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.

Systems that sense, adapt, and alleviate exposure to hypoxia (low O 2 ) are observed throughout evolution, reflecting the fundamental importance of O 2 in biology and its obligate role in multicellular life forms.In mammals, a sensing system comprised of a set of 2-oxoglutarate-dependent dioxygenase enzymes (Prolyl Hydroxylase Domain, PHD1, 2, and 3, otherwise known as EGLN 2,1, 3) and their Hypoxia-Inducible factor (HIF-1, -2, and 3α) target substrates is widespread and likely responsible for the majority of adaptive transcriptional responses to hypoxia (reviewed in (1,2)).Similarly, oxygen sensing in plants is orchestrated by plant cysteine dioxygenases (PCOs) which regulate the stability of ethylene response factor VII transcription factors (3)(4)(5).Both systems respond to changes in environmental O 2 by inducing a pattern of gene expression capable of reducing cellular O 2 usage, while simultaneously increasing O 2 delivery at the tissue level.
It was recently demonstrated that enzymatic N-cysteine dioxygenation also occurs in mammals through an orthologue of PCO, 2-aminoethanethiol dioxygenase (ADO) (6).ADO was first identified as an enzyme that catalyzes the oxidation of cysteamine to hypotaurine (7).However, recent work indicates that it also functions as an N-terminal cysteine dioxygenase that regulates protein stability via the Arg/Cys branch of the N-degron pathway (8) in a manner similar to the PCOs (6).Unlike PCO and PHD enzymes, the known substrates of ADO are not transcription factors but rather regulators of G-protein signaling (RGS4, 5, and 16) and the atypical cytokine interleukin-32 (IL-32).These RGS proteins interact with Gαq and i/o subunits of the heterotrimeric G-protein complex to increase the rate of GTP hydrolysis, thereby repressing downstream signaling through the associated G-protein coupled receptor (as reviewed in (9)).The physiological role of IL-32 remains to be elucidated, but strong associations have been made between its expression and the severity of non-alcoholic fatty liver disease (10,11).IL-32 has also been implicated in the host response to hepatitis B and C infections (12,13).Under normoxic conditions, ADO substrates are degraded via N-degron-mediated proteolysis, but reductions in cellular O 2 levels lead to a reduction in ADO activity and thus to substrate stabilization.Much like the PHD enzymes, recombinant ADO and PCO are highly sensitive to O 2 in vitro (6,14) and so could potentially transduce signals under physiological and pathophysiological levels of hypoxia (e.g., 0-10% O 2 ).However, in contrast to the detailed characterization of PHD/HIF, little is known about the analogous ADO/RGS system or how the two systems interface.
Here we compare and contrast the operation of these O 2 sensing pathways in mammalian cells.We show that both pathways operate with similar in cellulo sensitivity with respect to O 2 but have important biochemical and pharmacological distinctions.Understanding the constraints within which each pathway operates in cells will be important in deciphering their shared and distinct roles in mammalian hypoxia physiology and potentially in the therapeutic modulation of these systems.

Results
A defining feature of physiological O 2 -sensing mechanisms, exemplified by the HIF/PHD system, is a graded response to changes in cellular oxygenation within the physiological range.In in vitro studies, both the PHD enzymes and the ADO have been reported to be similarly sensitive to O 2 , with apparent K m O 2 values 200 to 500 μM O 2 (6,15,16).We have previously reported limited studies of the sensitivity of the ADO substrates, RGS4, and 5 to hypoxia in cell lines.In the present analysis, we first sought to extend this work by comparing the oxygen sensitivity of known ADO substrates to that of HIF over a wide range of physiological to pathophysiological oxygen concentrations.Since cellular oxygenation in culture is defined by a complex interaction between the ambient O 2 levels, monolayer confluency, and cellular O 2 consumption (17), these experiments were conducted under similar conditions across a range of cell lines (SH-SY5Y, RKO, HepG2, Kelly, and EA.hy926).To guide these analyses, RNA extracts from each cell line were initially screened for the presence of transcripts encoding the four known ADO substrates; RGS4, RGS5, RGS16, and IL32 (Fig. 1A).This revealed that, among the cell lines studied, RGS4 is the most widely expressed substrate, with RGS16 and IL32 being confined to the neuroblastomal (SH-SY5Y and Kelly) and the gastrointestinal cell lines (HepG2 and RKO), respectively.We then exposed the cells to different O 2 levels (18, 7.5, 3, 1, and 0.1% v/v O 2 ) for 4 h (for RGS4/5) or 18 h (for IL-32, for the time course of induction see Fig. 6) and measured the abundance of HIF-1α and expressed ADO substrates.These experiments revealed strikingly similar sensitivity of HIF-1α and RGS4/5 and/or IL-32 to hypoxia in all cell types studied.Data are illustrated for all proteins studied in each cell line in Figure 1B and quantified using non-linear regression analysis in Figure 1C and demonstrates a closely similar relationship between HIF-1α and ADO substrate protein levels and applied pO 2 .We were unable to detect RGS16 protein in Kelly cells nor was RGS5 protein detectable in EA.hy926 cells, despite the presence of the corresponding mRNA transcript in these cells.

A B
In previous studies, the destabilizing action of N-terminal cysteine dioxygenation in human cells was demonstrated by measuring the half-life of fusion proteins bearing the N-terminus of the plant ERF-VII transcription factor fused to a green fluorescent protein-V5 reporter protein, thereby targeting the reporter protein for N-degron dependent proteolysis in the presence of O 2 (6).HIF-1α has been reported as having a remarkably short half-life in oxygenated cells (18).Given similarities between HIF-1α, RGS4, and 5 with respect to protein accumulation in hypoxic cells, we next wanted to compare the relative level of these proteins in cells using assays of endogenous protein levels following re-oxygenation of hypoxic cells (Fig. 2).Whilst levels of RGS4 and RGS5 proteins remained stable under continued hypoxic exposure, reoxygenation resulted in rapid degradation of RGS4 and 5 with a T 1/2 (time for disappearance of half of the species) of approximately 4 min.These kinetics were very similar to those of HIF-1α (T 1/ 2 5 min), assayed under the same conditions.Notably, these experiments were performed in the absence of the protein synthesis inhibitor, cycloheximide, as this was found to reduce RGS4 and 5 expressions beyond reliable detection at all time points.Therefore, the figures for T 1/2 represent maximum values.
The HIF Prolyl-hydroxylases and Nt-Cysteine dioxygenases coordinate their catalytic Fe 2+ via His-Asp-His and His-His-His triads, respectively (19)(20)(21), and in previous studies, ADO appeared sensitive to a narrower range of Fe 2+ chelating small molecules than the PHDs (6).To investigate this further, we tested a range of iron chelating compounds for their ability to inhibit ADO using RGS4 1-11 GFP fusion protein that has been shown to report ADO-dependent proteolysis in cells (22).RKO cells stably expressing RGS4 1-11 GFP were treated with six different iron chelators (2,2DIP −2,2 0 -dipyridyl, DFO - Comparison of cysteine dioxygenation and prolyl-hydroxylation deferoxamine, Dp44mT -di-2-pyridyl ketone 4,4-dimethyl-3thiosemicarbazone, ICL670A -desferasirox, L1deferiprone, SIH -salicylaldehyde isonicotinoyl hydrazone) at a range of concentrations, and accumulation of the reporter assayed by monitoring GFP fluorescence.Of the compounds tested only 2,2DIP, Dp44 mT and SIH manifest inhibition of ADO at concentrations that did not elicit cytotoxicity (Fig. 3A).Similar results were obtained when the accumulation of endogenous RGS4 or 5 was analyzed in RKO or SH-SY5Y cells, respectively (Fig. 3B).In contrast, all compounds were broadly capable of stabilizing HIF-1α (Fig. 3B).Analysis of intracellular free Fe 2+ in RKO cells using a fluorescent indicator demonstrated that all compounds except L1 reduced intracellular Fe 2+ levels within 4 h of treatment (Fig. 3C).Whilst this was consistent with the presence or absence of HIF-1α stabilization in this cell type (Fig. 3B), the efficacy of ADO inhibition (as inferred from RGS4 expression) did not correlate with intracellular free Fe 2+ chelation, suggesting a more complex interface between chelators and the catalytic iron center.To investigate this further, we next analyzed the total Fe content of FLAG-tagged ADO and PHD2 proteins immunoprecipitated from SH-SY5Y cells treated with either 2,2DIP or DFO.In line with observations on RGS4/5 stability, 2,2DIP was potent at removing iron from both ADO and PHD2, whereas DFO had very little impact on ADO iron content despite effectively chelating all the iron from PHD2 (Fig. 3D).Interestingly, ADO consistently contained a higher relative iron content than PHD2 following immunoprecipitation, corroborating earlier observations noting a remarkably high iron occupancy of purified ADO relative to CDO1 (7).Finally, there was no further stabilization of RGS4 observed in ADO-deficient cells following treatment with 2,2DIP, Dp44mT, or SIH (Fig. 3E), consistent with action through ADO.
In addition to Fe 2+ , the PHDs and other 2-OG dependent dioxygenases are inhibited by transition metal ions such as Co 2+ (23-25) and activation of HIF target genes such as erythropoietin is an important feature of the medical toxicology of such metals.The proposed explanation for the inhibition of PHD enzymes by Co 2+ is the displacement of Fe 2+ within the catalytic domain, thus rendering the enzyme inactive (25).In view of the differences in the efficacy of Fe 2+ chelators observed earlier, we hypothesized that ADO might be differentially sensitive to Co 2+ , or other divalent cations, as compared to the PHD enzymes.Treatment of RGS4 1-11 GFP reporter cells with increasing concentrations of either CoCl 2 , MnCl 2 or NiCl 2 resulted in dose-dependent cytotoxicity, with minimal accumulation of GFP (Fig. 4A).However, CoCl 2 treatment did elicit striking induction of endogenous RGS4 in

