FR2688227A1 - PROTEINS FORMING COMPLEXES WITH CHAPERONES AND THEIR LIGANDS, THEIR FRAGMENTS, THEIR PRODUCTION AND THEIR BIOLOGICAL APPLICATIONS. - Google Patents

Abstract

Nucleotide sequences capable of hybridizing with the nucleotide sequence shown in figure 1, and the corresponding coded amino acid sequences, are disclosed. Said nucleotide sequences are particularly suitable for detecting complementary sequences in biological samples.

Description

PROTEINS FORMING COMPLEXES WITH CHAPERONES AND
THEIR LIGANDS, THEIR FRAGMENTS, THEIR OBTAINMENT AND THEIR
BIOLOGICAL APPLICATIONS.

 The subject of the invention is immunophilin proteins, p59 and related proteins, forming complexes with chaperones and their ligands.

 It relates more particularly to the nucleotide sequences coding for proteins related to those of these complexes, or to fragments of these proteins, as well as to the corresponding proteins and fragments.

 The invention also relates to the biological applications of these nucleotide and protein sequences, in particular for research and study of new pharmacological ligands of (des) immunophilin as well as for clinical diagnosis.

 The term chaperone protein denotes proteins associated with other biologically active proteins with which they interact and modify their functioning.

 The invention relates in particular to proteins forming complexes with the chaperone hsp90 which is a heat shock protein capable of binding with numerous ligands such as receptors for steroid hormones, thyroid hormones, metabolites of vitamins A and D, ligands called orphan receptors ", oncogenes in particular tyrosine kinases of viral origin (for example pp60src) r actin, tubulin and other proteins whose structural change and intracellular traffic are important for physiology and pathology of the cell.

In the context of work on steroid hormone receptors, some of the co-inventors of the present application have described the binding of hsp90 to steroid hormone receptors and its role in preventing the interaction of these receptors with DNA (see Joab et al., 1984, Nature, 308, 840, 853 and Catelli et al., 1989,
EMBO J.4, 3131-3135).

 A protein of molecular weight 59kDa has also been characterized in rabbits during the purification of these receptors. In the absence of a ligand, the receptors for all steroid hormones are found associated in the cell with other proteins which do not bind the hormone, constituting the non-activated form of these receptors. The separation of proteins will release the receptor itself which will then be able to interact with DNA (Baulieu et al., 1971, Rec. Progr. Horm. Res. 27, 351-419 and Sherman et al., 1984, Ann. Rev Physiol. 46, 83-125).

 As the structures and sequences of these p59 type proteins have not been elucidated to date, their function in the cell could not be defined precisely.

By screening a rabbit liver c-DNA library, the inventors succeeded in isolating and sequencing clones containing a nucleotide sequence capable of coding for a protein homologous to the rabbit p59 protein as it has was defined by its immunoreaction with the monoclonal antibody KN382 / EC1. (Nakao et al., 1985,
Can. J. Biochem. Cell. Biol. 63, 33-40).

 The object of the invention is therefore to provide such nucleotide sequences as well as the amino acid sequences expressed or deduced from the nucleotide sequences.

 It also aims to obtain these different sequences.

 The invention also aims to provide polyclonal and monoclonal antibodies directed against these proteins or protein fragments obtained.

 According to another aspect, the invention relates to the use of nucleotide or protein sequences, or antibodies, in biological applications, in particular for diagnostic purposes.

 The nucleotide sequences of the invention are characterized in that they comprise or are formed by a sequence of nucleotides capable of hybridizing under stringent conditions with one or more sequences of a gene whose cDNA has a sequence corresponding to the sequence (I) of nucleotides.

 The sequences of the nucleotide and protein sequences to which reference is made in the description and the claims are given at the end of the description.

The expression "stringent conditions" as used in this text means that a hybridization is obtained by operating according to the conditions described by
Sambrook, Fritsch Maniatis in "Molecular Cloning", 1989.

 According to one provision of the invention, the nucleotide sequences are characterized in that they contain the genetic information to code for all or part of the protein responding to sequence II.

 Sequences of this type are characterized in that they are formed, or that they comprise, at least part of the sequence I.

 The invention relates in particular to nucleotide sequences constituted by part or all of the open reading frame going in the sequence (I) from position 4 to position 1380.

 This sequence, without the leader sequence, has 2067 base pairs.

 Such sequences are further characterized in that they are capable of coding for a potential protein of 458 amino acids responding to sequence II.

 Sequences of great interest include the genetic information to code for one or more amino acid sequences of sequence II corresponding to a region of this sequence capable of binding with an immunosuppressant of the FK 506 type having the structural characteristics for a rotamase activity.

 Other preferred sequences include genetic information to code for one or more sequence II amino acid sequences corresponding to a region capable of binding ATP (adenosine triphosphate) / GTP (guanosine triphosphate).

Still other preferred sequences include the genetic information to code for one or more sequence II amino acid sequences corresponding to a region capable of binding to calmodulin.
According to another aspect, the invention relates to a recombinant sequence comprising one of the sequences defined above, optionally associated with a promoter capable of controlling the transcription of the sequence and a DNA sequence coding for the termination signals of the transcript.

 Such sequences advantageously contain sequences coding for chaperones.

