Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• the present invention relates to peptide fragments of proteins of the WASP family, or the peptides derived from these fragments, as well as their uses in particular in the context of methods for detecting molecules having an effect on cell mobility.
• the cells of our body are able to move and sometimes they round and divide into two sister cells. All of these movements are based on the actin cytoskeleton.
• the cytoskeleton plays an essential role for the organization of the body and for homeostasis. For example, cell migration is essential in embryogenesis and the immune response as well as in the repair of wounds where cells migrate to damaged regions. These movements are dependent on the normal functioning of the actin cytoskeleton. The consequences of disrupting the functioning of the cytoskeleton can be disastrous for the body. In metastatic processes, for example, the lack of control of the cytoskeleton of tumor cells can cause them to migrate outside their normal location, allowing them to proliferate in other parts of the body, which makes cancer treatment extremely difficult. .
• the first step in all cytoskeleton-dependent processes, such as movement, is the production of actin, or F-actin, filaments.
• actin or F-actin, filaments.
• the mechanism of the formation of these biological polymers in the cell is still unknown, despite the identification of numerous actin-binding proteins and the extensive study of the polymerization of actin in vitro.
• Wiskott-Aldrich syndrome is a disease of the cytoskeleton.
• the human WASP protein expressed from the WAS gene which is mutated in patients affected by this syndrome, as well as the N-WASP protein of bovine origin (which has approximately 45% of sequence identity with the human WASP protein), have therefore been the subject of studies with the aim of clarifying the mechanism of functioning of the cytoskeleton in the cell (Yarar et al., Current Biology, 9: 555 - 558 (1999); Rohatgi et al., Cell, 97: 221 - 231 (1999); Miki et al, The EMBO Journal, 15 ( 19): 5326 - 5335 (1996)).
• WASP and N-WASP proteins are composed of peptide domains having functional properties identifiable in biochemical tests.
• these two proteins mainly have the following domains:
• proline-rich region - a proline-rich region, - a domain of homology with verproline, a protein which also binds to proteins involved in the formation of actin filaments, this domain comprising a sequence homologous to verproline in the case of WASP human, and two sequences homologous to verproline in the case of N-WASP,
• WASP and N-WASP proteins have been shown to interact with the Arp2 / 3 complex (protein complex involved in the polymerization of actin), and thus induce the polymerization of actin.
• the WASP protein is sufficient to act on cellular motility based on actin, and that this function is dependent on the Arp2 / 3 complex (Yarar et al. 1999 mentioned above).
• the authors of this article prepared microspheres coated with WASP protein and demonstrated that these microspheres polymerize actin, form actin tails, and are endowed with actin-based motility in cell extracts.
• the microspheres coated with WASP protein no longer have motility and only have a residual actin polymerization activity.
• the C-terminal part of N-WASP namely the C-terminal fragment of 114 amino acids comprising the domain of homology with verproline (region V containing the two sequences homologous to verproline), the domain of homology with cofilin (region C), and the C-terminal acid segment (region A), or VCA fragment, binds to the Arp2 / 3 complex and strongly stimulates the capacity of the latter to nucleate the polymerization of actin (Rohatgi et al. 1999 mentioned above).
• the present invention follows from the discovery by the Inventors of the fact that, contrary to what the state of the art described above might have suggested, the fragments of these WASP and N-WASP proteins are sufficient to induce a actin polymerization such that it allows the displacement of supports to which said elements are linked in cell extracts.
• the object of the present invention is to provide new fragments, or derived polypeptides, of the WASP and N-WASP proteins, as well as the nucleotide sequences coding for these fragments.
• the invention also aims to provide new methods for detecting or screening molecules having an effect on the formation of the cytoskeleton, in particular cytotoxic molecules or drugs which can be used in the treatment of pathologies linked to an abnormal development of the cytoskeleton .
• the invention also aims to provide new reagents and kits for carrying out the above-mentioned methods.
• the subject of the present invention is the use:
• peptide fragments of proteins of the WASP family in eukaryotic cells, in particular human or other mammal cells, or insect cells, or of microorganisms such as yeasts, said peptide fragments having the property of proteins of the WASP family to polymerize actin by inducing cell motility, - Or of peptide sequences derived from the above-mentioned peptide fragments of proteins of the WASP family, in particular by substitution of one or more amino acids of these fragments, said derived sequences having the above-mentioned property of proteins of the WASP family and of said fragments of the latter , for the preparation of reagents usable within the framework of the implementation of a method of detection or screening of molecules having an effect of inhibiting or stimulating the formation of the actin cytoskeleton, and therefore an effect of inhibition or stimulation of cell motility.
• the subject of the invention is also the use of the peptide fragments or of the abovementioned derived sequences, within the framework of the implementation of a method of detection or screening of molecules capable of being able to be used as medicaments in the treatment pathologies linked to a dysfunction of the actin polymerization process in the context of the formation of the actin cytoskeleton.
