Lack of Activity of Recombinant HIF Prolyl Hydroxylases (PHDs) on Reported Non-HIF Substrates

Overview

Journal Elife

Specialty Biology

Date 2019 Sep 11

PMID 31500697

Citations 58

Authors

Matthew E Cockman

Kerstin Lippl

Ya-Min Tian

Hamish B Pegg

William D Figg Jnr

Martine I Abboud

Raphael Heilig

Roman Fischer

Johanna Myllyharju

Christopher J Schofield

Peter J Ratcliffe

Affiliations

Soon will be listed here.

Abstract

Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivity with recombinant PHD enzymes. Unexpectedly, we did not detect prolyl-hydroxylase activity on any reported non-HIF protein or peptide, using conditions supporting robust HIF-α hydroxylation. We cannot exclude PHD-catalysed prolyl hydroxylation occurring under conditions other than those we have examined. However, our findings using recombinant enzymes provide no support for the wide range of non-HIF PHD substrates that have been reported.

Citing Articles

Hypoxia-inducible factor in cancer: from pathway regulation to therapeutic opportunity.

Ortmann B BMJ Oncol. 2025; 3(1):e000154.

PMID: 39886164 PMC: 11203102. DOI: 10.1136/bmjonc-2023-000154.

Tumour hypoxia in driving genomic instability and tumour evolution.

Suvac A, Ashton J, Bristow R Nat Rev Cancer. 2025; 25(3):167-188.

PMID: 39875616 DOI: 10.1038/s41568-024-00781-9.

Hypoxia-induced increase in sphingomyelin synthase 2 aggravates ischemic skeletal muscle inflammation.

Mizugaki H, Nagane M, Sato-Akaba H, Kmiec M, Kuppusamy P, Yasui H FEBS J. 2024; 292(5):1086-1105.

PMID: 39739672 PMC: 11880985. DOI: 10.1111/febs.17379.

JmjC catalysed histone H2a N-methyl arginine demethylation and C4-arginine hydroxylation reveals importance of sequence-reactivity relationships.

Bonnici J, Oueini R, Salah E, Johansson C, Pires E, Abboud M Commun Biol. 2024; 7(1):1583.

PMID: 39604683 PMC: 11603075. DOI: 10.1038/s42003-024-07183-5.

Human prolyl hydroxylase domain 2 reacts with O and 2-oxoglutarate to enable formation of inactive Fe(III).2OG.hypoxia-inducible-factor α complexes.

Fiorini G, Marshall S, Figg Jr W, Myers W, Brewitz L, Schofield C Sci Rep. 2024; 14(1):26162.

PMID: 39478091 PMC: 11525979. DOI: 10.1038/s41598-024-75761-y.

References

Arsenault P, Heaton-Johnson K, Li L, Song D, Ferreira V, Patel N . Identification of prolyl hydroxylation modifications in mammalian cell proteins. Proteomics. 2014; 15(7):1259-67. PMC: 4438755. DOI: 10.1002/pmic.201400398. View

Bekker-Jensen D, Kelstrup C, Batth T, Larsen S, Haldrup C, Bramsen J . An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes. Cell Syst. 2017; 4(6):587-599.e4. PMC: 5493283. DOI: 10.1016/j.cels.2017.05.009. View

Gupta N, Wish J . Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors: A Potential New Treatment for Anemia in Patients With CKD. Am J Kidney Dis. 2017; 69(6):815-826. DOI: 10.1053/j.ajkd.2016.12.011. View

Kaelin Jr W, Ratcliffe P . Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell. 2008; 30(4):393-402. DOI: 10.1016/j.molcel.2008.04.009. View

Hirsila M, Koivunen P, Gunzler V, Kivirikko K, Myllyharju J . Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor. J Biol Chem. 2003; 278(33):30772-80. DOI: 10.1074/jbc.M304982200. View

