Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor induced by hypoxia. Under normoxic conditions, site-specific proline hydroxylation of the ␣ subunits of HIF allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets these subunits for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this hydroxylation is inhibited, allowing the ␣ subunits of HIF to escape VHL-mediated degradation. Three enzymes, prolyl hydroxylase domain-containing proteins 1, 2, and 3 (PHD1, -2, and -3; also known as HIF prolyl hydroxylase 3, 2, and 1, respectively), have recently been identified that catalyze proline hydroxylation of HIF ␣ subunits. These enzymes hydroxylate specific prolines in HIF ␣ subunits in the context of a strongly conserved LXXLAP sequence motif (where X indicates any amino acid and P indicates the hydroxylacceptor proline). We report here that PHD2 has the highest specific activity toward the primary hydroxylation site of HIF-1␣. Furthermore, and unexpectedly, mutations can be tolerated at the ؊5, ؊2, and ؊1 positions (relative to proline) of the LXXLAP motif. Thus, these results provide evidence that the only obligatory residue for proline hydroxylation in HIF-1␣ is the hydroxylacceptor proline itself.A critical means by which cells respond to stress is by the modulation of gene expression. The cellular response to hypoxia provides a cardinal example of this (1-3). Low oxygen tension (1% O 2 ) induces a battery of genes that allow cells to increase their uptake of glucose (through up-regulation of specific isoforms of the glucose transporter) and shift utilization of this energy source from aerobic to glycolytic metabolism (through up-regulation of genes such as phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, among others) (4). Low oxygen tension also induces the upregulation of genes involved in local and systemic responses to hypoxia. An example of the former is vascular endothelial growth factor, a potent angiogenic agent, whereas an example of the latter is erythropoietin, the central regulator of red blood cell maturation. Hypoxia-inducible factor (HIF)1 is a transcription factor that is the master regulator of all of the above genes (1, 2). HIF is heterodimeric and comprises an ␣ and a  subunit, both of which in turn are members of the PAS family of transcription factors. The  subunit is a member of the aryl hydrocarbon nuclear translocator family of proteins. Three ␣ subunit isoforms have been identified, HIF-1␣, HIF-2␣, and HIF-3␣ (5-9). The central mechanism by which HIF is activated by hypoxia is by regulated stabilization of the ␣ subunit. In the case of HIF-1␣, the most intensively studied isoform, two proline residues, Pro-402 and Pro-564, are constitutively hydroxylated under normoxic conditions (10 -13). This hydroxylation allows binding by the von Hippel-Lindau tumor suppressor protein (VHL) (14), the substrate recognition component of an E3 ubiquit...
A central means by which mammalian cells respond to low oxygen tension is through the activation of the transcription factor HIF-1 (hypoxia-inducible factor-1). Under normoxic conditions, HIF-1alpha (the alpha subunit of HIF-1) is targeted for rapid degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this degradation is inhibited, thereby leading to the stabilization and activation of HIF-1alpha. Here, we report the identification of IOP1 (iron-only hydrogenase-like protein 1), a protein homologous with enzymes present in anaerobic organisms that contain a distinctive iron-sulfur cluster. IOP1 is present in a broad range of cell types. Knockdown of IOP1 using siRNA (small interfering RNA) in mammalian cells increases protein levels of HIF-1alpha under both normoxic and hypoxic conditions, and augments hypoxia-induced HRE (hypoxia response element) reporter gene and endogenous HIF-1alpha target gene expressions. We find that IOP1 knockdown up-regulates HIF-1alpha mRNA levels, thereby providing a mechanism by which knockdown induces the observed effects. The results collectively provide evidence that IOP1 is a component of the protein network that regulates HIF-1alpha in mammalian cells.
BackgroundThe hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the α subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1α in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).ResultsPatients with IE were screened for changes in the HIF-1α coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.ConclusionThe Pro582Ser change represents a common polymorphism of HIF-1α that does not impair HIF-1α prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1α it does not diminish the association of HIF-1α with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.
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