Apo- and holo- transferrin differentially interact with ferroportin and hephaestin to regulate iron release at the blood-brain barrier

Background: Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanism of apo- and holo-Tf’s influence on iron release was largely unknown. Methods: Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism of apo- and holo-Tf’s influence over iron release. We placed our findings in physiological context by further deciphering how hepcidin played a role in this mechanism as well. Results: We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly binds to ferroportin, whereas apo-Tf directly binds to hephaestin. Only pathological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, and no amount of hepcidin disrupts the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin’s ability to rapidly internalize ferroportin compared to holo-Tf. Conclusions: These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin corporate to suppress iron release. We have established a more thorough understanding of the mechanisms behind iron release regulation with great clinical impact for a variety of neurological conditions in which iron release is dysregulated.

Free iron is released from cells, including ECs, through ferroportin (Fpn), the only know iron exporter. Fpn function is aided by a number of proteins, including hephaestin (Heph) 10,11 , a ferroxidase that converts ferrous (Fe2+) to ferric (Fe3+) iron. Heph is required for both the stability of Fpn in the plasma membrane and the e ux of iron through Fpn 10,11 . Inversely, Fpn can be inhibited by hepcidin 12 , a pro-in ammatory peptide hormone, primarily secreted by the liver 13 and in small amounts by astrocytes 14 . When hepcidin binds to Fpn, Fpn is ubiquitinated for internalization and subsequent degradation 12,15 . Simpson et al. found that, in addition to iron release, holo-Tf also decreases Fpn protein in EC culture models of the BBB 6 but the mechanism is unclear. Conversely, it has been proposed that apo-Tf participates in interactions with ferroxidases such as Heph and ceruloplasmin to facilitate iron release [16][17][18] . In the present study, we have determined the differential interactions that apo-and holo-Tf have with Fpn and Heph to control iron release. Moreover, we demonstrate the impact that hepcidin can have on these interactions. By understanding the regulatory mechanism of iron release into the brain, numerous neurological diseases with iron uptake dysregulation can be better studied and potentially treated.

Proximity Ligation Assay (PLA)
PLA is a technique that precisely demonstrates if two proteins directly interact with one another. When two proteins are in close enough proximity to be interacting, the secondary oligomer probes ligate together, allowing for the ampli cation of the oligomers and resulting in a uorescent signal. PLA was performed using a Duolink assay kit (Sigma-Aldrich, DUO92013) according to the manufacturer's instructions 21 . Chamber slides (Falcon, 354108) were coated with poly-D-lysine 2 hrs before HEK 293 cells were culture on the slides at a density of 15,000 cell/cm 2 . In order to remove an exogenous Tf, 24 hrs later the media was replaced with DMEM containing no FBS. Cells were exposed to apo-or holo-Tf (Sigma, T1147 and T4132) for 10 minutes and then washed to procced with PLA. PLA was performed the following day. Primary antibodies used were the following: myelin basic protein 1 (MBP1, Abcam, ab22460, 1:500), ferritin (Abcam, ab77127, 1:500), Tf (ProteinTech, 66161-1, 1:500), TfR (Cell Signaling, 13208S, 1:500), Tf (Abcam, ab82411, 1:500), Fpn (gift from M. Knutson, 1:500), and Heph (Santa Cruz, SC-365365, 1:500). Positive and negative controls used for assay optimization can be found in Supplemental Fig. 2. Imaging and analysis were performed using Revolve R4 microscope (Echo). The integrated density was calculated by summing the pixels from PLA signal and dividing by the eld of view area. The integrated density of background from negative controls were subtracted from these values. To determine the integrated density per cell, this was then divided by the number of cells in the eld of view. A minimum of three images were taken in different regions of the slides and then averaged for a single biological replicate. Image brightness was uniformly increased for the purposes of publication but not for quanti cation.

