WO2005093086A1 - PROCEDE DE CRIBLAGE D'AGENTS MODULANT L'UBIQUITINATION DE LA PROTEINE IkBα ET MOYENS DESTINES A LA MISE EN OEUVRE DUDIT PROCEDE - Google Patents
PROCEDE DE CRIBLAGE D'AGENTS MODULANT L'UBIQUITINATION DE LA PROTEINE IkBα ET MOYENS DESTINES A LA MISE EN OEUVRE DUDIT PROCEDE Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- the present invention relates to the field of screening for biologically active agents capable of modulating the ubiquitination of the IKBCC protein, in particular agents of therapeutic interest, and even more specifically therapeutic agents intended for preventing or treating conditions. inflammatory, autoimmune conditions or cancer.
- the transcription factors of the NF- ⁇ B family are part of the body's first defenses during viral, bacterial or fungal infections and also during physiological situations of stress. These transcriptional factors direct the expression of a large number of genes, and in particular many genes coding for mediators of inflammation. Among these genes, mention may be made of the genes encoding factor TNF- ⁇ , the interleukins IL-1, IL-6, IL-8, the molecules of adhesion ICAM-1, VCAM-1 and E-Selectin, NO synthase or even prostaglandin synthase Cox2.
- Factors in the NF- ⁇ B family are activated by a wide variety of pathogenic stimuli, both endogenous and exogenous, including bacterial proteins or lipids, cytokines, growth factors and molecules linked to stressful situations oxidative.
- pathogenic stimuli both endogenous and exogenous, including bacterial proteins or lipids, cytokines, growth factors and molecules linked to stressful situations oxidative.
- the activation of NF- ⁇ B factors, in response to these pathogenic stimuli, is observed for almost all cells involved in the immune response, such as epithelial cells, mesenchyme cells, lymphocytes, neutrophil cells and macrophages .
- factor NF- ⁇ B is closely linked to the destruction of factor l ⁇ B ⁇ by the ubiquitin proteasome pathway (Kroll et al, 1999; Winston et al, 1999).
- the factor NF- ⁇ B is sequestered in the cytoplasm of the cells.
- the factor NF- k B is therefore incapable of activating the expression of the target genes for this factor.
- the activation of target genes first requires the translocation of the NF k B factor of the cytoplasm towards the nucleus. This translocation is triggered by the degradation of factor l ⁇ B ⁇ by the ubiquitin proteasome pathway.
- Factor l ⁇ B ⁇ is indeed a protein which sequesters NF- ⁇ B factors in the cytoplasm of unstimulated cells (Hay et al., 1999).
- Exogenous inflammatory stimuli such as a viral or bacterial infection, activate a signaling pathway that causes phosphorylation of the IKBOC factor.
- This phosphorylation takes place specifically on the Serine residues at positions 32 and 36 of the amino acid sequence of factor l ⁇ B ⁇ .
- the factor l ⁇ B ⁇ is phosphorylated by the protein kinase complex l ⁇ .
- the factor l ⁇ B ⁇ is recognized by the ubiquitin ligase SCF ⁇ TrCp (Kroll et al, 1999; Winston et al, 1999).
- the recognition of the factor IKBCC by the ubiquitin ligase SCF ⁇ TrCp causes the poly-ubiquitination of this factor.
- the factor l ⁇ B ⁇ is then recognized and degraded by the proteasome.
- the destruction of factor l ⁇ B ⁇ causes the release of the cytoplasmic factor NF- ⁇ B.
- the factor NF- ⁇ B undergoes a translocation from the cytoplasm to the nucleus. Once localized in the nucleus of stimulated cells, the NF- ⁇ B factor specifically recognizes the promoters of target genes and strongly activates their transcription: the inflammatory response is installed (Ben Neriah, 2002).
- anti-inflammatory compounds which are both more effective and more specific than known anti-inflammatory compounds.
- anti-inflammatory compounds because of their specificity vis-à-vis a biological target, would be likely to have reduced undesirable side effects, or even to be free of any undesirable side effect.
- a method for screening agents of therapeutic interest, which are selected for their specificity of action on the ubiquitination of the human protein l ⁇ B by an ubiquitin ligase complex comprising the human protein ⁇ -TrCP.
- the applicant has shown that, surprisingly, it is possible to mimic, in yeast cells, the process of degradation of factor h B by the proteasome, a process which takes place naturally in human cells.
- an artificial ubiquitin ligase complex comprising yeast proteins with which the protein is associated has been reconstituted in yeast cells.
- human ⁇ -TrCP it has been shown that the human protein ⁇ -TrCP, when it is artificially expressed in yeast cells, associates with the yeast protein Skp1, which yeast protein Skp1 is contained in a protein complex ubiquitin ligase yeast.
- the ⁇ -TrCP protein associates with the yeast SCF protein complex which comprises (i) a catalytic core consisting of association of the proteins Skp1, Cdc53 and Hrt1, said catalytic heart being itself associated with the enzyme E2 Cdc34. It has been shown that this hybrid yeast / human protein complex is capable of mimicking, in yeast cells, the ubiquitin ligase activity exerted in human cells by the natural human SCF ⁇ ⁇ TrCp complex.
- this artificial protein complex having the ubiquitin ligase activity of the human SCF ⁇ 'TrC complex is only biologically active when this artificial complex is located in the cell nucleus.
- the natural SCF ⁇ TrCp complex exerts its biological activity in the cytoplasm of human cells, a cellular compartment in which it performs the ubiquitination of a second protein also localized in the cytoplasm, the factor IKBOC.
- the artificial ubiquitin ligase complex which has been developed is only active, in the process of degradation of factor l ⁇ B, when the factor IKBOC is co-located in the nucleus with said artificial complex ubiquitin ligase.
- the artificial protein complex ubiquitin ligase comprising the protein human ⁇ -TrCP is capable of achieving the ubiquitination of human factor l ⁇ B ⁇ , when the protein ⁇ -TrCP and factor IKBOC are artificially expressed in the cell nucleus.
- the subject of the invention is a method for the in vitro screening of agents modulating the ubiquitination of the l ⁇ B ⁇ protein by a functional protein complex ubiquitin ligase comprising the ⁇ -TrCP protein, said method comprising the following steps: (a) contacting a candidate agent to be tested with recombinant yeast cells which express in their nucleus: (i) a fusion protein comprising the polypeptide It B ⁇ and at least one first detectable protein; and (ii) a protein comprising the ⁇ -TrCP polypeptide; (b) quantifying said first detectable protein in yeast cells, at the end of at least a predetermined period of time after contacting the candidate agent with said cells; (c) compare the value obtained in step (b) with a control value obtained when step (a) is carried out in the absence of the candidate agent.
- the above method allows a person skilled in the art to determine whether an agent to be tested is capable of modifying the rate of degradation, or the degree of degradation, of the factor IKBOC by the proteasome, in yeast cells expressing both human ⁇ -TrCP protein and factor IkBoc.
- the above in vitro screening method because it implements a humanized artificial ubiquitination system in yeast cells, allows screening of agents which act specifically on the activity of human proteins only expressed in these cells.
- agents capable of inhibiting the rate or degree of degradation of factor IKBOC by the proteasome of yeast cells can be identified.
- Such inhibitory agents, identified by the method of the invention because they will also inhibit the degradation of factor IKBOC in human cells, constitute agents of therapeutic interest capable of inhibiting or blocking the translocation of the factor NF- ⁇ B in the cell nucleus and, in Consequently, inhibit or block the activation, by NF- ⁇ B, of various genes involved in inflammation, pathologies of autoimmunity or even cancers.
- the above in vitro screening method can comprise an additional step (d) which consists in positively selecting the inhibiting candidate agents for which the amount of detectable protein measured in step (b) is less than the control value of comparison.
- the method of the invention also makes it possible to identify agents capable of increasing the speed or the degree of degradation of the factor IKBOC by the proteasome of yeast cells.
- Such activating agents are capable of inducing or increasing the translocation of factor NF- ⁇ B in the cell nucleus and, consequently, of inducing or increasing the activation, by NF- ⁇ B, of various genes involved in inflammation, pathologies of autoimmunity or cancers.
- the in vitro screening method of the invention makes it possible to screen for pro-inflammatory agents.
- Some of the pro-inflammatory agents selected according to the method are likely to be of therapeutic interest when they are used in low doses or when they are administered for a short period, for example as agents inducing an early immune response.
- pro-inflammatory agents selected according to the in vitro screening method of the invention may consist of known active ingredients, in particular active drug ingredients, including a pro-inflammatory effect. undesirable is identified, and for which special precautions for use with regard to human health must be observed.
- the screening method according to the invention can comprise an additional step (d) which consists in positively selecting the activating candidate agents, for which the quantity of detectable protein measured in step (b) is greater to the comparison control value.
