EP1941281A1 - Method for screening a proteasome activity modulating agent and means for carrying out said method - Google Patents

Abstract

The invention relates to an in cellulo method for screening a proteasome activity modulating agent consisting a) in bringing a testable candidate agent into contact with recombinant yeast cells expressing in the core thereof a fusion protein comprising (i) a p21 polypeptide selected from p21<SUP>WAF1/Cip1 </SUP>and p21[6KR]<SUP>WAF1/Cip1</SUP> polypeptides and (ii) at least one type of detectable protein, b) in quantifying said first detectable protein in the yeast cells at the end of at least one predetermined time interval after bringing the candidate agent into contact with the yeast cells, and c) in comparing the value obtained at the stage (b) with a control value obtained when the stage (a) is performed in the absence of the candidate agent.

Description

 Method for screening agents that modulate proteasome activity and means for implementing said method

FIELD OF THE INVENTION

The present invention relates to the field of the screening of biologically active agents capable of modulating the proteolytic activity of the proteasome, in particular of agents of therapeutic interest intended to prevent or treat cancers, inflammatory syndromes, bacterial or fungal infections , muscle wasting or neurodegenerative diseases.

PRIOR ART

The ubiquitin proteasome pathway Within human cells, the concentration of any protein results from the balance between its rate of synthesis and its rate of degradation. The ubiquitin-proteasome pathway is the primary mechanism of protein degradation in human cells. The degradation of proteins by the ubiquitin-proteasome pathway is a biological process that is very precisely regulated and controlled over time. In the vast majority of cases, this process involves two major successive steps: (i) the labeling of the proteins to be degraded by the covalent addition of a ubiquitin polymer and (ii) the destruction by the proteasome of the proteins thus labeled, which will be cleaved into small inactive peptides (Glickman and Ciechanover, 2002). A number of proteins are known which are destroyed by the proteasome without being modified by the addition of a ubiquitin polymer. Similarly, peptide sequences have been identified that lead to proteasome degradation of the proteins that contain it (such as the peptide motif FAT10, Hipp et al., 2005) without ubiquitin intervention.

The ubiquitin-proteasome pathway plays a fundamental role in a very large number of biological processes. Indeed, the mechanisms of degradation of proteins by the proteasome are involved in important cellular mechanisms such as the repair of DNA, the control of gene expression, regulation of cell cycle progression, quality control of neosynthesized proteins, apoptosis or immune response (Glickman and Ciechanover, 2002).

Malfunctions of the ubiquitin-proteasome pathway causing the abnormal degradation of regulatory proteins are known to be at the origin of, or closely involved in, several genetic diseases and many pathologies such as cancers

(colorectal, lymphoma ...), inflammatory syndromes, neurodegenerative diseases such as Parkinson's disease or muscular atrophy (cachexia). The diversion of the ubiquitin-proteasome pathway by viruses and pathogenic bacteria (inducing the pathological degradation of human proteins) is at the root of several infection strategies.

The ubiquitin-proteasome domain therefore offers significant potential for the development of new drugs.

The proteasome

The proteasome present in human cells is a very large multi-protein complex (> 2.4 MDa) found in the cytoplasm and nucleus of human cells. The biochemical purifications of the proteasome present in human cells as well as the purifications of proteasomes made from other species of eukaryotic organisms, including yeasts, have shown that in all eukaryotic organisms, the purified forms of the proteasome include 2 major subunits, a proteolytic core called proteasome 2OS, and a 19S regulatory complex that binds to both ends of the 2OS proteasome (Coux et al., 1996, Glickman and Coux, 2001). The 2OS proteasome is a hollow cylinder particle, composed of 28 subunits, distributed in 4 heptameric rings. The peptidase activities are present on the inner surface of the cylinder and influence allosterically. Three proteolytic activities ("trypsin-, chymotrypsin- and caspase-like") have been associated with the 2OS proteasome and contribute to the destruction of proteins as inactive peptides of 3 to 20 amino acids. In addition to the 2OS proteasome, the 26S proteasome includes the regulatory complex 19S. This 19S regulator complex of 0.7 MDa comprises about 20 subunits, and can be dissociated into 2 major subdomains, a "lid" required for ubiquitinated protein recognition, and a base that binds to the 2OS proteasome. This base comprises 6 assembled ATPases in the form of rings, the hydrolysis of ATP is indeed necessary, not only for the active degradation of the ubiquitinated proteins, but also for the unfolding and linearization steps of these proteins, steps which precede the proteolytic degradation of these proteins. Regulatory cap subunits are involved in the recognition of multi-ubiquitinated proteins while others participate in the de-ubiquitination steps necessary to unfold the proteins to be degraded.

More recent studies, including immuno-purification techniques, have shown that the cellular proteasome comprises many proteins closely associated with the 2OS and 19S proteasome, strongly suggesting that the structural complexity of the cellular proteasome is greater than that of the 26S proteasome. Such proteins are, for example, PA200 human proteins (whose homologue in yeast is the 240 kDa BImIO protein (Schmidt et al., 2005), or the PA28αβ or PA28αγ activators (Wojcik et al., 1998; Ustrell et al. The functional roles of these proteins are not yet fully understood, but they could be proteins that transfer ubiquitinated proteins to the proteasome, proteins that regulate ubiquitinated protein recognition by the proteasome, or enzymes such as the Hul5 enzyme (Leggett et al., 2002), which are involved in the extension of polyubiquitin chains and serve to increase the rate of transfer of proteins to the peptidase sites. degradation of more than 80% of cellular proteins.

Proteome and cancer

It has recently been demonstrated that protein degradation by the proteasome is a biological process that is fundamental to the survival of cancer cells. Indeed, Numerous studies have clearly shown that many types of malignant cells are more sensitive to proteasome inhibitors than normal cells. Tests conducted with proteasome inhibitors, carried out either in vitro or in vivo, on different models of cancer cells have demonstrated that this class of molecules has particular properties that make them attractive candidates for developing new anti-cancer therapeutic weapons. . Ultimately, inhibition of proteasome activity induces apoptosis, increases the sensitivity of cancer cells to conventional chemotherapy and radiotherapy. Similarly, it has been observed that inhibition of the proteasome substantially reduces resistance to chemotherapy and radiotherapy (Adams 2004, Burger and Seth 2004).

Inhibition of regulatory degradation of important cycle and cellular homeostasis such as p21, p27 and p53 proteins has been implicated as a mechanism by which proteasome inhibition affects the growth and survival of tumor cells. Importantly, it has also been shown that the inhibition of the proteasome blocks the activation of the NF-κB cellular regulator whose aberrant activation is a characteristic of several blood cancers. Characterization of proteasome inhibitors has shown that they induce apoptosis (Pei et al., 2003), kill tumor cells (Beverly et al., 1999), increase sensitivity to radiation (Adams and Anderson, 2001), and overcome drug resistance (Hideshima et al., 2001). In addition, proteasome inhibition has been shown to block anti-apoptotic signals that are triggered by radiotherapy or chemotherapy.

It should also be noted that the specific sensitivity of malignant cells to proteasome inhibitors may result from their rapid proliferation which is associated with dysfunction of one or more "checkpoint" mechanisms. These alterations result in the rapid accumulation in malignant cells of a large number of defective proteins, and this much more markedly than in normal cells. This rapid and important accumulation of mutant proteins, and / or misfolding is most likely to significantly increase the dependence of malignant cells on an active proteasome and, therefore, make them very sensitive to proteasome inhibitors (Adams, 2004). Proteasome inhibitors can be synthetic or natural. Five major families of inhibitors have been described to date: peptide aldehydes, vinyl sulfone peptides, boronic peptide derivatives, peptide epoxicetone derivatives and β-lactones. To date, however, only two compounds have been developed to clinical stages following the demonstration of their anti-neoplastic properties; bortezomib (formerly PS-341) developed by the company Millennium (Cambridge, MA, USA) and the compound NPI-0052 developed by the company Nereus (San Diego, CA, USA)

Bortezomib, a boronic derivative of a dipeptide, has been shown to be effective against a broad spectrum of cancer lines, including colon cancer lines, central nervous system malignancies, prostate cancer lines, and prostate cancer. breast. Bortezomib is the first proteasome inhibitor to have been tested in clinical trials. The positive results of phase II and phase III trials in patients with myeloma who failed the first two therapies, demonstrated the efficacy of bortezomib and led to the approval of the marketing of bortezomib by the FDA in 2003 for the treatment of myeloma refractory to conventional therapies. Bortezomib is marketed under the name of Velcade ^® .

Bortezomib inhibits with great specificity one of the 3 proteolytic activities of the proteasome 2OS, ("chymotrypsin-like" activity). Its action induces apoptosis and its effects seem to be exerted as well by the activation of pro-apoptotic pathways as by the suppression of anti-apoptotic pathways (Hideshima et al., 2001, Beverly et al., 1999).

The appearance of Velcade®-resistant cells and the high toxicity of this compound which limits its application makes it necessary to develop new proteasome inhibitors. In particular, proteasome inhibitors which are known are catalytic inhibitors of the proteasome which can explain their toxicity and it would be interesting to develop non-catalytic inhibitors of the proteasome. Such non-catalytic inhibitors could be active by inhibiting the activity of 19S regulatory complexes and associated proteins that are necessary for the functioning of the cellular proteasome (ie, as it exists in cells). It is important to note that the inhibitors that are known and developed to date have been identified through in vitro biochemical tests that report the proteolytic activity of the purified proteasome against non-natural substrates such as peptides that diffuse freely in the catalytic cylinder 2OS. Such tests therefore do not report the activity of the cellular proteasome since they do not make use in particular of the functions of the regulatory complexes associated with the proteasome 2OS. In general, there is a need to identify therapeutic compounds that are capable of inducing the destruction of malignant cells.