 Preferred sequences of this type contain coding sequences for hsp90, in particular for the hydrophilic region A, of this highly negatively charged protein (Binart et al., J. Steroid. Biochem. 1989, vol. 34, n "1 , 369-374).

 The purine and pyrimidine bases of the nucleotide sequences under consideration may have a different order than that found in the cloned cDNA and / or if necessary, may be substituted. It is understood that such sequences fall within the scope of the invention, since a fragment of these sequences used as a probe gives rise to hybridization with a gene coding for proteins as defined above.

 The invention also relates to the RNAs and the complementary sequences of the various defined nucleotide sequences as well as the corresponding coded proteins.

 The invention also relates to recombinant cloning and expression vectors capable of transforming an appropriate host cell, comprising at least part of a nucleotide sequence as defined above, under the control of regulatory elements allowing his expression.

 The strains of transformed or transfected microorganisms also fall within the scope of the invention.

These strains comprise one of the nucleotide sequences defined above or also a recombinant vector as defined above.

 The invention also relates to the amino acid sequences deduced, according to the universal genetic code, from the nucleotide sequences defined above, and the proteins expressed by the genes comprising these sequences.

 Sequences according to the invention, which are of great interest with regard to the applications envisaged in diagnosis, are characterized in that they correspond to heat shock proteins or that they have the properties of such proteins.

 According to another advantageous arrangement of the invention, these are sequences capable of binding to hsp 90, even when hsp90 is involved in hetero-oligomeric complexes with other proteins as indicated above. They can be complexes involved in the formation of steroid receptors or in other complexes, for example with oncogenes and proteins of the cytoskeleton.

 The complexes of these protein sequences with hsp90 or with other heat shock proteins involved in these hetero-oligomers, or with regions of these different proteins, are also part of the invention.

 According to another arrangement of the invention, if necessary taken in combination with those which precede, the amino acid sequences are characterized in that they have one or more domains of the type of FKBP, identical or different from each other. other. The corresponding proteins will hereinafter be called HBI proteins, that is to say proteins of the immunophilin type binding heat shock proteins.

 FKBP is an immunophilin that binds at least two immunosuppressive drugs, namely FK506 and rapamycin.

 Such amino acid sequences are more particularly characterized in that they have the structural characteristics for binding with an immunosuppressant of the FK506 type and for peptidyl-prolyl isomerase cis-trans activity.

 The amino acid sequences of the invention, according to another provision of the invention, taken if appropriate in combination with the above, include an ATP binding site.

 According to another aspect, the amino acid sequences of the invention are characterized in that they comprise a consensus sequence for binding to calmodulin.

 According to yet another aspect, the amino acid sequences are characterized in that they correspond to the hinge regions between two domains.

 These are more specifically hydrophilic regions, accessible to proteolytic enzymes.

 In an alternative embodiment of the invention, amino acid sequences of the FKBP type comprise or are formed by only one of the domains of the FKBP type.

 In another variant, they comprise or are formed by two of these areas, identical or different, connected by a hinge.

 In yet another variant, the amino acid sequences comprise or are formed by a plurality of identical or different sequences corresponding to one of the domains of the FKBP type.

 In these different variants, these are domains having a very strong three-dimensional (3D) structure homology with FKPB of the order of 80 to 100%, or domains having a lower homology, in particular of the order of 35 to 80% or even 25 to 35% approximately.

 The amino acid sequences of the invention are also characterized in that they are such as obtained by transformation of host cells using a recombinant vector as defined above, cultured in an appropriate medium, transformed or transfected host cells and recovery of the protein from these cells or directly from the culture medium.

 The production of these proteins by such a process is also part of the invention.

 The proteins of the invention and their fragments, which can also be obtained by chemical synthesis, advantageously have a high degree of purity and are used to form, according to conventional techniques, polyclonal antibodies and monoclonal antibodies.

 Such polyclonal antibodies, as well as monoclonal antibodies capable of specifically recognizing an epitope of the above amino acid sequences, or of a fragment of these sequences by giving rise to an antigen-antibody type reaction, are also targeted by the invention.

 The invention further relates to the biological applications of nucleotide sequences, corresponding proteins and monoclonal or polyclonal antibodies.

 These applications include the development, from purified intragenic fragments, or from corresponding RNA, of molecular probes to search for the possible presence in various cell types of nucleotide sequences related to the gene capable of coding for proteins associated with hsp90 in hetero-oligomeric complexes as indicated above.

 The development of these probes includes, in particular the denaturation of double stranded sequences to obtain a single stranded sequence.

 The tests carried out to detect the presence of complementary sequences in various tumors and tissues in humans or animals have demonstrated the great specificity of these intragenic fragments.

 The use of these probes has thus made it possible to show that the gene containing the nucleotide sequences defined above is expressed in mammals.

 The invention therefore relates to detection probes characterized in that they comprise at least part of a nucleotide sequence defined above.

 Nucleotide substitutions or alterations can be made in these sequences, as soon as the corresponding probe is capable of hybridizing with the gene for the HBI protein as defined above.

 The construction of the probe is advantageously carried out according to conventional techniques, in particular a sufficient number of nucleotides is used to obtain the required specificity and the formation of a stable hybrid.

