Novel poly(adp-ribose) polymerase genes

Abstract

The invention relates to poly(ADP-ribose)polymerase (PARP) homologs which have an amino acid sequence which has a) a functional NAD + binding domain and b) no zinc finger sequence motif of the general formula CX 2 CX m HX 2 C in which m is an integral value from 28 or 30, and the X radicals are, independently of one another, any amino acid; and the functional equivalents thereof; nucleic acids coding therefor; antibodies with specificity for the novel protein; pharmaceutical and gene therapy compositions which comprise products according to the invention; methods for the analytical determination of the proteins and nucleic acids according to the invention; methods for identifying effectors or binding partners of the proteins according to the invention; novel PARP effectors; and methods for determining the activity of such effectors.

1 . A poly(ADP-ribose) polymerase (PARP) homolog derived from a human or non-human mammal which has an amino acid sequence which has
 a) a functional NAD +  binding domain   and   b) no zinc finger sequence motif of the general formula   
       
         
           
                 
                 
               
                     
                   CX 2 CX m HX 2 C 
                 
             
                
               
            
           
         
         in which 
         m is an integral value from 28 or 30, and the X radicals are, independently of one another, any amino acid. 
       
     
     
         2 . A PARP homolog as claimed in  claim 1 , wherein the functional NAD +  binding domain comprises one of the following general sequence motifs: 
       
         
           
                 
               
                   PX n (S/T)GX 3 GKGIYFA, 
                 
                     
                 
                   (S/T)XGLR(I/V)XPX n (S/T)GX 3 GKGIYFA 
                 
                   or 
                 
                     
                 
                   LLWHG(S/T)X 7 IL(S/T)XGLR(I/V)XPX n (S/T)GX 3 GKGIYFAX 3 SKS 
                 
                     
                 
                   AXY 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
         in which 
         n is an integral value from 1 to 5, and the X radicals are, independently of one another, any amino acid. 
       
     
     
         3 . A PARP homolog as claimed in  claim 1 , comprising at least another one of the following part-sequence motifs: 
       
         
           
                 
                 
               
                     
                   LX 9 NX 2 YX 2 QLLX (D/E)X 10/11 WGRVG, 
                 
                     
                     
                 
                     
                   AX 3 FXKX 4 KTXNXWX 5 FX 3 PXK, 
                 
                     
                     
                 
                     
                   QXL(I/L)X 2 IX 9 MX 10 PLGKLX 3 QIX 6 L, 
                 
                     
                     
                 
                     
                   FYTXIPHXFGX 3 PP; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   KX 3 LX 2 LXDIEXAX 2 L, 
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         in which the X radicals are, independently of one another, any amino acid. 
       
     
     
         4 . A PARP homolog as claimed in  claim 1 , selected from human PARP homologs, which has the amino acid sequence shown in SEQ ID NO: 2 (human PARP2) or SEQ ID NO: 4 or 6 (human PARP3 type 1 or 2); or murine PARP homologs which have the amino acid sequence shown in SEQ ID NO:8 (mouse PARP long form) or SEQ ID No:10 (mouse PARP short form). 
     
     
         5 . A binding partner for having specificity for PARP homologs as claimed in  claim 1 , selected from
 a) antibodies and fragments thereof,   b) protein-like compounds which interact with a part sequence of the protein, and   c) low molecular weight effectors which modulate the catalytic PARP activity or another biological function of a PARP molecule.   
     
     
         6 . A nucleic acid comprising
 a) a nucleotide sequence coding for at least one PARP homolog as claimed in  claim 1 , or the complementary nucleotide sequence thereof;   b) a nucleotide sequence which hybridizes with a sequence as specified in a) under stringent conditions; or   c) nucleotide sequences which are derived from the nucleotide sequences defined in a) and b) through the degeneracy of the genetic code.   
     
     
         7 . A nucleic acid as claimed in  claim 6 , comprising
 a) nucleotides +3 to +1715 shown in SEQ ID NO:1;   b) nucleotides +242 to +1843 shown in SEQ ID NO:3;   c) nucleotides +221 to +1843 shown in SEQ ID NO:5;   d) nucleotides +112 to +1710 shown in SEQ ID NO:7; or   e) nucleotides +1 to +1584 shown in SEQ ID NO:9.   
     
     
         8 . An expression cassette comprising, under the genetic control of at least one regulatory nucleotide sequence, at least one nucleotide sequence as claimed in  claim 6 . 
     
     
         9 . A recombinant vector comprising at least one expression cassette as claimed in  claim 8 . 
     
     
         10 . A recombinant microorganism comprising at least one recombinant vector as claimed in  claim 9 . 
     
     
         11 . A transgenic mammal comprising a vector as claimed in  claim 9 . 
     
     
         12 . A PARP-deficient mammal or PARP-deficient eukaryotic cell, in which functional expression of at least one gene which codes for a PARP homolog as claimed in  claim 1  is inhibited. 
     