• a more particular subject of the invention is the use of the above-mentioned peptide fragments or derived sequences, within the framework of the implementation of a method for detecting or screening for molecules having an effect of inhibiting the formation of the cytoskeleton actin, and therefore an inhibiting effect on cell motility, said molecules being capable of being used:
• a more particular subject of the invention is the use of the above-mentioned peptide fragments or derived sequences, within the framework of the implementation of a method for detecting side effects of molecules, in particular of drugs or molecules of the environment, namely a method for detecting molecules capable of having a cytotoxic effect corresponding to an inhibition or stimulation of the formation of the actin cytoskeleton.
• proteins of the WASP family is meant, in the above and what follows, the protein produced by the WAS gene mutated in the context of Wiskott-Aldrich syndrome in humans, as well as proteins of human origin or no, having at least about 45% homology with the aforementioned human WASP protein, and being involved in the process of polymerization of cellular actin, and, where appropriate, of cellular motility.
• this domain has structural characteristics similar to a domain of homology to pleckstrin (or pH domain), and is supposed to interact with the polymerized actin and with the phospholipids, a domain rich in prolines,
• WH2 / A domain which is divided into three sub-domains, namely the subprofile of homology with verproline, the subdomain of homology with cofilin, and the acid sub-domain mentioned above.
• the peptide fragments used in the context of the present invention are chosen from the fragments of the WASP, N-WASP, Scar and Las proteins, or the peptide sequences derived from the above-mentioned peptide fragments as defined above.
• a more particular subject of the invention is the aforementioned use of peptide fragments chosen from the fragments:
• N-WASP protein or other mammals, in particular bovine, or rat N-WASP protein,
• the Scar subfamily such as the Scar 1 / WAVE protein of Dictyostellium discoidewn, or of Caenorhabditis elegans, or of Drosophila melanogaster, of mice, or of humans,
• yeasts such as the Las17 / Beel protein of Saccharomyces cerevisiae, or the homologous protein
• WASP Wsplp of Schizosaccharomyces ponibe.
• the above-mentioned peptide fragments are chosen from those comprising:
• the domain of homology with verproline contained in the proteins of the WASP family or in a protein derived from the latter, or at least one of the two sequences homologous to verproline when said proteins of the WASP family contain two of these sequences , or a peptide sequence derived from the aforementioned domain, in particular by substitution, addition or deletion of one or more amino acids, and retaining the property of this domain to bind to actin,
• the aforementioned peptide fragments used in the context of the present invention also contain the C-terminal acid segment of said proteins
• the abovementioned peptide fragments do not contain the domain of homology with plekstrin, and / or the binding domain to Cdc42, and / or the proline-rich region, defined above of said proteins of the WASP family.
• a more particular subject of the invention is the aforementioned use of peptide fragments of proteins of the WASP family of human origin.
• the peptide fragments of proteins of the WASP family of human origin used are chosen from fragments of the human WASP protein comprising: the domain of homology with verproline delimited by the amino acids located at positions 430 and 446 of the peptide sequence SEQ ID NO 2 of the protein
• fragments of the human WASP protein used above are chosen from the following:
• Trp Asp Asp * the 84 amino acid fragment delimited by the amino acids located at positions 404 and 487 in FIG. 1, namely the following peptide SEQ ID NO 6:
• the peptide fragments of proteins of the WASP family of human origin used are chosen from fragments of the human N-WASP protein comprising: . the sequence homologous to verproline delimited by the amino acids located at positions 405 and 421 of the peptide sequence SEQ ID NO 12 of the human N-WASP protein represented in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above -above, .
• the domain of homology with the cofilin contained in the aforementioned N-WASP protein namely the domain delimited by the amino acids located at positions 470 and 488 of the peptide sequence SEQ ID NO 12 of the human N-WASP protein represented on Figure 2, or a peptide sequence derived from the above-mentioned domain as defined above.
• fragments of the human N-WASP protein used above are chosen from the following:
• the peptide fragments of proteins of the WASP family of human origin used are chosen from fragments of the human Scarl protein comprising:
• the fragments of the human Scarl protein used above are chosen from the following:
• a more particular subject of the invention is the aforementioned use of peptide fragments of proteins of the WASP family of non-human origin.
• the peptide fragments of the proteins of the WASP family of non-human origin used are chosen from:
• Asp Glu Trp Asp Asp * the fragment of 86 amino acids delimited by the amino acids located at positions 420 and 505 of FIG. 4, namely the following peptide SEQ ID NO 40:
• Bovine N-WASP represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above, * the fragments of which the N-terminal amino acid corresponds to that located in one of the positions 405 to 433 of FIG. 6, and the C-terminal amino acid corresponds to that located in one of the positions 488 to 505 in Figure 6,
• WASP family protein fragments from microorganisms such as:
• Wsplp WASP homologous protein
• the invention also relates to the aforementioned use of peptide fragments, or of sequences derived from the latter, as defined above, fused on the N-terminal or C-terminal side with one or more peptide sequences facilitating the detection and the purification of the aforementioned peptide fragments or derived sequences, without however affecting the aforementioned property of the latter of polymerizing actin by inducing cell motility.
