Figure 5.
The PPP is differentially regulated between E10.5 and E12.5 erythroid precursors. (A) Pathway map of the interplay between glycolysis and the PPP is shown. (B) Relative abundance of PPP enzymes between E10.5 and E12.5 populations. (C-D) Relative abundance of PPP metabolites (C) and reduced (GSH) and oxidized (GSSG) glutathione (D) in E10.5 and E12.5 populations. (E) Increased expression of hemoglobin (Hb) chains and band 3 (or solute carrier protein 4A1-SLC4A1). (F) These proteins are involved in the phenomenon of oxygen-dependent metabolic modulation, which regulates metabolic fluxes through glycolysis and the PPP as a function of Hb oxygenation status and competitive binding of deoxyhemoglobin and glycolytic enzymes for the N-terminus cytosolic domain of SLC4A1 band 3.59,73,74 (G-I) Switch-tag proteomic approach (G) revealed differing cysteine oxidation status (H) in the 2 cell populations; these differences were accompanied by altered expression levels of many redox enzymes (I), including CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1) and GPX4, glutathione reductase (GSR), and peroxiredoxin 1 (PRDX1), PRDX2, and PRDX6. Metabolites are graphed as violin plots and proteins as box-whisker plots. (J-K) Levels of ROS in E10.5 or E12.5 cells endogenously after collection (J) or after exogenous challenge (K) with 3, 10, or 30 μM H2O2. *P < .05, **P < .01, ***P < .001, ****P < .0001. ALDO, aldolase; ATP, adenosine triphosphate; AU, arbitrary unit; DPG, diphosphoglycerate; DTT, dithiothreitol; emPAI, exponentially modified protein abundance index; FBP, fructose-1,6-bisphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; IAM, iodoacetamide; LAC, lactate; MFI, mean fluorescence intensity; NADP, NAD phosphate; NADPH, reduced NADP; NEM, N-ethylmaleimide; ns, not significant; PFK, phosphofructokinase; PPP, Pentose phosphate pathway; 6PGD, 6-phosphogluconate dehydrogenase; 6PGL, 6-phosphogluconolactonase; RPE, ribulose-phosphate 3-epimerase; RPI, ribose-5-phosphate isomerase; RPIA, ribose-5-phosphate isomerase A; SEM, standard error of the mean; TALDO, transaldolase; TKT, transketolase.

The PPP is differentially regulated between E10.5 and E12.5 erythroid precursors. (A) Pathway map of the interplay between glycolysis and the PPP is shown. (B) Relative abundance of PPP enzymes between E10.5 and E12.5 populations. (C-D) Relative abundance of PPP metabolites (C) and reduced (GSH) and oxidized (GSSG) glutathione (D) in E10.5 and E12.5 populations. (E) Increased expression of hemoglobin (Hb) chains and band 3 (or solute carrier protein 4A1-SLC4A1). (F) These proteins are involved in the phenomenon of oxygen-dependent metabolic modulation, which regulates metabolic fluxes through glycolysis and the PPP as a function of Hb oxygenation status and competitive binding of deoxyhemoglobin and glycolytic enzymes for the N-terminus cytosolic domain of SLC4A1 band 3.59,73,74  (G-I) Switch-tag proteomic approach (G) revealed differing cysteine oxidation status (H) in the 2 cell populations; these differences were accompanied by altered expression levels of many redox enzymes (I), including CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1) and GPX4, glutathione reductase (GSR), and peroxiredoxin 1 (PRDX1), PRDX2, and PRDX6. Metabolites are graphed as violin plots and proteins as box-whisker plots. (J-K) Levels of ROS in E10.5 or E12.5 cells endogenously after collection (J) or after exogenous challenge (K) with 3, 10, or 30 μM H2O2. *P < .05, **P < .01, ***P < .001, ****P < .0001. ALDO, aldolase; ATP, adenosine triphosphate; AU, arbitrary unit; DPG, diphosphoglycerate; DTT, dithiothreitol; emPAI, exponentially modified protein abundance index; FBP, fructose-1,6-bisphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; IAM, iodoacetamide; LAC, lactate; MFI, mean fluorescence intensity; NADP, NAD phosphate; NADPH, reduced NADP; NEM, N-ethylmaleimide; ns, not significant; PFK, phosphofructokinase; PPP, Pentose phosphate pathway; 6PGD, 6-phosphogluconate dehydrogenase; 6PGL, 6-phosphogluconolactonase; RPE, ribulose-phosphate 3-epimerase; RPI, ribose-5-phosphate isomerase; RPIA, ribose-5-phosphate isomerase A; SEM, standard error of the mean; TALDO, transaldolase; TKT, transketolase.

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