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

TLDR

HIFs are key regulators of oxygen homeostasis, and hydroxylation of critical prolines by prolyl‑4‑hydroxylases (P4Hs) targets HIF‑α subunits for proteasomal degradation. The authors investigated the three human HIF‑P4Hs by analyzing isoenzyme transcripts and recombinant enzyme activity. They found that alternative splicing generates inactive isoenzyme transcripts, recombinant HIF‑P4Hs require >8‑residue peptides with isoenzyme‑specific activity, all hydroxylate Pro564 but differ on Pro402, hydroxylate sites in HIF‑2α and HIF‑3α, have O₂ Km just above atmospheric levels, and are inhibited by collagen P4H inhibitors with distinct Ki values, suggesting class‑specific inhibitor development.

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.