DESCRIPTION OF THE INVENTION

According to the invention, a method has been developed for screening agents of therapeutic interest, which are selected for their specificity of action on the cellular proteasome. This screening process is carried out in cellulo and, unlike existing biochemical tests, makes it possible to identify catalytic or non-catalytic inhibitors of the cellular proteasome.

The applicant has shown that, surprisingly, it is possible to mimic, in yeast cells, the proteasome-dependent, but independent of prior ubiquitination-dependent, degradation process of the human cellular regulator p2i ^{WAF1 / c} ' ^P1 by the proteasome, a process that takes place naturally in human cells. The _P 21 ^{WAF1 / Cιp1 protein} is a regulator of the dividing cycle of human cells that belongs to the family of "Cyclin Dependent Kinases" inhibitors, Cdk. The main function of the _P 21 ^{WAF1 / Cιp1 protein} is to regulate the activity of Cdk2 / cyclin complexes, the activation of which controls the progression of the cell cycle. The p2i protein ^{WAF1 / Cip1} is also known to regulate DNA synthesis by interacting with the PCNA factor (proliferating cell nuclear antigen), a subunit of DNA polymerase δ essential for chromosomal replication. The p2i protein ^{WAF1 / Cιp1} is a poorly structured, highly unstable protein (Kriwacki et al., 1996). There are two degradation pathways of the p2i protein ^{WAF1 / c} ' ^P1 . The former depends on ubiquitination and involves ^{SCP Skp2} ubiquitin ligase. This pathway is particularly induced during irradiation of cells with UV rays (Bendjennat et al 2003, Bomstein et al., 2003). The second pathway does not require the ubiquitination of p21 ^{WAF1 / C |} P ¹ but is nevertheless dependent on the activity of the proteasome. This second degradation pathway, which does not imply the ubiquitination of p2i ^{WAF1 / CIP1,} seems to ^imply in Mammalian cells the Mdm2 protein that would activate the degradation of p2i ^{WAF1 / CIP1} by promoting its physical interaction with the proteasome (Zhang et ^al. al., 2004). The ubiquitin ligase function of Mdm2 is not involved in this mechanism.

Since the yeast cells do not express orthologous protein or similar to the human Md m2 protein, it is therefore completely surprising that the applicant has shown that it is nevertheless possible to mime in yeast cells. degradation of the human p2i protein ^{WAF1 / Cιp1} by the proteasome, according to a proteasome degradation pathway which is not dependent on a ubiquitination step of the p2i protein ^{WAF1 / Cιp1} , prior to its degradation.

The invention relates to an in cellulo method for screening agents that modulate the activity of the proteasome, said method comprising the following steps: a) contacting a candidate agent to be tested with recombinant yeast cells that express in their nucleus a fusion protein comprising: (i) a p21 polypeptide selected from the polypeptide _P 21 ^{WAF1 / Cip1} and the polypeptide p21 [6KR] ^{WAF1 / Cip1} ; and (ii) at least one detectable protein. b) quantifying said first detectable protein in the yeast cells at the end of at least a predetermined period of time after contacting the candidate agent with said cells; c) comparing the value obtained in step (b) with a control value obtained when step (a) is performed in the absence of the candidate agent.

The process of the invention consists of a process which is carried out in cellulo, since the degradation of the target protein p2i ^{WAF1 / CIP1} or p21 [6KR] ^{WAF1 / CIP1} is carried out and visualized in yeast cells and not by reactions performed in a cell-free system.

In some embodiments of the above screening method, yeast cells which express a fusion protein comprising a mutant form of the p2i protein ^{WAF1 / Cιp1} in which the 6 lysine residues of the wild-type p21 protein have been substituted are used. by arginine residues. This mutant form of the _p21 w protein ^{AFi /} c ⁱ _P ⁱ _{is designated P} 21 [6KR] ^{WAF1 / Cip1} . These amino acid substitutions suppress all potential ubiquitination sites on the protein. The degradation of the derivative p21 [6KR] ^{WAF1 / Cip1} is therefore, because of the absence of ubiquitination sites in its amino acid sequence, exclusively and directly dependent on the proteolytic activity of the proteasome, and independent of a stage of ubiquitination.

Finally, it has also been shown that, in yeast cells, the degradation of the p21 polypeptide ( _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 / Cip1} ) by the proteasome takes place, even when the p21 polypeptide (p2i ^{WAF1 / c P1} or p21 [6KR] ^{WAF1 / Cip1} ) is fused to a detectable protein and in particular to a self-fluorescent protein. Surprisingly, in the yeast cells, the degradation of the fusion protein is complete, the polypeptides corresponding to the p21 protein ( _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 / Cip1} ) and to the detectable protein are both degraded. These results are surprising because previous experiments in biochemistry, performed in vitro, had demonstrated that when a fusion protein composed of p2 ^< | ^{WAFI /} c ^ιpi f _us j _onn Q _e Q _an autofluorescent protein is in the presence of the 26S proteasome or 2OS, then only the part _P21 ^{WAF1 / Cip1} is degraded, the portion corresponding to the auto-fluorescent moiety being released under a stable non-degraded form (Liu et al., 2003).

The total degradation of the p21 fusion protein ( _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 / Cip1} ), observed in the yeast cells, makes it possible to measure the amount of fusion protein present in the yeast by quantifying the signal generated by the detectable protein included in said fusion protein.

The above method enables one skilled in the art to determine whether a candidate agent to be tested is capable of modifying the rate of degradation, or the degree of degradation, of the p21 protein (p21 ^{WAF1 / Cιp1} or p21 [6KR] ^{WAF1 / Cip1} ) by the proteasome in yeast cells expressing the normal or mutated human p21 protein (p2i ^{WAF1 / Cιp1} or p21 [6KR] ^{WAF1 / Cip1} ).

The inventive cellulo screening method, in that it implements an artificial and humanized system for degradation of a target fusion protein in yeast cells, allows screening of agents which act specifically on the activity of the cellular proteasome, as it exists in all its complexity in the cell, including in the absence of a prior step of ubiquitination of the target protein in the cell.

In addition, by means of the above method, a physiological test for the screening of active agents on the proteasome has been developed, by constructing in yeast a metabolic pathway of protein degradation mimicking the pathway of degradation by the proteasome of the regulatory factor. p2i ^{WAF1 / c} ' ^P1 human. Thus, from the point of view of the metabolic pathway of protein degradation that is targeted, the method of the invention is carried out under physiological conditions very close to the physiological conditions of degradation of proteins by the human proteasome. For purposes of this disclosure, an agent that "modulates" proteasome activity includes, respectively, (i) an agent that inhibits or blocks proteasome activity, particularly that inhibits or blocks the activity of the proteasome independent of the proteasome. a step of ubiquitination prior to the degradation of the target protein, and (ii) an agent that activates or increases the activity of the proteasome, in particular that activates or increases the activity of the proteasome independent of a ubiquitination step prior to the degradation of the target protein.

By the above method, agents capable of inhibiting the rate or degree of intracellular degradation of the _p 2i polypeptide ^{WAF1 / CiP1} or the polypeptide p21 [6KR] ^{WAF1 / Cip1} by the proteasome yeast cells. Such inhibitory agents, which can be identified by the method of the invention, because they will also inhibit the activity of the proteasome in human cells, are agents of therapeutic interest capable of inhibiting or blocking the proliferation of tumor cells, to inhibit or block the activation of the expression of various genes involved in inflammation, autoimmune pathologies or cancers, to disrupt mechanisms for controlling cell death (apoptosis ). Thus, the above cellulo screening method may comprise an additional step (d) of positively selecting the inhibitory candidate agents for which the amount of detectable protein measured in step (b) is greater than the control value of comparison. The method of the invention also makes it possible to identify agents capable of increasing the rate or degree of degradation of the _p 2i polypeptide ^{WAF1 / CiP1} or of the p21 [6KR] ^{WAF1 / Cip1} polypeptide by the proteasome of yeast cells. Such activating agents, because they will also activate the proteasome activity in human cells, are agents of therapeutic interest capable of inducing or increasing the activation of the expression of various genes involved in inflammation, autoimmune diseases or cancers. Thus, according to this second aspect, the in vitro screening method of the invention makes it possible to screen pro-inflammatory agents. Some of the pro-inflammatory agents selected according to the method are likely to be of therapeutic interest when used at low dose or when administered for a short time, for example as inducing agents of an early immune response. , such as inducing a nonspecific resistance reaction to infection or such as activating antigen-presenting cells, for initiating an antigen-specific immune response, that it is either humoral or cell-mediated. Certain other 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, of which one adverse proinflammatory effect is identified, and for which particular precautions for use with respect to human health should be observed. Similarly, some of the agents selected according to the method are likely to have pro-apoptotic activities. Thus, according to another aspect, the screening method according to the invention may comprise an additional step (d) which consists in positively selecting the activating candidate agents for which the amount of detectable protein measured in step (b) is less than to the comparison control value. As will be understood, the agent modulating the activity of the proteasome can be of any kind. The agent may be any organic or inorganic compound, and may be either a naturally occurring agent or an agent produced, at least in part, by chemical or biological synthesis. Said agent may in particular be a peptide or a protein. Said agent also includes any molecule already known to have a biological effect, including a therapeutic effect, or conversely a toxic effect demonstrated or suspected for the body.

In the method of the invention, once the fusion protein, comprising the p2i polypeptide ^{WAF1 / c} ' ^P1 _or | _The p21 [6KR] ^{WAF1 / Cip1} polypeptide fused to a detectable protein is expressed in yeast cells, said fusion protein is recognized and undergoes proteolysis which is carried out by the proteasome. Quantification of the detectable protein contained in the yeast cell, at a given moment, makes it possible to determine the degree of degradation of said p21 fusion protein ^{WAF1 / Cip1-} detectable protein or p21 [6KR] ^{WAF1 / Cip1-} detectable protein, to this moment.

