Uranium-chelating peptides and uses thereof

Abstract

The invention concerns uranium-chelating peptides as well as their uses for decontaminating soils and water, and for detecting and treating people contaminated by uranium. Said peptides have a helix-loop-helix type structure comprising the sequence of a calmodulin loop including at least one mutation of neutral residues selected from the group consisting of S, T, C, H, Y, N and Q, of one, two or three residues of at least one of the four calmodulin calcium binding sites: site I: residues selected among D20, D22 and D24 residues; site II: residues selected among D56, D58 and N60 residues; site III: residues selected among D93, D95 and N97 residues; site IV: residues selected among D129, D131 and D133 residues; said positions being indicated with reference to the human calmodulin sequence.

1 . A peptide having a helix-loop-helix type structure comprising the sequence of a calmodulin loop including at least one mutation to neutral residues selected from the group consisting of Ser (S), Thr (T), Cys (C), His (H), Tyr (Y), Asn (N) and Gln (Q), of one, two or three residues of at least one of the four calcium-binding sites of calmodulin:
 site I: residues selected from residues D20, D22 and D24,   site II: residues selected from residues D56, D58 and N60,   site III: residues selected from residues D93, D95 and N97,   site IV: residues selected from residues D129, D131 and D133,   said positions being indicated with reference to the human calmodulin sequence (SWISSPROT P02593).   
     
     
         2 . The peptide as claimed in  claim 1 , wherein the mutation is a mutation to threonine (Thr), serine (Ser) or asparagine (Asn) neutral residues. 
     
     
         3 . The peptide as claimed in  claim 1  wherein the mutation is a mutation, to a threonine residue, of residue D20, D22 or D24, a mutation, to a threonine, serine or asparagine residue, of the two residues D20 and D24, of the two residues D20 and D22 or of the two residues D22 and D24, or a mutation, to a threonine, serine or asparagine residue, of the three residues D20, D22 and D24. 
     
     
         4 . The peptide as claimed in  claim 1 , wherein it is a calmodulin calcium-binding site mutant. 
     
     
         5 . The peptide as claimed in  claim 1 , wherein it is a cyclic peptide which has helices that each include a mutation of an amino acid residue to a residue that allows chemical bridging, in particular a cysteine residue, which cysteines are connected via a disulfide bridge. 
     
     
         6 . The peptide as claimed in  claim 5 , wherein it has the mutations F19C and V35C. 
     
     
         7 . The peptide as claimed in  claim 1 , wherein it also includes the mutation of an amino acid residue to a fluorescent amino acid residue. 
     
     
         8 . The peptide as claimed in  claim 7 , wherein said fluorescent amino acid residue is a tyrosine residue or a tryptophan residue. 
     
     
         9 . The peptide as claimed in  claim 8 , wherein it has a mutation selected from the group consisting of: T26Y, T26W, A15W and F16W. 
     
     
         10 . The peptide as claimed in  claim 1 , wherein it has one of the sequences SEQ ID Nos. 4-7 or SEQ ID Nos. 9-12. 
     
     
         11 . The peptide as claimed in  claim 1 , wherein it is conjugated to at least one fluorophore. 
     
     
         12 . The peptide as claimed in  claim 11 , wherein it is conjugated to two different fluorophores. 
     
     
         13 . The peptide as claimed in  claim 11  wherein said fluorophore is a fluorescent protein selected from: EBFP, ECFP, EYFP, EGFP, DsRed, CopGFP and PhiYFP. 
     
     
         14 . The peptide as claimed in  claim 11  wherein said fluorophore is selected from dansyl, coumarin, fluorescein and Alexa derivatives. 
     
     
         15 . The peptide as claimed in  claim 1 , wherein it is associated with at least one molecule that allows targeting to the kidney and/or to the bone. 
     
     
         16 . The peptide as claimed in  claim 1 , wherein it is associated with a molecule that promotes its excretion in vivo. 
     
     
         17 . A polypeptide comprising the concatenation of at least two identical or different peptides as claimed in  claim 1 . 
     
     
         18 . A peptide composition comprising at least one polypeptide as claimed in  claim 17  and at least one suitable vehicle. 
     
     
         19 . A fusion protein consisting of the in-frame fusion of the sequence of at least one peptide as claimed in  claim 1 , with the sequence of an appropriate protein. 
     
