# Patent

## Title
Wasp family protein fragments, and use thereof

## Abstract

The invention concerns peptide fragments of the WASP family proteins, or peptides derived from said fragments, and their uses in particular for preparing reagents for use in implementing a method for detecting or screening molecules with inhibiting or stimulating effect on the formation of the actin cytoskeleton, hence an inhibiting or stimulating effect on cellular motility.

## Background
The invention concerns peptide fragments of WASP family proteins or peptides derived from these fragments, and uses thereof in particular within the context of molecule detection processes having an effect on cellular motility.
The cells of our body are capable of moving and they sometimes become round and divide into two sister cells. All these movements are based on the actin cytoskeleton. At a multicellular stage, the cytoskeleton plays an essential role in the organization of the body and homeostasis. For example, cellular migration is essential in embryogenesis and the immune response, as well as in the healing of wounds, where the cells migrate to the damaged regions. These movements are dependent on the normal functioning of the actin cytoskeleton. The consequences of disturbing the functioning of the cytoskeleton can be disastrous to the organism. In metastatic processes, for example, the cytoskeleton's lack of control over tumorous cells can cause them to migrate outside their normal location, allowing them to proliferate in other parts of the body, which makes treatment of the cancer extremely difficult.
The characterisation of proteins capable of polymerising actin and the understanding of the mechanism whereby this polymerisation generates a force, represent the key elements in understanding the functioning of the cytoskeleton in the cell. However, the dynamic properties of the cytoskeleton make it extremely difficult to study. Moreover, the approaches currently available for analysing the cytoskeleton are complicated or tedious.
The first stage in all the processes dependent on the cytoskeleton, such as movement, is the production of filaments of actin, or F-actin. The mechanism of the formation of these biological polymers in the cell is still not known, despite the identification of numerous actin-binding proteins, and the extensive study of actin polymerisation in vitro.
Wiskott-Aldrich syndrome is a disease of the cytoskeleton. Human WASP protein, expressed on the basis of the WAS gene which is mutated in patients affected by this syndrome, and likewise N-WASP protein of bovine origin (which is approx. 45% identical in sequence with human WASP protein) have thus been the subject of investigations aimed at explaining the mechanism of the 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)).
These WASP and N-WASP proteins are made up of peptide domains having functional properties which are identifiable in biochemical tests. Thus, these two proteins mainly possess the following domains:

    
    
        a plekstrin homology domain which binds to phosphatidylinositol (4,5) biphosphate, the latter itself binding to proteins involved in the formation of actin filaments,
        a Cdc42 binding domain, a protein with GTPase activity inducing actin polymerisation, and hence regulating the cytoskeleton,
        a prolin-rich region,
        a verprolin homology domain, a protein also binding to proteins involved in the formation of actin filaments, said domain comprising a verprolin-homologous sequence in the case of human WASP, and two verprolin-homologous sequences in the case of N-WASP,
        a cofilin homology domain, an actin-binding protein, having an actin-depolymerisation activity dependent on the pH,
        a C-terminal acid segment.
    
    


Moreover, it has been shown that these WASP and N-WASP proteins interact with the Arp2/3 complex (a proteinic complex involved in actin polymerisation) and thus induce actin polymerisation.
On this basis, it has been demonstrated that 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). To demonstrate this, the authors of this article prepared microspheres covered with WASP protein and demonstrated that these microspheres polymerise actin, form actin tails, and are endowed with motility based on the actin in the cellular extracts. In cellular extracts in which the Arp2/3 complex has been eliminated, the microspheres covered with WASP protein no longer have any motility and only possess a residual actin polymerisation activity.
With regard to N-WASP protein, it has been demonstrated that the C-terminal part of N-WASP, i.e. the C-terminal fragment of 114 amino acids comprising the verprolin homology domain (V region containing the two verprolin homologous sequences), the cofilin homology domain (C region) and the C-terminal acid segment (A region), or VCA fragment, binds to the Arp2/3 complex and strongly stimulates the latter's capacity to nucleate actin polymerisation (Rohatgi et al. 1999, above-mentioned).
According to recent studies, whole WASP and N-WASP proteins would be required for cellular motility dependent on actin, to the extent that, not only does the C-terminal part of these proteins interact with actin, but also the N-terminal part which keeps the actin filament in the process of formation in proximity to the cellular surface (Loisel et al., Nature, 401: 613–616 (1999); Egile et al., The Journal of Cell Biology, 145: 1319–1332 (1999)).
The invention follows on from the highlighting by the Inventors of the fact that, contrary to what might be supposed from the prior art described above, fragments of these WASP and N-WASP proteins are sufficient to induce actin polymerisation as this allows the movement of the supports to which said fragments are bound in cellular extracts.
The invention aims to provide new fragments, or polypeptides derived from WASP and N-WASP proteins, as well as nucleotide sequences coding for these fragments.
The invention also aims to provide new molecule detection or screening processes having an effect on the formation of the cytoskeleton, in particular cytotoxic molecules or medicaments which can be used within the context of treatment of pathologies linked to abnormal development of the cytoskeleton.
The invention also aims to provide new reagents and kits for implementation of the above-mentioned processes.
The invention also concerns the use:

    
    
        of peptide fragments of WASP family proteins in eukaryotic cells, in particular in cells of humans or other mammals, or cells of insects or of micro-organisms such as yeasts, said peptide fragments having the property of WASP family proteins to polymerise actin, inducing cellular motility,
        or peptide sequences derived from the above-mentioned peptide fragments of WASP family proteins, in particular by substitution of one or more amino acids of these fragments, said derived sequences having the above-mentioned property of WASP family proteins and said fragments thereof,
        for the preparation of reagents which can be used within the framework of implementation of a process for the detection or screening of molecules having an inhibiting or stimulating effect on the formation of the actin cytoskeleton, and hence an inhibiting or stimulating effect on cellular motility.
    
    


The term “actin polymerisation property inducing cellular motility” above and below means the property of WASP family proteins, or fragments or sequences derived from these, to polymerise actin, inducing the movement of cells of the organism, or of appropriate supports, such as the microspheres described below, in vivo or in vitro.
The invention also concerns the use of the above-mentioned peptide fragments or derived sequences, within the context of implementation of a process for the detection or screening of molecules capable of being used as medicaments in the treatment of pathologies linked to a dysfunction of the actin polymerisation process within the context of formation of the actin cytoskeleton.
The invention concerns more particularly the use of above-mentioned peptide fragments or derived sequences, within the context of implementation of a process for the detection or screening of molecules having an inhibiting effect on the formation of the actin cytoskeleton, and thus an inhibiting effect on cellular motility, said molecules being capable of being used:

    
    
        as medicaments in the treatment of metastatic cancers,
        or as anti-parasitic antibiotics.
    
    


The invention concerns more particularly the use of above-mentioned peptide fragments or derived sequences, within the context of implementation of a process for the detection of secondary effects of molecules, in particular of medicaments or molecules from the environment, i.e. a process for the detection of molecules capable of having a cytotoxic effect corresponding to inhibition or stimulation of the formation of the actin cytoskeleton.
The term “WASP family proteins” above and below means the protein produced by the WAS gene, mutated within the context of Wiskott-Aldrich syndrome in humans, as well as proteins which may or may not be of human origin, having at least approx. 45% homology with the above-mentioned human WASP protein, and being involved in the cellular actin polymerisation process, and, possibly, in cellular motility.
The above-mentioned WASP family proteins also possess the common characteristic of possessing at least three major domains:

    
    
        a WH1/Scar domain in the N-terminal part; this domain has structural characteristics similar to a plekstrin homology domain (or pH domain), and is supposed to interact with polymerised actin and with phospholipids,
        a prolin rich domain,
        a WH2/A domain which is divided into three sub-domains, i.e. the above-mentioned verprolin homology sub-domain, cofilin homology sub-domain and acid sub-domain.
    
    


Advantageously, the peptide fragments used within the context of the present invention are chosen from WASP, N-WASP, Scar and Las protein fragments, or peptide sequences derived from the above-mentioned peptide fragments as defined above.
The invention concerns more particularly the above-mentioned use of peptide fragments chosen from fragments:

    
    
        of human or other mammalian WASP protein, in particular bovine or murine WASP protein,
        of human or other mammalian N-WASP protein, in particular bovine or rat N-WASP protein,
        of Scar sub-family proteins, such as Scar1/WAVE protein of Dictyostellium discoideum, or of Caenorhabditis elegans, or of Drosophila melanogaster, of mice or of humans.
        of proteins of the Las sub-family of micro-organisms, in particular yeasts such as the LAS17/Beel protein of Saccharomyces cerevisiae or the WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe. 
    
    


Advantageously, the above-mentioned peptide fragments are chosen from those including:

    
    
        the verprolin homology domain contained in WASP family proteins, or in a protein derived from these, or at least one of the two verprolin homologous sequences when said WASP family proteins contain two of these sequences, or a peptide sequence derived from the above-mentioned domain, in particular by substitution, addition or removal of one or more amino acids, and retaining the property of this domain to bind to actin,
        and the cofilin homology domain contained in WASP family proteins, or in a protein derived from these, or a peptide sequence derived from the above-mentioned domain, in particular by substitution, addition or removal of one or more amino acids, and retaining the property of this domain to intervene in actin polymerisation.
    
    


The above-mentioned peptide fragments used within the framework of the present invention possibly also contain the C-terminal acid segment of said WASP proteins or derivatives.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents the peptide sequence of a human WASP protein (SEQ ID NO: 2).
FIG. 2 represents the peptide sequence of a human N-WASP protein (SEQ ID NO: 12).
FIG. 3 represents the peptide sequence of a human Scar 1 protein (SEQ ID NO: 26).
FIG. 4 represents a peptide sequence of a murine WASP protein (SEQ ID NO: 36).
FIG. 5 represents a peptide sequence of a N-WASP protein (SEQ ID NO: 46).
FIG. 6 represents bovine N-WASP protein (SEQ ID NO: 56).
FIG. 7 represents a peptide sequence of a LAS 17 protein (SEQ ID NO: 66).
FIG. 8 represents a WASP homologous protein of Schizosaccharomyces pombe (SEQ ID NO: 76).



