Intracellular modulators of apoptopic cell death pathways

Abstract

A DNA sequence encoding a glucocorticoid-induced leucine-zipper family related protein (GILR), isoforms, fragments or analogs thereof, said GILR, isoforms, fragments or analogs thereof capable of inhibiting apoptosis and stimulating lymphocyte activity, GILR proteins, isoforms, analogs, fragments and derivatives thereof encoded by the aforesaid DNA sequence, their preparation and uses.

1 . A DNA sequence encoding a glucocorticoid-induced leucine-zipper family related (GILR) protein capable of inhibiting apoptosis and stimulating lymphocyte activity, which is selected from the group consisting of: 
 (a) a cDNA sequence encoding a native GILR protein;    (b) a DNA sequence which hybridizes to a sequence of (a) under moderately stringent conditions and which encodes a biologically active GILR protein; and    (c) a DNA sequence which is degenerate as a result of the genetic code to the DNA sequence defined in (b) and which encode a biologically active GILR protein.    
     
     
         2 . The DNA sequence of  claim 1 , wherein said GILR protein: 
 (a) is encoded by the nucleotide sequence of SEQ ID NO: 1; or    (b) contains no more than five amino acid changes from the amino acid sequence of SEQ ID NO:2, each of said changes being either alternative conservative substitutions within one of the following five groups of amino acid residues:    (1) Ala, Ser, Thr, Pro, Gly;    (2) Asp, Asn, Glu, Gln;    (3) His, Arg, Lys;    (4) Met, Leu, Ile, Val, Cys; and    (5) Phe, Tyr, Trp    or deletion of one or more of residues 123, 124, and 125 of SEQ ID NO:2.    
     
     
         3 . The DNA sequence of  claim 2 , wherein said GILR protein is encoded by the nucleotide sequence of SEQ ID NO: 1.  
     
     
         4 . The DNA sequence of  claim 2 , wherein said GILR protein contains no more than five amino acid changes from the amino acid sequence of SEQ ID NO:2, each of said changes being either alternative conservative substitutions within one of the following five groups of amino acid residues: 
 (1) Ala, Ser, Thr, Pro, Gly;    (2) Asp, Asn, Glu, Gln;    (3) His, Arg, Lys;    (4) Met, Leu, Ile, Val, Cys; and    (5) Phe, Tyr, Trp    or deletion of one or more of residues 123, 124, and 125 of SEQ ID NO:2.    
     
     
         5 . The DNA sequence of  claim 4 , wherein, in said GILR protein, said no more than five amino acid changes from the amino acid sequence of SEQ ID NO:2 are present at amino acid residue positions selected from the group consisting of residue positions 22, 50, 75, 84, 112, 122, 123, 124, 125, 127, and 128 of SEQ ID NO:2.  
     
     
         6 . The DNA sequence of  claim 4 , wherein said GILR protein contains no more than three amino acid changes from the amino acid sequence of SEQ ID NO:2.  
     
     
         7 . The DNA sequence of  claim 6 , wherein, in said GILR protein, said no more than three amino acid changes from the amino acid sequence of SEQ ID NO:2 are present at amino acid residue positions selected from the group consisting of residue positions 22, 50, 75, 84, 112, 122, 123, 124, 125, 127, and 128 of SEQ ID NO:2.  
     
     
         8 . The DNA sequence of  claim 4 , wherein said GILR protein contains a single amino acid change from the amino acid sequence of SEQ ID NO:2.  
     
     
         9 . The DNA sequence of  claim 8 , wherein, in said GILR protein, said single amino acid change from the amino acid sequence of SEQ ID NO:2 is present at an amino acid position selected from the group consisting of residue positions 22, 50, 75, 84, 112, 122, 123, 124, 125, 127, and 128 of SEQ ID NO:2.  
     
