The Principal Investigator's long term objective is to understand the molecular basis of the hypoxic response. A dramatic manifestation of this response is seen in solid tumors. The hypoxic conditions typically found in the core of such tumors activates the transcription factor, hypoxia inducible factor-laipha (HIF-1alpha), which in turn, induces the transcriptional upregulation of vascular endothelial growth factor, a potent angiogenic factor that promotes solid tumor growth. HIF-1alpha is regulated by proteolysis, and under normoxic conditions, is rapidly degraded by the ubiquitin-proteasome pathway. Under hypoxic conditions, this degradation process is markedly inhibited, and HIF-1alpha becomes stabilized and functionally active. The product of the von Hippel Lindau (VHL) tumor suppressor gene is a component of a ubiquitin ligase complex that targets HIF-1alpha for degradation under normoxic conditions. We have recently identified a region of HIF-1 alpha that mediates its binding to HIF-1 alpha and found that hydroxylation of a specific proline in this region is essential for its binding to VHL. Under conditions such as hypoxia or cobalt (a hypoxia mimic), this unusual modification is inhibited, allowing HIF-1 alpha to escape VHL-mediated degradation. In the first Specific Aim, we will first examine the effects of mutagenesis of additional residues in the vicinity of this proline on the stability and VHL-binding properties of HIF-1alpha. In the second Specific Aim, we will examine whether hypoxia induces hydroxyproline-independent changes; in particular, we will examine the effects of hypoxia or cobalt on the ubiquitinating activity of VHL. In the third Specific Aim, we will test the hypothesis that praline hydroxylation is not simply restricted to hypoxia-induced HIF-1alpha activation in two ways. First, we will examine whether another known inducer of HIF-1alpha, phorbol myristate acetate, induces changes in H IF-1 alpha praline hydroxylation. Second, we will perform an in vitro expression screen to identify and subsequently characterize navel proteins containing hydroxyproline. We anticipate that the proposed studies will enlarge our understanding of both the molecular response to hypoxia and the role of a novel postranslational modification--proline hydroxylation.
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