Biology:EGLN1

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Short description: Protein-coding gene in the species Homo sapiens

A representation of the 3D structure of the protein myoglobin showing turquoise α-helices.
Generic protein structure example


Hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2), or prolyl hydroxylase domain-containing protein 2 (PHD2), is an enzyme encoded by the EGLN1 gene. It is also known as Egl nine homolog 1.[1][2][3][4] PHD2 is a α-ketoglutarate/2-oxoglutarate-dependent hydroxylase, a superfamily non-haem iron-containing proteins. In humans, PHD2 is one of the three isoforms of hypoxia-inducible factor-proline dioxygenase, which is also known as HIF prolyl-hydroxylase.

The hypoxia response

HIF-1α is a ubiquitous, constitutively synthesized transcription factor responsible for upregulating the expression of genes involved in the cellular response to hypoxia. These gene products may include proteins such as glycolytic enzymes and angiogenic growth factors.[5] In normoxia, HIF alpha subunits are marked for the ubiquitin-proteasome degradation pathway through hydroxylation of proline-564 and proline-402 by PHD2. Prolyl hydroxylation is critical for promoting pVHL binding to HIF, which targets HIF for polyubiquitylation.[4]

Structure

The iron binding site of PHD2.

PHD2 is a 46-kDa enzyme that consists of an N-terminal domain homologous to MYND zinc finger domains, and a C-terminal domain homologous to the 2-oxoglutarate dioxygenases. The catalytic domain consists of a double-stranded β-helix core that is stabilized by three α-helices packed along the major β-sheet.[6] The active site, which is contained in the pocket between the β-sheets, chelates iron(II) through histidine and aspartate coordination. 2-oxoglutarate displaces a water molecule to bind iron as well.[7] The active site is lined by hydrophobic residues, possibly because such residues are less susceptible to potential oxidative damage by reactive species leaking from the iron center.[6]

PHD2 catalyses the hydroxylation of two sites on HIF-α, which are termed the N-terminal oxygen dependent degradation domain (residues 395-413, NODD) and the C-terminal oxygen dependent degradation domain (residues 556-574, CODD).[8][9] These two HIF substrates are usually represented by 19 amino acid long peptides in in vitro experiments.[10] X-ray crystallography and NMR spectroscopy showed that both peptides bind to the same binding site on PHD2, in a cleft on the PHD2 surface.[7][11] An induced fit mechanism was indicated from the structure, in which residues 237-254 adopt a closed loop conformation, whilst in the structure without CODD or NODD, the same residues adopted an open finger-like conformation.[7][11] Such conformational change was confirmed by NMR spectroscopy,[11] X-ray crystallography[7][11] and molecular dynamics calculations.[12] A recent study found a second peptide binding site on PHD2 although peptide binding to this alternative site did not seem to affect the catalytic activity of the enzyme.[13] Further studies are required to fully understand the biological significance of this second peptide binding site.

The enzyme has a high affinity for iron(II) and 2-oxoglutarate (also known as α-ketoglutarate), and forms a long-lived complex with these factors.[14] It has been proposed that cosubstrate and iron concentrations poise the HIF hydroxylases to respond to an appropriate "hypoxic window" for a particular cell type or tissue.[15] Studies have revealed that PHD2 has a KM for dioxygen slightly above its atmospheric concentration, and PHD2 is thought to be the most important sensor of the cell's oxygen status.[16]

Mechanism

The enzyme incorporates one oxygen atom from dioxygen into the hydroxylated product, and one oxygen atom into the succinate coproduct.[17] Its interactions with HIF-1α rely on a mobile loop region that helps to enclose the hydroxylation site and helps to stabilize binding of both iron and 2-oxyglutarate.[7] A feedback regulation mechanism that involves the displacement of HIF-1α by hydroxylated HIF-1α when 2-oxoglutarate is limiting was also proposed.[18]

PHD2 acts as a dioxygenase to hydroxylate proline and convert 2-oxoglutarate to succinate.