• the invention also relates to the abovementioned peptide fragments, or the sequences derived therefrom, as such, namely more particularly the peptide fragments of the proteins of the WASP family of eukaryotic cells chosen from those comprising:
• the domain of homology with verproline contained in the proteins of the WASP family or in a protein derived from the latter, or at least one of the two sequences homologous to verproline when said proteins of the WASP family contain two of these sequences , or a peptide sequence derived from the aforementioned domain, in particular by substitution, addition or deletion of one or more amino acids, and retaining the property of this domain to bind to actin,
• a more particular subject of the invention is the above-mentioned peptide fragments of the human WASP protein, or the peptide sequences derived from the latter, said fragments being chosen from those comprising:
• the invention relates more particularly still to the fragments of the human WASP protein chosen from the fragments whose N-terminal amino acid corresponds to that located at one of the positions 404 to 430 of FIG. 1, and the amino acid C -terminal corresponds to that located at one of positions 487 to 502 in FIG. 1, and more particularly the peptides SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, and SEQ ID NO 10 mentioned above, or the sequences derived from these as defined above.
• a more particular subject of the invention is the above-mentioned peptide fragments of the human N-WASP protein, or the peptide sequences derived therefrom, said fragments being chosen from those comprising:
• the domain of homology with the cofilin contained in the aforementioned N-WASP protein namely the domain delimited by the amino acids located at the positions 470 and 488 of the peptide sequence SEQ ID NO 12 of the human N-WASP protein shown in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above.
• the invention relates more particularly still to the fragments of the human N-WASP protein chosen from fragments whose N-terminal amino acid corresponds to that located at one of positions 392 to 433 in FIG. 2, and the acid C-terminal amino corresponds to that located at one of positions 488 to 505 in FIG. 2, and more particularly the peptides SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, and SEQ ID NO 24 above, or the sequences derived therefrom as defined above.
• a more particular subject of the invention is the above-mentioned peptide fragments of the human Scarl protein, or the peptide sequences derived from the latter, said fragments being chosen from those comprising:
• the invention relates more particularly still to the fragments of the human Scarl protein chosen from fragments whose N-terminal amino acid corresponds to that located at one of positions 443 to 497 in FIG. 3, and amino acid C -terminal corresponds to that located at one of positions 546 to 559 in FIG. 3, and more particularly the peptides SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, and SEQ ID NO 34 mentioned above, or the sequences derived from these as defined above.
• a more particular subject of the invention is the above-mentioned peptide fragments of the murine WASP protein, or the peptide sequences derived therefrom, said fragments being chosen from those comprising:
• the invention relates more particularly still to the fragments of the murine WASP protein chosen from the fragments whose N-terminal amino acid corresponds to that located at one of the positions 420 to 448 of FIG. 4, and the amino acid C -terminal corresponds to that located at one of the positions 505 to 520 of FIG. 4, and more particularly the peptides
• SEQ ID NO 38 SEQ ID NO 40, SEQ ID NO 42, and SEQ ID NO 44 above, or the sequences derived therefrom as defined above.
• a more particular subject of the invention is the above-mentioned peptide fragments of the rat N-WASP protein, or the peptide sequences derived therefrom, said fragments being chosen from those comprising:
• the invention relates more particularly still to the fragments of the rat N-WASP protein chosen from the fragments whose N-terminal amino acid corresponds to that located at one of positions 401 to 429 in FIG. 5, and the C-terminal amino acid corresponds to that located at one of positions 484 to 501 in FIG. 5, and more particularly the SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, and SEQ ID NO 54 peptides mentioned above, or the sequences derived therefrom as defined above.
• a more particular subject of the invention is the abovementioned peptide fragments of the bovine N-WASP protein, or the peptide sequences derived from the latter, said fragments being chosen from those comprising:
• Bovine N-WASP represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above,
• the invention relates more particularly still to the fragments of the bovine N-WASP protein chosen from the fragments whose N-terminal amino acid corresponds to that located at one of positions 405 to 433 in FIG. 6, and the acid C-terminal amine corresponds to that located at one of positions 488 to 505 in FIG. 6, and more particularly the peptides SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, and SEQ ID NO 64 mentioned above, or the sequences derived therefrom as defined above.
• a more particular subject of the invention is the above-mentioned peptide fragments of the Las 17 protein of Saccharomyces cerevisiae, or the peptide sequences derived from the latter, said fragments being chosen from those comprising:
• Las 17 represented in FIG. 7, or a peptide sequence derived from the above-mentioned domain as defined above,
• the invention relates more particularly still to the fragments of the Las 17 protein of Saccharomyces cerevisiae chosen from the fragments whose N-terminal amino acid corresponds to that located at one of positions 422 to 447 in FIG. 7, and the amino acid C-terminal corresponds to that located at one of positions 624 to 633 in FIG. 7, and more particularly the peptides SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, and SEQ ID NO 74 above-mentioned, or the sequences derived therefrom as defined above.
