Novel molecules of the card-related protein family and uses thereof

Abstract

Novel CARD-12 polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated CARD-12 proteins, the invention further provides CARD-12, fusion proteins, antigenic peptides and anti-CARD-12 antibodies. The invention also provides CARD-12 nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a CARD-12 gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

1 . An isolated polypeptide selected from the group consisting of: 
 a) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2;    b) a polypeptide comprising an amino acid sequence at least 90% identical to the CARD domain (amino acid residues 1-88 of SEQ ID NO:2) of CARD-12;    c) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 161-323 of SEQ ID NO:2 of CARD-12;    d) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 762-965 of SEQ ID NO:2 of CARD-12;    e) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 169-456 of SEQ ID NO:2) of CARD-12;    f) a polypeptide with a length of at least 25 amino acids, comprising at least one NACHT NTPase domain (amino acid residues 169-186, 196-220, 229-253, 261-282, 330-351, 414-430, or 438-457 of SEQ ID NO:2) of CARD-12;    g) a polypeptide with a length of at least 50 amino acids, comprising at least one leucine-rich repeat domain (amino acid residues 656-686, 687-708, 711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930, 936-962, 965-993, or 994-1021 of SEQ ID NO:2) of CARD-12; and    h) a polypeptide with a length of at least 25 amino acids, comprising the P-loop (amino acid residues 169-179 of SEQ ID NO:2) of CARD-12.    
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2 wherein up to 10 amino acids are replaced by conservative substitutions.  
     
     
         3 . A fusion protein comprising the polypeptide of  claim 1  and a non-CARD-12 polypeptide.  
     
     
         4 . The fusion protein of  claim 3 , wherein the non-CARD-12 polypeptide is glutathione S-transferase.  
     
     
         5 . A method for detecting the presence of a polypeptide of  claim 1  in a sample, comprising: 
 a) contacting the sample with a compound which selectively binds to a polypeptide of  claim 1;  and    b) determining whether the compound binds to the polypeptide in the sample.    
     
     
         6 . The method of  claim 5 , wherein the compound that binds to the polypeptide is an antibody.  
     
     
         7 . A method for identifying a compound which binds to CARD-12, the method comprising the steps of: 
 a) contacting a polypeptide, or a cell from the group consisting of: 
 i) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2;  
 ii) a polypeptide comprising an amino acid sequence at least 90% identical to the CARD domain (amino acid residues 1-88 of SEQ ID NO:2) of CARD-12;  
 iii) a polypeptide comprising an amino acid sequence at least 90% identical to an NBS domain (amino acid residues 161-323 of SEQ ID NO:2) of CARD-12;  
 iv) a polypeptide comprising an amino acid sequence at least 90% identical to a LRR domain (amino acid residues 762-965 of SEQ ID NO:2) of CARD-12;  
 v) a polypeptide comprising an amino acid sequence of a fragment of at least 200 amino acids of SEQ ID NO:2, wherein the fragment has at least one binding activity of CARD-12; and  
 vi) a cell expressing the polypeptide of i), ii), iii), iv) or v), with a test compound; and  
   b) determining whether the polypeptide binds to the test compound.    
     
     
         8 . The method of  claim 7 , wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: 
 a) detection of binding by direct detecting of test compound/polypeptide binding;    b) detection of binding using a competition binding assay;    c) detection of binding using an assay for CARD-12-mediated signal transduction;    d) detection of an effect on the binding of the polypeptide to a CARD domain;    e) detection of an effect on the binding of the polypeptide to another protein; and    f) detection of an effect on the binding of the polypeptide to a nucleotide.    
     
     
         9 . The method of  claim 7 , wherein the fragment includes a polypeptide selected from the group consisting of: 
 a) a polypeptide comprising an NBS domain (amino acid residues 169-456 of SEQ ID NO:2) of CARD-12;    b) a polypeptide comprising at least one NACHT NTPase domain (amino acid residues 169-186, 196-220, 229-253, 261-282, 330-351, 414-430, or 438-457 of SEQ ID NO:2) of CARD-12;    c) a polypeptide comprising a LRR domain (amino acid residues 656-1021 of SEQ ID NO:2) of CARD-12; and    d) a polypeptide comprising at least one leucine-rich repeat domain (amino acid residues 656-686, 687-708, 711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930, 936-962, 965-993, or 994-1021) of CARD-12.    
     
     
         10 . The method of  claim 7 , wherein the polypeptide is operatively linked to a non-CARD-12 polypeptide.  
     
     
         11 . The method of  claim 7 , wherein the test compound is contacted with a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a cell expressing a polypeptide comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         12 . The method of  claim 8 , wherein the detection of an effect on the binding of the polypeptide to a CARD domain comprises detection of an effect on the binding of CARD-12 to a CARD-5 polypeptide, wherein the CARD-5 polypeptide comprises the CARD domain of CARD-5.  
     
     
         13 . The method of  claim 8 , wherein the detection of an effect on the binding of the polypeptide to a CARD domain comprises detection of an effect on the binding of CARD-12 to a CARD-12 polypeptide, wherein the CARD-12 polypeptide comprises the CARD domain of CARD-12.  
     
     
         14 . The method of  claim 8 , wherein the detection of an effect on the binding of the polypeptide to another protein comprises detection of an effect on the binding of CARD-12 to caspase-1.  
     
     
         15 . A method for identifying a candidate compound for treating an apoptotic disorder or an inflammatory disorder, the method comprising: 
 a) measuring the binding of a first polypeptide or cell selected from the group consisting of:    i) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2;    ii) a polypeptide comprising an amino acid sequence at least 90% identical to the CARD domain (amino acid residues 1-88 of SEQ ID NO:2) of CARD-12;    iii) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 161-323 of SEQ ID NO:2 of CARD-12;    iv) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 762-965 of SEQ ID NO:2 of CARD-12;    v) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 169-456 of SEQ ID NO:2) of CARD-12;    vi) a polypeptide with a length of at least 25 amino acids, comprising at least one NACHT NTPase domain (amino acid residues 169-186, 196-220, 229-253, 261-282,330-351, 414-430, or 438-457 of SEQ ID NO:2) of CARD-12;    vii) a polypeptide with a length of at least 50 amino acids, comprising at least one leucine-rich repeat domain (amino acid residues 656-686, 687-708, 711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930, 936-962, 965-993, or 994-1021 of SEQ ID NO:2) of CARD-12; and    viii) a cell expressing the polypeptide of i), ii), iii), iv), v) vi) or vii), to a second molecule selected from the group consisting of: a polypeptide comprising a CARD domain, caspase-1 and a nucleotide, in the presence of a test compound; and    b) comparing the binding of the first polypeptide or cell to the second molecule measured in step (a) to the binding of the first polypeptide or cell to the second molecule in the absence of the test compound,    wherein altered binding of the first polypeptide or cell to the second molecule in the presence of the test compound compared the binding in the absence of the test compound indicates that the test compound is candidate compound treating a disorder.    
     
     
         16 . The method of  claim 15 , wherein the apoptotic disorder is a cancer, an autoimmune disorder, a viral infection, Alzheimer's disease, Parkinson's disease or aplastic anemia.  
     
     
         17 . The method of  claim 15 , wherein the inflammatory disorder is arthritis, Crohn's disease, psoriasis, allergy or asthma.  
     
     
         18 . The method of  claim 15 , wherein the test compound is contacted with a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a cell expressing a polypeptide comprising the amino acid sequence of SEQ ID NO:2.