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, NACHT domains and ARED 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 a NACHT 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: 66, 68, 70, 72, 74, 76 or 84; or    (b) a nucleic acid molecule that hybridizes to the complement of 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 , encoding SEQ ID NO: 66, 68, 70, 72, 74, 76 or 84.  
     
     
         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: 118, 121, 124 or 127; 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.    
     
     
         6 . A vector containing the nucleic acid molecule of  claim 5 .  
     
     
         7 . A recombinant cell containing the nucleic acid molecule of  claim 5 .  
     
     
         8 . An isolated nucleic acid molecule encoding a NACHT domain, comprising: 
 (a) a nucleic acid molecule encoding the NACHT domain amino acid set forth as any of SEQ ID NOS:107, 110, 113, 116, 119, 122, 125, 128;    (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 NACHT domain.    
     
     
         9 . A vector containing the nucleic acid molecule of  claim 8 .  
     
     
         10 . A recombinant cell containing the nucleic acid molecule of  claim 8 .  
     
     
         11 . An oligonucleotide comprising at least 17 nucleotides capable of specifically hybridizing with the nucleotide sequence set forth in any of SEQ ID NOS:69, 71, 73 or 75 or the complement thereof.  
     
     
         12 . An oligonucleotide comprising at least 50 nucleotides capable of specifically hybridizing with the nucleotide sequence set forth in any of SEQ ID NOS:69, 71, 73 or 75 or the complement thereof.  
     
     
         13 . 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:66, 68, 70, 72, 74, 76 or 84, wherein said polypeptide is a biologically active PAAD domain-containing polypeptide.  
     
     
         14 . The PAAD domain-containing polypeptide of  claim 13 , wherein said polypeptide comprises the amino acid sequence set forth as any of SEQ ID NOS:66, 68, 70, 72, 74, 76 or 84.  
     
     
         15 . 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: 118, 121, 124 or 127, wherein said polypeptide is a biologically active PAAD domain polypeptide.  
     
     
         16 . An isolated PAAD domain polypeptide, comprising the amino acid sequence set forth as any of SEQ ID NOS: 118, 121, 124 or 127.  
     
     
         17 . An isolated NACHT domain polypeptide, comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth as any of SEQ ID NOS:107, 110, 113, 116, 119, 122, 125, 128, wherein said polypeptide is a biologically active NACHT domain polypeptide.  
     
     
         18 . An isolated NACHT domain polypeptide, comprising an amino acid sequence set forth as any of SEQ ID NOS:107, 110, 113, 116, 119, 122, 125, 128.  
     
     
         19 . An isolated peptide comprising at least 10 contiguous amino acids of any of SEQ ID NOS:70, 72, 74 or 76.  
     
     
         20 . 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.  
     
     
         21 . An isolated anti-PAAD antibody having specific reactivity with the PAAD domain-containing polypeptide of  claim 14 .  
     
     
         22 . The antibody of  claim 21 , wherein said antibody is a monoclonal antibody.  
     
     
         23 . A cell line producing the monoclonal antibody of  claim 22 .  
     
     
         24 . The antibody of  claim 21 , wherein said antibody is a polyclonal antibody.  
     
     
         25 . 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 11 , wherein said contacting is effected under high stringency hybridization conditions, and identifying a nucleic acid molecule which hybridizes thereto.    
     
     
         26 . 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 21 , detecting the presence of an antibody:polypeptide complex, and thereby detecting the presence of a PAAD domain-containing polypeptide in said test sample.  
     
     
         27 . 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 13  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.    
     
     
         28 . A method of identifying a PAP comprising the steps of: 
 (a) contacting the PAAD domain polypeptide of  claim 16  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.    
     
     
         29 . A method of identifying a PAP comprising the steps of: 
 (a) contacting the NACHT domain polypeptide of  claim 18  with a candidate PAP;    (b) detecting association of said NACHT polypeptide with said candidate PAP,    wherein a candidate PAP that associates with said polypeptide is identified as a PAP.    
     
     
         30 . 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 13 , or a PAAD, NACHT or ARED 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.    
     
     
         31 . The method of  claim 30 , wherein said PAP is selected from the group consisting of ASC, ASC2, Caspase-1, Card10, Nod1, NIK, IKK-i, IκBα, IKAP, IKKα and IKKβ.  
     
     
         32 . 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 13  with a candidate agent; and    (b) detecting association of said PAAD domain-containing polypeptide with said agent.    
     
     
         33 . 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 15  with a candidate agent; and    (b) detecting association of said PAAD domain polypeptide with said agent.    
     
     
         34 . A method of identifying an agent that associates with a NACHT domain polypeptide, comprising the steps of: 
 (a) contacting the NACHT domain polypeptide of  claim 16  with a candidate agent; and    (b) detecting association of said NACHT domain polypeptide with said agent.    
     
     
         35 . 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, 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:118, 121, 124 or 127; 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.  
 
     
     
         36 . The method of  claim 35 , wherein said PAAD domain polypeptide comprises an amino acid sequence set forth as any of SEQ ID NOS:118, 121, 124 or 127, wherein said polypeptide is a biologically active PAAD domain polypeptide.  
     
     
         37 . The method of  claim 35 , wherein said cell is contacted with or recombinantly expresses an inducer of NFκB activity.  
     
     
         38 . A method of identifying an agent that modulates an activity of a NACHT domain of a PAAD domain-containing polypeptide, comprising the steps of: 
 (a) contacting the NACHT domain polypeptide of  claim 18  with a candidate agent; and    (b) detecting an activity of said NACHT domain, whereby an increase or decrease of said activity identifies said agent as an agent that modulates the activity of the NACHT domain of said PAAD domain-containing polypeptide;    wherein the detected activity of said NACHT domain is selected from homo-oligomerization, hetero-oligomerization, nucleotide hydrolysis, and nucleotide binding.    
     
     
         39 . 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.  
 
     
     
         40 . 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:69, 71, 73 or 75.