Nucleotide sequence encoding a modulator of NF-kappaB

Abstract

The present invention relates to nucleotide sequences encoding a modulator of NF-κB, and to the polypeptides encoded by the nucleotide sequences. In particular, the invention relates to nucleotide sequences and the polypeptides encoded thereby, wherein the polypeptides are involved in the response to NF-κB-activating stimuli, including HTLV-1 Tax, LPS, PMA and IL-1. The invention also relates to antibodies to the modulator of NF-κB, methods of detecting modulator of NF-κB using the antibodies, methods of treatment associated with NF-κB activation and to methods of identifying compounds which modulate the activity of the modulator of NF-κB.

What is claimed as new and is desired to be secured by letters patent of the United States is:  
     
         1 . A purified modulator of NF-κB, comprising a material selected from the group consisting of a protein, active fragments thereof, agonists thereof, mimics thereof, and combinations thereof, said modulator having the following characteristics: 
 a) an apparent molecular weight of approximately 48 kD;  
 b) a pI of approximately 5.66;  
 c) containing a leucine zipper motif; and  
 d) binding to a kinase involved in the activation of NF-κB.  
 
     
     
         2 . The purified modulator of NF-κB of  claim 1  which is a polypeptide comprising SEQ ID NO:1.  
     
     
         3 . The purified modulator of NF-κB of  claim 1  which is deficient in rat 5R cell line and in murine pre-B cell line 1.3E2.  
     
     
         4 . The purified modulator of NF-κB of  claim 1  labeled with a detectable label.  
     
     
         5 . The purified modulator of NF-κB of  claim 4  wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.  
     
     
         6 . An antibody to a purified modulator of NF-κB, the purified modulator of NF-κB to which said antibody is raised having the following characteristics: 
 a) an apparent molecular weight of approximately 48 kD;  
 b) a pI of approximately 5.66;  
 c) containing a leucine zipper motif; and  
 d) binding to a kinase involved in the activation of NF-κB.  
 
     
     
         7 . The antibody of  claim 6  which is a polyclonal antibody.  
     
     
         8 . The antibody of  claim 6  which is a monoclonal antibody.  
     
     
         9 . An immortal cell line that produces a monoclonal antibody according to  claim 8 .  
     
     
         10 . The antibody of  claim 6  labeled with a detectable label.  
     
     
         11 . The antibody of  claim 10  wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.  
     
     
         12 . A purified DNA sequence which encodes a purified modulator of NF-κB, or a fragment thereof which binds to a component of an IκB kinase, selected from the group consisting of: 
 (A) the DNA sequence SEQ ID NO:______;  
 (B) DNA sequences that hybridize to the DNA sequence of (A) under standard hybridization conditions; and  
 (C) DNA sequences that encode the protein of  claim 1 .  
 
     
     
         13 . A recombinant DNA molecule comprising the purified DNA sequence of  claim 12 .  
     
     
         14 . The recombinant DNA molecule of  claim 13 , wherein said DNA sequence is operatively linked to an expression control sequence.  
     
     
         15 . The recombinant DNA molecule of  claim 14 , wherein said expression control sequence is selected from the group consisting of the early or late promoters of CMV, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase and the promoters of the yeast α-mating factors.  
     
     
         16 . A probe capable of screening for a DNA sequence encoding a modulator of NF-κB in alternate species, wherein said probe is a fragment of the purified DNA sequence of  claim 12  which binds to the DNA sequence encoding a modulator of NF-κB in alternate species under standard hybridization conditions.  
     
     
         17 . A unicellular host transformed with the recombinant DNA molecule of  claim 14 .  
     
     
         18 . The unicellular host of  claim 17  wherein the unicellular host is selected from the group consisting of  E. coli , Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells in tissue culture.  
     
     
         19 . A method for producing the modulator of NF-κB of  claim 1  comprising: 
 (a) culturing the unicellular host of  claim 17;  and  
 (b) isolating the modulator of NF-κB from said cultured unicellular host.  
 
