Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor.
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.
Hirsilä
Maija
M
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland.
Koivunen
Peppi
P
Günzler
Volkmar
V
Kivirikko
Kari I
KI
Myllyharju
Johanna
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins; D017173:Caenorhabditis elegans; D002457:Cell Extracts; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D018411:Spodoptera; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
D051017:Apoptosis Regulatory Proteins; D001120:Arginine; D051792:Basic Helix-Loop-Helix Proteins; D002457:Cell Extracts; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D007656:Ketoglutaric Acids; D008239:Lysine; D009687:Nuclear Proteins; D011393:Procollagen-Proline Dioxygenase; D011392:Proline; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C116973:HIF3A protein, human