The present invention relates to a novel human protein which is involved in the targeting of proteins towards proteasome degradation pathways. This protein, called h-xcex2TrCP, is capable of interacting notably with the Vpu protein of HIV-1 virus and with the cell proteins IxcexaB, xcex2-catenin and Skp1p.
The degradation of proteins by proteasome, a multiprotein complex present in all cells, is involved in numerous essential cell phenomena such as the control of cell proliferation, the renewal of proteins and the removal of incorrectly folded proteins, particularly in the endoplasmic reticulum (CIECHANOVER A., Cell, 79, 13-21,1994). Numerous viruses, like HIV-1 virus, which degrades CD4 via one of its proteins Vpu (MRONO D., Cell, 82, 189-1992, 1995), exploit these cell pathways of protein degradation, in which the proteins are targeted towards proteasome by various interactions with other proteins before being degraded. To be targeted towards and degraded by proteasome, the proteins must generally be ubiquitinylated beforehand by ubiquitin-ligase complexes. Furthermore, to be ubiquitinylated, the proteins must often undergo modifications such as phosphorylations (CIECHANOVER A., Embo. J., 17, 7151-7160, 1998).
Several other proteins of the xcex2TrCP type are known at the present time.
the xcex2TrCP protein of Xenope, described by Spevak et al. (Mol. Cell. Biol., 13, 4953-4966, 1993);
the Slimb protein of drosophila, described by Jiang et al. Nature, vol. 391, Jan. 29, 1998); and
the KIAA 0696 protein identified by Ishikawa et al. (DNA Research, 5, 169-176, 1998) during a systematic analysis of sequences expressed in the brain.
Jiang et al. showed that the Slimb protein of drosophila is involved in the stability of the Armadillo protein and the signaling of two metabolic pathways essential for development, namely the Hedgehog and Wingless pathways. They also showed that the Slimb protein has a homology of about 80% with the xcex2TrCP protein of Xenope, none of whose functions was described by Spevak et al. As the xcex2-catenin of Xenope or man, which is the homolog of the Armadillo protein of drosophila, seems to be targeted towards proteasome degradation pathways in the absence of signaling of the Hedgehog and Wingless pathways, said authors suggest that, in man, the genes coding for the homologs of Slimb could be involved in the proteolytic degradation of xcex2-catenin, a protein which acquires oncogenic properties when it is not degraded (POLAKIS P., Biochim. Biophys. Acta, 1332, F127-47, 1997).
However, despite the fact that conservation of the Wingless and Hedgehog pathways in vertebrates is important, it is not certain that the functions of the homologous proteins will be totally conserved. Moreover, there are numerous examples which show that there are always significant differences between species.
Also, solely on the basis of genetic studies, Jiang et al,. established the involvement of Slimb in the control of the Wingless and Hedgehog pathways in drosophila. Proof that this control is dependent on a direct interaction between Slimb and Armadillo, for example, has neither been sought nor found.
The protein according to the invention, called h-xcex2TrCP, is capable of interacting with virus proteins or cell proteins which can act as mediators or be degraded by proteasome. In particular, the h-xcex2TrCP protein is capable of interacting notably with the Vpu protein of HIV-1 virus and with the cell proteins IxcexaB and xcex2-catenin.
It is particularly useful for screening therapeutic agents such as, in particular, antitumoral, antiviral, anti-inflammatory and anti-Alzheimer agents.
The Vpu protein is a small membrane protein of 81 amino acids which is expressed by the majority of isolates of HIV-1 virus but not by those of the considerably less pathogenic FUV-2 virus or by those of SIV simian virus (COHEN et al., Nature, 334, 532-534, 1988, and STREBEL et al., Science, 2, 1221-1223, 1988).
One of the functions of the Vpu protein is its capacity to induce degradation of the CD4 protein, a cell receptor of HIV-1 virus, so it participates in reducing the expression of the CD4 receptor on the cell surface (Willey et al., J. Virol., 68, 1207-1212, 1994).
