oxalylglycine and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Hypoxia-inducible factor-1 (HIF-1), the master regulator of transcriptional responses to reduced oxygen tension (hypoxia) in mammalian cells, consists of one HIF-1alpha and one HIF-1beta subunit. In normoxia, HIF-1alpha subunits are hydroxylated on specific proline residues; modifications that signal ubiquitination and degradation of HIF-1alpha by the proteasome. To test the effect of saturating HIF-1alpha degradation, we generated a construct, denoted the saturating domain (SD), based on a region surrounding proline 564 (Pro564) in HIF-1alpha. Expression of the SD led to accumulation of endogenous HIF-1alpha proteins in nuclei of normoxic cells. The induced HIF-1alpha was functional as it activated expression from a hypoxia-regulated reporter gene and from the endogenous vascular endothelial growth facor-a (Vegf-a) and carbonic anhydrase 9 (Ca9) genes. The effect of the SD was dependent on Pro564 since a mutated SD, in which Pro564 had been replaced by a glycine residue, failed to bind the von Hippel-Lindau protein (pVHL) and to stabilise HIF-1alpha. Treatment of cells with the prolylhydroxylase inhibitor dimethyloxalylglycine, or the proteasome inhibitor MG-132, mimicked the effect of the SD. In conclusion, we show that blocking HIF-1alpha degradation, either by saturation, or inhibition of prolyl hydroxylases or proteosomal degradation, leads to nuclear localisation of active HIF-1alpha proteins.

Topics: Amino Acids, Dicarboxylic; Animals; Antigens, Neoplasm; Carbonic Anhydrase IX; Carbonic Anhydrases; Cell Hypoxia; Cell Line; Cell Nucleus; Chlorocebus aethiops; COS Cells; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Microscopy, Confocal; Nuclear Proteins; Oxygen; Peptide Fragments; Procollagen-Proline Dioxygenase; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Transcription Factors; Transcription, Genetic; Transfection; Tumor Suppressor Proteins; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Vascular Endothelial Growth Factor A; Von Hippel-Lindau Tumor Suppressor Protein

Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)-HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.

Topics: Amino Acids, Dicarboxylic; Caspase 3; Caspases; Cell Hypoxia; Cell Line; Cysteine Proteinase Inhibitors; Deferoxamine; Enzyme Inhibitors; Ferric Compounds; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Iron; Leupeptins; Nitric Oxide; Nitric Oxide Donors; Procollagen-Proline Dioxygenase; Proteasome Endopeptidase Complex; Proteasome Inhibitors; S-Nitrosoglutathione; Transcription Factors; Triazenes; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein