically acceptable vehicle.

These pharmaceutical compositions are intended to be administered to subjects having an expression deficient in protein PRAX-1, associated with pathological conditions or having a pathological condition involving PRAX-1 or its receptor. The pharmaceutical compositions of the invention may also contain an inhibitor or inactivator of the activity of PRAX-1 and of any protein-protein interaction involving PRAX-1, in particular a polypeptide derived from a polypeptide as defined above.

Preferably, these compositions can be administered systemically, preferably intravenously, intramuscularly, intradermally or orally.

Their optimal modes of administration, dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a therapeutic treatment adapted to a patient such as for example the patient's age or body weight, the severity of general condition, tolerance to treatment and observed side effects, etc.

The invention is illustrated by the following examples for which reference is made to the appended figure which is a schematic representation with a partial restriction map of the plasmid pZeoSV2 (+) containing human PRAX-1 (2H).

EXAMPLE 1

Isolation and identification of PRAX-1 1) Double hybrid strategy

The double hybrid technique is a method of cloning by interaction in vivo in the yeast Saccharomyces cerevisiae. The object of this technique is to detect protein-protein interactions in vivo and search for the protein partners of a given protein used as bait. The bait used is defined on the protein sequence of PBR. Definition of PBR bait should be limited to regions hydrophilic protein according to the constraints of the double hybrid technique. As the PBR protein is strongly hydrophobic, with 5 transmembrane regions, located on the external mitochondrial membrane, we define the PBR bait from the C-terminal motif of the protein, a region demonstrated cytosolic and accessible to possible protein partners by l existence of an anti-PBR monoclonal antibody directed against this region of the protein. A PBR bait constructed on the basis of this C-terminal motif, by juxtaposition three times of the 14 C-terminal amino acids of PBR was selected.

2) Construction of the PBR bait

The PBR bait is a synthetic construct which juxtaposes three times the C-terminal motif of PBR, domain aa156 to 169, separated by glycine linkers: protein sequence bait:

RDNHGWHGGRRLPE-gly-gly-RDNHGWHGGRRLPE-gly-gly-RDNHGWHGGRRLPE or 46 amino acids.

The nucleotide sequence coding for the PBR bait is cloned into the vector pGBT9 (Clontech) at EcoRI and Xhol sites.

3) Screening of the Fusion Banks The HF7c line is first transformed by the bait plasmid then by the chosen fusion bank (fetal brain, Matchmaker library, Clontech) according to the method defined by Schiestl et al. Curr. Genet., 1989, 16, 339-346. The double hybrid screening is carried out on 2.10 cells. The HIS + and LacZ + clones are identified and tested for their specificity by replacing the PBR bait with other non-relevant constructions. Selection and specificity tests, carried out under the conditions defined in

Matchmaker Library User Manual (Clontech), allow to isolate 4 positive clones.

4) Identification of PRAX-1

The insert contained in each of the clones is sequenced with the ABI PRISM Dye Terminator cycle sequencing Ready reaction Kit, according to the instructions defined in the protocol of use, on an Applied sequencer.

Biosystems model 373A. The 4 clones contain an identical 2152bp sequence containing a polyA tail, a STOP codon, unknown in the databases.

This sequence was named PRAX-1 for Peripheral benzodiazepine Receptor Associated Protein 1. The sequence isolated by double hybrid encodes a polypeptide of 294 amino acids and is incomplete: it lacks the ATG codon coding for the initial methionine in 5 ′. Obtaining the complete sequence of PRAX-1 is described in Example II. EXAMPLE 2

Obtaining the entire sequence and cloning of the cDNA of the human protein PRAX-1 1) Screening of the library a) The library The selected library is a lamda Zap cDNA frontal cortex library

