Modulators of the function of FAS receptors and other proteins

Abstract

The present invention provides proteins capable of modulating or mediating the FAS receptor ligand or TNF effect on cells carrying FAS receptor or p55 receptor by binding or interacting with MORT-1 protein, which in turn binds to the intracellular domain of the FAS receptor or to another protein TRADD which binds to the p55 receptor. In addition, peptide inhibitors that interfere with the proteolytic activity of MORT-1-binding proteins having proteolytic activity are provided, as well as a method of designing them.

1 . A method for the modulation of the FAS-R ligand or TNF effect on cells carrying an FAS-R or p55 TNFR, comprising treating said cells with one or more polypeptides that bind to MORT-1 protein, each said polypeptide having a sequence comprising: 
 (a) residues 1-182 of SEQ ID NO: 5;    (b) an analog of (a), differing therefrom by no more than ten changes in the amino acid sequence thereof, each said change being a substitution, deletion and/or insertion of a single amino acid, which analog binds to MORT-1 protein;    (c) a fragment of (a) or (b), which fragment binds to MORT-1 protein; or    (d) a derivative of (a), (b) or (c) by modification of a functional group which occurs as a side chain or an N- or C-terminal group of one or more amino acid residues thereof without changing one amino acid to another of the twenty commonly occurring natural amino acids, which derivative binds to MORT-1 protein.    
     
     
         2 . A method in accordance with  claim 1 , wherein said treating of said cells comprises introducing into said cells said one or more polypeptides in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more polypeptides 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.  
     
     
         3 . A method for the modulation of the FAS-R ligand or TNF effect on cells according to  claim 2 , wherein said treating of cells comprises introducing into said cells said polypeptide, or a DNA sequence encoding said polypeptide 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.  
     
     
         4 . A method according to  claim 2 , 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 a FAS-R- or p55-R-carrying cell and a second sequence encoding said polypeptide, that when expressed in said cells is capable of modulating/mediating the activity of said FAS-R or p55-R; and    (b) infecting said cells with said vector of (a).    
     
     
         5 . A method according to  claim 1 , wherein said MORT-1-binding protein or said polypeptide is one which is capable of binding specifically to MORT-1 which in turn binds specifically to FAS-IC, or which is capable of binding to MORT-1 which in turn binds to TRADD and which in turn binds to the p55-IC.  
     
     
         6 . A method according to  claim 1 , wherein said polypeptide is the MACH isoform herein designated MACHα1, analogs, fragments and derivatives thereof.  
     
     
         7 . A method according to  claim 1 , wherein said polypeptide is the MACH isoform herein designated MACHβ1, analogs, fragments and derivatives thereof.  
     
     
         8 . A method according to  claim 1 , wherein said polypeptide is the MACH isoform herein designated MACHβ3, analogs, fragments and derivatives thereof.  
     
     
         9 . A method for modulating the FAS-R ligand or TNF effect on cells carrying a FAS-R or p55-R, comprising treating said cells with an antisense oligonucleotide consisting of a sequence complementary to at least a portion of the mRNA encoding a MORT-1-binding protein comprising residues 1-182 of SEQ ID NO: 5, said antisense oligonucleotide sequence being capable of blocking the expression of the MORT-1-binding protein.  
     
     
         10 . A method according to  claim 9 , wherein said antisense oligonucleotide is introduced to said cells via a recombinant animal virus vector, wherein said second sequence of said virus is said antisense oligonucleotide sequence.  
     
     
         11 . A method for treating tumor cells or HIV-infected cells or other diseased cells, comprising: 
 (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding a polypeptide that binds to MORT-1 protein, that when expressed in said tumor, HIV-infected, or other diseased cell is capable of killing said cell; and    (b) infecting said tumor or HIV-infected cells or other diseased cells with said vector of (a),    wherein said polypeptide that binds to MORT-1 protein has a sequence comprising 
 (a) residues 1-182 of SEQ ID NO: 5;  
 (b) an analog of (a), differing therefrom by no more than ten changes in the amino acid sequence thereof, each said change being a substitution, deletion and/or insertion of a single amino acid, which analog binds to MORT-1 protein;  
 (c) a fragment of (a) or (b), which fragment binds to MORT-1 protein; or  
 (d) a derivative of (a), (b) or (c) by modification of a functional group which occurs as a side chain or an N- or C-terminal group of one or more amino acid residues thereof without changing one amino acid to another of the twenty commonly occurring natural amino acids, which derivative binds to MORT-1 protein.  
   
