Oxygen Sensors: When is a target not a target?
Oxygen is essential for life – just count how many times you need to breathe while reading this article – and is used by virtually every cell in the human body. Most cells are able to sense a diminished oxygen supply (hypoxia) and respond by making changes to cellular metabolism, blood vessel formation and oxygen delivery. For example, physiological hypoxia, such as that encountered in anemia or at high altitudes, induces the production of a hormone called EPO, which causes the body to make more red blood cells to improve oxygen delivery.
Cells must have highly responsive oxygen sensors to regulate these processes, but to date researchers have found only one family of enzymes – the 2-oxoglutarate dioxygenase enzyme family – that is capable of sensing physiological levels of oxygen. These enzymes use molecular oxygen (O2), iron ions and 2-oxogluterate (a molecule with the chemical formula C5H6O5) to catalyze the transfer of oxygen onto amino acid or DNA substrates (Islam et al., 2018). They act as both hydroxylases, catalyzing the addition of a hydroxyl group (OH) to substrates, and dioxygenases, using O2 as a cosubstrate. Researchers have identified four enzymes from this family that act as oxygen sensors to regulate three transcription factors called HIF1α, HIF2α and HIF3α, which in turn regulate how cells express genes in response to hypoxia. Three of these enzymes are prolyl hydroxylase (PHD) enzymes, and the fourth is called factor inhibiting HIF (Epstein et al., 2001; Ivan et al., 2001; Hewitson et al., 2002; Lando et al., 2002).
The PHD enzymes use molecular oxygen to catalyze the hydroxylation of two proline amino acids in the HIFα proteins. When oxygen levels are normal, the HIFα proteins are hydroxylated, which causes them to be degraded by the cell (Figure 1). However, when oxygen levels decrease, leading to hypoxia, the HIFα proteins are not hydroxylated, so they are not degraded as rapidly. This allows them to migrate to the nucleus and activate the genes responsible for adapting to hypoxia (Kaelin and Ratcliffe, 2008).
Since the discovery of the PHD enzymes and factor inhibiting HIF, it has been unclear whether these enzymes could hydroxylate targets other than the HIFα proteins. If PHD enzymes hydroxylate other targets it would suggest that additional non-HIF pathways might be involved in the hypoxia response. Previous research efforts have identified many other potential targets for the PHD enzymes, including some with links to physiological responses to hypoxia (Strowitzki et al., 2019). However, many of these studies did not demonstrate that the PHD enzymes were directly catalyzing the hydroxylation of these proteins, raising doubts as to whether these proteins are bona fide targets for the PHD enzymes. Now, in eLife, Matthew Cockman (Francis Crick Institute), Peter Ratcliffe (University of Oxford) and co-workers – including Kerstin Lippl and Ya-Min Tian (both in Oxford) as joint first authors with Cockman, and other researchers from the Crick, Oxford and the University of Oulu – report on a fascinating study that seeks to clarify the situation (Cockman et al., 2019).
Cockman et al. undertook a rigorous array of in vitro biochemical and mass spectrometry experiments using purified enzymes and substrates. While they confirmed that the PHD enzymes robustly catalyze the hydroxylation of proline residues in HIFα proteins, they found no evidence for the hydroxylation of any of the other targets in vitro. Overall, they studied more than 20 different candidate target proteins and 40 potential modification sites. The substrates used in the experiments were short synthetic peptides and full-length recombinant proteins.
These results suggest that the HIFα proteins are the only primary targets of the oxygen-sensing PHD enzymes. If this is the case, then PHD enzymes have a more focused role in hypoxic signaling than previously thought. This is important for predicting the consequences of manipulating PHD activity for therapeutic purposes. However, while these well-controlled, designed and executed biochemical experiments show that targets other than HIFα proteins cannot be efficiently hydroxylated in vitro, they do not preclude the modification of these targets in vivo. This is because living cells might contain additional cofactors that the PHD enzymes need to hydroxylate non-HIFα targets. Furthermore, modulating the activity of the PHD enzymes affects HIF-independent processes, indirectly pointing to potential non-HIF targets. (Strowitzki et al., 2019).
If in vivo experiments confirm that the HIFα proteins are the only primary targets of the PHD enzymes, as Cockman et al. suggest, this would make these enzymes central to of one of the most specialized sensing and control systems in the cell.
References
-
Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural familyJournal of Biological Chemistry 277:26351–26355.https://doi.org/10.1074/jbc.C200273200
-
2-Oxoglutarate-dependent oxygenasesAnnual Review of Biochemistry 87:585–620.https://doi.org/10.1146/annurev-biochem-061516-044724
Article and author information
Author details
Publication history
- Version of Record published: September 12, 2019 (version 1)
Copyright
© 2019, Bersten and Peet
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,260
- views
-
- 99
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While d-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal d-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a d-serine transporter previously uncharacterized in the kidney and discovered d-serine as a non-canonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal d-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.
-
- Biochemistry and Chemical Biology
- Cell Biology
Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.