CLPX and CLPP proteins are required for regulation of ALAS2
Date
2021
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Publisher
University of Delaware
Abstract
Heme synthesis consists of a series of eight enzymatic steps that result in the synthesis of porphyrin products, which in turn can be used to generate other tetrapyrrole compounds. In late-stage erythropoiesis, these precursors can be converted into its terminal product, heme. Failure to convert one of the intermediate products results in buildup of heme precursors, ALA, and porphyrins, due to defects in regulation, which results in free reactive oxygen species due to autoxidation and photochemical reactions which are toxic and cause oxidative stress to the cell (Ryter & Tyrrell, 2000). It is therefore important to understand the mechanism of regulation for the precursors associated with the heme synthesis pathway to prevent the development of serious diseases such as porphyria. Here we focus on the ALAS2 enzyme which catalyzes the first, and rate-limiting, step of the heme synthesis pathway. To begin the synthesis of heme, glycine is imported and is condensed with succinyl-CoA to form δ-aminolaevulinic acid (ALA) catalyzed by ALA synthase, which has been identified as ALAS2 in erythroid cells (Hamza, I and Dailey HA, 2012). Studies demonstrate that ALAS is activated by mitochondrial ClpX by incorporating pyridoxal phosphate (Kardon, JR et al 2015). While this is an important breakthrough in heme synthesis studies, it is important to elucidate the role of CLPX and CLPP proteins in the mammalian erythroid cells. ☐ In this study, we hypothesized that loss of mammalian CLPX and CLPP will result in the disruption of hemoglobinization by altering the activity of ALAS2 as well as the regulation and stability of ALAS2 in erythroid cells. To test this hypothesis, we generated Clpx and Clpp knockout mouse erythroleukemia (MEL) cell lines using CRISPR/Cas9 mediated deletions. We first investigated alterations in hemoglobinization in knockout Clpx and Clpp MEL cell lines using benzidine staining to differentiate MEL cells and found that there were no changes in hemoglobinization. We then measured changes in expression of ALAS2 in knockout Clpx and Clpp MEL cell lines and we found that Clpx -/- demonstrates an increase in ALAS2 expression while Clpp -/- shows no change in ALAS2 expression in undifferentiated cells. Next, we examined alterations in Alas2 expression as a result of loss of Clpx and Clpp in both differentiated and undifferentiated MEL cell lines and we found that a loss of CLPX and CLPP decreases Alas2 mRNA expression. Finally, we will examine ALAS2 stability using Cycloheximide (CHX) pulse-chase assay in both differentiated and undifferentiated MEL cell lines and found in our Clpx -/- cell lines, we identified one line, Clpx B -/-, where there was a decrease in ALAS2 degradation and therefore increased ALAS2 stability in the undifferentiated cell line. Interestingly, we do see that ALAS2 degradation is decreased and therefore ALAS2 stability is prolonged throughout the 6-hour assay in differentiated cells for both the DS19 wildtype and knockout cell lines. ☐ Our data also demonstrates that CLPX may have a role in the regulation and overall stability of ALAS2 in early erythroid cells in culture as well as regulation of ALAS2 in differentiating cells is separate from that seen in non-differentiating cells. Due to the knock-out data, it is likely that ALAS2 transcriptional and post-translational activity is regulated primarily by CLPX expression in a feedback mechanism, and ALAS2 degradation is regulated by the expression of CLPP. Additionally, our data suggest that CLP proteins are required for the regulation of the initial steps and subsequent regulation of ALAS2 at the transcriptional and post-translational levels in the heme synthesis pathway. We also show that regulation of ALAS2, during differentiation, occurs with some uncharacterized addition to the regulatory pathway which results in increased stability of ALAS2 during differentiation. It still remains unanswered what additional players may have a role in the regulation of ALAS2 expression, activation, and its degradation and we must further investigate what drives the interactions between ALAS2 and CLPX and to what extend CLPP plays a role in the degradation of ALAS2 and how that degradation is mediated.
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Keywords
Erythroid, Erythropoiesis, Heme, Heme synthesis, Hemoglobin, Red blood cell