Advantageously, the above-mentioned peptide fragments do not contain the plekstrin homology domain, and/or the Cdc42 binding domain, and/or the prolin-rich region, defined above, of said WASP family proteins.
The invention concerns more particularly the above-mentioned use of peptide fragments of WASP family proteins of human origin.
Advantageously, the peptide fragments of WASP family proteins of human origin used are chosen from fragments of human WASP protein including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 430 and 446 of the peptide sequence SEQ ID NO: 2 of human WASP protein represented in FIG. 1, or a peptide sequence derived from the above-mentioned domain as defined above.
        and the cofilin homology domain delimited by the amino acids situated at positions 469 and 487 of the peptide sequence SEQ ID NO: 2 of human WASP protein represented in FIG. 1, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


Preferably, the above-mentioned fragments of human WASP protein used are chosen from the following:

    
    
        fragments of which the N-terminal amino acid corresponds to that situated at one of positions 404 to 430 in FIG. 1, and the C-terminal amino acid corresponds to that situated at one of positions 487 to 502 in FIG. 1,
        the fragment of 99 amino acids delimited by the amino acids situated at positions 404 and 502 in FIG. 1, i.e. the following SEQ ID NO: 4 peptide:
    
    










Pro Ser Ser Gly Asn Gly Pro Ala Pro Pro Pro Leu Pro Pro Ala Leu



 


Val Pro Ala Gly Gly Leu Ala Pro Gly Gly Gly Arg Gly Ala Leu Leu


 


Asp Gln Ile Arg Gln Gly Ile Gln Leu Asn Lys Thr Pro Gly Ala Pro


 


Glu Ser Ser Ala Leu Gln Pro Pro Pro Gln Ser Ser Glu Gly Leu Val


 


Gly Ala Leu Met His Val Met Gln Lys Arg Ser Arg Ala Ile His Ser


 


Ser Asp Glu Gly Glu Asp Gln Ala Gly Asp Glu Asp Glu Asp Asp Glu


 


Trp Asp Asp






    
    
        the fragment of 84 amino acids delimited by the amino acids situated at positions 404 and 487 in FIG. 1, i.e. the following SEQ ID NO: 6 peptide:
    
    










Pro Ser Ser Gly Asn Gly Pro Ala Pro Pro Pro Leu Pro Pro Ala Leu



 


Val Pro Ala Gly Gly Leu Ala Pro Gly Gly Gly Arg Gly Ala Leu Leu


 


Asp Gln Ile Arg Gln Gly Ile Gln Leu Asn Lys Thr Pro Gly Ala Pro


 


Glu Ser Ser Ala Leu Gln Pro Pro Pro Gln Ser Ser Glu Gly Leu Val


 


Gly Ala Leu Met His Val Met Gln Lys Arg Ser Arg Ala Ile His Ser


 


Ser Asp Glu Gly






    
    
        the fragment of 73 amino acids delimited by the amino acids situated at positions 430 and 502 in FIG. 1, i.e. the following SEQ ID NO: 8 peptide:
    
    










Gly Arg Gly Ala Leu Leu Asp Gln Ile Arg Gln Gly Ile Gln Leu Asn



 


Lys Thr Pro Gly Ala Pro Glu Ser Ser Ala Leu Gln Pro Pro Pro Gln


 


Ser Ser Glu Gly Leu Val Gly Ala Leu Met His Val Met Gln Lys Arg


 


Ser Arg Ala Ile His Ser Ser Asp Glu Gly Glu Asp Gln Ala Gly Asp


 


Glu Asp Glu Asp Asp Glu Trp Asp Asp






    
    
        the fragment of 58 amino acids delimited by the amino acids situated at positions 430 and 487 in FIG. 1, i.e. the following SEQ ID NO: 10 peptide:
    
    










Gly Arg Gly Ala Leu Leu Asp Gln Ile Arg Gln Gly Ile Gln Leu Asn



 


Lys Thr Pro Gly Ala Pro Glu Ser Ser Ala Leu Gln Pro Pro Pro Gln


 


Ser Ser Glu Gly Leu Val Gly Ala Leu Met His Val Met Gln Lys Arg


 


Ser Arg Ala Ile His Ser Ser Asp Glu Gly






    
    
        or the peptide sequences derived from the above-mentioned peptide fragments, in particular by substitution, addition or removal of one or more amino acids of these fragments, said derived sequences having the property defined above, of WASP family proteins and said fragments thereof.
    
    


Advantageously, the peptide fragments of WASP family proteins of human origin used are chosen from the fragments of human N-WASP protein including:

    
    
        the verprolin homologous sequence delimited by the amino acids situated at positions 405 and 421 of the peptide sequence SEQ ID NO: 12 of human N-WASP protein represented in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above,
        and/or the verprolin homologous sequence delimited by the amino acids situated at positions 433 and 449 of the peptide sequence SEQ ID NO: 12 of human N-WASP protein represented in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain contained in the above-mentioned N-WASP protein, i.e. the domain delimited by the amino acids situated at positions 470 and 488 of the peptide sequence SEQ ID NO: 12 of human N-WASP protein represented in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


Preferably, the above-mentioned fragments of the human N-WASP protein used are chosen from the following:

    
    
        fragments of which the N-terminal amino acid corresponds to that situated at one of positions 392 to 433 in FIG. 2, and the C-terminal amino acid corresponds to that situated at one of positions 488 to 505 in FIG. 2,
        the fragment of 114 amino acids delimited by the amino acids situated at positions 392 and 505 in FIG. 2, i.e. the following SEQ ID NO: 14 peptide:
    
    










Pro Ser Asp Gly Asp His Gln Val Pro Thr Thr Ala Gly Asn Lys Ala



 


Ala Leu Leu Asp Gln Ile Arg Glu Gly Ala Gln Leu Lys Lys Val Glu


 


Gln Asn Ser Arg Pro Val Ser Cys Ser Gly Arg Asp Ala Leu Leu Asp


 


Gln Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Ala Asp Gly Gln Glu


 


Ser Thr Pro Pro Thr Pro Ala Pro Thr Ser Gly Ile Val Gly Ala Leu


 


Met Glu Val Met Gln Lys Arg Ser Lys Ala Ile His Ser Ser Asp Glu


 


Asp Glu Asp Glu Asp Asp Glu Glu Asp Phe Glu Asp Asp Asp Glu Trp


 


Glu Asp






    
    
        the fragment of 97 amino acids delimited by the amino acids situated at positions 392 and 488 in FIG. 2, i.e. the following SEQ ID NO: 16 peptide:
    
    










Pro Ser Asp Gly Asp His Gln Val Pro Thr Thr Ala Gly Asn Lys Ala



 


Ala Leu Leu Asp Gln Ile Arg Glu Gly Ala Gln Leu Lys Lys Val Glu


 


Gln Asn Ser Arg Pro Val Ser Cys Ser Gly Arg Asp Ala Leu Leu Asp


 


Gln Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Ala Asp Gly Gln Glu


 


Ser Thr Pro Pro Thr Pro Ala Pro Thr Ser Gly Ile Val Gly Ala Leu


 


Met Glu Val Met Gln Lys Arg Ser Lys Ala Ile His Ser Ser Asp Glu


 


Asp






    
    
        the fragment of 101 amino acids delimited by the amino acids situated at positions 405 and 505 in FIG. 2, i.e. the following SEQ ID NO: 18 peptide:
    
    










Asn Lys Ala Ala Leu Leu Asp Gln Ile Arg Glu Gly Ala Gln Leu Lys



 


Lys Val Glu Gln Asn Ser Arg Pro Val Ser Cys Ser Gly Arg Asp Ala


 


Leu Leu Asp Gln Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Ala Asp


 


Gly Gln Glu Ser Thr Pro Pro Thr Pro Ala Pro Thr Ser Gly Ile Val


 


Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys Ala Ile His Ser


 


Ser Asp Glu Asp Glu Asp Glu Asp Asp Glu Glu Asp Phe Glu Asp Asp


 


Asp Glu Trp Glu Asp






    
    
        the fragment of 84 amino acids delimited by the amino acids situated at positions 405 and 488 in FIG. 2, i.e. the following SEQ ID NO: 20 peptide:
    
    










Asn Lys Ala Ala Leu Leu Asp Gln Ile Arg Glu Gly Ala Gln Leu Lys



 


Lys Val Glu Gln Asn Ser Arg Pro Val Ser Cys Ser Gly Arg Asp Ala


 


Leu Leu Asp Gln Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Ala Asp


 


Gly Gln Glu Ser Thr Pro Pro Thr Pro Ala Pro Thr Ser Gly Ile Val


 


Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys Ala Ile His Ser


 


Ser Asp Glu Asp






    
    
        the fragment of 73 amino acids delimited by the amino acids situated at positions 433 and 505 in FIG. 2, i.e. the following SEQ ID NO: 22 peptide:
    
    










Gly Arg Asp Ala Leu Leu Asp Gln Ile Arg Gln Gly Ile Gln Leu Lys



 


Ser Val Ala Asp Gly Gln Glu Ser Thr Pro Pro Thr Pro Ala Pro Thr


 


Ser Gly Ile Val Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys


 


Ala Ile His Ser Ser Asp Glu Asp Glu Asp Glu Asp Asp Glu Glu Asp


 


Phe Glu Asp Asp Asp Glu Trp Glu Asp






    
    
        the fragment of 56 amino acids delimited by the amino acids situated at positions 433 and 488 in FIG. 2, i.e. the following SEQ ID NO: 24 peptide:
    
    










Gly Arg Asp Ala Leu Leu Asp Gln Ile Arg Gln Gly Ile Gln Leu Lys



 


Ser Val Ala Asp Gly Gln Glu Ser Thr Pro Pro Thr Pro Ala Pro Thr


 


Ser Gly Ile Val Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys


 


Ala Ile His Ser Ser Asp Glu Asp






    
    
        or the peptide sequences derived from the above-mentioned peptide fragments, in particular by substitution, addition or removal of one or more amino acids of these fragments, said derived sequences having the property defined above, of WASP family proteins and said fragments thereof.
    
    


Advantageously, the peptide fragments of WASP family proteins of human origin used are chosen from the fragments of human Scar1 protein including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 497 and 513 of the peptide sequence SEQ ID NO: 26 of human Scar1 protein represented in FIG. 3, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain delimited by the amino acids situated at positions 531 and 546 of the peptide sequence SEQ ID NO: 26 of human Scar1 protein represented in FIG. 3, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


Preferably, the above-mentioned human Scar1 protein fragments used are chosen from the following:

    
    
        fragments of which the N-terminal amino acid corresponds to that situated at one of positions 433 to 497 in FIG. 3, and the C-terminal amino acid corresponds to that situated at one of positions 546 to 559 in FIG. 3,
        the fragment of 117 amino acids delimited by the amino acids situated at positions 443 and 559 in FIG. 3, i.e. the following SEQ ID NO: 28 peptide:
    
    











Val Thr Val Thr Ala Leu Ala His Pro Pro Ser Gly



 



Leu His Pro Thr Pro Ser Thr Ala Pro Gly Pro His



 



Val Pro Leu Met Pro Pro Ser Pro Pro Ser Gln Val



 



Ile Pro Ala Ser Glu Pro Lys Arg His Pro Ser Thr



 



Leu Pro Val Ile Ser Asp Ala Arg Ser Val Leu Leu



 



Glu Ala Ile Arg Ile Ala Val Glu Tyr Ser Asp Ser



 



Glu Asp Asp Ser Glu Phe Asp Glu Lys Gly Ile Gln



 



Leu Arg Lys Val Glu Glu Gln Arg Glu Gln Glu Ala



 