• a more particular subject of the invention is the above-mentioned peptide fragments of the Schizosaccharomyces pombe protein (Wsplp), or the peptide sequences derived from the latter, said fragments being chosen from those comprising: the domain of homology with verproline delimited by the amino acids located at positions 501 and 517 of the peptide sequence SEQ ID NO 76 of the homologous protein WASP (Wsplp) of Schizosaccharomyces pombe shown in FIG. 8, or a peptide sequence derived from above-mentioned domain as defined above,
• the invention relates more particularly still to the fragments of the WASP homologous protein (Wsplp) of Schizosaccharomyces pombe chosen from fragments whose N-terminal amino acid corresponds to that located at one of positions 477 to 501 in FIG. 8, and the C-terminal amino acid corresponds to that located at one of positions 565 to 574 in FIG. 8, and more particularly the peptides SEQ ID NO 78, SEQ ID NO 80,
• the invention also relates to the nucleotide sequences coding for the abovementioned peptide fragments, or for the proteins derived therefrom, or also for the fusion proteins as described above.
• the subject of the invention is more particularly: - the nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of positions 1244 to 1322 of the nucleotide sequence SEQ ID NO 1 shown in FIG. 1, and the 3' end corresponds to the nucleotide located at ' one of positions 1495 to 1540 in FIG. 1, said nucleotide sequences coding for the abovementioned fragments of the human WASP protein, the N-terminal amino acid of which corresponds to that located in one of positions 404 to 430 of FIG. 1, and the C-terminal amino acid corresponds to that located at one of positions 487 to 502 in FIG. 1,
• nucleotide sequence SEQ ID NO 3 delimited by the nucleotides located at positions 1244 and 1540 of FIG. 1, and coding for the peptide fragment of the human WASP protein corresponding to the peptide SEQ ID NO 4 mentioned above,
• nucleotide sequence SEQ ID NO 5 delimited by the nucleotides located at positions 1244 and 1495 of FIG. 1, and coding for the peptide fragment of the human WASP protein corresponding to the peptide SEQ ID NO 6 mentioned above,
• nucleotide sequence SEQ ID NO 7 delimited by the nucleotides located at positions 1322 and 1540 of FIG. 1, and coding for the peptide fragment of the protein
• nucleotide sequence SEQ ID NO 9 delimited by the nucleotides located at positions 1322 and 1495 of FIG. 1, and coding for the peptide fragment of the human WASP protein corresponding to the peptide SEQ ID NO 10 mentioned above, - the nucleotide sequences derived by degeneration the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
• nucleotide sequences derived from the abovementioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
• the invention relates more particularly still:
• nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of positions 1174 to 1299 of the nucleotide sequence SEQ ID NO 11 shown in FIG. 2, and the 3' end corresponds to the nucleotide located at ' one of positions 1464 to 1515 in FIG. 2, said nucleotide sequences coding for the abovementioned fragments of the protein N-WASP, the N-terminal amino acid of which corresponds to that located in one of positions 392 to 433 in FIG. 2 , and the C-terminal amino acid corresponds to that located at one of positions 488 to 505 in FIG.