     
     
         12 . A method of modulating the FAS-R ligand or TNF 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 a polypeptide comprising residues 1-182 of SEQ ID NO: 5, 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 resulting in the inhibition of expression of said polypeptide in said cells.  
     
     
         13 . A method for isolating and identifying proteins capable of binding to the MORT-1 protein, comprising applying the yeast two-hybrid procedure in which a sequence encoding said MORT-1 protein 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 MORT-1 protein, said protein being the MORT-1-binding proteins.  
     
     
         14 . A method for the modulation of the MORT-1-induced effect or MORT-1-binding protein-induced effect on cells comprising treating said cells with MORT-1-binding proteins, analogs, fragments or derivatives thereof or with sequences encoding MORT-1-binding proteins, analogs or fragments thereof, said treatment resulting in the enhancement or inhibition of said MORT-1-mediated effect and thereby also of the FAS-R or p55-R mediated effect.  
     
     
         15 . A method according to  claim 14 , wherein said MORT-1-binding protein, analog, fragment or derivative thereof is that part of the MORT-1-binding protein which is specifically involved in binding to MORT-1 or MORT-1-binding protein itself, or said MORT-1-binding protein sequence encodes that part of MORT-1-binding protein which is specifically involved in binding to MORT-1 or the MORT-1-binding protein itself.  
     
     
         16 . A method according to  claim 14 , wherein said MORT-1-binding protein is any one of the MACH isoforms selected from MACHα1, MACHβ1, and MACHβ3, said MACH isoforms capable of enhancing the MORT-1-assocaited effect on cells and thereby also of the FAS-R or p55-R associated effect on the cells.  
     
     
         17 . A method according to  claim 14 , wherein said MORT-1-binding protein is any one of the MACH isoforms selected from MACHα2 or MACHα3, said MACH isoforms being capable of inhibiting the activity of MACH isoforms intracellularly and thereby inhibiting the MROT-1-associated cellular effect and thereby also of the FAS-R or p55-R associated effect on the cells.  
     
     
         18 . A method of modulating apoptotic processes or programmed cell death processes comprising treating said cells with one or more polypeptides that link to MORT-1 protein, wherein said treating of said cells comprises introducing into said cells said one or more polypeptides in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more polypeptides 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, wherein each said polypeptide has a sequence comprising 
 (a) residues 1-182 of SEQ ID NO: 5;    (b) an analog of (a), differing therefrom by no more than ten changes in the amino acid sequence thereof, each said change being a substitution, deletion and/or insertion of a single amino acid, which analog binds to MORT-1 protein;    (c) a fragment of (a) or (b), which fragment binds to MORT-1 protein; or    (d) a derivative of (a), (b) or (c) by modification of a functional group which occurs as a side chain or an N- or C-terminal group of one or more amino acid residues thereof without changing one amino acid to another of the twenty commonly occurring natural amino acids, which derivative binds to MORT-1 protein.    
     
     
         19 . An antibody or an active fragment or a derivative of said antibody or fragment, which is capable of binding to a polypeptide that binds to MORT-1 protein and that has a sequence comprising: 
 (a) residues 1-182 of SEQ ID NO: 5;    (b) an analog of (a), differing therefrom by no more than ten changes in the amino acid sequence thereof, each said change being a substitution, deletion and/or insertion of a single amino acid, which analog binds to MORT-1 protein;    (c) a fragment of (a) or (b), which fragment binds to MORT-1 protein; or    (d) a derivative of (a), (b) or (c) by modification of a functional group which occurs as a side chain or an N- or C-terminal group of one or more amino acid residues thereof without changing one amino acid to another of the twenty commonly occurring natural amino acids, which derivative binds to MORT-1 protein.    
     
