Nobel Prize in Medicine 2019: How Cells Adapt to Oxygen Availability?

The Nobel Prize in Physiology/Medicine was awarded in the year 2019 to William G. Kaelin, Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza for their discoveries of how cells sense and adapt to oxygen availability.

We all know that Oxygen is essential for life. The human body functions intricately and reacts very astutely to changes in the level of oxygen to maintain homeostasis. So let us see what constitutes this Nobel Prize-winning discovery.

Basics To Understand The Paramountcy Of Oxygen:

          Respiration in humans is of two basic types, mechanical and cellular. Mechanical respiration consists of inspiration that is breathing in and expiration that is breathing out. Oxygen (O2) from the air taken in by the process of inhalation is absorbed and simultaneously carbon dioxide (CO2) is given out. This O2 is absorbed by the hemoglobin in the blood.

         Hemoglobin is stored in the red blood cells (RBC), hence RBC constitutes the oxygen-carrying capacity of the blood. O2 is further carried to the cells by the means of RBC in the blood vessels. This O2 is delivered to the cells where the cellular respiration begins, which involves complex cycles in cytoplasm and mitochondria.

The end product of this cellular respiration is ATP, which is the energy currency of the body. In normoxic (normal level of oxygen) states aerobic respiration occurs.

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           The reduced level of oxygen in the tissue is known as Hypoxia.  Hypoxia is detected by the peripheral chemoreceptors in the carotid body and aortic body, with the carotid body chemoreceptors being the major mediators of reflex responses to hypoxia-like hyperventilation. In the state of hypoxia the body shifts to anaerobic metabolism which produces and lactic acid and results in the production of lesser ATPs than in normal conditions. General symptoms of acute and chronic hypoxia are:

ACUTE CHRONIC
Breathlessness Blue discoloration (cyanosis
Reduced level of consciousness, confusion &disorientation Slow heart rate
Increased heart rate and pallor Low blood pressure
Muscle fatigue Heart failure eventually leading to shock and death
Vasodilation in body and Vasoconstriction in the lungs


RBCs are produced from the stem cells present in the bone marrow, present majorly within the long bones of the body.
Erythropoietin (EPO) is a glycoprotein with a molecular weight of about 34,000 which increases the level of RBC in the blood. 90% of all erythropoietin is formed in the kidneys, and the remainder is formed mainly in the liver.

EPO stimulates the stem cells to produce more RBC and as a result, increases the oxygen-carrying capacity on the blood. The above symptoms are a result of strenuous efforts of the human body adapting to maintain sufficient oxygen levels in the body. One such adaption is increased RBC production by Erythropoietin stimulation.

Discovery of HIF1:

In 1991, Semenza with his colleagues published a study showing that hypoxia induces the production of factor which enhances the production the EPO. He called this Hypoxia-Induced Factor-1 (HIF-1). He found that HIF-1 binds to a certain enhancer region close to the 3’ end of the EPO gene increasing its production. This lead to increased efforts towards isolating HIF-1 from the cells and studying its structure and function in detail.

Later in 1995, Semenza with his colleagues published another study delineating the structure of HIF-1 and that it is regulated by O2 levels. HIF-1 is a heterodimer consisting of the HIF-1α subunit and an HIF-1β (ARNT) subunit. There are three isoforms of the α-subunit (HIF-1α, HIF-2α, and HIF-3α) and two isoforms of the β-subunit. 

HIF serves as a mediator of transcriptional responses to hypoxia. HIF 1 in response to hypoxia acts as a transcriptional activator of the EPO gene, that is it promotes the production of EPO by stimulating the corresponding genes.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a cofactor that acts in association with HIF-1α as a heterodimer in the nucleus of a cell. HIF-1α is formed in the cytoplasm and moves into the nucleus where ARNT is already present and combines with it to carry out its function.

Along with this, it was found that in normoxic cells the HIF-1α subunit was highly unstable and got rapidly degraded in the cytoplasm of a cell. The rapid degradation was facilitated by the proteasome pathway, which is responsible for the physiological degradation of almost all proteins in the cell.