Biological role and disease relevance

PHD2 is the primary regulator of HIF-1α steady state levels in the cell. A PHD2 knockdown showed increased levels of HIF-1α under normoxia, and an increase in HIF-1α nuclear accumulation and HIF-dependent transcription. HIF-1α steady state accumulation was dependent on the amount of PHD silencing effected by siRNA in HeLa cells and a variety of other human cell lines.[4]

However, although it would seem that PHD2 downregulates HIF-1α and thus also tumorigenesis, there have been suggestions of paradoxical roles of PHD2 in tumor proliferation. For example, one animal study showed tumor reduction in PHD2-deficient mice through activation of antiproliferative TGF-β signaling.[19] Other in vivo models showed tumor-suppressing activity for PHD2 in pancreatic cancer as well as liver cancer.[20][21] A study of 121 human patients revealed PHD2 as a strong prognostic marker in gastric cancer, with PHD2-negative patients having shortened survival compared to PHD2-positive patients.[22]

Recent genome-wide association studies have suggested that EGLN1 may be involved in the low hematocrit phenotype exhibited by the Tibetan population and hence that EGLN1 may play a role in the heritable adaptation of this population to live at high altitude.[23]

As a therapeutic target

HIF's important role as a homeostatic mediator implicates PHD2 as a therapeutic target for a range of disorders regarding angiogenesis, erythropoeisis, and cellular proliferation. There has been interest both in potentiating and inhibiting the activity of PHD2.[5] For example, methylselenocysteine (MSC) inhibition of HIF-1α led to tumor growth inhibition in renal cell carcinoma in a PHD-dependent manner. It is thought that this phenomenon relies on PHD-stabilization, but mechanistic details of this process have not yet been investigated.[24] On the other hand, screens of small-molecule chelators have revealed hydroxypyrones and hydroxypyridones as potential inhibitors for PHD2.[25] Recently, dihydropyrazoles, a triazole-based small molecule, was found to be a potent inhibitor of PHD2 that is active both in vitro and in vivo.[26] Substrate analog peptides have also been developed to exhibit inhibitory selectivity for PHD2 over factor inhibiting HIF (FIH), for which some other PHD-inhibitors show overlapping specificity.[27] Gasotransmitters including carbon monoxide[28] and nitric oxide[29][30] are also inhibitors of PHD2 by competing with molecular oxygen for binding at the active site Fe(II) ion.

Additionally, PHD2 holds significant promise as a therapeutic target for ischemic conditions.[31] Ischemia, characterized by reduced blood flow and oxygen supply, can lead to severe tissue damage and dysfunction.[32] Modulating PHD2 activity in ischemic conditions can enhance tissue survival and recovery by stabilizing HIF-1α, which in turn activates genes that facilitate adaptive responses to hypoxia. This includes promoting angiogenesis, erythropoiesis, and metabolic reprogramming, crucial for cell survival under oxygen-deprived conditions.[33][34] Preclinical studies have suggested that inhibition of PHD2 can reduce tissue damage in models of myocardial infarction and cerebral ischemia, providing a foundation for future therapeutic strategies aimed at minimizing the consequences of acute ischemic events.[35] Ongoing research continues to explore the efficacy and safety of PHD2 inhibitors in various ischemic scenarios, with the potential to extend these findings to clinical applications.

