Caspase-8 interacting proteins

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.

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.

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.

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.