Regulators of type-1 tumor necrosis factor receptor and other cytokine receptor shedding

Abstract

The present invention provides compositions and methods for the regulation of cytokine signaling through the Tumor Necrosis Factor (TNF) pathway. Specifically, the invention provides a novel gene, polypeptide and related compositions and methods for the regulation of ectodomain shedding. In preferred embodiments, methods and compositions for the regulation of TNF Type-1 Receptor ectodomain shedding are provided. The present invention finds use in therapeutics, diagnostics, and drug screening applications.

1 . An isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1.  
     
     
         2 . An isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:2.  
     
     
         3 . An isolated nucleic acid encoding the polypeptide set forth in SEQ ID NO:2.  
     
     
         4 . A recombinant vector comprising the nucleic acid of  claim 3 .  
     
     
         5 . A host cell comprising the recombinant vector of  claim 4 , wherein said host cell is selected from the group consisting of prokaryotic host cells and eukaryotic host cells.  
     
     
         6 . An antibody directed against at least a portion of said polypeptide of  claim 2 , wherein said antibody is selected from the group consisting of monoclonal antibodies and polyclonal antibodies.  
     
     
         7 . An isolated nucleic acid substantially homologous to the nucleic acid of  claim 1 , wherein said nucleic acid is capable of hybridizing under high stringency conditions to the nucleic acid of  claim 1 .  
     
     
         8 . The isolated nucleic acid of  claim 7 , wherein said nucleic acid encodes a polypeptide having the ability to regulate the shedding of the extracellular domain of at least one cytokine receptor.  
     
     
         9 . The isolated nucleic acid of  claim 8 , wherein said cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80.  
     
     
         10 . A method for the isolated of an amplifiable nucleic acid substantially homologous to the nucleic acid of  claim 1 , comprising: 
 a) providing: 
 i) a sample comprising template nucleic acid suspected of encoding a gene substantially homologous to the nucleic acid of  claim 1 , and  
 ii) at least two primers;  
   b) annealing said primers to said template nucleic acid;    c) extending said primers with reiterated DNA synthesis under conditions such that said template nucleic acid is amplified to produce an amplified product; and    d) visualizing and isolating said amplified product.    
     
     
         11 . The amplified product of  claim 10 .  
     
     
         12 . The amplified product of  claim 11 , wherein said amplified product encodes a polypeptide having the ability to regulate the shedding of the extracellular domain of at least one cytokine receptor.  
     
     
         13 . The amplified product of  claim 12 , wherein said cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80.  
     
     
         14 . A method for regulating the shedding of the extracellular domain of at least one cytokine receptor, comprising the steps of: 
 a) providing: 
 i) a recombinant vector comprising the isolated nucleic acid of  claim 3  in sense orientation,  
 ii) a first tissue containing one or more cells expressing a cytokine receptor, and  
 iii) a second tissue comprising one or more cells capable of expressing the polypeptide encoded by said recombinant vectors; and  
   b) delivering said vector to said cells of said second tissue in the presence of said first tissue, under conditions which result in regulation of shedding of said cytokine receptor from cells of said first tissue.    
     
     
         15 . The method of  claim 14 , wherein said cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80.  
     
     
         16 . The method of  claim 14 , wherein said delivering said vector to said second tissue comprises a means of intracellular delivery selected from the group consisting of direct nucleic acid administration, liposome administration, viral vector delivery, and ex vivo gene delivery followed by transplantation.  
     
     
         17 . A method for regulating the shedding of the extracellular domain of at least one cytokine receptor, comprising the steps of: 
 a) providing: 
 i) the recombinant vector comprising at least a transcribeable portion of the isolated nucleic acid of  claim 1  in an antisense orientation,  
 ii) a first tissue comprising one or more cells expressing a cytokine receptor, and  
 iii) a second tissue comprising one or more cells expressing the endogenous polypeptide of SEQ ID NO:2, and one or more cells capable of transcribing said antisense nucleic acid; and  
   b) delivering said vector to said second tissue in the presence of said first tissue, under conditions that result in regulation of shedding of said cytokine receptor from the cells of said first tissue.    
     
     
         18 . The method of  claim 17 , wherein said cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80.  
     
     
         19 . The method of  claim 17 , wherein said delivering said vector to said second tissue comprises a means of intracellular delivery selected from the group consisting of direct nucleic acid administration, liposome administration, viral vector delivery, and ex vivo gene delivery followed by transplantation.  
     
     
         20 . A method for regulating the shedding of the extracellular domain of at least one cytokine receptor, comprising the steps of: 
 a) providing: 
 i) the antibody of  claim 6 , and  
 ii) a tissue comprising one or more cells expressing a cytokine receptor and the endogenous polypeptide of SEQ ID NO:2; and  
   b) delivering said antibody to said tissue under conditions such that said antibody regulates the shedding of said cytokine receptor from the surface of said cells of said tissue.    
     
     
         21 . The method of  claim 20 , wherein said cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80.  
     
     
         22 . The method of  claim 20 , wherein said means of delivery is selected from the group consisting of oral administration, intra-arterial injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, local surgical administration, systemic surgical administration, catheter, and any combination of these means of delivery.  
     