 It is possible to use fragments reaching several kb, results of high specificity being however also obtained with shorter fragments of approximately 20 to 40 nucleotides.

 These probes are advantageously labeled with a group allowing their recognition by being hybridized with the preparation containing the nucleotides to be studied.

 According to conventional techniques, these probes are brought into contact with the biological sample to be tested or their nucleic acids, under conditions allowing hybridization between the nucleotide sequence of the probe and a complementary sequence, when it is contained in the product studied.

 One can, for example, have recourse to the method of hybridization on spots or to the method of hybridization on replica, according to the technique of Southern. In the first method, according to the conventional technique, an aliquot of denatured DNA is deposited on nitrocellulose membranes. The second method comprises the electrophoretic separation in agarose gel of the DNA fragments generated after treatment of the DNA with restriction enzymes, the transfer after alkaline denaturation on appropriate membranes and their hybridization with the probe under the usual conditions.

These probes constitute markers allowing the early detection of the expression of the gene containing
HBI, that is to say capable of expressing the protein of the sequence II, which normally is little or not expressed in the corresponding normal tissues. The invention thus provides means for assessing tumor development and / or differentiation.

 The invention also relates to a method for invitro detection of the presence in a biological sample of sequences complementary to those defined above.

This process is characterized in that it comprises the stages of
contacting the sample to be studied with a nucleotide probe as defined above, under conditions allowing hybridization between the probe and the desired nucleotide sequence, and
- detection of the hybridization complex.

 This method allows rapid and highly specific detection of DNA and of the different species of transcription mRNA.

 It allows in particular the screening of a diseased tissue bank, the study of the development of tumor differentiation and the early detection of the expression of the gene for the HBI protein.

The invitro detection procedure defined above, based on the use of nucleotide probes, is advantageously implemented using kits comprising
- a determined quantity of a nucleotide probe according to the invention,
a medium suitable for hybridization between the probe used and the sequence to be detected, and advantageously,
- reagents for the detection of the hybridization complexes formed.

 The invention also relates to the immunological applications of the amino acid sequences defined above, more specifically for the development of specific antisera, as well as polyclonal and monoclonal antibodies.

 The polyclonal antibodies are as induced according to conventional techniques by injection of the amino acid sequences into animals, followed by the recovery of the antisera and then, from the latter, if desired antibodies, for example by chromatography of 'affinity.

Monoclonal antibodies are produced in the usual way by fusing myeloma cells with spleen cells from animals previously immunized with the proteins of the invention. In this respect, one operates advantageously according to the technique described by Köhler and
Milstein in Nature, vol 256, p 495, 1975.

 The antibodies of the invention make it possible to detect invitro the expression products of the nucleotide sequences defined above or those of the genes coding for a heat shock protein related to those of the heterooligomeric complexes defined above, in particular steroid receptors.

The in vitro screening method in a biological sample for the possible presence of these expression products is characterized in that it comprises
contacting the sample to be studied with an antibody according to the invention, under conditions allowing the formation of an immunological complex between all or part of the proteins expressed by the nucleotide sequences and this antibody, and
- detection of the immunological complex.

For this invitro screening method, use will advantageously be made of kits comprising - a determined quantity of a polyclonal or monoclonal antibody according to the invention,
a medium suitable for carrying out an immunological reaction between at least part of the products expressed and the antibody and, advantageously,
- reagents for the detection of immunological complexes formed.

 The amino acid sequences defined above also constitute tools of great interest for the study and / or regulation of the functions of proteins associated with said complexes.

 The invention thus provides very effective means for studying and preventing or treating pathologies linked to the dysfunction of proteins associated with the hetero-oligomeric complexes mentioned above.

 Mention will be made, for example, of diseases of the immune or autoimmune system, cancers, deficiencies in vitamin D responsible for rickets, or in retinoic acid or even intoxication by dioxin or related products.

 These means, whether polynucleotides, amino acid sequences or antibodies also allow the localization of proteins associated with the hetero-oligomeric complexes described above.

 It was thus possible to note that the natural protein associated with hsp90 in these complexes is localized in the nucleus of normal tissue cells whereas in tumor cells, it appears abundantly in the cytoplasm. The use of the means of the invention therefore makes it possible to make significant localization differences in pathologies.

 According to another aspect of great interest, they also make it possible to find natural ligands capable of occupying the binding sites recognized by the immunosuppressants.

 The HBI proteins are useful for the search for new reagents and drugs which bind to this protein, and which can influence the functioning of the heat shock protein and the proteins associated with it on receptors for steroid hormones and other ligands for receptors for the same family, on the activity of oncogenes having protein kinase activity, on the structure and functioning of cytoskeleton proteins and any protein associated with hsp90. This results in applications of great interest in the endocrine, cancer and immunological fields.

 In the examples which follow, other characteristics and advantages of the invention are reported.

In these examples, reference is made to FIGS. 1 to 7 which respectively represent
FIG. 1, the rabbit liver cDNA sequence coding for an HBI protein and the corresponding amino acid sequence,
FIG. 2, the representations established by the method of analysis of hydrophobic clusters (ACH) of the domains of an HBI protein compared to the FKBP and to FKBP type domains identified in various proteins,
FIGS. 3 and 4, the ACH representations of human FKBP and of the HBI protein and the alignments of the corresponding amino acid sequences,
FIG. 5, a representation of the structure of the domains of pHBI using as matrix the main chain of FKBP,
FIG. 6, a schematic representation of the main characteristics of the pHBI, and
- Figure 7, the results of a Western blot performed with a polyclonal anti-HBI antibody 1 invention.