     
     
         20 . A method for detecting the presence or activity of the modulator of NF-κB of  claim 1 , comprising the steps of: 
 A. contacting a biological sample from a mammal in which the presence or activity of said modulator of NF-κB is suspected with a binding partner of said modulator of NF-κB under conditions that allow binding of said modulator of NF-κB to said binding partner to occur; and  
 B. detecting whether binding has occurred between said modulator of NF-κB from said sample and the binding partner; and  
 C. correlating the detection of binding to the presence or activity of said modulator of NF-κB in said sample.  
 
     
     
         21 . A method for detecting the presence and activity of an activator of NF-κB comprising detecting the presence or activity of a modulator of NF-κB according to the method of  claim 20 , wherein detection of the presence or activity of the modulator of NF-κB indicates the presence and activity of an activator of NF-κB.  
     
     
         22 . The method of  claim 21  wherein said activator of NF-κB is lipopolysaccharide (LPS), phorbol myristate acetate (PMA), interleukin-1 (IL-1) and HTLV-I Tax.  
     
     
         23 . A method for detecting binding of a molecule to the modulator of NF-κB of  claim 1  comprising: 
 A. contacting a labeled molecule, the binding of which to the modulator of NF-κB is to be detected, with the purified modulator of NF-κB of  claim 1;   
 B. detecting the label bound to the purified modulator of NF-κB of  claim 1;   
 C. correlating the presence of said labeled molecule to the ability of said molecule to bind to the purified modulator of  claim 1 .  
 
     
     
         24 . A method of testing the ability of a drug or other entity to modulate the activity of the modulator of NF-κB of  claim 1  which comprises; 
 A. adding the drug or other entity to cultured cells;  
 B. measuring the level of binding of NF-κB to DNA following stimulation of the cultured cells; and  
 C. correlating a decrease in the level of binding of NF-κB to DNA with inhibition of said modulator of NF-κB.  
 
     
     
         25 . A test kit for the demonstration of the modulator of NF-κB of  claim 1  in a eukaryotic cellular sample, comprising: 
 A. a predetermined amount of a detectably labeled specific binding partner of the modulator of NF-κB of  claim 1;   
 B. other reagents; and  
 C. directions for use of said kit.  
 
     
     
         26 . A test kit for demonstrating the presence of the modulator of NF-κB of  claim 1  in a eukaryotic cellular sample, comprising: 
 A. a predetermined amount of the modulator of NF-κB of  claim 1;   
 B. a predetermined amount of a specific binding partner of said modulator of NF-κB of  claim 1;   
 C. other reagents; and  
 D. directions for use of said kit;  
 wherein either said modulator of NF-κB or said specific binding partner are detectably labeled.  
 
     
     
         27 . The test kit of  claim 25  or  26  wherein said labeled immunochemically reactive component is selected from the group consisting of polyclonal antibodies to the modulator of NF-κB, monoclonal antibodies to the modulator of NF-κB, fragments thereof, and mixtures thereof.  
     
     
         28 . A method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, wherein said cellular debilitations, derangements and/or dysfunctions and/or other disease states are caused by activation of NF-κB, comprising administering to a mammal a therapeutically effective amount of a material selected from the group consisting of the modulator of NF-κB of  claim 1 , an agent capable of modulating the production and/or activity of said modulator of NF-κB, an agent capable of mimicking the activity of said modulator of NF-κB, an agent capable of modulating the production of said modulator of NF-κB, and mixtures thereof.  
     
     
         29 . The method of  claim 28  wherein said cellular debilitations, derangements and/or dysfunctions and/or other disease states include immune responses, stress responses, inflammation or apoptosis.  
     
     
         30 . A pharmaceutical composition for the treatment of cellular debilitation, derangement and/or dysfunction in mammals, comprising: 
 A. a therapeutically effective amount of a material selected from the group consisting of the modulator of NF-κB of  claim 1 , an agent capable of modulating the production and/or activity of said modulator of NF-κB, an agent capable of mimicking the activity of said modulator of NF-κB, an agent capable of modulating the production of said modulator of NF-κB, and mixtures thereof, or a specific binding partner thereto; and    B. a pharmaceutically acceptable carrier.    
     
     
         31 . A recombinant virus transformed with the DNA molecule of  claim 12 .  
     
     
         32 . A method of inhibiting NF-κB activity in a mammal in need of such treatment, comprising administering to said mammal an effective amount of the modulator of NF-κB of  claim 1 , wherein said modulator of NF-κB inhibits the phosphorylation of intracellular IκB proteins.  
     