• nucleotide sequence delimited by the nucleotides located in positions 1174 to 1515 coding for the peptide fragment of the protein N-WASP delimited by acids amines located at positions 392 to 505 of the peptide sequence represented in FIG. 2 - the nucleotide sequence SEQ ID NO 15 delimited by the nucleotides located at positions 1174 and 1464 coding for the peptide fragment of the N-WASP protein corresponding to the peptide SEQ ID NO 16 mentioned above,
• nucleotide sequence SEQ ID NO 17 delimited by the nucleotides located at positions 1213 and 1515 coding for the peptide fragment of the protein N-WASP corresponding to the peptide SEQ ID NO 18 mentioned above,
• nucleotide sequence SEQ ID NO 19 delimited by the nucleotides located at positions 1213 and 1464 coding for the peptide fragment of the protein N-WASP corresponding to the peptide SEQ ID NO 20 mentioned above,
• nucleotide sequence SEQ ID NO 21 delimited by the nucleotides located at positions 1297 and 1515 coding for the peptide fragment of the protein N-WASP corresponding to the peptide SEQ ID NO 22 mentioned above,
• nucleotide sequence SEQ ID NO 23 delimited by the nucleotides located at positions 1297 and 1464 encoding the peptide fragment of the protein N-WASP corresponding to the peptide SEQ ID NO 24 above, - the nucleotide sequences derived by degeneration of the genetic code of the sequences aforementioned nucleotides, and coding for the aforementioned peptide fragments,
• nucleotide sequences derived from the abovementioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
• the subject of the invention is more particularly:
• nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of the positions 1327 to 1489 of the nucleotide sequence SEQ ID NO 26 shown in FIG. 3, and the 3' end corresponds to the nucleotide located at ' one of positions 1638 to 1677 in FIG. 3, said nucleotide sequences coding for the abovementioned fragments of the human Scarl protein whose N-terminal amino acid corresponds to that located at one of positions 546 to 497 in FIG. 3, and the C-terminal amino acid corresponds to that located at one of positions 487 to 559 in FIG. 3,
• nucleotide sequence SEQ ID NO 27 delimited by the nucleotides located at positions 1327 and 1677 of FIG. 3, and coding for the peptide fragment of the human Scarl protein corresponding to the peptide SEQ ID NO 28 mentioned above,
• nucleotide sequence SEQ ID NO 29 delimited by the nucleotides located at positions 1327 and 1638 in FIG. 3, and coding for the peptide fragment of the human Scarl protein corresponding to the peptide SEQ ID NO 30 mentioned above,
• nucleotide sequence SEQ ID NO 33 delimited by the nucleotides located at positions 1489 and 1638 of FIG. 3, and coding for the peptide fragment of the human Scarl protein corresponding to the peptide SEQ ID NO 34 mentioned above, - the nucleotide sequences derived by degeneration the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
• nucleotide sequences derived from the abovementioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
• the subject of the invention is more particularly:
• nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of positions 1282 to 1366 of the nucleotide sequence SEQ ID NO 36 shown in FIG. 4, and the 3' end corresponds to the nucleotide located at ' one of positions 1539 to 1584 in FIG. 4, said nucleotide sequences coding for the abovementioned fragments of the murine WASP protein, the N-terminal amino acid of which corresponds to that located in one of positions 420 to 448 in FIG. 4, and the C-terminal amino acid corresponds to that located at one of the positions 505 to 520 of FIG. 4,
• Murine WASP corresponding to the peptide SEQ ID NO 38 mentioned above the nucleotide sequence SEQ ID NO 39 delimited by the nucleotides located at positions 1282 and 1584 of FIG. 4, and coding for the peptide fragment of the murine WASP protein corresponding to the peptide SEQ ID NO 40 mentioned above,
• nucleotide sequence SEQ ID NO 43 delimited by the nucleotides located at positions 1366 and 1539 of FIG. 4, and coding for the peptide fragment of the murine WASP protein corresponding to the peptide SEQ ID NO 44 mentioned above, - the nucleotide sequences derived by degeneration the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
• nucleotide sequences derived from the abovementioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
• the subject of the invention is more particularly:
• nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of positions 1272 to 1356 of the nucleotide sequence SEQ ID NO 46 shown in FIG. 5, and the 3' end corresponds to the nucleotide located at one of positions 1523 to 1574 in FIG. 5, said nucleotide sequences coding for the abovementioned fragments of the rat N-WASP protein, the N-terminal amino acid of which corresponds to that located in one of positions 401 to 429 of the FIG. 5, and the C-terminal amino acid corresponds to that located at one of positions 484 to 501 in FIG. 5,
• nucleotide sequence SEQ ID NO 49 delimited by the nucleotides located at positions 1272 and 1523 in FIG. 5, and coding for the peptide fragment of the rat N-WASP protein corresponding to the peptide SEQ ID NO 50 mentioned above
• nucleotide sequence SEQ ID NO 51 delimited by the nucleotides located at positions 1356 and 1574 of FIG. 5, and coding for the peptide fragment of the rat N-WASP protein corresponding to the peptide SEQ ID NO 52 mentioned above
• nucleotide sequence SEQ ID NO 53 delimited by the nucleotides located at positions 1356 and 1523 of FIG. 5, and coding for the peptide fragment of the rat N-WASP protein corresponding to the peptide SEQ ID NO 54 mentioned above
• nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments
• nucleotide sequences derived from the abovementioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
• the subject of the invention is more particularly:
• nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of positions 1500 to 1584 of the nucleotide sequence SEQ ID NO 56 shown in FIG. 6, and the 3' end corresponds to the nucleotide located at ' one of positions 1751 to 1802 in FIG. 6, said nucleotide sequences coding for the abovementioned fragments of the bovine N-WASP protein, the N-terminal amino acid of which corresponds to that located in one of positions 405 to 433 in the figure 6, and the C-terminal amino acid corresponds to that located at one of positions 488 to 505 in FIG. 6,
• nucleotide sequence SEQ ID NO 57 delimited by the nucleotides located at positions 1500 and 1802 in FIG. 6, and coding for the peptide fragment of the bovine N-WASP protein corresponding to the peptide SEQ ID NO 58 mentioned above,
• nucleotide sequence SEQ ID NO 59 delimited by the nucleotides located at positions 1500 and 1751 in FIG. 6, and coding for the peptide fragment of the bovine N-WASP protein corresponding to the peptide SEQ ID NO 60 mentioned above,
• nucleotide sequence SEQ ID NO 61 delimited by the nucleotides located at positions 1584 and 1802 of FIG. 6, and coding for the peptide fragment of the protein
• nucleotide sequence SEQ ID NO 63 delimited by the nucleotides located at positions 1584 and 1751 in FIG. 6, and coding for the peptide fragment of the bovine N-WASP protein corresponding to the peptide SEQ ID NO 64 mentioned above, - the nucleotide sequences derived by degeneration of the genetic code of the abovementioned nucleotide sequences, and coding for the abovementioned peptide fragments, the nucleotide sequences derived from the abovementioned nucleotide sequences, and coding for the sequences derived from said peptide fragments as defined above.
