Regulation of target protein activity through modifier proteins

Abstract

The present invention is based on the discovery that a polypeptide containing the JAB subunit or the JAM domain has peptidase activity, e.g., isopeptidase activity. The present invention provides polypeptides and crystalline polypeptides containing the JAM domain and methods of using such polypeptides to screen for agents capable of affecting the peptidase activity of the polypeptides. The present invention also provides methods of using the JAM domain for rational drug design or identifying agents capable of affecting the peptidase activity of the JAM domain.

1 . A method of deconjugating a modifier protein from a target protein, wherein the modifier protein is conjugated to the target protein via a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein, the method comprising contacting the target protein to a polypeptide comprising a subunit characterized as JAB subunit.  
     
     
         2 . The method of  claim 1 , wherein the target protein is a cullin protein.  
     
     
         3 . The method of  claim 2 , wherein the target protein is Cul1, Cul2, Cul3, Cul4A, Cul4B, or Cul5.  
     
     
         4 . The method of  claim 1 , wherein the target protein has ubiquitin ligase activity.  
     
     
         5 . The method of  claim 1 , wherein the target protein is part of a protein complex having ubiquitin ligase activity.  
     
     
         6 . The method of  claim 1 , wherein the modifier protein is NEDD8, UBL1, SMT3H2, SMT3H1, APG12, FAT10, Fau, UCRP, URM1, or UBL5.  
     
     
         7 . The method of  claim 1 , wherein the polypeptide is a polypeptide complex of COP9/signalsome.  
     
     
         8 . The method of  claim 1 , wherein the polypeptide is AMSH, AMSH1, or AMSH2.  
     
     
         9 . The method of  claim 1 , wherein the target protein is exposed to the polypeptide in vitro.  
     
     
         10 . The method of  claim 1 , wherein the target protein is exposed to the polypeptide in vivo.  
     
     
         11 . A method for screening for an agent that affects deconjugation of a modifier protein from a target protein, wherein the modifier protein is conjugated to the target protein via a peptide bond between the carboxy terminus of the modifier protein and a free amino group of the target protein, the method comprising 
 incubating in the presence and absence of a test agent, the target protein and a polypeptide comprising a subunit characterized as JAB subunit,    determining the effect of the test agent, wherein an increase or decrease in the amount of the target protein not conjugated to the modifier protein caused by the test agent is indicative of an agent affecting deconjugation of the modifier protein from the target protein.    
     
     
         12 . The method of  claim 11 , wherein the target protein is a cullin protein.  
     
     
         13 . The method of  claim 12 , wherein the target protein is Cul1, Cul2, Cul3, Cul4A, Cul4B, or Cul5.  
     
     
         14 . The method of  claim 11 , wherein the target protein has ubiquitin ligase activity.  
     
     
         15 . The method of  claim 11 , wherein the target protein is part of a protein complex having ubiquitin ligase activity.  
     
     
         16 . The method of  claim 11 , wherein the modifier protein is NEDD8, UBL1, SMT3H2, SMT3H1, APG12, FAT10, Fau, UCRP, URM1, or UBL5.  
     
     
         17 . The method of  claim 11 , wherein the polypeptide is a polypeptide complex of COP9/signalsome.  
     
     
         18 . The method of  claim 11 , wherein the polypeptide is AMSH, AMSH1, or AMSH2.  
     
     
         19 . The method of  claim 11 , wherein a test agent decreasing the amount of the target protein not conjugated to the modifier protein is indicative of an agent decreasing deconjugation of the modifier protein from the target protein.  
     
     
         20 . The method of  claim 11 , wherein the target protein has the activity of peroxidase, alkaline phosphatase, or luciferase.  
     
     
         21 . The method of  claim 11 , wherein the target protein is a fluorescent protein.  
     
     
         22 . The method of  claim 21 , wherein the fluorescent protein is green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, or dsRed.  
     
     
         23 . The method of  claim 21 , wherein the target protein is a fluorescent protein via chemical modification.  
     
     
         24 . The method of  claim 11 , wherein the target-protein causes production of a detectable signal upon deconjugation from the modifier protein.  
     
     
         25 . The method of  claim 11 , wherein the polypeptide is a polypeptide complex of 26S proteasome.  
     
     
         26 . The method of  claim 11 , wherein the polypeptide is a polypeptide complex of 26S proteasome and the modifier protein is an ubiquitin.  
     
     
         27 . The method of  claim 25 , wherein the incubation is conducted in the presence and absence of the test agent, the target protein, the 26S proteasome, and a 20S inhibitor.  
     
     
         28 . The method of  claim 25 , wherein the incubation is conducted in the presence and absence of the test agent, the target protein, the 26S proteasome, a 20S inhibitor, and ATP.  
     
     
         29 . The method of  claim 27 , wherein the incubation further includes an inhibitor of deubiquitination by an ubiquitin isopeptidase.  
     
     
         30 . The method of  claim 25 , wherein the target protein not conjugated to the modifier protein is not degraded.  
     
     
         31 . The method of  claim 25 , wherein the target protein is Sic1.  
     
     
         32 . The method of  claim 25 , wherein the 26S proteasome is purified from  S. cerevisiae.    
     
     
         33 . The method of  claim 25 , wherein the 26S proteasome is purified from eukaryotic cells.  
     
     
         34 . The method of  claim 25 , wherein the 26S proteasome is purified from human cells.  
     
     
         35 - 47 . (cancelled)