Method for Screening Agents Modulating Ikbalpha Protein Ubiquitination and Means for Carrying out Said Method

Abstract

A method for screening agents modulating IκBα protein ubiquitination by a functional ubiquitin ligase protein complex containing β-TrCP protein, the method including the following steps:

Description

FIGURES

FIG. 1 illustrates the ability of Skp1 yeast proteins and β-TrCP human proteins to interact in yeast cells.

On the x-axis: plasmids present in the transformed yeast cells;On the y-axis, β-galactosidase activity, expressed in nanomoles of substrate transformed per minute per mg of cell protein.

FIG. 2 illustrates localisation, in yeast cells, of human proteins IκBα and β-TrCP according to whether or not they are fused to an NLS sequence of SV40. Top line: fluorescence microscopy images of cell nucleus DNA stained with Hoescht 333-42 dye.

Bottom line: fluorescence microscopy images showing the localisation of GFP expression in the cells.

A: Cells transformed by GFP-NLS-β-TrCP vector; B: Cells transformed by GFP-β-TrCP vector; C: Cells transformed by GFP-NLS-IκBα vector; D: Cells transformed by GFP-IκBα vector.

FIG. 3 shows how the presence of the human IKB(X protein in the nuclei of yeast cells leads to its phosphorylation at serines 32 and 36. The figure shows a gel electrophoresis image of cell proteins of recombinant yeast strains CYS22 and CYS126, respectively.

FIG. 4 shows, by epifluorescent microscopy, the degradation of the tripartite fusion protein GFP-NLS-IκBα in the yeast cells which, at the same time, express the tripartite fusion protein Flag-NLS-β-TrCP.

FIGS. 4A to 4D show fluorescence microscopy images: upper line, cell nucleus DNA stained with Hoescht 333-42 dye; lower line, fluorescence microscopy images showing the localisation of GFP expression in the cells.

FIG. 4A: results obtained with recombinant yeast strain CYS22;FIG. 4B: results obtained with recombinant yeast strain CYS61.

FIG. 4C: results obtained with recombinant yeast strain CYS126.

FIG. 4D: results obtained with recombinant yeast strain CYS135. On the x-axis: the different times in minutes after adding glucose to the cell cultures.

FIG. 5 shows, by measurement of the fluorescence produced, the degradation of the tripartite fusion protein GFP-NLS-IκBα in the yeast cells which express or do not express the tripartite fusion protein Flag-NLS-β-TrCP.

The results are given for the recombinant yeast strains CYS135, CYS126, CYS61 and CYS22, respectively, which are labelled in boxes on the graph.

On the x-axis: the time in minutes after adding glucose to the cell cultures;On the x-axis: average intensity of the fluorescence, expressed in arbitrary units of fluorescence.

FIG. 6 shows, by Western Blot type biochemical analysis, the degradation of the tripartite fusion protein GFP-NLS-IκBα in yeast cells which, at the same time, express the tripartite fusion protein Flag-NLS-β-TrCP.

Western blotting gel images revealed with anti-GFP antibodies and FLAG anti-peptide antibodies.

On the x-axis: the time in minutes after adding glucose to the cell cultures;

The results are shown for the following recombinant yeast strains; CYS22 (FIG. 6A), CYS61 (FIG. 6B), CYS126 (FIG. 6C) and CYS135 (FIG. 6D).

FIG. 7 shows, by Western Blot type biochemical analysis, the degradation of the mutated tripartite fusion protein GFP-NLS-IκBα[S3236A] in which the phosphorylation sites Ser32 and Ser36 have been replaced by Ala residues, mutations that, in human cells, make the protein non-degradable.

Western blotting gel images revealed with anti-GFP antibodies and FLAG anti-peptide antibodies.

On the x-axis: the time in minutes after adding glucose to the cell cultures;

The results are shown for the following recombinant yeast strains; CYS138 (FIG. 7A) and CYS139 (FIG. 7B).

FIG. 8 shows, by epifluorescent microscopy analysis, the degradation of the tripartite fusion protein GFP-NLS-IκBα[S3236A] in the yeast cells which, at the same time, express the tripartite fusion protein Flag-NLS-β-TrCP.

FIGS. 8A to 8B show fluorescence microscopy images: upper line, cell nucleus DNA stained with Hoescht 333-42 dye; lower line, fluorescence microscopy images showing the localisation of GFP expression in the cells.

FIG. 8A: results obtained with recombinant yeast strain CYS138;FIG. 8B: results obtained with recombinant yeast strain CYS139.

On the y-axis: the different times in minutes after adding glucose to the cell cultures.

FIG. 9 shows, by measurement of the fluorescence emitted, the degradation of the tripartite fusion protein GFP-NLS-IκBα[S3236A] in the strains of yeast described herein.

The results are given for the recombinant yeast strains CYS138 and CYS139, respectively, which are labelled in boxes on the graph.

On the x-axis: the time in minutes after adding glucose to the cell cultures;On the x-axis: average intensity of the fluorescence, expressed in arbitrary units of fluorescence.

EXAMPLES

Examples 1 to 3

Construction of Recombinant Vectors According to the Invention

A. Materials and Methods for Examples 1 to 3.

A.1. Summary of the Polynucleotide Sequences Used

The sequence of the IKcBo protein is that described in Strausberg et al. (PNAS (1999), 99(26): 16899-16903).

The sequence of the β-TrCP receptor sub-unit of the ubiquitin ligase complex SCF^β-TrCP is that described in Yaron et al. (Nature (1998) 396(6711): 590-594).

The sequence of the GFP gene from Aequora Victoria, optimised for expression in yeast (yEGFP3), and its product Green Fluorescent Protein mut3, (hereafter called GFP), is that described by Cormack et al. (Gene (1996) 173 (1): 33-38).

The nuclear localisation signal “NLS” sequence of the SV40 virus big-T antigen is a translation of the nucleic acid sequence,

5′-ACCTCCAAAAAAGAAGAGAAAGGTCGAATT-3′ (SEQ ID N^o25).