Comparison of cysteine dioxygenation and prolyl-hydroxylation
RKO cells and RGS5 in SH-SY5Y cells (Fig. 4B).Note, RGS4 protein level was not assessed in SH-SY5Y cells due to the contending influence of Co 2+ on HIF-mediated transcription in this cell line.Surprisingly, another ADO target, IL-32, was unaffected and induction of RGS4/5 was also clearly evident in ADO-deficient cells.Induction of RGS4 was not accompanied by detectable changes in RGS4 mRNA levels, as might be expected if it reflected a transcriptional response to HIF (Fig. 4C).
It was recently proposed that Co 2+ can cause "over-oxidation" of Nt-Cysteine residues, targeting them for lysosomal degradation (26).As was reported (26), we also observed an inhibitory effect of N-acetyl cysteine (NAC) on Co 2+ stimulated RGS4 and also on HIF-1α induction (Fig. 4D).However other compounds known to scavenge reactive oxygen species (ROS) such as ascorbate, the vitamin E analog Trolox (6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), or PEGylated superoxide dismutase had no effect on RGS4 levels.In contrast ascorbate did substantially reduce the level of HIF-1α, consistent with its known function in maintaining the activity of the catalytic Fe 2+ center of PHD enzymes (27).A persistent action of Co 2+ in the absence of ADO, in combination with the lack of effect on the accumulation of the RGS4 1-11 GFP reporter protein, suggested that Co 2+ does not interfere with N-terminal cysteine dioxygenation.In line with this hypothesis, Co 2+ induced protein levels of both RGS4:HA and a mutant that ablated the target cysteine residue RGS4(C2A):HA proteins in SH-SY5Y cells (Fig. 4E), with the latter being more abundant at baseline, as expected from a lack of action of ADO (6).It was previously reported that Co 2+ could directly bind to HIFα polypeptides and inhibit degradation irrespective of any effect on the PHD enzymes or HIF prolyl hydroxylation (28,29).Interestingly, we found significant Co 2+ -dependent enrichment of RGS4 in Hisimmunoprecipates from ADO-deficient SH-SY5Y lysates (Fig. 4F), strongly suggesting that RGS4 protein also binds Co 2+ .Taken together, this data indicates a hitherto undescribed effect of Co 2+ on RGS4 and RGS5 that is apparently independent of ADO and the action of reactive oxygen species.
Another characteristic of the HIF/PHD system is the operation of a feedback loop by which HIF-dependent upregulation of the HIF prolyl hydroxylases PHD2 and PHD3 serves to limit HIF activity over a period of hours (30).We observed that neither ADO protein nor mRNA levels were affected by periods of up to 48 h of hypoxia in SH-SY5Y cells, directly contrasting with a robust upregulation of PHD3 under those conditions (Fig. 5, A and B).Moreover, we found no evidence that other known components of the Arg/Cys Ndegron pathway were altered at the mRNA level by hypoxia (Fig. 5C).To address the effects on the time-dependent accumulation of ADO substrate proteins, cell lines were subjected to hypoxia (1% O 2 ), over periods ranging from 2 to 48 h, and the levels of substrate mRNAs and proteins were assessed (Fig. 6).These experiments revealed rapid accumulation of RGS4 and 5 in all cell lines tested, which was well-sustained even after 48 h continuous exposure to hypoxia, in contrast with HIF-1α.Most of these responses to hypoxia occurred without a change in the corresponding mRNA, consistent with an action of ADO on protein stability.In some cases, a modest induction of the corresponding mRNA was also observed (Fig. 6, A, B, and D) although this was nearly always delayed relative to the accumulation of protein.
Since all the ADO targets analyzed here have been reported to respond directly or indirectly to the HIF/PHD system in specific settings (31)(32)(33), we went on to investigate interactions between these systems.We first examined the expression of RGS4 mRNA in SH-SY5Y cells which it is been reported to be a target of HIF as well as ADO (31).siRNA targeting either HIF-1α or -2α was used to confirm the HIF dependency of this response, with successful knock-down confirmed by attenuation of hypoxia-induced CA9 and VEGFA transcripts, which were selected to report specific HIF-α isoform activity (Fig. 7A).Notably, increased hypoxic induction was observed when cells were treated with siRNA targeting the alternative HIF-α isoform (HIF-2α for CA9 and HIF-1α for VEGFA) which was repressed when both isoforms were targeted.Importantly, hypoxic induction of RGS4 mRNA was reduced in the presence of siRNA targeting HIF-2α and HIF-1+2α, whilst increased when cells were treated with HIF-1α siRNA alone (Fig. 7A), confirming an action of HIF-2α on RGS4 transcripts in SH-SY5Y cells.We then proceeded to address the relative contributions of ADO and HIF pathways to the accumulation of RGS4 protein in hypoxia.To this end, ADO-competent or -deficient SH-SY5Y cells were exposed to hypoxia (1% O 2 ) in the presence or absence of a HIF-prolyl hydroxylase inhibitor ([(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid, FG-2216 (34), Fig. 7B), a molecule previously shown not to inhibit ADO (6).As expected, hypoxia resulted in the accumulation of RGS4 in ADO-competent cells.Interestingly however, FG-2216 had no effect on RGS4 protein in normoxic cells whereas in ADO-deficient cells, in which RGS4 was constitutively stable, transcriptional upregulation of mRNA by

