Method for detecting acetyltransferase and deacetylase activities and method for screening inhibitors or enhancers of these enzymes

Abstract

A method for simply and conveniently detecting acetyltransferase and deacetylase activities of proteins by executing an acetylation reaction of a peptide substrate with an acetyltransferase, or a deacetylation reaction of an acetylated peptide substrate with a deacetylase, and after the completion of these reactions, detecting the acetyl group bound to the peptide substrate by using an anti-acetylated peptide antibody. This system for detecting acetyltransferase and deacetylase activities using the anti-acetylated peptide antibody enables screening inhibitors or enhancers of acetyltransferase and deacetylase. A system for screening deacetylase inhibitors or acetyltransferase enhancers using cultured cells is also provided.

1 - 15 . (canceled)  
     
     
         16 . A method for detecting the acetyltransferase activity or the deacetylase activity in a test peptide, to catalyze a reaction that transfers an acetyl group from one substrate to another with an acetyltransferase, or to release an acetyl group with a deacetylase, the method comprising: 
 (a) contacting a test peptide with a peptide substrate or acetylated peptide substrate, and,    (b) detecting an amount of an acetylated peptide substrate using an anti-acetylated peptide antibody, wherein an effective amount of the anti-acetylated peptide antibody recognizes with specificity only an acetylated form of the peptide substrate and does not recognize the peptide substrate in its unacetylated form.    
     
     
         17 . The method of  claim 16 , wherein the peptide substrate is p53.  
     
     
         18 . The method of  claim 16 , wherein the peptide substrate is labeled.  
     
     
         19 . The method of  claim 18 , wherein the label is biotin.  
     
     
         20 . The method of  claim 16 , wherein the peptide substrate is immobilized on a solid phase.  
     
     
         21 . The method of  claim 16 , wherein the anti-acetylated peptide antibody is labeled.  
     
     
         22 . The method of  claim 16 , wherein the amount of acetylated peptide substrate is detected by ELISA.  
     
     
         23 . A method for screening a compound that inhibits or enhances deacetylase activity, to catalyze a reaction that releases an acetyl group from a substrate, the method comprising: 
 (a) contacting the deacetylase with an acetylated peptide substrate in a presence of a test compound,    (b) detecting an amount of an acetylated peptide substrate using an anti-acetylated peptide antibody, wherein an effective amount of the anti-acetylated peptide antibody recognizes with specificity only an acetylated form of the peptide substrate and does not recognize the peptide substrate in its unacetylated form,    (c) comparing the amount of the acetylated peptide substrate detected in step (b) with a control amount defined as an amount of an acetylated peptide substrate detected in an absence of the test compound, and    (d) selecting the compound associated with an increase or decrease in the amount of the acetylated peptide substrate as compared to the control amount.    
     
     
         24 . The method of  claim 23 , wherein the peptide substrate is p53.  
     
     
         25 . The method of  claim 23 , wherein the peptide substrate is labeled.  
     
     
         26 . The method of  claim 25 , wherein the label is biotin.  
     
     
         27 . The method of  claim 23 , wherein the peptide substrate is immobilized on a solid phase.  
     
     
         28 . The method of claims  23 , wherein the anti-acetylated peptide antibody is labeled.  
     
     
         29 . The method of  claim 23 , wherein the amount of acetylated peptide substrate is detected by ELISA.  
     
     
         30 . A compound isolatable by the screening method of  claim 23 .  
     
     
         31 . A method for screening a compound that inhibits the deacetylase activity or a compound that enhances the activity or expression of acetyltransferase to catalyze a reaction that transfers an acetyl group from one substrate to another, comprising the steps of: 
 (a) contacting a test compound with cultured cells,    (b) immobilizing said cultured cells,    (c) detecting an acetyl group on a specific protein in said cultured cells using an anti-acetylated peptide antibody, and,    (d) screening a compound that increases the acetyl group on said specific protein in comparison with a control untreated with the test compound.    
     
     
         32 . A compound isolatable by the screening method of  claim 31 .  
     
     
         33 . A method for screening a compound that inhibits the deacetylase activity of histone or a compound that enhances the activity or expression of acetyltransferase of histone, comprising the steps of: 
 (a) providing cultured cells carrying a vector comprising a promoter that functions within cultured cells and a reporter gene ligated to the downstream of said promoter,    (b) contacting a test compound with said cultured cells, and,    (c) screening a compound that increases the reporter activity, in comparison with a control untreated with the test compound.    
     
     
         34 . A compound isolatable by the screening method of  claim 33 .  
     
     
         35 . A kit comprising an anti-acetylated peptide antibody, wherein said anti-acetylated peptide antibody recognizes with specificity an epitope including an acetylated amino acid residue of an acetylated form of a peptide substrate and does not recognize the peptide substrate in its unacetylated form.  
     
     
         36 . The kit of  claim 35 , further comprising an acetylated peptide substrate.  
     
     
         37 . The kit of  claim 35 , further comprising an unacetylated peptide substrate.  
     
     
         38 . The kit of  claim 36 , further comprising deacetyltransferase.  
     
     
         39 . The kit of  claim 37 , further comprising acetyltransferase.