Iren protein, its preparation and use

Abstract

There are provided DNA sequences encoding TRAF binding proteins, proteins encoded thereby, and their use in the treatment or prevention of pathological conditions associated with NF-κB induction, or an activity mediated by a TRAF.

1 . A DNA sequence encoding a protein capable of binding to TRAF selected from the group consisting of:
 (a) a cDNA sequence of the herein designated IREN comprising the nucleotide sequence of SEQ ID NO:5;   (b) a cDNA sequence of the herein designated IREN-10B comprising the nucleotide sequence of SEQ ID NO:6;   (c) a fragment of a sequence (a)-(b) which encodes a biologically active protein capable of binding to at least the 225-501 amino acid sequence of TRAF2;   (d) a DNA sequence capable of hybridization to a sequence of (a)-(c) under moderately stringent conditions and which encodes a biologically active protein capable of binding to at least the 225-501 amino acid sequence of TRAF2; and   (e) a DNA sequence which is degenerate as a result of the genetic code to the DNA sequences defined in (a)-(d) and which encodes a biologically active protein capable of binding to at least the 225-501 amino acid sequence of TRAF2.   
     
     
         2 . A DNA sequence according to  claim 1 , selected from the cDNA sequences herein designated IREN-10B and IREN-E. 
     
     
         3 . A DNA sequence according to  claim 1 , encoding a protein that modulates NF-κB activity. 
     
     
         4 . A vector comprising a DNA sequence according to  claim 1 . 
     
     
         5 . A vector according to  claim 4  capable of being expressed in a eukaryotic host cell. 
     
     
         6 . A vector according to  claim 4  capable of being expressed in a prokaryotic host cell. 
     
     
         7 . Transformed eukaryotic or prokaryotic host cells containing a vector according to  claim 4 . 
     
     
         8 . An IREN protein, isoforms, fragments, analogs and derivatives thereof, encoded by a DNA sequence according to  claim 1  or encoded by a cDNA sequence of SEQ ID NO:5, or a fragment thereof capable of binding to at least the 225-501 amino acid sequence of TRAF2, said protein, isoforms, fragments, analogs and derivatives thereof being capable of binding to at least the portion of the TRAF2 protein between amino acids 225-501 of TRAF2. 
     
     
         9 . A protein according to  claim 8  being the protein encoded by herein-designated clone 10B. 
     
     
         10 . A protein, isoforms, fragments, analogs and derivatives thereof according to  claim 8  being the protein IREN, isoforms, analogs, fragments and derivatives thereof. 
     
     
         11 . A protein IREN, isoforms, analogs, fragments and derivatives thereof according to  claim 10 , wherein said protein, isoforms, fragments and derivatives have at least part of the amino acid sequence of SEQ ID NO:7. 
     
     
         12 . A method for producing a protein, isoform, fragment, analog or derivative thereof capable of binding to TRAF, which comprises growing a transformed host cell according to  claim 10  under conditions suitable for the expression of said protein, isoform, fragment, analog or derivative thereof, effecting post-translational modification, as necessary, for obtaining said protein, isoform, fragment, analog or derivative thereof, isolating said expressed protein, isoform, fragment, analog or derivative. 
     
     
         13 . Antibodies or active fragments or derivatives thereof, specific for the IREN protein, isoform, analog, fragment or derivative thereof according to  claim 8 . 
     
     
         14 . A method for the modulation or mediation in cells of the activity of NF-κB or any other intracellular signaling activity modulated or mediated by TRAF2 or by other molecules to which an IREN protein, isoform, analog, fragment or derivative thereof according to  claim 8  binds, said method comprising treating said cells by introducing into said cells one or more of said protein, isoform, analog, fragment or derivative thereof in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more protein, isoform, analog, fragment or derivative thereof in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells. 
     
     
         15 . A method according to  claim 14 , wherein said treating of cells comprises introducing into said cells a DNA sequence encoding said protein, isoform, fragment, analog or derivative in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells. 
     