Comparison of cysteine dioxygenation and prolyl-hydroxylation
either hypoxia or FG-2216 resulted in a substantial additional increase in RGS4 protein levels.Thus, combined inhibition of ADO and PHD amplified the induction of RGS4 protein levels.Nevertheless, proteolytic control via ADO likely remains the dominant regulatory mechanism under many conditions, as witnessed by the absence of an effect of FG-2216 in ADOcompetent normoxic cells.Conversely, when proteolytic degradation is prevented, the accumulation of a given protein will be largely determined by the rate of de novo transcription/translation. We, therefore, wished to explore other interactions between transcriptional regulation and ADO-dependent proteolysis.IL32 has been shown to be regulated at the transcript level by inflammatory mediators such as TNFα (35,36).We, therefore, hypothesized that TNFα pre-treatment might influence the rate of IL-32 protein accumulation upon exposure to hypoxia, which was slower in HepG2 cells relative to other substrates in other cell lines (Fig. 6).To test this, HepG2 cells were pre-treated with TNFα for 16 h to elicit a robust induction of IL32 mRNA (Fig. 8A), and the effects of timed exposure to hypoxia (1% O 2 , 0.5-8 h) compared with and without this TNFα pre-treatment.These experiments revealed a marked interaction.Cells pretreated with TNFα demonstrated a greatly enhanced response of IL-32 protein levels to hypoxia (Fig. 8B) consistent with TNFα amplifying the effects of O 2 -regulated proteolysis transduced by ADO.To confirm this, ADO was inactivated in HepG2 cells by CRISPR/Cas9 gene editing, and these cells were treated with TNFα for 16 h, before a brief exposure to hypoxia (1% O 2 , 2 h).As ADO has been reported to influence TNFα signaling (37), cells were also treated with IL-1β (38).As shown in Figure 8C, induction of IL-32 protein by TNFα and IL-1β was much higher in ADO-deficient than parental HepG2 cells (Fig. 8C, compare lanes 1-3 and 7-9).As before, induction of IL-32 by hypoxia was greatly exaggerated in cytokine-treated cells, and observed even after brief (2 h) exposure.However, in ADO-deficient cells the very high levels of IL-32 were not further increased by hypoxia, consistent with ablation of ADO-dependent proteolysis.Taken together these results illustrate strong interactions in the multi-level control of substrates of the ADO pathway, with transcriptional induction amplifying O 2 -dependent regulation of ADO substrates at the protein level.