A polypeptide P2i ^{WAF1 / Cιp1} according to the invention consists of a polypeptide having at least 90% amino acid identity with the polypeptide _p 2i ^{WAF1 / CIP1} sequence SEQ ID N ⁰ 1. a polypeptide _p 2i ^{WAF1 / CIP1} according to invention can consist in the _p 2i ^{WAF1 / CiP1 polypeptide} of sequence SEQ ID N ⁰ 1.

A p21 polypeptide [6KR] ^{WAF1 / Cip1} according to the invention consists of a polypeptide having at least 90% amino acid identity with the polypeptide p21 [6KR] ^{WAF1 / Cip1} sequence SEQ ID N ⁰ 2. a polypeptide p21 [6KR] ^{WAF1 / Cιp1} according to the invention may consist of the p21 [6KR] ^{WAF1 / Cip1} polypeptide of sequence SEQ ID N ⁰ 2.

For purposes of this disclosure, the term "nucleotide sequence" may be used to denote either a polynucleotide or a nucleic acid. The term "nucleotide sequence" encompasses the genetic material itself and is therefore not restricted to information about its sequence.

The terms "nucleic acid", "polynucleotide", "oligonucleotide" or "nucleotide sequence" include RNA, DNA or cDNA sequences, or hybrid RNA / DNA sequences of more than one nucleotide, regardless of in the single-stranded form or in the form of duplex.

The term "nucleotide" refers to natural nucleotides (A, T, G, C and U). For purposes of the present invention, a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the complementary base of the second polynucleotide whose orientation is reversed. The complementary "bases" are A and T (or A and U), and C and G. According to the invention, a first nucleic acid having at least 90% identity with a second reference nucleic acid, will have at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99%, 6% nucleotide identity with said second reference nucleic acid. According to the invention, a first polypeptide having at least 90% identity with a second reference polypeptide, will have at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96% 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% amino acid identity with said second reference polypeptide. The "percent identity" between two nucleic acid sequences or between two polypeptide sequences in the sense of the present invention is determined by comparing the two optimally aligned sequences through a comparison window.

The part of the nucleotide or amino acid sequence in the comparison window may thus comprise additions or deletions (for example "gaps") relative to the reference sequence (which does not include these additions or deletions) so as to obtain an optimal alignment between the two sequences.

The percent identity is calculated by determining the number of positions at which an identical nucleotide base, or identical amino acid residue, is observed for the two compared sequences, and then dividing the number of positions at which there is identity between the two nucleic bases, or between the two amino acid residues, by the total number of positions in the comparison window, then multiplying the result by one hundred in order to obtain the percentage nucleotide identity of the two sequences between them, or the percentage amino acid identity of the two sequences between them ..

The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms. Most preferably, the percentage of sequence identity is determined using the CLUSTAL W software (version 1.82) with the parameters set as follows: (1) CPU MODE = ClustalW mp; (2) ALIGNMENT = "full"; (3) OUTPUT FORMAT = "aln w / numbers"; (4) OUTPUT ORDER = "aligned"; (5) COLOR ALIGNMENT = "no"; (6) KTUP (word size) = "default"; (7) WINDOW LENGTH = "default"; (8) SCORE TYPE = "percent"; (9) TOPDIAG = "default"; (10) PAIRGAP = "default"; (11) PHYLOGENETIC TREE / TREE TYPE = "none"; (12) MATRIX = "default"; (13) GAP OPEN = "default"; (14) END GAPS = "default"; (15) GAP EXTENSION = "default"; (16) GAP DISTANCES = "default"; (17) TREE TYPE = "cladogram" and (18) TREE GRAP DISTANCES = "hide".

According to the invention it has been shown that the sensitivity of the screening method described above is increased when, prior to contacting the yeast cells with the agent to be tested, the expression of the target p21 protein is stopped in yeast cells, that is to say either the p21 fusion protein ^{WAF1 /} Cip1-detectable protein or the p21 [6KR] fusion protein ^{WAF1 / Cip1-} detectable protein.

Thus, according to a first preferred embodiment of the method above, step (a) itself comprises the following steps: (ai) culturing yeast cells which express in their nucleus said fusion protein comprising the p21 polypeptide and at least one detectable protein;

(a2) stopping the expression of said fusion protein comprising the p21 polypeptide and at least one protein detectable by yeast cells;

(a3) contacting the yeast cells obtained at the end of step (a2) with the candidate agent to be tested.

Stopping the expression of the detectable protein p21 ^{WAF1 / Cip1-} protein protein at a chosen time can be easily achieved by ^those skilled in the art, using, to transform the yeast cells, a cassette of wherein the polynucleotide encoding said fusion protein is under the control of a promoter functional in yeast cells and 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.

In practice, the detectable protein-p21 fusion protein is rapidly recognized and degraded by the proteasome in yeast cells.

The optimum conditions for carrying out the screening method according to the invention are the conditions under which an intracellular accumulation of a large quantity of the detectable protein p21-protein which is expressed in the yeast cells is induced. The higher the intracellular quantity of detectable protein p21-protein found at the beginning of step a) of the method, the higher the value of the signal generated by the detectable protein, which can be measured with great precision at the time of detection. step b) of the process. Thus, the greater the intracellular quantity of detectable p21-protein fusion protein is large at the beginning of step a), the more the variations in the amount of this fusion protein can be measured accurately, in step b) of the method .

To obtain the accumulation of a large intracellular amount of detectable p21-protein fusion protein in yeast, it is advantageous to place the sequence coding for said fusion protein under the control of a strong repressible promoter and to use yeast strains having several copies of a polynucleotide comprising (i) a strong repressible promoter, (ii) a nucleic acid encoding the detectable p21-protein fusion protein. The presence of several copies of the polynucleotide coding of interest makes it possible to obtain a strong detection signal for the detectable protein in step a) of the method. These high signal conditions make it possible to quantify the detectable protein with great sensitivity throughout the duration of the process, as the protease-detectable protein p21-protein fusion protein is degraded. Obviously, the stronger the detectable signal of departure, the better the sensitivity of the measurements during the implementation of the method.

According to an advantageous embodiment of the screening method according to the invention, yeast cells in which the copy or copies of the polynucleotide encoding the detectable p21-protein fusion protein are (are) integrated into the chromosome are used. This advantageous embodiment allows the intracellular accumulation of the detectable p21 protein-protein at a high level in all cultured yeast cells for the implementation of the method. This embodiment of the screening method of the invention is an advantage over the use of plasmid-transformed yeast cells in each of which one or more copies of the polynucleotide encoding the detectable p21-protein fusion protein is ( are) inserted. Indeed, when the yeast cells which express the detectable p21-protein fusion protein consist of cells containing one or more recombinant vectors, in particular one or more recombinant plasmids, in which at least one copy of the polynucleotide encoding the protein is inserted. detectable protein fusion, it may happen that the transmission of said recombinant vectors of interest is not carried out homogeneously, with successive generations of yeast cells. In some cases, the level of intracellular expression of the detectable protein p21-proteine protein would be heterogeneous within the set of cells. cultured yeast, which would be likely to reduce the sensitivity of the screening method of the invention.

Description of the preferred embodiments of the screening method

The preferred embodiments of the screening method of the invention are described below, in particular in connection with the description of the functional and structural aspects of the various means for implementing said method. In general, the detectable protein that is included in the detectable p21-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 forms proteolysed proteins 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.

As is easily understood, the proteolytic activity of the proteasome is monitored, according to the method of the invention, by measuring its effect on the stability of the detectable protein p21-protein fusion. Thanks to the expression in yeast cells of the factor p21 in the form of a fusion protein, the degradation of the p21-containing fusion protein can be monitored in real time, by detecting the unproteolysed detectable protein.

Preferentially, the detectable protein p21-protein fusion protein comprises a mutant form of the p2i polypeptide ^{WAF1 / Cιp1} in which the 6 lysine residues of the p21 protein have been substituted by arginine residues, which is designated p21 [6KR] ^{WAF1 / Cιp1} . The stability of the detectable protein p21 [6KR] ^{WAF1 / Cip1-} protein protein depends exclusively on the proteolytic activity of the proteasome and does not imply its prior ubiquitination. Depending on the nature of the detectable protein fused to p21 _(p21 w ^{AF1 /} c ⁱ _P ⁱ _{or P} 21 [6KR] ^{WAF1 / Cip1} ), degradation of the fusion protein can be followed by techniques known per se, including fluorescence measurement with the aid of a flow cytometer, a microplate reader or a fluorimeter, or by means of a fluorescence microscope, or by colorimetric, enzymatic or immunological. By way of illustration, the detectable protein may be chosen from an antigen, a fluorescent protein or a protein having an enzymatic activity.