It is also known that the two phosphorylation serines of the Vpu protein, located in positions 52 and 56, are essential for the degradation of CD4 induced by Vpu (MARGOTIN et al., Virology, 223, 381-386, 1996). Moreover, during the process of infection by HIV-1 in the absence of the Vpu protein, the Gp160 envelope precursor and the newly synthesized CD4 protein combine in the endoplasmic reticulum to block the maturation of the Gp160 protein (BOUR et al., J. Virol., 65, 6387-6396, 1991. Degradation of the CD4 receptor mediated by the Vpu protein is essential for releasing the viral envelope protein which is held in the endoplasmic reticulum by being bound to CD4 through interaction with the Gp120 subunit, and for allowing the normal maturation of the envelope into the plasmic membrane and subsequently its integration into the virus particles, rendering them infectious. Recent studies have demonstrated the fact that degradation of the CD4 receptor mediated by the Vpu protein is sensitive to specific proteasome inhibitors and is dependent on the presence of an xe2x80x9cintact ubiquitinylation machineryxe2x80x9d (FUJITA et al., J. Gen. Virol., 78, 619-625, 1997).
Thus the Vpu protein participates in absolutely critical functions for assuring the production of large numbers of infectious virus particles, since it acts not only on the products of the gag gene, i.e. on the structural proteins, to increase the release of the virus particles, but also on the products of the env gene to allow the maturation of the envelope protein following degradation of the CD4 receptor. In 1996, MARGOTTIN et al. (supra) showed that the interaction between Vpu and CD4 took place via their cytoplasmic domain and that this interaction was not sufficient to trigger degradation of the CD4 receptor.
The Skp1p protein is a cell protein involved in the targeting of proteins towards proteasome degradation pathways, which depends on the ubiquitinylation of the proteins (PICKART C. M., The Faseb Journal, 11, 1055-1066,1997).
BAI et al. (Cell, 86, 263274, 1996) showed that the Skp1p protein was necessary for ubiquitin-mediated proteolysis and that this degradation took place due to the interaction of Skp1p with proteins containing a unit called F-box.
The Skp1p protein is an essential factor in the targeting of cell cycle regulatory proteins by proteasome. Targeting of the degradation of these regulators is particularly necessary when the cell cycle enters the S phase of DNA synthesis (PAGANO M., The Faseb Journal, 11, 1068-1075, 1997). Recent studies showed that the Skp1p protein and F-box proteins are the essential elements of high-molecular complexes called SCF (Skp1p-Cullin-F-box-protein complexes). These SCF complexes play the role of enzyme E3; through their ubiquitin-ligase activity, they allow the last step of the ubiquitinylation of substrate proteins, which are thus targeted towards degradation by proteasome (HOYT A., Cell, 91, 149-151, 1997). It is further pointed out that no Skp1p homolog has yet been identified in drosophila.
The IxcexaB protein, which exists in different forms (xcex1, xcex2, xcex5), is the major inhibitor of the NFxcexaB transcription factor, keeping it in the form of an inactive complex in the cytoplasm (Beg A. et al., Genes and Dev., 7, 2064-2070, 1993). After stimulation of the cells by factors such as interleukin-1 (IL1) and tumor necrosis factor (TNF), the IxcexaB protein is phosphorylated on serine residues S32 and S36. This phosphorylation leads very rapidly to the ubiquitinylation of the protein and to the targeting thereof towards degradation by proteasome. The active NFxcexaB factor, for example in the form of two subunits P50 and P65, is then released and imported into the nucleus, where it will be able to activate a very large number of genes and cause inflammatory phenomena in particular.
The xcex2-catenin protein is a cell protein controling the essential signal transduction pathways such as the Wingless pathways, which are very highly conserved in all vertebrates (MILLER et al., Genes and Dev., 10, 2527-2539, 1996, and POLAKIS P., Biochim. Biophys. Acta, 1332, F 127-47, 1997). xcex2-Catenin accumulates in cancerous cells, either as a result of mutations which prevent phosphorylation on serine residues 33 and 37 (mutated xcex2-catenin proteins), or as a result of mutations of its cofactor, the APC protein, which is necessary for its degradation.
The accumulation of xcex2-catenin due to its non-degradation leads to its importation into the nucleus and to the activation of genes controlled by TCF-LEF promoters, causing cell proliferation and transformation phenomena.
It was recently shown that mutations of presenilin-1 in patients suffering from Alzheimer""s disease caused a destabilization and enhanced degradation of xcex2-catenin (ZHANG et al., Nature, 395, 699-702, 1998). These authors showed that non-mutated presenilin-1 binds to xcex2-catenin and thereby contributes to its stability. In Alzheimer""s disease, the mutated presenilin is no longer capable of binding to xcex2-catenin, so the latter is degraded more rapidly. The level of xcex2-catenin is considerably reduced in the neuronal cells of patients suffering from Alzheimer""s disease. The loss of xcex2-catenin causes an enhanced apoptosis of the neuronal cells, which would account for the neuronal loss observed in this pathological condition.