Discussion
Despite a common and fundamental requirement to maintain oxygen homeostasis, many different systems for sensing and responding to hypoxia have been defined in different species (39).Somewhat surprisingly, several of these different systems deploy a common strategy of enzymatic protein oxidation coupled with proteolysis as the core signaling process.Of particular interest are the plant and animal systems that deploy N-terminal cysteine dioxygenation and prolyl hydroxylation to regulate transcriptional responses mediated by ERF-VII transcription factors and HIF respectively.Remarkably, the plant system of N-terminal cysteine dioxygenation is also represented in humans and other animal species by the N-terminal cysteine dioxygenase, ADO, which therefore has the potential to work alongside PHD/HIF.Since oxygen homeostasis must be maintained across different tissues, operating at different levels of oxygen and over different time scales, we sought to compare and contrast the characteristics of these pathways in human cells.
Our findings indicate that the two systems are similarly sensitive to oxygen concentrations, but that their temporal responses and pharmacological interactions display different characteristics.Importantly, the known action of RGS4 and 5 on Gα i/q activity is likely immediate and therefore the physiological impact directly reflects the rapid accumulation of RGS proteins upon exposure to hypoxia (Fig. 6), and also the very rapid degradation when cells are re-oxygenated (Fig. 2).This contrasts with the HIF-PHD pathway in which, although the HIFα subunits are rapidly stabilized, accumulation of the target proteins is typically delayed and, once expressed, are not inherently O 2 -sensitive and therefore function on a time-scale dependent on their own half-lives.This distinction may be predicted to be important during rapid cycling of oxygen levels such as occurs in disorders of breathing control including obstructive sleep apnea, in ischemia reperfusion, and in the tumor microenvironment.Similar sensitivity to oxygen also suggested that the PHD/HIF and ADO systems might interact should they share common targets.Remarkably, a number of reports have described transcriptional responses to HIF among the limited range of ADO substrates defined to date.Our findings confirmed this and revealed that in specific settings the two systems do indeed interact to amplify responses to hypoxia.These interactions may be important both physiologically and therapeutically where, for instance, HIF prolyl hydroxylase inhibition may be more effective in hypoxic regions where ADO activity is also reduced.Moreover, the interaction between inflammation and hypoxia in the regulation of IL-32 further support the established bidirectional relationship between the two (40)(41)(42), manifesting in diseases such as inflammatory bowel disease.
Although both PHD and ADO enzymes share similar kinetic properties relating to O 2 , the reactions they catalyze are quite distinct.HIF prolyl-hydroxylation requires 2oxoglutarate as co-substrate and hence is potentially affected by metabolic signals that modulate cellular levels of this metabolite.The N-terminal cysteine dioxygenase ADO

Comparison of cysteine dioxygenation and prolyl-hydroxylation
incorporates both oxygen atoms into the cysteine residue and has no such co-substrate requirement or reaction by-products.Moreover, both classes of enzymes coordinate their catalytic iron center using distinct motifs (His-Asp-His for PHDs, 3xHis for ADO).Although our studies revealed that ADO, like the PHDs, is sensitive to inhibition by iron chelating agents, these effects differed substantially between the two types of enzyme.Notably, while inhibition of the PHDs by chelating agents was broadly concordant with their action to reduce intracellular iron, this was not the case with inhibition of ADO, for which only selected chelators were effective and inhibitory activity did not correlate with simple depletion of intracellular iron.This implies that the catalytic Fe 2+ in ADO is not freely exchanging with cytosolic Fe 2+ and that the inhibitory chelators have a more specific interaction with the catalytic iron center of ADO.The Fe 2+ in ADO may also be better protected from oxidation to Fe 3+ , inactivating oxidation.This is supported by our observations that neither Co 2+ nor ascorbate, thought to promote (43) and prevent (27) this reaction respectively, influenced ADO activity.Moreover, those chelators demonstrated to be ineffective against ADO (DFO, ICL670A, and L1) are reported to preferentially bind Fe 3+ (44,45).However, it must be stated that the iron chelating compounds used vary considerably in their physiochemical properties (size, cell permeability, binding capacity, and affinity) and may also contribute to differential action on ADO versus PHDs.
Interestingly, although Co 2+ ions appeared incapable of inhibiting ADO activity with only minimal induction of an ADO-dependent reporter gene observed, a striking upregulation of RGS4 and RGS5 proteins was observed in response to Co 2+ treatment, as has recently been reported by others (26).It was proposed that Co 2+ -induced tri-oxidation of the N-terminal cysteine of RGS4/5 leads to lysosomal degradation rather than via the canonical 26S proteasome, with the former presumably being much less efficient to account for the observed large increase in accumulated RGS4/5 in cells exposed to Co 2+ .If true, a reduction in the steady-state RGS4/5 protein level in ADO-deficient cells treated with Co 2+ would be expected, as a portion of the constitutively stabilized RGS4/5 would now be targeted for autophagy and a new equilibrium achieved.We have presented several lines of evidence in contradiction to this hypothesis (Fig. 4).The near absence of action of Co 2+ on the RGS4 1-11 GFP reporter protein and the persistence of an increase in endogenous RGS4/5 in ADO-deficient cells and a C2A mutant of RGS4 all suggest that the action of Co 2+ on RGS4 and RGS5 are not, in the main, mediated by an effect on the oxidation status of Nterminal cysteine.We suggest that the action of N-acetyl cysteine to inhibit this proposed oxidation likely results from direct chelation of Co 2+ ions in solution (46,47).Indeed, NAC is clinically approved for the treatment of acute Co 2+ poisoning (48,49).Further work is needed to define the precise mechanism through which Co 2+ acts to increase RGS4/5 stabilization, and its relevance to the toxicology of cobalt.In summary, our data provide evidence that ADO acts as a physiological oxygen sensor operating in cells alongside, and interacting with, HIF.The ADO system responds at oxygen concentrations similar to those that induce HIF, but its direct action to promote degradation of RGS proteins over a time scale of minutes enables the transduction of responses to altered oxygen levels on a shorter time scale.In cellulo characterization of the ADO pathway revealed marked interactions with the transcriptional induction of specific targets and distinct divalent metal pharmacology that offers new insight into the biochemistry of cellular dioxygenases.

