Blk genes, gene products and uses thereof in apoptosis

Abstract

Genes and gene products of Blk, a pro-apoptotic protein in the Bcl-2 gene family, are provided. Effector molecules that either increase or decrease Blk and thus promote or inhibit apoptosis are described. The Blk genes and proteins and effector molecules may be used to treat diseases that have unwanted cell proliferation used to promote cell growth.

1 . An isolated nucleic acid molecule encoding mammalian Blk.  
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein mammalian Blk is human or murine Blk.  
     
     
         3 . The nucleic acid molecule of  claim 1 , wherein mammalian Blk has an amino acid sequence as presented in SEQ ID NO:2 or a variant thereof.  
     
     
         4 . The nucleic acid molecule of  claim 1 , wherein mammalian Blk is encoded by the nucleotide sequence presented in SEQ ID NO:1 or variant thereof.  
     
     
         5 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding a BH3 domain of a mammalian Blk protein.  
     
     
         6 . The nucleic acid molecule of  claim 5 , wherein the BH3 domain has the amino acid sequence Ala Leu Arg Leu Ala Cys Ile Gly Asp Glu Met Asp (SEQ ID NO:3).  
     
     
         7 . The nucleic acid molecule of  claim 6 , wherein the amino acid sequence has one or more amino acid substitutions, wherein the second Leu, the Gly, and the first Asp residues are not substituted.  
     
     
         8 . A nucleic acid vector comprising a nucleotide sequence encoding mammalian Blk.  
     
     
         9 . The vector of  claim 5 , wherein mammalian Blk has an amino acid sequence as presented in SEQ ID NO:2 or a variant thereof.  
     
     
         10 . The vector of  claim 5 , wherein the nucleotide sequence comprises the sequence presented in SEQ ID NO:1 or variant thereof.  
     
     
         11 . The vector of  claim 5 , wherein the nucleotide sequence encoding mammalian Blk is under control of a promoter.  
     
     
         12 . A nucleic acid vector comprising a nucleotide sequence encoding a BH3 domain of a mammalian Blk.  
     
     
         13 . The vector of  claim 12 , wherein the BH3 domain has the amino acid sequence Ala Leu Arg Leu Ala Cys Ile Gly Asp Glu Met Asp (SEQ ID NO:3).  
     
     
         14 . The vector of  claim 13 , wherein the amino acid sequence has one or more amino acid substitutions, wherein the second Leu, the Gly, and the first Asp residues are not substituted.  
     
     
         15 . A host cell containing a nucleic acid vector comprising a nucleotide sequence encoding mammalian Blk or a BH3 domain of a mammalian Blk.  
     
     
         16 . The host cell of  claim 15 , wherein the host cell is selected from the group consisting of a mammalian cell, a yeast cell and a bacterial cell.  
     
     
         17 . An isolated mammalian Blk protein.  
     
     
         18 . The protein of  claim 17 , wherein the protein comprises an amino acid sequence as presented in SEQ ID NO:2 or a variant thereof.  
     
     
         19 . An isolated BH3 domain of a mammalian Blk protein.  
     
     
         20 . The BH3 domain of  claim 19 , wherein the BH3 domain consists essentially of the sequence Ala Leu Arg Leu Ala Cys Ile Gly Asp Glu Met Asp (SEQ ID NO:3).  
     
     
         21 . The BH3 domain of  claim 20 , wherein the amino acid sequence has one or more amino acid substitutions, wherein the second Leu, the Gly, and the first Asp residues are not substituted.  
     
     
         22 . An antibody that selectively binds to a mammalian Blk protein.  
     
     
         23 . The antibody of  claim 22 , wherein the Blk protein is human or murine.  
     
     
         24 . The antibody of  claim 23 , wherein the Blk protein has the amino acid sequence presented in SEQ ID NO:2 or a variant thereof.  
     
     
         25 . The antibody of  claim 22 , wherein the antibody inhibits binding of Blk to either Bcl-2 or Bcl-xL.  
     