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, they can be prepared, according to any technique for obtaining antibodies, especially for polyclonal or monoclonal antibodies, well known to those skilled in the art. Preferentially, in this case, the detectable protein consists of a small antigen, which is not likely to interfere with the recognition of the p21 protein ( _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 / Cip1} ) by the proteasome. Thus, preferably, as antigen is used a peptide having a chain of 7 to 100 amino acids in length, better 7 to 50 amino acids in length, and more preferably 7 to 30 amino acids in length, for example 10 acids. amines of length. Illustratively, there may be used the antigen HA sequence [NH ₂ - YPYDVPDYA-COOH] SEQ ID ⁰ 7, or a sequence of FLAG antigen [NH ₂ -DYKDDDDK-COOH] SEQ ID ⁰ 8 (monomer FLAG ) or sequence [NH ₂ -MDYKDHDGDYKDHDIDYKDDDDK-COOH] SEQ ID NO 9 ⁰ (FLAG trimer) in this case, is used to quantify the detectable protein in step (b) of the method, an antibody that specifically recognizes the antigen included in the fusion protein, this antibody being labeled directly or indirectly. Quantification is then performed by measuring the detectable signal produced by the complexes formed in the yeast cells between the labeled antibody and the p21-antigen fusion protein. Thus, in step (b), when the first detectable protein is an antigen, said first detectable protein is quantified by detecting complexes formed between said protein and recognizing antibodies. When the detectable protein consists of an intrinsically fluorescent protein, it is especially chosen from the mAG protein, the GFP protein or one of its derivatives, the YFP protein or one of its derivatives, and the dsRED protein. Among the proteins derived from the GFP protein, it is possible to use in particular any of the proteins known under the names GFPMut3 (Cormack et al., Gene (1996) 173: 33-38)), Venus (Nagai et al., (Nat Biotechnol (2002) 20: 87-90)) or Sapphire (Zapata-Hommer and Griesbeck, BMC Biotechnol (2003) 3: 5). The intrinsically fluorescent protein may also be selected from auto-fluorescent proteins originating from various organisms, other than Aequorea Victoria. In particular, the intrinsically fluorescent protein may 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); PhiYFP protein from Phialidium sp. ; and described by DA Shagin et al (2004, Mol Biol Evol 21: 841-850); the AcGFP protein native to Aequorea coerulescens, as well as its variants, and described by NG Gurskaya, (2003, Biochem J. 373: 403-408); and the DsRed protein from Discosoma sp. ; and described by MV Matz et al (1999, Nature Biotech 17: 969-973). Preferably, in the p21 protein-detectable fusion protein, the mAG protein, also called "monomeric Azami- Green" originating from the coral of Galaxeidae, will be used; and described by Karasawa et al. (Karasawa et al., J. Biol Chem 2003 278: 34167-34171).

When the detectable protein consists of an intrinsically fluorescent protein, the detectable protein is quantified in step (b) of the method by measuring the fluorescence signal that is emitted by the p21-fluorescent protein fusion protein using any adapted device. Thus, in step (b), when the detectable protein is a fluorescent protein, said detectable protein is quantified by measuring the fluorescence signal emitted by said protein.

When the detectable protein consists of a protein with enzymatic activity, said detectable protein is chosen in particular from luciferase and β-lactamase. In this case, the detectable protein is quantified in step (b) of the process by measuring the amount of compound (s) produced by the conversion of the substrate by the enzyme. When the product of the enzymatic activity is colored, the measurement can be performed by colorimetry. When the product of the enzymatic activity is fluorescent, the intensity of the fluorescence signal emitted by said product is measured using any suitable fluorescence measuring device. Thus, in step (b), when 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.

According to a preferred embodiment, the protein comprising the _P 21 ^{WAF1 / Cιp1 polypeptide} consists of the amino acid sequence protein SEQ ID No. 3, which may be encoded by the nucleic acid of sequence SEQ ID No. 5. The protein of SEQ ID NO ^: 3 sequence consists, from the ₂ -terminal NH end to the COOH-terminal end, respectively in (i) the sequence of the detectable mAG protein ranging from the amino acid in position 1 to the at the amino acid at position 226, (ii) a first spacer peptide ranging from amino acid at position 227 to the amino acid at position 228 and (iii) the polypeptide _P 21 ^{WAF1 / Cip1} ranging from amino acid in position 229 to the amino acid in position 392. The nucleic acid of sequence SEQ ID No. 5 consists, from the 5 'end to the 3' end, respectively in (i) the coding sequence the detectable mAG protein ranging from the nucleotide at position 1 to the nucleotide at position 678, (ii) the sequence coding for a spacer peptide ranging from the nucleotide at position 679 to the nucleotide at position 684 and (iii) the sequence coding for the p2i polypeptide ^{WAF1 / CIP1} ranging from the nucleotide at position 685 to the nucleotide at position 1179 (cod we stop included). According to another preferred embodiment, the protein comprising the p21 [6KR] ^{WAF1 / Cιp1 polypeptide} consists of the amino acid sequence protein SEQ ID N ⁰ 4, which can be encoded by the nucleic acid of sequence SEQ ID N ⁰ 6. The protein of sequence SEQ ID NO ^: 4 consists, from the _2- terminal NH end to the COOH-terminal end, respectively in (i) the sequence of the detectable mAG protein ranging from the amino acid in the 1-position. to the amino acid at position 226, (ii) a first spacer peptide ranging from amino acid at position 227 to the amino acid at position 228 and (iii) the polypeptide p21 [6KR] ^{WAF1 / Cip1} ranging from the amino acid at position 229 to the amino acid at position 392. The nucleic acid of sequence SEQ ID No. 6 consists, from the 5 'end to the 3' end, respectively in (i) the sequence encoding the detectable mAG protein from the nucleotide at position 1 to the nucleotide at position 678, (ii) the sequence coding for a spacer peptide ranging from the nucleotide at position 679 to the nucleotide at position 684 and (iii) the sequence coding for the polypeptide p21 [6KR] ^{WAF1 / Cip1} ranging from the nucleotide at position 685 to the nucleotide at position 1179 (stop codon included).

In SEQ ID N ⁰ 5 polynucleotide sequence encoding the fusion protein comprising _P21 ^{WAF1 / Cip1} and of sequence SEQ ID N ⁰ 6 encoding the fusion protein comprising p21 [6KR] ^{WAF1 / Cip1,} the identity of the nucleobase has has been adapted to include, in these nucleic sequences, the codons which are preferentially used in yeast cells.

According to yet another aspect, the screening method according to the invention is characterized in that the recombinant yeast cells are transformed with a polynucleotide which comprises:

(a) an open reading frame encoding (i) the fusion protein comprising the p21 polypeptide ( _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 /} Cip1) and (ii) a detectable protein, and (b) a regulatory sequence functional in yeast cells that directs the expression of said open reading frame; The above polynucleotide may consist of the nucleic acid of sequence SEQ ID No. 5 or the nucleic acid of sequence SEQ ID No. 6.

Nucleic acids, expression vectors and transformed yeast cells preferred according to the invention.

Nucleic acids have been synthesized according to the invention, which, when introduced into yeast cells, cause the expression of the detectable protein p21-protein fusion protein, that is to say nucleic acids encoding the protein of the invention. p21 fusion ^{WAF1 / CIP1-} detectable protein and nucleic acids encoding the p21 fusion protein [6KR] ^{WAF1 / Cip1-} detectable protein.

Each of the synthesized nucleic acids comprises a coding sequence, which is also referred to as an "open reading frame" or "ORF" which encodes the p21 fusion protein - detectable protein of interest. Illustrative examples of the nucleic acids according to the invention are the nucleic acids of sequence SEQ ID N ⁰ 5 (p21 ^{WAF1 / Cip1} detectable -protein) and SEQ ID N ⁰ 6 (p21 [6KR] ^{WAF1 / Cip1} detectable -protein), including the structure has been described previously in the description.

Each of the nucleic acids also comprises a regulatory sequence comprising a promoter functional in yeast cells.

In a preferred embodiment, the functional promoter in yeast cells consists of a repressible promoter, that is to say a functional promoter in yeast cells and which is sensitive to the action of an inducing agent. When the inducing agent is added to the culture medium of the yeast cells, it induces the repression or the blocking of the expression of the sequence encoding the protein of interest placed under its control. This repressible promoter is advantageously chosen from the promoters CUP1, GAL1, GAL10,

MET3, MET25, PH05, PHO87 and THI4 of the yeast Saccharomyces cerevisae.

The promoter sequence of the GAL1 gene of the yeast S. cerevisiae, which can be used according to the invention may consist of that described by Johnston and Davis (Mol., C. Biol. (1984) 4: 1440-1448).

The promoter sequence of the S. cerevisiae yeast MET3 gene that can be used according to the invention may consist of that described by Cherest et al. (Gen. Gen. Genet (1987) 210 (2): 307-313).

The promoter sequence of the S. cerevisiae yeast MET25 gene that can be used according to the invention may consist of that described in Kerjan et al. (Nucleic Acids Res (1986) 14 (20): 7861-7871).

The promoter sequence of the S. cerevisiae yeast PHO5 gene that can be used according to the invention can consist of that described by Feldmann et al. (EMBO J. (1994) 13 (24): 5795-5809). The promoter sequence of the THI4 gene of S. cerevisiae yeast, which can be used according to the invention may consist of that described by Skala et al., Yeast (1995) 14: 1421-1427.

The promoter sequence of the CUP1 gene of S. cerevisiae yeast, which can be used according to the invention may consist of that described by Karin et al. (PNAS (1984) 81 (2): 337-341).

In a preferred embodiment, the nucleic acid or polynucleotide that encodes the detectable p21-protein fusion protein comprises the GAL1 regulatory sequence. This sequence activates expression of the open reading frame encoding the p21 polypeptide fusion protein when the yeast cells are cultured in the presence of galactose. On the other hand, this sequence represses the expression of the open reading frame encoding the fusion protein comprising the p21 polypeptide when the yeast cells are cultured in the presence of glucose.

Thus, in an advantageous embodiment of the screening method of the invention, the expression of the fusion protein comprising the p21 polypeptide is carried out temporarily during the screening test. After having been induced for a fixed time which can vary from 20 minutes to 24 hours, the expression of the protein containing p21 is specifically stopped (in an experiment known to those skilled in the art under the name of "promoter shut off") before to expose the cells to the molecules to be screened. This termination of the expression is obtained by the addition in the culture medium of a molecule that can repress the activity of the promoter controlling the expression of the p21-detectable protein fusion protein, or else by eliminating the presence, in the culture medium, of an agent or a molecule required for the activation of said promoter.