It is easy to see that there is an urgent need for means of modulating, namely activating or inhibiting, the targeting of proteins towards proteasome.
A novel human protein involved in the targeting of proteins towards proteasome degradation pathways has now been found which makes it possible to screen modulators of the targeting of proteins towards proteasome.
The present invention therefore relates to a novel human protein, called h-xcex2TrCP, which has SEQ ID No. 2 and which is involved in the targeting of proteins towards proteasome degradation pathways.
The h-xcex2TrCP protein possesses 569 amino acids and comprises one F-box and seven WD units having the following positions in the sequence SEQ ID No. 2:
Because of the homology of this novel protein with the xcex2TrCP of Xenope, a protein containing 0-transducin units and known as xe2x80x9cbeta transducin repeats containing proteinxe2x80x9d, the protein of the invention is called h-xcex2-ATrCP (human xcex2TrCP).
Via its WD units, the h-xcex2TrCp protein of the invention is capable of interacting with proteins degradable by proteasome, particularly with virus proteins and cell proteins which possess the phosphorylation unit comprising the amino acids Asp-Ser-Gly-Xaa-Xaa-Ser (SEQ ID NO: 9), in which Xaa is any natural amino acid and in which the serine residues are phosphorylated.
The phosphorylation of this unit Asp-Ser-Gly-Xaa-Xaa-Ser (SEQ ID NO: 9) is essential to the ubiquitinylation and subsequent degradation of proteins possessing this type of unit. The h-xcex2TrCP protein is only capable of interacting with proteins containing this unit when the two serine residues are phosphorylated, and it cannot interact with proteins containing a phosphorylation unit in which the serine residues are mutated to non-phosphorylatable amino acids. By interacting with the phosphorylated proteins on this unit, the h-xcex2TrCP protein controls their ubiquitinylation and their screening towards degradation by proteasome.
The virus protein Vpu and the cell proteins IxcexaB and xcex2-catenin may be mentioned in particular among these proteins.
It has also been found that the h-xcex2TrCP protein interacts via its F-box with the Skp1p protein, so it forms part of a novel SCF complex, SCF-h-xcex2TrCP, which selects certain cell or virus proteins for degradation by proteasome. Through its activity of targeting towards proteasome degradation pathways, the h-xcex2TrCP protein according to the invention acts as cell mediator of the Vpu protein in cells infected with HIV-1 virus.
Without wishing to exclude other theories, it is thought that, in cells infected with HIV-1 virus, the virus uses, via the Vpu protein, the SCF complex (of which the xcex2TrCP protein forms part) to induce degradation of the CD4 receptor, which will favor the replication of the virus and the release of the infectious virions.
The invention further relates to the peptide fragments of the h-xcex2TrCP protein which result from the addition, deletion and/or replacement of one or more amino acids, said peptide fragments having conserved the activity of interacting with proteins degradable by proteasome, particularly with the Vpu protein of HIV-1 virus, with the cell protein IxcexaB or the cell protein xcex2-catenin and/or with the Skp1p protein.
The invention relates in particular to the peptide fragments which comprise at least one of the following amino acid sequences of h-xcex2TrCP:
251-569,
292-569,
292-396,
292-545 and
1-291.
Very particularly preferred peptide fragments are those which are partially or totally devoid of the F-box or those which are partially or totally devoid of the WD units.
One particularly preferred peptide fragment is the mutant with residues 32-179 deleted, which is hereafter called xcex2TCPxcex94F.
The present invention further relates to the nucleic acid sequences, namely the genomic DNA sequences and the cDNA or mRNA sequences, which comprise or consist of a concatenation of nucleotides coding for the h-xcex2TrCP protein or for any one of its peptide fragments as defined above.
The invention relates notably to those nucleic acid sequences coding for the h-xcex2TrCP protein and its peptide fragments described above which are represented by:
a) the cDNA sequence SEQ ID No. 1 coding for said h-xcex2TrCP protein and the cDNA sequences of the nucleic acid fragments coding for said peptide fragments;
b) the DNA sequences which hybridize with the above sequences under strict conditions;
c) the DNA sequences which, due to the degeneracy of the genetic code, result from the sequences a) and b) above and code for the h-xcex2TrCP protein or its fragments; and
d) the corresponding mRNA and DNA sequences.
The proteins and peptide fragments according to the invention can be obtained by the genetic engineering technique comprising the following steps:
culture of a microorganism or eukaryotic cells which have been transformed with the aid of a nucleic acid sequence according to the invention; and
recovery of the protein or the peptide fragment produced by said microorganism or said eukaryotic cells.