Cell culture
The human cell lines SH-SY5Y, RKO and EA.hy926 were cultured as described previously (6).HepG2 was kindly gifted by Jane McKeating (University of Oxford, UK), and Kelly cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC 92110411).RKO, EA.hy926, and HepG2 cells were cultured in DMEM, and SH-SY5Y and Kelly cells in DMEM/F12, all supplemented with 10% fetal bovine serum, 2 mM L-Glutamine and 100 U/ml penicillin/10 μg/ml streptomycin.All cell lines were maintained at 37 C incubator containing 5% CO 2 , and hypoxic exposure was performed using an atmosphere-regulated workstation set to 0.1 to 7.5% O 2 : 5% CO 2 : balance N 2 (Invivo 400, Baker-Ruskinn Technologies).

RT-qPCR
mRNA was extracted from Tri-Reagent lysates by phase separation and equal amounts were used for cDNA synthesis using the High-Capacity cDNA Kit (Applied Biosystems).qPCR analysis was performed using Fast SYBR Green Master Mix on a StepOne thermocycler (Thermo Fisher Scientific) using the ΔΔCt method.Levels of the housekeeping gene Hypoxanthine-guanine phosphoribosyl transferase (HPRT) were used as a reference.Sequences for the primers used are as follows; RGS4 siRNA-mediated gene knockdown SH-SY5Y cells were seeded into 12-well plates and transfected twice on subsequent days with 40 nM of either scrambled siRNA (4390843) or siRNA sequences targeting HIF-1α (s6539), HIF-2α (s4700, Ambion) or a combination of both, diluted in OptiMEM and Lipofectamine RNAiMAX (Thermo Fisher Scientific).

Measurement of intracellular and ADO/PHD2-bound Fe 2+
Intracellular labile Fe 2+ levels were measured using Fer-roOrange, a fluorescent probe specific to Fe 2+ (52).RKO cells were seeded in black 96-well plates and treated with Fe 2+ chelators for 4 h in standard medium, which was subsequently replaced with imaging buffer (in mM; 117 NaCl, 4.5 KCl, 25 NaHCO 3 , 11 Glucose, 1 MgCl 2 , 1 NaH 2 PO 4 , 1 CaCl 2 ) containing 1 μM FerroOrange.Cells were incubated in a probe containing medium for 30 min at 37 C then fluorescence was measured at 544nm ex /590nm em using a FLUOstar Omega plate reader (BMG Labtech, UK).Fluorescence from a well containing medium alone (no FerroOrange) was used as a blank.To measure the total iron content of ADO and PHD2, N-terminally FLAG-tagged enzymes were immunoprecipitated from cells treated with either 2,2-dipyridyl or DFO (both 100 μM), and total Fe content was analyzed by Inductively coupled plasma mass spectrometry (ICP-MS).SH-SY5Y cells stably expressing an N-terminally FLAG-tagged HsADO or HsPHD2 were cultured to confluence in 15 cm plates, then treated for 4 h with 100 μM of either DFO or 2,2DIP and lysed in lysis buffer (see above).Enzymes were immunoprecipitated using anti-FLAG(M2) conjugated agarose beads overnight at 4 C, washed in PBS five times then eluted in 2% nitric acid.Beads incubated with lysis buffer (without cellular extract) were subjected to the same procedure as background readings for correction.Elutions were further diluted (2×) in 2% nitric acid and analyzed on a PerkinElmer NexION 2000B ICP-MS, calibrated using external calibration analysis with QMX standard dilutions, and spiked with 1 ng/g Rhodium to normalize any instrument drift.Values in immunoprecipitates were corrected for non-specific backgrounds by subtracting the iron detected in equivalent samples from cells that did not express any FLAG-tagged construct.

