Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux

Abstract

Edited by Ruma Banerjee Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe 3؉ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP) fusions of Heph and APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59 Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, (K/R)EWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.

Iron Responsive Element (IRE)-mediated responses to iron dyshomeostasis in Alzheimer’s disease

“…In that study, the authors extended investigation of the idea that the protein product of the fAD locus AβPP stabilizes the iron exporting protein ferroportin, providing a connection between AβPP and iron homeostasis in AD. The idea that AβPP interacts with ferroportin has been the subject of some contention [81,82]. Tsatsanis et al observed that inhibition of the amyloidogenic cleavage of AβPP by the enzyme β-secretase in mouse N2a neuroblastoma cells is associated with stabilization of ferroportin on the cell surface and a reduced, intracellular labile iron pool.…”

Ferroptosis Is Regulated by Mitochondria in Neurodegenerative Diseases

“…Tau tangles in AD may also be a potential factor leading to iron dyshomeostasis and ferroptosis [80]. APP plays an important role in regulating the iron content of neurons; it promotes iron export by maintaining the stability of FPN [81, 82]. Iron status may directly impact the translation of APP through an iron-responsive mRNA element (IRE) in its 5′-UTR [83].…”

Relationship between Down-Regulation of Copper-Related Genes and Decreased Ferroportin Protein Level in the Duodenum of Iron-Deficient Piglets

“…However, despite significant differences in the liver iron content of anemic and FeDex-supplemented piglets, the level of hepcidin expression did not differ within the studied groups ( Figure 4 B). Fpn is able to form a functional complex with a copper-dependent, membrane-like protein that possesses ferroxidase activity—hephaestin (Heph) [ 42 ]. We found that in the intestine of anemic piglets, the level of Heph expression, both at the mRNA and protein level, was significantly lower compared to FeDex-supplemented piglets ( Figure 4 C).…”

“…Therefore, even upon increased systemic iron demand, the transcriptional Hif-2α response can be reduced in the iron-rich duodenum. It has been proven in both in vitro and in vivo conditions that Smad4, an iron-dependent transcription factor, can limit Hif-2α transcriptional activity and iron deficiency leads to proteasomal degradation of Smad4 [ 42 ]. In the small intestine of mice fed iron-enriched, iron-replete, or low-iron diets, a dose-dependent decrease in Smad4 level has been demonstrated.…”