Co-immunoprecipitation
In order to remove an exogenous Tf, the media was replaced with DMEM containing no FBS 24 hrs before the start of experiments. Cells were exposed to apo-or holo-Tf (Sigma, T1147 and T4132) for 10 minutes and then washed on ice with cold PBS twice. Chilled 100µl Co-IP lysis buffer (20 mM Tris HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Triton x-100, and 2 mM EDTA) was added to each well. Cells were collected and incubated with rotation for 30 minutes at 4°C. Cell solutions were centrifuged at 14,000 x g for 20 minutes at 4°C. Supernatant was collected, and protein estimation was performed using Pierce BCA Protein Assay Kit (Thermo, 23227). Approximately 1 mg of protein was used for Co-IP using anti-HA magnetic beads (Thermo, 88837) or Protein G magnetic beads (Thermo, 10003D) complexed with anti-Heph antibody (Santa Cruz, SC-365365) according to manufacturer's instructions 22 . Brie y, magnetic beads were washed twice with PBS before adding lysates. The bead and lysate solutions were incubated with rotation for 30 minutes at room temperature. After washing with PBS, protein was eluted from beads by resuspending in non-reducing sample buffer and boiling at 90°C for 10 minutes. Magnet was used to isolate the magnetic beads from the protein solution, which was then reduced using 2 M DTT and then loaded for immunoblotting.

Membrane Protein Isolation
Cells were washed with PBS three time before incubating with 200µl digitonin buffer (20mM Tris-HCl, 250 mM sucrose, 0.007% digitonin, 1x protease inhibitor cocktail) 23 . Cells were gently lifted from the plate and collected in chilled glass mini homogenizers. Once homogenized, samples were spun at 1,500 x g for 10 minutes. The pellet was reserved and the supernatant was spun again at 10,000 x g for 10 minutes. The resulting pellet was combined with the pervious pellet and resuspended in RIPA buffer and 1x protease inhibitor cocktail. After immunoblotting was performed on the samples, the membranes were stained for total protein content using Ponceau S staining solution (Thermo, A40000279) to use as a loading control.

Statistical Analysis
Statistical analyses were performed using Prism 9.2 software (Graphpad Software Inc.). Data from at least three independent biological replicates were averaged and are expressed as the mean ± standard error of the mean (SEM). One-way ANOVA with Tukey post-hoc analysis, two-way ANOVA with Sidak's post hoc analysis, or unpaired t-tests were used to evaluate for statistical signi cance where appropriate. A p-value < 0.05 was considered signi cant.

Holo-Tf decreases Fpn levels through Fpn's degradation pathway
In the rst series of experiments, we examined the effects of apo-and holo-Tf on the cellular levels of Fpn by incubating iPSC-derived ECs with increasing concentrations of either apo-or holo-Tf in hESFM for 8 hours. ECs were cultured onto Transwell inserts and apo-or holo-Tf was placed in the basal chamber to represent the brain-side. The ECs were collected and probed for various iron transport proteins.
The degradation pathway for Fpn involves ubiquitination by E1 ubiquitin ligase, resulting in the internalization and degradation of Fpn 15,24 . To determine if this classic degradation pathway was the cause of the decreased Fpn induced by holo-Tf, we pretreated ECs with 50 µM PYR-41, an E1 ubiquitin ligase inhibitor, before exposure to either apo-or holo-Tf. The inhibition of Fpn ubiquitination resulted in a mitigation of holo-Tf's decrease of Fpn (Fig. 1D), while apo-Tf continued to have no impact on Fpn levels ( Fig. 1D). Hepcidin, a known inducer of Fpn ubiquitination, was used as a positive control to con rm the function of PYR-41's inhibition (Fig. 1G). ECs were exposed to 500nm of hepcidin following pretreatment with 50 µM PYR-41 for 30 minutes. Controls were either solely exposed to hepcidin or PYR-41. Hepcidin alone increased Fpn ubiquitination and PYR-41 pretreatment prevented this increase (Fig. 1G).