     
         16 . A method according to  claim 14 , wherein said treating of said cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of:
 (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cells to be treated and a second sequence encoding a protein selected from the said IREN protein, isoforms, analogs, fragments and derivatives, that when expressed in said cells is capable of modulating/mediating the activity of NF-κB or any other intracellular signaling activity modulated/mediated by TRAF2 or other said molecules; and   (b) infecting said cells with said vector of (a).   
     
     
         17 . A method for modulating TRAF2 modulated/mediated effect on cells comprising treating said cells with antibodies or active fragments or derivatives thereof, according to  claim 13 , said treating being by application of a suitable composition containing said antibodies, active fragments or derivatives thereof to said cells, wherein when the IREN protein or portions thereof of said cells are exposed on the extracellular surface, said composition is formulated for extracellular application, and when said IREN proteins are intracellular said composition is formulated for intracellular application. 
     
     
         18 . A method for modulating the TRAF2 modulated/mediated effect on cells comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence for at least part of the DNA sequence encoding an IREN protein according to  claim 1  or an IREN protein of SEQ ID NO:4, said oligonucleotide sequence being capable of blocking the expression of the IREN protein. 
     
     
         19 . A method according to  claim 18 , wherein said oligonucleotide sequence is introduced to said cells via a recombinant animal virus vector. 
     
     
         20 . A method for modulating the TRAF2 modulated/mediated effect on cells comprising applying the ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding an IREN protein according to  claim 8  is introduced into said cells in a form that permits expression of said ribozyme sequence in said cells, and wherein when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cleaves said mRNA sequence resulting in the inhibition of expression of said IREN protein in said cells. 
     
     
         21 . A method for isolating and identifying proteins, according to  claim 8 , capable of binding directly to TRAF2, comprising applying the yeast two-hybrid procedure in which a sequence encoding said TRAF2 is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said TRAF2. 
     
     
         22 . A method according to  claim 14 , wherein said protein is IREN or at least one of the IREN isoforms, analogs, fragments and derivatives thereof. 
     
     
         23 . A pharmaceutical composition for the modulation of the TRAF2 modulated/mediated effect on cells comprising, as active ingredient at least one IREN protein, according to  claim 8 , its biologically active fragments, analogs, derivatives or mixtures thereof. 
     
     
         24 . A pharmaceutical composition for modulating the TRAF2 modulated/mediated effect on cells comprising, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding at least one IREN protein, isoform, active fragments or analogs, according to  claim 8 . 
     
     
         25 . A pharmaceutical composition for modulating the TRAF2 modulated/mediated effect on cells comprising as active ingredient, an oligonucleotide sequence encoding an anti-sense sequence of the IREN protein mRNA sequence according to  claim 1  or corresponding to the cDNA of SEQ ID NO:4. 
     
     
         26 . A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-κB induction or with any other activity mediated by TRAF2 or by other molecules to which a protein according to  claim 8  binds, said composition comprising an effective amount of a protein encoded by IREN-10B or a DNA molecule coding therefor, or a molecule capable of disrupting the interaction of said protein encoded by IREN-10B with TRAF2 or any other molecule to which a protein encoded by IREN-10B binds. 
     
     
         27 . A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-κB induction or with any other activity mediated by TRAF2 or by other molecules to which a protein according to  claim 8  binds, said composition comprising an effective amount of a protein IREN, isoform, fragment, analog or derivative thereof, or a DNA molecule coding therefor, or a molecule capable of disrupting the interaction of said protein IREN, isoform, fragment, analog or derivative thereof with TRAF2 or any other molecule to which said protein IREN, isoform, fragment, analog or derivative binds. 
     
     
         28 . A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-κB induction or with any other activity mediated by TRAF2 or by other molecules to which the protein IREN binds, said composition comprising a molecule capable of interfering with the activity of the protein IREN. 
     
     
         29 . A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-κB induction or with any other activity mediated by TRAF2 or by other molecules to which a protein encoded by IREN-10B according to  claim 9  binds, said composition comprising an effective amount of a protein encoded by IREN 10B or a DNA molecule coding therefor, or a molecule capable of disrupting the interaction of said protein encoded by IREN 10B with TRAF2 or any other molecule to which said protein encoded by IREN-10B binds. 
     