The sequence of the pRS306 plasmid is that described by Sikorski and Hieter (Genetics (1989) 122(1): 19-27).

The sequence of the pRS304 plasmid is that described by Sikorski and Hieter (Genetics (1989) 122(1): 19-27).

The sequence of the pRS314 plasmid is that described by Sikorski and Hieter (Genetics (1989) 122(1): 19-27).

The sequence of the pRS316 plasmid is that described by Sikorski and Hieter (Genetics (1989) 122(1): 19-27).

The sequence of the plasmid pSH18-34, which contains four LexA operators upstream of the LacZ gene, is that described by Hanes et Brent (Cell (1989), 57:1275-1293)

The sequence of the pLexSkp1-1 plasmid, which expresses the Skp1 protein fused with the LexA protein, is that described in Patton et al. (Genes & Dev (1998), 12 :692-705)

The sequence of the pGADβTrCP plasmid, which expresses the human β-TrCP protein fused to the activator domain of the Gal4 yeast transcription factor is that described in Margottin et al. (Molec. Cell (1998), 1 :565-574).

The sequence of the GAL1 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Johnston and Davis (Mol. Cell. Biol. (1984) 4 (8): 1440-1448).

The sequence of the MET3 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Cherest et al. (Mol. Gen. Genet. (1987) 210 (2): 307-313).

The sequence of the MET28 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Kuras et al. (EMBO J. (1996) 15(10): 2519-2529).

The sequence of the TEF1 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Schaaff-Gerstenschlager et al. (Eur. J. Biochem. (1993) 217 (1): 487-492).

The sequence of the SAM4 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Thomas et al. (J. Biol. Chem. (2000) 275(52): 40718-40724).

The sequence of the MET25 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Keijan et al. (Nucleic Acids Res.(1986) 14(20): 7861-7871).

The sequence of the PHO5 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Feldman et al. (EMBO J. (1994) 13(24): 5795-5809).

The sequence of the CUP1 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Karin et al. (PNAS (1984) 81(2): 337-341).

The sequence of the PGK1 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Bolle et al. (Yeast (1992) 8(3): 205-213).

The sequence of the ADH1 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Bennetzen and Hall (J. Biol. Chem. (1982) 257(6): 3018-3025).

The sequence of the TDH3 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Arroyo et al. Unpublished (1996), direct submission to MIPS.

The sequence of the LEU2 promoter gene from the yeast S. cerevisiae used in the following descriptions is that described by Rad et al. (Yeast (1991) 7(5): 533-538).

A.2. Conventions Used

The descriptions use the nomenclature and typographical rules used by the Saccharomyces cerevisiae yeast biology community.

• • the name of the wild type gene is given in italicised upper case, for example: GAL1.
  • the name of the mutated form of the gene is given in italicised lower-case, the allele number, if known, follows after a hyphen; for example cup1-1.
  • the name of a non-functional allele in a gene is given in lower case followed by two colons followed by the name of the functional gene, e.g. ppr1::TRP1 (in this example the non-functional gene ppr1 has been interrupted by the functional gene TRP1).

Alternatively, a non-functional gene can be indicated by the “delta” symbol with the name, for example gal4Δ

• • the name of the protein and that of the gene coding for it is given in lower case except for the first letter, which is upper case, e.g. Gal4 (alternatively, one can use the same symbol followed by a p, for example Gal4p).

A.3. Preliminary Comments About Construction of the Plasmids

All the plasmids were constructed using classical molecular biology techniques according to the protocols described by Sambrook et al. (in Molecular Cloning, Laboratory Manual, 2nd edition, (1989), Cold Spring Harbor, N.Y.) and Ausubel et al., (in Current Protocols in Molecular Biology, (1990-2004), John Wiley and Sons Inc, N.Y.). Cloning, replication and generation of plasmid DNA were performed in the DH10B strain of Escherichia coli.

Example 1

Construction of Plasmids Able to Express the Fusion Proteins GFP-IκBα and GFP-NLS-IκBα in Yeast

The following plasmids can express derivatives of the human IκBα protein fused with a variant of Green Fluorescent Protein (GFP) from Aequora Victoria, in the yeast Saccharomyces cerevisiae. Depending on the plasmid construction, the fusion proteins do or do not contain the nuclear localisation sequence from the big-T antigen of the SV40 virus. The introduction of this sequence will cause proteins that contain it to be directed to the nuclear compartment of the cell. A 620 base-pair (bp) fragment corresponding to the GAL1 gene promoter (pGAL1l) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from the genomic DNA of a wild type S. cerevisiae strain, X2180-1A, using oligonucleotides “pGAL1(Asp)Forw”, sequence

• 5′-GCTGGGTACCTTAATAATCATATTACATGGCATTA-3′ [SEQ ID N^o6] and “pGAL1(EcoRI)Rev”, sequence
• 5′-GGCGGAATTCTATAGTTTTTTCTCCTTGACGTTA-3′ [SEQ ID N^o7].

The resulting fragment was digested with restriction enzymes Asp7181 and EcoRI and inserted into the S. cerevisiae-E. coli shuttle plasmid pRS306, previously digested with the enzymes Asp7181 and EcoRI, to produce the vector pRS306-pGAL1.

A 720 base-pair (bp) fragment from vector pUC19-yEGFP3, and corresponding to a variant of the gene coding for the Green Fluorescent Protein (GFP) of Aequora victoria, in which the sequence had been optimised for expression in yeast (yEGFP3), was amplified by Polymerase Chain Reaction (PCR), using the oligonucleotides “GFPEcoR15”, sequence 5′-GGTCGGAATTCATGTCTAAAGGTGAAGAATTATTC-3′ [SEQ ID N^o8] and “PBamHI(SmaI/SrfI PstI)3′”, sequence

5′-GGCGGGATCCGCCCGGGCTCTGCAGTTTGTACAATTCATCCATACC-3′ [SEQ ID N^o9]. The resulting fragment was digested with restriction enzymes BarnHI and EcoRI and inserted into plasmid pRS306-pGAL1, previously digested with the enzymes BamHI and EcoRI, to produce the vector pRS306-pGAL1-yEGFP3.

