Proteomic Screening for Redox State Dependent Protein-Protein Interactions

Abstract

This invention provides a modified yeast two-hybrid system in order to identify NO-dependent protein-protein interactions. Bait proteins implicated in apoptotic signaling pathways were used to identify NO-dependent interactions. The physiological relevance of these interactions is demonstrated by their occurrence and dependence on endogenous NO in mammalian cells, and by the functional interrelatedness of bait and prey.

1 . A method for identifying an RSMM inhibited protein complex comprising: 
 (a) culturing a first cell in a media comprising an RSMM;    (b) culturing a second cell in a media without the RSMM;    (c) identifying a first protein complex that exists in the second cell;    (d) analyzing the first cell to determine the existence of a protein complex analogous to the first protein complex;    wherein the RSMM inhibited protein complex is identified when the analogous protein complex does not exist or exists to a lesser degree than the first protein complex.    
   
   
       2 . A method for identifying an RSMM induced protein complex comprising: 
 (a) culturing a first cell in a media comprising an RSMM;    (b) culturing a second cell in a media without the RSMM;    (c) identifying a first protein complex that exists in the first cell;    (d) analyzing the second cell to determine the existence of a protein complex analogous to the first protein complex;    wherein the RSMM induced protein complex is identified when the analogous protein complex does not exist or exists to a lesser degree than the first protein complex.    
   
   
       3 - 4 . (canceled)  
   
   
       5 . The method of  claim 1  in which the RSMM is selected from the group consisting of nitric oxide, nitric dioxide, dinitrogen trixide, dinitrogen tetraoxide, S-nitrosothiol, nitroxyl anion, HNO, nitrite, nitrate, C—, N, O, S or metal-nitroso or nitro compounds, hydrogen peroxide, peroxynitrite, other peroxides, alkoxides, superoxide, hypochlorite ion, hydroxyl radical and physiological pO 2 .  
   
   
       6 . The method of  claim 1  in which the RSMM is an NO adduct.  
   
   
       7 . The method of  claim 6  wherein the NO adduct is selected from the group consisting of DETA-NO, S-nitrosothiol, SIN-1, angeli's salt, S-nitroso amino acids, S-nitroso-polypeptides, and nitrosoamines.  
   
   
       8 . The method of  claim 1  wherein the pO 2  is in a range from about 5 to about 100 mm Hg.  
   
   
       9 . The method of  claim 8  wherein the range is from about 10 to about 50 mm Hg.  
   
   
       10 . The method of  claim 9  wherein the range is from about 10 to about 30 mm Hg.  
   
   
       11 - 72 . (canceled)  
   
   
       73 . A method for culturing a cell in vitro comprising: 
 (a) providing a media comprising at least one RSMM; and    (b) culturing said cell in the media.    
   
   
       74 . The method of  claim 73  wherein the RSMM is selected from the group consisting of nitric oxide, nitric dioxide, hydrogen peroxide, superoxide, hypochlorite ion, hydroxyl radical and physiological pO 2 .  
   
   
       75 . The method of  claim 73  wherein the RSMM is an NO adduct.  
   
   
       76 . The method of  claim 75  wherein the NO adduct is DETA-NO, S-nitrosothiol, S-nitroso amino acids, S-nitroso-polypeptides, and nitrosoamines.  
   
   
       77 . The method of  claim 74  wherein the pO 2  is in a range from about 5 to about 100 mm Hg.  
   
   
       78 . The method of  claim 77  wherein the range is from about 10 to about 30 mm Hg.  
   
   
       79 . The method of  claim 78  wherein the range is from about 10 to about 30 mm Hg.  
   
   
       80 . The method of  claim 73  wherein the cell is a yeast cell.  
   
   
       81 . The method of  claim 80  wherein the yeast cell is  S. cerevisiae.    
   
   
       82 . The method of  claim 80  wherein the yeast cell does not express a functional flavohemoglobin gene.  
   
   
       83 . The method of  claim 73  wherein the concentration of the RSMM is between 100 nM to 1 μM and the time is a period of 24 hours.  
   
   
       84 . The method of  claim 73  wherein the media is a liquid media.  
   