 Example 1: Cloning and sequencing of the cDNA coding for an HBI protein.

In order to clone a rabbit liver cDNA library, probes are constructed from oligonucleotides whose sequence has been deduced from that of a fragment of 19 amino acids corresponding to the sequence
ALA GLU GLU MET LYS ALA THR GLU SER GLY ALA GLN X ALA
PRO LEU PRO MET GLU
It is the fragment of the N-terminal part of a protein identified in complexes of unprocessed steroid receptors described by Sanchez et al.

in Biochemistry 1990, 29, 5145-5152.

 The construction of the probe is carried out by operating according to the aforementioned work of Maniatis Volume 2, chapter 11 "Synthetic oligonucleotidic probes".

 The screening of the cDNA library is carried out according to the method described in chapter 8 of the aforementioned book "Construction and analysis of cDNA libraries".

 The longest positive clone cDNA is isolated and subcloned into pGEM 7Zf (Promega Biotec), transcribed and translated into a rabbit reticulocyte lysate system.

 Transcription and translation are carried out as described by the manufacturer of the plasmid in the presence of 35S methionine.

 The cDNA subcloned into the vector pGEM 7Zf is digested with exonuclease III to generate deletion subclones which are then sequenced using the T7 RNA polymerase promoter as a primer. The cDNA can also be subcloned into the vectors M13 MP 18/19 and sequenced according to the conventional technique as described for example in the aforementioned work by Maniatis.

 The nucleotide sequence and the amino acid sequence derived from the entire open reading frame are reported in FIG. 1. The underlined sequence corresponds to that used to generate the polyclonal antibody which is discussed below in Example 3 .

The dotted line indicates the region corresponding to the site of interaction with calmodulin.

Example 2: Study of the HBI protein, called below pHBI, by the method of analysis of hydrophobic clusters (abbreviated ACH)
MATERIAL AND METHODS
2D hydrophobic cluster analysis (ACH) strategies and their applications were first proposed by Gaboriaud et al., 1987, FEBS Lett. 224, 149, 155 and are described in detail by Lemesle-Varloot et al., 1990, Biochemistry, 72, 555-574.

 ACH is a method using the general principles of protein folding. It is based on the search for successive correspondences of hydrophobic clusters which are relevant to the secondary structure and the 3D folding of the protein domains.

 In related proteins, it is possible to deduce a precise alignment of sequences from ACH representations by proceeding from the hydrophobic nucleus of similar clusters towards regions linking clusters (loops) where insertions / deletions are allowed.

 For these HCA alignments, a numerical value (HCA score) can be calculated between clusters to determine the alignments.

3D studies were carried out with the program
MANOSK (Cherfils et al. 1988, J. Mol. Graph. 6, 155-160) on Evans & Sutherland PS390.

 Analysis of the pHBI shows that it, starting from the N-terminal end, three successive domains.

 Identification of immunophilin domains pHBI I and pHBI II domains.

Figure 2 gives aligned, ACH representations of human FKBP (FKBPh) and domains
I and II of pHBI. This figure also mentions the FKBP type domains already identified in other proteins. This is the RBP1 protein which binds rapamycin from Saccharomyces cerevisiae (Koltin et al. 1991,
Mol. Cell Biol. 11, 1718-1723), the FKBP of Neurospora crassa (Tropschug et al. 1990, Nature, 346, 674-677, a cryptic sequence of Neisseria meningiditis (Perry et al.

1988, J. Bact. 170, 1691-1697), a 25.3 kDa protein from
Pseudomonas aeruginosa (# JQ 0140), the cell surface protein mip of Legionella pneumophila (Engleberg et al.

1989 Inf. Immunity 57, 1263-1270), the L2 protein of
Chlamydia trachomatis (Lundemose et al. 1991, Mol.

 Microbiology 5, 109-115).

 Hatched hydrophobic clusters highlight anchor points for sequence alignments.

The secondary structure of the FKB h is indicated above its sequence in accordance with the description of the 3D structure. The sheets p are indicated by the symbols and the propellers a by
The black columns indicate the conserved residues, the gray columns and with dotted lines the hydrophobic and hydrophilic homologous residues and those open those particularly rich in P, G, S, T and / or A.

Amino acids (main and / or side chains) which are essential for the binding of FK506 to
FKBP and are fully stored for p59 I are indicated by arrows. Parentheses have been mentioned when alignment between amino acids is not possible.

 Analysis of this figure shows that pHBI comprises two successive domains of approximately 100 residues (pHBI I and pHBI II) having a strong homology with FKBP.

 The first domain has a sequence identity of 49% with FKBP without the introduction of deletion or insertion.

 The second domain has a sequence identity of 28% with FKBP and includes 4 insertion or deletion zones totaling 9 amino acids.

 These domains are assigned, in view of their structural homology with FKBP, a topology for folding anti-parallel p sheets, accompanied by an a helix and an additional p sheet.