(Stratagene). b) Preparation of the membranes

The bank clones are spread on LB agar medium, 0.2% maltose, 10 mM MgSO 4 (150 diameter petri dishes) coated with Hybond membranes (Amersham). After one night at 37 ° C., the clones are transferred by contact to new membranes. The latter are treated by placing them on Whatman 3 mm paper soaked with the following solutions: NaOH 0.5 N, NaCI 1.5 M for 2 minutes then Tris HCI 0.5 M pH 8, NaCI 1.5 M for 5 minutes, finally 30 sec on Tris HCI 0 , 2M pH 7.5, SSC 2X. The membranes are dried and then incubated in a vacuum oven at 80 ° C for 2 hours. c) Preparation of PRAX-1 probes

Different PRAX-1 probes are successively used defined on the

32 nucleotide sequence SEQ ID No. 1. The probes are labeled with P using the RTS Rad Prime DNA labeling System kit (Gibco BRL). The probes are prepared o with a specific activity of 5 10 dpm / μg on average. d) Prehybridization and hybridization

The membranes prepared in b) are prehybridized for 30 minutes at 42 ° C in 6 x SSC, 5 x Denhart's, 0.1% SDS and then hybridized overnight in the same buffer supplemented with the probe prepared in c) at a rate of 10 ⁶ dpm / ml. e) Washing and exposure of membranes

The membranes are washed twice at room temperature in the buffer 2 x SSC / SDS 0.1% then one hour at 56 ° C in 6 x SSC / SDS 0.1%. The hybrid clones are revealed by autoradiography and selected after multiple screening.

2) Sequencing of the clones obtained PRAX-1

The sequential screening of the lambda Zap library made it possible to isolate clones containing the cDNA coding for the whole protein PRAX-1. The sequence is obtained using the ABI PRISM Dye Terminator cycle sequencing Ready reaction Kit, according to the instructions defined in the operating protocol, on the Applied Biosystems model 373A sequencer.

The complete nucleotide sequence coding for PRAX-1 is 7033 bp, presented in the appendix. It codes for a polypeptide of 1857 amino acids, of molecular weight 200 kDa. The portion isolated by double hybrid is the C-terminal end of 294 C-terminal amino acids, corresponding on the coding sequence SEQ ID No. 1 to the region 4887 to 7033. The portion of PRAX-1 isolated in the double screening hybrid is referenced PRAX-1 (2H) and corresponds to the domain extending from amino acids 1564 to 1857 in SEQ ID No. 2.

EXAMPLE 3

1) Production of recombinant protein PRAX-1 in E. Coli a) Construction of the expression plasmid

It consists in inserting the coding sequence for the 2H portion of PRAX-1 (294 C-terminal amino acids of PRAX-1) in phase with glutathione S-transferase (GST) in the vector pGEX 4T2 (Pharmacia). For this, the corresponding insert (bp 4887 to 7033) is cloned at Smal site in the vector pGEX 4T2. The insert and the plasmid are digested and then purified after migration on 1% agarose gel using the Geneclean kit (Bio101). After ligation with 100 ng of insert and 10 ng of dephosphorylated vector and transformation by heat shock, the recombinant E. coli clones are verified by sequencing using the Applied Biosystem kit (Perkin Elmer). b) Expression and purification of the GST-PRAX-1 fusion protein (2H) This step was carried out using the "Bulk GST purification module" kit

(Pharmacia).

A recombinant clone was cultured at 37 ° C. in one liter of LB culture medium (bacterotryptone 10 g / l, yeast extract 5 g / l, NaCl 10 g / I) + ampicillin 100 μg / ml. At Dθ600nm 0.8, the expression is induced with 0.5mM IPTG for 2 h at 37 ° C. After centrifugation, the cell pellet is taken up in cold PBS and then sonicated by ultrasound. After centrifugation at 12000 g, for 10 min at 4 ° C, the supernatant is recovered. The purification is then carried out on a glutathione sepharose 4B affinity chromatography column. Fixation and washing are carried out in PBS buffer and elution is carried out by competition with reduced glutathione. The final concentration of GST-PRAX-1 fusion protein (2H) is variable between 0.5 and 1 mg / ml depending on the preparations.

