Caspase-8 interacting proteins

Abstract

There are provided polypeptides which bind to caspase-8. Production and use of such polypeptides is also provided, as well as DNA encoding them, and vectors and host cells having such DNA.

1 . A purified polypeptide that binds to caspase-8, said polypeptide comprising:
 (a) the amino acid sequence of SEQ ID NO:7;   (b) the amino acid sequence of an analog of (a), having no more than ten changes in the amino acid sequence of (a), each said change being a substitution, deletion or insertion of an amino acid, which analog binds to caspase-8;   (c) the amino acid sequence of a polypeptide encoded by a DNA sequence capable of hybridizing with the DNA sequence of the portion of RPCI5-1057I20 which encodes SEQ ID NO:7, under moderately stringent conditions, which polypeptide binds to caspase-8;   (d) the amino acid sequence of a fragment of (a), which fragment binds to caspase-8; or   (e) a derivative of (a), (b), (c), or (d) by modification of a side group of one or more amino acid residues thereof, which derivative binds to caspase-8.   
     
     
         2 . The polypeptide in accordance with  claim 1 , wherein said polypeptide consists of the polypeptide of (a) or (b) or a derivative of (a) or (b) by modification of a side 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 caspase-8. 
     
     
         3 . The polypeptide in accordance with  claim 1 , wherein said polypeptide comprises the polypeptide of (a) or a derivative of (a) by modification of a side group of one or more amino acid residues thereof, which derivative binds to caspase-8. 
     
     
         4 . A polypeptide in accordance with  claim 1 , which consists of the amino acid sequence of SEQ ID NO:7. 
     
     
         5 . A purified polypeptide that binds to caspase-8, said polypeptide consisting of the amino acid sequence of a fragment of SEQ ID NO:7, which fragment binds to caspase-8, or a derivative thereof by modification of a side group of one or more amino acid residues thereof, which derivative binds to caspase-8. 
     
     
         6 . A caspase-8 interacting peptide in accordance with  claim 5 , consisting of a derivative of said fragment by modification of a side group of one or more amino acid residues thereof, which derivative binds to caspase-8. 
     
     
         7 . A composition comprising a polypeptide according to  claim 1 , and a pharmaceutically acceptable excipient. 
     
     
         8 . A composition comprising a polypeptide according to  claim 5 , and a pharmaceutically acceptable excipient. 
     
     
         9 . A ribozyme specific for a DNA molecule comprising a nucleotide sequence coding for a polypeptide that binds to caspase-8, said polypeptide comprising an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:7; and   (b) the amino acid sequence of an analog of (a), having no more than ten changes in the amino acid sequence of (a), each said change being a substitution, deletion or insertion of an amino acid.   
     
     
         10 . An antibody directed at an epitope of an amino acid sequence of SEQ ID NO:6 or an amino acid sequence of SEQ ID NO:7. 
     
     
         11 . An immunoassay for the detection of a caspase-8 interacting protein, comprising the antibody according to  claim 10  as a reagent. 
     
     
         12 . A method of identifying caspase-8 interacting proteins, comprising the steps of
 a) providing a yeast cell having a reporter gene linked to a promoter comprising a DNA sequence motif;   b) expressing in said yeast cell a p20 subunit of said caspase-8;   c) expressing in said yeast cell a fusion protein of a DNA binding domain and the p10 and/or p20 subunit of said caspase-8, wherein said DNA binding domain is capable of binding to said DNA sequence motif;   d) optionally, expressing in said yeast cell an unfused p10 or p20 subunit of said caspase-8;   e) transforming a culture of said yeast cell with a library consisting of an expression vector driving expression of a fusion protein consisting of a cDNA library and a transcriptional activator;   f) screening the culture of transformed yeast cells for yeast cells wherein the reporter gene is activated, and   g) isolating a yeast cell from step f) and further isolating the caspase-8 interacting protein, which is expressed in its prey vector.   
     
     
         13 . A method of modulating caspase-8 activity comprising bringing a polypeptide capable of binding to caspase-8 into contact with caspase-8, said polypeptide comprising:
 (a) the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7;   (b) the amino acid sequence of an analog of (a), having no more than ten changes in the amino acid sequence of (a), each said change being a substitution, deletion or insertion of an amino acid, which analog binds to caspase-8;   (c) the amino acid sequence of a polypeptide encoded by a DNA sequence capable of hybridizing with the DNA sequence of SEQ ID NO:5 or of the portion of RPCI5-1057I20 which encodes SEQ ID NO:7, under moderately stringent conditions, which polypeptide binds to caspase-8;   (d) the amino acid sequence of a fragment of (a), which fragment binds to caspase-8; or   (e) a derivative of (a), (b), (c), or (d) by modification of a side group of one or more amino acid residues thereof, which derivative binds to caspase-8.   
     
     
         14 . A method of modulating TNF-receptor or Fas mediated effects in a cell, comprising bringing said cell into contact with a caspase-8 binding polypeptide comprising:
 (a) the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7;   (b) the amino acid sequence of an analog of (a), having no more than ten changes in the amino acid sequence of (a), each said change being a substitution, deletion or insertion of an amino acid, which analog binds to caspase-8;   (c) the amino acid sequence of a polypeptide encoded by a DNA sequence capable of hybridizing with the DNA sequence of SEQ ID NO:5 or of the portion of RPCI5-1057I20 which encodes SEQ ID NO:7, under moderately stringent conditions, which polypeptide binds to caspase-8;   (d) the amino acid sequence of a fragment of (a), which fragment binds to caspase-8; or   (e) a derivative of (a), (b), (c), or (d) by modification of a side group of one or more amino acid residues thereof, which derivative binds to caspase-8.   
     
     
         15 . A method for modulating apoptosis of a cell, comprising bringing a caspase-8 binding polypeptide into contact with said cell, said polypeptide comprising:
 (a) the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7;   (b) the amino acid sequence of an analog of (a), having no more than ten changes in the amino acid sequence of (a), each said change being a substitution, deletion or insertion of an amino acid, which analog binds to caspase-8;   (c) the amino acid sequence of a polypeptide encoded by a DNA sequence capable of hybridizing with the DNA sequence of SEQ ID NO:5 or of the portion of RPCI5-1057I20 which encodes SEQ ID NO:7, under moderately stringent conditions, which polypeptide binds to caspase-8;   (d) the amino acid sequence of a fragment of (a), which fragment binds to caspase-8; or   (e) a derivative of (a), (b), (c), or (d) by modification of a side group of one or more amino acid residues thereof, which derivative binds to caspase-8.   
     
     
         16 . An isolated DNA sequence comprising the DNA sequence of SEQ ID NO:1 or SEQ ID NO:3.