 I1 is referred to this sheet p as being of type A (chains A1 to A5).

 In FIG. 3, graphical representations according to ACH of the human FKBP and of the segmented pHBI are reported so as to show its internal symmetry. Above the representation of the FKBP, we indicate as in Figure 2, its secondary structure.

 The protein sequences are drawn on a conventional helix flattened on a cylinder, cut parallel to its axis and unwound to provide a two-dimensional representation.

 The representation is doubled vertically to restore a complete two-dimensional environment for each amino acid.

A group of hydrophilic amino acids (VILFWMY) are surrounded in clusters. The classic letter code is used for all amino acids, except for P (designated by a star) which is a cluster switch, G (designated by a diamond symbol) which shows great conformational flexibility and T and
S (open and dotted squares respectively) which are frequently encountered in loops, but can also be found in hydrophobic environments where the hydrophilicity of their hydroxyl groups is neutralized by hydrogen bonds with the main chains.

 The box reproducing the N-terminal part of the pHBI also indicates how the sequence is read linearly (see slightly oblique arrow).

 The vertical lines delimit the segmentation of the structures (S1 to S6) inside the domains.

 Hatched hydrophobic clusters highlight anchor points for sequence alignments.

 The examination of FIG. 3 shows that the classic segmentation of the globular domains into regular secondary structures (sheets p and helices a), statistically centered on the hydrophobic clusters of the representations according to ACH and in the loops linking these structures is clearly preserved for FKBP, pHBI I and pHBI II.

 There is an overall similarity centered on the S4 segment, between pHBI I, pHBI II and an extension of sequence according to pHBI II.

 This extension is attributed to a third globular domain of pHBI (pHBI III).

Using segments as anchors
S4, S5 and S6 graphical representations according to ACH of the pHBI I and pHBI II domains, an amino acid by amino acid alignment was carried out.

 These alignments are shown in Figure 4.

 The pressed lines represent the nuclei limits of the sequences of the third domain of pHBI (pHBI III) used to establish the alignments statistically.

 Their dimensions are 8, 7, 2, 23, 6, 12 amino acids for the pHBI III / pHBI II comparisons and 6,8,3,27,7,11 amino acids for the pHBI III / pHBI I comparisons, at namely 58 and 62 amino acids respectively.

 Above and below, indicated by solid circles and bars the sequence identities and conservative mutations for pHBI III / pHBI II and pHBI III / pHBI I.

 There is a sequence identity of about 13% between the pHBI III domain and pHBI II.

 The pHBI III domain has a 10 i sequence identity with the pHBI I domain.

 A comparison between pHBI III and the two other domains of pHBI makes it possible to establish a relationship between them, based on the following observations - the positions of the insertions and deletions present between the domains pHBI I and pHBI It are all preserved, - the pHBI III also shares 13% of sequence identity with the L2 protein of the FKBP type of Chlamydia trachomatis whose sequence is shown in FIG. 2. In this sequence, an extension of 21 amino acids (273-293 of pHBI III and 135 -152 L2) has 45% identity with pHBI III.

Main characteristics of the pHBI domains.

 I1 will be referred in the following to Figures 2 and 3 already considered and to Figure 5 which gives a representation of the complete structure of pHBI I using as matrix the main chain of FKBP in the form of ribbon (it will be noted that there are no insertions or deletions between the domains of these two proteins).

The numbering of the pHBI I residues is indicated five by five.

 The FK506 ligand linked to FKBP was kept as reference (balls).

 The side chains of amino acids which can be critical for a hypothetical binding of a ligand similar to FK506 are fully represented (see fig.

2).

The specificities of the pHBI domain are underlined
1 / shortening (4 amino acids) of the 38-45 loop (FKBP numbering),
2 / lengthening (3 amino acids) of the 5556 loop (ATP binding site,
3 / insertion (an amino acid) into the 87-90 loop (ATP phosphate stabilization),
4 / deletion (an amino acid) in loop 3135.

 pHBI I: No deletions or insertions are observed in pHBI I compared to FKBP.

 All the main or secondary chains involved in the binding of FK506 by FKBP are conserved in the domain of pHBI I.

Indeed, it can be noted that the replacement of
FKBP Q53 by a glycine in HBI I does not modify the interaction of the corresponding main chain CO with the ligand, if it exists.

 Similarly, the replacement of H87 with a serine seems to have only a weak influence since it interacts by a Van der Waals contact at a rather long distance (3,8-4A).

 A similar mutation (alanine) occurs in the same place for the human protein FKBP13. Consequently, all the structural requirements related to the catalytic site of FKBP appear preserved in pHBI I.

 pHBI II: Although the p59 II domain is clearly related to the FKBP matrix, it has specific characteristics as shown in Figures 2, 3 and 5.

 1/8 of the 12 essential amino acids for the binding of FK506 to FKBP are not conserved.

 2 / The loop V55 - I56 (loop of the type p / a numbering FKBP) of FKBP is elongated by 3 amino acids making it impossible to maintain the binding of a ligand similar to Fi506, since these residues are in contact with the very important pipecolinyl cycle of FK506 (deepest part of the active site).

 3 / The loop 87-90 (loop A2-A3) is largely modified with elongation of an amino acid and introduction of basic residues. (see snowflake symbol in Figure 5).

