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OVERVIEW / BACKGROUND
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BAKER RUSKINN RELEVANCE: In the 1999 Nature paper they in fact made a slight mistake. They initially reported that the association between VHL and HIF was sensitive to DFO and cobalt (DFO is an iron-chelator and stabilizes HIF-1alpha; cobalt induces HIF-1alpha in normoxia by preventing HIF binding to VHL) but not oxygen levels. They made this mistake because oxygen was getting into the buffers used in their cell culture, and because the cells were rapidly re-oxygenated when they were taken out of the tri-gas incubator. Only after collaborating with Andrew Skinn and getting a InVivO hypoxia workstation from him were they able to do the essential
experiments that showed the interaction between HIF and VHL was indeed oxygen sensitive. This is because they were able to produce stable hypoxic conditions without exposure to oxygen during vital experimentation.
In a similar way Baker Ruskinn Workstations have enabled a whole range of other techniques in Ratcliffe laboratory. Techniques such as chromatin IP (ChIP) absolutely require that the oxygen atmosphere is preserved as assay material is being prepared.
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Most life forms need oxygen for energy production but the fundamental oxygen sensing mechanisms that translate changes into action inside cells has been elusive.
During the last two decades William G. Kaelin, Sir Peter J. Ratcliffe and Gregg L. Semenza have worked towards understanding this and have now been awarded the 2019 Nobel Prize of Physiology or Medicine for the seminal discoveries of molecular machinery regulating the activity of genes in response to varying levels of oxygen. Their work revealed how cells respond to changes in oxygen levels. These findings are important in understanding and treating conditions and diseases like anemia, heart attacks, strokes and cancer.
Few decades later first evidence for oxygen sensing mechanism in animals came from the work describing erythropoietin (EPO) (1). EPO is a hormone produced by kidney and it stimulates red blood cell production in response to low blood oxygen levels. EPO was first purified in 1977 and the gene encoding it was cloned in 1985 (2) but how varied oxygen levels regulate EPO expression remained a mystery.
In early 1990s Gregg Semenza had studied the EPO gene and concluded that it must work like other genes, ie that a nearby stretch of DNA must activate it. He was interested in finding out how this worked and how varying oxygen levels can affect this. By using gene-modified
mice specific DNA segments located next to the EPO gene were shown to mediate the response to hypoxia. Eventually he was able to show that a region now called ‘hypoxia-responsive element’ or HRE at the other end of the EPO gene was able to bind nuclear factors and be induced by variations in oxygen levels (3).
At the same time Ratcliffe and his research group was studying O2-dependent regulation of the EPO gene. They found out that this HRE -element described by Semenza was present in cells that had nothing to do with EPO production. This lead Ratcliffe
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NOBEL PRIZE FOR PHYSIOLOGY AND MEDICINE 2019
Oxygen is essential for life. It is an electron acceptor and used by mitochondria to provide energy for the cells in an enzymatic process. To ensure adequate supply of oxygen to tissues and cells specific mechanisms have developed during evolution. The foundation for understanding these mechanisms was laid by Otto Warburg, the recipient of the 1931 Nobel Prize in Physiology or Medicine, revealed that this
conversion is an enzymatic process and a ‘mode of actions of a respiratory enzyme’. In 1938 this Nobel was awarded to Corneille Heymans for discovery of the role of sinus and aortic mechanisms in respiration regulation. He discovered how carotic body controls respiratory rate via blood oxygen sensing and that carotic body communicates directly with the brain.
BACKGROUND
THE BEGINNING – ERYHTROPOIETIN (EPO)
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REFERENCES
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Chan MC, Ilott NE, Schödel J, Sims D, Tumber A, Lippl K, Mole DR, Pugh CW, Ratcliffe PJ, Ponting CP, Schofield CJ. Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases. J Biol Chem. 2016 Sep 23;291(39):20661-73. Baker Ruskinn InvivO2 400
Frise MC, Cheng HY, Nickol AH, Curtis MK, Pollard KA, Roberts DJ, Ratcliffe PJ, Dorrington KL, Robbins PA. Clinical iron deficiency disturbs normal human responses to hypoxia. J Clin Invest. 2016 Jun 1;126(6):2139-50.
