Clpx plays pleiotropic roles in heme synthesis and erythropoiesis Free

Differentiating red cells synthesize large quantities of heme for hemoglobin production, requiring tight coordination between mitochondrial iron import and synthesis of protoporphyrin IX (PPIX). Accumulation of PPIX which is toxic and light sensitive, causes erythropoietic porphyria typically caused by loss of function mutations in FECH, or gain of function mutations in ALAS2. We previously reported that a dominant heterozygous point mutation in human CLPX (G298D) was a novel cause of erythropoietic protoporphyria (Yien et al. 2017, PNAS). CLPX encodes a ubiquitous ATPase-dependent mitochondrial protein unfoldase which is required for heme synthesis and incorporation of iron into the tetrapyrrole ring. It tightly regulates the stability of mitochondrial heme synthesis enzymes, ALAS1 and 2 and the activity of the terminal enzymes of the heme synthesis pathway, FECH and PPOX. CLPX also regulates mitochondrial iron utilization via mechanisms that have not been characterized (Rondelli et al. 2021, JBC).

Because the patient carrying the CLPX G298D mutation predominantly exhibited hematologic phenotypes, we asked if the role of CLPX in heme synthesis was erythroid-specific. We knocked out CLPX in 3T3 fibroblasts and quantitated heme synthesis using ¹⁴C-glycine and ¹⁴C-ALA. While ¹⁴C-glycine-labeled Clpx^-/-cells had normal heme synthesis, ¹⁴C-ALA-labled Clpx^-/-cells had decreased heme, indicating that there was a heme synthesis defect at the terminal steps of the pathway, which was compensated by excess ALA production caused by increased ALAS1 enzyme levels. These data indicated that CLPX is required for heme regulation in multiple cell types, but the extent to which it is required for the terminal steps of the heme synthesis pathway can vary. CLPX-EPP results from accumulation of PPIX due to excess ALA formation, and its symptoms in a patient can be ameliorated by iron supplementation (Ducamp et al. 2021, Hematologica).

To further interrogate the mechanism of the CLPX G298D in CLPX-EPP and the role of iron status in porphyrin accumulation, we generated G298D homozygous and G298D/- mutants in mouse erythroleukemia cells to investigate the role of the mutation and dosage dependence. Surprisingly, the G298D homozygous mutants did not exhibit porphyrin accumulation, while the G298D/- mutant did, suggesting a role for stoichiometry in heme regulation. Iron supplementation significantly decreased PPIX levels in G298D/- MEL cells, recapitulating previous observations in a patient (Ducamp et al. 2021, Hematologica) and suggesting that CLPX plays a role in coordinating iron utilization with heme synthesis. G298D/G298D and G298D/- mutants had a significant decrease in FECH activity, indicating that decreased FECH activity likely played a role in CLPX-EPP pathophysiology. We also knocked in the CLPX E360Q mutation into MEL cells which has deficient ATP binding but stabilizes the CLPXP multimer and retains proteolytic activity. The E360Q, like Clpx^-/-cells, had defective heme synthesis. Unlike Clpx^-/-, the E360Q appeared to be dominant, having a greater decrease in FECH activity. Further, iron was unable to rescue heme synthesis. On a molecular level, this data indicates that the proteolytic activity of CLPX can be uncoupled from its role in heme regulation.

Lastly, we generated Clpx^fl/flmice to determine its role in erythropoiesis in vivo. We crossed these mice into the Gata1:Cre-ER mice to knock out CLPX in erythroid lineages, and LyzM:Cre to knock it out in monocytes/macrophages, as macrophages are an important component of the erythropoietic niche. The Gata1:Cre-ER; Clpx^fl/flmice had decreased erythroid cell number, as expected from precious data.Both these mice had enlarged spleens and increased splenic erythropoiesis, suggesting compensation for erythropoietic stress. Our in vivo data indicate that CLPX plays both cell-intrinsic and cell-extrinsic roles in erythropoiesis. Collectively, our biochemical and in vivo data indicate that CLPX plays multiple roles in erythropoiesis and the erythropoietic niche, underscoring its importance in both iron regulation and erythroid development.