• the subject of the invention is more particularly: - the nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of the positions 2035 to 2110 of the nucleotide sequence SEQ ID NO 66 shown in FIG. 7, and the end 3 'corresponds to the nucleotide located at one of the positions 2643 to 2670 in FIG. 7, said nucleotide sequences coding for the abovementioned fragments of the Las 17 protein of Saccharomyces cerevisiae whose N-terminal amino acid corresponds to that located at l one of positions 422 to 447 in FIG. 7, and the C-terminal amino acid corresponds to that located in one of positions 624 to 633 in FIG. 7,
• nucleotide sequence SEQ ID NO 67 delimited by the nucleotides located at positions 2035 and 2670 of FIG. 7, and coding for the peptide fragment of the Las 17 protein of Saccharomyces cerevisiae corresponding to the peptide SEQ ID NO 68 mentioned above,
• nucleotide sequence SEQ ID NO 69 delimited by the nucleotides located at positions 2035 and 2643 of FIG. 7, and coding for the peptide fragment of the protein Las 17 of Saccharomyces cerevisiae corresponding to the peptide SEQ ID NO 70 above
• nucleotide sequence SEQ ID NO 71 delimited by the nucleotides located at positions 2110 and 2670 of FIG. 7, and coding for the peptide fragment of the protein Las 17 of Saccharomyces cerevisiae corresponding to the peptide SEQ ID NO 72 mentioned above
• nucleotide sequence SEQ ID NO 73 delimited by the nucleotides located at positions 2110 and 2643 of FIG. 7, and coding for the peptide fragment of the protein
• the subject of the invention is more particularly: - the nucleotide sequences whose 5 'end corresponds to the nucleotide located at one of the positions 1429 to 1501 of the nucleotide sequence SEQ ID NO 76 shown in FIG. 8, and the end 3 'corresponds to the nucleotide located at one of positions 1695 to 1722 in FIG. 8, said nucleotide sequences coding for the abovementioned fragments of the homologous protein WASP (Wsplp) of Schizosaccharomyces pombe whose N-terminal amino acid corresponds to that located at one of positions 477 to 501 in FIG. 8, and the C-terminal amino acid corresponds to that located at one of positions 565 to 574 in FIG. 8,
• nucleotide sequence SEQ ID NO 79 delimited by the nucleotides located at positions 1429 and 1695 of FIG. 8, and coding for the peptide fragment of the protein (Wsplp) of Schizosaccharomyces pombe corresponding to the peptide SEQ ID NO 80 mentioned above,
• nucleotide sequence SEQ ID NO 81 delimited by the nucleotides located at positions 1501 and 1722 of FIG. 8, and coding for the peptide fragment of the homologous protein WASP (Wsplp) of Schizosaccharomyces pombe corresponding to the peptide SEQ ID NO 82 mentioned above
• nucleotide sequence SEQ ID NO 83 delimited by the nucleotides located at positions 1501 and 1695 of FIG. 8, and coding for the peptide fragment of the homologous protein WASP (Wsplp) of Schizosaccharomyces pombe corresponding to the peptide SEQ ID NO 84 mentioned above
• the subject of the invention is also the vectors, in particular the plasmids, containing a nucleotide sequence as defined above.
• the invention also relates to host cells transformed with an above-mentioned vector, said cells expressing the above-mentioned peptide fragments, or the derivative sequences described above, in recombinant form.
• the above-mentioned host cells are chosen from the following: Escherichia coli D5 ⁇ and Escherichia coli BL21.
• a subject of the invention is also reagents for implementing a method for detecting or screening molecules having an inhibiting or stimulating effect on the formation of the actin cytoskeleton, and therefore an inhibiting effect. or for stimulating cell mobility, said reagent comprising at least one peptide fragment defined above, linked or adsorbed to a support capable of moving under the effect of actin polymerization, when said support linked to said fragment is placed in a medium containing the elements necessary for the polymerization of actin, in particular when said support is added to a medium containing mainly the Arp2 / 3 complex, the VASP protein (stimulated phosphoprotein vasodilator), cofilin, and styling proteins , this medium can for example be an extract prepared from supernatants of lysed cells of the organism.
