Hypoxia inducible factor (HIF) is an ab-heterodimer that was first recognized as a DNA-binding protein responsible for mediating the hypoxia-inducible activity of the gene encoding the hematopoietic growth factor erythropoietin. Both subunits contain basic helix-loop-helix (bHLH)-PAS domains. Whereas the bHLH domain defines a superfamily of eukaryotic transcription factors, the PAS domain was first defined in the PER, ARNT, and SIM proteins and defines a subset of the bHLH family. HIF-b subunits are constitu-tively expressed nuclear proteins that are involved via other dimerization partners in a variety of transcriptional responses. The HIF-a subunits exist as three isoforms, all of which are inducible by lack of oxygen (hypoxia). However, in oxygenated cells these a-subunits are rapidly degraded, with HIF-1a and HIF-2a having an exceptionally short half-life of just a few minutes. When the a-subunits are stabilized, HIF dimerizes and interacts with cis-acting hypoxia response elements (HREs) to induce transcriptional activity. A large and rapidly expanding array of genes have now been shown to contain HREs and to be transcription targets of HIF. Examples of these responses include not only erythro-poietin, but also endothelial nitric oxide synthase and endothelin, which control vascular tone; glucose transporters and many key enzymes involved in the metabolism of glucose; enzymes involved in catecholamine synthesis; proteins concerned with iron transport and handling; and checkpoints in cell proliferation and quiescence. However, most importantly with regard to the angiogenic phenotype of VHL disease, many genes that play an essential role in angiogenesis are HIF target genes.
HIF-a subunits contain two domains that regulate their activity in response to oxygen availability: an internal oxygen-dependent degradation domain (ODDD), which regulates protein destruction, and the C-terminal transacti-vation domain (CAD), which regulates transactivating ability through recruitment of the coactivator p300/CBP. In normoxia, enzymatic hydroxylation of two prolyl residues, within the HIF-a ODDD, by a newly defined prolyl hydro-xylase activity facilitates recognition of HIF-a by the VHL E3 ubiquitin ligase complex that targets HIF for destruction by the proteasome. In mammalian cells three closely related enzymes, each the product of a different gene, have been shown to have HIF prolyl hydroxylase activity. These proteins are all nonheme Fe (II) enzymes that are members of the superfamily of 2-oxoglutarate dependent dioxygenases. An absolute requirement for molecular oxygen renders their function oxygen sensitive. In the absence of pVHL, hydro-xylated HIF-a cannot be targeted for ubiquitination and subsequent proteasomal degradation, leading to constitutive upregulation of HIF-mediated gene transcription even in the presence of adequate oxygen for hydroxylase function.
Further oxygen-dependent control is regulated by factor inhibiting HIF (FIH) through hydroxylation of an aspar-aginyl residue in the C-terminal transactivation domain. Hydroxylation at this residue interferes with HIF transacti-vating ability by blocking interaction with the transcrip-tional coactivator CBP/p300. Interestingly, the fact that pVHL loss results in full, rather than partial, activation suggests that pVHL could be involved in aspects of the HIF response to oxygen beyond ODDD-mediated proteolysis. It has been suggested that pVHL might be involved directly in the process of transcriptional inactivation both by promoting the function of FIH and by recruitment of transcriptional repressors.
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