2) Production of recombinant protein PRAX-1 in Jurkat cells a) Construction of the expression plasmid The insert coding for the C-terminal end of PRAX-1 came out of the plasmid of the fetal brain bank pActll-PRAX-1 (2H) by digestion with Bglll. This digestion makes it possible to leave in 5 ′ of the insert coding for PRAX-1 (2H), the coding sequence for the HA epitope (hemagglutinin). The insert thus digested is cloned into pZeo SV2 + on site BamHI. The linearized cloning vector and the insert are ligated. Competent "thermal shock" bacteria are transfected by this construction. The construction is checked by sequencing (see figure in the appendix). b) Jurkat cells Jurkat cells (human leukemia T) are cultured in RPMI medium

1640 (Gibco) supplemented with 50 mg / ml gentamycin and 10% fetal calf serum (Gibco). c) Transformation of Jurkat cells

Jurkat cells are transfected by electroporation using the Biorad Gene Puiser device (Biorad), 320V, 250μFr, 5ms. The selection of the transfected clones is carried out with Zéocin 500 μg / ml in RPMI 1640 medium, 20% FCS. d) The transfected cells are cultured in RPMI1640 medium, 20% FCS, Zéocin 500 μg / ml. The production of recombinant HA-PRAX-1 (2H) protein is verified using the anti-HA (Boerhinger Mannheim) or anti-PRAX-1 antibody.

EXAMPLE 4

Preparation of specific polyclonal antibodies

Immunogenic peptides of 20 amino acids are selected from the protein sequence PRAX-1 SEQ ID No. 2, in the hydrophilic portions of the protein. 5 mg of each KLH coupled peptide are used to immunize 2 rabbits (male from 1.5 to 2 kg approximately, New Zealand). Four immunizations were carried out 15 days apart according to the protocol described by Vaitukaitis, Methods in enzymology, 1981, 73, 46. The sera are collected one month after the first immunization then 2 times successively at 1 month and 15 days after interval. Each serum collected is tested in western immunoblotting for the recognition of the PRAX-1 protein. The immune sera are purified on an Affigel affinity column (Biorad). The specificity of recognition of PRAX-1 by the antibodies was verified by displacement of the labeling obtained, in the presence of the corresponding immunogenic peptide. A polyclonal antibody was thus obtained. Its target is the peptide AA1772-1792 on the sequence SEQ ID No. 2.

EXAMPLE 5

Detection of the PRAX-1 protein by western immunoblotting 1) Materials used for immunoblotting a) Cell lines used for immunoblotting

The cell lines were cultivated, as described in the "Catalog of cell lines and hybridomas, 7th edition, 1992" of the ATCC (American Type Culture Collection). Human lines: U373 (astrocyte line), SHSY5Y (neuroblastoma), NHA (normal astrocytes), Jurkat, Molt-4 (T leukemia), Daudi, U937 (B lymphoma), SW480 (colon adenocarcinoma), DU145 (prostatic carcinoma) , Hacat, SVK14, WS1, NCTC2544, A431, VA ES BJ (keratinocytes). Murine line: J744-A1 (monocytes-macrophage). Rat line: C62b (Glioma). b) E. coli expressing GST-PRAX-1 (2H) or PRAX-1 protein (2H) translated in vitro The fusion protein GST-PRAX-1 (2H) is prepared as indicated in Example III. PRAX-1 (2H) is prepared as a reticulocyte lysate with the TNT T7 / T3 Coupled reticulocyte Lysate System kit (Promega ref. L5010) according to the protocol of use defined in the kit. The plasmid used in this system is a plasmid pBluescript-PRAX-1 (2H) constructed by insertion of the coding sequence for PRAX-1 (2H) (nuciéotides 4887 to 7033 on SEQ ID No. 1) in the vector pBluescript (Stratagene) in BamHI site.

2) Preparation of protein samples from cell cultures

After culture, the cells are washed with PBS and then lysed in the lysis buffer (6M urea, 50mM Tris HCl pH 6.8, 2% SDS, 10% glycerol, 100mM DTT, 0.1% bromophenol blue). The lysates are sonicated by ultrasound at 4 ° C for 2 min, heated 10 min at 100 ° C and stored at -20 ° C.