A small peptide, ubiquitin, is added to the HIF-1α protein. Ubiquitin functions as a tag for proteins destined for degradation in the proteasome by E3 ubiquitin ligase enzymes.

Genes Controlled by HIF:

  • Genes associated with VEGF (vascular endothelial growth factor) responsible for stimulating angiogenesis.
  • Erythropoietin (EPO) genes which stimulate RBC production.
  •  Mitochondrial genes involved with energy utilization. Glycolytic enzyme genes involved with anaerobic metabolism.
  • Genes that increase the availability of Nitric Oxide (NO), which causes pulmonary vasodilation.

Now the questions were what role oxygen plays and that leads to the degradation of this factor in normoxic cells also more importantly, what was the reason for the stabilization of the factor in hypoxic cells.

The Revelation Of The Role Of VHL:

 In 1999, Sir Peter J. Ratcliffe with his colleagues published the role of Von Hippel Lindau protein (pVHL) in the degradation of the HIF-1a subunit. This was an unexpected discovery.

Von Hippel Lindau disease is a rare genetic disorder due to a mutation in the tumor suppressor VHL gene present on chromosome 3, which results in the faulty production of pVHL. This manifests as a human cancer syndrome predisposing sufferers to highly angiogenic tumors. pVHL is a component of a multiprotein complex that bears structural and functional similarity to SCF ubiquitin ligase which helps in proteasomal degradation.

It was found that in VHL-defective cells, the HIF-1α subunit is constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored the degradation of the HIF-1.

It was seen that normally, the HIF-1α subunit is targeted for rapid degradation in normoxic cells by a proteasomal mechanism operating on an internal Oxygen-Dependent-Degradation (ODD) domain which required pVHL.

In the presence of oxygen, pVHL, in association with elongin B and elongin C (VBC), binds directly to HIF-1α subunit and targets them for polyubiquitination and destruction. This confirmed how HIF was inactivated in normoxic but what stabilized the factor in hypoxic cells still remained unknown.

PHD Aspect:

           A couple of studies published by William G. Kaelin Jr. in 2001 shed some light on this subject. The crux of these studies was the finding that pVHL interacted with a post-translationally modified HIF-1α. Some changes were found to be made in the presence of oxygen to the factor which allowed pVHL to form a complex with it by binding on the ODD.

Further, it said that this modification required the presence of iron as a cofactor along with oxygen.

         These studies also illustrated that this modification was the addition of hydroxyl (OH) residues(hydroxylation) on an amino acid named proline in the HIF peptide. It was seen that only those HIF peptides had the hydroxyproline residues combined with pVHL, in association with elongin B and elongin C to form a complex (VBC). This hydroxylation is carried out by enzymes known as HIF prolyl hydroxylases (HIF-PH/PHD).

These enzymes have absolute requirement molecular oxygen (O2) as cosubstrate and iron as a cofactor. They also require a tricarboxylic acid cycle intermediate, 2-oxoglutarate (α-ketoglutarate). Since the function of this enzyme depends completely on the presence of O2 (dioxygenase), it serves as a sensor for the level of oxygen in the tissue.

Higher Metazoans including humans contain three paralogous PHD genes (PHD1, PHD2, and PHD3). PHD1 is exclusively nuclear, PHD2 is mainly cytoplasmic, and PHD3 is found in both the cytoplasm and nucleus. Of the three PHD family members, PHD2 (also called EglN1) appears to be the primary HIF prolyl hydroxylase, contributing the majority of HIF prolyl hydroxylase activity in normoxic cells and hence setting normoxic HIF-1α levels.

          Factor inhibiting HIF -1 (FIH-1), like the PHD family members, is another iron and 2-oxoglutarate-dependent dioxygenase. When oxygen is available, FIH1 hydroxylates a conserved asparaginyl residue within the HIF-1α, which prevents its activation. FIH1 remains active at lower oxygen concentrations than the PHDs and so might suppress the activity of HIFα proteins that escape destruction in moderate hypoxia.