Cobalt-pull down
To measure the Co 2+ binding of RGS4/5, ADO-deficient SH-SY5Y cell lysates were incubated with HisBind resin (Millipore 69,670) pre-charged with Co 2+ ions, allowing the precipitation of Co 2+ -binding proteins as described previously (29).25 μl (packed volume) of HisBind resin was washed in PBS and then incubated with 50 mM CoCl 2 at room temperature for 1 h, then washed five times with lysis buffer.Cell lysate (15 cm dish per condition) was then incubated with Co 2+ (or control) loaded resin for 1 h under constant rotation at 4 C. Following this, the resin was pelleted (2000 rpm, 2 min) and a sample was taken and mixed with sample buffer to represent an unbound fraction.The resin was then further washed and subjected to the following elutions; 60 μl 60 mM imidazole for 10 min, 60 μl 1 M imidazole, then finally 70 μl SDS sample buffer.After each elution, the supernatant was taken and heated at 95 C for 5 min, then subjected to SDS-PAGE and immunoblotted as described above.

Figure 1 .
Figure 1.Response to graded hypoxia in mammalian cell lines.A, the expression pattern of ADO substrate mRNA transcripts in five cell lines.Color scale (white to blue) represents the Log 10 fold change in substrate mRNA level relative to HPRT.B, SH-SY5Y, RKO, HepG2, Kelly, and EA.hy926 cells were subjected to 4-h hypoxia (18 h for IL-32 in HepG2) at the indicated level of hypoxia and expression of ADO substrates present in each cell line assessed by immunoblotting.HIF-1α protein levels were analyzed in parallel for comparison.C, immunoblots from (B) were quantified and subjected to nonlinear regression analysis.In all but HepG2 cells, all data points were better represented statistically using one curve than individual curves, with p values 0.15 to 0.86.Data represent the mean ± SD from three independent experiments.

Figure 2 .
Figure 2. Degradation of RGS4, 5, and HIF-1α in the presence of oxygen.SH-SY5Y cells were cultured under hypoxic conditions (1% O 2 ) for 16 h then either maintained under hypoxia or reoxygenated by exposure to atmospheric levels of oxygen for 5, 15, or 45 min.Levels of RGS4, 5, and HIF-1α protein were assessed by immunoblotting, quantified, and subjected to nonlinear exponential decay regression to quantify the degradation.Data plotted are mean ± SD from three independent experiments.

Figure 3 .
Figure 3. Differential sensitivity of ADO and PHD enzymes to iron chelators.A, RKO cells stably expressing an RGS4 1-11 GFP fusion reporter protein were treated with various iron chelator compounds at the concentrations indicated for 24 h.Fluorescence (expressed as arbitrary units and closed squares) was measured and then cellular viability (open squares) was assessed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.B, RKO and SH-SY5Y cells were treated with the same six iron chelating compounds for 4 h at a maximal non-toxic concentration (all 100 μM except Dp44mT-3 μM and ICL670A-30 μM) and levels of the endogenous substrates RGS4 or RGS5 assessed by immunoblotting.Expression of HIF-1α was assayed in parallel for comparison.C, intracellular Fe 2+ levels were measured using FerroOrange in RKO cells treated with the indicated iron chelators for 4 h.D, measurement of total iron content in FLAG-ADO and FLAG-PHD2 immunoprecipitates from cells treated with DFO or 2,2DIP for 4 h, determined using ICP-MS.E, ADOdeficient RKO cells were treated with the Fe 2+ chelators shown to stabilize RGS4 (2,2DIP, Dp44mT, and SIH) for 4 h and levels of RGS4 protein assayed.Data in (A), (C), and (D) represent the mean ± SD from three independent experiments; all immunoblots are representative of at least three independent experiments.

