? Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutation of the VHL tumor suppressor gene. The VHL gene product, pVHL, is part of a multiprotein complex that polyubiquitinates the alpha subunits of the heterodimeric transcription factor called HIF (hypoxia-inducible factor) in the presence of oxygen, which results in their destruction by the proteasome. PVHL binds directly to a colinear 20 amino acid residue sequence located within a region of HIF called the Oxygen-Dependent Degradation Domain (ODD). This interaction is strictly regulated by oxygen-dependent, enzymatic hydroxylation of a conserved proline residue within this HIF 20 mer. I fused this 20 mer to firefly luciferase (ODD-Luciferase). In pilot experiments I established that ODD-luciferase binds to pVHL in vitro in a hydroxylation-dependent manner. In transfection experiments the ODD-luciferase chimera, but not wild-type luciferase, was induced following treatment with hypoxia mimetics. Finally, my preliminary data with tumor xenografts indicate that ODD-luciferase activity can be detected with a Xenogen camera in living animals and is restricted to the central regions of tumors, which are known to be hypoxic. I propose to use ODD-luciferase or an analogous fusion protein, ODD-GFP, as oxygen biosensors. Using these biosensors, I plan to develop cell based assays that might be used to identify compounds that either directly or indirectly disrupt the VHL/HIF interaction. Furthermore, I will develop a transgenic mouse expressing ODD-luciferase. Such mice could be used to study diseases characterized by the presence of hypoxic cells, such as cancer, and could also be used to monitor pharmacodynamic effects of agents that alter tissue oxygenation and/or modulate HIF. ? ?
