PAAD domain-containing polypeptides, encoding nucleic acids, and methods of use

Abstract

The invention provides isolated nucleic acid molecules encoding PAAD-domain containing polypeptides and functional fragments thereof, including fragments containing PAAD domains, NB-ARC domains and LRR domains, encoded polypeptides, and antibodies. Also provided are methods of identifying polypeptides and agents that associate with a PAAD-domain containing polypeptide or fragment thereof, or that alter an association of a PAAD domain-containing polypeptides. Further provided are methods of identifying agents that modulate PAAD domain-mediated inhibition of NFκB activity, or modulate an activity of an NB-ARC domain of a PAAD domain-containing polypeptide. Also provided are methods of modulating NFκB transcriptional activity in a cell, and methods of altering expression of a PAAD domain-containing polypeptide in a cell.

We claim:  
     
         1 . An isolated nucleic acid molecule encoding a PAAD-containing polypeptide, comprising: 
 (a) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence set forth as SEQ ID NOs: 16, 18, 20, 22, 24, 26 or 28; or    (b) a nucleic acid molecule that hybridizes to the nucleic acid molecule of (a) under highly stringent conditions, where the nucleic acid molecule encodes a biologically active PAAD domain-containing polypeptide.    
     
     
         2 . The nucleic acid molecule of  claim 1 , comprising a nucleotide sequence set forth as any of SEQ ID NO: 15, 17, 19, 21, 23, 25 and 27.  
     
     
         3 . A vector containing the nucleic acid molecule of  claim 1 .  
     
     
         4 . A recombinant cell containing the nucleic acid molecule of  claim 1 .  
     
     
         5 . An isolated nucleic acid molecule encoding a PAAD domain, comprising: 
 (a) a nucleic acid molecule encoding a PAAD domain amino acid sequence set forth as any of SEQ ID NOS: 2, 3, 4, 5, 6, 8, or 10; or    (b) a nucleic acid molecule that hybridizes to the nucleic acid molecule of (a) under highly stringent conditions, where the nucleic acid molecule encodes a biologically active PAAD domain.    
     
     
         7 . A vector containing the nucleic acid molecule of claim  6 .  
     
     
         8 . A recombinant cell containing the nucleic acid molecule of claim  6 .  
     
     
         9 . An isolated nucleic acid molecule encoding an NB-ARC domain, comprising: 
 (a) a nucleic acid molecule encoding the NB-ARC domain amino acid set forth as any of SEQ ID NOS:37, 60, 62 or 63;    (b) a nucleic acid molecule that hybridizes to the nucleic acid molecule of (a) under highly stringent conditions, where the nucleic acid molecule encodes a biologically active NB-ARC domain.    
     
     
         10 . A vector containing the nucleic acid molecule of  claim 9 .  
     
     
         11 . A recombinant cell containing the nucleic acid molecule of  claim 9 .  
     
     
         12 . An isolated nucleic acid molecule encoding an LRR domain, comprising: 
 (a) a nucleic acid molecule encoding the LRR domain amino acid set forth as any of SEQ ID NOS:39, 61 or 64; or    (b) a nucleic acid molecule that hybridizes to the nucleic acid molecule of (a) under highly stringent conditions, where the nucleic acid molecule encodes a biologically active LRR domain.    
     
     
         13 . A vector containing the nucleic acid molecule of  claim 12 .  
     
     
         14 . A recombinant cell containing the nucleic acid molecule of  claim 12 .  
     
     
         15 . An oligonucleotide comprising at least 17 nucleotides capable of specifically hybridizing with the nucleotide sequence set forth in any of SEQ ID NOs:15, 17, 19, 21, 23, 25 and 27 or the complement thereof.  
     
     
         16 . An oligonucleotide comprising at least 50 nucleotides capable of specifically hybridizing with the nucleotide sequence set forth in any of SEQ ID NOs:15, 17, 19, 21, 23, 25 and 27 or the complement thereof.  
     
     
         17 . An isolated PAAD domain-containing polypeptide, comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in any of SEQ ID NOS:16, 18, 20, 22, 24, 26 or 28, wherein said polypeptide is a biologically active PAAD domain-containing polypeptide.  
     
