p53-DEPENDENT APOPTOSIS-INDUCING PROTEIN AND METHOD OF SCREENING FOR APOPTOSIS REGULATOR

Abstract

p53-dependent Damage-Inducible Nuclear Protein 1 (p53DINP1 protein) is a p53-induced nuclear protein that induces p53-dependent apoptosis by regulating p53 function through Ser 46 phosphorylation. A DNA encoding p53DINP1 can be applied as anticancer agents for destroying neoplasms such as tumors, and as therapeutic or preventive agents for diseases associated with p53-mediated apoptosis abnormalities. It is also possible to apply the above protein and DNA in methods of screening for candidate compounds for regulating p53-mediated apoptosis.

1 . An isolated DNA of the following (a) or (b):
 (a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NOs: 2 or 4, or   (b) a DNA comprising the coding region of a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5.   
     
     
         2 . An isolated DNA of the following (a) or (b) encoding a protein having the activity to induce apoptosis:
 (a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NOs: 2 or 4, wherein one or more amino acids are substituted, deleted, inserted, and/or added, or   (b) a DNA hybridizing under stringent conditions to a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5.   
     
     
         3 . A DNA containing at least 15 nucleotides, wherein said DNA is complementary to a DNA comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5, or to the complementary strand thereof. 
     
     
         4 . An isolated protein encoded by the DNA of  claim 1  or  2 . 
     
     
         5 . A vector comprising the DNA of  claim 1  or  2 . 
     
     
         6 . A host cell carrying the DNA of  claim 1  or  2 , or the vector of  claim 5 . 
     
     
         7 . A method for producing the protein of  claim 4 , wherein said method comprises the steps of culturing the host cell of  claim 6 , and recovering the protein expressed by said host cell. 
     
     
         8 . An antibody binding to the protein of  claim 4 . 
     
     
         9 . An antisense polynucleotide to the DNA comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 3, and 5. 
     
     
         10 . A method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:
 (a) contacting a test sample with the protein of  claim 4 ,   (b) detecting the binding activity of the test sample to said protein, and   (c) selecting a compound having the activity to bind to said protein.   
     
     
         11 . A method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:
 (a) contacting a test sample with the protein of  claim 4  in the presence of p53,   (b) measuring the phosphorylation level of said p53 protein at the Ser46 residue after the contact, and   (c) selecting a compound capable of regulating apoptosis based on the phosphorylation level measured in step (b).   
     
     
         12 . A method of screening for a candidate compound for an apoptosis-regulating agent, wherein said method comprises the steps of:
 (a) contacting a test sample with a cell containing a vector having the structure in which the p53-binding sequence of SEQ ID No: 8 and a reporter gene are operably linked, or an extract of said cell,   (b) measuring the expression level of said reporter gene, and   (c) selecting a compound capable of decreasing or increasing the expression level of said reporter gene measured in step (b), compared to that measured in the absence of the test sample.   
     
     
         13 . An apoptosis-regulating agent comprising as an effective ingredient a compound selected by a method of any one of  claims 10  to  12 .