A 340 base-pair (bp) fragment corresponding to the ADH1 gene terminator signal (tADH1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from the genomic DNA of a wild type S. cerevisiae strain, X2180-1A, using oligonucleotides “TermADH1(NotIBstXI)5′”, sequence

5′-GGCGGCGGCCGCCACCGCGGTGGGCGAATTTCTTATGATTTATG-3′ [SEQ ID N^o10] and “TermADH1(SacI)3′”, sequence

5′-GGCGGAGCTCTGGAAGAACGATTACAACAG-3′ [SEQ ID N^o11].

The resulting fragment was digested with restriction enzymes SacI and NotI and inserted into plasmid pRS306-p Gal1-yEGFP3, previously digested with the enzymes SacI and NotI, to produce the vector pCSY226.

The gene coding for the protein IκBα was purified from the plasmid pGad1318-IkBa by digestion with the restriction enzyme XbaI followed by treatment with Klenow DNA polymerase I in order to remove the overhang and give a blunt 3′ end, and then a second digestion with BamHI for the 5′ end of the gene. The fragment was cloned into plasmid pCSY226, prepared by a KpnI restriction digest, followed by treatment with Klenow fragment and then digestion with restriction enzyme BamHI. The resulting vector has been called pCSY226-IκBα.

A version of this vector also includes the nuclear location sequence NLS. This was obtained by synthesising a pair of oligonucleotides complementary to the sequences “NLS-5′”: 5′ACCTCCAAAAAAGAAGAGAAAGGTCGAATT-3′ [SEQ ID N^o12], and

“NLS-3′”: 5′-AATTCGACCTTTCTCTFCTCTTGGAGGT-3′ [SEQ ID N^o26].

and rehybridising them to form a double-stranded DNA. This DNA fragment was then incorporated into the vector pCSY226-IκBα digested with restriction enzyme ScrFI, to give the vector pCSY226-NLS-IκBα.

Example 2

Construction of Plasmids Able to Express the Fusion Proteins GFP-β-TrCP and GFP-NLS-β-TrCP in Yeast

The following plasmids express derivatives of the human β-TrCP protein fused with a variant of Green Fluorescent Protein (GFP) from Aequora victoria, in the yeast Saccharomyces cerevisiae. Depending on the plasmid construction, the fusion proteins do or do not contain the nuclear localisation sequence from the big-T antigen of the SV40 virus. The introduction of this sequence will cause proteins that contain it to be directed to the nuclear compartment of the cell.

A 620 base-pair (bp) fragment corresponding to the GAL1 gene promoter (pGAL1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from the genomic DNA of a wild type S. cerevisiae strain, X2180-1A, using oligonucleotides “pGAL1(Asp)Forw”, sequence

5′-GCTGGGTACCTTAATAATCATATTACATGGCATTA-3′ [SEQ ID N^o6] and “pGAL1(EcoRI)Rev”, sequence

5′-GGCGGAATTCTATAGTTTTTTCTCCTTGACGTTA-3′ [SEQ ID N^o7].

The resulting fragment was digested with restriction enzymes Asp7181 and EcoRI and inserted into the S. cerevisiae-E. coli shuttle plasmid pRS306, previously digested with the enzymes Asp718I and EcoRI, to produce the vector pRS306-pGAL1.

A 720 base-pair (bp) fragment from vector pUC19-yEGFP3, and corresponding to a variant of the gene coding for the Green Fluorescent Protein (GFP) of Aequora victoria, in which the sequence had been optimised for expression in yeast (yEGFP3), was amplified by Polymerase Chain Reaction (PCR), using the oligonucleotides “GFPEcoR15′”, sequence

5′-GGTCGGAATTCATGTCTAAAGGTGAAGAATTATTC-3′ [SEQ ID N^o8] and “GFPBamHI(SmaI/SrfI PstI)3′”, sequence

5′-GGCGGGATCCGCCCGGGCTCTGCAGTTTGTACAATTCATCCATACC-3′ [SEQ ID N9].

The resulting fragment was digested with restriction enzymes BamHI and EcoRi and inserted into plasmid pRS306-pGALI, previously digested with the enzymes BamHI and EcoRl, to produce the vector pRS306-pGAL1-yEGFP3.

A 340 base-pair (bp) fragment corresponding to the ADHI gene promoter (tADH1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from the genomic DNA of a wild type S. cerevisiae strain, X2180-1A, using oligonucleotides “TermADH1(NotIBstXI)5′”, sequence 5′-GGCGGCGGCCGCCACCGCGGTGGGCGAATTTCTTATGATTTATG-3′ [SEQ ID N^o10] and “TermADH1(SacI)3′”, sequence 5′-GGCGGAGCTCTGGAAGAACGATTACAACAG-3′ [SEQ ID N^o11].

The resulting fragment was digested with restriction enzymes SacI and NotI and inserted into plasmid pRS306-pGAL1-yEGFP3, previously digested with the enzymes SacI and NotI, to produce the vector pCSY226. The gene coding for the βTrCP protein was purified from the plasmid pGad 1318-βTrCP by digestion with the restriction enzymes BamHI and NotI. The fragment was cloned in the plasmid pCSY226 prepared by digestion with the restriction enzymes BamHI and NotI. The resulting vector has been called pCSY226-βTrCP.

A version of this vector also includes the nuclear location sequence NLS. This was obtained by synthesising a pair of oligonucleotides complementary to the sequences “NLS-5′”: 5′-ACCTCCAAAAAAGAAGAGAAAGGTCGAATT-3′ [SEQ ID N^o12], and “NLS-3′”: 5′-AATTCGACCTTTCTCTTCTTlTTTGGAGGT-3′ [SEQ ID N^o26]and rehybridising them to form a double-stranded DNA. This DNA fragment was then incorporated into the vector pCSY226-βTrCP digested with restriction enzyme ScrFI, to give the vector pCSY226-NLS-βTrCP.

