An Iron Sensing E3 Ubiquitin Ligase Regulates Iron Homeostasis

Human iron homeostasis must be tightly regulated to provide sufficient iron for vital cellular processes while preventing the toxic accumulation of free iron. IRP2 plays a critical role in cellular iron homeostasis by coordinating the posttranscriptional regulation of a variety of genes involved in iron metabolism. Posttranslational regulation of IRP2 is essential for its ability to maintain cellular iron homeostasis. The protein is stabilized under iron deficient conditions but polyubiquitinated and degraded by the proteasome when iron is plentiful. However, the E3 ubiquitin ligase that targets IRP2 for degradation is unknown. Moreover, the mechanisms cells use to sense iron levels and correlate changes of this metabolite to differences in IRP2 stability remain poorly understood. To identify the E3 ubiquitin ligase responsible for IRP2 degradation, a high throughput RNAi screen was conducted. The top hit from the screen, FBXL5, interacts with and polyubiquitinates IRP2. Interestingly, FBXL5 is inversely regulated to IRP2. The protein is stabilized under conditions of excess iron and destabilized when iron is limiting. Deletion experiments identified the N terminus of FBXL5 as the region of the protein required for its iron dependent regulation. Bioinformatics predicted the N terminus encodes an iron binding hemerythrin domain. Consistent with this prediction X-ray crystallography demonstrated that the FBXL5 N-terminal domain adopts a hemerythrin fold with a diiron center. Mutation of iron ligating residues in the hemerythrin domain to abolish iron binding leads to constitutive destabilization of FBXL5. Collectively, these findings indicate that the hemerythrin domain acts as a ligand dependent regulatory switch controlling FBXL5Õs expression. Moreover, these data suggest that iron dependent regulation of FBXL5 exerts reciprocal effects on IRP2 stability. Thus, FBXL5 possesses an iron binding hemerythrin domain enabling cells to gauge bioavailable iron levels and control IRP2 expression accordingly, resulting in a tightly regulated circuit in the maintenance of iron homeostasis.