Moreover, HIF2α is relatively resistant to FIH1-mediated inactivation compared to HIF-1α. As a result, the depth of hypoxia required to activate a given HIF-responsive gene can be differentiated and recognized.

          To summarise the above discoveries, in hypoxic conditions, HIF-PH is not able to function and hence not able to degrade the HIF-1α subunit with the help of pVHL. HIF-1α then crosses into the nucleus and binds with HIF-1β/ARNT. This heterodimer then binds to the enhancer region of the EPO gene and starts the production of EPO.

EPO then increases RBC production, which carries hemoglobin. This increases the oxygen-carrying capacity of the blood and helps absorb more oxygen to maintain homeostasis in hypoxic conditions.

Important Applications Of The Discoveries:

Tissue ischemia is a major cause of morbidity and mortality (Anaemia, Stroke, Myocardial infarction). In principle, drugs that stabilize HIF may augment angiogenesis (formation of new blood vessels) and the adaptation to chronic hypoxia.

Several small-molecule proline hydroxylase inhibitors such as daprodustat, roxadustat, molidustat, vadadustat, and desidustat have been developed as antifibrotic agents and can now be tested in ischemia models.

Solid tumors often contain hypoxic regions due to the chaotic architecture leading to transient interruptions in tumor blood flow. Consistent with these findings, increased HIFα levels have been documented in many solid tumors.

Moreover, both hypoxia and high HIFα levels are usually linked to poor prognosis in cancer patients. An alternative explanation, however, would be that highly aggressive tumors are more likely to outgrow their blood supply, and hence become hypoxic, than more indolent tumors. In this scenario, HIFα is a marker, rather than a cause, of malignant cell behavior.

A causal role for HIFα, and particularly HIF2α, in the formation of a tumor, has perhaps been most firmly established through studies of pVHL-defective tumors. Notably, VHL/ clear cell renal carcinomas usually overproduce both HIF-1α and HIF2α or HIF2α alone.

In preclinical tumor models, HIF2α (but not HIF-1α) overexpression can override pVHL’s tumor suppressor activity whereas elimination of HIF2α in VHL/ renal carcinoma cells is sufficient to suppress tumor growth. These observations suggest that drugs that inhibit HIF, or critical downstream HIF targets, might be useful for treating cancers such as VHL/clear cell carcinomas.


References and Key Publications:

Oxygen Sensing by Metazoans: The Central Role of the HIF Hydroxylase Pathway William G. Kaelin, Jr. and Peter J. Ratcliffe

Kaelin, W.G. (2005). Proline hydroxylation and gene expression. Annu. Rev. Biochem.74.

Schofield, C.J., and Ratcliffe, P.J. (2004). Oxygen sensing by HIF hydroxylases. Nat. Rev. Mol. Cell Biol. 5.

Semenza, G.L. (2003). Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3.

Kaelin, W.G., Jr (2007). Von Hippel-Lindau disease. Annu. Rev. Pathol. 2.

Semenza, G.L, Ne Semenza, G.L, Nejfelt, M.K., Chi, S.M. & Antonarakis, S.E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer felt, M.K., Chi, S.M. & Antonarakis, S.E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer element located 3’ to the human erythropoietin gene. Proc NatlAcad Sci USA, 88

Wang, G.L., Jiang, B.-H., Rue, E.A. & Semenza, G.L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad SciUSA, 92

Maxwell, P.H., Wiesener, M.S., Chang, G.-W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R. & Ratcliffe, P.J. (1999). The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature, 399,

Mircea, I., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S. & Kaelin Jr., W.G. (2001) HIFa targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing. Science, 292

Jakkola, P., Mole, D.R., Tian, Y.-M., Wilson, M.I., Gielbert, J., Gaskell, S.J., von Kriegsheim, A., Heberstreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W. & Ratcliffe, P.J. (2001). Targeting of HIF-a to the von Hippel-Lindau ubiquitylation complex by O2- regulated prolyl hydroxylation. Science, 292

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