This technique is well known to those skilled in the art. Further details on this subject may be obtained by reference to the following work: Recombinant DNA Technology 1, Editors Ales Prokop, Raskesh K. Bajpai; Annals of the New York Academy of Sciences, volume 646, 1991.
They can also be prepared by the conventional peptide syntheses well known to those skilled in the art.
The nucleic acids according to the invention can be prepared by chemical synthesis and genetic engineering using the techniques well known to those skilled in the art, as described e.g. by SAMBROOK et al. (supra).
For example, the cDNA sequences according to the invention can be synthesized by amplifying the mRNAs of human cells by the PCR (Polymerase Chain Reaction) method, as described e.g. by GOBLET et al. (Nucleic Acid Research, 17, 2144, 1989), using, as primers, synthetic oligonucleotides defined from the DNA sequence SEQ ID No. 1.
The amplified nucleic acid fragment can then be cloned by the techniques described by AU SUBEL et al. (Current Protocols in Molecular Biology, chapter 3, supra).
The invention further relates to transgenic animals which express a transgene for the h-xcex2TrCP protein of the invention, or transgenic animals in which the xcex2TrCP gene has been invalidated.
These transgenic animals or animals in which the h-xcex2TrCP protein gene has been invalidated may be used as models for the in vivo study of perturbation of the cell cycle and proliferation by the absence or overexpression of the gene for the h-xcex2TrCP protein or for truncated or mutated forms of this protein, the Skp1p protein, the Vpu protein, the IxcexaB protein or the xcex2-catenin protein.
These transgenic animals are obtained by techniques well known to those skilled in the art, such as those described in Manipulating the mouse embryo; a laboratory manual. HOGAN B., BEDDINGTON R., COSTANNNI F. and LACY E., Cold Spring Harbor laboratory press, second edition, 1994.
The preferred animals are mammals such as mice or rats.
The invention further relates to the prokaryotic microorganisms and eukaryotic cells transformed with the aid of an expression vector containing a DNA sequence according to the invention. This expression vector, which can be e.g. in the form of a plasmid, must contain, in addition to the DNA sequence of the invention, the means necessary for its expression, such as, in particular, a promoter, a transcription terminator, an origin of replication and, preferably, a selection marker. The transformation of microorganisms and eukaryotic cells is a technique well known to those skilled in the art, who will easily be able to determine, as a function of the microorganism to be transformed, the means necessary for the expression of the DNA sequence according to the invention.
The preferred microorganism for the purposes of the invention is E. coli, while the yeast used is preferably Saccharomyces cerevisiae. 
COS, CHO, SF9, Jurkat and other cells, all of which are listed in the ATCC, may be mentioned in particular as examples of eukaryotic cells which are suitable for the purposes of the invention.
The invention further relates to the eukaryotic cells cotransformed with expression vectors containing on the one hand the DNA sequence coding for the Vpu protein, for the Skp1p protein, for the IxcexaB protein or for the mutated xcex2-catenin proteins, and on the other hand a sequence coding for the h-xcex2TrCP protein, said expression vectors also containing means useful for their expression, including in the yeast two-hybrid system.
The present invention therefore further relates to the anti-HIV-1 antiviral agents consisting of the peptide fragments of the h-xcex2TrCP protein of the invention which have conserved the properties of interaction of the h-xcex2TrCP protein either with the Vpu protein or with the Skp1p protein. These peptide fragments are devoid of the F-box or the WD units, so they are no longer able to interact with the Skp1p protein or, respectively, the Vpu protein.
Other antiviral agents, antitumoral agents or anti-inflammatory agents which may be mentioned are antibodies directed against the h-xcex2TrCP protein of the invention and its peptide fragments, said antibodies being a further subject of the invention.
These antibodies can be monoclonal antibodies obtained by the well-known method of KOHLER and MILSTEIN (Nature, 256, 495-497, 1975) or polyclonal antibodies obtained by the conventional methods of animal immunization (Antibodies, a laboratory manual. E. Harlow and D. Lane. Cold Spring Harbor laboratory press, 1988).
Finally, antiviral agents, antitumoral agents or anti-inflammatory agents which may be mentioned are antisense oligonucleotides which block the transcription or translation of the h-xcex2TrCP protein of the invention and which hybridize with a nucleic acid sequence as defined above, said oligonucleotides also forming a further subject of the present invention.