References

  1. ↑ "Mapping, characterization, and expression analysis of the SM-20 human homologue, c1orf12, and identification of a novel related gene, SCAND2". Genomics 69 (3): 348–354. November 2000. doi:10.1006/geno.2000.6343. PMID 11056053. 
  2. ↑ "Characterization and comparative analysis of the EGLN gene family". Gene 275 (1): 125–132. September 2001. doi:10.1016/S0378-1119(01)00633-3. PMID 11574160. 
  3. ↑ "Entrez Gene: EGLN1 egl nine homolog 1 (C. elegans)". https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=ShowDetailView&TermToSearch=54583. 
  4. ↑ 4.0 4.1 4.2 "HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia". The EMBO Journal 22 (16): 4082–4090. August 2003. doi:10.1093/emboj/cdg392. PMID 12912907. 
  5. ↑ 5.0 5.1 "The prolyl hydroxylase enzymes that act as oxygen sensors regulating destruction of hypoxia-inducible factor alpha". Advances in Enzyme Regulation 44: 75–92. 2004. doi:10.1016/j.advenzreg.2003.11.017. PMID 15581484. 
  6. ↑ 6.0 6.1 "Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2)". Proceedings of the National Academy of Sciences of the United States of America 103 (26): 9814–9819. June 2006. doi:10.1073/pnas.0601283103. PMID 16782814. Bibcode: 2006PNAS..103.9814M. 
  7. ↑ 7.0 7.1 7.2 7.3 7.4 "Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases". Structure 17 (7): 981–989. July 2009. doi:10.1016/j.str.2009.06.002. PMID 19604478. 
  8. ↑ "Chemical basis for the selectivity of the von Hippel Lindau tumor suppressor pVHL for prolyl-hydroxylated HIF-1alpha". Biochemistry 49 (32): 6936–6944. August 2010. doi:10.1021/bi100358t. PMID 20695530. 
  9. ↑ "Studies on the Substrate Selectivity of the Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain". ChemBioChem 19 (21): 2262–2267. November 2018. doi:10.1002/cbic.201800246. PMID 30144273. 
  10. ↑ "Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases". The Journal of Biological Chemistry 283 (7): 3808–3815. February 2008. doi:10.1074/jbc.M707411200. PMID 18063574. 
  11. ↑ 11.0 11.1 11.2 11.3 "Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases". Nature Communications 7. August 2016. doi:10.1038/ncomms12673. PMID 27561929. Bibcode: 2016NatCo...712673C. 
  12. ↑ "Structural insight into the prolyl hydroxylase PHD2: a molecular dynamics and DFT study". Eur J Inorg Chem 2012 (31): 4973–4985. Nov 2012. doi:10.1002/ejic.201200391. 
  13. ↑ "Non-competitive cyclic peptides for targeting enzyme-substrate complexes". Chemical Science 9 (20): 4569–4578. May 2018. doi:10.1039/C8SC00286J. PMID 29899950. 
  14. ↑ "Hypoxia-inducible factor prolyl hydroxylase 2 has a high affinity for ferrous iron and 2-oxoglutarate". Molecular BioSystems 1 (4): 321–324. October 2005. doi:10.1039/b511249b. PMID 16880998. 
  15. ↑ "Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay". The Biochemical Journal 401 (1): 227–234. January 2007. doi:10.1042/BJ20061151. PMID 16952279. 
  16. ↑ "Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor". The Journal of Biological Chemistry 278 (33): 30772–30780. August 2003. doi:10.1074/jbc.M304982200. PMID 12788921. 
  17. ↑ "The use of dioxygen by HIF prolyl hydroxylase (PHD1)". Bioorganic & Medicinal Chemistry Letters 12 (12): 1547–1550. June 2002. doi:10.1016/S0960-894X(02)00219-6. PMID 12039559. 
  18. ↑ "2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2". Chemical Communications 54 (25): 3130–3133. March 2018. doi:10.1039/C8CC00387D. PMID 29522057. 