Thus, when the detectable protein p21-protein fusion protein is expressed under the control of the GAL1 gene promoter, then the expression of this promoter is repressed by adding glucose to the final concentration of 2% in the culture medium. Stopping the neosynthesis of the fusion protein comprising p21 makes it possible to measure its stability in real time, for example by determining the fluorescence of the yeast cells over time after stopping synthesis, in the wherein said fusion protein contains a detectable intrinsically fluorescent protein, such as mAG or a GFP-derived protein.

Thus, the invention also relates to a functional expression cassette in yeast cells comprising a polynucleotide which comprises an open reading frame encoding the fusion protein comprising the p21 polypeptide and at least one detectable protein, and a regulatory sequence functional in yeast cells that directs the expression of said open reading frame. Such an expression cassette may be characterized in that the p21 polypeptide is chosen from the p2i ^{WAF1 / Cip1} polypeptide and the p21 [6KR] ^{WAF1 / Cip1} polypeptide.

Such an expression cassette may in particular be characterized in that the p21 polypeptide is selected from the polypeptide P2i ^{WAF1 / Cιp1} sequence SEQ ID N ⁰ 1 and the p21 polypeptide [6KR] ^{WAF1 / Cip1} sequence SEQ ID N ⁰ 2.

Such expression cassette may in particular comprise or consist of the nucleic acid of sequence SEQ ID No. 5 according to the invention, which encodes the mAG- _P 21 ^{WAF1 / Cip1} fusion protein of sequence SEQ ID No. 3.

Such an expression cassette may also comprise or consist of the nucleic acid of sequence SEQ ID N ⁰ 6 according to the invention, which encodes the fusion protein MAG- p21 [6KR] ^{WAF1 / Cip1} sequence SEQ ID N ⁰ 4 According to a preferred embodiment of such an expression cassette, the regulatory sequence comprises a repressible promoter functional in yeast cells, and which is sensitive to the action of an inducing agent, such as a chosen promoter. among the promoters CUP1, GAU, GAUO, MET3, MET25, PHO5, and THI4 of the yeast Saccharomyces cerevisiae.

According to another advantageous embodiment of the screening method according to the invention, the recombinant yeast cells possess the nucleic acid or the polynucleotide comprising the sequence coding for the p21-proteine fusion protein detectable in a form integrated in their genome, such as this is illustrated in the examples In yet another embodiment of the screening method according to the invention, the yeast cells are transformed with the nucleic acid or the polynucleotide comprising the sequence coding for the detectable p21 protein-protein which is in a non-integrated form in the chromosomes, for example in the form of functional vectors in yeast cells and which carry at least one origin of functional replication in yeast cells.

In general, for the implementation of the screening method of the invention, it is advantageous to use yeast cells which have good membrane permeability, including good membrane permeability for the agents to be tested by the method.

For the implementation of the preferred embodiment of the screening method of the invention in which the expression of the fusion protein is carried out under the control of a repressible promoter, it is also advantageous to use yeast cells which have good membrane permeability for the "inducing" compounds against which said repressible promoters are sensitive.

Thus, in another preferred embodiment of the screening method of the invention, yeast strains whose genome comprises one to several mutations which increase the permeability to the products to be tested, such as mutations inactivating the PDR1 and PDR3, two genes encoding transcriptional factors that in yeast control the expression of transporters inserted into the plasma membrane (Vidal et al., 1999, Nourani et al., 1997). Preferentially, yeast strains possessing the genetic background of the strain W303 of the yeast Saccharomyces cerevisiae described by Bailis et al. (1990), or any other characterized strain of said yeast Saccharomyces cerevisiae.

Transformation of yeast cells with exogenous DNA is preferably carried out using techniques known to those skilled in the art, in particular the technique described by Schiestl et al. (1989). The constructs of the various yeast strains were made using genetic techniques (cross, sporulation dissection of asci and phenotypic analysis of spores) known and described in particular by Sherman et al. (1979) and reverse genetics techniques described in particular by Rothstein (1991).

According to the invention, the yeasts are preferably converted 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).

Thus, another subject 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 according to the invention is the vector pCSYAQ6-p21wt which is described in the examples, and which was used for the construction of the yeast strain CYS343.

A second vector according to the invention is the vector pCSYAQ6-p21 [6KR] which is described in the examples, and which was used for the construction of the yeast strain CYS344.

The present invention also relates to a recombinant yeast strain comprising, in an integrated form in its genome, a polynucleotide which comprises (a) an open reading frame encoding the fusion protein comprising a p21 polypeptide (p2i ^{WAF1 / c} ' ^P1 or p21 [6KR] ^{WAF1 / Cip1} ) and at least one detectable protein, and (b) a regulatory sequence functional in yeast cells that directs the expression of said open reading frame.

A recombinant yeast strain according to the invention may consist of a recombinant yeast strain comprising, in an integrated form in its genome, a polynucleotide which comprises (a) an open reading frame encoding the fusion protein comprising a _p21 w ^AFi polypeptide ^/ c ⁱ _P ⁱ _{of sequence} SEQ ID N ⁰ 1 and at least one detectable protein, and (b) a regulatory sequence functional in yeast cells which directs the expression of said open reading frame. A recombinant yeast strain according to the invention may consist of a recombinant yeast strain comprising, in a form integrated in its genome, a polynucleotide which comprises (a) an open reading frame encoding the fusion protein comprising a p21 [6KR polypeptide ] ^{WAF1 / Cip1} of sequence SEQ ID N ⁰ 2 and at least one protein detectable, and (b) a functional regulatory sequence in yeast cells that directs the expression of said open reading frame.

In particular, the invention relates to a recombinant yeast strain according to the above definition, which consists of the yeast strain CYS343, which expresses the fusion protein comprising the _P 2i polypeptide ^{WAF1 / CiP1} and the detectable protein mAG. , ie the fusion protein of sequence SEQ ID N ⁰ 3.

In particular, the invention relates to a recombinant yeast strain according to the above definition, which consists of the yeast strain CYS344, which expresses the fusion protein comprising the p21 [6KR] ^{WAF1 / Cip1} polypeptide and the protein detectable mAG, ie the fusion protein sequence SEQ ID N ⁰ 4.

The invention also relates to a kit or kit for screening agents that modulate the activity of the proteasome, characterized in that it comprises an expression vector comprising an expression cassette encoding the fusion protein comprising the p21 polypeptide

_(p21 w ^{AFi /} c ⁱ _P ⁱ _{or P} 21 [6KR] ^{WAF1 / Cip1} ) as defined above.

The invention also relates to a kit or kit for screening agents that modulate the activity of the proteasome, characterized in that it comprises recombinant yeast cells comprising, in a form inserted in their genome, an expression cassette. encoding the fusion protein comprising the p21 polypeptide _(P21 ^{WAF1 / C |} P ¹ OR p21 [6KR] ^{WAF1 / Cip1)} as defined above.

Preferentially, the kit or kit above comprises recombinant yeast cells of the yeast strain CYS343 or recombinant yeast cells of the yeast strain CYS344.

The screening method according to the invention makes it possible to visualize the activity of the proteasome vis-à-vis a human target protein in yeast cells, and more precisely with respect to a target protein p21, c either the target protein consisting of a fusion protein comprising the human p2i protein ^{WAF1 / Cιp1} fused to a detectable protein, or the mutated protein p21 [6KR] ^{WAF1 / Cip1} fused to a detectable protein.

This method is particularly advantageous for screening molecules or agents capable of acting on the pathologies associated with a excessive or insufficient degradation of cellular proteins, such as certain cancers, inflammatory and immune syndromes, fungal, bacterial and viral infections or certain diseases of the central nervous system. The main advantages of the screening method of the invention include the following: the simplicity of implementation: the activity of the proteasome as it exists in the cells is simply visualized thanks to the controlled expression of the wild or human factor mutant p ₂ ^< | ^{WAFi /} c ^ιpi _in | _this || _u | _es (I | _e VING In addition, when the factor p2 ^{<|. WAFI /} c ^ιpi j _are expressed as fusion hybrid protein with an intrinsically fluorescent protein, such as GFP or mAG, proteasome activity with respect to the p2i factor ^{WAF1 / c} ' ^P1 or p21 [6KR] ^{WAF1 / Cip1} is directly measured by the quantification of the fluorescence emitted by the hybrid protein, likewise when the factor _p 2i ^{WAF1 / CiP1} or factor p21 [6KR] ^{WAF1 / Cip1} is expressed as a fusion protein with a protein such as luciferase, proteasome activity against p2i factor ^{WAF1 / Cip1} or factor p21 [6KR] ^{WAF1 / Cip1} is directly measured by the quantification of the luminescence emitted by the hybrid protein in the presence of a substrate such as fluorescein, the adequacy with a therapeutic context: the activity of the proteasome is monitored according to a functional test carried out in whole cells. in cellulo screening according to the vention thus makes it possible to select molecules capable of activating or inhibiting the activity of the proteasome in a context similar to that of their final therapeutic use. the specificity: although produced in cellulo in the cell, the screening method according to the invention is specific because it is based on the expression, in an organism heterologous to the human organism, of the human protein p21. The molecules selected by the screening method of the invention will be specific for the activity of the proteasome, and therefore will not be selected molecules because, for example, of their ability to interfere with one of the many signaling pathways inducing the degradation of p2i factor ^{WAF1 / c} ' ^P1 in human cells. Indeed, during the implementation of the screening method according to the invention, the degradation of _P 21 ^{WAF1 / Ci} e ¹ or p21 [6KR] ^{WAF1 / Cip1} by the proteasome is induced by a completely artificial metabolic pathway and fully reproducible, such as, for example, the addition of glucose to block the activity of the GAL1 promoter, when _p21 w ^{AF1 /} c ⁱ _P ⁱ _{or P} 21 [6KR] ^{WAF1 / Cip1} is expressed under the control of this promoter. The use of the p21 [6KR] ^{WAF1 / CiP1 protein, of} which the 6 lysine residues of the native p2i protein ^{WAF1 / Cιp1} were substituted with arginine residues, suppressing the internal sites of ubiquitination, further ensures that the molecules selected according to The invention does not interfere with the other components of the ubiquitin-proteasome pathway such as ubiquitin ligases, ubiquitin conjugation enzymes and ubiquitin activating enzyme. the stability of recombinant yeast strains: integration techniques in a selected location of a yeast chromosome and targeted replacement of genes allow the construction of recombinant yeast strains expressing the fusion protein containing either _p 2i ^{WAF1 / CiP1} , either p21 [6KR] ^{WAF1 / Cip1} from the chromosomes of the yeast. These recombinant yeast strains are therefore genetically stable and can be multiplied and stored indefinitely, the speed of growth and screening: yeast is a fast-growing and high-yielding microorganism. In particular, 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 is particularly high, which makes it possible to obtain a large amount of recombinant yeast cells for the simultaneous realization 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 method of the invention: the yeast is a microorganism whose culture, carried out in a low volume of medium, at low temperature, in a conventional atmosphere, in the air, is particularly suitable for automation (robotization) screening methods.

