Fig 2: The CBS inhibitor AOAA selectively increases ATP generation in DS hippocampal tissue. Hippocampal tissues from control or Dup(Rno20)Yah rats were obtained with punch biopsies and subjected to Extracellular Flux Analysis after incubation were treated with 0 or 100 μM AOAA for 1h at 37 °C. Data, expressed as mean ± SEM of 6 animals per experimental condition, were analyzed by two-way ANOVA followed by post hoc Bonferroni multiple comparison t-tests. ##p ≤ 0.01 shows the stimulatory effect of 100 μM AOAA on ATP generation in the Dup(Rno20)Yah group.

Fig 3: PHD2 is persulfidated by CBS and persulfidation of PHD2 at zinc finger domain maintains its activation.(A) Modified biotin switch assay for detecting persulfidation of PHD2 in untreated and NaHS-treated (1 mM) HUVEC and HAOECs. Dithiothreitol (DTT) treatment (1 mM) for 30 min reversed PHD2 persulfidation. (B) Modified biotin switch assay to determine the persulfidation status of PHD2 in si-Control and si-CBS–treated HUVEC and HAOECs after 48 hours transfections. (C) HUVEC and HAOEC were transfected with either scrambled (si-Control) or two PHD2 siRNA (si-PHD2_08 and si-PHD2_09) for 48 hours. Expression of PHD2 was determined by immunoblotting. β-Actin was used as a loading control. (D) HUVECs were first transfected with either si-Control or si-PHD2 for 24 hours and further transfected with HA–empty vector (EV) or cDNAs encoding HA-PHD2 [wild type (WT), C21S (M1), C33S (M2), C127S (M3), C201S (M4), or C208S (M5)] for another 36 hours, and mRNA expression levels of PHD2 gene was determined. 18S rRNA was used as an internal control. si-Control–pretreated and EV-transfected mRNA levels were set to 1, and data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when comparing between EV-treated groups in both si-Control or si-PHD2–pretreated group. (E) HUVECs were first transfected with si-PHD2 for 24 hours and further transfected with HA–empty vector (EV) or cDNAs encoding HA-PHD2 [WT, C21S (M1), C33S (M2), C127S (M3), C201S (M4), or C208S (M5)] for another 36 hours, and lysates from each treatment conditions were incubated with recombinant biotinylated HIF-1α peptide and further captured using streptavidin-conjugated magnetic beads. Next, these peptide-coated beads were incubated with in vitro–translated [35S] pVHL and finally quantified by SDS-PAGE and autoradiography as a measurement PHD2 activity. (F) HUVEC cells were transfected with either si-Control, si-PHD2, or si-PHD2 along with reexpression of WT or mutant PHD2 constructs. Cellular lysates served as a source of PHD2 and were incubated with GST or GST–HIF-1α prebound to GSH-agarose for 2 hours at 4°C. Respective agarose pull-down were then subjected to immunoblotting for PHD2 and HIF-1α, while cellular lysates served as input control for PHD2 and β-actin.

Fig 4: Alterations in cysteine catabolism followingsulfite oxidase (SO) deficiency. Cysteine catabolism in WT and SO-deficient cells. Steps and enzymes that localize to mitochondria are depicted by the light blue background. The area outside refers to the cytosol. Cytosolic glutamate oxaloacetate transaminase 1 (GOT1) is responsible for cellular sulfite production, whereas mitochondrial GOT (GOT2), plays an important role in H2S production in SO deficiency. In absence of SO, sulfite (SO32−) and hydrogen sulfide (H2S) accumulate. SO deficiency furthermore further impacts cellular cysteine catabolism resulting in lower intracellular cysteine levels and downregulation of many involved enzymes (depicted in gray letters). Moderate increase in GOT2 and strong induction of sulfide:quinone oxidoreductase (SQR) suggest an increased flux of sulfur via the GOT2-MPST-SQR axis. Abbreviations: CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; CDO, cysteine dioxygenase; GOT, glutamate oxaloacetate transaminase; MPST, mercaptopyruvate sulfurtransferase; SQR, sulfide:quinone oxidoreductase; ETHE1, persulfide dioxygenase; TST, thiosulfate sulfurtransferase.

Fig 5: Expression levels of proteins involved in H2S metabolism. A. Expression levels of proteins involved in cysteine catabolism (SO, SQR, CBS, CSE, TST, GOT1, MPST, GOT2 and ETHE1) in WT, SUOX−/−, GOT1−/−/SUOX−/− and GOT2−/−/SUOX−/− cells. Tubulin was used as a loading control. B. Quantification of expression changes of SQR in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.C. Quantification of expression changes of ETHE1 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. D. Quantification of expression changes of CSE in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. E. Quantification of expression changes of CBS in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. F. Quantification of expression changes of GOT1 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. G. Quantification of expression changes of GOT2 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. H. Quantification of expression changes of MPST (cytosolic and mitochondrial isoforms) in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.