Molecular imaging of hypoxia-inducible factor 1 alpha and von Hippel-Lindau interaction in mice.
Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1 alpha is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1 alpha and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1 alpha and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1 alpha and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1 alpha are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.
Pubmed ID: 19123984 RIS Download
Animals | Cell Line, Tumor | Diagnostic Imaging | HCT116 Cells | Humans | Hypoxia-Inducible Factor 1, alpha Subunit | Luciferases, Firefly | Luminescent Measurements | Mice | Mice, Nude | Models, Molecular | Mutation | Proline | Recombinant Fusion Proteins | Von Hippel-Lindau Tumor Suppressor Protein | Whole Body Imaging