Parkin substrate and assay

Abstract

The invention provides in vitro, ex vivo, and in vivo assays for Parkin activity, in which Parkin-mediated ubiquitination of a Sept4 protein is measured. The assays may be used to screen for agents that modulate Parkin protein ligase activity.

1 - 20 . (canceled) 
     
     
         21 . A method for identifying modulators of Parkin activity, said method comprising:
 (a) providing a non-human animal co-expressing Parkin and Sept4 in neuronal cells;   (b) administering a test agent to the animal;   (c) comparing the rate or extent of Sept4 ubiquitination in said neuronal cells of the animal in to which the test agent was administered with the rate or extent of Sept4 ubiquitination in a control animal not administered the test agent, wherein a relative increase in Sept4 ubiquitination in the presence of the test the agent indicates that the test agent enhances Parkin ubiquitination activity and a relative decrease in Sept4 ubiquitination in the presence of the test agent indicates that the test agent inhibits Parkin ubiquitination activity.   
     
     
         22 . The method of  claim 21 , wherein the animal is a rodent or non-human primate. 
     
     
         23 . The method of  claim 22 , wherein the animal is a mouse. 
     
     
         24 . The method of  claim 21 , wherein the animal expresses a transgene or transgenes in said cells, said transgene(s) encoding either or both of Sept4 and Parkin. 
     
     
         25 . The method of  claim 21 , wherein the Parkin is expressed recombinantly in said neuronal cells. 
     
     
         26 . The method of  claim 25 , wherein the Parkin is human Parkin. 
     
     
         27 . The method of  claim 21 , wherein the Parkin is expressed endogenously in said neuronal cells. 
     
     
         28 . The method of  claim 21 , wherein the Sept4 is expressed recombinantly in said neuronal cells. 
     
     
         29 . The method of  claim 28 , wherein the Sept4 is human Sept4. 
     
     
         30 . The method of  claim 21 , wherein the Sept4 is expressed endogenously in said neuronal cells.