Example 3

Construction of Plasmids Able to Express the Fusion Proteins GFP-β-TrCP and GFP-NLS-β-TrCP in Yeast

The following plasmids express, in the yeast Saccharomyces cerevisiae, derivatives of the human β-TrCP protein containing a repetition of three antigenic Flag motifs at their amino-terminal end. The expression of these fusion proteins is induced by growing the yeast cells containing plasmid for 1 to 10 hours in culture medium containing 2 to 5% galactose.

A 700 base-pair (bp) fragment corresponding to the PGK1 gene promoter (pPGK1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from the genomic DNA of a wild type S. cerevisiae strain, X2180-1A, using oligonucleotides “pPGK1-Asp718-5′”, sequence 5′-GGCGGGTACCGTGAGTAAGGAAAGAGTGAGG-3′ [SEQ ID N^o13] and “pPGK-EcoRI-3′”, sequence 5′-GGCGGAATTCTGTTTTATATTTGTTGTAAAAAG-3′ [SEQ ID N^o14].

The resulting fragment was digested with restriction enzymes Asp718I and EcoRI and inserted into the S. cerevisiae-E. coli shuttle plasmid pRS304, previously digested with the enzymes Asp718I and EcoRI, to produce the vector pRS304-pPGK1.

A 100 base-pair (bp) fragment corresponding a string of 3 FLAG reporter sequences (3FLAG) was amplified by Polymerase Chain Reaction (PCR) from the vector p3XFLAG-myc-CMV-24 5Sigma Aldrich, using oligonucleotides “3FLAG-EcoRI-5′”, sequence 5′-GGCGGAATTCATGGACTACAAAGACCATGACGG-3′ [SEQ ID N^o15] and “3FLAGBamHI(SmaI/SrfI PstI)3′”, sequence 5′-GGCGGGATCCGCCCGGGCTCTGCAGCTTGTCATCGTCATCCTTGTA-3′ [SEQ ID N^o16].

The resulting fragment was digested with restriction enzymes BamHI and EcoRI and inserted into plasmid pRS304-pPGK1, previously digested with the enzymes BamHI and EcoRI, to produce the vector pRS304-pPGK1-3FLAG.

A 340 base-pair (bp) fragment corresponding to the ADH1 gene terminator signal (tADH1) of the yeast Saccharornyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from the genomic DNA of a wild type S. cerevisiae strain, X2180-1A, using oligonucleotides “TermADH1 (NotIBstXI)5′”, sequence5′-GGCGGCGGCCGCCACCGCGGTGGGCGAATTTCTTATGATTTATG-3′[SEQ ID N^o10] and “TermADH1(SacI)3′”, sequence 5′-GGCGGAGCTCTGGAAGAACGATTACAACAG-3′ [SEQ ID N^o11].

The resulting fragment was digested with restriction enzymes SacI and NotI and inserted into plasmid pRS304-pPGK1-3FLAG, previously digested with the enzymes SacI and NotI, to produce the vector pCSY614.

The gene coding for the TrCP protein was purified from the plasmid pGad1318-βTrCP by digestion with the restriction enzymes BamHI and NotI. The fragment was cloned in the plasmid pCSY614 prepared by digestion with the restriction enzymes BamHI and NotI. The resulting vector has been called pCSY614-βTrCP.

A version of this vector also includes the nuclear location sequence NLS. This was obtained by synthesising a complementary pair of oligonucleotides, for the sequence 5′ACCTCCAAAAAAGAAGAGAAAGGTCGAATT-3′ [SEQ ID N^o12] and rehybridising them to form a double-stranded DNA. This DNA fragment was then incorporated into the vector pCSY614-βTrCP digested with restriction enzyme ScrFI, to give the vector pCSY614-NLS-βTrCP.

Examples 4 to 12

Development of the Screening Method According to the Invention

Example 4

Interaction Between Yeast Skp1 and Human β-TrCP Proteins in Yeast Cells

The interaction between Skp1 and β-TrCP proteins is visualised using the two-hybrid method Bartel et al. (in Cellular Interactions in Development: a practical approach (1991), Oxford University Press, Oxford, pp153-179). Yeast cells are simultaneously transformed with the pGAD-βTrCP plasmid which expresses the human β-TrCP protein fused with the activator domain Gal4, with the plasmid pLexSkp1-1 which expresses the yeast protein Skp1 fused to the DNA-binding domain of the bacterial protein LexA, and with the plasmid pSH18-34 which includes the LacZ reporter gene coding for β-galactosidase, under the control of LexA operators. Measurement of β-galactosidase activity in cellular extracts from such cells shows that expression of this reporter gene increases by a factor of 15 when compared to its expression in cells expressing only one of the two fusion proteins described herein. This induction of reporter gene expression indicates that the Skp1 protein from Saccharomyces cerevisiae is capable of interacting with the human β-TrCP protein. β-galactosidase activity is expressed in nmoles of substrate transformed per minute per mg of protein (nmole/min/mg).

Example 5

Localisation in Yeast Cells of Human Proteins IκBα and β-TrCP According to Whether or not They are Fused to an NLS Sequence of SV40

The yeast cells containing the plasmids able to express the hybrid proteins, either GPF-IκBα, GFP-NLS-IκBα, GFP-β-TrCP, or GFP-NLS-β-TrCP under the GAL1 promoter, are grown in the presence of 2% galactose for 2 hours and then observed with a fluorescence microscope. The position of the nucleus is revealed using a nuclear-specific dye, Hoescht 333-42.

Example 6

Phosphorylation of the IκBα Protein in Yeast Cell Nuclei

Example 6 shows how presence of the human IκBα protein in the nuclei of yeast cells leads to its phosphorylation at serines 32 and 36.