Apo-and holo-Tf differentially interact with Fpn and Heph
We next aimed to determine if holo-Tf interacted directly with Fpn. We used HEK 293 cells transfected with an HA-tagged Fpn plasmid to selectively pull-down HA-Fpn. We incubated the cells with 0.25 µM of either apo-or holo-Tf (physiological level in CSF 25 ) in media containing no FBS for 10 minutes prior to coimmunoprecipitation (co-IP). Regardless if the cells were incubated with either apo-or holo-Tf, Tf was coimmunoprecipitated with HA-Fpn ( Fig. 2A). This indicates that apo-and holo-Tf bind to the Fpn complex of proteins. Because Heph aids Fpn in the export of iron 11 , we hypothesized that apo-Tf could bind to Heph, leading to its co-immunoprecipitation with HA-Fpn. To con rm this, we incubated ECs, which have greater Heph expression than HEK 293 cells, with either apo-of holo-Tf, and performed co-IP with Heph antibody Again, in cells incubated with either apo-of holo-Tf, Tf was co-immunoprecipitated ( Fig. 2B) further con rming that Fpn, Heph, and Tf complex together.
Because co-IP precipitates the entire complex of Fpn, Heph, apo-Tf, and holo-Tf, we aimed to better differentiate if apo-and holo-Tf directly interact with Fpn and Heph by employing proximity ligation assay (PLA), a highly sensitive method of detecting protein-protein interactions. HEK 293 cells were incubated with 0.25 µM of either apo-or holo-Tf in media containing no FBS for 10 minutes. Cells incubated with holo-Tf showed PLA signal when probing for a Tf and Fpn interaction (Fig. 2D). While cells incubated with apo-Tf showed PLA signal when probing for a Tf and Heph interactions (Fig. 2F). Thus, holo-Tf directly interacts with Fpn while apo-Tf does not (***p < 0.001, Fig. 2E). Conversely, apo-Tf directly interacts with Heph, while holo-Tf does not (****p < 0.0001, Fig. 2H).

High levels of hepcidin interrupt the interaction between holo-Tf and Fpn
Hepcidin is a well-known regulator and binding partner of Fpn, therefore we aimed to understand how the novel interaction between holo-Tf and Fpn could be impacted by physiological conditions that contribute to iron release. To do so, we used PLA to examine if hepcidin competed with holo-Tf for binding to Fpn. HEK 293 cells were co-incubated with 500 nM hepcidin and varying concentrations of holo-Tf (Fig. 3A-F).
To determine if the amount of hepcidin was crucial to the interruption of the holo-Tf and Fpn interaction, we performed the reverse competition experiment and co-incubated HEK 293 cells with 0.25 µM holo-Tf and varying concentrations of hepcidin ( Fig. 3G-K). Hepcidin interrupted the interaction between holo-Tf and Fpn in a dose dependent manner. Only the highest concentration of 500 nM signi cantly interrupted the interaction between holo-Tf and Fpn (*p < 0.05, Fig. 3G, K). As the concentration of hepcidin decreased, the PLA signals for holo-Tf and Fpn interactions, increased ( Fig. 3G-J). The physiological baseline concentration of hepcidin 26 , 25 nM, had no impact on the holo-Tf-Fpn interaction (Fig. 3J).

Hepcidin does not interrupt the interaction between apo-Tf and Heph
Apo-Tf has been shown to stimulate iron release despite the presence of hepcidin 4 , thus we hypothesized that hepcidin would have no impact on the interaction between apo-Tf and Heph using PLA. HEK 293 cells were co-incubated with 500 nM hepcidin and varying concentrations of apo-Tf (Fig. 4B-F). Unlike with holo-Tf, 500 nM hepcidin did not interrupt the interaction between any amount of apo-Tf and Heph ( Fig. 4B-E), as indicated by the unchanged PLA signal (Fig. 4F). In the reverse competition experiment, we co-incubated HEK 293 cells with 0.25 µM apo-Tf and varying concentrations of hepcidin and indicated in (Fig. 4G-J). Again, no concentration of hepcidin was su cient to alter the interaction between apo-Tf and Heph (Fig. 4K).