     
         18 . The PAAD domain-containing polypeptide of  claim 17 , wherein said polypeptide comprises the amino acid sequence set forth as any of SEQ ID NOS:16, 18, 20, 22, 24, 26 or 28.  
     
     
         19 . An isolated PAAD domain polypeptide, comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth as any of SEQ ID NOS: 2, 3, 4, 5, 6, 8, or 10, wherein said polypeptide is a biologically active PAAD domain polypeptide.  
     
     
         20 . An isolated PAAD domain polypeptide, comprising the amino acid sequence set forth as any of SEQ ID NOS: 2, 3, 4, 5, 6, 8, or 10.  
     
     
         21 . An isolated NB-ARC domain polypeptide, comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth as any of SEQ ID NOS:37, 60, 62 or 63, wherein said polypeptide is a biologically active NB-ARC domain polypeptide.  
     
     
         22 . An isolated NB-ARC domain polypeptide, comprising an amino acid sequence set forth as any of SEQ ID NOS:37, 60, 62 or 63.  
     
     
         23 . An isolated LRR domain polypeptide, comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth as any of SEQ ID NOS:39, 61 or 64, wherein said polypeptide is a biologically active LRR domain polypeptide.  
     
     
         24 . An isolated LRR domain polypeptide, comprising an amino acid sequence set forth as any of SEQ ID NOS:39, 61 or 64.  
     
     
         25 . An isolated peptide comprising at least 10 contiguous amino acids of any of SEQ ID NOS:16, 18, 20, 22, 24, 26 or 28.  
     
     
         26 . A method of producing a PAAD domain-containing polypeptide comprising expressing the nucleic acid molecule of  claim 1  in vitro or in a cell under conditions suitable for expression of said polypeptide.  
     
     
         27 . An isolated anti-PAAD antibody having specific reactivity with the PAAD domain-containing polypeptide of  claim 18 .  
     
     
         28 . The antibody of  claim 27 , wherein said antibody is a monoclonal antibody.  
     
     
         29 . A cell line producing the monoclonal antibody of  claim 29 .  
     
     
         30 . The antibody of  claim 27 , wherein said antibody is a polyclonal antibody.  
     
     
         31 . A method of identifying a nucleic acid molecule encoding a PAAD-containing polypeptide in a sample, said method comprising: 
 contacting a sample containing nucleic acids with an oligonucleotide according to  claim 15 , wherein said contacting is effected under high stringency hybridization conditions, and identifying a nucleic acid molecule which hybridizes thereto.    
     
     
         32 . A method of detecting the presence of a PAAD domain-containing polypeptide in a sample, said method comprising contacting a test sample with an antibody according to  claim 27 , detecting the presence of an antibody:polypeptide complex, and thereby detecting the presence of a PAAD domain-containing polypeptide in said test sample.  
     
     
         33 . A method of identifying a PAAD domain-containing polypeptide-associated polypeptide (PAP) comprising the steps of: 
 (a) contacting the PAAD domain-containing polypeptide of  claim 17  with a candidate PAP;    (b) detecting association of said PAAD domain-containing polypeptide with said candidate PAP, 
 wherein a candidate PAP that associates with said polypeptide is identified as a PAP.  
   
     
     
         34 . A method of identifying a PAP comprising the steps of: 
 (a) contacting the PAAD domain polypeptide of  claim 19  with a candidate PAP;    (b) detecting association of said PAAD domain polypeptide with said candidate PAP, 
 wherein a candidate PAP that associates with said polypeptide is identified as a PAP.  
   
     
     
         35 . A method of identifying a PAP comprising the steps of: 
 (a) contacting the NB-ARC domain polypeptide of  claim 21  with a candidate PAP;    (b) detecting association of said NB-ARC polypeptide with said candidate PAP, 
 wherein a candidate PAP that associates with said polypeptide is identified as a PAP.  
   
     
     
         36 . A method of identifying a PAP comprising the steps of: 
 (a) contacting the LRR domain polypeptide of  claim 23  with a candidate PAP;    (b) detecting association of said LRR polypeptide with said candidate PAP, 
 wherein a candidate PAP that associates with said polypeptide is identified as a PAP.  
   