These antisense oligonucleotides are prepared by techniques well known to those skilled in the art, such as those described by AUSUBEL et al. (Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Interscience, New York, 1989, published up to 1997).
The peptide fragments of h-xcex2TrCP which possess the F-box or which have conserved both the WD units and the F-box can be used as antitumoral or anti-inflammatory agents.
The peptide fragments of h-xcex2TrCP which are devoid of the F-box can be used in gene therapy for the treatment of osteoarticular inflammatory diseases or acute inflammatory syndromes which are accompanied by NFxcexaB activation induced by the massive release of TNFxcex1 during these processes.
As illustrated in FIG. 7, the expression of h-xcex2TrCPxcex94F is capable of massively inhibiting, by a factor of about 20, the transcription activation induced by TNFxcex1 Therefore, h-xcex2TrCPxcex94F could act as a potent anti-inflammatory agent in any pathological condition associated with an intense inflammatory reaction due to a release of TNFxcex1. For example, several attempts are currently being made to apply gene therapy to rheumatoid polyarteritis by injecting recombinant viruses into the damaged joints. Vectors expressing h-xcex2TrCPxcex94F can be used in these gene therapy experiments on inflammatory syndromes. These vectors may be of several types (retroviruses, adenoviruses; ANDERSON F., Nature, 392, 25-30, 1998). The expression of h-xcex2TrCPxcex94F may be monitored by its effects on the inhibition of NFxcexaB activation by TNF.
The present invention further relates to the use of the h-xcex2TrCP protein, or the nucleic acid sequences coding for this protein or for its peptide fragments, for the screening of therapeutic agents which are capable of modulating the interaction of the h-xcex2TrCP protein with proteins degradable by proteasome, and particularly for the screening of:
anti-HIV-1 antiviral agents capable of inhibiting the interaction between the h-TrCP protein and the Vpu protein and/or inhibiting the interaction between the h-xcex2TrCP protein and the Skp1p protein;
antitumoral agents capable of perturbing the regulation of the cell cycle or the protein degradation processes in tumoral human cells by modulating (inhibiting or activating) the interaction between the h-xcex2TrCP protein and the Skp1p protein, and by reactivating the interaction between the h-xcex2TrCP protein and the mutated xcex2-catenin proteins in tumoral cells, or between the TrCp protein and the normal xcex2-catenin protein in tumoral cells devoid of the APC protein;
anti-inflammatory agents capable of perturbing the activation of the NFxcexaB transcription factor by inhibiting the interaction between the h-xcex2TrCP protein and the IxcexaB protein; and
anti-Alzheimer agents capable of reducing the degree of degradation of xcex2-catenin in neuronal cells by inhibiting the interaction between the h-xcex2TrCP protein and the xcex2-catenin protein.
In fact, by perturbing the Vpu/h-xcex2TrCP and/or Skp1p/h-xcex2TrCP interactions, it is possible:
either to inhibit the replication and production of HIV-1 virus by infected cells;
or to inhibit the entry of the cell cycle into the S phase and to have an antiproliferative effect.
By perturbing the IxcexaB/h-xcex2TrCP and/or Skp1p/h-xcex2TrCP interactions, it is possible to inhibit the degradation of the IxcexaB protein by proteasome and hence to inhibit the activation of the NFxcexaB transcription factor.
Finally, by activating the mutated xcex2-catenin/h-xcex2TrCP interaction, it is possible to activate the degradation of the xcex2-catenin which has accumulated in tumoral cells. By inhibiting the xcex2-catenin/h-xcex2TrCP interaction in patients suffering from Alzheimer""s disease, it is possible to reduce the apoptosis of neuronal cells.
The antiviral agents can be selected either from random peptide banks on the surface of phages (SCOTT J. et al., Science, 249, 386-390, 1990) or by using random synthetic oligonucleotides according to the technique of the SELEX type (TUERK and GOLD, Science, 249, 505-510, 1990). This technique makes it possible to isolate, from a very large pool of oligonucleotides, those which have a high affinity for the protein of interest, namely the h-xcex2TrCP protein in the present case. They are called aptamers. From these aptamers it will be possible, using the screening method below, to select those which inhibit both the Vpu/h-xcex2TrCP and Skp1p/h-xcex2TrCP interactions.
The screening method defined above can be carried out e.g. by using the yeast two-hybrid system in which yeast cells co-expressing the h-xcex2TrCP protein according to the invention and one of the proteins Vpu, IxcexaB, xcex2-catenin or Skp1p are cultivated on appropriate selective media in the presence of the test substance; the selective media are the media commonly used in this field and hence are well known to those skilled in the art.