Cells expressing either the fusion protein GFP-IκBα or tripartite fusion protein GFP-NLS-IκBα under the GAL1 promoter, are grown in Minimum Essential Medium in the presence of 2% galactose for 2 hours. The proteins from these cells are then extracted according to the protocol described by Kuras et al. (Mol. Cell (2002), 10:69-80). The proteins are then analysed by Western blotting firstly using a specific antibody to the GFP protein (called “GFP-IκBα”) and secondly an antibody which specifically recognises human IκBα protein phosphorylated at serine 32 (called “P-IκBα”). As a control for the total amount of protein loaded in each well, the same proteins are analysed with an antibody specific for yeast Lysy1-tRNA-synthase (called “LysRS”). The proteins made by the parental strain of yeast which does not express any fusion protein (called “control”) serve as a test for specificity.

Example 7

Degradation of the GFP-NLS-IκBα Protein

Example 7 shows, by epifluorescent microscopy, the degradation of the tripartite fusion protein GFP-NLS-IκBα in the yeast cells which, at the same time, express the tripartite fusion protein Flag-NLS-β-TrCP.

All the strains used are grown and analysed by fluorescence microscopy in an identical manner. The cells are grown for 120 minutes in galactose-rich medium as the source of carbon. At time t=0, 2% glucose is added to the culture and the cells are observed by epifluorescent microscope (Nikon Eclipse fluorescent microscope equipped with an Omega XF116 filter). All the images were recorded using a Hamamastu® camera identically adjusted and analysed with LUCIA G software, just before (t=0) and 10, 20, 30 and 60 minutes after addition of the glucose. The fluorescence of the fusion proteins GFP-IκBα or GFP-NLS-IκBα is called “GFP”. The position of the nucleus (called “DNA”) in the cells was revealed using a nuclear-specific dye, Hoescht 333-42.

A) yeast strain CYS22 (MATa, his3, leu2, trp1, ura3::pGAL1-GFP-IκBα::URA3) expressing the fusion protein GFP-IκBα without NLS and localised in the cytoplasm of yeast cells;

B) yeast strain CYS61 (MATa, his3, leu2, ura3::pGAL1-GFP-IκBα::URA3, trp1:.pGAL1-3Flag-βTrCP::TRP1) expressing the fusion proteins GFP-IκBα and Flag-β-TrCP, localised in the cytoplasm of yeast cells;

C) yeast strain CYS126 (MATa, his3, leu2, trp1, ura3::pGAL1-GFP-NLS-IκBα::URA3) expressing the fusion protein GFP-NLS-IκBα localised in the nucleus of yeast cells;

D) yeast strain CYS135 (MATa, his3, leu2, ura3::pGAL1-GFP-NLS-IκBα::URA3, trp1::pGAL1-3Flag-NLS-βTrCP::TRP1) expressing the fusion proteins GFP-NLS-IκBα and Flag-NLS-β-TrCP, localised in the nucleus of yeast cells.

Example 8

Degradation of GFP-NLS-IκBα With or Without Co-Expression of Flag-NLS-β-TrCP (Results from Fluorescence)

Example 8 shows, by measurement of the fluorescence produced, the degradation of the tripartite fusion protein GFP-NLS-IκBα in the yeast cells which, at the same time, do or do not express the tripartite fusion protein Flag-NLS-β-TrCP.

Strains of yeast identical to those described in FIG. 4, and grown under the same conditions as described in FIG. 4, were analysed by fluorescence microscopy. For each strain, the fluorescence of 200 cells (at least) was measured just before (t=0) and 10, 20, 30 and 60 minutes after the addition of glucose, using the LUCIA G software. The results are given, in arbitrary units, as the amount of fluorescence measured per cell.

Example 9

Degradation of GFP-NLS-IκBα with or Without Co-Expression of Flag-NLS-β-TrCP (Results from Immunoblotting)

Example 9 shows, by Western Blot type biochemical analysis, the degradation of the tripartite fusion protein GFP-NLS-IκBα in yeast cells which, at the same time, express the tripartite fusion protein Flag-NLS-β-TrCP. All the strains used were grown and analysed in an identical manner. The cells were grown for 120 minutes in galactose-rich medium as the source of carbon. At time t=0, 2% glucose is added to the culture and the total protein is extracted just before (t=0) and 10, 20, 30 and 60 minutes after the addition of glucose. These proteins are analysed by Western blotting using an antibody to the GFP part of the fusion proteins including IκBα (called “GFP-NLS-IκBα”) and an antibody to the Flag part of the fusion protein Flag-NLS-β-TrCP (called “Flag-NLS-β-TrCP”). As a control for the total amount of protein loaded in each well, the same proteins are analysed with an antibody specific for yeast Lysyl-tRNA-synthase (called “LysRS”). The proteins made by the parental strain of yeast which does not express any fusion protein (called “control”) serve as a test for specificity.

A) yeast strain CYS22 (MATα, his3, leu2, trp1, ura3::pGAL1-GFP-IκBα::URA3) expressing the fusion protein GFP-IκBα without NLS and localised in the cytoplasm of yeast cells;

B) yeast strain CYS61 (MATa, his3, leu2, ura3::pGAL1-GFP-IκBα::URA3, trp1::pGAL1-3Flag-βTrCP::TRP1) expressing the fusion proteins GFP-IκBα and Flag-β-TrCP, localised in the cytoplasm of yeast cells;

C) yeast strain CYS126 (MATa, his3, leu2, trp1, ura3::pGAL1-GFP-NLS-IκBα::URA3) expressing the fusion protein GFP-NLS-IκBα localised in the nucleus of yeast cells;

D) yeast strain CYS135 (MATa, his3, leu2, ura3::pGAL1-GFP-NLS-IκBα::URA3, trp1::pGAL1-3Flag-NLS-βTrCP::TRP1) expressing the fusion proteins GFP-NLS-IκBα and Flag-NLS-β-TrCP, localised in the nucleus of yeast cells.

