Novel cyclin-selective ubiquitin carrier polypeptides

Abstract

Disclosed are novel human and clam ubiquitin carrier polypeptides involved in the ubiquitination of cyclins A and/or B. Also disclosed are inhibitors of such polypeptides, nucleic acids encoding such polypeptides and inhibitors, antibodies specific for such polypeptides, and methods of their use.

1 . An isolated and purified, non-xenopal, ubiquitin carrier polypeptide (Ubc) involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension.  
     
     
         2 . The Ubc of  claim 1  involved in the ubiquitination of cyclin A.  
     
     
         3 . The Ubc of  claim 1  involved in the ubiquitination of cyclin B.  
     
     
         4 . The Ubc of  claim 1  which is recombinantly produced.  
     
     
         5 . An enzymatically active fragment of the Ubc of  claim 1 .  
     
     
         6 . The Ubc of  claim 1  which is a human polypeptide  
     
     
         7 . The Ubc of  claim 6  having an amino acid sequence with about 61-100% homology to the amino acid sequence set forth as SEQ ID NO:1.  
     
     
         8 . The Ubc of  claim 7  having an amino acid sequence with at least 94-100% homology to the amino acid sequence set forth as SEQ ID NO:1.  
     
     
         9 . The Ubc of  claim 8  having the amino acid sequence set forth as SEQ ID NO:1.  
     
     
         10 . The Ubc of  claim 6  encoded by a nucleic acid hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:2.  
     
     
         11 . The Ubc of  claim 1 , wherein the N-terminal extension has an amino acid sequence set forth as SEQ ID NO:9.  
     
     
         12 . The Ubc of  claim 1 , wherein the Ubc is a clam polypeptide.  
     
     
         13 . The Ubc of  claim 12  having an amino acid sequence with at least 61-100% homology to the amino acid sequence set forth as SEQ ID NO:3.  
     
     
         14 . The Ubc of  claim 13  having an amino acid sequence with at least 94-100% homology to the amino acid sequence set forth as SEQ ID NO:3.  
     
     
         15 . The Ubc of  claim 14  having the amino acid sequence set forth as SEQ ID NO:3.  
     
     
         16 . The Ubc of  claim 13  encoded by a nucleic acid hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:4.  
     
     
         17 . The Ubc of  claim 1 , wherein the N-terminal extension has an amino acid sequence set forth as SEQ ID NO:10.  
     
     
         18 . A nucleic acid encoding the Ubc of  claim 1 .  
     
     
         19 . The nucleic acid of  claim 18  encoding an enzymatically active fragment of the Ubc of  claim 1 .  
     
     
         20 . The nucleic acid of  claim 18  encoding a human Ubc having an amino acid sequence with about 61-100% homology with the amino acid sequence set forth as SEQ ID NO:1.  
     
     
         21 . The nucleic acid of  claim 20  encoding a human Ubc having an amino acid sequence with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1.  
     
     
         22 . The nucleic acid of  claim 18  encoding a clam Ubc having an amino acid sequence with about 61-100% homology with the amino acid sequence set forth as SEQ ID NO:3.  
     
     
         23 . The nucleic acid of  claim 22  encoding a clam Ubc having an amino acid sequence with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:3.  
     
     
         24 . The nucleic acid of  claim 18  hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:2.  
     
     
         25 . The nucleic acid of  claim 18  hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:4.  
     
     
         26 . The nucleic acid of  claim 18  encoding a human Ubc having an amino acid sequence with about 61-100% homology with the amino acid sequence set forth as SEQ ID NO:1.  
     
     
         27 . The nucleic acid of  claim 26  encoding a human Ubc having an amino acid sequence with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1.  
     
     
         28 . The nucleic acid of  claim 18  encoding a clam Ubc having an amino acid sequence with about 61-100% homology with the amino acid sequence set forth as SEQ ID NO:3.  
     
     
         29 . The nucleic acid of  claim 28  encoding a clam Ubc having an amino acid sequence with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:3.  
     
     
         30 . The nucleic acid of  claim 18  hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:2.  
     
     
         31 . The nucleic acid of  claim 18  hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:4.  
     
     
         32 . The nucleic acid of  claim 18  which is a cDNA.  
     
     
         33 . The nucleic acid of  claim 32  which is cDNA having the nucleic acid sequence set forth as SEQ ID NO:2.  
     
