Characterization of the Human Prolyl 4-Hydroxylases That Modify the Hypoxia-inducible Factor

• Published in: The Journal of biological chemistry 2003-08, Vol.278 (33), p.30772-30780
• Main Authors: Hirsilä, Maija, Koivunen, Peppi, Günzler, Volkmar, Kivirikko, Kari I., Myllyharju, Johanna
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Amino Acid Sequence; Amino Acid Substitution; Animals; Apoptosis Regulatory Proteins; Arginine - genetics; Arginine - metabolism; Basic Helix-Loop-Helix Transcription Factors; Caenorhabditis elegans; Cell Extracts; Cloning, Molecular; DNA, Complementary; DNA-Binding Proteins - metabolism; Enzyme Inhibitors - pharmacology; Gene Expression Regulation, Enzymologic; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Isoenzymes - antagonists & inhibitors; Isoenzymes - genetics; Isoenzymes - metabolism; Ketoglutaric Acids - metabolism; Lysine - genetics; Molecular Sequence Data; Nuclear Proteins - metabolism; Procollagen-Proline Dioxygenase - antagonists & inhibitors; Procollagen-Proline Dioxygenase - genetics; Procollagen-Proline Dioxygenase - metabolism; Proline - genetics; Proline - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Repressor Proteins; RNA, Messenger - analysis; Spodoptera; Substrate Specificity; Trans-Activators - metabolism; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M304982200

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-α 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-1α, 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-2α and one in HIF-3α. 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.