Ubiquitination of the Ferroportin Q248H Mutation Disregulates Autophagy, Hipoxia and Generates Lysosomal and Mitochondrial Damage
Authors: Vazquez-Meves Guelaguetza , Andrey Ivanov , Kumari Namita , Lin Xionghao , Nekhai Sergei A. 
Published: October 2019

Abstract
The interplay between hepcidin and its only known receptor, the iron exporter ferroportin (FPN), is fundamental for iron homeostasis. FPN is downregulated by hepcidin, which causes its endocytosis and degradation followed by increased intracellular H-ferritin . To date, fourty five mutations of FPN gene have been reported, with FPN Q248H mutant being a variant present in African and African American populations with a prevalence that ranges from 2.2% to 13.4%. Although at physiologic hepcidin concentrations, this mutation has shown a hepcidin resistant phenotype, the effect of this mutation on iron homeostasis and cellular metabolism is not fully understood. Thus FPN Q248H analysis is of great interest not only as fundamental for iron physiology in Africans and African Americans but also for possible drug development.
To determine the effect of FPN Q248H on intracellular iron, we expressed FPN-wt-EGFP and FPN-Q248H-EGFP in HEK293 cells and measured cellular ferritin and labile iron. Iron-leaded cells that expressed WT FPN showed increase in labile iron levels when treated with, whereas there was no chage in lable iron in cells expressing FPN-Q248H while for ferritin we found an increase in FPN-Q248H compared to its normal counterpart. To determin whether Q248H mutation affects FPN ubiquitination, we immunoprecipited EGFP-fused FPN with anti-EGFP antibodies and analzyed its ubiquinitation using MS/MS analysis. Unexpectedly, and contrary to previous reports that only showed hepcidin-resistant FPN mutations preventing its ubiquitination, we found increased ubiquitination of FPN Q248H on K240, and validated this finding by immunoprecipitation and Western Blot analysis.
To visualize the effect of FPN-Q248H-GFP mutation in the cells, we used confocal microscopy to detect FPN and lysosomes with Lisotracker. Cells that expressed FPN Q248H mutant showed two times more lysosomes than the cells expressing WT FPN, when hepcidin was added the number of lysosomes was further increased. We validated this results by RFP-fused lysosmal membrane-incorportated protein, LAMP1. These results indicated that FPN Q248H may induce autophagy. Previous studies indicated that ubiquitinated FPN is trafficked to and degradated by proteasomes. Thus we next assesed the effect of FPN Q248H mutation on autophagy by analyzing the expression of GABARAP1, LC3, p62, TRIM5 and TRIM22 in the presence and absence of the proteasome inhibitor MG-132 and the autophagy inhibitor Bafylomicin. We also treated cells with hepcidin and measured Ferritin levels. We found that the components of the autophagy pathway were significaly upregulated in the cells expressing FPN Q248H comparing to the WT FPN which still showed some upregulation. The induction of autophagy was even more prominent when hepcidin was added. Similar results were observed for NCOA4 protein, which mediates the selective cargo receptor for autophagic turnover of ferritin (ferritinophagy). The levels of expression of the autophagy related proteins were higher when proteasome was blocked and the levels of ferritin were slightly lower. Together, our results show that expression of FPN-Q248H induces autophagy/lysosome and proteasome pathways that can work alternatively to eliminate iron.
Finally, we measured the expression of active cathepsin D, a lysosome-enclozed enzyme and found more in FPN-Q248H expressing cells, indicating problems on the lysosomes integrity. Next, we measured cytochrome C expression to check mitochondrial integrity and HIF-1α and HIF-2α expression, observing that in cells expressing FPN-Q248H, all three proteins were upregulated compared with FPN-wt. Indicating that mitochondria are getting damaged in FPN Q248H-expressing cells by the Cathepsin D (t its presence, changes the polarization of the mitochondrial membrane) present in cytosol which consecuently generates hypoxia. Our results extended our previous finding of hepcidin resistance by FPN-Q248H to show that this mutaton induces autophagy. This pathway could be turned by premature ubiquitination of FPN Q248H and function as an "emergency pathway" to eliminate iron by selective ferritinophagy, bypassing proteasomes. As autophagy is a pro-survival mechanism that provides intermittent energy source and continuously disposes damaged organelles, this pathway could also increase cell survival and prevents iron toxicity.

Disclosures
No relevant conflicts of interest to declare.