Hepcidin internalizes Fpn faster than holo-Tf
The PLA experiments showed there was competition between holo-Tf and hepcidin, but did not differentiate if this was due to hepcidin directly competing with holo-Tf for a binding site on Fpn or by internalizing Fpn faster than holo-Tf. To answer these questions, we utilized pretreatment with PYR-41, which prevents the degradation of Fpn and thus removes internalization dynamics as a factor in the binding of holo-Tf and hepcidin to Fpn. We performed PLA on HEK 293 cells exposed to 0.25 µM holo-Tf alone (Fig. 5A), 0.25 µM holo-Tf and 500 nM hepcidin (Fig. 5B), and pretreatment of 50 µM PYR-41 and then 0.25 µM holo-Tf and 500 nM hepcidin (Fig. 5C). As before, hepcidin interrupts the interaction between holo-Tf and Fpn (*p < 0.05), however, this decrease in interaction is prevented when with the PYR-41 pretreatment (***p < 0.001, Fig. 5A-D). This nding indicates that hepcidin decreases the interaction between holo-Tf and Fpn due to its ability to rapidly internalize Fpn. We further con rmed a decrease of Fpn membrane presence by isolating membrane bound proteins for immunoblotting (Fig. 5E-F). The coincubation of 0.25 µM holo-Tf and 500 nM hepcidin results in a signi cant decrease of membrane Fpn protein (*p < 0.05, Fig. 5E-F). This decrease in membrane Fpn is prevented when pretreated with PYR-41 (*p < 0.01, Fig. 5E-F). These data align with the PLA results and suggests that hepcidin prevents holo-Tf from binding to Fpn by inducing the rapid internalization of Fpn.