3) Western Blot

The samples calibrated for their protein content are deposited on 4-12% or 4-20% SDS-PAGE gel and then electrotransfered onto a nitrocellulose membrane.

4) Revelation with the anti-PRAX-1 antibody

The membrane is incubated for at least 1 h in TBST blocking buffer (Tris HCI 10mM pH 8, NaCl 150mM, 0.1% Tween 20) added with 10% milk at room temperature. The membrane is successively placed in the presence of the anti-PRAX-1 antibody in the same buffer, for 3 h at room temperature, washed 3 times for 10 min with TBST, then incubated for 45 min with a second anti-immunoglobulin antibody. of rabbit coupled to peroxidase (SIGMA) in TBST 5% milk buffer. After 3 washes in TBST of 15 min, the revelation is carried out using the ECL kit (Amersham) by chemiluminescence.

5) Results

Figure in annex: The immunoblots obtained with the anti-PRAX-1 antibody show that it specifically recognizes the recombinant proteins and endogenous. Analysis of human cell lines shows an expression of the PRAX-1 protein detected in all of the lines tested.

EXAMPLE 6 Chromosome localization

The chromosomal localization of the PRAX-1 gene was carried out by in situ hybridization according to the procedure described by Heng et al. Proc. Natl Acad. Sci. USA (1992), 89, 9509-9513 and Chromosoma (1993), 102, 325-332. The cDNA probe used is a 3.5Kb probe defined on the sequence SEQ ID No. 1, comprising the 3 ′ end of the cDNA isolated by double hybrid. 100 mitoses were analyzed, 73 mitotic images show double spots located on a pair of chromosomes. The results make it possible to locate the PRAX-1 gene in the q22-q23 region on chromosome 17.

EXAMPLE 7

Expression of human PRAX-1 mRNA

Analysis of the expression of the PRAX-1 mRNA was carried out on a Multiple Tissue Northern Blot membrane and RNA Master Blot (Clontech) carrying poly A + RNA from human tissues separated by electrophoresis or deposited in dot. The membranes are prehybridized for 1 hour at 65 ° C in Church buffer (1% BSA, SDS

7%, 0.5 M NaHPθ4, 1 mM EDTA), hybridized in the same buffer containing the cDNA PRAX-1 probe, at 65 ° C overnight. The membranes are washed for 5 min in 5% SDS buffer, 0.5% BSA, 40 mM NaH2Pθ4, 1 mM EDTA. The markings are revealed by autoradiography after 24 hours of exposure. An 837bp PRAX-1 probe is prepared by PCR using the primers SEQ ID No. 3 and SEQ ID No. 8. The probe purified on gel by geneClean (Bio101) is

32 radiolabelled with P using the Radprime labeling kit (Gibco) under the conditions defined by the kit's labeling protocol.

The results show a majority expression of the PRAX-1 transcript in the human brain. The visible majority transcript is 7.5Kb in agreement with the size of the cDNA determined after screening of the cDNA libraries.

The PRAX-1 transcript therefore exhibits strong expression in the central nervous system, with expression varying according to the regions of the brain. On the periphery the thymus and the pituitary gland are distinguished from other tissues where expression is weaker. Among fetal tissues, the fetal brain gives a strong signal, followed by the heart, kidney, thymus, and lung. EXAMPLE 8

Functionality of the PRAX-1 protein

New tracks concerning the functionality of this protein were brought by experiments showing modulations of its expression. The experiments were carried out on cell lines and on tissues. Modulations of the expression of the PRAX-1 protein have been observed associated with proliferative processes on PBL cells, associated with neurological lesions following treatment with kainate or focal ischemia on tissues. In rats treated with Kainate (11 mg / kg I.P.), a drastic decrease in the expression of PRAX-1 is observed 9 days later in this hippocampal region. A decrease is also apparent in the other brain regions sensitive to kainate such as the amygdala, the piriform cortex or the cerebral cortex.