Vukovic M, Sepulveda C, Subramani C, Guitart AV, Mohr J, Allen L, Panagopoulou TI, Paris J, Lawson H, Villacreces A, Armesilla-Diaz A, Gezer D, Holyoake TL, Ratcliffe PJ, Kranc KR. Adult hematopoietic stem cells lacking Hif-1α self-renew normally. Blood. 2016 Jun 9;127(23):2841-6.
Chan MC, Holt-Martyn JP, Schofield CJ, Ratcliffe PJ. Pharmacological targeting of the HIF hydroxylases--A new field in medicine development. Mol Aspects Med. 2016 Feb-Mar;47-48:54-75.
Vukovic M, Guitart AV, Sepulveda C, Villacreces A, O’Duibhir E, Panagopoulou TI, Ivens A, Menendez-Gonzalez J, Iglesias JM, Allen L, Glykofrydis F, Subramani C, Armesilla-Diaz A, Post AE, Schaak K, Gezer D, So CW, Holyoake TL, Wood A, O’Carroll D, Ratcliffe PJ, Kranc KR. Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance. J Exp Med. 2015 Dec 14;212(13):2223-34.
Hodson EJ, Nicholls LG, Turner PJ, Llyr R, Fielding JW, Douglas G, Ratnayaka I, Robbins PA, Pugh CW, Buckler KJ, Ratcliffe PJ, Bishop T. Regulation of ventilatory sensitivity and carotid body proliferation in hypoxia by the PHD2/HIF-2 pathway. J Physiol. 2016 Mar 1;594(5):1179-95.
Schödel J, Grampp S, Maher ER, Moch H, Ratcliffe PJ, Russo P, Mole DR. Hypoxia, Hypoxia-inducible Transcription Factors, and Renal Cancer. Eur Urol. 2016 Apr;69(4):646-657.
Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001, 292, 468-472. Baker Ruskinn InvivO2 400
A.C.R.Epstein, J.M.Gleadle, L.A.McNeill, K.S.Hewitson, J.F.O’Rourke, D.R.Mole, M.Mukherji, E.Metzen, M.I.Wilson, A.Dhanda, Y.-M.Tian, N.Masson, D.L.Hamilton, P.Jaakkola, R.Barstead, J.Hodgkin, P.H.Maxwell, C.W.Pugh, C.J.Schofield, P.J.Ratcliffe. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107 2001 43-54.
P.H.Maxwell, M.S.Wiesener, G.-W.Chang, S.C.Clifford, E.C.Vaux, M.E.Cockman, C.C.Wykoff, C.W.Pugh, E.R.Maher, P.J.Ratcliffe. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399 1999 271-275.
Masson N, Singleton RS, Sekirnik R, Trudgian DC, Ambrose LJ, Miranda MX, Tian YM, Kessler BM, Schofield CJ, Ratcliffe PJ. The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity.. EMBO Rep. 13; 2012 251-7.
Schödel J, Bardella C, Sciesielski L, Brown JM, Pugh CW, Buckle V, Tomlinson IP, Ratcliffe PJ, Mole DR. Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1expression.. Nature Genetics 44 2012; 420-5.
Adam J, Hatipoglu E, O’Flaherty L, Ternette N, Sahgal N, Lockstone H, Baban D, Nye E, Stamp GW, Wolhuter K, Stevens M, Fischer R, Carmeliet P, Maxwell PH, Pugh CW, Frizzell N, Soga T, Kessler BM, El-Bahrawy M, Ratcliffe PJ, Pollard PJ. Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling. Cancer Cell. 20 2011; 524-37.
M.E.Cockman, J.D.Webb, H.B.Kramer, B.M.Kessler, P.J.Ratcliffe. Proteomics-based identification of novel factor inhibiting HIF (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins. Molecular & Cellular Proteomics 8, 2009; 535-546.
