Interaction Between the VHL Tumour Suppressor and Hypoxia Inducible Factor, and Assay Methods Relating Thereto

Abstract

The invention relates to the finding that the VHL tumour suppressor protein regulates hypoxia inducible factor α subunits, by targeting HIF α for destruction in normoxic, but not hypoxic cells. The invention provides assays for modulators of this interaction, and peptides based upon HIF α subunit sequences which may modulate this interaction.

1 . An assay for a modulator of VHL-HIF α subunit interaction, which comprises:
 a) bringing into contact a VHL protein, a HIF α subunit protein and a putative modulator compound under conditions where the VHL protein and the HIF α subunit protein, in the absence of modulator, are capable of forming a complex; and 
 b) measuring the degree of inhibition of complex formation caused by said modulator compound. 
 
     
     
         2 . An assay according to  claim 1  in the form of a two-hybrid assay. 
     
     
         3 . An assay according to  claim 1  in the form of an immunoprecipitation. 
     
     
         4 . An assay according to  claim 1  wherein at least one of said proteins is labelled with a detectable label. 
     
     
         5 . An assay according to any one of the preceding claims wherein at least one of said proteins is in the form of a fusion protein. 
     
     
         6 . An assay according to any one of the preceding claims wherein the ubiquitylation of the HIF α subunit is determined. 
     
     
         7 . An assay a modulator of VHL-HIF α subunit interaction, which comprises:
 a) bringing into contact a VHL protein, a HIF α subunit protein and a putative modulator compound under conditions where the VHL protein and the HIF α subunit protein, in the absence of modulator, are capable of forming a complex; 
 b) providing an HIF response element to which the HIF α subunit protein is capable of binding and transcriptionally activating; and 
 c) measuring the degree of modulation of binding of the α subunit to, or transcriptional activation of, the response element caused by said modulator compound. 
 
     
     
         8 . An assay according to  claim 6  wherein said response element is operably linked to a reporter gene. 
     
     
         9 . An assay according to any one of the preceding claims wherein said VHL protein is human VHL (Genbank accession number AF010238) or a fragment thereof comprising at least residues 63-156. 
     
     
         10 . An assay according to any one of the preceding claims wherein said HIF α subunit protein is human HIF α subunit protein (Genbank accession number U22431) or a fragment thereof comprising at least residues 549-572. 
     
     
         11 . An isolated polypeptide which consists of from 5 to 50 amino acids whose sequence is found in region 549-652, particularly 549-572 of human HIF-1α (Genbank accession number U22431). 
     
     
         12 . An isolated polypeptide which consists of from 5 to 50 amino acids, said polypeptide being capable of forming a complex with VHL, and characterised by the presence of a sequence selected from: 
       
         
           
                 
                 
                 
               
                     
                   LAPYIPMD; 
                   SEQ ID NO: 1 
                 
                     
                     
                 
                     
                   LAPYISMD; 
                   SEQ ID NO: 2 
                 
                     
                     
                 
                     
                   LLPYIPMD; 
                   SEQ ID NO: 3 
                 
                     
                     
                 
                     
                   LVPYIPMD; 
                   SEQ ID NO: 4 
                 
                     
                     
                 
                     
                   IAPYIPMD; 
                   SEQ ID NO: 5 
                 
                     
                     
                 
                     
                   IAPYIPME; 
                   SEQ ID NO: 6 
                 
                     
                   and 
                     
                 
                     
                     
                 
                     
                   LVPYISMD. 
                   SEQ ID NO: 7 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         13 . A polypeptide according to  claim 12  which comprises the sequence: 
       
         
           
                 
                 
                 
               
                     
                   DLDLEMLAPYIPMDDDFQL; 
                   (SEQ ID NO: 8) 
                 
             
                
               
            
           
         
       
       and
 variants thereof in which there are from 1 to 4 substitutions. 
 
     
     
         14 . A polypeptide according to  claim 12  which comprises the sequence: 
       
         
           
                 
                 
               
                   PFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSP; 
                   (SEQ ID NO: 9) 
                 
             
                
               
            
           
         
       
       and
 variants thereof in which there are from 1 to 4 substitutions. 
 
     
     
         15 . A polypeptide comprising the polypeptide of any one of  claims 11  to  14  fused to a membrane translocation sequence. 
     
     
         16 . A polypeptide according to any one of  claims 11  to  14  for use in a method of inhibiting the interaction of a HIF α subunit with VHL. 
     
     
         17 . A method of promoting angiogenesis and/or cellular survival in a cell exposed to a hypoxic environment, said method comprising blocking the interaction between VHL and a HIF α subunit. 
     
     
         18 . An assay for a modulator which promotes VHL-HIF α subunit interaction, which assay comprises:
 a) bringing into contact a HIF α subunit protein and a putative modulator compound in the presence or absence of a VHL protein, 
 b) providing a VHL protein where said protein is absent in step (a); and 
 c) determining whether the VHL-HIF α subunit interaction has been promoted by the presence of the modulator.