The yeast two-hybrid system is described by FIELDS and SONG in Nature, 340, 245-246, 1989, and in patent U.S. Pat. No. 5,667,973. This two-hybrid system is based on detection of the protein-protein interactions by activation of the His or LacZ reporter gene under the control of Gal4 transcription activation domains in the yeast.
In this two-hybrid system, a yeast is cotransformed with a two-hybrid vector containing the cDNA of one of the proteins and a vector containing the cDNA of the other protein, each of said vectors containing either a DNA binding domain or a transcription activation domain. The two proteins are then expressed by the yeast in an appropriate culture medium, for example a histidine-free culture medium. The interaction between the two hybrid proteins allows on the one hand activation of the His3 gene and growth of the yeasts on a histidine-free medium, as well as activation of the LacZ gene, which is disclosed by a color reaction specific for xcex2-galactosidase. It is therefore possible to verify the interaction when the yeasts grow on a histidine-free medium and when a color reaction is observed.
A further possibility is to use the halo test as described by Valtz and Peter (Meth. Enzymol., 283, 350-365, 1997) to detect whether there is any interaction.
It is also possible to use variants of the two-hybrid system, such as the three-hybrid system described by TIRODE et al. (J. Biol. Chem., 272, 22995-22999, 1997) or by COLAS et al. (Nature, 380, 548-550, 1996), in which a peptide inhibiting the interaction can be expressed as a third partner to inhibit the interaction of the other two. A random peptide bank can also be used in this way.
A further possibility is to use the reverse-hybrid system described by VIDAL et al. (Proc. Natl. Acad. Sci., 93, 10315-10320), in which selection is carried out against an interaction and not for an interaction. In this system, as in the conventional two-hybrid system, it is possible to screen banks of small chemical molecules, including those derived from chemical synthesis, in order to bring yeasts cotransformed with two-hybrid or reverse-hybrid vectors carrying fusions with the Vpu protein, the IxcexaB protein, xcex2-catenin, the h-xcex2TrCP protein or the Skp1p protein into contact with these small molecules in the search for an inhibitor of the Vpu/h-xcex2TrCP, Skp1p/h-xcex2TrCP, xcex2-catenin/h-xcex2TrCP or IxcexaB/h-xcex2TrCP interactions.
The screening assays for interaction inhibitors may also be carried out using the conjugative two-hybrid system (FROMONT-RACINE et al., Nature Genetics, 16, 277-282, 1997), the membrane two-hybrid system (BRODER Y. C. et al., Curr. Biol., 8, 1121-1124, 1998) and optionally, if phosphorylations can take place in the bacteria, the bacterial two-hybrid system (KARIMOVA et al., Proc. Natl. Acad. Sci., 95, 5752-5756, 1998).
This screening can also be effected in vitro by using one of the proteins Vpu, IxcexaB, xcex2-catenin or Skp1p and the h-xcex2TrCP protein, one of the proteins being immobilized on an appropriate support and the other being labeled by any means used in the methods of detecting biological substances, it being possible for this labeling means to be e.g. a radioactive isotope, a luminescent agent, biotin or a specific antibody.
One of the proteins will preferably be immobilized in the form of a fusion protein with glutathione S-transferase (GST) on agarose-glutathione beads or in microtiter plates, the GST serving as an agent for coupling said protein with the beads or with the wells of the plates.
This can be done particularly using the scintillation proximity assay (SPA) described by BOSWORTH et al. (Nature, 341, 167-168, 1989) and marketed by Amersham. This assay consists in labeling one of the proteins with a radioactive element, for example tritium, and immobilizing the other protein on magnetic beads or agarose-glutathione beads. The inhibitory effect of the test substances on interactions involving the h-xcex2TrCP protein can easily be detected, without separation of the bound or free radioactive species, according to the protocols described by BOSWORTH et al. (supra).
Another possible technique is that of surface plasmon resonances described by KARLSSON et al. (J. Immunol. Methods, 145, 229-233, 1991), using Biacore, marketed by Pharmacia, to isolate the inhibitors of interactions involving the h-xcex2TrCP protein according to the invention.
The inhibitory activity of the antiviral agents selected in this way may be verified by assays on CD4+T cells or on chimpanzees infected with HIV-1 virus or SIV Cpz.
The antitumoral agents and anti-inflammatory agentsxe2x80x94ligands of the h-xcex2TrCP protein of the inventionxe2x80x94can also be isolated by the two-hybrid techniques or related techniques or by interaction in vitro with combinatorial banks of peptides or other chemical products, as described above.