- an agent which “modulates” the ubiquitination of the ⁇ -TrCP protein consists (i) of an agent which increases or on the contrary consists (ii) of an agent which inhibits or blocks the degradation of the ⁇ -TrCP protein which is detected in step (b) of the screening method of the invention, compared with a control situation of degradation of this protein, when the method is carried out in the absence of the agent tested.
- the agent modulating the ubiquitination of the ⁇ -TrCP protein can be of any kind.
- Said agent can be any organic or mineral compound, and can be either an agent of natural origin, or an agent produced, at least in part, by chemical or biological synthesis.
- Said agent can in particular be a peptide or protein.
- Said agent also includes any molecule already known to have a biological effect, in particular a therapeutic effect, or conversely a toxic effect demonstrated or suspected for the organism.
- the fusion protein IKBOC-detectable protein is ubiquitinylated by the artificial SCF complex comprising the ⁇ -TrCP polypeptide
- said fusion protein undergoes proteolysis which is carried out by the multi-catalytic complex of proteasome.
- the quantification of the detectable protein contained in the yeast cell, at a given time makes it possible to determine the degree of degradation of said l ⁇ B ⁇ -detectable protein fusion, at this given time.
- the sensitivity of the screening method described above is increased when, prior to bringing the yeast cells into contact with the agent to be tested, the accumulation of the target fusion protein is promoted.
- l ⁇ B ⁇ -protein detectable in the cell nucleus is promoted.
- step (a) itself comprises the following steps:
- step (ai) culturing yeast cells which express in their nucleus a fusion protein comprising the IKBOC polypeptide and at least one first detectable protein; (a2) stopping the expression of said fusion protein comprising the polypeptide I KBOC and at least one first protein detectable by yeast cells; (a3) bringing the yeast cells obtained at the end of step (a2) into contact with the candidate agent to be tested.
- Stopping the expression of the l ⁇ B ⁇ -detectable protein fusion, at a chosen time, can be easily carried out by a person skilled in the art, by using, to transform the yeast cells, an expression cassette in which the polynucleotide encoding said fusion protein is placed under the control of a functional promoter in yeast cells and whose activation, or conversely repression, is induced by an inducing agent.
- a functional promoter in yeast cells whose activation, or conversely repression, is induced by an inducing agent.
- Many inducible promoters active in yeast cells are known to those skilled in the art, some of which are described later in the description, including in the examples.
- step (ai) of the process allows a strong detection signal of the detectable protein to be obtained, at the start of the process. .
- These strong signal conditions make it possible to quantify with great sensitivity the detectable protein throughout the duration of the process, as the fusion protein ItcBoc-detectable protein by the proteasome degrades, after it has been ubiquitinylated. by the artificial SCF complex comprising the protein ⁇ -TrCP.
- the stronger the detectable starting signal the better the sensitivity of the measurements during the implementation of the method.
- the yeast cells express the protein comprising the ⁇ -TrCP polypeptide during all of the steps (ai), (a2) and (a3).
- the yeast cells express the protein comprising the ⁇ -TrCP polypeptide during all of the steps (a2) and (a3) and do not express the protein comprising the ⁇ -TrCP polypeptide during step (ai).
- the control of the expression of the protein comprising the ⁇ -TrCP polypeptide is easily achieved, using, to transform the yeast cells, an expression cassette in which the polynucleotide encoding the protein comprising the ⁇ polypeptide -TrCP is placed under the control of a functional promoter in yeast cells and whose activation, or conversely repression, is induced by an inducing agent.
- a functional promoter in yeast cells whose activation, or conversely repression, is induced by an inducing agent.
- Many inducible promoters active in yeast cells are known to those skilled in the art, some of which are described later in the description, including the examples.
- the inducible promoter included in the expression cassette coding for the protein comprising the ⁇ -TrCP polypeptide is distinct from the inducible promoter included in the expression cassette coding for the fusion protein I KBOC-detectable protein.
- a separate control is carried out respectively (i) of the expression of the l ⁇ Boc-detectable protein fusion protein and (ii) of the expression of the protein comprising the ⁇ -TrCP polypeptide.
- the It B ⁇ -detectable protein fusion protein accumulates in the nucleus of yeast cells during step (ai), in the absence of the ⁇ -TrCP polypeptide. Then, in step (a2), the l ⁇ Boc-detectable protein fusion protein which is no longer produced is brought into the presence, in the cell nucleus, of the artificial SCF complex which comprises the ⁇ -TrCP protein whose expression has been induced.
- the target fusion protein comprising IKBOC is first accumulated, then the ubiquitination effector protein is expressed, namely the protein comprising the ⁇ -TrCP polypeptide, which will initiate the degradation process. of the l ⁇ Boc-detectable protein fusion protein. And the degradation process of the Iv B ⁇ -detectable protein fusion protein, which can be modulated by the agent to be tested, is measured in step (b) of the screening method of the invention.
- the yeast cells express the protein comprising the ⁇ -TrCP polypeptide during all of the steps (a2) and (a3), and (i) n ' do not express the protein comprising the ⁇ -TrCP polypeptide for a predetermined duration, at the start of step (ai); (ii) (ii) express the protein comprising the ⁇ -TrCP polypeptide for the remaining duration of step (ai).
- the l ⁇ Bcc-detectable protein fusion protein is expressed during the whole of step (ai) of the method, and the expression of said fusion protein is stopped in step (a2) of the method.
- the expression of the protein comprising the ⁇ -TrCP polypeptide is activated at a chosen time during the duration of step (ai). Under these conditions, in part (ii) of step (ai), the l ⁇ Boc-detectable protein fusion protein and the protein comprising the ⁇ -TrCP polypeptide are simultaneously expressed in yeast cells.
- the fusion protein l ⁇ B ⁇ -detectable protein accumulates in large quantity in the nucleus of yeast cells during the whole of step (ai) and the effector protein comprising the polypeptide ⁇ -TrCP is expressed early in the during step (ai) and continues to accumulate during steps (a2) and (a3) during which the target fusion protein is no longer synthesized.
- the expression of the fusion protein l ⁇ Bcc-detectable protein is activated for a duration T1 of between 0.25 hours and 10 hours, better between 0.5 hour and 6 hours, and even better between 1 hour and 4 hours.
- the expression of the effector protein comprising the ⁇ -TrCP polypeptide is activated.
- the instant t2 is located between [T1 - 8 hours] and [T1 - 0.1 hours], better between [T1 - 5 hours] and [T1 - 0.25 hours], and even better between [T1 - 3 hours] and [T1 - 0.5 hour], time t2 being, by definition, chosen within the limits of the duration T1 previously selected.
- the expression of the fusion protein l ⁇ Boc-detectable protein is stopped. From this moment, only the expression of the effector protein comprising the ⁇ -TrCP polypeptide is kept activated in the yeast cells, and this for the entire duration of the screening process, ie up to at the end of the process.
- the detectable protein which is included in the l ⁇ Boc-detectable protein fusion protein can be of any kind, since its presence can be specifically detected in yeast cells before its proteolysis, and that the presence of proteolysed forms of the detectable protein, in particular peptide fragments produced by proteolysis of said detectable protein, are not detected by the specific detection means which is chosen.
- the ubiquitin ligase activity of the artificial protein complex comprising the ⁇ -TrCP protein is followed, according to the method of the invention, by measuring its effect on the stability of the l ⁇ B ⁇ -detectable protein fusion protein.
- the addition of poly-ubiquitin chains to the IKBOC factor by the artificial human / yeast SCF complex results in the recognition of the factor l k Boc ubiquitin by the proteasome and its rapid degradation by the latter. Thanks to the expression in yeast cells of the IKBOC factor in the form of a fusion protein, the degradation of the fusion protein containing IKBOC can be monitored in real time, by detection of the detectable protein which is not proteolysed.
- the degradation of the fusion protein can be followed by techniques known per se, in particular techniques for measuring fluorescence using either a flow cytometer or a microplate reader, either with a fluorimeter, or with a fluorescence microscope, or by colorimetric, enzymatic or immunological techniques.
- the detectable protein can be chosen from an antigen, a fluorescent protein or a protein having an enzymatic activity.
- the detectable protein when the detectable protein consists of an antigen, it can be any type of antigen, since antibodies specific for this antigen are already accessible or, alternatively, can be prepared, according to any technique for obtaining antibodies, in particular d polyclonal or monoclonal antibodies, well known to those skilled in the art job.
- the detectable protein consists of a small antigen, which is not likely to interfere with the recognition of the factor IKBOC by the polypeptide ⁇ -TrCP.
- a peptide having a chain of 7 to 100 amino acids in length, better from 7 to 50 amino acids in length, and even better still from 7 to 30 amino acids in length, for example 10 acids is used as antigen.
