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

By Maija Hirsilä, P. Koivunen, V. Günzler, K. Kivirikko, J. Myllyharju. Journal of Biological Chemistry, 2003

795 citations recorded by the source corpus.

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.

Subfields: Cancer Research, Genetics, Physiology

View Paper: https://www.semanticscholar.org/paper/bbc1397fdffe2803fa0685c32bcabe84d034a351

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In Cancer Research

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2. Navdeep S. Chandel
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In Physiology

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Citations in the corpus

• Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation. (2005, positive): “Because of their O2 dependence (McNeill et al., 2002) and relatively high Km for O2 in vitro (Hirsila et al., 2003), it has been proposed that the HIFspecific hydroxylases may directly sense O2 deprivation to stabilize HIF.”
• Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. (2005, background): “9% O2) in vitro, indicating that the prolyl hydroxylases are decreasing their enzymatic activity throughout the physiological range of PO2 (Hirsila et al., 2003).”
• Ascorbic acid: Chemistry, biology and the treatment of cancer (2012, background): “All three of the HIF prolyl hydroxylases (PHD1, PHD2, and PHD3) recognize the C-terminal hydroxylation site with a higher affinity than the N-terminal hydroxylation site [75].”
• Influence of Metabolism on Epigenetics and Disease (2013, neutral): “Certain 2-oxoglutarate-dependent dioxygenases, such as the EglN prolyl hydroxylases that mark the HIF transcription factor for destruction, have O2 Km values at or near atmospheric oxygen levels and hence their activity is sensitive to changes in oxygen availability within a physiologically relevant”
• Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing. (2005, background): “PHDs require O2 as a substrate and exhibit a relatively high Km for O2 in vitro (Hirsilae et al., 2003), so it has been proposed that PHDs function as O2 sensors because the decreasing availability of O2 during hypoxia could limit their ability to hydroxylate HIF (Hagen et al., 2003; Epstein et…”
• ADAPTIVE AND MALADAPTIVE CARDIORESPIRATORY RESPONSES TO CONTINUOUS AND INTERMITTENT HYPOXIA MEDIATED BY HYPOXIA-INDUCIBLE FACTORS 1 AND 2 (2012, background): “The observed reduction in hydroxylase activity under hypoxic conditions may be explained by substrate (O2) limitation (49, 80, 108, 115, 225) and/or by an increase in mitochondrial production of reactive oxygen species (ROS) that may oxidize Fe(II) and inactivate the PHDs (29, 107, 212).”
• Oxygen sensing by mitochondria at complex III: the paradox of increased reactive oxygen species during hypoxia (2006, background): “Moreover, these in vitro studies utilized a small peptide derived from HIF-α as a substrate rather than the full-length protein, which may have introduced important differences in the activity of the enzyme (Hirsilae et al. 2003).”
• Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. (2008, background): “Studies of transcriptional selectivity have defined exclusive HIF1a targets, exclusive HIF2a targets, and genes that are responsive to both HIF1a and HIF2a (Holmquist-Mengelbier et al., 2006; Hu et al., 2003; Raval et al., 2005).”
• Prolyl 4-Hydroxylase (2003, methodology): “Furthermore, experiments with various smallmolecule inhibitors with respect to 2-oxoglutarate indicated that there must be distinct differences in the structures of the binding sites between the two classes of prolyl 4hydroxylase, and even between the HIF prolyl 4-hydroxylase isoenzymes (Hirsilä et ”