   
       85 . The method of  claim 73  wherein the media is a semisolid media.  
   
   
       86 . The method of  claim 73  wherein the media comprises between 0.3% to 10% of a solidifying agent.  
   
   
       87 . The method of  claim 86  wherein the solidifying agent is agar or agarose.  
   
   
       88 - 163 . (canceled)  
   
   
       164 . A method of detecting differences between a protein-protein interaction within a first cell and a second cell: 
 (a) culturing the first cell in a media comprising an RSMM;    (b) isolating a protein complex from the first cell; and    (c) comparing the protein complex to a protein complex from the second cell grown in media without an RSMM.    
   
   
       165 . The method of  claim 164  wherein the first cell and second cell are mammalian cells.  
   
   
       166 . The method of  claim 164  wherein the isolating of the protein complex is by immunoprecipitation.  
   
   
       167 . The method of  claim 164  wherein the protein complex comprises multiple members and at least one member is labeled with a detectable label.  
   
   
       168 . The method of  claim 167  wherein the detectable label is selected from the group consisting of biotin, chemiluminescence, digoxigenin, fluorescence, iodination, kinase, ubiquitin and oligosaccharide.  
   
   
       169 . The method of  claim 164  wherein the RSMM is selected from the group consisting of nitric oxide, nitric dioxide, hydrogen peroxide, superoxide, hypochlorite ion, hydroxyl radical and physiological pO 2 .  
   
   
       170 . The method of  claim 164  wherein the RSMM is an NO adduct.  
   
   
       171 . The method of  claim 170  wherein the NO adduct is DETA-NO, S-nitrosothiol, S-nitroso amino acids, S-nitroso-polypeptides, and nitrosamines.  
   
   
       172 . The method of  claim 164  wherein the RSMM is pO 2  in a range from about 0 to about 100 mm Hg.  
   
   
       173 . The method of  claim 172  wherein the range is from about 10 to about 50 mm Hg.  
   
   
       174 . The method of  claim 173  wherein the range is about 10 to about 30 mm Hg.  
   
   
       175 . The method of  claim 173  wherein the range is about 0 to about 20 mm Hg.  
   
   
       176 . The method of  claim 164  wherein the RSMM is produced by stimulation of an enzyme.  
   
   
       177 . The method of  claim 176  wherein the stimulation is provided by addition of calcium/L-arginine, bradykinin, EGF or other cytokine, growth factor, neuroheumal, or developmental stimulus or activator of a G protein coupled receptor.  
   
   
       178 . The method of  claim 164  wherein the RSMM is produced from an RSMM-generating enzyme.  
   
   
       179 . The method of  claim 178  wherein the RSMM-generating enzyme is produced from a recombinant RSMM-generating enzyme vector.  
   
   
       180 . The method of  claim 178  wherein the RSMM-generating enzyme is NO synthase, NADPH oxidase, or a constitutively active rac G-protein.  
   
   
       181 - 184 . (canceled)  
   
   
       185 . The method of  claim 2  in which the RSMM is selected from the group consisting of nitric oxide, nitric dioxide, dinitrogen trixide, dinitrogen tetraoxide, S-nitrosothiol, nitroxyl anion, HNO, nitrite, nitrate, C—, N, O, S or metal-nitroso or nitro compounds, hydrogen peroxide, peroxynitrite, other peroxides, alkoxides, superoxide, hypochlorite ion, hydroxyl radical and physiological pO 2 .  
   
   
       186 . The method of  claim 2  in which the RSMM is an NO adduct.  
   
   
       187 . The method of  claim 186  wherein the NO adduct is selected from the group consisting of DETA-NO, S-nitrosothiol, SIN-1, angeli's salt, S-nitroso amino acids, S-nitroso-polypeptides, and nitrosoamines.  
   
   
       188 . The method of  claim 2  wherein the pO 2  is in a range from about 5 to about 100 mm Hg.  
   
   
       189 . The method of  claim 188  wherein the range is from about 10 to about 50 mm Hg.  
   
   
       190 . The method of  claim 189  wherein the range is from about 10 to about 30 mm Hg.