• Styling protein is an essential part of WASP-dependent motility. These proteins are capable of converting the polymerization of actin into an actin-dependent movement by spatially limiting the polymerization to certain sites of the cell cytoskeleton. In comparison, in the absence of styling proteins the actin polymerization still takes place, but since uncontrolled, can no longer generate a force. Styling proteins are composed of two subunits, alpha (p34) and beta (p32) which are both required for styling activity. The dimer associates with the plus end of the filament which is normally favored for the addition of actin monomers and thus blocks the addition of monomers. Genes coding for styling proteins have been identified in all the species studied to date and have been shown to be essential for the survival of organisms. In humans, Cap beta 1, beta2, G and CapZ have been identified to date. Also know that multifunctional proteins such as gelsoline and villin also have styling activity in addition to their other actin-modifying activities.
• a more particular subject of the invention is the reagents as defined above, chosen from microspheres whose diameter is advantageously between approximately
• the material constituting the microspheres itself being preferably chosen from polystyrenes or latex, said microspheres each advantageously containing approximately 5,000 to approximately 50,000 molecules of the abovementioned peptide fragment or of a sequence derived according to the invention.
• the above-mentioned peptide fragment, or its derived sequence are adsorbed on the surface of said microspheres, said reagent being obtained by simple mixing of said microspheres with said peptide fragment or with its derived sequence.
• a more particular subject of the invention is the reagents as defined above, chosen from drops of oil, in particular oil from C14 to C18, such as palmitic acid, the diameter of which is advantageously between approximately 1 ⁇ m and approximately
• the subject of the invention is also any method of detecting or screening molecules as defined above, having an effect of inhibiting or stimulating the formation of the actin cytoskeleton, and therefore an effect of inhibiting or stimulation of cell motility, said method comprising:
• the abovementioned medium in which the molecule tested is placed in the presence of said reagent contains a compound marked in particular by fluorescence, making it possible to detect the displacement of said reagent.
• the compound marked above is a fluorescent derivative of actin, such as actin-rhodamine (commercially available), making it possible to visualize the polymerization of actin by epifluorescence microscopy.
• a subject of the invention is also the application of the method as defined above, to the detection or screening of molecules, as defined above, capable of being able to be used as medicaments in the treatment of related pathologies to a dysfunction of the actin polymerization process in the context of the formation of the actin cytoskeleton, or likely to have a cytotoxic effect corresponding to an inhibition or stimulation of the formation of the actin cytoskeleton.
• the subject of the invention is also a kit or kit for implementing a process mentioned above, comprising
• the invention will be further illustrated by means of the following detailed description of the preparation of microspheres coated with a peptide fragment of the invention, and of the detection of the polymerization of actin on the surface of these microspheres in a cell supernatant extract.
• the sequence coding for the WH2 / A domain of the human WASP protein (namely the 99 amino acid fragment delimited by the amino acids located at positions 404 and 502 in FIG. 1, also designated peptide SEQ ID NO 4), and that encoding the epitope myc9E10 were amplified by PCR.
• the myc epitope fused to the carboxy-terminal portion of the protein serves as a molecular label, allowing detection of the protein by immunological approaches without the need for an anti-WASP antibody.
• This DNA sequence was introduced into the vector pGEX2T (Pharmacia), downstream of the sequence coding for glutathione-S-transferase (GST), generating the plasmid WH2 / A- pGEX2T.
• the GST domain was chosen in this work because it facilitates the purification of the recombinant protein, and it has been shown that this domain does not inhibit the ability to polymerize the actin of the GST-VCA protein derived from N-WASP in a pyrene actin test (Rohatgi et al., 1999).
• Figure 3 shows the organization of GST-WH2 domains. This recombinant protein is composed of GST and WH2 / A domains, of 237 and
• E. coli bacteria (strain BL21) were transformed with the plasmid WH2 / 1 pG ⁇ X2T.
• the bacteria were cultured in standard LB medium containing the antibiotic ampicillin to maintain the bacteria comprising the plasmid under selection pressure.
• the bacteria were grown in suspension at 37 ° C until the culture reached an optical density of 0.8 at 600 nm.
• isopropylthio- ⁇ -D-galactoside (IPTG) was added to the medium at a final concentration of 1 mM to induce the production of the protein. After 2 hours, the bacteria were collected by centrifugation and the pellets were stored at -80 ° C.
• the pellets were thawed and added to extraction buffer (phosphate-buffered saline pH 7.2, 200 NaCl, 2 mM EDTA (ethylenediamine tetra acid), containing 1 ⁇ g / ml of each of the following protease inhibitors, leupeptin, benzamidine, pepstatin, at a ratio of 1 g of pellet per 10 volumes of extraction buffer.