Example 10

Degradation of GFP-NLS-IκBα Mutated at Serine Residues 32 and 36, With or Without Co-Expression of Flag-NLS-D-TrCP (Results From Immunoblotting)

Example 10 shows, by Western Blot type biochemical analysis, the degradation of the mutated tripartite fusion protein GFP-NLS-Iκα[S3236A] in which the phosphorylation sites Ser32 and Ser36 have been replaced by Ala residues, mutations that, in human cells, make the protein non-degradable. Analysis was carried out also in yeast cells either expressing or not expressing the tripartite fusion protein Flag-NLS-β-TrCP. All the strains used were grown and analysed in an identical manner. The cells were grown for 120 minutes in galactose-rich medium as the source of carbon. At time t=0, 2% glucose is added to the culture and the total protein extracted just before (t=0) and 10, 20, 30 and 60 minutes after the addition of glucose. These proteins are analysed by Western blotting using an antibody to the GFP part of the fusion proteins including IκBα[S3236A] (called “GFP-NLS-IκBα[S3236A]”) and an antibody to the Flag part of the fusion protein Flag-NLS-β-TrCP (called “Flag-NLS-β-TrCP”). As a control for the total amount of protein loaded in each well, the same proteins are analysed with an antibody specific for yeast Lysyl-tRNA-synthase (called “LysRS”). The proteins made by the parental strain of yeast which does not express any fusion protein (called “control”) serve as a test for specificity.

A) yeast strain CYS138 (MATα, his3, leu2, trp1, ura3::pGAL1-GFP-NLS-IκBα[S3236A]::URA3) expressing the mutated fusion protein GFP-NLS-IκBα[S3236A] localised in the nucleus of yeast cells;

B) yeast strain CYS139 (MATα, his3, leu2, ura3:.pGAL1-GFP-NLS-IκBα[S3236A]::URA3, trp1::pGAL1-3Flag-NLS-βTrCP::TRP1) expressing the fusion proteins GFP-NLS-IκBα[S3236A] and Flag-NLS-β-TrCP, localised in the nucleus of yeast cells.

Example 1

Degradation of GFP-NLS-IκBα with or Without Co-Expression of Flag-NLS-β-TrCP (Results from Fluorescence)

Example 11 shows, by epifluorescent microscopy, the degradation of the tripartite fusion protein GFP-NLS-IκBα[S3236A] in the yeast cells which, at the same time, express the tripartite fusion protein Flag-NLS-β-TrCP. The 2 strains used (CYS138 and CYS139) were grown, and analysed by fluorescence microscopy, in an identical manner. The cells are observed by epifluorescent microscopy (Nikon Eclipse fluorescent microscope equipped with an Omega XF116 filter). All the images were recorded using a Hamamastu® camera identically adjusted and analysed with LUCIA G software, just before (t=0) and 10, 20, 30 and 60 minutes after addition of the glucose. The fluorescence of the fusion proteins GFP-IκBα or GFP-NLS-IκBα is called “GFP”. The position of the nucleus (called “DNA”) in the cells is revealed using a nuclear-specific dye, Hoescht 333-42.

Example 12

Degradation of GFP-NLS-IκBα with or Without Co-Expression of Flag-NLS-β-TrCP (Results from Fluorescence)

Example 12 shows, by measurement of the fluorescence emitted, the degradation of the tripartite fusion protein GFP-NLS-IκKα[S3236A] in the strains of yeast described herein. For each strain, the fluorescence of 200 cells (at least) was measured just before (t=0) and 10, 20, 30 and 60 minutes after the addition of glucose, using the LUCIA G software. The results are given, in arbitrary units, as the amount of fluorescence measured per cell.

TABLE 1
Genotype of the strains of yeast Saccharomyces cerevisiae prepared for
use in the present invention.
Strain   Genotype
CC788-2B MATa, his3, leu2, ura3, trp1.
CYS22    MATa, his3, leu2, trp1, ura3::pGAL1-GFP-IκBα::URA3
CYS61    MATa, his3, leu2, ura3::pGAL1-GFP-IκBα::URA3,
         trp1::pGAL1-3Flag-βTrCP::TRP1
CYS122   MATa, his3, leu2, trp1, ura3::pGAL1-GFP-βTrCP::URA3
CYS123   MATa, his3, leu2, trp1, ura3::pGAL1-GFP-
         NLS-βTrCP::URA3
CYS126   MATa, his3, leu2, trp1, ura3::pGAL1-GFP-NLS-IκBα::URA3
CYS135   MATa, his3, leu2, ura3::pGAL1-GFP-NLS-IκBα::URA3,
         trp1::pGAL1-3Flag-NLS-βTrCP::TRP1
CYS138   MATa, his3, leu2, trp1, ura3::pGAL1-GFP-NLS-
         IκBα[S3236A]::URA3
CYS139   MATa, his3, leu2, ura3::pGAL1-GFP-NLS-IκBα[S3236A]
         ::URA3, trp1::pGAL1-3Flag-NLS-βTrCP::TRP1

TABLE 2
(SEQUENCES)
SEQ ID No	Type	Description
1	DNA	GFP-NLS-IkBα
2	Protein	GFP-NLS-IkBα
3	DNA	GFP-NLS-βTrCP
4	Protein	GFP-NLS-βTrCP
5	DNA	NLS sequence of the SV40
		big-T antigen
6-16	DNA	Primers
17	Protein	HA antigen
18	Protein	FLAG monomer
19	Protein	FLAG trimer
20	Protein	Nucleoplasmin NLS
21	Protein	NLS repressor alpha 2 (1)
22	Protein	NLS repressor alpha 2 (2)
23	Protein	Gal4 NLS
24	DNA	SV40 T-Ag NLS
25	DNA	Primer

TABLE 3

List of GFPs usable according to the invention

λ

λ

Residues

excitation

emission

26

46

64

65

66

67

68

69

70

72

80

145

146

153

163

164

167

168

175

203

212

(nm)

(nm)

Références

wtGFP

Lys

Phe

Phe

Ser

Tyr

Gly

Val

Gln

Cys

Ser

Gln

Tyr

Asn

Met

Val

Asn

Ile

Ile

Ser

Thr

Asn

395-470

509-540

Heim et al., 1994

BFP

Leu

His

Ala

387

450

Quantum

CFP

Leu

Trp

Ile

Thr

Ala

His

436

480

(YRC)

