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      Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor.

      The Journal of Biological Chemistry

      Alternative Splicing, Amino Acid Sequence, Amino Acid Substitution, Animals, Arginine, genetics, metabolism, Basic Helix-Loop-Helix Transcription Factors, Caenorhabditis elegans, Cell Extracts, Cloning, Molecular, DNA, Complementary, DNA-Binding Proteins, Enzyme Inhibitors, pharmacology, Gene Expression Regulation, Enzymologic, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Isoenzymes, antagonists & inhibitors, Ketoglutaric Acids, Lysine, Molecular Sequence Data, Nuclear Proteins, Procollagen-Proline Dioxygenase, Proline, RNA, Messenger, analysis, Recombinant Proteins, Spodoptera, Substrate Specificity, Trans-Activators, Transcription Factors

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          The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. Hydroxylation of one or two critical prolines by specific hydroxylases (P4Hs) targets their HIF-alpha subunits for proteasomal degradation. By studying the three human HIF-P4Hs, we found that the longest and shortest isoenzymes have major transcripts encoding inactive polypeptides, which suggest novel regulation by alternative splicing. Recombinant HIF-P4Hs expressed in insect cells required peptides of more than 8 residues, distinct differences being found between isoenzymes. All the HIF-P4Hs hydroxylated peptides corresponding to Pro564 in HIF-1alpha, whereas a Pro402 peptide had 20-50-fold Km values for two isoenzymes but was not hydroxylated by the shortest isoenzyme at all; this difference was not explained by the two prolines being in a -Pro402-Ala- and -Pro564-Tyr-sequence. All the HIF-P4Hs-hydroxylated peptides corresponding to two of three potential sites in HIF-2alpha and one in HIF-3alpha. The Km values for O2 were slightly above its atmospheric concentration, indicating that the HIF-P4Hs are effective oxygen sensors. Small molecule inhibitors of collagen P4Hs also inhibited the HIF-P4Hs, but with distinctly different Ki values, indicating that it should be possible to develop specific inhibitors for each class of P4Hs and possibly even for the individual HIF-P4Hs.

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