  19. ↑ "Inhibition of HIF prolyl hydroxylase-2 blocks tumor growth in mice through the antiproliferative activity of TGFβ". Cancer Research 71 (9): 3306–3316. May 2011. doi:10.1158/0008-5472.CAN-10-3838. PMID 21436457. 
  20. ↑ "Prolyl hydroxylase-2 (PHD2) exerts tumor-suppressive activity in pancreatic cancer". Cancer 118 (4): 960–972. February 2012. doi:10.1002/cncr.26344. PMID 21792862. 
  21. ↑ "Effect of prolyl hydroxylase domain-2 haplodeficiency on the hepatocarcinogenesis in mice". Journal of Hepatology 57 (1): 61–68. July 2012. doi:10.1016/j.jhep.2012.02.021. PMID 22420978. 
  22. ↑ "Prolyl hydroxylase domain 2 protein is a strong prognostic marker in human gastric cancer". Pathobiology 79 (1): 11–17. Jan 2012. doi:10.1159/000330170. PMID 22236543. 
  23. ↑ "Genetic evidence for high-altitude adaptation in Tibet". Science 329 (5987): 72–75. July 2010. doi:10.1126/science.1189406. PMID 20466884. Bibcode: 2010Sci...329...72S. 
  24. ↑ "Prolyl hydroxylase 2 dependent and Von-Hippel-Lindau independent degradation of Hypoxia-inducible factor 1 and 2 alpha by selenium in clear cell renal cell carcinoma leads to tumor growth inhibition". BMC Cancer 12. July 2012. doi:10.1186/1471-2407-12-293. PMID 22804960. 
  25. ↑ "Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH)". Journal of Inorganic Biochemistry 113: 25–30. August 2012. doi:10.1016/j.jinorgbio.2012.03.002. PMID 22687491. 
  26. ↑ "Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain". PLOS ONE 10 (7). Jul 2015. doi:10.1371/journal.pone.0132004. PMID 26147748. Bibcode: 2015PLoSO..1032004C. 
  27. ↑ "Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides". Bioorganic & Medicinal Chemistry Letters 21 (14): 4325–4328. July 2011. doi:10.1016/j.bmcl.2011.05.050. PMID 21665470. 
  28. ↑ "Carbon Monoxide is an Inhibitor of HIF Prolyl Hydroxylase Domain 2". ChemBioChem 22 (15): 2521–2525. August 2021. doi:10.1002/cbic.202100181. PMID 34137488. 
  29. ↑ "Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases". Molecular Biology of the Cell 14 (8): 3470–3481. August 2003. doi:10.1091/mbc.E02-12-0791. PMID 12925778. 
  30. ↑ "Nitric oxide modulates oxygen sensing by hypoxia-inducible factor 1-dependent induction of prolyl hydroxylase 2". The Journal of Biological Chemistry 282 (3): 1788–1796. January 2007. doi:10.1074/jbc.M607065200. PMID 17060326. 
  31. ↑ "Mechanistic insights into gut microbe derived siderophores and PHD2 interactions with implications for HIF-1α stabilization". Scientific Reports 15 (1). January 2025. doi:10.1038/s41598-024-83730-8. PMID 39774022. Bibcode: 2025NatSR..15.1113P. 
  32. ↑ "Mechanisms of neuronal damage in brain hypoxia/ischemia: focus on the role of mitochondrial calcium accumulation". Pharmacology & Therapeutics 80 (2): 203–229. November 1998. doi:10.1016/S0163-7258(98)00029-1. PMID 9839772. 
  33. ↑ "Biology of HIF-1alpha". Cell Death and Differentiation 15 (4): 621–627. April 2008. doi:10.1038/cdd.2008.12. PMID 18259201. 
  34. ↑ "Sequence Analysis and Phylogenetic Studies of Hypoxia-Inducible Factor-1α". Cancer Informatics 16. 2017-01-01. doi:10.1177/1176935117712242. PMID 28615919. 
  35. ↑ "Neuroprotective strategies for acute ischemic stroke: Targeting oxidative stress and prolyl hydroxylase domain inhibition in synaptic signalling". Brain Disorders 5. March 2022. doi:10.1016/j.dscb.2022.100030. ISSN 2666-4593. 

Further reading

  • Overview of all the structural information available in the PDB for UniProt: Q9GZT9 (Egl nine homolog 1) at the PDBe-KB.



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