Assays for identifying ubiquitin agents and for identifying agents that modify the activity of ubiquitin agents

Abstract

Provided are methods and compositions for assaying for ubiquitin agents that are enzymatic components of ubiquitin-mediated proteolysis and, more particularly, methods and compositions for assaying for agents that modulate the activity of such ubiquitin agents.

1 . A method of assaying for an agent that modulates the attachment of a ubiquitin moiety to at least one ubiquitin agent, said method comprising: 
 a) combining: 
 i) a first ubiquitin agent;  
 ii) a candidate agent; and  
 iii) a ubiquitin moiety; and  
   b) assaying for the attachment of said ubiquitin moiety to said first agent.    
     
     
         2 . The method according to  claim 1 , wherein said first ubiquitin agent is an ubiquitin activating agent.  
     
     
         3 . The method according to  claim 2 , wherein said ubiquitin activating agent is an E1.  
     
     
         4 . The method according to  claim 1 , further comprising including a second ubiquitin agent in said combining step.  
     
     
         5 . The method according to  claim 4 , wherein said first agent is a ubiquitin conjugating agent and said second agent is a ubiquitin activating agent.  
     
     
         6 . The method according to  claim 5 , wherein said ubiquitin conjugating agent is an E2 and said ubiquitin activating agent is an E1.  
     
     
         7 . The method according to  claim 4 , wherein said first agent is a ubiquitin ligating agent and said second agent is a ubiquitin conjugating agent comprising said ubiquitin moiety.  
     
     
         8 . The method according to  claim 7 , wherein said ubiquitin ligating agent is an E3 and said ubiquitin conjugating agent is an E2 comprising said ubiquitin moiety.  
     
     
         9 . The method according to  claim 5 , wherein said ubiquitin conjugating agent is an E2 and said ubiquitin activating agent is an E1 comprising said ubiquitin moiety.  
     
     
         10 . The method according to  claim 9  further comprising including a third ubiquitin agent in said combining step.  
     
     
         11 . The method according to  claim 10 , wherein said third agent is a ubiquitin ligating agent.  
     
     
         12 . The method according to  claim 11 , wherein said ubiquitin ligating agent is an E3.  
     
     
         13 . The method according to  claim 1 , wherein said first ubiquitin agent is attached to a solid support.  
     
     
         14 . The method according to claims  4 , wherein said second ubiquitin agent is attached to a solid support.  
     
     
         15 . The method according to  claim 7 , wherein said first ubiquitin agent is attached to a solid support.  
     
     
         16 . The method according to any of claims  13 ,  14 , and  15 , wherein said solid support is a microtiter plate.  
     
     
         17 . The method according to any of claims  13 ,  14 , and  15 , wherein said solid support is a bead.  
     
     
         18 . The method according to claims  1  or  7 , wherein said first agent comprises a tag.  
     
     
         19 . The method according to  claim 4 , wherein said second agent comprises a tag.  
     
     
         20 . The method according to any of claims  1 ,  4 , and  7 , wherein said ubiquitin moiety comprises a tag.  
     
     
         21 . The method according to claims  1  or  7 , wherein said first agent comprises an attachment tag.  
     
     
         22 . The method according to  claim 4 , wherein said second agent comprises an attachment tag.  
     
     
         23 . The method according to claims  1  or  7 , wherein said first agent comprises a label  
     
     
         24 . The method according to  claim 4 , wherein said second agent comprises a label.  
     
     
         25 . The method according to claims  1  or  7 , wherein said first agent comprises an epitope tag.  
     
     
         26 . The method according to  claim 4 , wherein said second agent comprises an epitope tag.  
     
     
         27 . The method according to any of claims  1 ,  4 , and  7 , wherein at least a first and a second ubiquitin moiety is used, wherein said first and second ubiquitin moiety moieties comprise different fluorescent labels, and wherein said labels form a fluorescence resonance energy transfer (FRET) pair.  
     
     
         28 . A method of assaying for an agent that modulates the attachment of a ubiquitin moiety to at least one ubiquitin agent, said method comprising: 
 a) combining: 
 i) a first ubiquitin agent comprising a ubiquitin ligating agent;  
 ii) a second ubiquitin agent;  
 iii) a candidate agent;  
 iv) a ubiquitin moiety; and  
 v) a target protein; and  
 assaying for the attachment of said ubiquitin moiety to said first agent.  
   
     
     
         29 . The method according to  claim 28 , wherein said second agent is a ubiquitin conjugating agent comprising said ubiquitin moiety.  
     
     
         30 . The method according to  claim 28  further comprising including a third ubiquitin agent in said combining step, wherein said third agent is a ubiquitin activating agent; wherein said substrate and said ubiquitin moiety comprise different fluorescent labels, and wherein said labels form a fluorescence resonance energy transfer (FRET) pair.  
     
     
         31 . The method according to  claim 1  or  28 , wherein said ubiquitin moiety is a mammalian ubiquitin moiety.  
     
     
         32 . The method according to  claim 28 , wherein said ubiquitin moiety comprises a label.  
     
     
         33 . The method according to  claim 28 , wherein said label comprises an epitope tag.