     
     
         37 . A method of identifying an effective agent that alters the association of a PAAD domain-containing polypeptide with a PAAD domain-containing polypeptide-associated polypeptide (PAP), comprising the steps of: 
 (a) contacting the PAAD domain-containing polypeptide of  claim 17 , or a PAAD, NB-ARC or LRR domain therefrom, and said PAP under conditions that allow said PAAD domain-containing polypeptide or said fragment and said PAP to associate, with a candidate agent; and    (b) detecting the altered association of said PAAD domain-containing polypeptide or domain with said PAP, 
 wherein an agent that alters said association is identified as an effective agent.  
   
     
     
         38 . The method of  claim 37 , wherein said PAP is selected from the group consisting of ASC, ASC2, Caspase-1, Card10, Nod1, NIK, IKK-i, IκBα and IKAP.  
     
     
         39 . A method of identifying an agent that associates with a PAAD domain-containing polypeptide, comprising the steps of: 
 (a) contacting the PAAD domain-containing polypeptide of  claim 17  with a candidate agent; and    (b) detecting association of said PAAD domain-containing polypeptide with said agent.    
     
     
         40 . A method of identifying an agent that associates with a PAAD domain polypeptide, comprising the steps of: 
 (a) contacting the PAAD domain polypeptide of  claim 19  with a candidate agent; and    (b) detecting association of said PAAD domain polypeptide with said agent.    
     
     
         41 . A method of identifying an agent that associates with a NB-ARC domain polypeptide, comprising the steps of: 
 (a) contacting the NB-ARC domain polypeptide of  claim 21  with a candidate agent; and    (b) detecting association of said NB-ARC domain polypeptide with said agent.    
     
     
         42 . A method of identifying an agent that associates with a LRR domain polypeptide, comprising the steps of: 
 (a) contacting the LRR domain polypeptide of  claim 23  with a candidate agent; and    (b) detecting association of said LRR domain polypeptide with said agent.    
     
     
         43 . A method of identifying an agent that modulates PAAD domain-mediated inhibition of NFκB activity, comprising the steps of: 
 (a) contacting a cell that recombinantly expresses a PAAD domain-containing polypeptide with a candidate agent; and  
 (b) detecting NFκB activity in said cell, whereas increased or decreased NFκB activity in said cell compared to a control cell indicates that said candidate agent is an agent that modulates PAAD domain-mediated inhibition of NFκB activity.  
 
     
     
         44 . The method of  claim 43 , wherein said PAAD domain polypeptide comprises an amino acid sequence set forth as any of SEQ ID NOS:1-14.  
     
     
         45 . The method of  claim 43 , wherein said PAAD domain polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth as any of SEQ ID NOS: 2, 3, 4, 5, 6, 8, or 10, wherein said polypeptide is a biologically active PAAD domain polypeptide.  
     
     
         46 . The method of  claim 43 , wherein said cell is contacted with or recombinantly expresses an inducer of NFκB activity.  
     
     
         47 . A method of identifying an agent that modulates an activity of a NB-ARC domain of a PAAD domain-containing polypeptide, comprising the steps of: 
 (a) contacting the NB-ARC domain polypeptide of  claim 21  with a candidate agent; and    (b) detecting an activity of said NB-ARC domain, whereby an increase or decrease of said activity identifies said agent as an agent that modulates the activity of the NB-ARC domain of said PAAD domain-containing polypeptide; 
 wherein the detected activity of said NB-ARC domain is selected from homo-oligomerization, hetero-oligomerization, nucleotide hydrolysis, and nucleotide binding.  
   
     
     
         48 . A method of modulating NFκB transcriptional activity in a cell, comprising the steps of: 
 (a) introducing the nucleic acid molecule of  claim 5  into a cell; and  
 (b) expressing said nucleic acid molecule in said cell, whereby the expression of said nucleic acid modulates NFκB transcriptional activity in said cell.  
 
     
     
         49 . A method of decreasing expression of a PAAD domain-containing polypeptide in a cell, comprising introducing an antisense or dsRNA nucleic molecule into a cell, wherein said antisense or dsRNA nucleic molecule binds to any of SEQ ID NOS:15, 17, 19, 21, 23, 25 and 27.