• extraction buffer phosphate-buffered saline pH 7.2, 200 NaCl, 2 mM EDTA (ethylenediamine tetra acid)
• the suspension was sonicated until it was no longer viscous.
• the extract was centrifuged at 20,000 xg for 10 minutes at 4 ° C.
• the protein GST-WH2 / A was purified from the bacterial extract by affinity chromatography on resin coupled with Glutathione (Pharmacia) and eluted with 20 mM reduced glutathione according to manufacturers' recommendations. Purification was confirmed by analysis of GST-
• WH2 / A by electrophoresis on acrylamide gel.
• the presence of the myc epitope in the GST-WH2 / A sequence was confirmed by immunoblotting with an antibody directed against this epitope.
• the GST-WH2 / A protein was absorbed on 500 nm latex beads (Polyscience
• a water-oil emulsion is prepared as follows: 100 ⁇ l of oil are mixed with 900 ⁇ l of borate buffer
• 0.1 MpH 8.5 (boric acid buffered with NaOH).
• the solution is sonicated (probe) for a few seconds and a white foam is obtained.
• the size of the oil drops is between 20 ⁇ m and l ⁇ m.
• 20 ⁇ l of this solution are mixed with 100 ⁇ l of the aforementioned GST-WH2 / A protein (also designated GST-WA) (1 mg / ml in borate buffer). This solution is incubated at room temperature for 12 hours
• GST-WA according to the supplier's protocol.
• the particles are resuspended to 1% solid in PBS (Phosphate Buffer Saline) and stored on ice.
• PBS Phosphate Buffer Saline
• Each sample contains 15 ⁇ l of HeLa extracts (supplemented with ATP, creatine phosphate and rhodamine-labeled actin), 0.5 ⁇ l of GST-WA particles and 1.5 ⁇ l of PBS (A ), or 1.5 ⁇ l of GST-WA protein at 1 mg / ml (B) or 1.5 ⁇ l of GST-PRO protein (PRO corresponding to the fragment delimited by the amino acids located at positions 235 to 584 of the ActA protein Listeria monocytogenes) at 1 mg / ml (C). 6 ⁇ l of the final mixture of these three solutions are sealed between slide and coverslip.
• the present invention provides an in vitro test for testing the polymerization of actin on the surface of beads, and shows that this system reproduces the essential characteristics of the polymerization of actin in human cells.
• these beads recruit proteins important for the polymerization of actin in cells, such as the Arp2 / 3 complex and cofilin.
• components recruited from the surface of the beads are targets for signaling pathways involving tyrosine kinases. Thanks to this in vitro system, it is now possible to study the conditions necessary for the polymerization of actin in the cell, a process which until now was not accessible to direct experimental manipulation. a) Method for screening anti-metastatic components
• the bead system described here relies on human proteins which are necessary for the polymerization of actin in human cells. However, these proteins are kept during evolution, from yeast to humans, including amoebas. Despite the conservation of their basic function, these proteins also exhibit divergences in their primary sequence, suggesting significant functional differences. Analogous to metastatic cancers, the actin cytoskeleton of parasites is rarely the target of drugs used in the treatment of parasitoses, despite the important role it plays during the infectious cycle of many parasites (amoebae for example). It is conceivable that drugs which affect the human actin cytoskeleton only when used in high concentrations, affect that of parasites at much lower concentrations. This is why, thanks to the universal nature of the evolution of the actin cytoskeleton, the bead system of the present invention also makes it possible to search for drugs which can be used in the treatment of parasitoses. c) Detection of drug side effects
• the beads are used to check and confirm that drugs have no side effects on the polymerization of cellular actin.
• drugs have no side effects on the polymerization of cellular actin.
• the most effective screening tests for testing new drugs have the following properties: they are simple, inexpensive and rapid.
• the polymerization test of cellular actin of the present invention has all of these characteristics. 200-500nm beads can be produced by absorbing the purified recombinant protein GST-WH2 / A on their surface. Once prepared, the beads are stable for several months at 4 ° C. The beads are then added to extracts prepared from supernatants of lysed culture cells. The volume of extract necessary for an experiment is of the order of a few microliters, reducing the cost of the experiment. These extracts have the advantage of being able to be produced in large quantities and stored in the long term, at -80 ° C.
• a fluorescent actin derivative (commercial actin-rhodamine) is added to the extract in order to visualize the polymerization of actin by epifluorescence microscopy. Actin polymerization is observed 15 min after the addition of the beads, resulting in an accumulation of fluorescent actin around the beads.
• the experiment takes less than 30 min and one person is able to process several samples in parallel. This procedure can be automated, allowing rapid screening of a large number of samples.
• the inventors have shown that the small amounts of solvents used to dissolve these products (water, ethanol, dimethylsulphoxide) do not disturb the system of the invention.
• this screening test has been validated by showing that it is sensitive to currently known drugs for inhibiting the polymerization of actin, such as latrunculin and cytochalasin D.

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