- amines in length a peptide having a chain of 7 to 100 amino acids in length, better from 7 to 50 amino acids in length, and even better still from 7 to 30 amino acids in length, for example 10 acids.
- HA antigen of sequence [NH 2 -YPYDVPDYA-COOH] SEQ ID No. 17 or else a FLAG antigen of sequence [NH 2 -DYKDDDDK- COOH] SEQ ID No. 18 (monomer FLAG ) or of sequence [NH 2 - MDYKDHDGDYKDHDIDYKDDDDK-COOH] SEQ ID No. 19 (trimer FLAG)
- an antibody which specifically recognizes the is used to quantify the protein detectable in step (b) of the process antigen included in the fusion protein, this antibody being labeled directly or indirectly.
- step (b) when the first detectable protein is an antigen, said first detectable protein is quantified by detection of the complexes formed between said protein and antibodies recognizing it.
- the detectable protein consists of an intrinsically fluorescent protein
- it is in particular chosen from the GFP protein or one of its derivatives, the YFP protein or one of its derivatives, and the dsRED protein.
- the proteins derived from the GFP protein use may in particular be any of the proteins known under the names GFPMut3, Venus, Sapphire etc.
- An illustrative list of GFP proteins capable of being used in the process of the invention is presented in Table 3, at the end of this description.
- the intrinsically fluorescent protein can also be chosen from auto-fluorescent proteins originating from various organisms, other than Aequorea Victoria.
- the intrinsically fluorescent protein can be chosen from the following proteins: - the CopGFP protein originating from Pontellina plumata, and described by DA Shagin et al. (2004, Mol. Biol. Evol. 21: 841-850); TurboGFP protein, a variant of CopGFP; and described by DA Shagin et al., 2004 (Mol. Biol. Evol. 21: 841 -850); the J-Red protein, originating from Anthomedusae); and described by DA Shagin et al., 2004 (Mol. Biol. Evol. 21: 841 -850); the protein PhiYFP originating from Phialidium sp.
- the detectable protein in step (b) of the method is quantified by measuring the fluorescence signal which is emitted by the fusion protein l ⁇ B ⁇ -fluorescent protein using any adapted device.
- the detectable protein in step (b) of the method is quantified by measuring the fluorescence signal emitted by said protein.
- the detectable protein consists of a protein with enzymatic activity
- said detectable protein is chosen in particular from luciferase and ⁇ -lactamase.
- the protein detectable in step (b) of the method is quantified by measuring the amount of the compound or compounds produced by the conversion of the substrate by the enzyme.
- the product of the enzymatic activity is colored, the measurement can be carried out by colorimetry.
- the product of the enzymatic activity is fluorescent, the intensity of the fluorescence signal which is emitted by said product is measured, using any suitable fluorescence measurement device.
- the first detectable protein is a protein having an enzymatic activity
- said detectable protein is quantified by measuring the amount of substrate transformed by said protein.
- the protein comprising the ⁇ -TrCP polypeptide also consists of a fusion protein comprising, in addition to the ⁇ -TrCP polypeptide, also a detectable protein.
- the level of expression of the ⁇ -TrCP polypeptide in the yeast cells can be monitored over time, by detecting, and optionally by quantifying, the detectable protein contained in the protein comprising the ⁇ -TrCP polypeptide.
- This particular embodiment is mainly implemented when the expression of the protein comprising the ⁇ -TrCP polypeptide is controlled positively or negatively, at the different substeps of step (a) of the method.
- the detectable protein contained in the polypeptide comprising the ⁇ -TrCP polypeptide is chosen from an antigen, a fluorescent protein and a protein having enzymatic activity.
- the detectable protein contained in the protein comprising the ⁇ -TrCP polypeptide is distinct from the detectable protein contained in the l ⁇ Boc-detectable protein fusion protein, which allows the expression of factor I BCC and the expression of the ⁇ -TrCP polypeptide to be monitored separately in yeast cells.
- the degradation of the human IKBOC target polypeptide by the proteasome of yeast cells is carried out only when the fusion protein l ⁇ Boc-detectable protein and the protein comprising the human ⁇ -TrCP polypeptide are both co-located in the nucleus of yeast cells.
- the factor IKBOC is phosphorylated on the serine residue at position 32 exclusively in the nucleus of yeast cells, whereas it does not undergo phosphorylation in the cytoplasm .
- a posteriori the event of phosphorylation of the serine residue in position 32 of the factor l ⁇ B ⁇ , in the yeast cells, makes it possible to explain, at least partially, the reason why, in the yeast cells, the ubiquitination of this factor can only be performed in the cell nucleus.
- any means is used allowing nuclear localization of both the l ⁇ Bcc-detectable protein fusion protein and the protein comprising the ⁇ -TrCP polypeptide.
- the l ⁇ B-detectable protein fusion protein and the protein comprising the ⁇ -TrCP polypeptide both comprise a peptide making it possible to localize these two proteins in the nucleus of yeast cells.
- the l ⁇ Bcc-detectable protein fusion protein and the protein comprising the ⁇ -TrCP polypeptide both comprise, in their amino acid sequence, at least one nuclear localization peptide (“NLS”) which is functional in eukaryotic cells, and more particularly in yeast cells.
- NLS nuclear localization peptide
- Each of the proteins comprises, independently of one another, 1, 2, 3 or 4 nuclear localization peptides.
- each of these proteins comprises, independently of one another, from 1 to 4 copies of a nuclear localization peptide.
- the nuclear localization peptide (s) are chosen from the following peptides: the NLS peptide derived from the large antigen of the SV40 virus having the amino acid sequence SEQ ID No. 24; the nucleoplasmin NLS peptide having the amino acid sequence SEQ ID No. 20; an NLS peptide of the yeast alpha 2 repressor chosen from the sequences SEQ ID No. 21 and SEQ ID No. 22; - an NLS peptide of the yeast Gal4 protein having the amino acid sequence SEQ ID No. 23.
- the l ⁇ B-detectable protein fusion protein and the protein comprising the ⁇ -TrCP polypeptide both comprise the nuclear localization peptide with sequence SEQ ID No. 24.
- the l k Boc-detectable protein fusion polypeptide consists of an amino acid chain which comprises, from the NH 2 - terminal end to the COOH-terminal end, respectively (i) the sequence of the detectable protein , (ii) the NLS nuclear localization sequence and (iii) the sequence of l k B ⁇ .
- the GFP sequence and the NLS sequence can be linked directly to each other, by a peptide bond.
- the NLS sequence and the 1 k Boc sequence can be linked directly to each other, by a peptide bond.
- the GFP sequence and the NLS sequence can be separated, in the sequence of the fusion polypeptide, by a first spacer peptide.
- the NLS sequence and the 1 k B ⁇ sequence can be separated, in the sequence of the fusion polypeptide, by a second spacer peptide.
- the spacer peptide (s), when it is (are) present, has (have) a size ranging from 1 to 30 amino acids, preferably from 1 to 15 amino acids, and most preferably 2 to 10 amino acids in length.
- the protein comprising the IKBOC polypeptide consists of the amino acid sequence protein
- SEQ ID N ° 2 which can be encoded by the nucleic acid of sequence SEQ
- the protein of sequence SEQ ID No. 2 consists, from the end
- the protein comprising the ⁇ TrCP polypeptide consists of an amino acid chain which comprises, from the NH 2 - terminal end to the COOH-terminal end, respectively (i) the sequence of a second detectable protein, (ii ) the nuclear localization NLS sequence, and (iii) the ⁇ TrCP sequence.
- the protein comprising the ⁇ -TrCP polypeptide consists of the amino acid sequence protein SEQ ID No. 4, which is encoded by the nucleic acid of sequence
- the protein with sequence SEQ ID No. 4 consists, from the NH 2 -terminal end towards the COOH-terminal end, respectively in (i) the sequence of the detectable protein GFP (yEGFP3) ranging from the amino acid in position 1 to the amino acid at position 240, (ii) a first spacer peptide ranging from amino acid at position 241 to amino acid at position 243, (iii) the NLS peptide of the large T antigen of SV40 going from amino acid at position 244 to amino acid at position 250, (iv) a second spacer peptide going from amino acid at position 251 to amino acid at position
- the nucleic acid of sequence SEQ ID No. 3 consists, from the 5 ′ end towards the 3 ′ end, respectively in (i) the sequence coding for the detectable protein GFP ( yEGFP3) going from the nucleotide in position 1 to the nucleotide in position 714, (ii) the sequence coding for a first spacer peptide going from the nucleotide in position 715 to the nucleotide in position 729, (iii) the sequence coding for the NLS peptide of SV40 large T antigen ranging from the nucleotide at position 730 to the nucleotide at position 750, (iv) the sequence coding for a second spacer peptide ranging from the nucleotide at position 751 to the nucleotide at position 765 and (v) the sequence coding for ⁇ -Tr
- the screening method according to the invention is characterized in that the recombinant yeast cells are transformed with:
- a first polynucleotide which comprises (a) an open reading frame encoding (i) the fusion protein comprising the IKBOC polypeptide, (ii) a nuclear localization sequence and (iii) a first detectable protein, and (b) a functional regulatory sequence in yeast cells which directs expression of said open reading frame; and
- a second polynucleotide which comprises (a) an open reading frame encoding (i) a protein comprising the ⁇ -TrCP polypeptide, (ii) a nuclear localization sequence, and (iii) a functional regulatory sequence in cells of yeast which directs the expression of said open reading frame;
- the above polynucleotide (1) can consist of the nucleic acid of sequence SEQ ID No. 1.