     
         23 . A method for treating a subject, comprising the steps of: 
 a) providing: 
 i) a composition selected from the group consisting of a recombinant vector comprising an isolated nucleic acid encoding the polypeptide set forth in SEQ ID NO:2 in the sense orientation, a recombinant vector comprising at least a portion of an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 in the antisense orientation, an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:2, and an antibody directed to at least a portion of the polypeptide having the amino acid set forth in SEQ ID NO:2,  
 ii) a subject, and  
 iii) a means of delivery of said composition to at least one tissue of said subject; and  
   b) delivering said composition to said subject using said means of delivery.    
     
     
         24 . The method of  claim 23 , wherein said subject is selected from the group consisting of a subject displaying pathology resulting from abnormal cytokine activity, a subject suspected of displaying pathology resulting from abnormal cytokine activity, and a subject at risk of displaying pathology resulting from abnormal cytokine activity.  
     
     
         25 . The method of  claim 24 , wherein said cytokine activity is mediated by a cytokine selected from the group consisting of tumor necrosis factor a, interleukin-1 alpha, interleukin-1 beta, and interleukin-6.  
     
     
         26 . The method of  claim 23 , wherein said subject is a human.  
     
     
         27 . The method of  claim 23 , wherein said means of delivery is selected from the group consisting of oral administration, intra-arterial injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, local surgical administration, systemic surgical administration, catheter, and any combination of these means of delivery.  
     
     
         28 . The method of  claim 27 , wherein said means of delivery is further selected from the group consisting of direct nucleic acid administration, liposome administration, viral vector delivery, and ex vivo gene delivery followed by transplantation.  
     
     
         29 . A means for detecting an ARTS-1 mRNA in a sample, wherein said means comprises at least a portion of the nucleic acid of  claim 1  complementary to at least a portion of said ARTS-1 mRNA, and further wherein said nucleic acid is a probe.  
     
     
         30 . The means of  claim 29 , wherein said means comprises Northern blotting.  
     
     
         31 . The means of  claim 29 , wherein said sample is a tissue sample from a subject.  
     
     
         32 . A means for detecting an ARTS-1 polypeptide in a sample, wherein said means comprises the antibody of  claim 6 .  
     
     
         33 . The means of  claim 32 , wherein said means comprises Western immunoblotting.  
     
     
         34 . The means of  claim 32 , wherein said means comprises an enzyme-linked immunosorbent assay.  
     
     
         35 . The means of  claim 32 , wherein said sample is a tissue sample from a subject.  
     
     
         36 . A diagnostic kit comprising a means to measure ARTS-1 expression, wherein said means is selected from the group consisting of a probe comprising at least a portion of an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 complementary to at least a portion of ARTS-1 mRNA and a monoclonal or polyclonal antibody directed to at least a portion of an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:2.  
     
     
         37 . A method for drug screening to identify drugs having the ability regulate ARTS-1 expression comprising the steps of: 
 a) providing: 
 i) a drug,  
 ii) cultured cells, and  
 iii) a means to measure ARTS-1 expression, wherein said means is selected from the group consisting of a probe comprising at least a portion of an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 complementary to at least a portion of ARTS-1 mRNA and a monoclonal or polyclonal antibody directed to at least a portion of an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:2;  
   b) exposing said cells to said drug; and    c) using said means to measure ARTS-1 expression.    
     
     
         38 . The method of  claim 37 , wherein said cultured cells are human NCI-H292 pulmonary mucoepidermoid carcinoma cells.  
     
     
         39 . A method for drug screening to identify drugs capable of regulating the peptidase activity of ARTS-1, comprising the steps of: 
 a) providing: 
 i) purified ARTS-1 polypeptide,  
 ii) an amino acid p-nitroaniline,  
 iii) a means to measure amino acid p-nitroaniline cleavage, and  
 iv) a drug;  
 b) exposing said purified ARTS-1 polypeptide to said amino acid p-nitroaniline in the absence and presence of said drug; and  
 c) measuring amino acid p-nitroaniline cleavage in the absence and presence of said drug.  
   
     
     
         40 . The method of  claim 39 , wherein said purified ARTS-1 polypeptide comprises glutathione-S-transferase.  
     
     
         41 . The method of  claim 39 , wherein said amino acid p-nitroaniline is selected from the group consisting of isoleucine p-nitroanilide, phenylalanine p-nitroanilide and glycine p-nitroanilide.  
     
     
         42 . The method of  claim 39 , wherein said means to measure amino acid p-nitroaniline cleavage comprises measuring absorbance at 380 nm.  
     
     
         43 . A method for drug screening to identify drugs capable of regulating the shedding of a cytokine receptor, comprising the steps of: 
 a) providing: 
 i) cultured cells expressing a cytokine receptor,  
 ii) a means to quantitate the concentration of the soluble form of said cytokine receptor in the supernatants of said cultured cells, and  
 iii) a drug;  
   b) culturing said cells in the absence and presence of said drug;    c) quantitating the concentration of said soluble form of said cytokine receptor in said supernatants of said cultured cells using said means; and    d) comparing the concentrations of said soluble cytokine receptor in said supernatants of said cell cultures in the absence and presence of drug.    
     
     
         44 . The method of  claim 43 , wherein said cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80.  
     
     
         45 . The method of  claim 43 , wherein said means to quantitate the concentration of the soluble form of a cytokine receptor is an enzyme-linked immunosorbent assay.  
     
     
         46 . The method of  claim 43 , wherein said cultured cells are cultured human NCI-H292 pulmonary mucoepidermoid carcinoma cells.