     
         33 . A method of determining the ability of a pharmacological agent to enhance the activity of a modulator of NF-κB comprising: 
 administering the pharmacological agent to a first mammal;  
 comparing the activity of the modulator of NF-κB to a sample from a second mammal to which the pharmacological agent is not administered.  
 
     
     
         34 . An antisense nucleic acid against an mRNA encoding the modulator of NF-κof  claim 1  comprising a nucleic acid sequence hybridizing to said mRNA.  
     
     
         35 . The antisense nucleic acid of  claim 34  which is RNA.  
     
     
         36 . The antisense nucleic acid of  claim 35  which is DNA.  
     
     
         37 . The antisense nucleic acid of  claim 35  which binds to the initiation codon of said mRNA.  
     
     
         38 . A recombinant DNA molecule having a DNA sequence which, on transcription, produces an antisense ribonucleic acid against an mRNA encoding the modulator of NF-κB of  claim 1 , said antisense ribonucleic acid comprising an nucleic acid sequence capable of hybridizing to said mRNA.  
     
     
         39 . A cell line transfected with the recombinant DNA molecule of  claim 38 .  
     
     
         40 . A method for creating a cell line which exhibits reduced transcriptional activity, comprising transfecting an NF-κB-producing cell line with the recombinant DNA molecule of  claim 38 .  
     
     
         41 . A ribozyme that cleaves mRNA encoding the modulator of NF-κB of  claim 1 .  
     
     
         42 . The ribozyme of  claim 41  which is a Tetrahymena-type ribozyme.  
     
     
         43 . The ribozyme of  claim 41  which is a Hammerhead-type ribozyme.  
     
     
         44 . A recombinant DNA molecule having a DNA sequence which, upon transcription, produces the ribozyme of  claim 41 .  
     
     
         45 . An NF-κB-producing cell line transfected with the recombinant DNA molecule of  claim 44 .  
     
     
         46 . A method for creating a cell line which exhibits reduced transcriptional activity, comprising transfecting an NF-κB-producing cell line with the recombinant DNA molecule of  claim 44 .  
     
     
         47 . A compound which competes with a purified modulator of NF-κB, wherein said modulator of NF-κB comprises a material selected from the group consisting of a protein, active fragments thereof, agonists thereof, mimics thereof, and combinations thereof, said modulator having the following characteristics: 
 a) an apparent molecular weight of approximately 48 kD;  
 b) a pI of approximately 5.66;  
 c) containing a leucine zipper motif; and  
 d) binding to a kinase involved in the activation of NF-κselected by the process of  claim 20 .  
 
     
     
         48 . A compound which competes with a purified modulator of NF-κB, wherein said modulator of NF-κB comprises a material selected from the group consisting of a protein, active fragments thereof, agonists thereof, mimics thereof, and combinations thereof, said modulator having the following characteristics: 
 a) an apparent molecular weight of approximately 48 kD;  
 b) a pI of approximately 5.66;  
 c) containing a leucine zipper motif; and  
 d) binding to a kinase involved in the activation of NF-κB, selected by the process of  claim 21 .  
 
     
     
         49 . A compound which competes with a purified modulator of NF-κB, wherein said modulator of NF-κB comprises a material selected from the group consisting of a protein, active fragments thereof, agonists thereof, mimics thereof, and combinations thereof, said modulator having the following characteristics: 
 a) an apparent molecular weight of approximately 48 kD;  
 b) a pI of approximately 5.66;  
 c) containing a leucine zipper motif; and  
 d) binding to a kinase involved in the activation of NF-κB, selected by the process of  claim 23 .  
 
     
     
         50 . A compound which competes with a purified modulator of NF-κB, wherein said modulator of NF-κB comprises a material selected from the group consisting of a protein, active fragments thereof, agonists thereof, mimics thereof, and combinations thereof, said modulator having the following characteristics: 
 a) an apparent molecular weight of approximately 48 kD;  
 b) a pI of approximately 5.66;  
 c) containing a leucine zipper motif; and  
 d) binding to a kinase involved in the activation of NF-κB, selected by the process of  claim 24.