     
         13 . An in vitro detection method for PARP inhibitors, which comprises
 a) incubating an unsupported or supported polyADP
 ribosylatable target with a reaction mixture comprising 
 a1) a PARP homolog as claimed in  claim 1 , 
 a2) a PARP activator; and 
 a3) a PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected; 
   b) carrying out the polyADP ribosylation reaction; and   c) determining the polyADP ribosylation of the target qualitatively or quantitatively.   
     
     
         14 . The method as claimed in  claim 13 , wherein the PARP homolog is preincubated with the PARP activator and the PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected, before the polyADP ribosylation reaction is carried out. 
     
     
         15 . The method as claimed in  claim 13 , wherein the polyADP-ribosylatable target is a histone protein. 
     
     
         16 . The method as claimed in  claim 13 , wherein the PARP activator is activated DNA. 
     
     
         17 . The method as claimed in  claim 13 , wherein the polyADP ribosylation reaction is started by adding NAD + . 
     
     
         18 . The method as claimed in  claim 13 , wherein the polyADP ribosylation of the supported target is determined using anti-poly(ADP-ribose) antibodies. 
     
     
         19 . The method as claimed in  claim 13 , wherein the unsupported target is labeled with an acceptor fluorophore. 
     
     
         20 . The method as claimed in  claim 19 , wherein the polyADP ribosylation of the unsupported target is determined using anti-poly(ADP-ribose) antibody which is labeled with a donor fluorophore which is able to transfer energy to the acceptor fluorophore. 
     
     
         21 . The method as claimed in  claim 19 , wherein the target is biotinylated histone, and the acceptor fluorophore is coupled thereto via avidin or streptavidin. 
     
     
         22 . The method as claimed in  claim 20 , wherein the anti-poly(ADP-ribose) antibody carries a europium cryptate as donor fluorophore. 
     
     
         23 . An in vitro screening method for binding partners for a PARP molecule, which comprises
 a1) immobilizing at least one PARP homolog as claimed in  claim 1  on a support;   b1) contacting the immobilized PARP homolog with an analyte in which at least one binding partner is suspected; and   c1) determining, where appropriate after an incubation period, analyte constituents bound to the immobilized PARP homolog;   or   a2) immobilizing on a support an analyte which comprises at least one possible binding partner for a PARP molecule;   b2) contacting the immobilized analyte with at least one PARP homolog as claimed in  claim 1  for which a binding partner is sought; and   c2) examining the immobilized analyte, where appropriate after an incubation period, for binding of the PARP homolog.   
     
     
         24 . A method for the qualitative or quantitative determination of nucleic acids encoding a PARP homolog as claimed in  claim 1 , which comprises
 a) incubating a biological sample with a defined amount of an exogenous nucleic acid as claimed in  claim 6 , hybridizing under stringent conditions, determining the hybridizing nucleic acids and, where appropriate, comparing with a standard; or   b) incubating a biological sample with a pair of oligonucleotide primers with specificity for a PARP homolog-encoding nucleic acid, amplifying the nucleic acid, determining the amplification product and, where appropriate, comparing with a standard.   
     
     
         25 . A method for the qualitative or quantitative determination of a PARP homolog as claimed in  claim 1 , which comprises
 a) incubating a biological sample with a binding partner specific for a PARP homolog,   b) detecting the binding partner/PARP complex and, where appropriate,   c) comparing the result with a standard.   
     
     
         26 . The method as claimed in  claim 25 , wherein the binding partner is an antibody or a binding fragment thereof, which carries a detectable label where appropriate. 
     
     
         27 . The method as claimed in  claim 24  for diagnosing energy deficit-mediated illnesses. 
     
     
         28 . A method for determining the efficacy of PARP effectors, which comprises
 a) incubating a PARP homolog as claimed in  claim 1  with an analyte which comprises an effector of a physiological or pathological PARP activity; removing the effector again where appropriate; and   b) determining the activity of the PARP homolog, where appropriate after adding substrates or cosubstrates.   
     
     
         29 . A gene therapy composition, which comprises in a vehicle acceptable for gene therapy a nucleic acid construct which
 a) comprises an antisense nucleic acid against a coding nucleic acid as claimed in  claim 6 ; or   b) a ribozyme against a nucleic acid as claimed in  claim 6 ; or   c) codes for a specific PARP inhibitor.   
     
     
         30 . A pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one PARP protein as claimed in  claim 1 , at least one PARP binding partner as claimed in  claim 5  or at least one coding nucleotide sequence as claimed in  claim 6 . 
     
     
         31 . The use of low molecular weight PARP binding partners as claimed in  claim 5  for the manufacture of a pharmaceutical agent for the diagnosis or therapy of pathological states in the development and/or progress of which at least one PARP protein, or a polypeptide derived therefrom, is involved. 
     
     
         32 . The use of low molecular weight PARP binding partners as claimed in  claim 5  for the manufacture of a pharmaceutical agent for the diagnosis or therapy of pathological states mediated by an energy deficit.