Nobel Prize for Physiology and Medicine 2019 Awarded for Discovery in Oxygen Related Mechanisms of Disease
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. Semenzahave worked towards understanding this and have since 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 attack, stroke and cancer.
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. He 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.
The Beginning - Eryhthropoietin (EPO)
A few decades later , the first evidence of oxygen sensing mechanisms in animals came from the work describing erythropoietin (EPO) (1). EPO is a hormone produced by the 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 the relationship between oxygen levels and EPO expression remeained unclelar.
In the early 1990s Gregg Semenza had studied the EPO gene and concluded that it must work like other genes, such 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-response element’ or HRE at the other end of the EPO gene was able to bind nuclear factors and be induced by altering oxygen levels (3).
At the same time Ratcliffe and his research group were 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 to understand that oxygen sensing mechanisms might in fact affect expression of other genes as well (4) and quite quickly they found HRE -elements in the genes encoding glycolytic enzymes (5). These findings pointed out that changes in oxygen levels, and especially hypoxia, could confer adaptations at the cellular level by regulating glycolysis for oxygen-independent energy production. Finally, it was discovered that hypoxia also induced the expression of angiogenic growth factors. It became clear that there was a wide oxygen sensing network in cells that governed expression of a number of fundamental genes is response to oxygen availability (6) and that the oxygen sensing process operated in mammalian cells.
Relevance Fact #1:
In studying this they did face some problems. For instance, that not all cells responded well at low oxygen levels (1% O2). As it is now understood, this was because cell types differ greatly in oxygen consumption and it was (and still is) easy to miss responses without directly measuring oxygen (7). At this time Ratcliffe and colleagues were using oxygen -controlled incubators (Baker Ruskinn chambers were not available yet) where low oxygen conditions were lost at each door opening. At this time, however, this was not an issue because 1) it was not understood how rapid the changes oxygen caused were 2) they were using stable reporter gene constructs and not HIF (as you see below, HIF had not been discovered yet).
Discovery of The HIF System
It was now known that there were oxygen-regulated nuclear factors that bound the EPO gene and it was important to understand what those were. Hypoxia-inducible factor 1 or HIF-1 was purified in 1995 by Semenza and colleagues (8). HIF-1 is essential for the formation of the vascular system as well as red blood cell production (8). It is composed of two subunits, namely, HIF-1alpha and HIF-1beta. They both were expressed in both ambient and low oxygen conditions but only HIF-1beta seemed to be stably expressed; HIF-1alpha was constantly degraded in ambient oxygen conditions. This was a clear indication that somehow HIF-1alpha -levels were regulated in the presence of oxygen.
William Kaelin had read about the identification of a gene for hereditary cancer called von Hippel-Lindau (VHL) disease. Patients with this cancer often develop tumors in the kidneys, adrenal glands, or pancreas. They also often have tumors in the central nervous system that look like nests of blood vessels. Kaelin thought knew that tumors would produce EPO (and thus stimulate red blood cell productions) and saw that this greatly resembled the behavior of a tissue that is short of oxygen. Thus he wondered whether the VHL disease itself and especially the gene behind the condition (VHL) involved an abnormal response to ambient oxygen levels.
Knowing that in VHL disease, the VHL gene is often deleted or mutated, Kaelin and his team were able to show that this VHL loss could lead to stabilization of genes that increased the formation of the vasculature, one such gene being vascular endothelial growth factor (VEGF) (10). As it was already shown by Ratcliffe that hypoxia also induced the expression of angiogenic growth factors, the likes of VEGF, Kaelin soon discovered that loss of VHL leads to upregulation of VEGF even in ambient oxygen conditions (11).
Ratcliffe and his research group then made a key discovery: demonstrating that VHL can physically interact with HIF-1α and is required for its degradation at normal oxygen levels (10,12). This conclusively linked VHL to HIF-1α. The only thing left that was not known was how oxygen levels regulated the interaction between HIF and VHL.
In a 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 an InVivO hypoxia workstation, 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’s laboratory. Techniques such as chromatin IP (ChIP) absolutely require that the oxygen atmosphere is preserved as assay material is being prepared.
Alot of information had been gained the search focused on the part of HIF-1alpha protein that was known to be important for VHL-dependent degradation. It was likely that the oxygen sensitive bit would reside in this area of the protein and indeed in 2001 they published the seminal findings that under ambient oxygen hydroxyl groups are added to HIF-1alpha (a protein modification called prolyl hydroxylation), leads to its recognition by VHL protein and its subsequent degradation (13,14,15,16,17).Together Kaelin, Ratcliffe and Semenza were able to compose the oxygen sensing system in cells. Working independently they were able to build from different angles (Kaelin being an oncologist, Ratcliffe kidney specialist and Semenza a medical geneticist) this great contribution to medical science that now awarded them with 2019 Nobel Prize for Physiology or Medicine.
More info about the Baker Ruskinn workstations here
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