Zheng X, Zhai B, Koivunen P, Shin S, Lu G, Liu J . Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase. Genes Dev. 2014; 28(13):1429-44. PMC: 4083087. DOI: 10.1101/gad.242131.114. View

Xie L, Pi X, Townley-Tilson W, Li N, Wehrens X, Entman M . PHD2/3-dependent hydroxylation tunes cardiac response to β-adrenergic stress via phospholamban. J Clin Invest. 2015; 125(7):2759-71. PMC: 4563684. DOI: 10.1172/JCI80369. View

German N, Yoon H, Yusuf R, Murphy J, Finley L, Laurent G . PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2. Mol Cell. 2016; 63(6):1006-20. PMC: 5040345. DOI: 10.1016/j.molcel.2016.08.014. View

Semenza G . Hypoxia-inducible factors in physiology and medicine. Cell. 2012; 148(3):399-408. PMC: 3437543. DOI: 10.1016/j.cell.2012.01.021. View

10.

Guo J, Chakraborty A, Liu P, Gan W, Zheng X, Inuzuka H . pVHL suppresses kinase activity of Akt in a proline-hydroxylation-dependent manner. Science. 2016; 353(6302):929-32. PMC: 5326551. DOI: 10.1126/science.aad5755. View

11.

Davis S, Charles P, He L, Mowlds P, Kessler B, Fischer R . Expanding Proteome Coverage with CHarge Ordered Parallel Ion aNalysis (CHOPIN) Combined with Broad Specificity Proteolysis. J Proteome Res. 2017; 16(3):1288-1299. PMC: 5363888. DOI: 10.1021/acs.jproteome.6b00915. View

12.

Chowdhury R, McDonough M, Mecinovic J, Loenarz C, Flashman E, Hewitson K . Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases. Structure. 2009; 17(7):981-9. DOI: 10.1016/j.str.2009.06.002. View

13.

Koditz J, Nesper J, Wottawa M, Stiehl D, Camenisch G, Franke C . Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor. Blood. 2007; 110(10):3610-7. DOI: 10.1182/blood-2007-06-094441. View

14.

Niwa T, Ying B, Saito K, Jin W, Takada S, Ueda T . Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins. Proc Natl Acad Sci U S A. 2009; 106(11):4201-6. PMC: 2657415. DOI: 10.1073/pnas.0811922106. View

15.

Huo Z, Ye J, Chen J, Lin X, Zhou Z, Xu X . Prolyl hydroxylase domain protein 2 regulates the intracellular cyclic AMP level in cardiomyocytes through its interaction with phosphodiesterase 4D. Biochem Biophys Res Commun. 2012; 427(1):73-9. DOI: 10.1016/j.bbrc.2012.09.005. View

16.

Koivunen P, Hirsila M, Kivirikko K, Myllyharju J . The length of peptide substrates has a marked effect on hydroxylation by the hypoxia-inducible factor prolyl 4-hydroxylases. J Biol Chem. 2006; 281(39):28712-20. DOI: 10.1074/jbc.M604628200. View

17.

Maxwell P, Eckardt K . HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond. Nat Rev Nephrol. 2015; 12(3):157-68. DOI: 10.1038/nrneph.2015.193. View

18.

Rodriguez J, Pilkington R, Munoz A, Nguyen L, Rauch N, Kennedy S . Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways. Cell Rep. 2016; 14(11):2745-60. PMC: 4805855. DOI: 10.1016/j.celrep.2016.02.043. View

19.

Elkins J, Hewitson K, McNeill L, Seibel J, Schlemminger I, Pugh C . Structure of factor-inhibiting hypoxia-inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha. J Biol Chem. 2002; 278(3):1802-6. DOI: 10.1074/jbc.C200644200. View

20.

Scotti J, Leung I, Ge W, Bentley M, Paps J, Kramer H . Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation. Proc Natl Acad Sci U S A. 2014; 111(37):13331-6. PMC: 4169948. DOI: 10.1073/pnas.1409916111. View