     
         26 . An antibody that selectively binds to a BH3 domain of a mammalian Blk protein.  
     
     
         27 . The antibody of  claim 26 , wherein the Blk protein is human or murine.  
     
     
         28 . The antibody of  claim 26 , wherein the Blk protein has the amino acid sequence Ala Leu Arg Leu Ala Cys lie Gly Asp Glu Met Asp (SEQ ID NO:3).  
     
     
         29 . The antibody of  claim 26 , wherein the antibody inhibits binding of Blk to either Bcl-2 or Bcl-xL.  
     
     
         30 . The antibody of either of claims  22  or  26 , wherein the antibody is an antibody fragment, a single chain antibody, or a humanized antibody.  
     
     
         31 . A pharmaceutical composition comprising mammalian Blk protein and a physiologically acceptable carrier.  
     
     
         32 . A pharmaceutical composition comprising a BH3 domain of a mammalian Blk protein and a physiologically acceptable carrier.  
     
     
         33 . A method for delivering a nucleic acid molecule encoding a mammalian Blk protein to a cell, comprising contacting the cell with a gene delivery vehicle comprising a nucleic acid molecule comprising a Blk nucleotide sequence under control of a promoter.  
     
     
         34 . The method of  claim 33 , wherein the Blk protein comprises an amino acid sequence as presented in SEQ ID NO:2 or a variant thereof.  
     
     
         35 . The method of  claim 33 , wherein the Blk protein comprises residues 52 to 63 as presented in SEQ ID NO:2 or a variant thereof, wherein the variant has one or more amino acid substitutions at a residue other than Leu55, Gly59, and Asp60.  
     
     
         36 . A method for treating unwanted cell proliferation, comprising administering to a patient an effector molecule that increases unbound Blk protein in a cell.  
     
     
         37 . The method of  claim 36 , wherein the effector molecule is a peptide that comprises residues 52 to 63 as presented in SEQ ID NO:2 or a variant thereof, wherein the variant has one or more amino acid substitutions at a residue other than Leu55, Gly59, and Asp60.  
     
     
         38 . A method for treating unwanted cell proliferation, comprising administering to a patient a gene delivery vehicle that encodes a Blk protein that comprises residues 52 to 63 as presented in SEQ ID NO:2 or a variant thereof, wherein the variant has one or more amino acid substitutions at a residue other than Leu55, Gly59, and Asp60.  
     
     
         39 . A method for screening for effector molecules that increase unbound Blk protein, comprising: 
 (a) contacting a candidate effector molecule with a solution comprising Blk and Bcl-2 or Bcl-xL; and    (b) detecting unbound Blk;    whereby an increase in the amount of unbound Blk when the candidate effector molecule is present compared to the amount of unbound Blk when no candidate effector molecule is present indicates that the effector molecule increases unbound Blk protein.    
     
     
         40 . A method for screening for effector molecules that increase unbound Blk protein in a cell, comprising: 
 (a) contacting a candidate effector molecule with a cell that expresses Blk and Bcl-2 or Bcl-xL; and    (b) detecting unbound Blk;    whereby an increase in the amount of unbound Blk when the candidate effector molecule is present compared to the amount of unbound Blk when no candidate effector molecule is present indicates that the effector molecule increases unbound Blk protein.    
     
     
         41 . The method of  claim 40 , wherein the Blk and Bcl-2 or Bcl-xL are expressed as fusion proteins with a DNA-binding protein and a transcriptional activator protein, and wherein the transcriptional activator controls expression of a reporter gene, and wherein the increased amount of unbound Blk is detected by a decrease in the amount of the reporter gene.  
     
     
         42 . The method of  claim 41 , wherein the cell is a yeast cell.  
     
     
         43 . The method of either  claim 38  or  40 , wherein the Blk is a fragment thereof that comprises amino acids 52 to 63 as presented in SEQ ID NO:2 or variant thereof, wherein the variant has one or more amino acid substitutions at a residue other than Leu55, Gly59, and Asp60.