Val Asp Trp Leu Glu






    
    
        the fragment of 104 amino acids delimited by the amino acids situated at positions 443 and 546 in FIG. 3, i.e. the following SEQ ID NO: 30 peptide:
    
    











Val Thr Val Thr Ala Leu Ala His Pro Pro Ser Gly



 



Leu His Pro Thr Pro Ser Thr Ala Pro Gly Pro His



 



Val Pro Leu Met Pro Pro Ser Pro Pro Ser Gln Val



 



Ile Pro Ala Ser Glu Pro Lys Arg His Pro Ser Thr



 



Leu Pro Val Ile Ser Asp Ala Arg Ser Val Leu Leu



 



Glu Ala Ile Arg Lys Gly Ile Gln Leu Arg Lys Val



 



Glu Glu Gln Arg Glu Gln Glu Ala Lys His Glu Arg



 



Ile Glu Asn Asp Val Ala Thr Ile Leu Ser Arg Arg



 



Ile Ala Val Glu Tyr Ser Asp Ser






    
    
        the fragment of 63 amino acids delimited by the amino acids situated at positions 497 and 559 in FIG. 3, i.e. the following SEQ ID NO: 32 peptide:
    
    











Ala Arg Ser Val Leu Leu Glu Ala Ile Arg Lys Gly



 



Ile Gln Leu Arg Lys Val Glu Glu Gln Arg Glu Gln



 



Glu Ala Lys His Glu Arg Ile Glu Asn Asp Val Ala



 



Thr Ile Leu Ser Arg Arg Ile Ala Val Glu Tyr Ser



 



Asp Ser Glu Asp Asp Ser Glu Phe Asp Glu Val Asp



 



Trp Leu Glu






    
    
        the fragment of 50 amino acids delimited by the amino acids situated at positions 497 and 546 in FIG. 3, i.e. the following SEQ ID NO: 34 peptide:
    
    











Ala Arg Ser Val Leu Leu Glu Ala Ile Arg Lys Gly



 



Ile Gln Leu Arg Lys Val Glu Glu Gln Arg Glu Gln



 



Glu Ala Lys His Glu Arg Ile Glu Asn Asp Val Ala



 



Thr Ile Leu Ser Arg Arg Ile Ala Val Glu Tyr Ser



 



Asp Ser






    
    
        or the peptide sequences derived from the above-mentioned peptide fragments, in particular by substitution, addition or removal of one or more amino acids of these fragments, said derived sequences having the property defined above, of WASP family proteins and said fragments thereof.
    
    


The invention concerns more particularly the above-mentioned use of peptide fragments of WASP family proteins of non-human origin.
Advantageously, the peptide fragments of WASP family proteins of non-human origin used are chosen from:

    
    
        fragments of non-human mammalian WASP family proteins, such as:
        
            fragments of murine WASP protein, themselves chosen from:
            
                those including:
                
                    the verprolin homology domain delimited by the amino acids situated at positions 448 and 465 of the peptide sequence SEQ ID NO: 36 of murine WASP protein represented in FIG. 4, or a peptide sequence derived from the above-mentioned domain as defined above,
                    and the cofilin homology domain delimited by the amino acids situated at positions 487 and 505 of the peptide sequence SEQ ID NO: 36 of murine WASP protein represented in FIG. 4, or a peptide sequence derived from the above-mentioned domain as defined above,
                
                
                fragments of which the N-terminal amino acid corresponds to that situated at one of positions 420 to 448 in FIG. 4, and the C-terminal amino acid corresponds to that situated at one of positions 505 to 520 in FIG. 4,
                the fragment of 101 amino acids delimited by the amino acids situated at positions 420 and 520 in FIG. 4, i.e. the following SEQ ID NO: 38 peptide:
            
            
        
        
    
    











Pro Pro Pro Cys Pro Gly Ser Gly Pro Ala Pro Pro



 



Pro Leu Pro Pro Thr Pro Val Ser Gly Gly Ser Pro



 



Ala Pro Gly Gly Gly Arg Gly Ala Leu Leu Asp Gln



 



Ile Arg Gln Gly Ile Gln Leu Asn Lys Thr Pro Gly



 



Ala Leu Glu Asn Ser Val Gln Gln Pro Pro Ala Gln



 



Gln Ser Glu Gln Leu Val Gly Ala Leu Met His Val



 



Met Gln Lys Arg Ser Arg Val Ile His Ser Ser Asp



 



Glu Gly Glu Asp Gln Thr Gly Glu Asp Glu Glu Asp



 



Asp Glu Trp Asp Asp






    
    
        
        
            
            
                the fragment of 86 amino acids delimited by the amino acids situated at positions 420 and 505 in FIG. 4, i.e. the following SEQ ID NO: 40 peptide:
            
            
        
        
    
    











Pro Pro Pro Cys Pro Gly Ser Gly Pro Ala Pro Pro



 



Pro Leu Pro Pro Thr Pro Val Ser Gly Gly Ser Pro



 



Ala Pro Gly Gly Gly Arg Gly Ala Leu Leu Asp Gln



 



Ile Arg Gln Gly Ile Gln Leu Asn Lys Thr Pro Gly



 



Ala Leu Glu Asn Ser Val Gln Gln Pro Pro Ala Gln



 



Gln Ser Glu Gly Leu Val Gly Ala Leu Met His Val



 



Met Gln Lys Arg Ser Arg Val Ile His Ser Ser Asp



 



Glu Gly






    
    
        
        
            
            
                the fragment of 73 amino acids delimited by the amino acids situated at positions 448 and 520 in FIG. 4, i.e. the following SEQ ID NO: 42 peptide:
            
            
        
        
    
    











Gly Arg Gly Ala Leu Leu Asp Gln Ile Arg Gln Gly



 



Ile Gln Leu Asn Lys Thr Pro Gly Ala Leu Glu Asn



 



Ser Val Gln Gln Pro Pro Ala Gln Gln Ser Glu Gly



 



Leu Val Gly Ala Leu Met His Val Met Gln Lys Arg



 



Ser Arg Val Ile His Ser Ser Asp Glu Gly Glu Asp



 



Gln Thr Gly Glu Asp Glu Glu Asp Asp Glu Trp Asp



 



Asp






    
    
        
        
            
            
                the fragment of 58 amino acids delimited by the amino acids situated at positions 448 and 505 in FIG. 4, i.e. the following SEQ ID NO: 44 peptide:
            
            
        
        
    
    











Gly Arg Gly Ala Leu Leu Asp Gln Ile Arg Gln Gly



 



Ile Gln Leu Asn Lys Thr Pro Gly Ala Leu Glu Asn



 



Ser Val Gln Gln Pro Pro Ala Gln Gln Ser Glu Gly



 



Leu Val Gly Ala Leu Met His Val Met Gln Lys Arg



 



Ser Arg Val Ile His Ser Ser Asp Glu Gly






    
    
        
        
            fragments of rat N-WASP protein, themselves chosen from:
            
                those including:
                
                    the verprolin homologous sequence delimited by the amino acids situated at positions 401 and 417 of the peptide sequence SEQ ID NO: 46 of rat N-WASP protein represented in FIG. 5, or a peptide sequence derived from the above-mentioned domain as defined above,
                    and the verprolin homologous sequence delimited by the amino acids situated at positions 429 and 444 of the peptide sequence SEQ ID NO: 46 of rat N-WASP protein represented in FIG. 5, or a peptide sequence derived from the above-mentioned domain as defined above,
                    and the cofilin homology domain contained in the above-mentioned N-WASP protein, i.e. the domain delimited by the amino acids situated at positions 466 and 484 of the peptide sequence SEQ ID NO: 46 of rat N-WASP protein represented in FIG. 5, or a peptide sequence derived from the above-mentioned domain as defined above,
                
                
                fragments of which the N-terminal amino acid corresponds to that situated at one of positions 401 to 429 in FIG. 5, and the C-terminal amino acid corresponds to that situated at one of positions 484 to 501 in FIG. 5,
                the fragment of 101 amino acids delimited by the amino acids situated at positions 401 and 501 in FIG. 5, i.e. the following SEQ ID NO: 48 peptide:
            
            
        
        
    
    











Asn Lys Ala Ala Leu Leu Asp Gln Ile Arg Glu Gly



 



Ala Gln Leu Lys Lys Val Glu Gln Asn Ser Arg Pro



 



Val Ser Cys Ser Gly Arg Asp Ala Leu Leu Asp Gln



 



Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Ser Asp



 



Gly Gln Glu Ser Thr Pro Pro Thr Pro Ala Pro Thr



 



Ser Gly Ile Val Ser Asp Glu Asp Glu Asp Asp Asp



 



Asp Glu Glu Asp Phe Gln Asp Asp Asp Glu Trp Glu



 



Asp






    
    
        
        
            
            
                the fragment of 84 amino acids delimited by the amino acids situated at positions 401 and 484 in FIG. 5, i.e. the following SEQ ID NO: 50 peptide:
            
            
        
        
    
    











Asn Lys Ala Ala Leu Leu Asp Gln Ile Arg Glu Gly



 



Ala Gln Leu Lys Lys Val Glu Gln Asn Ser Arg Pro



 



Val Ser Cys Ser Gly Arg Asp Ala Leu Leu Asp Gln



 



Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Ser Asp



 



Gly Gln Glu Ser Thr Pro Pro Thr Pro Ala Pro Thr



 



Ser Gly Ile Val Gly Ala Leu Met Glu Val Met Gln



 



Lys Arg Ser Lys Ala Ile His Ser Ser Asp Glu Asp






    
    
        
        
            
            
                the fragment of 73 amino acids delimited by the amino acids situated at positions 429 and 501 in FIG. 5, i.e. the following SEQ ID NO: 52 peptide:
            
            
        
        
    
    










Gly Arg Asp Ala Leu Leu Asp Gln Ile Arg Gln Gly



 


Ile Gln Leu Lys Ser Val Ser Asp Gly Gln Glu Ser


 


Thr Pro Pro Thr Pro Ala Pro Thr Ser Gly Ile Val


 


Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys


 


Ala Ile His Ser Ser Asp Glu Asp Glu Asp Asp Asp


 


Asp Glu Glu Asp Phe Gln Asp Asp Asp Glu Trp Glu


 


Asp






    
    
        
        
            
            
                the fragment of 56 amino acids delimited by the amino acids situated at positions 429 and 484 in FIG. 5, i.e. the following SEQ ID NO: 54 peptide:
            
            
        
        
    
    










Gly Arg Asp Ala Leu Leu Asp Gln Ile Arg Gln Gly



 


Ile Gln Leu Lys Ser Val Ser Asp Gly Gln Glu Ser


 


Thr Pro Pro Thr Pro Ala Pro Thr Ser Gly Ile Val


 


Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys


 


Ala Ile His Ser Ser Asp Glu Asp






    
    