EBFP

Lys

Thr

His

Phe

380

440

Yang et al., 1996

(Clontech)

Cormack et al., 1996

ECFP

Leu

Thr

Ile

Thr

Ala

475

501

Heim et al., 1994-1996

(Clontech)

ECFP

Arg

Lys

Thr

Trp

Ile

Thr

Ala

His

Lys

434

474

Miyawaki et al.,

1997

EGFP =

Leu

Thr

488

508

Yang et al., 1998

GFPmut1

(Clontech)

Cormack et al., 1996

EYFP

Gly

Leu

Ala

Tyr

514

527

Ormö et al., 1997

(Clontech)

GFP405

405

510

Clontech

“SuperBright”

GFPmut3

Gly

Ala

501

511

Cormack et al., 1996

GFPuv

395

408

Crameri et al., 1996

mCFP

Trp

Ala

Gly

440

485

Haseloff et al., 1999

mGFP5

Ala

Thr

Gly

400-475

508

Haseloff et al., 1997

Siemering et al., 1996

mYFP

Gly

Ala

Ala

Tyr

Gly

Tyr

514

527

Haseloff et al., 1999

PA-GFP

Ala

His

413-488

520

Patterson et al., 2002

rsGFP

Leu

Cys

Thr

473

509

Quantum

RsGFP

Gly

Ala

Trp

505

522

Reed et al., 2001

S65T

Thr

488

507

Heim et al., 1995

T-Sapphire

Met

Val

Ala

Gly

Ile

399

511

O. Zapata-Hommer

and O. Griesbeck,

2003

yEGFP3

Gly

Ala

501

511

Cormack et al., 1997

(Cormack)

YFP

Gly

Ala

Tyr

500

535

Ormö et al., 1996

(YRC)

YFP-

Gly

Leu

Met

Ala

Tyr

490-510

515

Griesbeck et al., 2001

citrine

YFP-Venus

Leu

Leu

Gly

Leu

Ala

Thr

Ala

Gly

Tyr

488

514-527

Nagai et al., 2002

(YRC)

REFERENCES

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Rothstein, in Methods in Enzymnology, (1991), 194:281-301

Sambrook et al. (in Molecular Cloning, Laboratory Manual, 2^nd edition, (1989), Cold Spring Harbor, N.Y.)

Schiestl et al. (Curr. Genet. (1989), 16:339-346

Sherman et al., in Methods in Yeast Genetics: a Laboratory Manual, (1979), Cold Spring Harbor, N.Y.

Sikorski and Hieter (Genetics (1989) 122(1): 19-27).

Strausberg et al. (PNAS (1999), 99(26): 16899-16903

Thomas et al. (J. Biol. Chem. (2000) 275(52): 40718-40724).

Vidal et al., Trends Biotechnol., (1999), 17:374-381

Winston et al., Genes Dev. (1999), 13:270-283

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Yaron et al. (Nature (1998) 396(6711): 590-594).

Claims

1. A method for screening agents modulating IκBα protein ubiquitination by a functional ubiquitin ligase protein complex containing β-TrCP protein, said method comprising the following steps: (a) bringing into contact a candidate agent to be tested with recombinant yeast cells that express in their nucleus:(i) a fusion protein containing the polypeptide IκBα and at least one first detectable protein; and(ii) a protein containing the polypeptide β-TrCP;(b) quantifying said first detectable protein in the yeast cells, at the end of at least one predetermined period of time after bringing the candidate agent into contact with said cells;(c) comparing the result obtained in step (b) with a control result obtained when step (a) is carried out in the absence of the candidate agent.

2. A method according to claim 1, characterised in that step (a) includes the following steps: (a1) growing yeast cells which express in their nucleus a fusion protein containing the polypeptide IκBα and at least one first detectable protein;(a2) stopping the expression of said fusion protein containing the polypeptide IκBα and at least one first protein detectable by the yeast cells;(a3) bringing the yeast cells obtained at the end of step (a2) into contact with the candidate agent to be tested.

3. A method according to claim 2, characterised in that the yeast cells express the protein containing the polypeptide β-TrCP throughout all the steps (a1), (a2) and (a3).

4. A method according to claim 2, characterised in that the yeast cells express the protein containing the polypeptide β-TrCP throughout all the steps (a2) and (a3) and do not express the protein containing the polypeptide β-TrCP in step (a1).

5. A method according to claim 2, characterised in that the yeast cells express the protein containing the polypeptide β-TrCP throughout all the steps (a2) and (a3), and (i) do not express the protein containing the polypeptide β-TrCP for a predetermined time at the start of step (a1);(ii) do express the protein containing the polypeptide β-TrCP for the remainder of step (a1).

6. A method according to claim 1, characterised in that the detectable protein in the polypeptide containing IκBα polypeptide is chosen from an antigen, a fluorescent protein and a protein having enzymatic activity.

7. A method according to claim 6 characterised in that the detectable protein is a fluorescent protein selected from the GFP protein or one of its derivatives, the YFP protein or one of its derivatives, and the dsRED protein.

8. A method according to claim 6, characterised in that the detectable protein is a protein having enzymatic activity selected from luciferase and β-lactamase.

9. A method according to claim 6, characterised in that the detectable protein is an antigen selected from the Ha peptide and the Flag peptide.

10. A method according to claim 1, characterised in that the protein containing the polypeptide β-TrCP a fusion protein also containing a second detectable protein.

11. A method according to claim 10, characterised in that the second detectable protein included in the fusion protein containing the β-TrCP polypeptide is chosen from an antigen, a fluorescent protein and a protein having enzymatic activity.

12. A method according to claim 10, characterised in that (i) the first detectable protein included in the fusion protein containing the polypeptide IκBα and (ii) the second detectable protein included in the fusion protein containing the polypeptide β-TrCP are different from each other.