The specificity of the antiviral, antitumoral or anti-inflammatory agents selected by the two-hybrid assay can then be determined by the culture of mammalian cells, for example human cells transfected with the xcex2TrCp protein or a fragment thereof, in the presence of a reporter gene specific for the protein involved in the pathological condition which it is desired to treat.
Thus, for the IxcexaB protein, it will be possible to use human cells originating from the cell lines Hela, 293, 293T, etc. and the reporter gene dependent on NFxcexaB sites (3Enh-xcexaB-ConA Luc), which controls the expression of luciferase.
In non-stimulated human cells, the human xcex2TrCP protein is transitorily expressed from a eukaryotic expression vector such as pCDNA3 (Invitrogen), or any other eukaryotic expression vector, which has inserted the DNA coding for the xcex2TrCP protein under the control of a strong promoter of the cytomegalovirus, CMV, or the like. An amount of the order of 3 xcexcg of this vector permitting the expression of the xcex2TrCP protein will be cotransfected by one of the common transfection techniques (calcium phosphate, lipofectamine (Life Technologies), electroporation (Ausubel and Sambrook, cf. below) etc.) with 1 xcexcg of a reporter vector dependent on NFxcexaB sites (3Enh-xcexaB-ConA luc) or independent of NFxcexaB sites (RSV Luc or ConA Luc) which control the expression of the luciferase reporter gene. Molecules capable of inhibiting the h-xcex2TrCP/IxcexaB interaction will inhibit the increase in the expression of lucifersse in this assay. These inhibitors will be added to the culture medium for at least 6 hours, 24, 36 or 48 hours after transfection. The specificity of these inhibitors may be checked by verifying that they have no effect on RSV Luc or ConA Luc. Another possible alternative will be to use the dual luciferase system from Promega, in which two different reporter vectors can be assayed at the same time.
According to one experimental protocol similar to that described above, but with stimulated cells, it will be possible to verify that the inhibition induced by the expression of the h-xcex2TrCPxcex94F fragment on the TNF-dependent transcription activation has been nullified.
Thus, in this second assay, the human cells are cotransfected with 1 xcexcg of reporter vector, i.e. either 3Enh-xcexaB-ConA Luc, ConA Luc or RSV Luc, and with 3 xcexcg of pCDNA3 expressing the h-xcex2TrCPxcex94F peptide fragment, which is a mutant of xcex2TrCP with its F-box deleted. 24 to 48 h after transfection, the cells are treated for 6 h with TNF or okadaic acid (OKA), which are potent NFxcexaB activators (BAUERLE et al., Cell, 1996, 87, 13-20). The h-xcex2TrCPxcex94F mutant has a massive inhibitory effect on the expression of the luciferase reporter compared with a control plasmid transfected under the same conditions. This effect is due to the inhibition of IxcexaB degradation induced by the binding of the h-xcex2TrCPxcex94F mutant in place of the endogenous wild-type h-xcex2TrCP protein. Therefore an inhibitor of the h-xcex2TrCP/IxcexaB interaction will also inhibit the h-xcex2TrCPxcex94F/IxcexaB interaction and hence will reverse the inhibitory effect of the h-xcex2TrCPxcex94F fragment. The potential inhibitors are added to the medium under the same conditions as those indicated above. From the cells stimulated with TNF or OKA, those inhibitors are chosen which induce an increase in the expression of the reporter gene.
After the selection of inhibitors in the previous two assays, a third assay can be carried out to verify that they are capable of inhibiting the activation of NFxcexaB induced by stimulation of the cells with TNF or OKA.
The cells transfected only with 1 ng of reporter vector (3Enh-KB-ConA Luc) and stimulated for 6 h with TNF or OKA are treated with the potential inhibitors. To be specific, these inhibitors must have an effect only on the IxcexaB-dependent reporter vectors and not on the other reporter vectors (ConA or RSV).
In the case of xcex2-catenin, it will be possible to use human cells originating from the above lines transformed with mutated xcex2-catenin or the peptide fragment of xcex2TrCP devoid of the F-box, in the presence of vector Top-TK-Luci, which contains a multimer of TCF-LEF sites responding to xcex2-catenin, patenin, or Fop-tk Luci, which contains an inactive mutated multimer and no longer responds to xcex2-catenin.