Discussion
This study addresses the molecular mechanisms by which apo-and holo-Tf regulate iron release at the BBB. More speci cally, this study demonstrates that apo-and holo-Tf differentially interact with Heph and Fpn. Through its interaction, holo-Tf reduces Fpn protein levels, and this is through Fpn's established degradation pathway as shown when Fpn degradation is inhibited. Holo-Tf directly interacts with Fpn as shown by orthogonal techniques. Furthermore, when incubated together, hepcidin can interrupt this interaction at high levels that correspond with in ammation or high systemic iron levels, but not at levels that correspond with baseline levels. Hepcidin's interruption is likely due to its ability to internalize Fpn faster than holo-Tf and not due to direct competition for the same binding site, as we additionally demonstrate herein. On the other hand, hepcidin does not interrupt the interaction between apo-Tf and Heph. These ndings offer a glimpse at the mechanism of free iron release into the brain, a crucial process for neurological health.
Fpn is the only known iron exporter, thus control of membrane Fpn is control of free iron release. The internalization and subsequent degradation of Fpn has been extensively studied in the context of hepcidin 12,15,24,27 . Brie y, once hepcidin binds to Fpn, it triggers the ubiquitination of the Fpn, thus signaling for its internalization and lysosomal degradation. Simpson et al. showed that by incubating BRECs with 12.5 µM holo-Tf, the levels of Fpn decreased 6 . Here we have replicated those ndings in iPSCderived ECs but at a physiological level; transferrin is found in CSF at about 2 mg/dL, or 0.25 µM 25 . We demonstrate that a basal incubation of as low at 0.1 µM holo-Tf results in a 50% decrease of membrane Fpn. These data provide a mechanistic explanation for why holo-Tf suppresses iron release from ECs.
What's more, other iron-related proteins, such as Heph, DMT1, and TfR, are unchanged with basal holo-Tf exposure. Interestingly, even when exposed to high amounts of holo-Tf, the levels of Fpn do not decrease beyond 50%, suggesting there is a plateaued effect of holo-Tf within the 8-hour experimental time window. The holo-Tf-mediated internalization of Fpn is blocked when the ubiquitination of Fpn is inhibited, suggesting that holo-Tf exerts its effect through the established degradation pathway, similar to hepcidin.
To complete the process of iron export, Fpn works in a complex with many proteins, including Heph 10,11 . Heph is a ferroxidase that converts the Fpn-exported ferrous (Fe2+) to ferric (Fe3+) that can bind to apo-Tf and be utilized by cells. Numerous studies have shown that Heph is required to stabilize Fpn in the plasma membrane and to enable iron export 10,11,28,29 . We have replicated these ndings, by demonstrating that Fpn and Heph can be co-immunoprecipitated from ECs. Furthermore, we demonstrate the novel nding that both apo-and holo-Tf independently are co-immunoprecipitated with Fpn and Heph. These results suggest that apo-and holo-Tf bind to Fpn and Heph in a complex of iron export proteins. In order to narrow down which protein holo-Tf bound to in the membrane that resulted in decreasing Fpn, we employed PLA. We found that holo-Tf directly interacts with Fpn, while apo-Tf does not. On the other hand, apo-Tf interacts with Heph, while holo-Tf does not, a nding that is supported in the literature 16,18,30 . It is hypothesized that apo-Tf binds to Heph to accept the ferric iron that Heph converts from ferrous iron. This stimulates the release of more iron as long as there is apo-Tf to accept it. Taken together these data suggest that apo-and holo-Tf differentially interact with iron export proteins, likely due to their structural differences 31 . The exact binding sites, conformation changes, and catalysts for these interactions are an exciting unexplored area that could pave the way for clinical manipulation.
For example, as has been done experimental 7 , Tf could be infused to modulate iron accumulation in diseases in which it is dysregulated. Additionally, pharmaceuticals could be designed to facilitate or inhibit the endogenous protein interactions in an effort to correct brain iron accumulation.
Prior to the discovery that elevated holo-Tf could suppress iron release, hepcidin was the primary focus of iron release regulation 13 . Hepcidin is a pro in ammatory hormone peptide primarily secreted by the liver and upregulated in environments of in ammation and high iron levels 32 . Astrocytes 33,34 and the choroid plexus 35,36 have also been shown to secrete hepcidin, though in much smaller amounts that cannot account for total brain hepcidin levels 36,37 , suggesting much of the brain hepcidin comes from systemic levels when pathologically necessary, though this has not yet been proven. A number of groups have shown that astrocytic hepcidin reduces Fpn levels and subsequent iron release 14,38,39 . However, we have previously demonstrated that supraphysiological levels of hepcidin are not capable of blocking iron release from ECs 3,4 . These data suggest that hepcidin cannot be the sole regulator of iron release in the brain. In support of this notion, Enculescu et al. modeled iron levels, and when compared to their experimental results, the study found that hepcidin control over iron uptake was necessary, but not su cient 40 . Once a secondary regulatory mechanism was added to the model, their experimental results aligned with the model 40 . Thus, our data directly support that hepcidin is not the sole regulator of iron release and indicate the additional regulators are apo-and holo-Tf.
Our data offer an opportunity to explore the concept of regulation of iron uptake in general by hepcidin. We found that hepcidin competes with holo-Tf for binding to Fpn at low holo-Tf and high hepcidin concentrations. However, when there was more holo-Tf or less hepcidin present, this effect was reduced.
Notably, when hepcidin was only present at physiological baseline levels 26 , there was no interruption of the interaction between holo-Tf and Fpn. These ndings suggest that hepcidin is only effective at controlling Fpn levels at levels consistent with in ammation or high iron. In observing competition between holo-Tf and hepcidin for Fpn binding, the internalization of Fpn was inhibited to determine if the competition was for binding site availability or rate of internalization. By preventing the internalization of Fpn, hepcidin had no impact on the interaction between holo-Tf and Fpn. This suggests that hepcidin internalizes Fpn faster than holo-Tf, which was con rmed by isolating membrane Fpn. Hepcidin reduces membrane Fpn by nearly 50% in 5 minutes, whereas holo-Tf only starts to reduce membrane Fpn at 60 minutes. On the other hand, no amount of hepcidin impacts the interaction between apo-Tf and Heph.
These data offer the intriguing suggestion that if apo-Tf is present, it will bind to Heph even in pathological states and may be an explanation for iron accumulation in neurodegenerative disease. It has been postulated that in Alzheimer's disease 41 and Parkinson's disease 42 the brain may start as functional iron de cient, along with elevated levels of apo-Tf, which triggers increased iron uptake until the excess iron detrimentally damages the BBB and surrounding cells. The question remains however, if the binding of apo-Tf to Heph will continue to stimulate iron release in the presence of hepcidin.
The model of apo-and holo-Tf regulation of iron release from ECs works as a feedback loop. As cells, such as neurons or astrocytes, need iron for metabolic processes, myelin synthesis, or dopamine synthesis, they take up holo-Tf through TfR 43 . Once endocytosed, the iron is removed and the resulting apo-Tf is released 43 . The communication of brain iron status via apo-and holo-Tf allows cells to signal their iron needs based on their iron consumption. Numerous studies have shown higher regional iron uptake that correspond to areas with higher iron needs 9,44,45 . Our pervious data suggest that as the apoto holo-Tf ratio changes in the extracellular uid, more iron is released locally from the BBB. In support of this notion are data showing CSF from iron de cient monkeys and iron chelated astrocytes increase iron release from cultured bovine retinal ECs (BRECs), while iron loaded biological samples resulted in decreased iron release 6 . These data have been replicated when cells are exposed to apo-or holo-Tf directly 3,4,6 or when apo-or holo-Tf is directly infused into the brain 7 . In all studies mentioned here, apo-Tf increased iron release while holo-Tf decreased iron release.
The data in this study expand the model for brain iron uptake by suggesting that apo-Tf stimulates iron release by binding to Heph to access exported free iron (Fig. 6A). Once loaded with iron, the now holo-Tf becomes available to surrounding cells. If the levels of holo-Tf in the extracellular uid rise, holo-Tf binds to Fpn to suppress more iron release (Fig. 6B). The internalization of Fpn by holo-Tf is not rapid, unlike hepcidin. When upregulated and present in high amounts, hepcidin can rapidly internalize Fpn (Fig. 6C). Thus, we propose that hepcidin is likely used as a fast acting, immediate stop to iron release in environments of in ammation and very high iron. However, for moment-by-moment regional control of iron release, holo-Tf may be a better candidate to regulate regional iron supply