- the above polynucleotide (2) may consist of the nucleic acid of sequence SEQ ID No. 3.
- Preferred nucleic acids, expression vectors and transformed yeast cells according to the invention are Preferred nucleic acids, expression vectors and transformed yeast cells according to the invention.
- Nucleic acids are synthesized according to the invention, which, when introduced into yeast cells, cause the expression of the l ⁇ Boc-detectable protein fusion protein and of the protein comprising the ⁇ -TrCP polypeptide respectively in these cells. , and more particularly in the nucleus of yeast cells.
- each of the nucleic acids synthesized comprises a coding sequence, which is also designated “open reading frame” or “ORF”. which codes for the protein of interest, respectively the l ⁇ Boc-detectable protein fusion protein, or the protein comprising the ⁇ -TrCP polypeptide, said protein of interest also comprising in its sequence at least the sequence of a nuclear localization peptide .
- Illustrative examples of the nucleic acids according to the invention are the nucleic acids of sequence SEQ ID No. 1 and SEQ ID No. 3, the structure of which has been described previously in the description.
- Each of the nucleic acids also comprises a regulatory sequence comprising a promoter functional in yeast cells.
- the promoter functional in yeast cells consists of a constitutive promoter which can be chosen from the promoters PGK1, ADH1, TDH3, LEU2 and TEFL
- each of the nucleic acids comprises, as promoter, a promoter called "inducible” , ie a promoter which is functional in yeast cells and which is sensitive to the action of an inducing agent.
- a promoter can be used which, when the inducing agent is added to the culture medium of yeast cells, activates the expression of the sequence coding for the protein of interest placed under its control. It is also possible to use a promoter which, when the inducing agent is added to the culture medium of yeast cells, represses or blocks the expression of the sequence coding for the protein of interest placed under its control.
- the inducible promoter which is contained in the nucleic acids of the invention is chosen from CUP1, GAL1, MET3, MET25, MET28, SAM4 and PH05.
- the nucleic acid or the polynucleotide which codes for the fusion protein l ⁇ B ⁇ -detectable protein comprises the regulatory sequence GAL1, which activates the expression of the open reading frame coding for the fusion protein comprising the polypeptide the polypeptide IKBOC in the presence of glucose.
- the expression of the fusion protein comprising the factor IKBOC is carried out temporarily during the screening test.
- the expression of the protein containing l ⁇ B ⁇ is specifically stopped (in an experiment known to those skilled in the art as name of "promoter shut off") before exposing the cells to the molecules to be screened.
- This stopping of expression is obtained by the addition (or deletion) in the culture medium of a molecule capable of repressing the activity of the promoter controlling the expression of the tripartite protein containing IKBOC.
- the l ⁇ B ⁇ -detectable protein fusion protein when expressed under the control of the promoter of the GAL1 gene, then the expression of this promoter is repressed by adding glucose to the final concentration of 2% in the culture medium.
- the stopping of the neosynthesis of the fusion protein comprising IKBOC makes it possible to measure its stability in real time, by determining for example the fluorescence of the yeast cells over time after the stopping of synthesis, in the embodiment in which said fusion protein contains a detectable intrinsically fluorescent protein, such as GFP or a protein derived from GFP.
- the temporary expression of the fusion protein comprising the factor IKBOC is associated with an also temporary expression of the protein comprising the ⁇ -TrCP polypeptide.
- the fusion protein comprising the IKBOC polypeptide is expressed during the chosen time period T1, for example by using yeast cells which express the fusion protein comprising the IKBOC polypeptide UNDER the control of the GAL1 promoter and which are cultivated in the presence of 0.5 to 4% galactose for the duration T1.
- the expression of the protein comprising the ⁇ -TrCP polypeptide is induced.
- This induction is, for example, obtained in cells expressing the protein containing ⁇ -TrCP under the control of the promoter of the CUP1 gene, by adding in the medium of culture a concentration of copper sulphate between 0.05 mM and 5 mM.
- the expression of the fusion protein comprising IKBOC is stopped by adding glucose to the culture medium at a concentration of between 0.5 and 2%.
- This addition of glucose has no effect on the expression of the protein comprising ⁇ -TrCP from the promoter of the CUP1 gene.
- the accumulation of ubiquitin ligase comprising ⁇ -TrCP is continued, while the neo-synthesis of the fusion protein comprising IKBOC is stopped.
- the nucleic acid or the polynucleotide which codes for the protein comprising the ⁇ -TrCP polypeptide comprises the regulatory sequence CUP1, which activates the expression of the open reading frame encoding a protein comprising the ⁇ -TrCP polypeptide in the presence of copper sulfate.
- the subject of the invention is also a cassette for expression functional in yeast cells comprising a coding polynucleotide which comprises an open reading frame coding the fusion protein comprising the polypeptide the polypeptide IKBOC and at least one first detectable protein, and a functional regulatory sequence in yeast cells which directs expression of said open reading frame.
- Such an expression cassette may in particular consist of the nucleic acid of sequence SEQ ID No. 1 according to the invention, which codes for the fusion protein GFP-NLS-1 k B ⁇ of sequence SEQ ID No. 2.
- the invention also relates to a functional expression cassette in yeast cells comprising a polynucleotide which comprises an open reading frame encoding a protein comprising the ⁇ -TrCP polypeptide and a regulatory sequence functional in yeast cells which directs expression. said open reading frame.
- Such an expression cassette can in particular comprise the nucleic acid of sequence SEQ ID No. 3 according to the invention, which codes for the fusion protein GFP-NLS- ⁇ TrCP of sequence SEQ ID No. 4.
- the regulatory sequence comprises an inducible promoter functional in yeast cells, such as a promoter chosen from the promoters PGK1, ADH1, TDH3, LEU2 and TEFL
- the regulatory sequence contained in said polynucleotide, the regulatory sequence contained in the second polynucleotide, or the two regulatory sequences comprises (s) a promoter which is functional in yeast cells and which is sensitive to the action of an inducing agent, which is also called an inducible promoter.
- the inducible functional promoter in yeast cells is chosen from CUP1, GAL1, MET3, MET25, MET28, SAM4 and PH05.
- the yeast cells are transformed with (i) the nucleic acid or the polynucleotide comprising the sequence coding for the fusion protein l ⁇ B ⁇ -detectable protein as well as with (ii ) acid nucleic acid or polynucleotide encoding the protein comprising the ⁇ -TrCP polypeptide, which are in a non-integrated form, for example in the form of functional vectors in yeast cells and which carry at least one origin of functional replication in the cells of yeast.
- the recombinant yeast cells have the nucleic acid or the polynucleotide comprising the sequence coding for the l ⁇ Boc-detectable protein fusion protein as well as the nucleic acid or the polynucleotide encoding the protein comprising the ⁇ -TrCP polypeptide in a form integrated into their genome, as illustrated in the examples.
- yeast cells which have good membrane permeability, in particular good membrane permeability for the agents to be tested by the method.
- yeast cells which have good membrane permeability for inducing compounds to which said inducible promoters are sensitive
- yeast strains are used whose genome comprises one or more mutations which increase the permeability to the products to be tested, such as mutations inactivating the PDR1 and PDR3, two genes encoding transcriptional factors which in the yeast control the expression of transporters inserted into the plasma membrane (Vidal et al, 1999, Nourani et al, 1997).
- yeast strains having the genetic background of the strain W303 of the yeast Saccharomyces cerevisiae described by Bailis et al are used. (1990), or any other characterized strain of the so-called Saccharomyces cerevisiae yeast.
- the transformation of yeast cells with exogenous DNA is preferably using techniques known to those skilled in the art, in particular the technique described by Schiestl et al. (1989).
- the constructions of the different yeast strains were carried out using genetic techniques (crossing, sporulation, dissection of asci and phenotypic analysis of spores) known and described in particular by Sherman et al. (1979) and the reverse genetics techniques described in particular by Rothstein (1991).
