Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Alternative Splicing
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Amino Acid Sequence
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Amino Acid Substitution
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Animals
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Apoptosis Regulatory Proteins
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Arginine / genetics
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Arginine / metabolism
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Basic Helix-Loop-Helix Transcription Factors
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Caenorhabditis elegans
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Cell Extracts
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Cloning, Molecular
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DNA, Complementary
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DNA-Binding Proteins / metabolism*
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Enzyme Inhibitors / pharmacology
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Gene Expression Regulation, Enzymologic
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Humans
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Isoenzymes / antagonists & inhibitors
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Isoenzymes / genetics*
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Isoenzymes / metabolism*
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Ketoglutaric Acids / metabolism
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Lysine / genetics
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Molecular Sequence Data
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Nuclear Proteins / metabolism*
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Procollagen-Proline Dioxygenase / antagonists & inhibitors
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Procollagen-Proline Dioxygenase / genetics*
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Procollagen-Proline Dioxygenase / metabolism*
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Proline / genetics
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Proline / metabolism
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RNA, Messenger / analysis
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Repressor Proteins
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Spodoptera
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Substrate Specificity
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Trans-Activators / metabolism
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Transcription Factors / metabolism
Substances
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Apoptosis Regulatory Proteins
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Basic Helix-Loop-Helix Transcription Factors
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Cell Extracts
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DNA, Complementary
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DNA-Binding Proteins
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Enzyme Inhibitors
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HIF1A protein, human
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HIF3A protein, human
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Isoenzymes
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Ketoglutaric Acids
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Nuclear Proteins
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RNA, Messenger
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Recombinant Proteins
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Repressor Proteins
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Trans-Activators
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Transcription Factors
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endothelial PAS domain-containing protein 1
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Arginine
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Proline
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Procollagen-Proline Dioxygenase
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Lysine