Furthermore, as oncogenic mutated xcex2-catenin can easily be distinguished from wild-type xcex2-catenin by the fact that the former, in contrast to the latter, is incapable of binding to xcex2TrCP in the two-hybrid assay, xcex2-catenin mutations can be detected in human tumors by measuring the interaction with xcex2TrCP in the two-hybrid assay.
This assay is valuable because xcex2-catenin mutations are found in numerous cancers such as colon cancer, melanomas, hepatocarcinomas, etc. The only way of detecting these mutations hitherto was to sequence the xcex2-catenin by carrying out RT-PCR on the RNA of the tumors studied. For greater reliability, several double-stranded sequences have to be made in this assay of the prior art. Also, the existence of a mutation does not in itself indicate the oncogenic character of this mutation. It could be a case of polymorphism unconnected with tumorigenicity.
The advantage of the two-hybrid assay with the xcex2TrCP protein is that, in times equivalent to those required to obtain a sequence, it is possible to obtain a clear answer regarding the percentage of oncogenic mutated xcex2-catenin sequences detected from the tumoral RNA. Over a large number of colonies, the percentage of oncogenic forms of xcex2-catenin which are incapable of interacting with xcex2TrCP, compared with the wild-type forms which do interact with xcex2TrCP, can be determined precisely. The assay can be performed in a time equivalent to that required to obtain a few sequences, and at a reduced cost.
This assay comprises the following steps:
1xe2x80x94Preparation of the total RNA from a biopsy of a tumor and of the surrounding healthy tissue, as control, using one of the various RNA preparation techniques or kits (AUSUBEL et al., Current Protocols in Molecular Biology).
2xe2x80x94Amplification of the catenin sequences of the tumor and of the surrounding healthy tissue by carrying out RT-PCR on the RNA samples using a pair of oligonucleotides which permit amplification either of the N-terminal part only (1-130), which contains the most frequently encountered oncogenic mutations (RUBINFELD B. et al., Science, 275, 1790-1792, 1997; DE LA COSTE et al., Proc. Natl. Acad. Sci. USA, 95, 8847-8851, 1998), or of the whole of the xcex2-catenin coding sequence.
3xe2x80x94Insertion of these amplified fragments, by ligation, into one of the two-hybrid vectors, for example pGAD1318, to give a frame fusion with the Gal4 transcription activation domain or the equivalent activation domain for transcription or binding to the DNA coded for by the two-hybrid vector used.
4xe2x80x94ransformation of bacteria of various appropriate strains and plating of the whole of the transformant on LB-ampicillin medium.
5xe2x80x94Harvesting of all the colonies and plasmid minipreparation (AUSUBEL, supra).
6xe2x80x94L40 yeasts or any other appropriate strain of yeast will be cotransformed by the plasmid containing the xcex2-catenin sequences of the above minipreparation with a fusion hybrid containing xcex2TrCP, for example pLexA-xcex2TrCP, in which the xcex2TrCP is fused to the LexA DNA binding domain. A two-hybrid assay is performed on all the colonies obtained, for example by plating the cotransformed yeasts on DO-W-L medium and then transferring the colonies to selective medium for detection of the interactions, i.e. DOW-L-H medium, or in the presence of X-Gal for detection of the interactions by xcex2-galactosidase production (BARTEL P. and FIELDS S., Meth. Enzymol., 254, 241-263, 1995).
This assay requires the following reagents:
1xe2x80x94Vector pGAD1318 predigested at the appropriate sites for inserting the amplified fragment obtained by RT-PCR.
2xe2x80x94The appropriate oligonucleotides for amplifying the xcex2-catenin sequence and then inserting the amplified xcex2-catenin sequences. The oligonucleotide primers for amplification will be chosen according to the mode of insertion of the amplified fragment and the chosen sites.
3xe2x80x94Plasmid pBTM116-xcex2TrCP expressing xcex2TrCP fused to the LexA DNA binding domain.
4xe2x80x94As control, plasmids coding for fusion hybrids with control proteins, for example pLexRas and pGAD1318Raf.
The gap repair technique (SCHWARTZ H. et al., Mutation detection by a two-hybrid assay, Hum. Mol. Gen., 7, 1029-1032, 1998) may also be applied for this assay in order to insert the sequence of the amplified xcex2-catenin fragment into the two-hybrid vector and transform yeasts directly without proceeding via the step of prior transformation in bacteria.
The invention will now be described in detail with the aid of the following account of experiments.
A large part of the techniques described in these Examples, which are well known to those skilled in the art, is explained in detail in the book by SAMBROOK et al. (supra) or in the book by AUSUBEL et al. (supra).