     
         34 . The nucleic acid of  claim 32  which is cDNA having the nucleic acid sequence set forth as SEQ ID NO:4.  
     
     
         35 . A selective inhibitor of ubiquitin carrier polypeptide (Ubc) function.  
     
     
         36 . The selective inhibitor of  claim 35  which inhibits an isolated and purified, non-xenopal, Ubc involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension.  
     
     
         37 . The inhibitor of  claim 35  which is a dominant negative mutant.  
     
     
         38 . The dominant negative mutant of  claim 39  which inhibits an isolated and purified, non-xenopal, Ubc involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension.  
     
     
         39 . The dominant negative mutant of  claim 37  which is recombinantly produced.  
     
     
         40 . The dominant negative mutant of  claim 37  comprising a serine residue at position 114 substituted for a cysteine residue.  
     
     
         41 . A fragment of the dominant negative mutant of  claim 37  which inhibits Ubc function.  
     
     
         42 . The dominant negative mutant of  claim 37  which inhibits the function of a human Ubc.  
     
     
         43 . The dominant negative mutant of  claim 42  having an amino acid sequence with about 94-100% homology to the amino acid sequence set forth as SEQ ID NO:5.  
     
     
         44 . The dominant negative mutant of  claim 42  encoded by a nucleic acid hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6.  
     
     
         45 . The dominant negative mutant of  claim 37  which inhibits the function of a clam Ubc.  
     
     
         46 . The dominant negative mutant of  claim 45  having an amino acid sequence with about 94-100% homology to the amino acid sequence set forth as SEQ ID NO:7.  
     
     
         47 . The dominant negative mutant of  claim 46  encoded by a nucleic acid hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:8.  
     
     
         48 . A nucleic acid encoding the dominant negative mutant of  claim 37 .  
     
     
         49 . The nucleic acid of  claim 48  hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6.  
     
     
         50 . The nucleic acid of  claim 48  hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:8.  
     
     
         51 . The nucleic acid of  claim 48  which is a cDNA.  
     
     
         52 . The cDNA of  claim 51  having the nucleotide sequence set forth as SEQ ID NO:6.  
     
     
         53 . The cDNA of  claim 51  having the nucleotide sequence set forth as SEQ ID NO:8.  
     
     
         54 . A kit useful for the ubiquitination and degradation of a cyclin comprising: 
 (a) a ubiquitin-ubiquitin carrier polypeptide complex, wherein the ubiquitin carrier polypeptide is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof; and    (b) a ubiquitin ligase (E3).    
     
     
         55 . The kit of  claim 54  wherein the cyclin to be ubiquitinated is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier polypeptide complex comprises human UbcH10.  
     
     
         56 . The kit of  claim 55  wherein the Ubc in the complex has an amino acid sequence set forth as SEQ ID NO:1.  
     
     
         57 . The kit of  claim 54  wherein the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier polypeptide complex comprises clam E2-C.  
     
     
         58 . The kit of  claim 57  wherein the Ubc in the complex has an amino acid sequence set forth as SEQ ID NO:3.  
     
     
         59 . A kit useful for the ubiquitination and degradation of a cyclin comprising: 
 (a) ubiquitin;    (b) a ubiquitin activating enzyme (E1);    (c) ATP;    (d) a ubiquitin carrier polypeptide selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active portion thereof; and    (e) a ubiquitin ligase (E3).    
     
     
         60 . The kit of  claim 59  wherein the cyclin to be degraded is cyclin A or cyclin B, and the ubiquitin-ubiquitin carrier polypeptide complex comprises human UbcH10 having an amino acid sequence set forth as SEQ ID NO:1.  
     
     
         61 . The kit of  claim 59  wherein the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier polypeptide complex comprises clam E2-C having an amino acid sequence set forth as SEQ ID NO:3.  
     
     
         62 . A method of ubiquitinating a cyclin and/or targeting a cyclin for destruction, comprising the step of contacting the cyclin with: 
 (a) a ubiquitin-ubiquitin carrier protein complex, wherein the ubiquitin carrier protein is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof; and    (b) a ubiquitin ligase (E3).    
     
     
         63 . A method of inhibiting the proliferation of a cell comprising the step of contacting the cell with an inhibitor of ubiquitin carrier polypeptide function, in an amount sufficient to inhibit the ubiquitination of a cyclin.  
     
     
         64 . The method of  claim 63 , wherein the inhibitor administered is a dominant negative mutant of a ubiquitin carrier polypeptide (Ubc).  
     