4 / The well exposed omega loop from 38 to 45 (loop
A5-p) is shortened by 4 amino acids (large star), leading to a direct link between positions 39 and 45 via a continuous p chain connecting A5 and p (see Figures 2 and 5).

5 / Loop 31-35 (loop A4-A5) is slightly modified by a deletion (G33; small star)
pHBI III: this domain shares with pHBI II the same distribution of insertions and deletions compared to the first domain and has a sequence significantly related to this first domain. However, it lacks many of the characteristics of FKBP that appear in the first two areas. All essential amino acids for binding to FK506 have been mutated and the sequence identity with FKBP is only 5.7%.

Hinge regions - N and C terminal regions.

 The hinge regions linking the above globular domains are indicated in FIGS. 2, 3 and 6.

 Figure 6 gives a schematic representation of the main structural characteristics of pHBI with indication of the sequence identities of pHBI II (26%) and pHBI I (13%) compared to pHBI I (100 *).

 As shown in the figures above, the hinge 1 connects pHBI I and pHBI II via 10 strongly hydrophilic and acidic amino acids. Hinge 2 links pHBI II and pHBI III via 13 mainly acidic residues.

 The pHBI has a short N-terminal part before pHBI I and is after pHBI III a longer C-terminal part.

Example 3 Preparation of Anti-HBI Antibodies
We use the amino acid sequence of the carboxy terminal part of the sequence II going from position 441 to 458.

 This peptide fragment is coupled to KLH and injected into rabbits according to the usual techniques.

 The IgG fractions recovered are purified by precipitation with ammonium sulphate and ion exchange chromatography with each bleeding.

 Polyclonal antibody preparations are used either for immunoblot analysis or for density gradient analysis to study their ability to recognize pHBI in different tissues and species, as well as in the heterooligomeric structure of progesterone.

 The Western blot results are reported in FIG. 7, with different samples, namely rabbit uterus cytosol (lane 1, 10 p1), rat hepatoma cells (lane 2) and human cell cytosols HeLa (lane 3) subjected to electrophoresis after ion exchange chromatography.

 The position of the markers is indicated on the left of the figure. HC designates the heavy chains of rabbit immunoglobulins detected by a second anti-rabbit antibody.

 With the anti-HBI antibody used, a single band at 59 kDa is observed at the same position as that recognized by EC1 (see reference by Nakao et al. Above).

 This antibody also reacts with human protein and recognizes a protein of the same size in rats, which is not detected by EC-1.