        
        
            fragments of bovine N-WASP protein, themselves chosen from:
            
                those including:
                
                    the verprolin homology domain delimited by the amino acids situated at positions 405 and 421 of the peptide sequence SEQ ID NO: 56 of bovine N-WASP protein represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above,
                    and/or the verprolin homology domain delimited by the amino acids situated at positions 433 and 488 of the peptide sequence SEQ ID NO: 56 of bovine N-WASP protein represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above,
                    and the cofilin homology domain delimited by the amino acids situated at positions 470 and 488 of the peptide sequence SEQ ID NO: 56 of bovine N-WASP protein represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above,
                
                
                fragments of which the N-terminal amino acid corresponds to that situated at one of positions 405 to 433 in FIG. 6, and the C-terminal amino acid corresponds to that situated at one of positions 488 to 505 in FIG. 6,
                the fragment of 101 amino acids delimited by the amino acids situated at positions 405 and 505 in FIG. 6, i.e. the following SEQ ID NO: 58 peptide:
            
            
        
        
    
    










Ser Lys Ala Ala Leu Leu Asp Gln Ile Arg Glu Gly



 


Ala Gln Leu Lys Lys Val Glu Gln Asn Ser Arg Pro


 


Val Ser Cys Ser Gly Arg Asp Ala Leu Leu Asp Gln


 


Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Thr Asp


 


Ala Pro Glu Ser Thr Pro Pro Ala Pro Ala Pro Thr


 


Ser Gly Ile Val Gly Ala Leu Met Glu Val Met Gln


 


Lys Arg Ser Lys Ala Ile His Ser Ser Asp Glu Asp


 


Glu Asp Glu Asp Asp Asp Glu Asp Phe Glu Asp Asp


 


Asp Glu Trp Glu Asp






    
    
        
        
            
            
                the fragment of 84 amino acids delimited by the amino acids situated at positions 405 and 488 in FIG. 6, i.e. the following SEQ ID NO: 60 peptide:
            
            
        
        
    
    










Ser Lys Ala Ala Leu Leu Asp Gln Ile Arg Glu Gly



 


Ala Gln Leu Lys Lys Val Glu Gln Asn Ser Arg Pro


 


Val Ser Cys Ser Gly Arg Asp Ala Leu Leu Asp Gln


 


Ile Arg Gln Gly Ile Gln Leu Lys Ser Val Thr Asp


 


Ala Pro Glu Ser Thr Pro Pro Ala Pro Ala Pro Thr


 


Ser Gly Ile Val Gly Ala Leu Met Glu Val Met Gln


 


Lys Arg Ser Lys Ala Ile His Ser Ser Asp Glu Asp






    
    
        
        
            
            
                the fragment of 73 amino acids delimited by the amino acids situated at positions 433 and 488 in FIG. 6, i.e. the following SEQ ID NO: 62 peptide:
            
            
        
        
    
    










Gly Arg Asp Ala Leu Leu Asp Gln Ile Arg Gln Gly



 


Ile Gln Leu Lys Ser Val Thr Asp Ala Pro Glu Ser


 


Thr Pro Pro Ala Pro Ala Pro Thr Ser Gly Ile Val


 


Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys


 


Ala Ile His Ser Ser Asp Glu Asp Glu Asp Glu Asp


 


Asp Asp Glu Asp Phe Glu Asp Asp Asp Glu Trp Glu


 


Asp






    
    
        
        
            
            
                the fragment of 56 amino acids delimited by the amino acids situated at positions 433 and 488 in FIG. 6, i.e. the following SEQ ID NO: 64 peptide:
            
            
        
        
    
    










Gly Arg Asp Ala Leu Leu Asp Gln Ile Arg Gln Gly



 


Ile Gln Leu Lys Ser Val Thr Asp Ala Pro Glu Ser


 


Thr Pro Pro Ala Pro Ala Pro Thr Ser Gly Ile Val


 


Gly Ala Leu Met Glu Val Met Gln Lys Arg Ser Lys


 


Ala Ile His Ser Ser Asp Glu Asp






    
    
        fragments of WASP family proteins of micro-organisms, such as:
        
            fragments of Saccharomyces cerevisiae Las17 protein, themselves chosen from:
            
                those including:
                
                    the verprolin homology domain delimited by the amino acids situated at positions 447 and 466 of the peptide sequence SEQ ID NO: 66 of Las17 protein represented in FIG. 7, or a peptide sequence derived from the above-mentioned domain as defined above,
                    and the cofilin homology domain delimited by the amino acids situated at positions 607 and 624 of the peptide sequence SEQ ID NO: 66 of Las17 protein represented in FIG. 7, or a peptide sequence derived from the above-mentioned domain as defined above,
                
                
                fragments of which the N-terminal amino acid corresponds to that situated at one of positions 422 to 447 in FIG. 7, and the C-terminal amino acid corresponds to that situated at one of positions 624 to 633 in FIG. 7,
                the fragment of 212 amino acids delimited by the amino acids situated at positions 422 and 633 in FIG. 7, i.e. the following SEQ ID NO: 68 peptide:
            
            
        
        
    
    










Ser Asn Met Ser Ser Pro Pro Pro Pro Pro Val Thr



 


Thr Phe Asn Thr Leu Thr Pro Gln Met Thr Ala Ala


 


Thr Gly Gln Pro Ala Val Pro Leu Pro Gln Asn Thr


 


Gln Ala Pro Ser Gln Ala Thr Asn Val Pro Val Ala


 


Pro Pro Pro Pro Pro Ala Ser Leu Gly Gln Ser Gln


 


Ile Pro Gln Ser Ala Pro Ser Ala Pro Ile Pro Pro


 


Thr Leu Pro Ser Thr Thr Ser Ala Ala Pro Pro Pro


 


Pro Pro Ala Phe Leu Thr Gln Gln Pro Gln Ser Gly


 


Gly Ala Pro Ala Pro Pro Pro Pro Pro Gln Met Pro


 


Ala Thr Ser Thr Ser Gly Gly Gly Ser Phe Ala Glu


 


Thr Thr Gly Asp Ala Gly Arg Asp Ala Leu Leu Ala


 


Ser Ile Arg Gly Ala Gly Gly Ile Gly Ala Leu Arg


 


Lys Val Asp Lys Ser Gln Leu Asp Lys Pro Ser Val


 


Leu Leu Gln Glu Ala Arg Gly Glu Ser Ala Ser Pro


 


Pro Ala Ala Ala Gly Asn Gly Gly Thr Pro Gly Gly


 


Pro Pro Ala Ser Leu Ala Asp Ala Leu Ala Ala Ala


 


Leu Asn Lys Arg Lys Thr Lys Val Gly Ala His Asp


 


Asp Met Asp Asn Gly Asp Asp Trp






    
    
        
        
            
            
                the fragment of 203 amino acids delimited by the amino acids situated at positions 422 and 624 in FIG. 7, i.e. the following SEQ ID NO: 70 peptide:
            
            
        
        
    
    










Ser Asn Met Ser Ser Pro Pro Pro Pro Pro Val Thr



 


Thr Phe Asn Thr Leu Thr Pro Gln Met Thr Ala Ala


 


Thr Gly Gln Pro Ala Val Pro Leu Pro Gln Asn Thr


 


Gln Ala Pro Ser Gln Ala Thr Asn Val Pro Val Ala


 


Pro Pro Pro Pro Pro Ala Ser Leu Gly Gln Ser Gln


 


Ile Pro Gln Ser Ala Pro Ser Ala Pro Ile Pro Pro


 


Thr Leu Pro Ser Thr Thr Ser Ala Ala Pro Pro Pro


 


Pro Pro Ala Phe Leu Thr Gln Gln Pro Gln Ser Gly


 


Gly Ala Pro Ala Pro Pro Pro Pro Pro Gln Met Pro


 


Ala Thr Ser Thr Ser Gly Gly Gly Ser Phe Ala Glu


 


Thr Thr Gly Asp Ala Gly Arg Asp Ala Leu Leu Ala


 


Ser Ile Arg Gly Ala Gly Gly Ile Gly Ala Leu Arg


 


Lys Val Asp Lys Ser Gln Leu Asp Lys Pro Ser Val


 


Leu Leu Gln Glu Ala Arg Gly Glu Ser Ala Ser Pro


 


Pro Ala Ala Ala Gly Asn Gly Gly Thr Pro Gly Gly


 


Pro Pro Ala Ser Leu Ala Asp Ala Leu Ala Ala Ala


 


Leu Asn Lys Arg Lys Thr Lys Val Gly Ala His






    
    
        
        
            
            
                the fragment of 187 amino acids delimited by the amino acids situated at positions 447 and 633 in FIG. 7, i.e. the following SEQ ID NO: 72 peptide:
            
            
        
        
    
    










Gly Gln Pro Ala Val Pro Leu Pro Gln Asn Thr Gln



 


Ala Pro Ser Gln Ala Thr Asn Val Pro Val Ala Pro


 


Pro Pro Pro Pro Ala Ser Leu Gly Gln Ser Gln Ile


 


Pro Gln Ser Ala Pro Ser Ala Pro Ile Pro Pro Thr


 


Leu Pro Ser Thr Thr Ser Ala Ala Pro Pro Pro Pro


 


Pro Ala Phe Leu Thr Gln Gln Pro Gln Ser Gly Gly


 


Ala Pro Ala Pro Pro Pro Pro Pro Gln Met Pro Ala


 


Thr Ser Thr Ser Gly Gly Gly Ser Phe Ala Glu Thr


 


Thr Gly Asp Ala Gly Arg Asp Ala Leu Leu Ala Ser


 


Ile Arg Gly Ala Gly Gly Ile Gly Ala Leu Arg Lys


 


Val Asp Lys Ser Gln Leu Asp Lys Pro Ser Val Leu


 


Leu Gln Glu Ala Arg Gly Glu Ser Ala Ser Pro Pro


 


Ala Ala Ala Gly Asn Gly Gly Thr Pro Gly Gly Pro


 


Pro Ala Ser Leu Ala Asp Ala Leu Ala Ala Ala Leu


 


Asn Lys Arg Lys Thr Lys Val Gly Ala His Asp Asp


 


Met Asp Asn Gly Asp Asp Trp






    
    
        
        
            
            
                the fragment of 178 amino acids delimited by the amino acids situated at positions 447 and 624 in FIG. 7, i.e. the following SEQ ID NO: 74 peptide:
            
            
        
        
    
    










Gly Gln Pro Ala Val Pro Leu Pro Gln Asn Thr Gln



 


Ala Pro Ser Gln Ala Thr Asn Val Pro Val Ala Pro


 


Pro Pro Pro Pro Ala Ser Leu Gly Gln Ser Gln Ile


 


Pro Gln Ser Ala Pro Ser Ala Pro Ile Pro Pro Thr


 


Leu Pro Ser Thr Thr Ser Ala Ala Pro Pro Pro Pro


 


Pro Ala Phe Leu Thr Gln Gln Pro Gln Ser Gly Gly


 


Ala Pro Ala Pro Pro Pro Pro Pro Gln Met Pro Ala


 