The screening methods according to the invention are particularly useful for selecting and characterizing active agents such as anti-cancer agents, anti-inflammatory agents, anti-viral agents, agents against fungal infections, bacterial agents or agents against diseases of the central nervous system. .

The present invention is further illustrated, without being limited, by the following figures and examples.

DESCRIPTION OF THE FIGURES FIG. 1 represents the maps of the recombinant plasmids encoding a detectable p21-protein fusion protein. Figure 1A is a schematic of plasmid pCSYAQ6-p21 comprising an expression cassette encoding the mAG-p21 fusion protein. Figure 1B is a schematic of plasmid pCSYAQ6-p21-6KR comprising an expression cassette encoding the mAG-p21 [6KR] fusion protein.

Figure 2 shows the nucleotide sequence and amino acid sequence of the p21 [6KR] ^{WAF1 / Cip1} protein.

Figure 3 depicts immunoblotting ("Western Blotting") images illustrating the degradation of fusion proteins comprising _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 / Cip1} fused with mAG, in the presence or absence of a proteasome inhibitory agent, compound MG132. The results are illustrated for the following recombinant yeast strains: CYS343 (Figure 2A) and CYS344 (Figure 2B).

Figure 4 illustrates the results of epifluorescence microscopy analysis, degradation of mAG-p21 [6KR] ^{WAF1 / Cip1} fusion protein in yeast cells of recombinant strain CYS344. Figure 3A illustrates the epifluorescence results obtained with cells of the CYS344 strain cultured in the presence of glucose and at time 0, 60 minutes, 120 minutes and 180 minutes (from the top to the bottom of the figure) after stopping. the induction of the expression of the protein fusion. FIG. 3B illustrates the epifluorescence results obtained with cells of the CYS344 strain cultured in the presence of glucose and at the time of 0, 60 minutes, 120 minutes and 180 minutes (from the top to the bottom of the figure) after stopping induction of expression of the fusion protein.

FIG. 5 illustrates the quantification, by flow cytometry of the yeast cells of each of the strains CYS343 and CYS344, of the fluorescence signal emitted by these cells during the culture time, respectively (i) during the induction of the production. of the fusion protein in the presence of galactose and (ii) in the presence of glucose after stopping the production of the fusion protein. The yeast cells were cultured in the presence or in the absence of the proteasome inhibitor MG 132. FIG. 4A illustrates flow cytometry quantification (FACS) of the fluorescence emitted by the mAG-p21 fusion protein ^{WAF1 / Cip1} produced by strain CYS343, in the presence and absence of the MG132 proteasome inhibitor. FIG. 4B illustrates flow cytometry quantification (FACS) of the fluorescence emitted by the mAG-p21 [6KR] ^{WAF1 / Cip1} fusion protein produced by the CYS344 strain, in the presence and absence of the MG132 proteasome inhibitor.

EXAMPLES EXAMPLES 1 TO 3.

A. MATERIAL AND METHODS OF EXAMPLES 1 TO 3. A.1. Summary of the polynucleotide sequences used

In the descriptions that follow

The sequence of the p2i protein ^{Waf1 / Cιp1} is that deposited in the EMBL databank, accession number BC001935.1

The sequence of the p21 [6KR] ^{Waf1 / Cip1} protein is that deposited in the EMBL databank, BC001935.1 accession number whose

6 Lysine residues were transformed into Arginine residues.

The sequence of the mAG (monomeric Azami-Green) coral gene

Galaxeidae encoding the fluorescent protein hereinafter referred to as mAG is that deposited at GenBankTM / EBI Data Bank with accession number AB108447. The sequence of the plasmid pRS306 is that deposited in the EMBL database, identifier "PRS306" accession number U03438.

The promoter sequence of the S. cerevisiae yeast GAL1 gene used in the following descriptions is fully included in the sequence deposited in the EMBL databank, identifier "SCGAL10", accession number K02115.

The promoter sequence of the GAL1 gene of the yeast S. cerevisiae, which can be used according to the invention may consist of that described by Johnston and Davis (Mol., C. Biol., (1984) 4 (8): 1440-1448). ).

The promoter sequence of the S. cerevisiae yeast MET3 gene that can be used according to the invention may consist of that described by Cherest et al. (Gen. Gen. Genet (1987) 210 (2): 307-313).

The promoter sequence of the S. cerevisiae yeast MET25 gene that can be used according to the invention may consist of that described in Kerjan et al. (Nucleic Acids Res (1986) 14 (20): 7861-7871). The promoter sequence of the PHO5 gene of yeast

S. cerevisiae, which may be used according to the invention may consist of that described by Feldmann et al. (EMBO J. (1994) 13 (24): 5795-5809).

The promoter sequence of the THI4 gene of yeast S. cerevisiae, which can be used according to the invention may consist of that described by Skala et al., Yeast (1995) 14: 1421-1427.

The promoter sequence of the CUP1 gene of S. cerevisiae yeast, which can be used according to the invention may consist of that described by Karin et al. (PNAS (1984) 81 (2): 337-341).

A.2. Conventions used

Descriptions are made using the nomenclature and typographical rules used in the community of yeast biologists Saccharomyces cerevisiae. the name of the wild form of a gene is given in uppercase italic; example: GAL1. the name of a mutant form of a gene is given in lowercase italic, the allele number if known is specified following a dash; example cup1-1. the name of an inactivated allele of a gene is given in lowercase followed by 2 times colon followed by the name of the inactivating gene ex ppr1 :: TRP1 (in this example the inactivated gene ppr1 was interrupted by the active gene TRP1).

Alternatively, 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 that the code is given in lowercase, except the first letter which is capitalized ex Gal4

(alternatively we can use the same symbol followed by a p, example Gal4p).

A3. Preliminary remarks concerning plasmid construction

All plasmids were constructed by standard molecular biology techniques according to protocols described by Sambrook et al. (in Molecular Cloning, Laboratory Manual, 2 ^nd Edition, (1989), CoId Spring Harbor, NY) and Ausubel et al., (in Current Protocols in Molecular Biology, (1990-2004), John Wiley and Sons Inc., NY) . Cloning, propagation and production of plasmid DNAs were carried out in Escherichia coli strain DH 10B.

EXAMPLE 1 Construction of Plasmids for the Expression in Yeast of the mAG-p21 Fusion Proteins ^{WAF1 / Cip1} and mAG-

The following plasmids allow the expression in yeast Saccharomyces cerevisiae of derivatives of the human protein _p21 w ^{AFi /} c ⁱ _P ⁱ _{or of} | _{a protein huma} i _{do mu} aunt p21 [6KR] ^{WAF1 / Cip1} fused to the fluorescent protein mAG coral Galaxeidae. A fragment of 614 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 following oligonucleotides: (i) "pGAL1 (Asp) Forw" sequence δ'-GCTGGGTACCTTAATAATCATATTACATGGCATTA-S '[SEQ ID N ⁰ 10], and (ii) "pGAL1 (Xho) Rev" sequence δ -CGACCTCGAGTATAGTTTTTTCTCCTTGACGTTAA-S 'SEQ ID NO ^: 11].

The fragment obtained was digested with the restriction enzymes Asp7 'and XhoI and inserted into the shuttle plasmid S.cerevisiae-E.coli pRS306 previously digested with the enzymes Λsp7181 and Xho1, producing the vector pRS306-pGAL1.

A fragment of 678 base pairs (bp) corresponding to a variant of the gene coding for the Azami Green monomeric fluorescent protein (mAG) of the coral Galaxeidae, whose sequence is optimized for yeast expression, was obtained by enzymatic digestion with XhoI and EcoR restriction enzymes from the pPCR-Script-mAG vector. The fragment obtained was inserted into the plasmid pRS306-pGAL1 previously digested with the XhoI and EcoRI enzymes, producing the pRS306-pGAL1-mAG vector.

A 340 base pair (bp) fragment 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 following oligonucleotides: (i) "TermADH1 (NotIBstXI) 5 '" of sequence 5'-

GGCGGCGGCCGCCACCGCGGTGGGCGAATTTCTTATGATTTATG- 3 '[SEQ ID NO ^: 12] and (ii) "TermADH1 (SacI) 3'" of sequence δ'-GGCGGAGCTCTGGAAGAACGATTACAACAG-S '[SEQ ID NO ^: 13]. The fragment obtained was digested with the restriction enzymes SacI and / ofI and inserted into the plasmid pRS306-pGAL1-mAG previously digested with the enzymes SacI and NotI, producing the vector PCSYAQ6. The gene coding for the p2i protein ^{WAF1 / Cιp1} , the sequence of which was optimized for expression in yeast, was purified from the plasmid pPCR-Script-p21WAF1 / Cip1 [wt] by digestion with the restriction enzymes EcoR \ and Λ / ofl. The fragment was cloned into the plasmid pCSYAQβ prepared by digestion with restriction enzymes EcoR1 and Λ / ofl. The product vector was named pCSYAQ6-p21 [wt], which is shown in Figure 1 A.

