Oxygen Sensors: When is a target not a target?
Cells rely on prolyl hydroxylase enzymes to sense low levels of oxygen, but they might act on fewer targets than previously thought.
- University of Adelaide, Australia
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).
Figure 1
PHD enzymes, HIFα protein and the hypoxia response.
When oxygen levels are normal (normoxia, left), a PHD enzyme (green) can use molecular oxygen (O2), iron ions (Fe2+) and 2-oxogluterate (2-OG) to hydroxylate (ie, add an OH group to) two proline amino acids (P) on a HIFα protein. Hydroxylation destabilizes the HIFα protein, causing it to be degraded by the cell, and the genes involved in the hypoxic response of the cell are not expressed. When oxygen levels are low (hypoxia, right), the PHD enzyme is not able to hydroxylate the HIFα protein, so this protein can migrate into the nucleus and bind to a protein called ARNT. Together, they interact with hypoxia response elements (HREs) in the genome to activate the transcription of hypoxia response genes. ARNT: aryl hydrocarbon receptor nuclear translocator or hypoxic inducible factor-β (HIFβ); HIF: hypoxic inducible transcription factor; PHD: prolyl hydroxylase.
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
-
- 1,275
- views
-
- 100
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
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)
Oxygen Sensors: When is a target not a target?
eLife 8:e50585.
https://doi.org/10.7554/eLife.50585
Further reading
-
- Biochemistry and Chemical Biology
- Plant Biology
Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.
-
- Biochemistry and Chemical Biology
- Neuroscience
Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer’s disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17–42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.
