Apaf-1, an activator of caspase-3

Abstract

Novel polypeptides, designated Apaf-1, which are capable of modulating apoptosis are provided. Compositions including Apaf-1 chimeras, nucleic acid encoding Apaf-1, and antibodies to Apaf-1 are also provided.

We claim:  
     
         1 . An isolated Apaf-1 protein having at least about 80% amino acid sequence identity with the sequence of SEQ ID NO:2 or the sequence of SEQ ID NO: 16.  
     
     
         2 . The Apaf-1 protein of  claim 1  having at least about 90% amino acid sequence identity with the sequence of SEQ ID NO:2 or the sequence of SEQ ID NO: 16.  
     
     
         3 . The Apaf-1 protein of  claim 2  having at least about 95% amino acid sequence identity with the sequence of SEQ ID NO:2 or the sequence of SEQ ID NO: 16.  
     
     
         4 . An isolated Apaf-1 protein comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:16.  
     
     
         5 . A chimeric molecule comprising the Apaf-1 protein of  claim 1  fused to a heterologous amino acid sequence.  
     
     
         6 . The chimeric molecule of  claim 5 , wherein said heterologous amino acid sequence is an epitope tag sequence.  
     
     
         7 . The chimeric molecule of  claim 5 , wherein said heterologous amino acid sequence is an immunoglobulin sequence.  
     
     
         8 . The chimeric molecule of  claim 7 , wherein said immunoglobulin sequence is an IgG.  
     
     
         9 . An antibody which binds to the Apaf-1 protein of  claim 1 .  
     
     
         10 . The antibody of  claim 9 , wherein said antibody is a monoclonal antibody.  
     
     
         11 . The antibody of  claim 9 , which is an agonist antibody.  
     
     
         12 . An isolated nucleic acid comprising a nucleotide sequence encoding the Apaf-1 protein of  claim 1 .  
     
     
         13 . The isolated nucleic acid of  claim 12 , comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:15.  
     
     
         14 . The nucleic acid of  claim 12 , wherein said nucleotide sequence encodes an Apaf-1 protein comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:16.  
     
     
         15 . An isolated nucleic acid comprising a nucleotide sequence which hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:15.  
     
     
         16 . A vector comprising the nucleic acid of  claim 12 .  
     
     
         17 . The vector of  claim 16  operably linked to control sequences recognized by a host cell transformed with the vector.  
     
     
         18 . A host cell comprising the vector of  claim 16 .  
     
     
         19 . A process for producing Apaf-1 protein comprising: 
 culturing the host cell of  claim 18  to express Apaf-1 protein in the host cell.    
     
     
         20 . A non-human, transgenic animal containing cells that expresses an inserted nucleotide sequence encoding Apaf-1 protein.  
     
     
         21 . The animal of  claim 20  which is a mouse or rat.  
     
     
         22 . A non-human, knockout animal which contains cells having an altered gene encoding Apaf-1 protein.  
     
     
         23 . The animal of  claim 22  which is a mouse or rat.  
     
     
         24 . An article of manufacture, comprising a container and a composition contained within said container, wherein the composition includes Apaf-1 protein or anti-Apaf-1 antibodies.  
     
     
         25 . The article of manufacture of  claim 24  further comprising instructions for using the Apaf-1 protein or anti-Apaf-1 antibodies in vivo or ex vivo.  
     
     
         26 . A method for screening compounds for agents which modulate apoptosis, the method comprising: 
 analyzing the ability of a candidate compound to competitively displace cytochrome c binding to an Apaf-1 protein; and    correlating competitive displacement of cytochrome c binding to said Apaf-1 protein by the candidate compound with the ability of the candidate compound to modulate apoptosis.    
     
     
         27 . A method for identifying compounds which modulate apoptosis, the method comprising, 
 incubating a candidate compound in a mixture containing Apaf-1 protein, Apaf-3 protein, dATP, and cytochrome c;    adding a caspase precursor to the incubating mixture; and    correlating a change in cleavage of the caspase precursor in the presence of the candidate compound as compared with cleavage in the absence of the compound with the candidate compound's ability to modulate apoptosis.