EXAMPLE 9 The PRAX-1 / PBR interaction was demonstrated in animals by the co-localization of PRAX-1 and PBR in the thymus, the adrenal gland and the skin.

The promoter of the human gene encoding PRAX-1 is known. Its characterization reveals the existence of potential sites for regulating the expression of PRAX-1. The human PRAX-1 gene is around 26Kb and is divided into 32 exons separated by 31 introns, the size of the exons varies from 21 to 840bp while the size of the introns ranges from 79 to 3300bp. The 2.6Kb analysis preceding the translation initiation site shows the presence of potential modulation sites for gene transcription: 3 Sp 1 sites, 1 AP-1 site, 3 AP-2 sites, 2 SRE, 10 GRE, 1 Zre site, 15 CACCC sites, 5 CT boxes and binding sites for different regulatory factors MAZ (two), C / EBPα (one), c-Ets-2 (one), Puf (three), GATA -1 (one), MyoD (one), USF (three), GC rich binding factor (one), SRY (three) and SOX-5 (one). Sp1 sites are part of the basic transcription machinery and provide a level of expression. Growth factors can modulate the expression of PRAX-1 through the SRE sites. AP-2 sites are involved in particular during the development of the central nervous system, proliferation and oncogenesis phenomena. GRE sites are glucocorticoid response sites and are either activators or repressors. They frequently act in conjunction with other related factors. USF is an activator of ubiquitous transcription. Puf is involved in the removal of tumors. The CACCC sites have in particular been described active on blood cells. The factor C / EBPcc is involved in energy phenomena, cell multiplication and differentiation. Other factors have specific expressions: MyoD is involved in myogenesis, MAZ, GATA-1, c-ets-2 are involved in hematopoiesis and lymphocyte maturation, the SRY and SOX-5 sites are particularly active during development and embryogenesis.

Different constructions are possible allowing expression of the whole protein: - CFP-PRAX-1, expression in a cellular system in the form of a GFP associated fusion protein. Cloning in pEGFP-C1 Clontech ref 6084-1 and p-EGFP-N2, Clontech ref 6081-1. The transfection of U937 and THP1 cells with these plasmids is carried out according to the conditions defined in EXAMPLE 3 of the present application.

- Bluescript-PRAX-1, in vitro expression of the whole protein PRAX-1. The expression of the protein is carried out using in vitro transcription / translation kits (Riboprobe Promega kit, Flexi-rabbit Promega kit).

A construct is also available containing a PRAX-1 mouse insert of 1410 bp corresponding to the N terminal end of PRAX-1, cloning into the vector pcDNA3.

Claims

I. Purified polypeptide, comprising an amino acid sequence chosen from: a) the sequence SEQ ID No. 2; b) a biologically active sequence derived from SEQ ID No. 2; and c) a fragment of the sequence SEQ ID No. 2 having a biological activity of the same nature as that of the polypeptide of sequence SEQ ID No. 2, or immunologically related to the polypeptide of sequence SEQ ID No. 2.

2. Polypeptide according to claim 1, characterized in that it comprises the sequence between: residue 1564 and residue 1857 of SEQ ID No. 2.

3. Polypeptide according to claim 1, characterized in that it results from an alternative splicing of the messenger RNA of the corresponding gene.

4. Polypeptide according to any one of the preceding claims, characterized in that it is a recombinant polypeptide produced in the form of a fusion protein.

5. An isolated nucleic acid sequence encoding a polypeptide according to any one of the preceding claims.

6. Isolated nucleic acid sequence according to claim 5, characterized in that it is chosen from: a) the sequence SEQ ID No. 1; b) the sequence complementary to the sequence SEQ ID No. 1; c) the nucleic acid sequences capable of hybridizing specifically to the sequence SEQ ID No. 1, or to their complementary sequences, or to hybridizing specifically to their proximal sequences. d) sequences derived from sequences a), b) and c), due to the degeneracy of the genetic code, mutation, deletion, insertion, alternative splicing or allelic variability.

