The Role of TCA Cycle Compounds in Erythroid Heme Synthesis and Its Regulation During an Inflammatory Response

Abstract One early hallmark event of terminal erythroid differentiation is the induction of heme and globin synthesis. Onset of heme synthesis occurs when the first pathway enzyme, erythroid-specific 5-aminolevulinate synthase (ALAS2), is induced. The steps of heme biosynthesis are identical between non-erythroid (housekeeping) and differentiating erythroid cells, but two genes encode the first enzyme, 5-aminolevulinate synthase (ALAS). The housekeeping gene (ALAS1) is expressed in non-erythroid cells while the erythroid-specific gene (ALAS2) is expressed only in the erythron. Heme synthesis in metazoan cells requires iron, glycine, and succinyl CoA. The supply of iron required for heme synthesis has been well studied and a reasonable view of the components involved in this process exists. Glycine is abundant in plasma (~250 µM) and studies of glycine transporters are consistent with glycine being supplied from extracellular sources. For succinyl CoA the general assumption is that it is produced by the TCA cycle. This may be possible for housekeeping heme synthesis where only modest levels of heme are made at a given time, but this mechanism would clearly place cells under strain during erythroid differentiation when cells make ~109 molecules of heme in a span of 2-3 days. ALAS2, but not ALAS1, physically interacts with the succinyl CoA synthetase (SCS) ATP-dependent subunit SUCLA2. Since ALAS utilizes succinyl CoA to synthesize aminolevulinic acid (ALA), and because SUCLA2 as a subunit of SCS may be involved in the ATP-dependent reverse reaction of SCS to generate succinyl CoA from succinate, one popular hypothesis is that the ALAS2-SUCLA2 interaction exists to provide succinyl CoA for ALAS2. While the evidence for an association between ALAS2 and SUCLA2 is strong, there is no experimental data showing that SUCLA2 has an impact on ALAS2 activity or that there is a role for this interaction in the supply of succinyl CoA for ALAS2. In the current work we have sought to determine if succinyl CoA for heme synthesis during erythropoiesis in MEL cells originates from the TCA cycle or more directly from metabolism of glutamine via transaminases and α-ketoglutarate dehydrogenase (α-KGDH). Employing 13C-labeled glucose, diethyl succinate, or glutamine we determined that succinate is a poor source of carbons for erythroid heme synthesis, providing a maximum of 5 carbons out of a possible 26 (by isotopomer analysis) and labeling only 2.5% of carbons of isolated heme. Glucose contributes up to 18 carbons and a total of 8.3%, and glutamine contributes to 23 carbons and a total of 23%. Enzyme assays of TCA cycle enzymes revealed that during erythropoiesis only aconitase, isocitrate dehydrogenase, and α-KGDH are induced, which is consistent with glutamine, and not succinate, being the major source of carbon for heme. We also examined the impact of itaconate, a compound produced from the TCA cycle in LPS-induced macrophages, on erythroid differentiation. Itaconate is known to inhibit succinate dehydrogenase, and so might be expected to induce heme synthesis if succinyl CoA is being supplied from accumulating succinate via ATP-dependent SCS. However, dimethyl itaconate (a cell permeable form of itaconate) inhibits heme synthesis in differentiating erythroid K562 cells and this inhibition is reversed by ALA. It has long been assumed that SCS has broad specificity and is capable of making itaconyl CoA (Adler J. et al. 1957, JBC 229:865). However, we found that purified human and porcine SCS do not synthesize itaconyl CoA, but that the enzyme succinyl CoA:glutarate CoA transferase (SUGCT) generates itaconyl CoA from itaconate and succinyl CoA. Additionally we demonstrated that itaconyl CoA inhibits purified ALAS2 at µM concentrations. Previously published data along with our studies on K562 cells in the presence of LPS-induced RAW cells suggest that during the inflammatory response macrophages resident in erythroblastic islands produce itaconate as well as cytokines that induce proximal developing red cells to express a plasmalemmal transporter (possibly SLC13A5) required for itaconate import. Intracellular itaconate is then converted to itaconyl CoA via SUGCT, resulting in ALAS2 inhibition and a concomitant decrease in heme synthesis. This process restricts the amount of protoporphyrin synthesized during infection and, thereby, complements mechanisms that restrict the availability of iron to microbial pathogens. Disclosures No relevant conflicts of interest to declare.