Conclusions
The regulation of brain iron uptake is not in uenced by systemic levels 46 , thus a local source is needed. The data herein provide insights into a local regulatory process. This study is the rst demonstration that apo-and holo-Tf differentially interact with Fpn and Heph to regulate iron release from ECs of the BBB. Moreover, we have identi ed a physiologically relevant dynamic between hepcidin and holo-Tf and their in uence on membrane Fpn levels. Hepcidin interrupts the interaction between holo-Tf and Fpn by internalizing Fpn much faster than holo-Tf. Furthermore, we show that hepcidin does not interrupt the interaction between apo-Tf and hepcidin. These data suggest the mechanism of free iron release from ECs at the BBB and provide opportunity for further studies in neurological disease models to understand how this mechanism may be disrupted in each disease.  Figure 1 Modulation of Fpn protein levels in ECs by holo-Tf iPSC-derived ECs were cultured on bi-chamber plates, incubated with apo-or holo-Tf in the basal chamber, and collected after 8 hours for immunoblotting. Fpn protein levels were normalized to cyclophilin B as a loading control. All quanti cations were further normalized to untreated control to account for cell count variability. Holo-Tf decreased Fpn protein levels by 50% at concentrations as low as 0.1 μM, while apo-Tf did not (A-C). Holo-Tf-mediated internalization and degradation of Fpn was inhibited by a ubiquitination inhibitor, PYR-41, (D-F) con rming that holo-Tf's decreases Fpn through the established degradation pathway. PYR-41's inhibition of ubiquitination was validated using hepcidin to induce Fpn ubiquitination (C). Exposure to hepcidin alone for 30 minutes increases ubiquitination of Fpn.

