Characterizing the Role of FBXL5 in Intracellular Iron Homeostasis

Iron is an essential trace element found in all forms of life. Despite its critical role in the cell involving redox reactions, many aspects of its intracellular regulation remain unknown. In the following work, we outline extensive research performed on two proteins that were previously uncharacterized with respect to iron homeostatic systems: the iron-binding ubiquitin ligase FBXL5 and the osmotic stress regulatory kinase SPAK. We discovered that FBXL5 controls the protein stability of the mRNA-binding IRP proteins IRP1 and IRP2 in an iron-dependent manner that is controlled by iron bound directly to FBXL5 via a novel hemerythrin-like domain found on the N-terminus. This hemerythrin-like domain is the first mammalian domain of its type to be identified and performs a critical role in iron sensing. When FBXL5 binds iron its structure is stabilized and it assembles into an E3 ubiquitin ligase complex that targets IRP1 and IRP2 for degradation; without iron, FBXL5 is itself destabilized and degraded.We also studied the osmotic stress regulatory kinase SPAK and its role in iron homeostasis. SPAK has been well-characterized for its role in salt homeostasis: it is activated by an upstream salt-sensing kinase WNK1 and then phosphorylates membrane-bound sodium-potassium-chloride co-transporters to equilibrate changes in osmotic pressure. In addition to this role, we have revealed that SPAK controls the stability of FBXL5 and NCOA4, both important proteins in iron homeostasis. While FBXL5 post-translationally regulates the protein expression of IRP proteins, NCOA4 controls the trafficking of iron-containing ferritin cages to the autophagosome to mediate release of iron.We discovered that SPAK binding and activity affects the poly-ubiquitination and steady-state abundance of both FBXL5 and NCOA4. SPAK coordinates with HERC2, an E3 ubiquitin ligase, to form a regulatory complex to perform these functions. We believe that it may allow for an additional layer of regulation for these two pathways and can aid in coordinately regulating both ferritin expression and degradation. This complex system has been the subject of our research for years now, and given the role of SPAK in iron regulation and the severity of iron-mediated illnesses SPAK could prove to be an attractive subject for pharmaceutical study