Dbc1, a novel native inhibitor of the anti-aging protein sirt1

Abstract

A novel complex is identified between the NAD-dependent deacetylase, SIRT1 and its novel inhibitor, DBC1. Provided herein are methods to indentify a compound that inhibits the complexation between SIRT1 and DBC1. Exemplary methods comprise contacting either the complexation between DBC1 and SIRT1 with an agent being tested for its ability to inhibit the complexation between SIRT1 and DBC1. Also, provided are methods to identify a compound that increases the complexation between SIRT1 and DBC1. Exemplary methods comprise contacting either the complexation between DBC1 and SIRT1 with an agent being tested for its ability to increase the complexation between SIRT1 and DBC1. Further, methods are provided to increase or decrease SIRT1 activity by contacting the complexation between SIRT1 and DBC1 with a peptide that either decreases or increases the complexation between SIRT1 and DBC1. Further, methods are provided for the treatment of patients suffering from diseases including metabolic diseases including obesity and diabetes, and neurodegenerative disorders including Alzheimer's disease and Huntington's disease using compounds that inhibit the complexation between SIRT1 and DBC1.

1 - 2 . (canceled) 
     
     
         3 . A method for identifying a compound which inhibits the complexation between DBC1 and SIRT1 comprising:
 (a) contacting DBC1 bound to SIRT1 with an agent to be tested for its ability to inhibit the complexation between DBC1 and SIRT1;   (b) determining whether the level of unbound SIRT1 increases or the level of the complexation between SIRT1 and DBC1 decreases is different in the sample containing the agent being tested as compared to the control; and   (c) if the level of unbound SIRT1 is increased or the level of the complexation between SIRT1 and DBC1 is decreased, then the agent being tested inhibits the complexation between SIRT1 and DBC1.   
     
     
         4 . The method of  claim 3 , step a where the agent to be tested for its ability to inhibit the complexation between DBC1 and SIRT1 is tested in vitro. 
     
     
         5 . The method of  claim 3 , step a where the agent is a peptide. 
     
     
         6 . The method of  claim 5 , where the peptide can hybridize with the target under stringent conditions. 
     
     
         7 . The method of  claim 6  where the peptide is comprised of amino acids 210 to 500 of the SIRT1 protein. 
     
     
         8 . The method of  claim 4  where the difference in the levels is determined by differential centrifugation; chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis; immunoprecipitation; pulldown assay, ELISA assays fluorescence energy transfer; surface plasmon resonance; or in vitro tubulin deacetylation assays. 
     
     
         9 . The method of  claim 3 , step a where the agent to be tested for its ability to inhibit the complexation between DBC1 and SIRT1 is tested on a cell. 
     
     
         10 . The method of  claim 3 , step a where the agent being tested for its ability to inhibit complexation between DBC1 and SIRT1 is inside the cell. 
     
     
         11 . The method of  claim 3 , step a where the agent being tested for its ability to inhibit complexation between DBC1 and SIRT1 is outside the cell and has a cascade effect. 
     
     
         12 . The method of  claim 9  where the cell is a yeast cell. 
     
     
         13 . The method of  claim 9  where the cell is from human osteosarcoma U2OS cells 
     
     
         14 . The method of  claim 9  where the difference in levels is determined by yeast two hybrid, adipoctye differentiation assay, or a deacetylation assay. 
     
     
         15 . The method of  claim 9  where the agent is an siRNA or and shRNA. 
     
     
         16 . A method for identifying a compound which increases the complexation between DBC1 and SIRT1 comprising:
 (a) contacting DBC1 or SIRT1 with an agent being tested for its ability to increase the complexation between DBC1 and SIRT;   (b) determining whether the level of unbound SIRT1 decreases or the level of the complexation between SIRT1 and DBC1 increases is different in the sample containing the agent being tested as compared to the control; and   (c) if the level of unbound SIRT1 is decreased or the level of the complexation between SIRT1 and DBC1 is increased, then the agent being tested increases the complexation between SIRT1 and DBC1.   
     
     
         17 . The method of  claim 16 , step (a) where the agent being tested for its ability to increase the complexation between DBC1 and SIRT1 is tested in vitro. 
     
     
         18 . The method of  claim 16 , step (b) where the level of unbound SRT1 or the level of complexation between SIRT1 and DBC1 is determined by differential centrifugation; chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis; immunoprecipitation; pulldown assay, ELISA assays fluorescence energy transfer; surface plasmon resonance; or in vitro tubulin deacetylation assays. 
     
     
         19 . The method of  claim 16 , step (a) where the agent being tested for its ability to increase the complexation between DBC1 and SIRT1 is tested on a cell. 
     
     
         20 . The method of  claim 19  where the agent being tested for its ability to increase complexation between DBC1 and SIRT1 is inside the cell. 
     
     
         21 . The method of  claim 19  where the agent being tested for its ability to increase complexation between DBC1 and SIRT1 is outside the cell where it has a cascade effect. 
     
     
         22 . The method of  claim 19  where the cell is a yeast cell. 
     
     
         23 . The method of  claim 19  where the cell is from human osteosarcoma U2OS cells. 
     
     
         24 . The method of  claim 16 , step (b) where the difference in levels is determined by yeast two hybrid, adipoctye differentiation assay, or a deacetylation assay. 
     
     
         25 . A method for increasing SIRT1 activity by contacting the complexation between SIRT1 and DBC1 with an agent which inhibits DBC1 activity. 
     
     
         26 . The method of  claim 25  where the agent is a peptide 
     
     
         27 . The method of  claim 26  where the peptide hybridizes to its target under stringent conditions. 
     
     
         28 . The method of  claim 27  where the peptide is derived from amino acids 210 to 500 of the SIRT1 protein. 
     
     
         29 - 33 . (canceled)