M.Mazzone, D.Dettori, R.L.deOliveira, S.Loges, T.Schmidt, B.Jonckx, Y.-M.Tian, A.A.Lanahan, P.Pollard, C.R.deAlmodovar, F.DeSmet, S.Vinckier, J.Aragones, K.Debackere, A.Luttun, S.Wyns, B.Jordan, A.Pisacane, B.Gallez, M.G.Lampugnani, E.Dejana, M.Simons, P.Ratcliffe, P.Maxwell, P.Carmeliet. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell 136, 2009; 839-851.
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Sir Peter J Ratcliffe
Cockman ME, Lippl K, Tian YM, Pegg HB, Figg WD Jnr, Abboud MI, Heilig R, Fischer R, Myllyharju J, Schofield CJ, Ratcliffe PJ. Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates. Elife. 2019 Sep 10;8.
Schödel J, Ratcliffe PJ. Mechanisms of hypoxia signalling: new implications for nephrology. Nat Rev Nephrol. 2019 Oct;15(10):641-659.
Masson N, Keeley TP, Giuntoli B, White MD, Puerta ML, Perata P, Hopkinson RJ, Flashman E, Licausi F, Ratcliffe PJ. Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants. Science. 2019 Jul5;365(6448):65-69. Baker Ruskinn InvivO2 400
Yamamoto A, Hester J, Macklin PS, Kawai K, Uchiyama M, Biggs D, Bishop T, Bull K, Cheng X, Cawthorne E, Coleman ML, Crockford TL, Davies B, Dow LE, Goldin R, Kranc K, Kudo H, Lawson H, McAuliffe J, Milward K, Scudamore CL, Soilleux E, Issa F, Ratcliffe PJ, Pugh CW. Systemic silencing of PHD2 causes reversible immune regulatory dysfunction. J Clin Invest. 2019 Jun 4;130:3640-3656.
Smythies JA, Sun M, Masson N, Salama R, Simpson PD, Murray E, Neumann V, Cockman ME, Choudhry H, Ratcliffe PJ, Mole DR. Inherent DNA-binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin. EMBO Rep. 2019 Jan;20(1). Baker Ruskinn InvivO2 400
Wang Y, Zhong S, Schofield CJ, Ratcliffe PJ, Lu X. Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively. J Cell Sci. 2018 Nov 19;131(22). Baker Ruskinn InvivO2 400
Markolovic S, Zhuang Q, Wilkins SE, Eaton CD, Abboud MI, Katz MJ, McNeil HE, Leśniak RK, Hall C, Struwe WB, Konietzny R, Davis S, Yang M, Ge W, Benesch JLP, Kessler BM, Ratcliffe PJ, Cockman ME, Fischer R, Wappner P, Chowdhury R, Coleman ML, Schofield CJ. The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases. Nat Chem Biol. 2018 Jul;14(7):688-695.
Kaelin WG Jr, Ratcliffe PJ, Semenza GL. Pathways for Oxygen Regulation and Homeostasis: The 2016 Albert Lasker Basic Medical Research Award. JAMA. 2016 Sep 27;316(12):1252-3.
Tel: +44 (0) 1656 645988 | Web: bakerco.com/grow | Email: [email protected]
Yeh TL, Leissing TM, Abboud MI, Thinnes CC, Atasoylu O, Holt-Martyn JP, Zhang D, Tumber A, Lippl K, Lohans CT, Leung IKH, Morcrette H, Clifton IJ, Claridge TDW, Kawamura A, Flashman E, Lu X, Ratcliffe PJ, Chowdhury R, Pugh CW, Schofield CJ. Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials. Chem Sci. 2017 Nov 1;8(11):7651-7668.
Sadiku P, Willson JA, Dickinson RS, Murphy F, Harris AJ, Lewis A, Sammut D, Mirchandani AS, Ryan E, Watts ER, Thompson AAR, Marriott HM, Dockrell DH, Taylor CT, Schneider M, Maxwell PH, Chilvers ER, Mazzone M, Moral V, Pugh CW, Ratcliffe PJ, Schofield CJ, Ghesquiere B, Carmeliet P, Whyte MK, Walmsley SR. Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses. J Clin Invest. 2017 Sep 1;127(9):3407-3420.