7. Cloning and / or expression vector containing a nucleic acid sequence according to any one of claims 5 and 6.

8. Vector according to claim 7, characterized in that it is the plasmid pZeoSV2 +.

9. Host cell transfected with a vector according to claim 7 or 8.

10. Host cell transfected according to claim 9, characterized in that it is E. coli MC 1061.

II. Nucleotide sequence usable as a probe or primer characterized in that it hybridizes specifically with any of the sequences according to the claims 5 and 6 or their complementary sequences or the corresponding messenger RNAs or the corresponding genes.

12. Nucleotide sequence according to claim 11 usable as probe or primer, characterized in that it comprises at least 16 nuciéotides.

13. Nucleotide sequence according to claim 12 which can be used as a probe, characterized in that it comprises the entire sequence of the gene coding for one of the polypeptides of claim 1.

14. Nucleotide probe or primer chosen from the following oligonucleotides or their complement: SEQ ID No. 3: GCT CTG TTT GAC TAT GAC CC

SEQ ID n ° 4: AAC TTG AAT CCA ATG AGC AA

SEQ ID n ° 5: TCT GCT GAC CTG AAA GCT CC

SEQ ID n ° 6: CCA TCA GGG GGC ACA GGC TC

SEQ ID n ° 7: TGG GCC TTG TAC CCC CAG CT SEQ ID n ° 8: TCC TCC TGA GGA GCT GCT TC

SEQ ID # 9: CCC CTG CCC GGA AGG GCA GC

SEQ ID n ° 10: TTG ACC AAG GTC ACT GAG CC

SEQ ID n ° 1 1: GGG CCT GCT GGC CCC TGT AA

SEQ ID # 12: GCC ATC GGC ATC CTT GTC CC SEQ ID # 13: CCT CTC CGG TCT CTG TTG CCC G

SEQ ID n ° 14: AGG CCC AGG GGG CCT CCG AAG GC

SEQ ID n ° 15: CGC TGC CCG CCT CCT GCT CAT CCT C

SEQ ID # 16: TCC TCC AGG GCT GGA GAC TGC CT

SEQ ID n ° 17: GGT TCC AGG ACT TGC TCC TTC CAG AAG SEQ ID n ° 18: CAG CTC CCT CAC TGG CCA AG

SEQ ID n ° 19: TGG CTC CGG CCA GCC TGC C

SEQ ID No. 20: TCC GGG TCA CAG GCT ATG CCA T

SEQ ID n ° 21: CC CAG TAC CTA CTG GGC CAC CT

SEQ ID # 22: GCT TCC ATA TCT GTG TGA ATG GG SEQ ID # 23: GGT AAG GCA CGG CAG GAG GCT C

SEQ ID n ° 24: GCT GGC TCC TTC TCA CAC TG

SEQ ID # 25: GGA GAG CTC ATG GAT GGC CGA AG

SEQ ID # 26: CCA GTG AGG TAG AGG AGC TGG

SEQ ID # 27: TCC ATT CCC CAG CCT TGC CGG GG SEQ ID # 28: GCT CGA AGA ACA GTG CCG CAG CC

SEQ ID # 29: TCG CAA GAG CCA GGG CCT GCA GA

SEQ ID # 30: GCT GGA ATC GGA GCT CAG CAA G

SEQ ID # 31: CCC GTG ATT CTA GGG GAG CCA GAG SEQ ID n ° 32: GAG TGC AGG GAG CTG CAG GCC A SEQ ID n ° 33: GGC TGA AGC GGA AGA ACG CCG AG SEQ ID n ° 34: CAA TCT GGA GCT GCT GAG GG SEQ ID n ° 35: TGA GGT CTG AGG AGA GTT CCA AG SEQ ID n ° 36: ATG GAG CCA TGG GCA CTG CCC

15. Use of a sequence according to any one of claims 5 and 6 for the manufacture of oligonucleotide primers for specific sequencing or amplification reactions according to the PCR technique or any variant thereof.

