Methods and Compositions for Enhancing Proteasome Activity

Abstract

This invention relates to methods and compositions for enhancing proteasome activity in a cell. The methods and compositions for enhancing the activity of the proteasome in cells modulate the activity of Ubp6 (yeast) or Usp14 (human), an endogenous inhibitor of the proteasome. The methods and compositions partially or completely reduce the inhibitory activity of Usp14 on a proteasome, thereby specifically enhancing the protein-degradation activity of the proteasome. The invention also provides methods of screening to identify inhibitors of Ubp6, Usp14, and/or both Ubp6 and Usp14.

1 . A method of enhancing proteasome activity in a cell comprising contacting said cell with a Usp14 inhibitor in an amount sufficient to reduce inhibition of proteasome-mediated protein degradation. 
     
     
         2 . The method of  claim 1 , wherein said Usp14 inhibitor is a small-molecule inhibitor. 
     
     
         3 . The method of  claim 1 , wherein said Usp14 inhibitor interacts with the catalytic site of Usp14. 
     
     
         4 . The method of  claim 1 , wherein said Usp14 inhibitor increases the rate of proteasome-mediated degradation of ubiquitin or a ubiquitinated polypeptide as compared to the rate of proteasome-mediated degradation in the absence of said Usp14 inhibitor. 
     
     
         5 . A method of treating a proteinopathy in a subject comprising administering to said subject a Usp14 inhibitor in an amount effective to reduce endogenous inhibition of proteasome-mediated protein degradation. 
     
     
         6 . The method of  claim 5 , wherein said proteinopathy is a neurodegenerative disorder or a cancer. 
     
     
         7 . The method of  claim 6  wherein said neurodegenerative disorder is Huntington's disease. 
     
     
         8 . The method of  claim 5 , wherein said Usp14 inhibitor is a small-molecule inhibitor. 
     
     
         9 . The method of  claim 5 , wherein said Usp 14 inhibitor interacts with the catalytic site of Usp14. 
     
     
         10 . The method of  claim 5 , wherein said Usp 14 inhibitor increases the rate of proteasome-mediated degradation of ubiquitin or a ubiquitinated polypeptide as compared to the rate of proteasome-mediated degradation in the absence of said Usp14 inhibitor. 
     
     
         11 . A method of screening for a Usp14 inhibitor comprising:
 (a) providing a Usp14 polypeptide and a proteasome under conditions sufficient to allow the Usp14 polypeptide and proteasome to form a complex;   (b) contacting said Usp14 polypeptide/proteasome complex with a test compound; and   (c) determining whether said test compound dissociates the Usp14 polypeptide/proteasome complex,   whereby dissociation of the Usp14 polypeptide/proteasome complex indicates that said test compound is a Usp14 inhibitor.   
     
     
         12 . The method of  claim 11 , wherein said test compound is a small molecule. 
     
     
         13 . The method of  claim 11 , wherein said proteasome is selected from a yeast proteasome, a murine proteasome and a human proteasome. 
     
     
         14 . The method of  claim 11 , wherein said Usp14 inhibitor interacts with the catalytic site of Usp14. 
     
     
         15 . The method of  claim 11 , wherein said Usp14 inhibitor increases the rate of proteasome-mediated degradation of ubiquitin or a ubiquitinated polypeptide as compared to the rate of proteasome-mediated degradation in the absence of said Usp14 inhibitor. 
     
     
         16 . The method of  claim 11 , wherein said test compound is detectably labeled. 
     
     
         17 . A method of screening for an inhibitor of the catalytic site of Usp14 comprising:
 (a) providing a test solution comprising Usp14 polypeptide and a proteasome under conditions sufficient to allow the Usp14 polypeptide and proteasome to form a complex;   (b) contacting said test solution with a test compound and a substrate of said Usp14 polypeptide/proteasome, wherein said substrate is coupled to a reporter that is detectable after cleavage by a deubiquitinating enzyme;   (c) determining whether said substrate is deubiquitinated in the presence of the test compound and the Usp14 polypeptide/proteasome complex,   whereby an absence of deubiquitination of the substrate in the presence of the test compound and the Usp14 polypeptide/proteasome complex indicates that said test compound is an inhibitor of the Usp14 catalytic site.   
     
     
         18 . The method of  claim 17 , wherein said substrate is ubiquitin-AMC. 
     
     
         19 . The method of  claim 17 , wherein said test compound is a small molecule. 
     
     
         20 . The method of  claim 17 , wherein said reporter is fluorescently labeled. 
     
     
         21 . A method of screening for inhibitors of non-catalytic proteasome inhibition by Usp14, comprising:
 (a) providing a test solution comprising Usp14 polypeptide and a proteasome under conditions sufficient to allow the Usp14 polypeptide and proteasome to form a complex;   (b) contacting said test solution with a test compound and a ubiquitinated cyclin B polypeptide;   (c) determining the rate of degradation of said ubiquitinated cyclin B polypeptide in the presence of the test compound; and   (d) comparing the rate of degradation of said ubiquitinated cyclin B polypeptide in the absence of the test compound;   whereby an increased rate of degradation of ubiquitinated cyclin B in the presence of the test compound as compared to the rate of degradation in the absence of the test compound indicates that said test compound is an inhibitor of non-catalytic proteasome inhibition by Usp14.   
     
     
         22 . The method of  claim 21 , wherein said Usp14 is a catalytically inactivated form of Usp14. 
     
     
         23 . The method of  claim 21 , wherein said test compound is a small molecule. 
     
     
         24 . The method of  claim 21 , wherein said proteasome is selected from a yeast proteasome, a murine proteasome and a human proteasome. 
     
     
         25 . The method of  claim 24 , wherein said proteasome is a murine proteasome. 
     
     
         26 . The method of  claim 21 , wherein said ubiquitinated cyclin B polypeptide is a ubiquitinated form of a cyclin B-luciferase fusion protein.