Hypoxia-Inducible Factors

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Hypoxia-inducible factors (HIFs) are transcription factors responsible of the cellular adaptation to hypoxia, which is a condition of low oxygen availability. Among the genes regulated by HIF, we can find those involved in erythropoiesis, angiogenesis and metabolism.

Contents

Structure [1][2]

HIFs were discovered more than 30 years ago thanks to the Erythropoietin (EPO) gene, an hormone that is transcribed in hypoxic conditions and stimulates erythrocyte proliferation [3]. EPO presents an upstream Hypoxia Response Element (HRE) that resulted to be bound by HIF [4]. HIFs form part of the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) family of proteins[5]. Active HIFs are achieved by heterodimerization of a constitutively expressed subunit HIF-β and an oxygen-regulated subunit, which can be HIF-1α or its paralogs HIF-2α and HIF-3α (Table 1.). HIF-β is also known as the aryl hydrocarbon nuclear translocator (ARNT)[6]. Their structure can contain the following domains from the N-terminus to the C-terminus:

  • basic Helix-Loop-Helix (bHLH): essential for heterodimer formation and DNA binding to HRE
  • Per-Arnt-Sim (PAS): their surface forms the key core of the heterodimer.
  • Oxygen-dependent degradation domain (ODDD): mediates oxygen-regulated stability in the α subunits. This domain contains two Proline residues susceptible of hydroxylation and one Lysine that can be acetylated
  • N-terminal and C-terminal transactivating domains (N-TAD and C-TAD): bind different coactivators to promote gene expression, such as p300/CBP[7]. HIF-3α lacks one of the TAD domains.
Member Gene Protein
HIF-1α HIF1A hypoxia-inducible factor 1, α subunit
HIF-2α EPAS1 endothelial PAS domain protein 1
HIF-3α HIF3A hypoxia-inducible factor 3, α subunit
HIF-1β ARNT aryl hydrocarbon receptor nuclear translocator
HIF-2β ARNT2 aryl hydrocarbon receptor nuclear translocator 2
HIF-3β ARNT3 aryl hydrocarbon receptor nuclear translocator 3
Table 1: Members of the HIF family

thumb | left | alt= HIF members structure

Physiological Function

Both HIF-α and β subunits are constitutively and ubiquitously expressed in almost every single cell, although some isoforms may be predominant in some specific tissues[8]. For example, HIF-2α is mostly expressed in the endothelium, kidney, lung, heart and small intestine . While HIF-β protein levels in the cell are constant, HIF-α has a short half-life (~ 5 mins) and is highly regulated by oxygen through its ODDD domain. With normal oxygen levels (normoxia), HIF-α protein levels are rapidly degraded, resulting in essentially no detectable HIF-α protein. However, in hypoxic conditions HIF-α becomes stabilized and is translocated from the cytoplasm to the nucleus, where it dimerizes with HIF-β and the HIF complex formed becomes transcriptionally active. More than 100 genes with varying functions have been described to be transcribed by the action of HIF. Moreover, HIF regulates up to 2% of all human genes in arterial endothelial cells, either directly or indirectly[9]. In response to hypoxia, the capacity of red blood cells to transport oxygen is upregulated by the expression of genes like EPO, essential in erythropoiesis, and genes involved in iron metabolism. In addition, a large number of genes involved in different steps of angiogenesis have been shown to increase by hypoxia challenge, being the Vascular Endothelial Growth Factor (VEGF) among them. VEGF directly recruits endothelial cells into hypoxic areas to generate new blood vessels by stimulating their proliferation. Furthermore, HIF complexes have been shown to induce pro-survival factors such as Insulin-like Growth Factor-2 (IGF2) and Transforming Growth Factor-α (TGF-α), which in turn enhances expression of HIFα itself.