16. Couples of nucleotide primers chosen from the following sequences

- couple n ° 1: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n ° 3) antisense primer: AAC TTG AAT CCA ATG AGC AA (SEQ ID n ° 4)

- couple n ° 5: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n ° 3) antisense primer: TCC TCC TGA GGA GCT GCT TC (SEQ ID n ° 8)

17. Use of a sequence according to any one of claims 5 and 6, for the manufacture of a medicament usable in gene therapy.

18. Use of a sequence according to any one of claims 5 and 6, for the production of diagnostic nucleotide probes or primers, or antisense sequences usable for the manufacture of a medicament usable in gene therapy.

19. Use of nucleotide primers according to any one of claims 11 and 12 for sequencing.

20. Use of a probe or primer according to any one of claims 11 to 16, as an in vitro diagnostic tool for the detection, by hybridization procedures, of nucleic acid sequences coding for a polypeptide according to any one of claims 1 to 4, in biological samples, or for the detection of aberrant syntheses or genetic abnormalities.

21. In vitro diagnostic method for the detection of aberrant syntheses or genetic anomalies in the nucleic acid sequences coding for a polypeptide according to any one of claims 1 to 4, characterized in that it comprises:

- bringing a nucleotide probe according to any one of claims 11 to 16 into contact with a biological sample under conditions allowing the formation of a hybridization complex between said probe and said nucleotide sequence, optionally after a prior step amplifying said nucleotide sequence;

- detection of the hybridization complex possibly formed;

- optionally, sequencing of the nucleotide sequence forming the hybridization complex with the probe of the invention.

22. Use of a nucleic acid sequence according to any one of claims 5 and 6, for the production of a recombinant polypeptide according to any one of claims 1 to 4.

23. Method for producing a recombinant protein PRAX-1 or fragment or biologically active derivative thereof or fragment immunologically related thereto, characterized in that cultured transfected cells according to claim 9 or 10 in conditions allowing the expression of a recombinant polypeptide of sequence SEQ ID No. 2 or fragment or biologically active derivative thereof, and that said recombinant polypeptide is recovered.

24. Mono or polyclonal antibodies or their fragments, chimeric or immunoconjugate antibodies, characterized in that they are specifically directed against a polypeptide according to any one of claims 1 to 4.

25. Use of the antibodies according to the preceding claim, for the purification or the detection of a polypeptide according to any one of claims 1 to 4 in a biological sample.

26. A method of in vitro diagnosis of pathologies correlated with an abnormal expression or accumulation of PRAX-1 proteins, from a biological sample, characterized in that at least one antibody according to claim 24 is brought into contact with said biological sample, under conditions allowing the formation of specific immunological complexes between a PRAX-1 protein and the or said antibodies and in that the specific immunological complexes possibly detected are detected.

27. Kit for the in vitro diagnosis of an abnormal expression or accumulation of PRAX-1 proteins in a biological sample and / or for measuring the level of expression of these in said sample comprising:

- at least one antibody according to claim 24, optionally attached to a support,

- Means for revealing the formation of specific antigen / antibody complexes between a PRAX-1 protein and said antibody and / or means for quantifying these complexes.

28. Method for determining an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic abnormality in the PRAX-1 gene, characterized in that it implements at least one nucleotide having a nucleotide sequence according to any one of claims 5 or 6.

29. Pharmaceutical composition comprising, as active principle, a polypeptide according to any one of claims 1 to 4.

30. Pharmaceutical composition according to the preceding claim, characterized in that it comprises a polypeptide according to claim 2.

31. Pharmaceutical composition containing an inhibitor or activator of the activity of PRAX-1 and of any protein-protein interaction involving PRAX-1.

32. Pharmaceutical composition containing a polypeptide derived from a polypeptide according to any one of claims 1 to 4 characterized in that it is an inhibitor or activator of PRAX-1, or of any protein-protein interaction involving PRAX- 1.