Grampp S, Schmid V, Salama R, Lauer V, Kranz F, Platt JL, Smythies J, Choudhry H, Goppelt-Struebe M, Ratcliffe PJ, Mole DR, Schödel J. Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway. PLoS Genet. 2017 Jul 17;13(7):e1006872.
Pugh CW, Ratcliffe PJ. New horizons in hypoxia signaling pathways. Exp Cell Res. 2017 Jul 15;356(2):116-121.
Grampp S, Platt JL, Lauer V, Salama R, Kranz F, Neumann VK, Wach S, Stöhr C, Hartmann A, Eckardt KU, Ratcliffe PJ, Mole DR, Schödel J. Genetic variation at the 8q24.21 renal cancer susceptibility locus affects HIF binding to a MYC enhancer. Nat Commun. 2016 Oct 24;7:13183.
Chowdhury R, Leung IK, Tian YM, Abboud MI, Ge W, Domene C, Cantrelle FX, Landrieu I, Hardy AP, Pugh CW, Ratcliffe PJ, Claridge TD, Schofield CJ. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nat Commun. 2016 Aug 26;7:12673.
Platt JL, Salama R, Smythies J, Choudhry H, Davies JO, Hughes JR, Ratcliffe PJ, Mole DR. Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters. EMBO Rep. 2016 Oct;17(10):1410-1421. Baker Ruskinn InvivO2 400
“This type of apparatus (InvivO2) is very important for work in hypoxia, . . . it’s easy to allow inadvertent re-oxygenation to confound your experimental results. Using a controlled environment work station InvivO2 greatly reduces that risk.”
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“Research into the molecular cell biology of responses to hypoxia carries the (apparent) advantage of allowing the representation of an important disease complication in a tissue culture dish; hypoxia complicates most human diseases, and oxygen levels can be reduced in tissue culture to those that simulate disease and induce adaptive responses.
This apparent simplicity belies a number of traps for the unwary researcher. Oxygen diffuses rapidly across plastic ware, and into buffers and any materials that are used to make biochemical preparations from hypoxia tissue culture cells. So any attempt to study the hypoxic status of the cell must take account of this.
I remember Andrew Skinn visiting the laboratory (I think in late 1999 or early 2000). He was showing some very nice data from Darren Richards using the new Baker-Ruskinn InvivO2 controlled environment chamber. To be honest I wasn’t absolutely convinced on the necessity straightaway; just a gut feeling that if we were working on the biochemistry of hypoxia signalling surely we might need to control oxygen through all phases of the experiment.
And indeed that was correct. Critically it enabled us to correct a small mistake in our work connecting HIF to VHL. We were somewhat surprised that although the interaction (between HIF and VHL) was necessary for degradation of HIF and the interaction could readily be suppressed by iron chelators and cobalt, we apparently could not see suppression in hypoxic cells.
That work also apparently explained a paradox in the field. When HIF was induced by hypoxia, then displayed by electrophoretic
mobility shift assay, it generally appeared as a double band, whereas when HIF was induced by cobalt or iron chelators, it generally appeared as a single band. These results were widely observed but unexplained. We were very pleased to sort this out; the double band contained HIF complexed to VHL, as well as HIF alone, (hence two distinct mobilities) which we proudly showed with super-shift assays.
But we were always worried by this result. Despite the rapid harvest, might oxygen have got into the cells? This is where the BR chamber came in – enabling Panu Jaakkola and David Mole to revisit the position using IVTT proteins and IP-IB from human cells, respectively. This was actually at the time a real ‘tour de force’. By excluding oxygen from all the buffers and performing the whole ‘pull-down’ procedures in the Inviv02 chamber they were able to show that hypoxia did indeed suppress formation of the complex – a very important result.
This type of apparatus is very important for work in hypoxia, aside from the issue of control (of the oxygen level) it is all too easy to make a mistake. Unlike for pH we don’t use a visible oxygen indicator, so it’s easy to allow inadvertent re-oxygenation to confound your experimental results. Using a controlled environment work station greatly reduces that risk.”
Sir Peter J Ratcliffe
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