Oxygen is one of the most vital molecules required for existence. We know the macroscopic picture – Oxygen from the air, taken in by the lungs, is carried by haemoglobin present in the red blood cells to the different organs, where they diffuse into the cells, directly partaking in the enzymatic reaction required to derive energy. But what happens after the Oxygen enters the cell? How does the cell sense its presence? This was the exact question, pursued by the 2019 Nobel laureates – Gregg L. Semenza, Sir Peter J. Ratcliffe and Willian G. Kaelin, Jr.
The understanding of the cell’s Oxygen sensing abilities first began when a correlation was made between low Oxygen levels in the blood (hypoxia) and the activation of the erythropoietin gene in rodent kidney and liver cells. Hypoxic environment or anaemia stimulated the production of a glycoprotein cytokine – erythropoietin (EPO), which stimulated the erythroid progenitor cells to make more RBC, thereby increasing the Oxygen carrying ability of the blood. The cells clearly showed the ability to sense the abundance of oxygen in them, but what was the molecular mechanism behind this?
It was HIF1 all along!! – Gregg Semenza, 1991
In the effort to understand how the erythropoietin gene is regulated, Gregg Semenza in 1991 hypothesised that the gene must contain sequences that bind factors that activate it. He successfully identified using mice models and human liver cell lines, that a 256bp region in the 3’ flanking sequence of the EPO gene, bound to at least two nuclear factors that were induced by anaemia and hypoxia. They called this factor, the Hypoxia Inducible Factor (HIF). On exploring further, Gregg Semenza and group identified that the RNA and protein levels of the two HIF1 subunits – HIF1α and HIF1β were induced by hypoxia (1% O2) in human liver cell line (Hep-3B) and these levels rapidly decayed when the Oxygen levels were restored back to 20%. We were getting closer to deciphering the cell’s Oxygen sensing abilities, but it wasn’t clear how Oxygen regulated the degradation of HIF1 (1, 2).
pVHL enters the picture – Sir Peter J. Ratcliffe, 1999
Around the same time, Peter J. Ratcliffe and group were working on the Von Hippel-Lindau syndrome which is characterised by the formation of highly angiogenic tumours. These tumours lacked the Von Hippel-Lindau tumour suppressor protein (pVHL) and they identified that in these VHL defective cells, HIF1 alpha was constitutively active and on re introducing the pVHL into these cells, the oxygen dependent degradation of HIF 1 alpha was restored. They also identified using immunoprecipitation that HIF 1 alpha interacts with pVHL in the presence of oxygen. Additionally, in cells exposed to iron chelators, this interaction was lost. They hence concluded, that the degradation of HIF1 alpha was dependant on its interaction with pVHL, which in turn was dependant on the presence of Oxygen and Iron. But the question remained as to how these molecules enabled the interaction between HIF1 alpha and pVHL and how this interaction enabled the degradation of HIF1 alpha (3).
HIF-PH does the job – William G. Kaelin, Jr, 2001 and Sir Peter J. Ratcliffe, 2001
All the pieces of the puzzle finally came together when it was identified by both – the William G. Kaelin and the Peter Ratcliffe groups, almost simultaneously, that it was the hydroxylation of a Proline residue in the HIF1 alpha protein (HIF 1 α – P564) that facilitated interaction between HIF1 alpha and pVHL. Peter Ratcliffe and team identified an enzyme responsible for this hydroxylation, which required Oxygen as a cosubstrate and Iron as a cofactor, and they termed it HIF 1 α proryl hydroxylase (HIF-PH). The pVHL then ubiquitylates HIF1 alpha and thereby targets it towards proteasomal degradation (4, 5).
How has this helped us?
These discoveries helped put all the pieces together, but in what way is it of significance to modern science and medicine? Firstly, it’s helped us better understand basic physiological processes including metabolism, immune response and embryonic development. Secondly, it’s helped us take huge leaps towards progressing certain therapeutic interventions such as in wound healing, anaemia and cancer treatment. Indeed, their efforts deserve much credit!
- 1.G. Semenza, M. Nejfelt, S. Chi, S. Antonarakis, Hypoxia-inducible nuclear factors bind to an enhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad Sci U S A. 88, 5680–4 (1991).
- 2.G. Wang, B. Jiang, E. Rue, G. Semenza, Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 92, 5510–4 (1995).
- 3.P. Maxwell, M. Wiesener, G. Chang, S. Clifford, E. Vaux, M. Cockman, C. Wykoff, C. Pugh, E. Maher, P. Ratcliffe, The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 399, 271–5 (1999).
- 4.P. Jaakkola, D. Mole, Y. Tian, M. Wilson, J. Gielbert, S. Gaskell, K. von, H. Hebestreit, M. Mukherji, C. Schofield, P. Maxwell, C. Pugh, P. Ratcliffe, Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 292, 468–72 (2001).
- 5.M. Ivan, K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, A. Salic, J. Asara, W. Lane, W. Kaelin, HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 292, 464–8 (2001).