Use of grf1 protein for screening molecules

Abstract

The invention concerns the use of whole or part of the GRF1 protein, or of cells expressing whole or part of the GRF1 protein, in methods for detecting compounds for preventing and/or treating pathologies or disorders of the central nervous system involving neuronal death, such as apoptosis, or related to leptin metabolism. The pathologies of the central nervous system are in particular cerebral ischemia, Parkinson's disease or Alzheimer's disease.

1 . The use of all or part of the GRF1 protein, or of cells expressing all or part of the GRF1 protein, in a method for detecting compounds intended for the prevention and/or treatment of pathological conditions or of disorders of the central nervous system involving neuronal death, or associated with obesity or with leptin metabolism.  
     
     
         2 . The use as claimed in  claim 1 , characterized in that said detection method comprises a step consisting of measuring phosphorylation of the GRF1 protein.  
     
     
         3 . The use as claimed in  claim 1 , characterized in that said method comprises a step consisting of measuring exchange activity of the GRF1 protein on proteins of the family of small G proteins, in particular Ras, Rac and Cdc42.  
     
     
         4 . The use as claimed in  claim 1 , characterized in that said method comprises a step consisting of measuring the interaction between the GRF1 protein and the beta-gamma subunits of the heterotrimeric G proteins.  
     
     
         5 . The use as claimed in  claim 1 , characterized in that said detection method comprises a step consisting of bringing the compounds to be screened into contact with yeasts transformed with grf1.  
     
     
         6 . The use as claimed in  claim 5 , characterized in that said detection method comprises a step consisting of bringing the compounds to be screened into contact with yeast transformed with grf1 and in which the CDC25 and/or CDC24 genes have been inactivated or mutated.  
     
     
         7 . The use as claimed in  claim 1 , characterized in that said detection method comprises a step consisting of bringing the compounds to be screened into contact with yeast expressing a first hybrid protein consisting of a fusion protein between Ras, CDC42Hs or Rac and a DNA-interacting domain, and a second hybrid protein consisting of a fusion protein between all or part of GRF1 and a transactivating domain.  
     
     
         8 . The use as claimed in  claim 1 , characterized in that said detection method comprises a step consisting of bringing the compounds to be screened into contact with yeast expressing all or part of GRF1, PAK1 (CRIB domain) fused with a transactivating domain, and Rac or CDC42Hs fused with a DNA-interacting domain.  
     
     
         9 . The use as claimed in  claim 1 , characterized in that said detection method comprises a step consisting of bringing the compounds to be screened into contact with yeast expressing all or part of GRF1, Raf1 (RBD) fused with a transactivating domain and Ras fused with a DNA-interacting domain.  
     
     
         10 . The use as claimed in any one of the preceding claims, characterized in that the pathological condition is cerebral ischemia, Parkinson's disease or Alzheimer's disease.  
     
     
         11 . A method for screening or for detecting compounds intended for the prevention and/or treatment of pathological conditions of the central nervous system involving neuronal death or associated with leptin metabolism, comprising the steps consisting in 
 (i) culturing cells consisting of cells expressing the GRF1 protein in the presence of a phosphorylated substrate and of a test compound,    (ii) lyzing said cells, and    (iii) measuring the amount of labeled GRF1 protein.    
     
     
         12 . A method for screening or for detecting compounds intended for the prevention and/or treatment of pathological conditions of the central nervous system involving neuronal death or associated with leptin metabolism, comprising the prior steps consisting in: 
 (i) culturing cells consisting of cells expressing the GRF1 protein in the presence of a test compound,    (ii) lyzing said cells, and    (iii) measuring the amount of phosphorylated GRF1 using an antibody specific for a phosphorylated amino acid.    
     
     
         13 . A method for screening or for detecting compounds intended for the prevention and/or treatment of pathological conditions of the central nervous system involving neuronal death or associated with leptin metabolism, comprising the steps consisting in: 
 (i) bringing into contact the GRF1 protein, all or part of a protein of the small G protein family loaded with labeled GDP or GTP, and a test compound,    (ii) adding respectively unlabeled GTP or GDP, and    (iii) measuring the amount of labeled G protein.    
     
     
         14 . A method for detecting, by cell screening and in vitro, compounds intended for the prevention and/or treatment of pathological conditions of the central nervous system involving neuronal death or associated with leptin metabolism, comprising the steps consisting in: 
 (i) transfecting immortalized mammalian cells with a vector expressing a grf1 transgene and imparting on said cells resistance to a selection agent,    (ii) selecting and cloning said cells transfected and expressing the transgene and cloning them in an agar growth support,    (iii) adding said test compounds to a suspension of said cells, and    (iv) detecting said GRF1-inhibiting compounds by a decrease in the number of clones.