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Explicating cellsí use of oxygen
STOCKHOLM—On Oct. 7, the Nobel Assembly at Karolinska Institutet awarded the 2019 Nobel Prize in Physiology or Medicine jointly to William G. Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza for their work on how cells sense and adapt to changing oxygen availability. The researchers identified a molecular circuit that regulates the activity of genes in response to varying levels of oxygen.
“I’m honored and delighted at the news,” Ratcliffe said in a press release from the University of Oxford. “I’ve had great support from so many people over the years. It’s a tribute to the lab, to those who helped me set it up and worked with me on the project over the years, to many others in the field, and not least to my family for their forbearance of all the up and downs.”
Oxygen (O2) is used by the mitochondria present in virtually all animal cells in order to convert food into useful energy. During evolution, certain mechanisms developed to ensure a sufficient supply of oxygen to tissues and cells. For example, the carotid body contains specialized cells that sense blood’s oxygen levels.
The researchers established the basis for understanding how oxygen levels affect cellular metabolism and physiological function. In addition to the carotid body-controlled rapid adaptation to low oxygen levels (hypoxia), there are other fundamental physiological adaptations. A rise in levels of erythropoietin (EPO) is a key physiological response to hypoxia, which leads to increased production of red blood cells (erythropoiesis).
Semenza studied the EPO gene and how it’s regulated by oxygen levels. Using gene-modified mice, specific DNA segments located next to the EPO gene were shown to mediate the response to hypoxia. Ratcliffe also studied O2-dependent regulation of the EPO gene, and both research groups found that the oxygen-sensing mechanism was present in virtually all tissues, not only in the kidney cells where EPO is normally produced. These findings showed that the mechanism was general and functional in many different cell types.
Semenza wanted to identify the cellular components mediating this response, and in cultured liver cells he discovered a protein complex that binds to the identified DNA segment in an oxygen-dependent manner. He named this complex the hypoxia-inducible factor (HIF). Extensive efforts to purify the HIF complex began, and in 1995, Semenza was able to publish some of his key findings, including identification of the genes encoding HIF.
HIF was found to consist of two different DNA-binding proteins: transcription factors now named HIF-1α and ARNT. When oxygen levels are high, cells contain very little HIF-1α. But when oxygen levels are low, the amount of HIF-1α increases so that it can bind to and regulate the EPO gene and other genes with HIF-binding DNA segments. Several research groups have shown that HIF-1α, which generally degrades rapidly, is protected from degradation in hypoxia.
At normal oxygen levels, the proteasome degrades HIF-1α. Under such conditions a small peptide, ubiquitin, is added to the HIF-1α protein. Ubiquitin functions as a tag for proteins destined for degradation in the proteasome. How ubiquitin binds to HIF-1α in an oxygen-dependent manner remained a central question.
Around the same time that Semenza and Ratcliffe were exploring the regulation of the EPO gene, Harvard University professor and cancer researcher Kaelin was researching von Hippel-Lindau’s disease (VHL disease). This genetic disease leads to a dramatically increased risk of certain cancers in families with inherited VHL mutations.
Kaelin showed that the VHL gene encodes a protein that prevents the onset of cancer. Kaelin also showed that cancer cells lacking a functional VHL gene express abnormally high levels of hypoxia-regulated genes, but when the VHL gene was reintroduced into cancer cells, normal levels were restored.
This proved that VHL was involved somehow in controlling responses to hypoxia. Additional clues came from several research groups showing that VHL is part of a complex that labels proteins with ubiquitin, marking them for proteasome degradation. Ratcliffe and his research group demonstrated that VHL can physically interact with HIF-1α and is required for its degradation at normal oxygen levels. This conclusively linked VHL to HIF-1α.
In order to understand how O2 levels regulate the interaction between VHL and HIF-1α, the search focused on a specific portion of the HIF-1α protein known to be important for VHL-dependent degradation. Both Kaelin and Ratcliffe suspected the key to O2-sensing resided somewhere in this protein domain.
In 2001, in two simultaneously published articles Ratcliffe and Kaelin showed that under normal oxygen levels, hydroxyl groups are added at two specific positions in HIF-1α. This protein modification, called prolyl hydroxylation, allows VHL to recognize and bind to HIF-1α and thus explained how normal oxygen levels control rapid HIF-1α degradation.
Further research by Ratcliffe and others identified the responsible prolyl hydroxylases. It was also shown that the gene-activating function of HIF-1α was regulated by oxygen-dependent hydroxylation. The Nobel laureates had finally elucidated the oxygen sensing mechanism and shown how it works.
Oxygen sensing allows cells to adapt their metabolism to low oxygen levels. It’s been shown to be essential during fetal development for controlling normal blood vessel formation and placenta development. And our immune system and other physiological functions are also fine-tuned by O2 sensing.
Oxygen sensing is also central to a large number of diseases. Patients with chronic renal failure often suffer from severe anemia, due to decreased EPO expression. The oxygen-regulating pathway also has an important role in cancer. In tumors, the molecular circuit is utilized to stimulate blood vessel formation and reshape metabolism for effective proliferation of cancer cells.
“What we discovered was a molecular pathway or a molecular circuit that all multicellular organisms use, so this is something that’s highly conserved throughout evolution that allows cells and tissues to know whether they’re getting enough oxygen, and to respond accordingly,” Kaelin said in an AP video. “One thing this has enabled us to do is … to develop drugs that will either activate or inactivate this particular molecular circuit.”
Academic laboratories and pharmaceutical companies are now focused on developing drugs that can interfere with different disease states by either activating or blocking the oxygen-sensing molecular pathway.