Fig 2: Nt-acetylation abrogates oxidation by ADO in vitro and vice versa.a Deconvoluted MS spectra showing oxidation (32 Da increase) of RGS42-15 (RGS4) and N-terminally acetylated RGS42-15 (Ac-RGS4) before (blue, 0 min) and after (black, 30 min) incubation with HsADO at 37 °C (100 µM peptide, 0.1 µM ADO). b Oxidation of other acetylated ADO substrate peptides (Fig. 2a) compared to oxidation of RGS42-15 and IL322-15 peptides after 30 min (100 µM peptide, 0.1 µM HsADO, 37 °C). c Deconvoluted MS spectra showing acetylation (42 Da increase) of JUNB2-15 (JUNB) and N-terminally oxidised JUNB2-15 (Ox-JUNB) before (blue, 0 min) and after (black, 30 min) incubation with NatA at 37 °C (100 µM peptide, 0.15 µM NatA). d Acetylation of other oxidised NatA substrates peptides (Fig. 6a) compared to acetylation of non-oxidised NatA substrates peptides after 90 min (100 µM peptide, 0.15 µM NatA, 37 °C). In (b, d), data are presented as mean ± standard deviation, n = 3. Statistical significance determined using 2-way ANOVA with Bonferroni’s multiple comparison post-test: ****p ≤ 0.0001. Source data are provided as a Source Data file.

Fig 3: Modulation of mGluR5-associated calcium signaling by RGS4 overexpression in astrocytes. Astrocyte cultures were infected with a control lentivirus or as RGS4-lentivirus, both carrying an mCherry expression cassette, allowing for the detection of infected cells (bar 400 µm) (A). RGS4 expression was confirmed by immunoblotting (B). Astrocytes were exposed to 50 µM DHPG for 120 s, then to 10 µM of MTEP for 60 s, and changes in the cytosolic calcium concentration were monitored using Fura2. Representative traces indicating a Fura2 fluorescence ratio from 4 to 8 different cells are shown for astrocyte cultures infected with control lentiviruses (C) or with lentiviruses encoding RGS4 (D). Panel (E) illustrates the proportion of cells showing calcium oscillations and peak–plateau responses for each condition. Panels (F,G) are representative of the signaling recorded in cells responding with a peak–plateau profile after infection with the control and RGS4 lentiviruses, respectively, and the red traces represent the average of the signal measured for these cells. The histogram in (H) shows the slope depicting the decay of the plateau from these cells. Descriptive statistics (mean, SEM) were obtained from at least three different experiments. Statistical tests were performed through a hierarchical mixed logistic regression fitted by maximum likelihood, with lentiviral infection as fixed and experiment repetition as the random effect, along with Wald z-test statistics for comparisons between fixed effects (E) or a one-way ANOVA followed by Student pairwise comparisons (H) (* p < 0.05, *** p < 0.001, ### p < 0.001).

Fig 4: Differential sensitivity of ADO and PHD enzymes to iron chelators.A, RKO cells stably expressing an RGS41–11GFP 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 Fe2+ 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, ADO-deficient RKO cells were treated with the Fe2+ 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.

Fig 5: Regulation of RGS4 and 5 by Co2+.A, RKO cells stably expressing an RGS41–11GFP fusion reporter gene were treated with increasing concentrations of CoCl2, MnCl2 or NiCl2 for 24 h. Accumulation of the reporter gene was monitored by fluorescence, and viability was assessed in parallel. B, Co2+ but not Mn2+ or Ni2+ 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 CoCl2 for 4 or 24 h, and accumulation of RGS4 protein or mRNA transcript was assessed. D, RKO cells were treated for 24 h with Co2+ 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 HA-tagged WT or C2A mutant RGS4 were treated with CoCl2 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 CoCl2 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.