The gene coding for the p21 [6KR] ^{WAF1 / Cip1} protein, the sequence of which was optimized for expression in yeast, was purified from the plasmid pPCR-Script-p21WAF1 / Cip1 [6KR] by enzyme digestion. of restriction EcoR \ and Λ / ofl. The fragment was cloned into plasmid pCSYAQ6 prepared by digestion with restriction enzymes EcoR1 and Λ / ofl. The product vector was named pCSYAQβ-p21 [6KR], the scheme of which is shown in Figure 1B.

Construction of yeast strains CYS343 and CYS344

For obtaining the yeast strain CYS343, the plasmid pCSYAQ6-p21 [wt]. described above was linearized using the Sfu1 enzyme. The Sfu1 enzyme cleaves plasmid pCSYAQ6-p211 [wt] at a unique position located in the URA3 gene sequence. The linearized plasmid pCSYAQ6-p21 [wt] integrates with the URA3 locus located on the left arm of chromosome 5 of the yeast Saccharomyces cerevisiae.

To obtain the yeast strain CYS344, the plasmid pCSYAQ6-p21 [6KR] described above was linearized using the enzyme SM. The Sfu1 enzyme cleaves plasmid pCSYAQ6-p21 [6KR] at a unique position localized in the URA3 gene sequence. The linearized plasmid pCSYAQ6-p21 [6KR] integrates with the URA3 locus located on the left arm of chromosome 5 of the yeast Saccharomyces cerevisiae.

Yeast strains CYS343 and CYS344 were obtained as described above, according to the general protocol described by Rohthstein (1991). The respective genotypes of the yeast strains according to the invention are shown in Table 1 below.

Table 1: Genotype of Saccharomyces cerevisiae yeast strains constructed for the purposes of the present invention.

EXAMPLES 2 TO 6: Development of the Screening Process According to the Invention

EXAMPLE 2: Nucleotide and Protein Sequences of the Mutant Form of the Human Factor p21 r6KR1 ^{WAF1 / Cip1} Expressed in Yeast Cells

The amino acid sequence coding for the fusion protein comprising the p21 [6KR] ^{WAF1 / Cιp1 protein} consists of the polypeptide of sequence SEQ ID N ⁰ 4 which may be encoded by the polynucleotide of sequence SEQ ID N ⁰ 6, of which Codon identity has been specifically adapted to optimal expression in yeast cells.

The nucleotide sequence and the amino acid sequence of the p21 [6KR] ^{WAF1 / Cip1} protein are shown in Figure 2.

In Figure 2, the 6 lysine residues (K) that have been replaced by arginine (R) residues are highlighted in black, as well as the corresponding codons. Optimized codons for the expression of p21 [6KR] ^{WAF1 / Cιp1} in the yeast Saccharomyces cerevisiae are highlighted in gray.

Example 3: Degradation cellular proteasome human factors _P21 ^{WAF1 / CJ} P ¹ and r6KR1 p21 ^{WAF1 / Cip1} expressed in yeast (immunoblot results) The degradation, by the proteasome of the recombinant yeast cells of strains CYS343 and CYS344, of fusion proteins comprising _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 / Cip1} fused with mAG, was followed by an immunoblot technique , in the presence or absence of a proteasome inhibitory agent, the compound Mg132. A. Materials and Methods

All yeast strains employed were grown and analyzed identically.

The cells were cultured in a minimum medium in the presence of galactose as carbon source for 120 minutes.

At the end of these 120 minutes, 2% glucose is added to the culture, in the presence and in the absence of 50 μM of a known proteasome inhibitor, Mg132.

The results are shown in Figure 3 (Figure 3A and Figure 3B). Figure 3A shows the results obtained with the recombinant yeast strain CYS344.

The total proteins are prepared before the addition of galactose (0), 60 and 120 minutes after the addition of galactose (+ galactose 0, 60, 120) and 30, 60 and 120 minutes (+ glucose, 30, 60, 120 ) after the addition of glucose. Figure 3B shows the results obtained with the recombinant yeast strain CYS343. The total proteins are prepared before the addition of galactose (0), 60 and 120 minutes after the addition of galactose (+ galactose 0, 60, 120) and 30, 60, 120 and 150 minutes (+ glucose, 30, 60 , 120, 150) after the addition of glucose. These proteins are analyzed by the Western blotting technique using an antibody recognizing the p2i ^{WAF1 / CIP1 portion} of the fusion proteins (indicated as "mAG-p21 ^{WAF1 / Cip1} " or "mAG-p21 [6KR] ] ^{WAF1 / Cip1} "in Figure 3).

The presence of MG132 in the culture is indicated "+ MG132" in FIG. 3. A control of the quantity of proteins deposited in each well is carried out by analyzing the same proteins using antibodies recognizing Lysyl-tRNA-synthase. yeast (indicated

"LysRS" in Figure 3). Immunoblot gel images

("Western blotting") revealed with anti-p21 antibodies and anti-Lys antibodies. B. Results

The results are illustrated for the following recombinant yeast strains: CYS343 (Figure 3A) and CYS344 (Figure 3B). The results presented in FIG. 3A illustrate, by a Western blotting biochemical analysis, the degradation of the mAG-p21 [6KR] ^{WAF1 / Cip1} fusion protein in which the 6 Lysine residues of the p2i factor ^{WAF1 / Cιp1} have been replaced by Arginine residues.

The results presented in FIG. 3B illustrate, by a Western blotting biochemical analysis, the degradation of the mAG-p21 [wt] ^{WAF1 / Cip1} fusion protein in yeast cells.

In FIG. 3A and FIG. 3B, it is observed that the presence of galactose (indicated "+ Galactose" in FIG. 3) during the entire culture time causes a continuous induction of the production of the fusion protein comprising p21 [6KR ] ^{WAF1 / Cip1} (Figure 3A) or comprising p21 [wt] ^{WAF1 / Cip1} (Figure 3B), which mitigates fusion protein degradation by the proteasome that occurs simultaneously in yeast cells.

In FIG. 3A and FIG. 3B, it is observed that the culture of the cells in the presence of glucose (indicated "+ Glucose" in FIG. 3), since the production of the fusion protein is no longer induced, makes it possible to visualize degradation of the fusion protein by the proteasome.

In FIG. 3A and FIG. 3B, it is observed that the culture of the cells in the presence of glucose and of a proteasome inhibitor, compound MG132 (indicated "+ Glucose + 50 μM MG132" in FIG.

3) makes it possible to inhibit the degradation of the fusion protein which is observed in the absence of the compound MG132.

EXAMPLE 4 Degradation of the mAG-p21 f6KR1 protein ^{WAF1 / Cip1} (epifluorescence microscopy)

Example 4 illustrates the results of an epifluorescence microscope analysis, the degradation of the mAG-p21 [6KR] ^{WAF1 / Cιp1} fusion protein in yeast cells of the recombinant strain CYS344. A. Materials and Methods

The yeast cells of strain CYS344 were cultured in a minimum medium in the presence of galactose as carbon source for 120 minutes.

At the end of these 120 minutes, 2% glucose is added to the culture, in the presence and in the absence of 50 μM of a known proteasome inhibitor, MG 132.

The cells are observed under an epifluorescence microscope (Nikon Eclipse fluorescence microscope equipped with an Omega filter

XF116). All images were recorded using a Hamamastu® camera using identical settings and analyzed with LUCIA G software, just before the addition of glucose (0 minutes), and 60, 120 and 180 minutes (60, 120, 180 minutes) after adding glucose. The presence of MG132 in culture is indicated "+

MG132 ".

B. Results

The results are shown in Figure 4 (Figure 4A and Figure 4B). FIG. 4A illustrates the epifluorescence results obtained with the cells of the CYS344 strain cultured in the presence of glucose and at the time 0, 60 minutes, 120 minutes and 180 minutes (from the top to the bottom of the figure) after the stopping of induction of expression of the fusion protein. FIG. 4B illustrates the epifluorescence results obtained with the cells of the CYS344 strain cultured in the presence of glucose and at the time 0, 60 minutes, 120 minutes and 180 minutes (from the top to the bottom of the figure) after stopping induction of expression of the fusion protein.

In FIG. 4A and FIG. 4B, the left column represents the fluorescence microscopy images making it possible to locate in the cells the expression of the mAG-p21 [6KR] ^{WAF1 / Cip1} fusion protein. In Figure 4A and Figure 4B, the right column represents a visualization of the same cells in visible light.

In FIG. 4A, there is a progressive reduction of the fluorescence signal with the duration of culture, as and when the progression of degradation of the fusion protein by the proteasome (left column), while the cell density in the observed field is identical (right column).

In FIG. 4B, a maintenance of the intensity of the fluorescence signal is observed with the culture time (left column), whereas the cell density in the observed field is identical (right column), which illustrates the blocking degradation activity of the fusion protein by the proteasome, a blockade that is induced by the MG132 inhibitor.

EXAMPLE 5 Degradation of the mAG-p21 f6KR1 protein ^{WAF1 / Cip1} (quantification by flow cytometry)

Example 5 illustrates a quantification, by flow cytometry of the yeast cells of each of the strains CYS343 and CYS344, of the fluorescence signal emitted by these cells, during the culture time, respectively (i) during the induction of the production of the fusion protein in the presence of galactose and (ii) in the presence of glucose after stopping the production of the fusion protein. The yeast cells were cultured in the presence or absence of the MG 132 proteasome inhibitor.