- the yeasts are preferably transformed by plasmids constructed according to conventional molecular biology techniques, in particular according to the protocols described by Sambrook et al. (1989) and Ausubel et al. (1990-2004).
- Another object of the invention consists of an expression vector characterized in that it comprises an expression cassette as defined in the present description.
- a first vector in accordance with the invention is the vector pCSY226-NLS-IKBOC which is described in the examples, and which was used for the construction of the yeast strain CYS135 deposited in the National Collection of Cultures of Microorganisms of the Institut Pasteur from Paris under the access number 1-3187.
- a second vector in accordance with the invention is the vector pCSY226-NLS- ⁇ -TrCP which is described in the examples, and which was used for the construction of the yeast strain CYS135 deposited in the National Collection of Cultures of Microorganisms of the Institut Pasteur de Paris under access number 1-3187.
- the present invention also relates to a recombinant yeast strain comprising, in a form integrated into its genome,
- a first polynucleotide which comprises an open reading frame coding for the fusion protein comprising the polypeptide the polypeptide IKBOC and at least a first detectable protein, and a regulatory sequence functional in yeast cells which directs the expression of said framework open reading;
- a second polynucleotide which comprises an open reading frame encoding a protein comprising the ⁇ -TrCP polypeptide and a functional regulatory sequence in yeast cells which directs the expression of said open reading frame;
- the invention relates to a recombinant yeast strain conforming to the definition above, which consists of the yeast strain CYS135 deposited in the National Collection of Cultures of Microorganisms of the Institut Pasteur de Paris (CNCM) under access number 1-3187.
- the invention also relates to a kit or kit for the screening of agents modulating the ubiquitination of the IkBoc protein by a functional protein complex ubiquitin ligase comprising the protein ⁇ -TrCP, characterized in that it comprises: (i) a first expression vector comprising an expression cassette encoding the fusion protein comprising the IkBoc polypeptide as defined above; and
- the invention also relates to a kit or kit for the screening of agents modulating the ubiquitination of the IkBoc protein by a functional protein complex ubiquitin ligase comprising the protein ⁇ -TrCP, characterized in that it comprises yeast cells recombinant comprising, in a form inserted into their genome, respectively:
- the above kit or kit comprises recombinant yeast cells of the yeast strain CYS 135 deposited at the CNCM under the access number 11-3187.
- the screening method according to the invention makes it possible to visualize the activity of the ubiquitin ligase SCF ⁇ "TrCP with respect to the human IKBOC factor, substrate of the ubiquitin proteasome pathway for protein degradation.
- This method is particularly advantageous for screening molecules or agents capable of acting on pathologies linked to the activation of NF- ⁇ B factors and to dysfunctions of the NF- ⁇ B pathway in humans such as inflammatory and immune syndromes, certain cancers, certain diseases like reperfusion injury and fungal, bacterial and viral infections.
- the main advantages of the screening method of the invention are in particular the following: the simplicity of implementation: the induction of the activity of the ubiquitin ligase SCF ⁇ 'TrCP with respect to the factor IKBOC is carried out simply thanks to the controlled expression of human factors IKBOC and ⁇ -TrCP in yeast cells.
- the IKBOC factor is expressed as a fusion fusion protein with an intrinsically fluorescent protein, such as GFP, the activity of artificial SCF ⁇ TrCP ubiquitin ligase with respect to the IKBOC factor is directly measured by the. quantification of the fluorescence emitted by the hybrid protein.
- the IKBOC factor when expressed as a fusion protein fusion with a protein such as luciferase, the activity of the artificial SCF ⁇ "TrCP ubiquitin ligase with respect to the IKBOC factor is directly measured by the quantification. of the luminescence emitted by the hybrid protein in the presence of a substrate such as fluorescein.
- adequacy with a therapeutic context the activity of ubiquitin ligase SCF ⁇ 'TrCP artificial with respect to the IKBOC factor is monitored according to a functional test carried out on whole cells
- the in vitro screening method according to the invention therefore makes it possible to select molecules capable of activating or inhibiting the degradation of l ⁇ Boc in a context similar to that of their final therapeutic use.
- specificity although carried out in vitro in the cell, the screening method according to the invention is specific, because it is based on co-expression, in a heterologous organism with the human organism, of the two human proteins l ⁇ Bcc and ⁇ -TrCP.
- the molecules selected using the screening method of the invention will be specific for this ubiquitin ligase ⁇ -TrCP / IKBOC substrate protein pair, and will therefore not be molecules selected because of, for example, their ability to interfere with one numerous signaling pathways inducing the degradation of l ⁇ Bcc in human cells.
- the degradation of IB via the artificial ubiquitin ligase SCF ⁇ TrCP , is induced by a completely artificial and totally metabolic pathway reproducible, such as the addition of glucose to block the activity of the GAL1 promoter, when IKBOC is expressed under the control of this promoter, the stability of the recombinant yeast strains: integration techniques in a chosen place of a yeast chromosome, and targeted replacement of genes allow the construction of recombinant yeast strains expressing hybrid human proteins containing either IKBOC or ⁇ -TrCP from the yeast chromosomes. These recombinant yeast strains are therefore genetically stable and can be multiplied and stored indefinitely.
- yeast is a microorganism with rapid growth and high yield.
- the screening method of the invention is preferably carried out by culturing the yeast cells in a complete culture medium, in which the growth of the yeast cells is particularly rapid and the yield particularly high, which makes it possible to obtain a large quantity of recombinant yeast cells for the simultaneous performance of a large number of screening tests.
- the low cost yeast is a microorganism whose culture, storage and characterization are inexpensive
- the automation of the screening process of the invention yeast is a microorganism whose culture, carried out in a small volume of medium , at low temperature, in a conventional atmosphere, in the air, is particularly suitable for the automation (robotization) of screening processes.
- the screening methods according to the invention are useful in particular for selecting and characterizing active agents such as anti-inflammatory, anti-cancer, anti-viral agents, agents against fungal, bacterial or viral infections.
- FIG. 1 illustrates the possibility for the yeast Skp1 proteins and human ⁇ -TrCP to interact in yeast cells.
- FIG. 2 illustrates the localizations in yeast cells of the human proteins IKBOC and ⁇ -TrCP depending on whether or not they are fused to an NLS sequence of SV40.
- Upper line fluorescence microscopy images of DNA staining of cell nuclei with the dye Hoechst 333-42.
- Lower line fluorescence microscopy images allowing the localization of GFP expression in the cells.
- A Cells transformed by the vector GFP-NLS- ⁇ -TrCP; B: cells transformed with the vector GFP- ⁇ -TrCP; C: cells transformed with the vector GFP-NLS-1 ⁇ Bcc; D: cells transformed by the vector GFP-IKBOC.
- FIG. 3 illustrates how the addressing of the human protein IKBOC in the nucleus of yeast cells induces its phosphorylation on serines 32 and 36.
- the figure represents an electrophoresis gel of the cellular proteins of the recombinant yeast strains CYS22 and CYS126, respectively.
- FIG. 4 illustrates by an epifluorescence microscope analysis, the degradation of the tripartite fusion protein GFP-NLS-1 ⁇ B ⁇ in yeast cells when these also express the tripartite fusion protein Flag-NLS- ⁇ -TrCP.
- FIGS. 4A to 4D represent pictures of fluorescence microscopy: upper line, staining of the DNA of the cell nuclei with the dye Hoechst 333-42; lower line, fluorescence microscopy images allowing the localization of GFP expression in the cells.
- Figure 4A results obtained with the recombinant yeast strain CYS22;
- Figure 4B results obtained with the recombinant yeast strain CYS61.
- Figure 4C results obtained with the recombinant yeast strain CYS 126.
- Figure 4D results obtained with the recombinant yeast strain CYS 135.
- FIG. 5 illustrates, by quantification of the fluorescence emitted, the degradation of the tripartite fusion protein GFP-NLS-1 ⁇ Boc in yeast cells when these latter express or not the tripartite fusion protein Flag-NLS- ⁇ -TrCP.
- FIG. 6 illustrates by a Western Blotting type biochemical analysis, the degradation of the tripartite fusion protein GFP-NLS-1 ⁇ Bcc in yeast cells when these also express the tripartite fusion protein Flag-NLS- ⁇ -TrCP.
- Immunoblotting gel shots (“Western blotting”) revealed with anti-GFP antibodies and anti-FLAG peptide antibodies
- FIG. 7 illustrates by a Western blotting biochemical analysis, the degradation of the mutant tripartite fusion protein GFP-NLS-1 ⁇ Bcc [S3236A] in which the phosphorylation sites Ser32 and Ser36 have been replaced by Ala residues, mutations which in human cells make the protein non-degradable.