     
         30 . A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a protein IREN, isoform, fragment, analog or derivative according to  claim 10  binds, said composition comprising an effective amount of a protein IREN, isoform, fragment, analog or derivative thereof, or a DNA molecule coding therefor, or a molecule capable of disrupting the interaction of said protein IREN, isoform, fragment, analog or derivative thereof with TRAF2 or any other molecule to which said protein IREN, isoform, fragment, analog or derivative binds. 
     
     
         31 . A method for the prevention or treatment of a pathological condition associated with NF-κB induction or with any other activity mediated by TRAF2 or by other molecules to which an IREN protein according to  claim 8  binds, said method comprising administering to a patient in need an effective amount of said IREN protein, or a DNA molecule coding therefor, or a molecule capable of disrupting the interaction of said IREN protein or isoform, fragment, analog and derivative thereof or a mixture of any thereof with TRAF2 or any other molecule to which said IREN protein or isoform, fragment, analog and derivative thereof or a mixture of any thereof binds. 
     
     
         32 . A method according to  claim 31 , wherein said protein is encoded by IREN. 
     
     
         33 . A method according to  claim 31 , wherein said protein is IREN. 
     
     
         34 . A method for screening of a ligand capable of binding to a protein according to  claim 8 , comprising contacting an affinity chromatography matrix to which said protein is attached with a cell extract whereby the ligand is bound to said matrix, and eluting, isolating and analyzing said ligand. 
     
     
         35 . A method for screening of a DNA sequence coding for a ligand capable of binding to an IREN protein according to  claim 8 , comprising applying the yeast two-hybrid procedure in which a sequence encoding said protein is carried by one hybrid vector and sequences from a cDNA or genomic DNA library are carried by the second hybrid vector, transforming yeast host cells with said vectors, isolating the positively transformed cells, and extracting said second hybrid vector to obtain a sequence encoding said ligand. 
     
     
         36 . A method for identifying and producing a ligand capable of modulating the cellular activity modulated/mediated by TRAF2, comprising:
 (a) screening for a ligand capable of binding to a polypeptide comprising at least a portion of TRAF2 having the amino acid residues 225-501 of TRAF2;   (b) identifying and characterizing a ligand, other than TRAF2 or portions of a receptor of the TNF/NGF receptor family, found by said screening step to be capable of said binding; and   (c) producing said ligand in substantially isolated and purified form.   
     
     
         37 . A method for identifying and producing a ligand capable of modulating the cellular activity modulated or mediated by a protein according to  claim 11 , comprising:
 (a) screening for a ligand capable of binding to a polypeptide comprising at least a portion of the IREN sequence of SEQ ID NO:7;   (b) identifying and characterizing a ligand, other than TRAF2 or portions of a receptor of the TNF/NGF receptor family, found by said screening step to be capable of said binding; and   (c) producing said ligand in substantially isolated and purified form.   
     
     
         38 . A method for identifying and producing a ligand capable of modulating the cellular activity modulated/mediated by the protein IREN, comprising:
 (a) screening for a ligand capable of binding to at least a portion of the IREN sequence of SEQ ID NO:7;   (b) identifying and characterizing a ligand, other than TRAF2 or portions of a receptor of the TNF/NGF receptor family, found by said screening step to be capable of said binding; and   (c) producing said ligand in substantially isolated and purified form.   
     
     
         39 . A method for identifying and producing a molecule capable of directly or indirectly modulating the cellular activity modulated/mediated by the protein IREN, comprising:
 (a) screening for a molecule capable of modulating activities modulated/mediated by the protein IREN;   (b) identifying and characterizing said molecule; and   (c) producing said molecule in substantially isolated and purified form.   
     
     
         40 . A method for identifying and producing a molecule capable of directly or indirectly modulating the cellular activity modulated/mediated by an IREN protein according to  claim 8 , comprising:
 (a) screening for a molecule capable of modulating activities modulated/mediated by said IREN protein;   (b) identifying and characterizing said molecule; and   (c) producing said molecule in substantially isolated and purified form.