     
         20 . A method for modulating the FAS-R ligand or TNF effect on cells carrying a FAS-R or a p55R comprising treating said cells with antibodies or active fragments or derivatives according to  claim 19 , said treating being by application of a suitable composition containing said antibodies, active fragments or derivatives to said cells, wherein when the MORT-1-binding protein or portions thereof of said cells are exposed on the extracellular surface, said composition is formulated for extracellular application, and when said MORT-1-binding proteins are intracellular, said composition is formulated for intracellular application.  
     
     
         21 . A pharmaceutical composition for the modulation of the FAS-R ligand- or TNF-effect on cells comprising, as active ingredient a polypeptide that binds to MORT-1 protein and that has a sequence comprising: 
 (a) residues 1-182 of SEQ ID NO: 5;    (b) an analog of (a), differing therefrom by no more than ten changes in the amino acid sequence thereof, each said change being a substitution, deletion and/or insertion of a single amino acid, which analog binds to MORT-1 protein;    (c) a fragment of (a) or (b), which fragment binds to MORT-1 protein; or    (d) a derivative of (a), (b) or (c) by modification of a functional group which occurs as a side chain or an N- or C-terminal group of one or more amino acid residues thereof without changing one amino acid to another of the twenty commonly occurring natural amino acids, which derivative binds to MORT-1 protein.    
     
     
         22 . A pharmaceutical composition for modulating the FAS-R ligand or TNF effect on cells, comprising, as active ingredient, an antisense oligonucleotide consisting of a sequence complementary to at least a portion of the mRNA encoding a MORT-1-binding protein comprising residues 1-182 of SEQ ID NO: 5, said antisense oligonucleotide sequence being capable of blocking the expression of the MORT-1-binding protein.  
     
     
         23 . An inhibitor of the proteolytic activity of a MACH protease, comprising a compound which binds to the protease active site of said MACH protease so as to interfere with the proteolytic activity of said MACH protease.  
     
     
         24 . An inhibitor in accordance with  claim 23 , comprising a peptide having a sequence of at least the four amino acids which are the minimum length peptide substrate for said MACH protease, said peptide further including a moiety which interferes with proteolytic activity of said MACH protease.  
     
     
         25 . A polypeptide that binds to MORT-1, MORT-1 being a protein which binds to the intracellular domain of the FAS-R and which binds to the protein TRADD which binds to the intracellular domain of p55 TNFR, which polypeptide affects the intracellular signaling process initiated by the binding of FAS ligand to its receptor or the binding of TNF to p55 TNFR, said polypeptide having: 
 (a) a sequence comprising residues 1-182 and 221-479 of SEQ ID NO: 7;    (b) a sequence comprising an analog of (a), having no more than ten changes in the amino acid sequence of a) or b), each said change being a substitution, deletion or insertion of an amino acid, which analog binds to MORT-1 and affects the intracellular signaling process initiated by the binding of FAS ligand to its receptor or the binding of TNF to p55 TNFR;    (c) a sequence comprising a fragment of (a) or (b), which fragment binds to MORT-1 and affects the intracellular signaling process initiated by the binding of FAS ligand to its receptor or the binding of TNF to p55 TNFR; or    (d) a derivative of (a), (b) or (c) which binds to MORT-1 and affects the intracellular signaling process initiated by the binding of FAS ligand to its receptor or the binding of TNF to p55 TNFR.    
     
     
         26 . A polypeptide in accordance with  claim 25 , wherein the polypeptide of paragraph (a) of  claim 25  is a native protein that binds to MORT-1 and affects the intracellular signaling process initiated by the binding of FAS ligand to its receptor or the binding of TNF to p55 TNFR.  
     
     
         27 . A polypeptide according to  claim 25 , wherein the polypeptide of paragraph (a) of  claim 25  is a MACH protein isoform selected from the group consisting of MACHα1, MACHα2, and MACHα3.  
     
     
         28 . A polypeptide in accordance with  claim 25 , encoding a native protein that binds to MORT-1 and affects the intracellular signaling process,initiated by the binding of FAS ligand to its receptor or the binding of TNF to p55 TNFR.  
     
     
         29 . A polypeptide in accordance with  claim 28 , encoding a MACH protein isoform selected from the group consisting of MACHα1, MACHα2, and MACHα3.