Regulation of HIF-α subunits is achieved by pos-translational modifications such as hydroxylation, ubiquitination, acetylation and phosphorylation. De novo synthesized cytoplasmic HIF-α is rapidly hydroxylated on Pro402 and Pro564 (in HIF-1α) or Pro405 and Pro531 (in HIF-2α) by a family of Prolyl Hydroxylases (PHD) that recognize the consensus sequence LXXLAP . These PHD require an oxygen molecule that will be splitted to catalyze the hydroxylation of the target prolines[10]. Once the two proline residues of HIF-α are converted to hydroxyproline, the Von Hippel-Lindau tumor suppressor (pVHL) will recognize it. pVHL acts as the substrate recognition component of a complex with E3 ubiquitin ligase activity that will then polyubiquitinate HIF-1α on Lysine 532 for proteasomal degradation[11]. In hypoxic conditions, PHDs will be inactive due to the lack of oxygen, so HIF-α can escape proteasomal degradation and translocate to the nucleus to active gene expression. Additional modifications can also modulate HIF-α properties: acetylation of Lys532 by Arrest-Defective-1 (ARD1) favors the interaction of HIF-1α with pVHL[12]; hydroxylation of Asn803 or 851 within the C-CAD domain (in HIF-1α or HIF-2α, respectively) by Factor Inhibiting HIF-1 (FIH-1) prevents interaction with p300/CBP, thus inhibiting transcription[13]. Furthermore, Mitogen-Activated Protein Kinase (MAPK) pathway seems to be able to phosphorylate HIF-α in response to growth factors. This phosphorylation would increase HIF complex transcriptional activity rather than affecting its stability or DNA-binding ability[14].

Clinical Significance

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Function

Disease

Relevance

Structural highlights

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References

  1. Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol. 2006 Nov;70(5):1469-80. doi: 10.1124/mol.106.027029. Epub 2006 Aug, 3. PMID:16887934 doi:http://dx.doi.org/10.1124/mol.106.027029
  2. Wu D, Potluri N, Lu J, Kim Y, Rastinejad F. Structural integration in hypoxia-inducible factors. Nature. 2015 Aug 5. doi: 10.1038/nature14883. PMID:26245371 doi:http://dx.doi.org/10.1038/nature14883
  3. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science. 1988 Dec 9;242(4884):1412-5. doi: 10.1126/science.2849206. PMID:2849206 doi:http://dx.doi.org/10.1126/science.2849206
  4. Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE. Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5680-4. doi: 10.1073/pnas.88.13.5680. PMID:2062846 doi:http://dx.doi.org/10.1073/pnas.88.13.5680
  5. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5510-4. PMID:7539918
  6. Reyes H, Reisz-Porszasz S, Hankinson O. Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science. 1992 May 22;256(5060):1193-5. PMID:1317062
  7. Dames SA, Martinez-Yamout M, De Guzman RN, Dyson HJ, Wright PE. Structural basis for Hif-1 alpha /CBP recognition in the cellular hypoxic response. Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5271-6. PMID:11959977 doi:http://dx.doi.org/10.1073/pnas.082121399
  8. Loboda A, Jozkowicz A, Dulak J. HIF-1 and HIF-2 transcription factors--similar but not identical. Mol Cells. 2010 May;29(5):435-42. doi: 10.1007/s10059-010-0067-2. Epub 2010 Apr, 12. PMID:20396958 doi:http://dx.doi.org/10.1007/s10059-010-0067-2
  9. Manalo DJ, Rowan A, Lavoie T, Natarajan L, Kelly BD, Ye SQ, Garcia JG, Semenza GL. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood. 2005 Jan 15;105(2):659-69. doi: 10.1182/blood-2004-07-2958. Epub 2004 Sep , 16. PMID:15374877 doi:http://dx.doi.org/10.1182/blood-2004-07-2958
  10. Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science. 2001 Nov 9;294(5545):1337-40. doi: 10.1126/science.1066373. Epub 2001, Oct 11. PMID:11598268 doi:http://dx.doi.org/10.1126/science.1066373
  11. Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW. Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci U S A. 2000 Sep 12;97(19):10430-5. PMID:10973499 doi:http://dx.doi.org/10.1073/pnas.190332597
  12. Jeong JW, Bae MK, Ahn MY, Kim SH, Sohn TK, Bae MH, Yoo MA, Song EJ, Lee KJ, Kim KW. Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation. Cell. 2002 Nov 27;111(5):709-20. PMID:12464182
  13. Lando D, Peet DJ, Whelan DA, Gorman JJ, Whitelaw ML. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science. 2002 Feb 1;295(5556):858-61. doi: 10.1126/science.1068592. PMID:11823643 doi:http://dx.doi.org/10.1126/science.1068592
  14. Richard DE, Berra E, Gothie E, Roux D, Pouyssegur J. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1. J Biol Chem. 1999 Nov 12;274(46):32631-7. doi: 10.1074/jbc.274.46.32631. PMID:10551817 doi:http://dx.doi.org/10.1074/jbc.274.46.32631
  15. Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
  16. Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644
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