Thr Ser Thr Ser Gly Gly Gly Ser Phe Ala Glu Thr


 


Thr Gly Asp Ala Gly Arg Asp Ala Leu Leu Ala Ser


 


Ile Arg Gly Ala Gly Gly Ile Gly Ala Leu Arg Lys


 


Val Asp Lys Ser Gln Leu Asp Lys Pro Ser Val Leu


 


Leu Gln Glu Ala Arg Gly Glu Ser Ala Ser Pro Pro


 


Ala Ala Ala Gly Asn Gly Gly Thr Pro Gly Gly Pro


 


Pro Ala Ser Leu Ala Asp Ala Leu Ala Ala Ala Leu


 


Asn Lys Arg Lys Thr Lys Val Gly Ala His






    
    
        
        
            fragments of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe, themselves chosen from:
            
                those including:
                
                    the verprolin homology domain delimited by the amino acids situated at positions 501 and 517 of the peptide sequence SEQ ID NO: 76 of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe represented in FIG. 8, or a peptide sequence derived from the above-mentioned domain as defined above,
                    and the cofilin homology domain delimited by the amino acids situated at positions 548 and 565 of the peptide sequence SEQ ID NO: 76 of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe represented in FIG. 8, or a peptide sequence derived from the above-mentioned domain as defined above,
                
                
                fragments of which the N-terminal amino acid corresponds to that situated at one of positions 477 to 501 in FIG. 8, and the C-terminal amino acid corresponds to that situated at one of positions 565 to 574 in FIG. 8,
                the fragment of 98 amino acids delimited by the amino acids situated at positions 477 and 574 in FIG. 8, i.e. the following SEQ ID NO: 78 peptide:
            
            
        
        
    
    










Pro Pro Ala Pro Ala Pro Ala Pro Ala Ala Pro Val Ala Ser Ile Ala



 


Glu Leu Pro Gln Gln Asp Gly Arg Ala Asn Leu Met Ala Ser Ile Arg


 


Ala Ser Gly Gly Met Asp Leu Leu Lys Ser Arg Lys Val Ser Ala Ser


 


Pro Ser Val Ala Ser Thr Lys Thr Ser Asn Pro Pro Val Glu Ala Pro


 


Pro Ser Asn Asn Leu Met Asp Ala Leu Ala Ser Ala Leu Asn Gln Arg


 


Lys Thr Lys Val Ala Gln Ser Asp Glu Glu Asp Glu Asp Asp Asp Glu


 


Trp Asp






    
    
        
        
            
            
                the fragment of 89 amino acids delimited by the amino acids situated at positions 477 and 565 in FIG. 8, i.e. the following SEQ ID NO: 80 peptide:
            
            
        
        
    
    










Pro Pro Ala Pro Ala Pro Ala Pro Ala Ala Pro Val Ala Ser Ile Ala



 


Glu Leu Pro Gln Gln Asp Gly Arg Ala Asn Leu Met Ala Ser Ile Arg


 


Ala Ser Gly Gly Met Asp Leu Leu Lys Ser Arg Lys Val Ser Ala Ser


 


Pro Ser Val Ala Ser Thr Lys Thr Ser Asn Pro Pro Val Glu Ala Pro


 


Pro Ser Asn Asn Leu Met Asp Ala Leu Ala Ser Ala Leu Asn Gln Arg


 


Lys Thr Lys Val Ala Gln Ser Asp Glu






    
    
        
        
            
            
                the fragment of 74 amino acids delimited by the amino acids situated at positions 501 and 574 in FIG. 8, i.e. the following SEQ ID NO: 82 peptide:
            
            
        
        
    
    










Ala Asn Leu Met Ala Ser Ile Arg Ala Ser Gly Gly Met Asp Leu Leu



 


Lys Ser Arg Lys Val Ser Ala Ser Pro Ser Val Ala Ser Thr Lys Thr


 


Ser Asn Pro Pro Val Glu Ala Pro Pro Ser Asn Asn Leu Met Asp Ala


 


Leu Ala Ser Ala Leu Asn Gln Arg Lys Thr Lys Val Ala Gln Ser Asp


 


Glu Glu Asp Glu Asp Asp Asp Glu Trp Asp






    
    
        
        
            
            
                the fragment of 65 amino acids delimited by the amino acids situated at positions 501 and 565 in FIG. 8, i.e. the following SEQ ID NO: 84 peptide:
            
            
        
        
    
    










Ala Asn Leu Met Ala Ser Ile Arg Ala Ser Gly Gly Met Asp Leu Leu



 


Lys Ser Arg Lys Val Ser Ala Ser Pro Ser Val Ala Ser Thr Lys Thr


 


Ser Asn Pro Pro Val Glu Ala Pro Pro Ser Asn Asn Leu Met Asp Ala


 


Leu Ala Ser Ala Leu Asn Gln Arg Lys Thr Lys Val Ala Gln Ser Asp


 


Glu






    
    
        the peptide sequences derived from the above-mentioned peptide fragments, in particular by substitution, addition or removal of one or more amino acids of these fragments, said derived sequences having the property defined above, of WASP family proteins and said fragments thereof.
    
    


The invention also concerns the above-mentioned use of above-defined peptide fragments, or sequences derived from these, fused on the N-terminal or C-terminal side with one or more peptide sequences facilitating the detection and purification of the above-mentioned peptide fragments or derived sequences, without greatly affecting the above-mentioned property of the latter to polymerise actin, inducing cellular motility. Among such peptide sequences fused to peptide fragments, or to sequences derived from the latter, of the invention, we may cite that of glutathion-S-transferase (GST, described in Smith D. B. and Johnson K. S., Gene 67: 31–141 (1988)) fused to the N-terminal part of the above-mentioned peptide fragments or derived sequences, or that of epitopes recognised by specific antibodies, such as that of the myc9E10 epitope (described in Evan G. I. et al., Molecular and Cellular Biology 5: 3610–3616 (1985)) fused to the C-terminal part of the above-mentioned peptide fragments or derived sequences.
The invention also concerns the above-mentioned peptide fragments, or sequences derived from these, as such, i.e. more particularly peptide fragments of WASP family proteins of eukaryotic cells chosen from those including:

    
    
        the verprolin homology domain contained in WASP family proteins, or in a protein derived from the latter, or at least one of the two verprolin homologous sequences when said WASP family proteins contain two of these sequences, or one peptide sequence derived from the above-mentioned domain, in particular by substitution, addition or removal of one or more amino acids, and retaining the property of this domain to bind to actin,
        and the cofilin homology domain contained in WASP family proteins, or in a protein derived from the latter, or a peptide sequence derived from the above-mentioned domain, in particular by substitution, addition or removal of one or more amino acids, and retaining the property of this domain to intervene in actin polymerisation,
    
    


or peptide sequences derived from the above-mentioned peptide fragments of WASP family proteins, in particular by substitution, addition or removal of one or more amino acids of these fragments, said derived sequences having the above-mentioned property of WASP protein and said fragments,
to the exclusion:

    
    
        of the peptide fragment of human WASP protein delimited by the amino acids situated at positions 429 to 503 of the peptide sequence represented in FIG. 1,
        of the peptide fragment of human WASP protein delimited by the amino acids situated at positions 187 to 489 of the peptide sequence represented in FIG. 1,
        of the peptide fragment of human WASP protein delimited by the amino acids situated at positions 422 to 489 of the peptide sequence represented in FIG. 1,
        of the peptide fragment of human N-WASP protein delimited by the amino acids situated at positions 392 to 505 of the peptide sequence represented in FIG. 2,
        of the peptide fragment of human Scar1 protein delimited by the amino acids situated at positions 443 to 559 of the peptide sequence represented in FIG. 3,
        of the peptide fragment of bovine N-WASP protein delimited by the amino acids situated at positions 392 to 505, and that delimited by the amino acids situated at positions 392 to 485 of the peptide sequence represented in FIG. 6.
    
    


The invention concerns more particularly the above-mentioned peptide fragments of human WASP protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 430 and 446 of the peptide sequence SEQ ID NO: 2 of human WASP protein represented in FIG. 1, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain delimited by the amino acids situated at positions 469 and 487 of the peptide sequence SEQ ID NO: 2 of human WASP protein represented in FIG. 1, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of human WASP protein, chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 404 to 430 in FIG. 1, and the C-terminal amino acid corresponds to that situated at one of positions 487 to 502 in FIG. 1, and more particularly the above-mentioned SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 peptides, or the sequences derived from the latter as defined above.
The invention concerns more particularly the above-mentioned peptide fragments of human N-WASP protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homologous sequence delimited by the amino acids situated at positions 405 and 421 of the peptide sequence SEQ ID NO: 12 of human N-WASP protein represented in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above,
        and/or the verprolin homologous sequence delimited by the amino acids situated at positions 433 and 449 of the peptide sequence SEQ ID NO: 12 of human N-WASP protein represented in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain contained in the above-mentioned N-WASP protein, i.e. the domain delimited by the amino acids situated at positions 470 and 488 of the peptide sequence SEQ ID NO: 12 of human N-WASP protein represented in FIG. 2, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of human N-WASP protein, chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 392 to 433 in FIG. 2, and the C-terminal amino acid corresponds to that situated at one of positions 488 to 505 in FIG. 2, and more particularly the above-mentioned SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24 peptides, or the sequences derived from the latter as defined above.
The invention concerns more particularly the above-mentioned peptide fragments of human Scar1 protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 497 and 513 of the peptide sequence SEQ ID NO: 26 of human Scar1 protein represented in FIG. 3, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain delimited by the amino acids situated at positions 531 and 546 of the peptide sequence SEQ ID NO: 26 of human Scar1 protein represented in FIG. 3, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of human Scar1 protein, chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 443 to 497 in FIG. 3, and the C-terminal amino acid corresponds to that situated at one of positions 546 to 559 in FIG. 3, and more particularly the above-mentioned SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 34 peptides, or the sequences derived from the latter as defined above.
The invention concerns more particularly the above-mentioned peptide fragments of murine WASP protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 448 and 465 of the peptide sequence SEQ ID NO: 36 of murine WASP protein represented in FIG. 4, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain delimited by the amino acids situated at positions 487 and 505 of the peptide sequence SEQ ID NO: 36 of murine WASP protein represented in FIG. 4, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of murine WASP protein, chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 420 to 448 in FIG. 4, and the C-terminal amino acid corresponds to that situated at one of positions 505 to 520 in FIG. 4, and more particularly the above-mentioned SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44 peptides, or the sequences derived from the latter as defined above.
The invention concerns more particularly the above-mentioned peptide fragments of rat N-WASP protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homologous sequence delimited by the amino acids situated at positions 401 and 417 of the peptide sequence SEQ ID NO: 46 of rat N-WASP protein represented in FIG. 5, or a peptide sequence derived from the above-mentioned domain as defined above,
        and/or the verprolin homologous sequence delimited by the amino acids situated at positions 429 and 444 of the peptide sequence SEQ ID NO: 46 of rat N-WASP protein represented in FIG. 5, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain contained in the above-mentioned N-WASP protein, i.e. the domain delimited by the amino acids situated at positions 466 and 484 of the peptide sequence SEQ ID NO: 46 of rat N-WASP protein represented in FIG. 5, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of rat N-WASP protein chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 401 to 429 in FIG. 5, and the C-terminal amino acid corresponds to that situated at one of positions 484 to 501 in FIG. 5, and more particularly the above-mentioned SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO: 54 peptides, or the sequences derived from the latter as defined above.
The invention concerns more particularly the above-mentioned peptide fragments of bovine N-WASP protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 405 and 421 of the peptide sequence SEQ ID NO: 56 of bovine N-WASP protein represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above,
        and/or the verprolin homology domain delimited by the amino acids situated at positions 433 and 488 of the peptide sequence SEQ ID NO: 56 of bovine N-WASP protein represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain delimited by the amino acids situated at positions 470 and 488 of the peptide sequence SEQ ID NO: 56 of bovine N-WASP protein represented in FIG. 6, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of bovine N-WASP protein chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 405 to 433 in FIG. 6, and the C-terminal amino acid corresponds to that situated at one of positions 488 to 505 in FIG. 6, and more particularly the above-mentioned SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 and SEQ ID NO: 64 peptides, or the sequences derived from the latter as defined above.
The invention concerns more particularly the above-mentioned peptide fragments of Saccharomyces cerevisiae Las17 protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 447 and 466 of the peptide sequence SEQ ID NO: 66 of Las17 protein represented in FIG. 7, or a peptide sequence derived from the above-mentioned domain as defined above.
        and the cofilin homology domain delimited by the amino acids situated at positions 607 and 624 of the peptide sequence SEQ ID NO: 66 of Las17 protein represented in FIG. 7, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of Saccharomyces cerevisiae Las17 protein chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 422 to 447 in FIG. 7, and the C-terminal amino acid corresponds to that situated at one of positions 624 to 633 in FIG. 7, and more particularly the above-mentioned SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 74 peptides, or the sequences derived from the latter as defined above.
The invention concerns more particularly the above-mentioned peptide fragments of Schizosaccharomyces pombe (Wsp1p) protein, or peptide sequences derived from the latter, said fragments being chosen from those including:

    
    
        the verprolin homology domain delimited by the amino acids situated at positions 501 and 517 of the peptide sequence SEQ ID NO: 76 of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe represented in FIG. 8, or a peptide sequence derived from the above-mentioned domain as defined above,
        and the cofilin homology domain delimited by the amino acids situated at positions 548 and 565 of the peptide sequence SEQ ID NO: 76 of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe represented in FIG. 8, or a peptide sequence derived from the above-mentioned domain as defined above.
    
    


The invention concerns still more particularly fragments of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe chosen from fragments of which the N-terminal amino acid corresponds to that situated at one of positions 477 to 501 in FIG. 8, and the C-terminal amino acid corresponds to that situated at one of positions 565 to 574 in FIG. 8, and more particularly the above-mentioned SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 and SEQ ID NO: 84 peptides, or the sequences derived from the latter as defined above.
The invention also concerns the nucleotide sequences coding for the above-mentioned peptide fragments, or for the proteins derived from the latter, or for the fusion proteins as described above.
The invention concerns more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 1244 to 1322 of the nucleotide sequence SEQ ID NO: 1 represented in FIG. 1, and the 3′ end corresponds to the nucleotide situated at one of positions 1495 to 1540 in FIG. 1, said nucleotide sequences coding for the above-mentioned fragments of human WASP protein, of which the N-terminal amino acid corresponds to that situated at one of positions 404 to 430 in FIG. 1, and the C-terminal amino acid corresponds to that situated at one of positions 487 to 502 in FIG. 1,
        nucleotide sequence SEQ ID NO: 3 delimited by the nucleotides situated at positions 1244 and 1540 of FIG. 1, and coding for the peptide fragment of human WASP protein corresponding to the above-mentioned SEQ ID NO: 4 peptide,
        nucleotide sequence SEQ ID NO: 5 delimited by the nucleotides situated at positions 1244 and 1495 of FIG. 1, and coding for the peptide fragment of human WASP protein corresponding to the above-mentioned SEQ ID NO: 6 peptide,
        nucleotide sequence SEQ ID NO: 7 delimited by the nucleotides situated at positions 1322 and 1540 of FIG. 1, and coding for the peptide fragment of human WASP protein corresponding to the above-mentioned SEQ ID NO: 8 peptide,
        nucleotide sequence SEQ ID NO: 9 delimited by the nucleotides situated at positions 1322 and 1495 of FIG. 1, and coding for the peptide fragment of human WASP protein corresponding to the above-mentioned SEQ ID NO: 10 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention concerns still more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 1174 to 1299 of the nucleotide sequence SEQ ID NO: 11 represented in FIG. 2, and the 3′ end corresponds to the nucleotide situated at one of positions 1464 to 1515 in FIG. 2, said nucleotide sequences coding for the above-mentioned fragments of N-WASP protein, of which the N-terminal amino acid corresponds to that situated at one of positions 392 to 433 in FIG. 2 and the C-terminal amino acid corresponds to that situated at one of positions 488 to 505 in FIG. 2, to the exclusion of the nucleotide sequence delimited by the nucleotides situated at positions 1174 to 1515 coding for the peptide fragment of N-WASP protein delimited by the amino acids situated at positions 392 to 505 of the peptide sequence represented in FIG. 2,
        nucleotide sequence SEQ ID NO: 15 delimited by the nucleotides situated at positions 1174 and 1464 coding for the peptide fragment of N-WASP protein corresponding to the above-mentioned SEQ ID NO: 16 peptide, nucleotide sequence SEQ ID NO: 17 delimited by the nucleotides situated at positions 1213 and 1515 coding for the peptide fragment of N-WASP protein corresponding to the above-mentioned SEQ ID NO: 18 peptide,
        nucleotide sequence SEQ ID NO: 19 delimited by the nucleotides situated at positions 1213 and 1464 coding for the peptide fragment of N-WASP protein corresponding to the above-mentioned SEQ ID NO: 20 peptide,
        nucleotide sequence SEQ ID NO: 21 delimited by the nucleotides situated at positions 1297 and 1515 coding for the peptide fragment of N-WASP protein corresponding to the above-mentioned SEQ ID NO: 22 peptide,
        nucleotide sequence SEQ ID NO: 23 delimited by the nucleotides situated at positions 1297 and 1464 coding for the peptide fragment of N-WASP protein corresponding to the above-mentioned SEQ ID NO: 24 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention concerns still more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 1327 to 1489 of the nucleotide sequence SEQ ID NO: 26 represented in FIG. 3, and the 3′ end corresponds to the nucleotide situated at one of positions 1638 to 1677 in FIG. 3, said nucleotide sequences coding for the above-mentioned fragments of human Scar1 protein, of which the N-terminal amino acid corresponds to that situated at one of positions 546 to 497 in FIG. 3 and the C-terminal amino acid corresponds to that situated at one of positions 487 to 559 in FIG. 3,
        nucleotide sequence SEQ ID NO: 27 delimited by the nucleotides situated at positions 1327 and 1677 in FIG. 3, and coding for the peptide fragment of human Scar1 protein corresponding to the above-mentioned SEQ ID NO: 28 peptide,
        nucleotide sequence SEQ ID NO: 29 delimited by the nucleotides situated at positions 1327 and 1638 in FIG. 3, and coding for the peptide fragment of human Scar1 protein corresponding to the above-mentioned SEQ ID NO: 30 peptide,
        nucleotide sequence SEQ ID NO: 31 delimited by the nucleotides situated at positions 1489 and 1677 in FIG. 3, and coding for the peptide fragment of human Scar1 protein corresponding to the above-mentioned SEQ ID NO: 32 peptide,
        nucleotide sequence SEQ ID NO: 33 delimited by the nucleotides situated at positions 1489 and 1638 in FIG. 3, and coding for the peptide fragment of human Scar1 protein corresponding to the above-mentioned SEQ ID NO: 34 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention concerns still more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 1282 to 1366 of the nucleotide sequence SEQ ID NO: 36 represented in FIG. 4, and the 3′ end corresponds to the nucleotide situated at one of positions 1539 to 1584 in FIG. 4, said nucleotide sequences coding for the above-mentioned fragments of murine WASP protein, of which the N-terminal amino acid corresponds to that situated at one of positions 420 to 448 in FIG. 4 and the C-terminal amino acid corresponds to that situated at one of positions 505 to 520 in FIG. 4,
        nucleotide sequence SEQ ID NO: 37 delimited by the nucleotides situated at positions 1282 and 1584 in FIG. 4, and coding for the peptide fragment of murine WASP protein corresponding to the above-mentioned SEQ ID NO: 38 peptide,
        nucleotide sequence SEQ ID NO: 39 delimited by the nucleotides situated at positions 1282 and 1584 in FIG. 4, and coding for the peptide fragment of murine WASP protein corresponding to the above-mentioned SEQ ID NO: 40 peptide,
        nucleotide sequence SEQ ID NO: 41 delimited by the nucleotides situated at positions 1366 and 1584 in FIG. 4, and coding for the peptide fragment of murine WASP protein corresponding to the above-mentioned SEQ ID NO: 42 peptide,
        nucleotide sequence SEQ ID NO: 43 delimited by the nucleotides situated at positions 1366 and 1539 in FIG. 4, and coding for the peptide fragment of murine WASP protein corresponding to the above-mentioned SEQ ID NO: 44 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention concerns still more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 1272 to 1356 of the nucleotide sequence SEQ ID NO: 46 represented in FIG. 5, and the 3′ end corresponds to the nucleotide situated at one of positions 1523 to 1574 in FIG. 5, said nucleotide sequences coding for the above-mentioned fragments of rat N-WASP protein, of which the N-terminal amino acid corresponds to that situated at one of positions 401 to 429 in FIG. 5 and the C-terminal amino acid corresponds to that situated at one of positions 484 to 501 in FIG. 5,
        nucleotide sequence SEQ ID NO: 47 delimited by the nucleotides situated at positions 1272 and 1574 in FIG. 5, and coding for the peptide fragment of rat N-WASP protein corresponding to the above-mentioned SEQ ID NO: 48 peptide,
        nucleotide sequence SEQ ID NO: 49 delimited by the nucleotides situated at positions 1272 and 1523 in FIG. 5, and coding for the peptide fragment of rat N-WASP protein corresponding to the above-mentioned SEQ ID NO: 50 peptide,
        nucleotide sequence SEQ ID NO: 51 delimited by the nucleotides situated at positions 1356 and 1574 in FIG. 5, and coding for the peptide fragment of rat N-WASP protein corresponding to the above-mentioned SEQ ID NO: 52 peptide,
        nucleotide sequence SEQ ID NO: 53 delimited by the nucleotides situated at positions 1356 and 1523 in FIG. 5, and coding for the peptide fragment of rat N-WASP protein corresponding to the above-mentioned SEQ ID NO: 54 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention concerns still more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 1500 to 1584 of the nucleotide sequence SEQ ID NO: 56 represented in FIG. 6, and the 3′ end corresponds to the nucleotide situated at one of positions 1751 to 1802 in FIG. 6, said nucleotide sequences coding for the above-mentioned fragments of bovine N-WASP protein, of which the N-terminal amino acid corresponds to that situated at one of positions 405 to 433 in FIG. 6 and the C-terminal amino acid corresponds to that situated at one of positions 488 to 505 in FIG. 6,
        nucleotide sequence SEQ ID NO: 57 delimited by the nucleotides situated at positions 1500 and 1802 in FIG. 6, and coding for the peptide fragment of bovine N-WASP protein corresponding to the above-mentioned SEQ ID NO: 58 peptide,
        nucleotide sequence SEQ ID NO: 59 delimited by the nucleotides situated at positions 1500 and 1751 in FIG. 6, and coding for the peptide fragment of bovine N-WASP protein corresponding to the above-mentioned SEQ ID NO: 60 peptide,
        nucleotide sequence SEQ ID NO: 61 delimited by the nucleotides situated at positions 1584 and 1802 in FIG. 6, and coding for the peptide fragment of bovine N-WASP protein corresponding to the above-mentioned SEQ ID NO: 62 peptide,
        nucleotide sequence SEQ ID NO: 63 delimited by the nucleotides situated at positions 1584 and 1751 in FIG. 6, and coding for the peptide fragment of bovine N-WASP protein corresponding to the above-mentioned SEQ ID NO: 64 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention concerns still more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 2035 to 2110 of the nucleotide sequence SEQ ID NO: 66 represented in FIG. 7, and the 3′ end corresponds to the nucleotide situated at one of positions 2643 to 2670 in FIG. 7, said nucleotide sequences coding for the above-mentioned fragments of Saccharomyces cerevisiae Las17 protein, of which the N-terminal amino acid corresponds to that situated at one of positions 422 to 447 in FIG. 7 and the C-terminal amino acid corresponds to that situated at one of positions 624 to 633 in FIG. 7,
        nucleotide sequence SEQ ID NO: 67 delimited by the nucleotides situated at positions 2035 and 2670 in FIG. 7, and coding for the peptide fragment of Saccharomyces cerevisiae Las17 protein corresponding to the above-mentioned SEQ ID NO: 68 peptide,
        nucleotide sequence SEQ ID NO: 69 delimited by the nucleotides situated at positions 2035 and 2643 in FIG. 7, and coding for the peptide fragment of Saccharomyces cerevisiae Las 17 protein corresponding to the above-mentioned SEQ ID NO: 70 peptide,
        nucleotide sequence SEQ ID NO: 71 delimited by the nucleotides situated at positions 2110 and 2670 in FIG. 7, and coding for the peptide fragment of Saccharomyces cerevisiae Las17 protein corresponding to the above-mentioned SEQ ID NO: 72 peptide,
        nucleotide sequence SEQ ID NO: 73 delimited by the nucleotides situated at positions 2110 and 2643 in FIG. 7, and coding for the peptide fragment of Saccharomyces cerevisiae Las17 protein corresponding to the above-mentioned SEQ ID NO: 74 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention concerns still more particularly:

    
    
        the nucleotide sequences of which the 5′ end corresponds to the nucleotide situated at one of positions 1429 to 1501 of the nucleotide sequence SEQ ID NO: 76 represented in FIG. 8, and the 3′ end corresponds to the nucleotide situated at one of positions 1695 to 1722 in FIG. 8, said nucleotide sequences coding for the above-mentioned fragments of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe, of which the N-terminal amino acid corresponds to that situated at one of positions 477 to 501 in FIG. 8 and the C-terminal amino acid corresponds to that situated at one of positions 565 to 574 in FIG. 8,
        nucleotide sequence SEQ ID NO: 77 delimited by the nucleotides situated at positions 1429 and 1722 in FIG. 8, and coding for the peptide fragment of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe corresponding to the above-mentioned SEQ ID NO: 78 peptide,
        nucleotide sequence SEQ ID NO: 79 delimited by the nucleotides situated at positions 1429 and 1695 in FIG. 8, and coding for the peptide fragment of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe corresponding to the above-mentioned SEQ ID NO: 80 peptide,
        nucleotide sequence SEQ ID NO: 81 delimited by the nucleotides situated at positions 1501 and 1722 in FIG. 8, and coding for the peptide fragment of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe corresponding to the above-mentioned SEQ ID NO: 82 peptide,
        nucleotide sequence SEQ ID NO: 83 delimited by the nucleotides situated at positions 1501 and 1695 in FIG. 8, and coding for the peptide fragment of WASP homologous protein (Wsp1p) of Schizosaccharomyces pombe corresponding to the above-mentioned SEQ ID NO: 84 peptide,
        the nucleotide sequences derived by degeneration of the genetic code of the above-mentioned nucleotide sequences, and coding for the above-mentioned peptide fragments,
        the nucleotide sequences derived from the above-mentioned nucleotide sequences and coding for the sequences derived from said peptide fragments as defined above.
    
    


The invention also concerns vectors, in particular plasmids, containing a nucleotide sequence as defined above.
The invention also concerns host cells transformed by an above-mentioned vector, said cells expressing the above-mentioned peptide fragments, or the derived sequences described above, in recombinant form. Advantageously, the above-mentioned host cells are chosen from the following: Escherichia coli D5α and Escherichia coli BL21.
The invention also concerns reagents for the implementation of a process for the detection or screening of molecules having an inhibiting or stimulating effect on the formation of the actin cytoskeleton, and hence an inhibiting or stimulating effect on cellular motility, said reagent comprising at least one peptide fragment defined above, bound or adsorbed to a support capable of moving under the effect of actin polymerisation, when said support bound to said fragment is placed in a medium containing the elements necessary for actin polymerisation, in particular when said support is added to a medium containing mainly Arp2/3 complex, VASP protein (vasodilator-stimulated phosphoprotein), colifin, and capping proteins, this medium possibly being, for example, an extract prepared from supernatants of lysed cells from the organism.
Capping protein is an element essential for WASP-dependent motility. These proteins are capable of converting actin polymerisation into actin-dependent movement, spatially limiting polymerisation to certain sites in the cellular cytoskeleton. By comparison, in the absence of capping proteins, actin polymerisation still takes place, but since it is uncontrolled, it cannot generate any force. Capping proteins are made up of two sub-units, alpha (p34) and beta (p32) which are both required for capping activity. The dimer is associated with the plus end of the filament which is normally favoured for the addition of actin monomers and thus blocks the addition of monomers. Genes coding for capping proteins have been identified in all species studied to date, and have been shown to be indispensable for the survival of organisms. In humans, Cap beta 1, beta2, G and CapZ have been identified to date. It is also known that multifunctional proteins such as gelsolin and villin also have a capping activity in addition to their other actin-modifying activities.
The invention concerns more particularly reagents as defined above, chosen from microspheres whose diameter is advantageously between approx. 100 nm and approx. 10,000 nm, the material constituting the microspheres itself being chosen preferably from polystyrenes or latex, said microspheres each advantageously containing approx. 5,000 to approx. 50,000 molecules of above-mentioned peptide fragment or a sequence derived according to the invention.
Advantageously, the above-mentioned peptide fragment, or its derived sequence, are adsorbed onto the surface of said microspheres, said reagent being obtained by simple mixing of said microspheres with said peptide fragment, or with its derived sequence.
The invention concerns more particularly reagents as defined above, chosen from drops of oil, in particular oil of C14 to C18, such as palmitic acid, whose diameter is advantageously between approx. 1 μm and approx. 20 μm.
The invention also concerns any process for the detection or screening of molecules as defined above, having an inhibiting or stimulating effect on the formation of the actin cytoskeleton, and hence an inhibiting or stimulating effect on cellular motility, said process comprising:

    
    
        a stage of placing the tested molecule in the presence of a reagent as described above, into a medium containing actin and the elements necessary for actin polymerisation, in particular into an extract of cellular supernatant or other medium as defined above,
        followed by the eventual detection of inhibition or activation of the actin polymerisation process on the surface of said reagent, compared with a control (i.e. a medium as described above not containing the tested molecule, and containing said reagent), respectively demonstrating an inhibiting or stimulating effect on the formation of the actin cytoskeleton.
    
    


Advantageously, the above-mentioned medium into which the tested molecule is placed in the presence of said reagent, contains a compound marked in particular by fluorescence, making it possible to detect movement of said reagent. By way of illustration, the above-mentioned marked compound is a fluorescent derivative of actin, such as actin-rhodamine (available commercially), enabling actin polymerisation to be made visible by epifluorescence microscopy.
The invention also concerns the use of the process as defined above, for detection or screening of molecules, as defined above, capable of being used as medicaments in the treatment of pathologies linked to a dysfunction of the actin polymerisation process within the context of formation of the actin cytoskeleton, or capable of having a cytotoxic effect corresponding to inhibition or stimulation of the formation of the actin cytoskeleton.
The invention also concerns a kit for implementation of an above-mentioned process, comprising

    
    
        a reagent as defined above,
        possibly a marked compound defined above enabling polymerisation of actin to be made visible, in particular actin marked with fluorescence,
        possibly an appropriate medium containing the elements necessary for actin polymerisation, in particular an extract of cellular supernatant as defined above.
    