- Immunoblotting gel shots (“Western blotting”) revealed with anti-GFP antibodies and anti-FLAG peptide antibodies
- FIG. 8 illustrates by an epifluorescence microscope analysis, the degradation of the tripartite fusion protein GFP-NLS-l ⁇ Boc [S3236A] in yeast cells when these also express the tripartite fusion protein Flag-NLS- ⁇ -TrCP .
- FIGS. 8A and 8B represent fluorescence microscopy images: upper line, staining of the DNA of the cell nuclei with the dye Hoechst 333-42; lower line, fluorescence microscopy images allowing the localization of GFP expression in the cells.
- Figure 8A results obtained with the recombinant yeast strain CYS138
- Figure 8B results obtained with the recombinant yeast strain CYS139.
- FIG. 9 illustrates, by quantification of the fluorescence emitted, the degradation of the tripartite fusion protein GFP-NLS-1 ⁇ Bcc [S3236A] in the yeast strains described above.
- Examples 1 to 3 Construction of the recombinant vectors according to the invention.
- the sequence of the IKBOC protein is that described in Strausberg et al. (PNAS (1999), 99 (26): 16899-16903).
- the sequence of the ⁇ -TrCP receptor subunit of the ubiquitin ligase SCF ⁇ "TrCP complex is that described in Yaron et al. (Nature (1998) 396 (6711): 590-594).
- GFP Green Fluorescent Protein mut3
- the nuclear localization sequence “NLS” of the large T antigen encoded by the SV40 virus is the translation of the nucleic sequence
- the sequence of the plasmid pRS306 is that described in Sikorski and Hieter (Genetics (1989) 122 (1): 19-27).
- the sequence of the plasmid pRS304 is that described in Sikorski and Hieter (Genetics (1989) 122 (1): 19-27).
- the sequence of the plasmid pRS314 is that described in Sikorski and Hieter (Genetics (1989) 122 (1): 19-27).
- the sequence of the plasmid pRS316 is that described in Sikorski and Hieter (Genetics (1989) 122 (1): 19-27).
- the sequence of the plasmid pSH 18-34 which contains four LexA operators placed upstream of the LacZ gene is that described by Hanes and Brent (Cell (1989), 57: 1275-1293)
- the sequence of the plasmid pLexSkp1-1, which allows the expression of the yeast protein Skp1 fused to the bacterial protein LexA, is that described in Patton et al. (Genes & Dev (1998), 12: 692-705)
- the sequence of the plasmid pGAD ⁇ TrCP, which allows the expression of the human protein ⁇ -TrCP fused to the activating domain of the yel Gal4 transcription factor nel is that described in Margottin et al. (Molec. Cell (1998), 1: 565-574).
- TEF1 gene promoter from the yeast S. cerevisiae used in the descriptions which follow is that described by Schaaff-Gerstenschlager et al. (Eur. J. Biochem. (1993) 217 (1): 487-492).
- the sequence of the promoter of the MET25 gene of the yeast S. cerevisiae used in the descriptions which follow is that described in Kerjan et al. (Nucleic Acids Res. (1986) 14 (20): 7861-7871).
- the sequence of the promoter of the PH05 gene of the yeast S. cerevisiae used in the descriptions which follow is that described by Feldmann et al. (EMBO J. (1994) 13 (24): 5795-5809).
- the names are made using the nomenclature and the typographic rules in use in the community of biologists specialized in the yeast Saccharomyces cerevisiae.
- the name of the wild form of a gene is given in capital letters; example: GAL1.
- the name of a mutant form of a gene is given in lowercase italics, the allele number if known is specified after a dash; example cup1-1.
- the name of an inactivated allele of a gene is given in lower case followed by 2 colon followed by the name of the inactivating gene ex ppr1 :: TRP1 (in this example the inactivated gene pprl was interrupted by the active gene TRP1).
- the name of an inactivated gene can be given by the symbol "delta"; example gal4A the name of the protein is that of the gene which codes it is given in lower case, except the first letter which is in upper case ex Gal4 (alternatively one can use the same symbol followed by a p; example Gal4p).
- EXAMPLE 1 Construction of the plasmids allowing expression in yeast of the fusion proteins GFP-l ⁇ Boc and GFP-NLS-l ⁇ B ⁇ .
- the following plasmids allow the expression in yeast Saccharomyces cerevisiae of derivatives of the human protein IKBOC fused with a variant of Green Fluorescent Protein (GFP) to Aequora Victoria.
- GFP Green Fluorescent Protein
- the fusion proteins constructed may or may not include the nuclear localization sequence of the large T antigen of the SV40 virus. The introduction of this sequence makes it possible to address fusion proteins which comprise it in the nuclear compartment.
- a fragment of 620 base pairs (bp) corresponding to the promoter of the GAL1 gene (pGAL1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from genomic DNA of a wild strain of S. cerevisiae X2180 -1A, using the oligonucleotides "pGAL1 (Asp) Forw" of sequence
- the fragment obtained was digested with the restriction enzymes Asp718l and EcoRI and inserted into the shuttle plasmid S.ce ⁇ ev / s / ae-E.co // pRS306 previously digested with the enzymes AspT'IB and EcoRI, producing the vector pRS306- pGAL1.
- the fragment obtained was digested with the restriction enzymes SamHI and EcoRI and inserted into the plasmid pRS306-pGAL1 previously digested with the enzymes SamHI and EcoRI, producing the vector pRS306-pGAL1-yEGFP3.
- a fragment of 340 base pairs (bp) corresponding to the terminator of the ADH1 gene (tADH1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from genomic DNA of a wild strain of S. cerevisiae X2180 -1A, using the oligonucleotides "TermADH1 (NotlBstXI) 5 '" of sequence 5'- GGCGGCGGCCGCCACCGCGGTGGGCGAATTTCTTATGATTTATG-3' [SEQ ID N ° 10] and "TermADHI (Sacl) 3 '" of sequence 5'-GGCGGACAGAGCTGA' SEQ ID No. 11].
- PCR Polymerase Chain Reaction
- the fragment obtained was digested with the restriction enzymes Sac1 and Noti and inserted into the plasmid pRS306-pGAL1-yEGFP3 previously digested with the enzymes Sac1 and Noti, producing the vector pCSY226.
- the gene coding for the protein IKBOC was purified from the plasmid pGad1318-lkBa by digestion with the restriction enzyme Xba ⁇ followed by treatment with the Klenow fragment of DNA polymerase I in order to transform the cohesive end in 3 ′ of the blunt-ended fragment, then of a second digestion with the Bam ⁇ restriction enzyme for the 5 ′ end of the gene.
- the fragment was cloned into the plasmid pCSY226 prepared by digestion with the restriction enzyme Kpn ⁇ , followed by treatment with the Klenow fragment, then digestion with the restriction enzyme Bam ⁇ .
- the vector produced was called pCSY226-l ⁇ Bcc.
- a version of this vector additionally contains the NLS nuclear addressing sequence. This was obtained by synthesizing a pair of oligonucleotides complementary to sequence
- EXAMPLE 2 Construction of the plasmids allowing expression in yeast of the fusion proteins GFP- ⁇ -TrCP and GFP-NLS- ⁇ -TrCP.
- the following plasmids allow the expression in yeast Saccharomyces cerevisiae of derivatives of the human protein ⁇ -TrCP fused with a variant of the Green Fluorescent Protein (GFP) to Aequora Victoria.
- GFP Green Fluorescent Protein
- the fusion proteins constructed may or may not include the nuclear localization sequence of the large T antigen of the SV40 virus. The introduction of this sequence makes it possible to address fusion proteins which comprise it in the nuclear compartment.
- a fragment of 620 base pairs (bp) corresponding to the promoter of the GAL1 gene (pGAL1) from the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from genomic DNA from a wild strain of S.cerevisiae X2180-1A, using the oligonucleotides "pGAL1 (Asp) Forw” of sequence 5'-GCTGGGTACCTTAATAATCATATTACATGGCATTA-3 '[SEQ ID N ° 6] and
- the fragment obtained was digested with the restriction enzymes Asp7 ⁇ 8 and EcoRI and inserted into the shuttle plasmid S.cerevisiae-E.coli pRS306 previously digested with the enzymes Asp7181 and EcoRI, producing the vector pRS306-pGAL1.
- the fragment obtained was digested with the restriction enzymes ⁇ amHI and EcoRI and inserted into the plasmid pRS306-pGAL1 previously digested with the enzymes SamHI and EcoRI, producing the vector pRS306-pGAL1-yEGFP3.
- a fragment of 340 base pairs (bp) corresponding to the terminator of the ADH1 gene (tADH1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from genomic DNA of a wild strain of S.
- the fragment obtained was digested with the restriction enzymes Sac1 and Noti and inserted into the plasmid pRS306-pGAL1-yEGFP3 previously digested with the enzymes Sac1 and Noti, producing the vector pCSY226.