The results are illustrated by the curves shown in Figure 5.

Figure 5A illustrates flow cytometric quantification (FACS) of the fluorescence emitted by the mAG-p2i fusion protein ^{WAF1 / c} ' ^P1 produced by strain CYS343, in the presence and absence of the proteasome inhibitor MG132.

FIG. 5B illustrates flow cytometric quantification (FACS) of the fluorescence emitted by the mAG-p21 [6KR] ^{WAF1 / Cip1} fusion protein produced by the CYS344 strain, in the presence and absence of the proteasome inhibitor MG 132. .

A. Materials and Methods

All cells were cultured in a minimum medium in the presence of galactose as carbon source for 120 minutes. At the end of these 120 minutes, 2% glucose is added to the culture, in the presence and in the absence of 50 μM of a known proteasome inhibitor, MG 132.

The fluorescence emitted by the cells was quantified using a FacsCalibur flow cytometer (Beckton-Dickinson), just before the addition of galactose (10), 1 and 2 hours after the addition of galactose (11 and 12) and 1, 2 and 3 hours (D1, D2 and D3) after the addition of glucose. The presence of Mg132 in the culture is indicated "+ MG132". Fluorescence is reported in arbitrary units

B. Results

The results of Figures 5A and 5B show that the fusion protein is actively produced by the yeast strains CYS343 and CYS344, when the cells are cultured in the presence of galactose (time lo, M h, I2h / Dθ).

The results of FIGS. 5A and 5B show that the fusion protein, which has accumulated in the cells in the presence of galactose, is progressively degraded during the culture time in the absence of galactose and in the presence of glucose, in the presence of glucose. absence of compound MG132 (time D1 h, D2h, D3h).

The results of FIGS. 5A and 5B show that, in the presence of the proteasome inhibitor MG132, the degradation of the fusion protein is strongly inhibited (Time D1 h, D2h, D3h).

EXAMPLE 6: Localization, in Yeast Cells, of the Fusion Proteins mAG-p2ir6KR1 ^{WAF1 / Cip1}

In Example 6, the localization, in the yeast cells of strain CYS344, of the protein mAG-p21 [6KR] ^{WAF1 / Cip1 was determined} .

Yeast cells of strain CYS344 comprising a polynucleotide allowing the expression of the mAG-p21 [6KR] ^{WAF1 / Cip1} fusion protein under the control of the GAL1 promoter were cultured in the presence of 2% galactose for 2 hours and were then observed by fluorescence microscopy.

The position of the nucleus was revealed by using a specific color indicator of the nucleus, the Hoescht 333-42. Visible light microscopy and fluorescence microscopy were taken to stain the DNA of the cell nuclei with the Hoechst 333-42 stain (not shown). Visible light microscopy and fluorescence microscopy (not shown) were superimposed.

Co-localization of the cell nuclei and the mAG-p21 [6KR] ^{WAF1 / Cip1} fusion protein was observed.

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Table 2: Sequences

Claims

An in cellulo method for screening agents for modulating proteasome activity, said method comprising the steps of: a) contacting a candidate agent to be tested with recombinant yeast cells that express in their nucleus a fusion protein comprising:

(i) a p21 polypeptide selected from the p2i ^{WAF1 / Cιp1} polypeptide and the p21 [6KR] ^{WAF1 / Cip1} polypeptide, and (ii) at least one detectable protein. b) quantifying said first detectable protein in the yeast cells at the end of at least a predetermined period of time after contacting the candidate agent with said cells; c) comparing the value obtained in step (b) with a control value obtained when step (a) is performed in the absence of the candidate agent.

2. Method according to claim 1, characterized in that step (a) comprises the following steps:

(ai) culturing yeast cells which express in their nucleus said fusion protein comprising the p21 polypeptide and at least one detectable protein;

(a2) stopping the expression of said fusion protein comprising the p21 polypeptide and at least one protein detectable by yeast cells; (a3) contacting the yeast cells obtained at the end of step (a2) with the candidate agent to be tested.

3. Method according to one of claims 1 and 2, characterized in that the detectable protein included in the fusion protein comprising the p21 polypeptide is selected from an antigen, a fluorescent protein and a protein having an enzymatic activity.

4. Method according to claim 3, characterized in that the detectable protein consists of a fluorescent protein chosen from the mAG protein or one of its derivatives, and the GFP protein or one of its derivatives.

5. Method according to claim 3, characterized in that the detectable protein consists of a protein having an enzymatic activity selected from luciferase and β-lactamase.

6. Method according to claim 3, characterized in that the detectable protein consists of an antigen selected from the peptide Ha and the peptide Flag.

7. Method according to one of claims 1 to 6, characterized in that the protein comprising the p2i polypeptide ^{WAF1 / Cιp1} consists of the protein sequence SEQ ID No. 3.

8. Method according to one of claims 1 to 6, characterized in that the protein comprising the polypeptide p21 [6KR] ^{WAF1 / Cιp1} consists of the protein sequence SEQ ID No. 4.

9. Method according to one of claims 1 to 8, characterized in that in 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 antibodies recognizing it.

10. Method according to one of claims 1 to 8, characterized in that in step (b), when the first detectable protein is a fluorescent protein, said detectable protein is quantified by measuring the fluorescence signal emitted by said protein.

11. Method according to one of claims 1 to 8, characterized in that in step (b), when the first detectable protein is a protein having an enzymatic activity, said detectable protein is quantified by a measurement of the quantity substrate transformed by said protein.

12. Method according to one of claims 1 to 11, characterized in that the recombinant yeast cells are transformed with a polynucleotide which comprises:

(a) an open reading frame encoding (i) the fusion protein comprising the p21 polypeptide ( _p 2i ^{WAF1 / CiP1} or p21 [6KR] ^{WAF1 /} Cip1) and (ii) a detectable protein, and

(b) a functional regulatory sequence in yeast cells that directs the expression of said open reading frame;

13. The method of claim 12, characterized in that the regulatory sequence contained in the first polynucleotide comprises a functional promoter in yeast cells and which is sensitive to the action of an inducing agent.

14. The method of claim 13, characterized in that the promoter responsive to the action of an inducing agent consists of a functional repressible promoter in yeast cells selected from CUP1, GAL1, GAUO, MET3, MET25, PHO5, and THI4. yeast Saccharomyces cerevisae.

15. The method of claim 14, characterized in that said polynucleotide encoding the fusion protein comprises the regulatory sequence GAL1, which activates the expression of the open reading frame encoding said fusion protein.

16. Method according to one of claims 1 to 15, characterized in that the recombinant yeast cells possess the polynucleotide in a form inserted in their genome.

17. Method according to one of claims 1 to 16, characterized in that the recombinant yeast cells have in their genome an inactivated form of one or more genes controlling the expression of transporter proteins inserted into the plasma membrane.

18. The method of claim 17, characterized in that the inactivated gene or genes are selected from the PDR1 and PDR3 genes.

19. Functional expression cassette in yeast cells comprising a coding polynucleotide which comprises an open reading frame encoding the fusion protein comprising a p21 polypeptide and at least one detectable protein, and a regulatory sequence functional in yeast cells which directs the expression of said open reading frame.

20. functional expression cassette according to claim 19, characterized in that the p21 polypeptide is chosen from the _p21 w polypeptide ^{AFi /} c ⁱ _P ⁱ _{and p} 21 [6KR] ^{WAF1 / Cip1} poiypeptjde polypeptide.

21. Functional expression cassette according to claim 20, characterized in that the p21 polypeptide is chosen from the _p21 wAFi / ci _P i polypeptide _{of sequence SEQ} . _DN o _{1 and} , _e poiypeptjde of _P 21 [6KR] ^{WAF1 / Cip1} sequence SEQ ID N ⁰ 2.

22. Expression cassette according to one of claims 19 to 21, characterized in that the regulatory sequence contained in said polynucleotide comprises a promoter functional in yeast cells and which is sensitive to the action of an inducing agent.

23. An expression cassette according to claim 22, characterized in that the inducible promoter functional in yeast cells is selected from CUP1, GAU, GAUO, MET3, MET25, PHO5, and THI4 from the yeast Saccharomyces cerevisiae.

24. Expression vector characterized in that it comprises an expression cassette according to one of claims 19 to 23.

25. Expression vector according to claim 24, characterized in that it is the vector pCSYAQ6-p21 [wt].

26. Expression vector according to claim 24, characterized in that it is the vector pCSYAQ6-p21 [6KR].

A recombinant yeast strain comprising, in an integrated form in its genome, a polynucleotide which comprises (a) an open reading frame encoding the fusion protein comprising a p21 polypeptide and at least one detectable protein, and (b) a sequence functional regulator in yeast cells that directs the expression of said open reading frame.

28. A recombinant yeast strain according to claim 27, characterized in that the p21 polypeptide is chosen from the _p21 w polypeptide ^{AFi /} c ⁱ _P ⁱ _{and the p} 21 [6 KR] ^{WAF1 / Cip1} polypeptide.

29. A recombinant yeast strain according to claim 28, characterized in that the p21 polypeptide is chosen from the _p21 wAFi / ci _P i polypeptide _{of sequence SEQ} . _DN o _{1 and} , _e poiypeptjde of _P 21 [6KR] ^{WAF1 / Cip1} sequence SEQ ID N ⁰ 2.

30. Kit or kit for the screening of agents modulating the activity of the proteasome, characterized in that it comprises an expression vector comprising an expression cassette according to one of claims 19 to 23.

31. Kit or kit for screening agents modulating the activity of the proteasome, characterized in that it comprises recombinant yeast cells comprising, in a form inserted in their genome, an expression cassette according to one of the claims. 19 to 23.