Sequences 1: nucleotide sequences: sequence I
10 20 30 40 50 60 GCCATGACCG CCGAGGAGAT GAAGGCGGCC GAGAGCGGGG CGCAGTCGGC GCCGCTGCCC
70 80 90 100 110 120 CTCGAGGGCG TGGACATCAG CCCCAAGCAA GACGAAGGCG TGCTGAAGGT CATCAAGCGA
130 140 150 160 170 180 GAGGGCACAG GCACCGAGAC ACCCATGATC GGGGACCGAG TCTTTGTCCA CTACACTGGC
190 200 210 220 230 240
TGGCTCTTGG ATGGCACGAA GTTTGACTCC AGTCTGGACC GCAAGGACAA ATTCTCCTTT
250 260 270 280 290 300 GACCTGGGAA AAGGGGAGGT CATCAAGGCT TGGGACATTG CTGTTGCAAC CATGAAGGTG
310 320 330 340 350 360
GGGGAATTGT GCCGCATCAC CTGCAAACCC GAATATGCCT ACGGTTCGGC AGGCAGCCCT
370 380 390 400 410 420 CCAAAGATCC CCCCCAACGC CACACTTGTG TTCGAGGTGG AATTGTTTGA GTTCAAGGGA
430 440 450 460 470 480
GAGGATCTGA CAGACGACGA AGACGGCGGA ATCATCCGCA GAATACGGAC TCGGGGTGAA
490 500 510 520 530 540 GGCTATGCTA GGCCCAACGA TGGTGCTATT GTGGAGGTCG CACTGGAAGG GTACTACAAG
550 560 570 580 590 600
GACCGGCTCT TTGACCAGCG GGAACTCCGC TTTGAGGTCG GCGAGGGGGA GAGTCTGGAT
610 620 630 640 650 660
CTGCCTTGTG GGCTAGAGAA GGCCATTCAG CGCATGGAGA AAGGCGAACA TTCCATCTTG
670 680 690 700 710 720
TACCTCAAGC CCAGTTACGC GTTTGGCAAT GCTGGGAAGG AAAAGTTTCA GATCCCGCCA
730 740 750 760 770 780
TATGCTGAGC TGAAATATGA AGTCCACCTC AAGAGTTTTG AGAAGGCCAA GGAGTCCTGG
790 800 810 820 830 840
GAGATGAGCT CGGAGGAGAA GCTGGAGCAG AGCGCCATCG TGAAAGAGCG AGGCACCGTG
850 860 870 880 890 900
TACTTCAAGG AAGGCAAGTA CAAGCAGGCT CTGCTACAGT ACAAGAAGAT TGTGTCTTGG
910 920 930 940 950 960
CTGGAATACG AGTCAAGTTT TTCCAGTGAG GAAGTGCAAA AGGCACAGGC CCTGCGCCTG
970 980 990 1000 1010 1020
GCCTCCCACC TCAACCTGGC TATGTGTCAC CTGAAGCTAC AGGCCTTCTC GGCAGCCGTG
1030 1040 1050 1060 1070 1080
GAAAGCTGTA ACAAGGCCCT GGAACTGGAC AGCAACAACG AGAAGGGCCT CTTCCGCCGG
1090 1100 1110 1120 1130 1140
GGAGAGGCCC ACCTGGCTGT GAACGACTTT GACCTGGCAC GGGCTGACTT CCAGAAGGTC
1150 1160 1170 1180 1190 1200
CTGCAGCTCT ACCCCAGCAA CAAAGCGGCT AAGGCCCAGC TGGCTGTGTG CCAGCAGCGG
1210 1220 1230 1240 1250 1260
ATCCGCAAGC AGATTGCCCG GGAGAAGAAG CTCTACGCCA ACATGTTTGA GAGGCTGGCA
1270 1280 1290 1300 1310 1320
GAGGAGGAGA ACAAGGCGAA GGCAGAAGTG GCCGCAGGCG ACCATCCCAT GGACACAGAG
1330 1340 1350 1360 1370 1380
ATGAAGGATG AGCGGAACGA CGTGGCTGGA AGCCAGTCTC AGGTGGAGAC AGAAGCATAG
1390 1400 1410 1420 1430 1440
CCTCTCTGGC CTGACTCCTG CGACTGCCCG CCTCCTGCTC CCCTGCCCTA CTCCACCCTG
1450 1460 1470 1480 1490 1500
TTAGTTTTGT AAAAACTGAA GAATTTTGAG TGACTTAGAC CTTTATTTTT CTATCTGGTT
1510 1520 1530 1540 1550 1560
GGATGGTGGC TTTGCGAGGA GGGGGGAAAG ACTAGGCTGG GAACGCTGAG GTAGGGCTGA
1570 1580 1590 1600 1610 1620
TTCTAGGGAG TGTCACCCGC CCCCCTTCCC CCATCACACG TGAATGTACA TCCATCCACA
1630 1640 1650 1660 1670 1680
CACACGCAAC TGAATGTTCG TGCATTTTGC TCCCTCTGTT AGGTCTACTC TGCAAATGGT
Chain I continued
1690 1700 1710 1720 1730 1740 AGAAGGGGGC AAGTGGTGGG GATGGGGTCT GATGTGAAAC CAGGGTGGAG AGGGAGACCG
1750 1760 1770 1780 1790 1800 ACTCCTGGGC AGCTGCTTTC CTGATCCTTG TCCTCTCCCA GTCCCTTTCA AACTGTGGCC
1810 1820 1830 1840 1850 1860 TCCAGGTGGG GGGTGCTGGG GGTGGGGAAA CCATTGCTGT GCCTGTTACC TCTCAGTCCC
1870 1880 1890 1900 1910 1920
TTCCTCACCC CAGATGGTGT GGCTGACTGT GTCTGGTGTC CAAGACCACC CCCGTCCCCC
1930 1940 1950 1960 1970 1980
ATTCTGGTAT TGGCCTTCCT AGCAGTTTCC CTTCTCAGCC AGGTTGTATC CCCACCCTCC
1990 2000 2010 2020 2030 2040
CACCCTGTCA GCCTCTTCTC TGCACGTTGC TGAAAGTCCA GGCTCGCCTC AAGTTCTGTG
2050 2060 2070
CTTGAGCAAT AAAGTGGAAA CAATAAAAAA 2. Sequence of amino acid sequences
10 20 30
MTAEEMKAAE SGAQSAPLPL EGVDISPP
40 50 60
EGVLKVIKRE GTGTETPMIG DRVFVHYTGW
70 80 90
LLDGTKFDSS LDRKDKFSFD LGXGEVIKAW
100 110 120
DIAVATMKVG ELCRITCKPE YAYGSAGSPP
130 140 150
KIPPNATLVF EVELFEFKGE DLTDDEDC
160 170 180
IRRIRTRGEG YARPNDGAIV EVALEGYYKD
190 200 210
RLFDQRELRF EVGEGESLDL PCGLEKAIQR
220 '230 - 240
MEKGEHSILY LKPSYAFGNA GKEKFQIPPY
250 260 270
AELKYEVHLK SFEKAKESWE MSSEEKLI
280 290 300
AIVKERGTVY FKEGKYKQAL LQYHKIVSWL
310 320 330 EYESSFSSEE VQKAQALRLA SHLNLAMCHL
340 350 360
KLQAFSAAVE SCNKALELDS NNEKGLFRRG
370 380 390
EAHLAVNDFD LARADFQKVL QLYPSNKAAK
400 410 420
AQLAVCQQRI RKQIAREKKL YANMFERLAE
430,440
EENKAKAEVA AGDHPMDTEM KDERNDVAGS
458
QSQVEI

Claims (24)

