Extension of a protein-protein interaction surface to inactivate the function of a cellular protein

Abstract

Acidic amino acid extensions to multimeric proteins, particularly nucleic acid (e.g., DNA or RNA) binding proteins, provide novel acidically modified proteins which can inhibit the function of cellular proteins, thereby regulating and controlling cell growth. The acidically modified nucleic acid binding proteins are engineered to contain a plurality of acidic amino acids appended to the proteins, generally as extensions of the multimerization or dimerization domain at the amino terminus, and can replace the basic region DNA binding domain of a DNA binding protein. The acidically extended nucleic acid binding proteins act as potent dominant negatives which were demonstrated to inhibit the activation of endogenous transactivators, such as AP1. The invention provides novel methods to create such acidically modified DNA binding proteins which can specifically and stably heterodimerize with cellular regulatory proteins and control cell growth. Suitable nucleic acid binding proteins for acidic extensions include members of transcription regulatory protein families, e.g., bZIP and HLH proteins, having characteristic leucine zipper motifs and helix-loop-helix motifs, respectively. The amino terminal extensions of the basic regions of acidically modified nucleic acid binding proteins are comprised of a sequence of amino acid residues, all or some of which are acidic in nature, and produce robust dominant negatives to the native counterpart proteins in the cell. The acidic amino terminal extension affords a unique protein-protein interaction surface and allows stable multimerization or dimerization between a native protein and the acidically extended protein, thereby controlling, via inhibition or inactivation, the functions of cellular protein products of diverse species, including plants, animals, microorganisms, and viruses.

What is claimed is:  
     
         1 . An isolated and purified nucleic acid binding protein having appended amino-terminally thereto an extension of amino acid residues comprising a plurality of acidic amino acid residues.  
     
     
         2 . The protein according to  claim 1 , wherein said protein is a DNA binding protein.  
     
     
         3 . The protein according to  claim 1 , wherein said protein is an RNA binding protein.  
     
     
         4 . The protein according to  claim 1 , wherein said acidic amino acid residues dimerize with the basic region of a cellular DNA binding protein to inhibit the binding of said protein to DNA.  
     
     
         5 . The protein according to  claim 1 , wherein said acidic amino acid residues dimerize with the basic region of a cellular RNA binding protein to inhibit the binding of said protein to RNA.  
     
     
         6 . The protein according to  claim 4 , said protein being a dominant negative to a naturally occurring cellular protein.  
     
     
         7 . The protein according to  claim 2 , wherein said protein is a bZIP protein.  
     
     
         8 . The protein according to  claim 7 , wherein said bZIP protein is selected from the group consisting of Fos, Jun, GCN4, VBP, GBF, opaque, CREB, C/EBP, PAR, ATF2 and plant G-box protein.  
     
     
         9 . The protein according to  claim 2 , wherein said protein is a bHLH protein.  
     
     
         10 . The protein according to  claim 8 , wherein said bHLH protein is selected from the group consisting of Myc, Max, and Mad.  
     
     
         11 . The protein according to claims  1 ,  2 , or  3 , wherein the acidic amino acid residues are glutamic acid or aspartic acid.  
     
     
         12 . The protein according to  claim 1 ,  2 , or  3 , wherein said acidic extension comprises from two to one-hundred amino acid residues.  
     
     
         13 . The protein according to  claim 12 , wherein said acidic extension comprises from three to fifty amino acid residues.  
     
     
         14 . The protein according to  claim 13 , wherein said acidic extension comprises from four to thirty amino acid residues.  
     
     
         15 . The protein according to  claim 14 , wherein said acidic extension comprises twenty-eight amino acid residues.  
     
     
         16 . An isolated DNA molecule consisting essentially of the sequence as shown in SEQ ID NOS:1-52.  
     
     
         17 . An isolated DNA molecule encoding a nucleic acid binding protein having appended N-terminally thereto a plurality of acidic amino acid residues.  
     
     
         18 . A plasmid vector construct comprising the DNA molecule according to  claim 16  or  claim 17 , a promoter, a transcription initiation site, a transcription termination site, an origin of replication site, and a polyadenylation site, for expression in eukaryotic cells.  
     
     
         19 . The vector according to  claim 18 , wherein said eukaryotic cells are selected from the group consisting of plant cells, yeast cells, and mammalian cells.  
     
     
         20 . A plasmid vector construct comprising the DNA molecule according to  claim 16  or  claim 17 , a promoter, a transcription initiation site and a transcription termination site, for expression in prokaryotic cells.  
     
     
         21 . The construct according to  claim 18 , wherein said promoter is tissue specific.  
     
     
         22 . The DNA molecule according to  claim 16  or  17 , wherein said nucleic acid binding protein is a DNA binding protein.  
     
     
         23 . A method for producing a dominant negative nucleic acid binding protein for inhibiting cell growth and proliferation, comprising: 
 (a) preparing a sequence of amino acids, wherein at least one amino acid of the sequence is acidic to produce an acidic amino acid extension; and    (b) appending said acidic extension to the N-terminus of a multimerization or a dimerization domain of said nucleic acid binding protein to create said dominant negative protein.    
     
     
         24 . The method according to  claim 23 , wherein said dominant negative protein is a DNA binding protein.  
     
     
         25 . The method according to  claim 23 , wherein said acidic extension comprises from two to one-hundred amino acids.  
     
     
         26 . The method according to  claim 25 , wherein said acidic extension comprises from three to fifty amino acids.  
     
     
         27 . The method according to  claim 26 , wherein said acidic extension comprises from four to thirty amino acids.  
     
     
         28 . A method of controlling cell growth by inhibiting the function of a naturally occurring cellular protein, comprising: 
 (a) introducing into a cell the construct according to  claim 18  under conditions allowing for the expression of said acidically extended nucleic acid binding protein;    (b) inhibiting the binding of a cognate naturally occurring cellular nucleic acid binding protein to its target nucleic acid sequence by multimeric or dimeric complexation between said expressed acidically extended nucleic acid binding protein and said naturally occurring cellular protein.