     
         10 . The DNA sequence of  claim 1 , wherein said GILR protein contains no more than ten amino acid changes from the amino acid sequence of SEQ ID NO:2, each of said changes being either alternative conservative substitutions of amino acid residue 122, 127, and 128 of SEQ ID NO:2 within one of the following five groups of amino acid residues: 
 (1) Ala, Ser, Thr, Pro, Gly;    (2) Asp, Asn, Glu, Gln;    (3) His, Arg, Lys;    (4) Met, Leu, Ile, Val, Cys; and    (5) Phe, Tyr, Trp,    or deletion of one or more of residues 123, 124, and 125 of SEQ ID NO:2.    
     
     
         11 . The DNA sequence of  claim 10 , wherein, in said GILR protein, said alternative conservative substitutions of amino acid residue positions are selected from the group consisting of residue positions 50, 75, 84, 122, and 128.  
     
     
         12 . A vector comprising the DNA sequence of  claim 1 .  
     
     
         13 . The vector of  claim 12 , which is an expression vector capable of being expressed in a eukaryotic cell.  
     
     
         14 . The vector of  claim 12 , which is an expression vector capable of being expressed in a prokaryotic cell.  
     
     
         15 . A host cell transformed with the vector of  claim 12 .  
     
     
         16 . A process for the preparation of a GILR protein, comprising: 
 growing the transformed host cell of  claim 15  under conditions suitable for the expression of said protein;    effecting post-translational modifications as necessary for obtaining of said protein, or a fragment, analog or derivative thereof; and    isolating said expressed protein or fragment, analog or derivative thereof.    
     
     
         17 . A pharmaceutical composition for enhancing apoptosis in cells by inhibiting GILR activity in said cells, comprising as active ingredient, an anti-sense oligonucleotide sequence which is complementary to and is capable of blocking expression from GILR mRNA transcribed from the DNA sequence of  claim 3 .  
     
     
         18 . A method for enhancing apoptosis in cells by inhibiting the activity of GILR proteins in said cells, comprising treating said cells with an antisense oligonucleotide sequence which is complementary to and which is capable of blocking expression from GLR mRNA transcribed from the DNA sequence of  claim 3 .  
     
     
         19 . A method for enhancing apoptosis in cells by inhibiting the activity of GILR proteins in said cells, comprising applying the ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding a GILR protein and transcribed from the DNA sequence of  claim 3 , is introduced into said cells in a form that permits expression of said ribozyme sequence in said cells, and wherein when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA.  
     
     
         20 . A method for inhibiting apoptosis in cells mediated by the Fas/Fas L system, CD3/TCR system or other intracellular mediators of apoptosis, comprising: treating said cells by introducing into said cells the vector comprising the DNA sequence of  claim 3 , wherein said vector is capable of effecting the insertion of said DNA sequence into said cells in a way that said sequence is expressed.  
     
     
         21 . The method of  claim 20 , wherein said cells are transfected with a recombinant animal virus vector comprising the steps of: 
 (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cells to be treated and a second sequence encoding the GILR protein of SEQ ID NO:2, that when expressed in said cells is capable of inhibiting apoptosis; and    (b) infecting said cells with said vector of (a).    
     
     
         22 . A method for treating tumor cells, HIV-infected cells or other diseased cells to enhance apoptosis in said cells by inhibiting the activity of GILR proteins comprising: 
 (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding an inactive GILR mutant protein, said mutant protein, when expressed in said tumor, HIV-infected, or other diseased cell is capable of inhibiting the activity of normal endogenous GILR and enhancing apoptosis in said cells; and    (b) infecting said tumor or HIV-infected cells or other diseased cells with said vector of (a).    
     
     
         23 . A pharmaceutical composition for enhancing apoptosis in cells by inhibiting GILR activity in said cells, comprising, as active ingredient, an inactive mutant GILR protein or DNA sequence encoding said inactive mutant GILR protein, which GILR mutant, when introduced into or expressed in said cells, inhibits the activity of the normal endogenous GILR protein.