- the gene coding for the protein ⁇ TrCP was purified from the plasmid pGad1318- ⁇ TrCP by digestion with the restriction enzymes SamHI and Noti.
- the fragment was cloned into the plasmid pCSY226 prepared by digestion with the restriction enzymes ⁇ amHI and Noti.
- the vector produced was called pCSY226- ⁇ TrCP.
- a version of this vector additionally contains the NLS nuclear addressing sequence. This was obtained by synthesizing a pair of oligonucleotides complementary to sequence
- NLS-3 ' 5'-AATTCGACCTTTCTCTTCTTTTTTGGAGGT-3' [SEQ ID No. 26] rehybridized to form double stranded DNA.
- This DNA fragment was then incorporated into the vector pCSY226- ⁇ TrCP digested with the restriction enzyme ScrFI to give the vector pCSY226-NLS- ⁇ TrCP.
- EXAMPLE 3 Construction of the plasmids allowing expression in yeast of the fusion proteins GFP- ⁇ -TrCP and GFP-NLS- ⁇ -TrCP.
- the following plasmids allow the expression in yeast Saccharomyces cerevisiae of derivatives of the ⁇ -TrCP protein comprising a repetition of three Flag antigenic motifs at their amino terminus.
- the expression of these fusion proteins is induced by culturing the yeast cells hosting these plasmids in the presence of 2 to 5% of galactose in the culture medium for 1 to 10 hours.
- a 700 base pair (bp) fragment corresponding to the promoter of the PGK1 gene (pPGK1) from the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from genomic DNA of a wild strain of S. cerevisiae X2180 -1A, using the oligonucleotides "pPGK1-Asp718-5 '" of sequence 5'-GGCGGGTACCGTGAGTAAGGAAAGAGTGAGG-3' [SEQ ID No. 13] and "pPGK-EcoRI-3 '" of sequence 5'-GGCGGAATTCTGTTTTATATTTGTTGTAAAAAG-3 SEQ ID No. 14].
- the fragment obtained was digested with the restriction enzymes Asp718l and EcoRI and inserted into the shuttle plasmid S.cerevisiae-E.coli pRS304 previously digested with the enzymes Asp718l and EcoRI, producing the vector pRS304-pPGK1.
- a fragment of 100 base pairs (bp) corresponding to a succession of 3 FLAG reporter sequences (3FLAG) was amplified by Polymerase Chain Reaction (PCR) from the vector p3XFLAG-myc-CMV-24 5Sigma Aldrich), using the "3FLAG- EcoRI-5 '" oligonucleotides of sequence 5'-GGCGGAATTCATGGACTACAAAGACCATGACGG-3 '[SEQ ID N ° 15] and “3FLAGBamHI (Smal / Srfl Pstl) 3'” of sequence 5'-
- the fragment obtained was digested with the restriction enzymes ⁇ amHI and EcoRI and inserted into the plasmid pRS304-pPGK1 previously digested with the enzymes SamHI and EcoRI, producing the vector pRS304-pPGK1-3FLAG.
- a fragment of 340 base pairs (bp) corresponding to the terminator of the ADH1 gene (tADH1) of the yeast Saccharomyces cerevisiae was amplified by Polymerase Chain Reaction (PCR) from genomic DNA of a wild strain of S. cerevisiae X2180 -1A, using the oligonucleotides "TermADH1 (NotlBstXI) 5 '" of sequence 5'- GGCGGCGGCCGCCACCGCGGTGGGCGAATTTCTTATGATTTATG-3' [SEQ ID No. 10] and "TermADH1 (Sacl) 3 '" of sequence 5'-GGCGAAGAGATTG SEQ ID No. 11].
- PCR Polymerase Chain Reaction
- the fragment obtained was digested with the restriction enzymes Sac1 and Noti and inserted into the plasmid pRS304-pPGK1 -3FLAG previously digested with the enzymes Sac1 and Noti, producing the vector pCSY614.
- the gene coding for the protein ⁇ TrCP was purified from the plasmid pGad1318- ⁇ TrCP by digestion with the restriction enzymes ⁇ amHI and Noti. The fragment was cloned into the plasmid pCSY614 prepared by digestion with the restriction enzymes BamHI and Noti. The vector produced was called pCSY614- ⁇ TrCP. A version of this vector additionally contains the NLS nuclear addressing sequence. This was obtained by synthesizing a pair of oligonucleotides complementary to sequence
- EXAMPLE 4 Interaction between the yeast Skp1 proteins and human ⁇ -TrCP in yeast cells.
- ⁇ -galactosidase activity in cellular extracts produced in such cells demonstrates that the expression of this reporter gene is induced by a factor 15, in comparison with its expression in cells expressing only one of the two fusion proteins described above.
- This induction of expression of the reporter gene indicates that the Skp1 protein of the yeast Saccharomyces cerevisiae is capable of interacting with the human ⁇ -TrCP protein.
- the ⁇ -galactosidase activity is expressed in nmoles of substrate transformed per minute and per mg of proteins (nmoles / min / mg).
- EXAMPLE 5 localization, in yeast cells, of human proteins l ⁇ B ⁇ and ⁇ -TrCP depending on whether or not they are fused to an NLS sequence of SV40.
- Yeast cells comprising plasmids allowing expression of the hybrid proteins either GPF-1 ⁇ Boc, or GFP-NLS-1 ⁇ Bcc, or GFP- ⁇ -TrCP, or GFP-NLS- ⁇ -TrCP from the promoter GAL1 were cultured in presence of 2% galactose for 2 hours and were then observed under fluorescence microscopy. The position of the nucleus was revealed using a specific color indicator of the nucleus, the Hoescht 333-42.
- EXAMPLE 6 Phosphorylation of the IKBOC protein in the nucleus of yeast cells.
- Example 6 illustrates how the addressing of the human protein IKBOC in the nucleus of yeast cells induces its phosphorylation on serines 32 and 36.
- Example 7 illustrates, by an epifluorescence microscope analysis, the degradation of the tripartite fusion protein GFP-NLS-1 ⁇ Bcc in yeast cells when these also express the tripartite fusion protein Flag-NLS- ⁇ -TrCP.
- yeast strain CYS22 MA Ta, his3, Ieu2, trpl, ura3 :: pGAL1- GFP-l ⁇ Ba :: URA3 expressing the fusion protein GFP-l ⁇ Bcc without NLS and localized in the cytoplasm of yeast cells;
- yeast strain CYS61 (MATa, his3, Ieu2, ura3 :: pGAL1-GFP- l ⁇ B ⁇ :: URA3, trp1 :: pGAL1-3Flag- ⁇ TrCP :: TRP1) expressing the fusion proteins GFP-l ⁇ Boc and Flag- ⁇ -TrCP , located in the cytoplasm of yeast cells;
- yeast strain CYS126 (MATa, his3, Ieu2, trpl, ura3 :: pGAL1- GFP-NLS-l ⁇ B ⁇ :: URA3) expressing the fusion protein GFP-NLS-l ⁇ Bcc located in the nucleus of yeast cells;
- yeast strain CYS135 (MATa, his3, Ieu2, ura3 :: pGAL1-GFP- NLS-l ⁇ B ⁇ :: URA3, trp1 :: pGAL1-3Flag-NLS- ⁇ TrCP :: TRP1) expressing the fusion proteins GFP-NLS-l ⁇ Bcc and Flag-NLS- ⁇ -TrCP localized in the nucleus of yeast cells.
- Example 8 illustrates, by quantification of the fluorescence emitted, the degradation of the tripartite fusion protein GFP-NLS-1 ⁇ Boc in yeast cells when these latter express or not the tripartite fusion protein Flag-NLS- ⁇ -TrCP.
- the yeast strains identical to those described in FIG. 4, and cultivated under the same conditions as those described in FIG. 4, were analyzed 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 quantity of fluorescence measured per cell in arbitrary unit.
- proteins are analyzed by the Western blotting technique using an antibody recognizing the GFP part of the fusion proteins containing I KBOC (indicated “GFP-NLS-l ⁇ B ⁇ ”) and an antibody recognizing the Flag part of the fusion protein Flag-NLS- ⁇ -TrCP (indicated "Flag-NLS- ⁇ -TrCP”).
- the quantity of proteins deposited in each well is checked by analyzing the same proteins using antibodies recognizing the yeast Lysyl-tRNA synthase (indicated “LysRS”). Proteins from the parental yeast strain expressing no fusion protein (indicated as "control”) serve as a control for specificity.