    


The invention will be further illustrated by means of the following detailed description of preparation of microspheres coated with a peptide fragment of the invention, and of detection of actin polymerisation on the surface of these microspheres in an extract of cellular supernatant.
I) Material and Methods
The sequence coding for the WH2/A domain of human WASP protein (i.e. the fragment of 99 amino acids delimited by the amino acids situated at positions 404 and 502 in FIG. 1, also designated peptide SEQ ID NO: 4) and that coding for the myc9E10 epitope were amplified by PCR. The myc epitope fused to the carboxy-terminal portion of the protein serves as a molecular tag, making possible the detection of the protein by immunological approaches without needing an anti-WASP antibody. This DNA sequence was introduced into the pGEX2T vector (Pharmacia) downstream of the sequence coding for glutathion-S-transferase (GST), generating the WH2/A-pGEX2T plasmid. The GST domain was chosen in this work, as it facilitates purification of the recombinant protein, and it has been shown that this domain does not inhibit the actin-polymerisation capacity of the GST-VCA protein derived from N-WASP in a pyrene-actin test (Rohatgi et al., 1999). FIG. 3 shows the organisation into domains of GST-WH2. This recombinant protein is made up of GST and WH2/A domains, of 237 and 99 residues and of a myc9E10 epitope of 9 residues, respectively.
Purification and characterisation of the GST-WH2/A protein were carried out as follows.
E. coli bacteria (strain BL21) were transformed with WH2/1-pGEX2T plasmid. The bacteria were cultivated in standard LB medium containing the antibiotic ampicillin to keep bacteria comprising the plasmid under selection pressure. The bacteria were cultivated in suspension at 37° C. until the culture reached an optical density of 0.8 to 600 nm. Subsequently, isopropylthio-β-D-galactoside (IPTG) was added to the medium, with a final concentration of 1 mM to induce 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 (saline solution buffered with phosphate pH 7.2, 200 NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid)) containing 1 μg/ml of each of the following protease inhibitors: leupeptin, benzamidin, pepstatin, in a ratio of 1 gr of pellet to 10 volumes of extraction buffer. The suspension was sonicated until it was no longer viscous. The extract was centrifuged at 20,000×g for 10 minutes at 4° C. and the supernatant containing the GST-WH2/A protein was conserved. The GST-WH2/A protein was purified from the bacterial extract by affinity chromatography on resin coupled to glutathione (Pharmacia) and eluated with 20 mM glutathione reduced according to the manufacturers' recommendations. The purification was confirmed by analysis of the GST-WH2/A by electrophoresis on acrylamide gel. The presence of the myc epitope in the GST-WH2/A sequence was confirmed by immunological impression with an antibody directed against this epitope.
The GST-WH2/A protein was absorbed onto 500 nm latex beads (Polyscience Inc., 400 Valley Road, Warrington Pa., USA) following the manufacturers' instructions. These beads, added to the extracts prepared from cells, are capable of polymerising actin sufficiently to bring about the movement of these beads.
Adsorption of the GST-WA protein onto drops of oil: a water-oil emulsion was prepared as follows: 100 μl of oil are mixed with 900 μl of borate buffer 0.1 MpH=8.5 (boric acid buffered with NaOH). The solution is sonicated (wave) for a few seconds and a white foam is obtained. The size of the oil drops is between 20 μm and 1 μm. In a 1.5 ml Eppendorf, 20 μl of this solution are mixed with 100 μl of the above-mentioned GST-WH2/A protein (also designated GST-WA) (1 mg/ml in borate buffer). This solution is left to incubate at ambient temperature for 12 hours (overnight) on a rotating agitator. After incubation, the solution is centrifuged for approx. one minute at 5000 rpm with a small table-top centrifuge. Drops of oil are located in the upper part of the solution. A too strong and/or too prolonged centrifugation brings about fusion of the oil drops. The bottom part of the solution (buffer alone) is removed with a pipette and the top part of the solution is resuspended in PBS (Phosphate Buffer Saline). The oil drops are cleaned at least four times in this manner and finally resuspended in 100 μl of PBS. 1 μl of this solution is mixed with 10 μl of HeLa extracts.
Inhibition of actin polymerisation induced by GST-WA protein: 10 μm polystyrene particles (Polysciences inc.) are covered to saturation with GST-WA protein according to the manufacturer's protocol. The particles are resuspended to 1% solid in PBS (Phosphate Buffer Saline) and stored on ice. To test the independence of the actin polymerisation mechanism induced by GST-WA we prepared three solutions. Each sample contained 15 μl of HeLa extracts (supplemented with ATP, creatine phosphate and actin marked with rhodamine), 0.5 μl of GST-WA particles and 1.5 μl of PBS (A), or 1.5 μl of GST-WA protein at 1 mg/ml (B) or 1.5 μl of GST-PRO protein (PRO corresponding to the fragment delimited by the amino acids situated at positions 235 to 584 of the ActA protein of Listeria monocytogenes) at 1 mg/ml (C). 6 μl of the final mixture of these three solutions were sealed between slide and coverglass. We observed fluorescence (rhodamine actin) around the particles for thirty minutes and measured the intensity of fluorescence on a population of forty beads using a microscope (Leica) and a linear digital camera (Micromax Princeton instrument). The average intensity M (arbitrary unit) was 95,000±13,000 for sample A (positive control). For sample B, M=0±1000, the actin polymerisation on the surface of the particles was inhibited by the addition of GST-WA in solution. For sample C, M=4000±1500; the addition to the solution of the GST-PRO protein did not inhibit the actin polymerisation on the surface of the particles. The intensity was lower than in case A (PBS); this is probably due to the activity induced by GST-PRO in the extracts which results in a very high consumption of rhodamine actin. These results show that the actin polymerisation mechanism induced by the GST-WA protein is independent of the GST-PRO actin polymerisation mechanism.
B) Uses
1) Uses of GST-WH2/A Beads and of the Cellular Actin Polymerisation Test
Cellular movement dependent on the actin cytoskeleton occurs during embryonic development, immune response and healing of wounds. However, the molecular mechanism whereby the cytoskeleton participates in physiological or physio-pathological processes are still poorly understood. This is due to the fact that few in vitro experimental systems are available for studying these processes.
The invention proposes an in vitro test for actin polymerisation on the surface of beads, and shows that the system reproduces the essential characteristics of actin polymerisation in human cells. For example, these beads recruit large proteins for actin polymerisation in cells, such as Arp2/3 complex and cofilin. Moreover, the components recruited onto the surface of the beads, are targets for tyrosine kinase signalling pathways. Thanks to this in vitro system, it is now possible to study the conditions necessary for actin polymerisation in cells, a process which up to now has not been accessible to direct experimental manipulation.
a) Anti-metastatic Component Screening Process
At present, it has not been possible to develop anti-metastatic medicaments targeted at the actin cytoskeleton, as simple tests for screening banks of chemicals have not been available. The beads of the invention “mimic” the dynamic cytoskeleton of cells in motion and thus constitute an ideal test for researching molecules affecting the dynamics of the cytoskeleton. Chemicals thus identified will subsequently be used to develop medicaments for treatment of metastatic cancers.
b) Anti-parasitic Antibiotic Screening Process
The system of beads described here is based on human proteins which are necessary for actin polymerisation in human cells. However, these proteins have been conserved throughout evolution, from yeast to humans, via amoebae. Despite the conservation of their basic function, these proteins also diverge at the level of their primary sequence, suggesting particular functional differences. As in the case of metastatic cancers, the actin cytoskeleton of parasites is rarely the target of medicaments used in the treatment of parasitoses, despite the important role which it plays in the infectious cycle of numerous parasites (for example amoebae). It is conceivable that medicaments which affect the human actin cytoskeleton only when they are used in a strong concentration, affect that of parasites at much lower concentrations. This is why, thanks to the universal nature of the evolution of the actin cytoskeleton, the system of beads of the invention also enables research into medicaments which can be used in the treatment of parasitoses.
c) Detection of Secondary Effects of Medicaments
In another test, the beads are used to verify and confirm that medicaments have no secondary effects on cellular actin polymerisation. Thus it is possible to avoid the disastrous consequences of drug treatments unintentionally affecting cellular migration during the development of the embryo in pregnant women. For example after some years of use, it has been shown that the teratogenic properties of valproic acid, an anti-epileptic medicament, are due to its secondary effects on the actin cytoskeleton.
2) Process for Screening Molecules Affecting Cellular Actin Polymerisation
The most effective screening tests carried out on new medicaments have the following properties: they are simple, cheap and quick. The cellular actin polymerisation test of the invention has all these characteristics. Beads measuring 200–500 nm can be produced by absorbing purified GST-WH2/A recombinant protein 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 cells in culture. The volume of extract necessary for an experiment is of the order of a few microlitres, reducing the cost of the experiment. These extracts have the advantage that they can be produced in a large quantity and stored for a long period, at −80° C. Before the addition of these beads, a fluorescent actin derivative (commercial actin-rhodamine) is added to the extract to make the actin polymerisation visible by epifluorescence microscopy. The actin polymerisation is observed 15 min after the addition of the beads, resulting in an accumulation of fluorescent actin around the beads. Starting with a stock of standardised reagents, the experiment takes less than 30 min and one person is capable of dealing with several samples in parallel. This process can be automated, enabling the rapid screening of a large number samples.
To identify the active components, it is necessary to test the chemical products at variable concentrations. The inventors have shown that low quantities of solvents used to dissolve these products (water, ethanol, dimethyl sulphoxide) do not disturb the system of the invention. Moreover, this screening test has been validated by showing that it is sensitive to known medicaments inhibiting actin polymerisation, such as latrunculin and cytochalasin D.

## Claims

1. A process for the detection or screening of molecules, having a modulating effect on the formation of actin cytoskeleton, and hence a modulating effect on cellular motility, said process comprising:
a stage of placing a test molecule in the presence of a reagent in a medium containing actin and elements necessary for actin polymerization,
followed by detecting any inhibition or activation of the actin polymerization process on the surface of said reagent, compared with a control, respectively demonstrating an inhibiting or stimulating effect on the formation of the actin cytoskeleton by the test molecule, and wherein said reagent comprises the WASP peptide fragment consisting of SEQ ID NO: 4, bound or adsorbed to a support capable of moving under the effect of actin polymerization, when said support bound to said fragment is placed in a medium containing the elements necessary for actin polymerization.

2. A process for the detection or screening of molecules, having a modulating effect on the formation of actin cytoskeleton, and hence a modulating effect on cellular motility, said process comprising:
placing a test molecule in the presence of a reagent in a medium containing actin and the elements necessary for actin polymerization,
detecting any inhibition or activation of the actin polymerization process on the surface of said reagent, compared with a control, respectively demonstrating an inhibiting or stimulating effect on the formation of the actin cytoskeleton by the test molecule, and wherein said reagent comprises the WASP peptide fragment consisting of SEQ ID NO: 4, bound or adsorbed to a support capable of moving under the effect of actin polymerization, when said support bound to said fragment is placed in a medium containing the elements necessary for actin polymerization, and wherein said reagent is in a form chosen from microspheres.

3. A process for the detection or screening of molecules, having a modulating effect on the formation of actin cytoskeleton, and hence a modulating effect on cellular motility, said process comprising:
placing a test molecule in the presence of a reagent in a medium containing actin and the elements necessary for actin polymerization, detecting any inhibition or activation of the actin polymerization polymerisation process on the surface of said reagent, compared with a control, respectively demonstrating an inhibiting or stimulating effect on the formation of the actin cytoskeleton by the test molecule, and wherein said reagent contains a WASP peptide fragment consisting of SEQ ID NO: 4, bound or adsorbed to a support capable of moving under the effect of actin polymerization, when said support bound to said fragment is placed in a medium containing the elements necessary for actin polymerization.

4. The process according to claim 3, wherein said reagent is in the form of microsphere.

5. The process according to claim 4, wherein said microspheres have a diameter approximately between 100 nm and approximately 10,000 nm.

6. The process according to claim 4, wherein said microspheres comprise a material selected from the group consisting of polystyrene and latex.

7. The process according to claim 5, wherein said microspheres comprise a material selected from the group consisting of polystyrene and latex.

8. The process according to claim 3, wherein said support is a microsphere.