The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover.

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx-/- cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism.

Radiolabeling Heme and Porphyrin with 14C-Glycine or 14C δ-Aminolevulinic Acid.

Radiolabeling enables the quantitation of newly synthesized heme and porphyrin, allowing us to distinguish heme synthesis rates from total cellular heme. Here, we describe a protocol for labeling heme with 14C-glycine or ALA and the sequential extraction of heme and porphyrin from the same samples for quantitation by liquid scintillation.

Preschool children involve a third party in a dyadic sharing situation based on fairness.

To investigate whether preschool children would involve third parties in dyadic sharing situations, we created unequal sharing situations in which one agent had no resources and a child participant and a third-party individual had unequal resources. We analyzed children's tendency to share with the indigent agent and to involve the wealthy agent, whereas the indigent agent elicited sharing from them by means of progressive levels of request. The results showed that 5-year-olds involved the third party to a greater extent than 3.5-year-olds when they had fewer resources than the third party (Experiment 1). Yet, when 5-year-olds had the largest amount of resources, they were less likely to involve a third party but more likely to share themselves (Experiment 2). This study provides the first evidence that the tendency to involve third-party individuals in resource allocation situations develops at the preschool age and that it is based on fairness considerations.

FAM210B is dispensable for erythroid differentiation in adult mice.

Iron plays a central role in cellular redox processes, but its ability to adopt multiple oxidation states also enables it to catalyze deleterious reactions. The requirement for iron in erythropoiesis has necessitated the evolution of mechanisms with which to handle the iron required for hemoglobinization. FAM210B was identified as a regulator of mitochondrial iron import and heme synthesis in erythroid cell culture and zebrafish models. In this manuscript, we demonstrate that while FAM210B is required for erythroid differentiation and heme synthesis under standard cell culture conditions, holotransferrin supplementation was sufficient to chemically complement the iron-deficient phenotype. As the biology of FAM210B is complex and context specific, and whole-organism studies on FAM210 proteins have been limited, we sought to unravel the role of FAM210B in erythropoiesis using knockout mice. We were surprised to discover that Fam210b -/- mice were viable and the adults did not have erythropoietic defects in the bone marrow. In contrast to studies in C. elegans, Fam210b -/- mice were also fertile. There were some modest phenotypes, such as a slight increase in lymphocytes and white cell count in Fam210b -/- females, as well as an increase in body weight in Fam210b -/- males. However, our findings suggest that FAM210B may play a more important role in cellular iron homeostasis under iron deficient conditions. Here, we will discuss the cell culture conditions used in iron metabolism studies that can account for the disparate finding on FAM210B function. Moving forward, resolving these discrepancies will be important in identifying novel iron homeostasis genes.