     
         65 . The method of  claim 64 , wherein the dominant negative mutant has an amino acid sequence which is about 94-100% homologous with the amino acid sequence set forth as SEQ ID NO:5.  
     
     
         66 . The method of  claim 64 , wherein the dominant negative mutant has a serine residue at position 114 substituted for a cysteine residue.  
     
     
         67 . The method of  claim 63  wherein the dominant negative mutant is encoded by a nucleic acid which is hybridizable with the nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6.  
     
     
         68 . The method of  claim 63  wherein the dominant negative mutant is encoded by a nucleic acid which is hybridizable with the nucleic acid having the nucleotide sequence set forth as SEQ ID NO:8.  
     
     
         69 . A method of screening for compounds which inhibit Ubc function, comprising the steps of: 
 (a) providing an assay for measuring Ubc function, wherein the assay comprises a ubiquitin carrier polypeptide selected from the group consisting of a non-xenopal ubiquitin carrier polypeptide involved in the ubiquitination of cyclin A and/or B and having a Ubc-specific N-terminal extension, and an enzymatically active fragment thereof;    (b) performing the assay in the presence and absence of a compound to-be-tested; and    (c) determining the amount of change in Ubc function measured in the presence of the compound as compared to Ubc function measured in the absence of the compound,    a reduction of Ubc function measured in the presence of the compound indicating that the compound is an inhibitor of Ubc function.    
     
     
         70 . The method of  claim 69 , wherein the ubiquitin carrier polypeptide is isolated and purified.  
     
     
         71 . The method of  claim 70 , wherein the ubiquitin carrier polypeptide is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof.  
     
     
         72 . An inhibitor of cyclin ubiquitination identified by the method of  claim 69 .  
     
     
         73 . A method of screening for compounds which inhibit the ubiquitination of a cyclin, comprising the steps of: 
 (a) incubating the cyclin with: 
 (i) ubiquitin;  
 (ii) a ubiquitin activating enzyme (E1);  
 (iii) ATP;  
 (iv) a ubiquitin carrier polypeptide selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof;  
 (v) a ubiquitin ligase (E3); and  
 (vi) Cdc2,  
   in the presence and in the absence of a compound-to-be-tested; and    (b) measuring the amount of cyclin-ubiquitin-Cdc2 complex formed in step (a) in the presence and in the absence of the compound,    a reduction in the amount of complex formed in the presence of the compound indicating that the compound inhibits cyclin ubiquitination.    
     
     
         74 . The method of  claim 73 , wherein the ubiquitin carrier polypeptide is isolated and purified.  
     
     
         75 . The method of  claim 74 , wherein the ubiquitin carrier polypeptide is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof.  
     
     
         76 . An inhibitor of cyclin ubiquitination identified by the method of  claim 73 .  
     
     
         77 . A therapeutic formulation comprising a selective inhibitor of ubiquitin carrier protein function in an amount sufficient to inhibit the ubiquitination of a cyclin, and a pharmaceutically acceptable carrier.  
     
     
         78 . The therapeutic formulation of  claim 77 , wherein the inhibitor comprises a dominant negative mutant of a ubiquitin carrier protein, or a fragment thereof capable of inhibiting Ubc function.  
     
     
         79 . The therapeutic formulation of  claim 78 , wherein the dominant negative mutant has a serine residue at position 114 substituted for a cysteine residue.  
     
     
         80 . The therapeutic formulation of  claim 77 , wherein the dominant negative mutant has an amino acid sequence which is at least 90-95% homologous with the amino acid sequence set forth as SEQ ID NO:5.  
     
     
         81 . The therapeutic formulation of  claim 80 , wherein the dominant negative mutant is encoded by a nucleic acid hybridizable with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6.  
     
     
         82 . The therapeutic formulation of  claim 77 , wherein the dominant negative mutant has an amino acid sequence which is at least 90-95% homologous with the amino acid sequence set forth as SEQ ID NO:7.  
     
     
         83 . The therapeutic formulation of  claim 82 , wherein the dominant negative mutant is encoded by a nucleic acid which is hybridizable with the nucleic acid having a nucleotide sequence set-forth as SEQ ID NO:8.  
     
     
         84 . A Ubc of  claim 1  having amino acids 33 to 179 of SEQ ID NO:1.  
     
     
         85 . A Ubc of  claim 1  having amino acids 33 to 177 of SEQ ID NO:3