     
         20 . The fusion protein as claimed in  claim 19 , wherein the sequence of said peptide is fused to the sequence of a protein selected from the group consisting of calmodulin, chameleon proteins derived from the latter and proteins having a helix-loop-helix type motif, capable of binding calcium. 
     
     
         21 . The fusion protein as claimed in  claim 19  wherein it is conjugated to at least one fluorophore as defined in  claim 13 . 
     
     
         22 . The fusion protein as claimed in  claim 21 , wherein one of the ends of said protein is coupled to a fluorescence donor, and the other is coupled to a fluorescence acceptor. 
     
     
         23 . The fusion protein as claimed in  claim 22 , wherein it comprises, at one of its ends, the sequence of EBFP or ECFP and, at the other end, the sequence of EGFP or of EYFP. 
     
     
         24 . A method of using a product selected from the group consisting of: a peptide as claimed in  claim 1 , a polypeptide derived from said peptide, a peptide composition comprising said peptide and a fusion protein derived from said peptide, said method comprising preparing a reagent for detecting soils and water contaminated with uranium from said product. 
     
     
         25 . A method of using a product selected from the group consisting of: a peptide as claimed in  claim 1 , a polypeptide derived from said peptide, a peptide composition comprising said peptide and a fusion protein derived from said peptide, said method comprising preparing a reagent for diagnosing individuals contaminated with uranium from said product. 
     
     
         26 . An isolated nucleic acid molecule comprising a sequence encoding a peptide as claimed in  claim 1 . 
     
     
         27 . Probes and primers capable of specifically detecting/amplifying the nucleic acid molecules as claimed in  claim 26 , which probes and primers comprise a sequence of approximately 10 to 30 nucleotides that is adjacent to a helix-loop-helix type motif or else to one of the helices or to the loop of this motif, of a calcium ion-binding protein. 
     
     
         28 . A eukaryotic or prokaryotic recombinant vector comprising an insert consisting of a nucleic acid molecule as claimed in  claim 26 . 
     
     
         29 . Eukaryotic or prokaryotic cells modified with a recombinant vector as claimed in  claim 28 . 
     
     
         30 . A transgenic nonhuman animal organism comprising cells modified with a nucleic acid molecule as claimed in  claim 26 . 
     
     
         31 . A transgenic comprising cells modified with a nucleic acid molecule as claimed in  claim 26 . 
     
     
         32 . Prokaryotic or eukaryotic cells modified with a regulatory system that includes a peptide as claimed in  claim 1  as a regulator or repressor of a gene encoding a bioluminescent protein. 
     
     
         33 . A method for the remediation of soil or water contaminated with uranium, comprising contacting soil or mater contaminated with uranium with the modified prokaryotic or eukaryotic cells as claimed in  claim 29 . 
     
     
         34 . A method of using the modified prokaryotic or eukaryotic cells as claimed in  claim 32  which comprises preparing a reagent for detecting soils and water contaminated with uranium from said cells. 
     
     
         35 . A method of using the modified prokaryotic or eukaryotic cells as claimed in  claim 32  which comprises preparing a reagent for diagnosing individuals contaminated with uranium from said cells. 
     
     
         36 . An antibody that binds selectively to the peptide as claimed in  claim 1 . 
     
     
         37 . A kit for detecting a contamination with uranium comprising at least one of: a peptide as claimed in  claim 1 , a polypeptide derived from said peptide, a peptide composition comprising said peptide, a fusion protein derived from said peptide, an antibody that binds selectively to said peptide, or modified prokaryotic or eukaryotic cells modified with a regulatory system that includes said peptide as a regulator or repressor of a gene encoding a bioluminescent protein. 
     
     
         38 . The fusion protein as claimed in  claim 19  wherein it is conjugated to at least one fluorophore as defined in  claim 14 . 
     
     
         39 . An isolated nucleic acid molecule comprising a sequence encoding a polypeptide as claimed in  claim 17 . 
     
     
         40 . An isolated nucleic acid molecule comprising a sequence encoding a fusion protein as claimed in  claim 19 . 
     
     
         41 . A method for the remediation of soil or water contaminated with uranium, comprising contacting soil or mater contaminated with uranium with the of transgenic plants as claimed in  claim 31 .