Monitoring ADO dependent proteolysis in cells using fluorescent reporter proteins.

2-Aminoethanethiol dioxygenase (ADO) is the mammalian orthologue of the plant cysteine oxidases and together these enzymes are responsible for catalysing dioxygenation of N-terminal cysteine residues of certain proteins. This modification creates an N-degron motif that permits arginylation and subsequent proteasomal degradation of such proteins via the Arg-branch of the N-degron pathway. In humans 4 proteins have been identified as substrates of ADO; regulators of G-protein signalling (RGS) 4, 5 and 16, and interleukin-32 (IL-32). Nt-cysteine dioxygenation of these proteins occurs rapidly under normoxic conditions, but ADO activity is very sensitive to O2 availability and as such the stability of substrate proteins is inversely proportional to cellular O2 levels. Much is still to understand about the biochemistry and physiology of this pathway in vitro and in vivo, and Cys N-degron targeted fluorescent proteins can provide a simple and effective tool to study this at both subcellular and high-throughput scales. This chapter describes the design, production and implementation of a fluorescent fusion protein proteolytically regulated by ADO and the N-degron pathway.

Comparative analysis of N-terminal cysteine dioxygenation and prolyl-hydroxylation as oxygen-sensing pathways in mammalian cells.

In animals, adaptation to changes in cellular oxygen levels is coordinated largely by 2-oxoglutarate-dependent prolyl-hydroxylase domain (PHD) dioxygenase family members, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of non-transcription factor substrates; the regulators of G-protein signaling 4, 5. and 16 and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their co-evolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2-sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO, and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia. Collectively, our comparative analysis provides further insight into ADO/N-degron-mediated oxygen sensing and its integration into established mechanisms of oxygen homeostasis.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.

Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation.

Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O2 levels. Using an O2-sensitive phosphorescent nanoparticle probe, we measured an intracellular O2 level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O2. Induction of HO-1 and GCLM by SFN (2.5 µM) was significantly attenuated in cells adapted to 5 kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O2 and subjected to hypoxia (1 kPa O2, 1 h) and reoxygenation. In cells adapted to 18 kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 µM). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX™ Red and FeRhoNox™-1, was diminished in cells cultured under 5 kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O2 levels.

Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.

Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels.

The effects of physiological oxygen tension on Nuclear Factor-E2-Related Factor 2 (Nrf2)-regulated redox signaling remain poorly understood. We report the first study of Nrf2-regulated signaling in human primary endothelial cells (EC) adapted long-term to physiological O2 (5%). Adaptation of EC to 5% O2 had minimal effects on cell ultrastructure, viability, basal redox status or HIF1-α expression. Affymetrix array profiling and subsequent qPCR/protein validation revealed that induction of select Nrf2 target genes, HO-1 and NQO1, was significantly attenuated in cells adapted to 5% O2, despite nuclear accumulation and DNA binding of Nrf2. Diminished HO-1 induction under 5% O2 was stimulus independent and reversible upon re-adaptation to air or silencing of the Nrf2 repressor Bach1, notably elevated under 5% O2. Induction of GSH-related genes xCT and GCLM were oxygen and Bach1-insensitive during long-term culture under 5% O2, providing the first evidence that genes related to GSH synthesis mediate protection afforded by Nrf2-Keap1 defense pathway in cells adapted to physiological O2 levels encountered in vivo.