Figure 4 .
Figure 4. Regulation of RGS4 and 5 by Co 2+ .A, RKO cells stably expressing an RGS4 1-11 GFP fusion reporter gene were treated with increasing concentrations of CoCl 2 , MnCl 2 or NiCl 2 for 24 h.Accumulation of the reporter gene was monitored by fluorescence, and viability was assessed in parallel.B, Co 2+ but not Mn 2+ or Ni 2+ induced RGS4 or 5 in RKO and SH-SH5Y cells, respectively.2,2DIP and HIF-1α immunoblots were used as positive controls.All chemicals were applied at 100 μM.C, ADO-competent or -deficient RKO cells were treated with CoCl 2 for 4 or 24 h, and accumulation of RGS4 protein or mRNA transcript was assessed.D, RKO cells were treated for 24 h with Co 2+ alone or in the presence of ascorbate (asc, 100 μM), N-acetyl-L-cysteine (NAC, 1 mM), PEGylated superoxide dismutase (SOD, 50 U/ml) or the vitamin E analogue Trolox (200 μM).E, SH-SY5Y cells stably expressing a C-terminal HAtagged WT or C2A mutant RGS4 were treated with CoCl 2 for 24 h as before.Endogenous RGS5 levels are shown for comparison.F, RGS4 precipitation from ADO-deficient SH-SY5Y cell extracts using His-beads pre-charged with CoCl 2 and successive elutions of increasing strength to assess the affinity of binding.β-actin was used as a negative control.All data are representative of at least three independent experiments.

Figure 5 .
Figure 5. Lack of apparent feedback regulation in the ADO pathway during hypoxic exposure.A, levels of ADO, arginyl transferase 1 (ATE1), and HIF prolyl-hydroxylase 3 (PHD3) protein in SH-SY5Y exposed to hypoxia for 4, 24, or 48 h.B, hypoxic upregulation of mRNA transcript encoding PHD3, but not ADO, in SH-SY5Y cells.C, no effect of hypoxia was observed on the transcript levels of other components of the Arg/Cys N-degron pathway (METAP1, methionine aminotransferase 1, UBR1/2, Ubiquitin Protein Ligase E3 Component N-Recognin 1/2).All data represent the mean ± SD from three independent experiments.

Figure 6 .
Figure 6.Time course of induction of known hypoxia-inducible proteins.Five cell lines; SH-SY5Y (A), RKO (B), HepG2 (C), Kelly (D), and EA.hy926 (E) were exposed to 1% O 2 for the indicated periods of time.Representative immunoblots (top) and mRNA levels of the ADO substrates (RGS5, RGS4, and IL-32) are shown below.All data represent the mean ± SD from three independent experiments.

Figure 7 .
Figure 7. Coordinated regulation of RGS4 by HIF-2α and ADO under hypoxia.A, SH-SY5Y cells were transfected with siRNA targeting HIF-1α and/or HIF-2α, or scrambled control (scr), and subjected to 24 h of hypoxia (1% O 2 ).RGS4 mRNA levels were assessed alongside canonical HIF-1α (CA9) or HIF-2α (VEGF) target genes as positive controls.The color scale represents mean fold change relative to cells treated with scr control (normoxia) from three independent experiments.B, ADO-competent or -deficient SH-SY5Y cells were exposed to hypoxia (1% O 2 ) or treated with the PHD inhibitor FG-2216 (100 μM), or both, for 4 h and samples blotted for RGS4 and HIF-1α protein.RGS4 mRNA levels were assessed in parallel, confirming transcript upregulation.Immunoblots are representative of three separate experiments, and data in the histogram are the mean ± SD, n = 3.All treatments significantly induced RGS4 mRNA, 2-way ANOVA with Holm-Sidak post-hoc analysis, p < 0.001.

Figure 8 .
Figure 8. Interplay between transcriptional and proteolytic regulation of IL-32.A, induction of IL32 mRNA in HepG2 cells treated for 16 h with TNFα (20 ng/μl).B, Accumulation of IL-32 protein in HepG2 cells treated with TNFα prior to exposure to hypoxia for the times indicated.C, ADO-competent or -deficient HepG2 cells were treated with TNFα or IL-1β (20 ng/μl) for 16 h then exposed to hypoxia for a further 2 h, and levels of IL-32 protein were analyzed.HIF-1α protein levels were assayed for comparison.All data represent the mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney t test (A) or 2-way ANOVA with Holm-Sidak post hoc analysis (B and C).