13. A method according to claim 1, characterised in that the protein containing the polypeptide IκBα also contains a nuclear localisation peptide.

14. A method according to claim 1, characterised in that the protein containing the polypeptide β-TrCP also contains a nuclear localisation peptide.

15. A method according to claim 1, characterised in that the protein containing the polypeptide IκBα is the protein with the sequence SEQ ID No2.

16. A method according to claim 1, characterised in that the protein containing the polypeptide β-TrCP is the protein with the sequence SEQ ID 4.

17. A method according to claim 1, characterised in that at step (b), when the first detectable protein is an antigen, said first detectable protein is quantified by detecting the complexes formed between said protein and the antibodies which recognise it.

18. A method according to claim 1, characterised in that at step (b), when the first detectable protein is a fluorescent protein, said detectable protein is quantified by measuring the fluorescent signal emitted by said protein.

19. A method according to claim 1, characterised in that at step (b), when the first detectable protein is a protein having enzymatic activity, said detectable protein is quantified by measuring the quantity of substrate modified by said protein.

20. A method according to claim 1, characterised in that the recombinant yeast cells are transformed with: respectively: (1) a first polynucleotide that contains (a) an open reading frame coding for (i) the fusion protein containing, the IκBα polypeptide and (iii) a first detectable protein, and a regulatory sequence functional in yeast cells which controls expression of said open reading frame; and(2) a second polynucleotide that contains (a) an open reading frame coding for (i) the protein containing the β-TrCP polypeptide, ii) a nuclear localisation sequence and (iii) a regulatory sequence functional in yeast cells which controls expression of said open reading frame;

21. A method according to claim 20, characterised in that the regulatory sequence contained in the first polynucleotide, the regulatory sequence contained in the second polynucleotide, or both regulatory sequences, contain a promoter functional in yeast cells and sensitive to the action of an inducing agent.

22. A method according to claim 21 characterised in that the inducible promoter functional in yeast cells is chosen from PGK1, ADH1, TDH3, LEU2 and TEF1.

23. A method according to claim 21 characterised in that the inducible promoter functional in yeast cells is chosen from CUP1, GAL1, MET3, MET25, MET28, SAM4 and PHO5.

24. A method according to claim 20, characterised in that the first polynucleotide contains the regulatory sequence GAL1, which activates the expression of the open reading frame coding for the fusion protein containing the polypeptide the IκBα polypeptide in the presence of glucose.

25. A method according to claim 20, characterised in that the second polynucleotide contains the regulatory sequence CUP1, which activates the expression of the open reading frame coding for a protein containing the polypeptide β-TrCP in the presence of copper sulphate.

26. A method according to claim 20, characterised in in that the recombinant yeast cells have the first and second polynucleotide in a form integrated into their genome.

27. A method according to claim 1, characterised in that the recombinant yeast cells have in their genome an inactivated form of one or several genes which controls the expression of transporter proteins in the plasma membrane.

28. A method according to claim 27, characterised in that the inactivated genes are chosen from genes PDR1 and PDR3.

29. An expression cassette functional in yeast cells containing a coding polynucleotide which includes an open reading frame encoding the fusion protein which contains the polypeptide the IκBα polypeptide and at least one first detectable protein, and a regulatory sequence functional in yeast cells which controls the expression of said open reading frame.

30. An expression cassette functional in yeast cells including a polynucleotide which contains an open reading frame encoding a protein containing the β-TrCP polypeptide and a regulatory sequence functional in yeast cells which controls the expression of said open reading frame.

31. An expression cassette according claim 29, characterised in that the regulatory sequence contained in said polynucleotide, the regulatory sequence contained in the second polynucleotide, or both regulatory sequences, contain a promoter functional in yeast cells and sensitive to the action of an inducing agent.

32. An expression cassette according to claim 31 characterised in that the inducible promoter functional in yeast cells is chosen from PGK1, TEF1, PHO5, MET3, MET28, CUP1, GAL1 and SAM4.

33. An expression vector characterised in that it contains an expression cassette according to claim 29.

34. An expression vector according to claim 33, characterised in that it is the vector pCSY226-NLS-IκBα.

35. An expression vector according to claim 33, characterised in that it is the vector pCSY226-NLS-β-TrCP.

36. A recombinant yeast strain containing, in a form integrated into its genome, (i) a first polynucleotide that contains an open reading frame coding for the fusion protein containing the polypeptide the IκBα polypeptide, and at least one first detectable protein, and a regulatory sequence functional in yeast cells which controls expression of said open reading frame; and (ii) a second polynucleotide that contains an open reading frame coding for a protein containing the β-TrCP polypeptide and a regulatory sequence functional in yeast cells which controls expression of said open reading frame.

37. A recombinant yeast strain according to claim 36, characterised in that it consists of the yeast strain CYS135 deposited in the Collection Nationale de Cultures of microorganismes at the Institut Pasteur de Paris (CNCM) under accession number I-3187.

38. The tools or kit for screening agents modulating the ubiquitination of the IκBα protein by a functional ubiquitin ligase protein complex containing the β-TrCP protein, characterised in that it contains (i) a first expression vector containing an expression cassette according to claim 29; and(ii) a second expression vector containing an expression cassette functional in yeast cells including a polynucleotide which contains an open reading frame encoding a protein containing the β-TrCP polypeptide and a regulatory sequence functional in yeast cells which controls the expression of said open reading frame.

39. The tools or kit for screening agents modulating the ubiquitination of the IκBα protein by a functional ubiquitin ligase protein complex containing the β-TrCP protein, characterised in that it includes recombinant yeast cells containing, in a form integrated into their genome, respectively (i) an expression cassette according to claim 29; and(ii) an expression cassette functional in yeast cells including a polynucleotide which contains an open reading frame encoding a protein containing the β-TrCP polypeptide and a regulatory sequence functional in yeast cells which controls the expression of said open reading frame.

40. The tools or kit according to claim 39, characterised in that it contains recombinant yeast cells of the strain CYs135 deposited at the CNCM under accession number I-3187. _