 1 / Nucleotide sequences, characterized in that they include or are formed by a sequence of nucleotides capable of hybridizing, under stringent conditions, with one or more sequences of a gene whose cDNA has a sequence corresponding to the sequence (I) of nucleotides.  2 / Nucleotide sequences, characterized in that they contain the genetic information to code for all or part of the protein corresponding to the sequence II.  3 / Nucleotide sequences, characterized in that they are formed, or that they comprise, at least part of the sequence I.  4 / Nucleotide sequences according to claim 3, constituted by part or all of the open reading frame going in the sequence (I) from position 4 to position 1380.  5 / Nucleotide sequences according to claim 3, capable of coding for a potential protein of 458 amino acids responding to sequence II.  6 / Nucleotide sequences according to one of the preceding claims, characterized in that they include the genetic information to code for one or more amino acid sequences of the sequence II corresponding to a region capable of binding with a immunosuppressant type FK 506 and having the structural characteristics for rotamase activity.  7 / Nucleotide sequences according to one of claims 1 to 6, characterized in that they include the genetic information to code for one or more amino acid sequences of sequence II, corresponding to a region capable of link to ATP / GTP.  8 / nucleotide sequences according to one of claims 1 to 7, characterized in that they include the genetic information to code for one or more amino acid sequences of the sequence II corresponding to a region capable of binding calmodulin.  9 / Recombinant sequence comprising one of the nucleotide sequences defined in any one of claims 1 to 8, optionally associated with a promoter capable of controlling the transcription of the sequence and a DNA sequence coding for the signals of termination of transcription.  10 / Nucleotide sequences according to one of claims 1 to 9, characterized in that they contain coding sequences for chaperone proteins, in particular sequences coding for hsp9O, in particular for the hydrophilic region A of this protein.  11 / The RNAs and the complementary sequences of the various nucleotide sequences defined in one of claims 1 to 10, as well as the corresponding coded proteins.  12 / Recombinant cloning and expression vectors capable of transforming an appropriate host cell, comprising at least part of a nucleotide sequence according to any one of claims 1 to 11, under the control of regulatory elements allowing its expression.  13 / Strains of transformed or transfected microorganisms, characterized in that they comprise at least one of the nucleotide sequences defined in one of claims 1 to 11, or also a recombinant vector as defined in claim 12.  14 / Amino acid sequences deduced, according to the universal genetic code, from the nucleotide sequences according to one of claims 1 to 11 and the proteins expressed by the genes comprising these sequences.  15 / Amino acid sequences according to claim 14, characterized in that they correspond to heat shock proteins or that they have the properties of these proteins, and that in particular they are coding sequences for chaperone proteins , in particular coding sequences for hsp90, in particular for the hydrophilic A region of this protein.  16 / Amino acid sequences according to one of claims 14 or 15, characterized in that they have one or more domains of the type of FKBP, identical or different, in particular that they have the structural characteristics for a bond with an immunosuppressant of the FK506 type and for rotamase activity, and / or that they have an ATP binding site, and / or a consensus binding sequence to calmodulin, and / or that they correspond to the hinge regions between two areas.  17 / Amino acid sequences according to one of claims 10 to 16, characterized in that they are obtained by transformation of host cells using a recombinant vector according to claim 12, cultured in an appropriate medium, transformed or transfected host cells and recovery of the protein from these cells, or directly from the culture medium.  18 / The complexes of the proteins of the amino acid sequences according to one of claims 1, 4 to 17 with hsp90 or with other heat shock proteins involved in these hetero-oligomers, or with regions of these different proteins .  19 / Polyclonal and monoclonal antibodies capable of specifically recognizing the amino acid sequences and the complexes according to one of claims 14 to 18.  20 / Application of the nucleotide sequences according to one of claims 1 to 11 for the development of probes for detecting genes coding for proteins forming complexes with chaperones and their ligands.  21 / A method of invitro detection of the presence in a biological sample of nucleotide sequences complementary to those defined in one of claims 1 to 11, comprising the steps of  bringing the sample to be studied into contact with a nucleotide probe produced using a sequence according to one of claims 1 to 11, under conditions allowing hybridization between the probe and the desired nucleotide sequence, and  - detection of the hybridization complex.  22 / Kits for the in vitro detection of the presence in a biological sample of nucleotide sequences complementary to those defined in one of claims 1 to 11, characterized in that they comprise  a determined quantity of a nucleotide probe produced from a nucleotide sequence according to one of claims 1 to 11,  a medium suitable for hybridization between the probe used and the sequence to be detected, and advantageously,  - reagents allowing the. detection of the hybridization complexes formed.  23 / Method for in vitro screening in a biological sample of the possible presence of the expression products of the nucleotide sequences according to one of claims 1 to 11 or those of the genes coding for a heat shock protein, characterized in that she understands  contacting the sample to be studied with an antibody according to claim 19, under conditions allowing the formation of an immunological complex between all or part of the proteins expressed by the nucleotide sequences and this antibody, and  - detection of the immunological complex.  24 / Kits for the invitro detection in a biological sample of the possible presence of the expression products of the nucleotide sequences according to one of claims 1 to 11 or those of the genes coding for a heat shock protein, characterized in that they understand  - a determined quantity of a polyclonal or monoclonal antibody according to claim 19,  a medium suitable for carrying out an immunological reaction between at least part of the products expressed and the antibody and, advantageously,  - reagents for the detection of the immunological complexes formed.