- yeast strain CYS22 (MATa, his3, Ieu2, trpl, ura3 :: pGAL1- GFP-l ⁇ B ⁇ :: URA3) expressing the fusion protein GFP-l ⁇ Bcc without NLS and localized in the cytoplasm of yeast cells
- yeast strain CYS61 (MATa, his3, Ie ⁇ 2, ura3 :: pGAL1-GFP- l ⁇ B ⁇ :: URA3, trp1 :: pGAL1-3Flag- ⁇ TrCP :: TRP1) expressing the fusion proteins GFP-l ⁇ Boc and Flag- ⁇ -TrCP , located in the cytoplasm of yeast cells
- yeast strain CYS126 (MATa, his3, Ieu2, trpl, ura3 :: pGAL1- GFP-NLS-l ⁇ B ⁇ :: URA3) expressing the fusion protein GFP-NLS-l ⁇ Boc located in the nu
- Example 10 illustrates by a Western blotting biochemical analysis, the degradation of the mutant tripartite fusion protein GFP-NLS-1 ⁇ Boc [S3236A] in which the phosphorylation sites Ser32 and Ser36 have been replaced by Ala residues, mutations which in human cells make the protein non-degradable.
- the analysis was carried out in yeast cells also or not expressing the flag-NLS- ⁇ -TrCP tripartite fusion protein. All the strains used were cultivated and analyzed in an identical manner. The cells were cultured in a rich medium in the presence of galactose as a carbon source for 120 minutes.
- yeast strain CYS138 (MATa, his3, Ieu2, trpl, ura3 :: pGAL1- GFP-NLS-l ⁇ B ⁇ [S3236A] :: URA3) expressing the mutant fusion protein GFP-NLS-l ⁇ B ⁇ [S3236A] located in the nucleus of yeast cells;
- yeast strain CYS 139 (MATa, his3, Ieu2, ura3 :: pGAL1-GFP- NLS-l ⁇ B ⁇ [S3236A] :: URA3, trp1 :: pGAL1-3Flag-NLS- ⁇ TrCP :: TRP1) expressing the GFP fusion proteins -NLS-l ⁇ B ⁇ [S3236A] and Flag- NLS- ⁇ -TrCP located in the nucleus of yeast cells.
- Example 11 illustrates, by an epifluorescence microscope analysis, the degradation of the tripartite fusion protein GFP-NLS-l ⁇ B ⁇ [S3236A] in yeast cells when these also express the tripartite fusion protein Flag-NLS- ⁇ - TrCP.
- the fluorescence of the fusion proteins GFP-1ccBcc or GFP-NLS-1 ⁇ Boc is indicated “GFP”.
- the position of the nucleus (indicated "DNA") in the cells was revealed by using a specific nucleus colored indicator, Hoescht 333-42.
- Example 12 illustrates, by quantification of the fluorescence emitted, the degradation of the tripartite fusion protein GFP-NLS-1 ⁇ Bcc [S3236A] in the yeast strains described above.
- Table 1 Genotype of the Saccharomyces cerevisiae yeast strains constructed for the purposes of the present invention.
- Kuras et al. (EMBO J. (1996) 15 (10): 2519-2529). Kuras et al. (Mol. Cell (2002), 10: 69-80).
- Patton et al. (Genes & Dev (1998), 12: 692-705) Patton et al. Genes & Dev (1998), 12: 692-705.
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JP2007503388A JP2007535314A (ja) | 2004-03-16 | 2005-03-15 | IκBαタンパク質ユビキチン化を調節する薬剤のスクリーニング方法および該方法を実施する手段 |
CA002559867A CA2559867A1 (fr) | 2004-03-16 | 2005-03-15 | Procede de criblage d'agents modulant l'ubiquitination de la proteine ikb.alpha. et moyens destines a la mise en oeuvre dudit procede |
US10/592,944 US20080057522A1 (en) | 2004-03-16 | 2005-03-15 | Method for Screening Agents Modulating Ikbalpha Protein Ubiquitination and Means for Carrying out Said Method |
EP05739597A EP1730297A1 (fr) | 2004-03-16 | 2005-03-15 | Procede de criblage d'agents modulant l'ubiquitination de la proteine lkb alpha et moyens destines a la mise en oeuvre dudit procede |
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FR0450528A FR2867783B1 (fr) | 2004-03-16 | 2004-03-16 | Procede de criblage d'agent modulant l'ubiquitination de la proteine ikbalpha et moyens destines a la mise en oeuvre dudit procede |
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WO2009088991A2 (fr) * | 2008-01-03 | 2009-07-16 | Los Alamos National Security, Llc | Système de criblage par double hybride en levure à base gfp avec cytométrie de flux |
Citations (4)
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WO1998036070A1 (fr) * | 1997-02-18 | 1998-08-20 | Signal Pharmaceuticals, Inc. | COMPOSITIONS ET PROCEDES SERVANT A MODULER L'ACTIVATION DE λB-NF CELLULAIRE |
WO1999004033A1 (fr) * | 1997-07-16 | 1999-01-28 | Mitotix, Inc. | ESSAI PERMETTANT DE DECELER LES INHIBITEURS DE LA PROTEOLYSE DU I KAPPA B (IλB) |
WO1999038969A1 (fr) * | 1998-01-30 | 1999-08-05 | Institut National De La Sante Et De La Recherche Medicale | PROTEINE HUMAINE β-TrCP |
EP1182251A1 (fr) * | 2000-08-11 | 2002-02-27 | Yissum Research Development Company of the Hebrew University of Jerusalem | Procédé pour l'identification de composés inhibant la protéolyse de IkB par l'ubiquitine |
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2004
- 2004-03-16 FR FR0450528A patent/FR2867783B1/fr not_active Expired - Fee Related
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- 2005-03-15 CA CA002559867A patent/CA2559867A1/fr not_active Abandoned
- 2005-03-15 JP JP2007503388A patent/JP2007535314A/ja active Pending
- 2005-03-15 EP EP05739597A patent/EP1730297A1/fr not_active Withdrawn
- 2005-03-15 US US10/592,944 patent/US20080057522A1/en not_active Abandoned
- 2005-03-15 WO PCT/FR2005/050165 patent/WO2005093086A1/fr not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1998036070A1 (fr) * | 1997-02-18 | 1998-08-20 | Signal Pharmaceuticals, Inc. | COMPOSITIONS ET PROCEDES SERVANT A MODULER L'ACTIVATION DE λB-NF CELLULAIRE |
WO1999004033A1 (fr) * | 1997-07-16 | 1999-01-28 | Mitotix, Inc. | ESSAI PERMETTANT DE DECELER LES INHIBITEURS DE LA PROTEOLYSE DU I KAPPA B (IλB) |
WO1999038969A1 (fr) * | 1998-01-30 | 1999-08-05 | Institut National De La Sante Et De La Recherche Medicale | PROTEINE HUMAINE β-TrCP |
EP1182251A1 (fr) * | 2000-08-11 | 2002-02-27 | Yissum Research Development Company of the Hebrew University of Jerusalem | Procédé pour l'identification de composés inhibant la protéolyse de IkB par l'ubiquitine |
Non-Patent Citations (4)
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KROLL MATHIAS ET AL: "Inducible degradation of IkappaBalpha by the proteasome requires interaction with the F-box protein h-betaTrCP", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 12, 19 March 1999 (1999-03-19), pages 7941 - 7945, XP002308507, ISSN: 0021-9258 * |
LEWIS A J ET AL: "New targets for anti-inflammatory drugs", CURRENT OPINION IN CHEMICAL BIOLOGY, CURRENT BIOLOGY LTD, LONDON, GB, vol. 3, 1999, pages 489 - 494, XP002197383, ISSN: 1367-5931 * |
STRACK P ET AL: "SCFBETA-TRCP AND PHOSPHORYLATION DEPENDENT UBIQUITINATION OF IKAPPABALPHA CATALYZED BY UBC3 AND UBC4", ONCOGENE, BASINGSTOKE, HANTS, GB, vol. 19, 20 July 2000 (2000-07-20), pages 3529 - 3536, XP000991550, ISSN: 0950-9232 * |
WINSTON JEFFREY T ET AL: "The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro", GENES AND DEVELOPMENT, vol. 13, no. 3, 1 February 1999 (1999-02-01), pages 270 - 283, XP002308533, ISSN: 0890-9369 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009088991A2 (fr) * | 2008-01-03 | 2009-07-16 | Los Alamos National Security, Llc | Système de criblage par double hybride en levure à base gfp avec cytométrie de flux |
WO2009088991A3 (fr) * | 2008-01-03 | 2009-12-30 | Los Alamos National Security, Llc | Système de criblage par double hybride en levure à base gfp avec cytométrie de flux |
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FR2867783B1 (fr) | 2008-02-01 |
US20080057522A1 (en) | 2008-03-06 |
CA2559867A1 (fr) | 2005-10-06 |
EP1730297A1 (fr) | 2006-12-13 |
FR2867783A1 (fr) | 2005-09-23 |
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