Figures
When pretreated with PYR-41 for an additional 30 minutes, this increase of ubiquitination of Fpn is blocked. Total Fpn levels are unchanged. n=3 for all experiments, means of biological replicates ± SEM were evaluated for statistical signi cance using one-way ANOVA with Tukey's posttest for signi cance. Apo-and holo-Tf interactions with Fpn and Heph HEK 293 cells were transfected with HA-tagged Fpn and subsequently incubated with 0.25 μM apo-or holo-Tf. Immunoprecipitate (IP) and 50% of cell lysate (input) was processed for immunoblotting. Co-IP of HA-Fpn shows that both apo-and holo-Tf are pulled down along with the Fpn complex (A). Co-IP of Heph in iPSC-derived ECs replicated these data (B). HEK 293 cells were used to determine direct protein interactions using PLA, reported as integrated density per cell in the eld of view per image. Holo-Tf interacts with Fpn (D), while apo-Tf does not (C). Alternatively, apo-Tf interacts with Heph (F), while holo-Tf does not (G). n=4 for all experiments, means of biological replicates ± SEM were evaluated for statistical signi cance using unpaired t test. ***p<0.001, ****p<0.0001 Hepcidin impact on interaction between holo-Tf and Fpn HEK 293 cells were used to determine the impact of hepcidin on holo-Tf and Fpn interactions using PLA, reported as integrated density per cell in the eld of view per image. High levels of hepcidin interrupt the interaction between holo-Tf and Fpn when holo-Tf is present in physiological levels (D and G), but not when holo-Tf concentrations are higher (B and C) or hepcidin concentrations are closer to baseline physiological (H-J). n=3 for all experiments, means of biological replicates ± SEM were evaluated for statistical signi cance using one-way ANOVA with Tukey's post-test for signi cance. *p<0.05, **p<0.01 Figure 4 Hepcidin impact on interaction between apo-Tf and Heph HEK 293 cells were used to determine the impact of hepcidin on apo-Tf and Heph interactions using PLA, reported as integrated density per cell in the eld of view per image. Hepcidin has no impact on the interaction between apo-Tf and Heph at any apo-Tf concentrations (B-E) or at any hepcidin concentrations (G-J). n=3 for all experiments, means of biological replicates ± SEM were evaluated for statistical signi cance using one-way ANOVA with Tukey's post-test for signi cance.  (C) prevented the hepcidin induced reduction of interaction between holo-Tf and Fpn (B). The isolation of membrane bound Fpn con rms that hepcidin and holo-Tf co-incubation greatly reduces membrane Fpn levels, and this is prevented with PYR-41 (E-F). Hepcidin reduces membrane Fpn at a faster rate than holo-Tf (G-H). n=3 to 5 for all experiments, means of biological replicates ± SEM were evaluated for statistical signi cance using one-way ANOVA with Tukey's posttest for signi cance (D) and (F) or two-way ANOVA with Sidak's post-test for signi cance (H). *p<0.05, ***p<0.001 Figure 6 Model of Iron Release Regulation In our proposed model, in areas that have higher ratios of apo-to holo-Tf (A), apo-Tf binds to Heph in order to accept the exported free iron and further stimulates iron release through Fpn. Alternatively, areas of lower ratios of apo-to holo-Tf (B), excessive holo-Tf binds to Fpn to facilitate the internalization and degradation of Fpn, and thus suppressing iron release through Fpn. In environments of in ammation or high iron levels, hepcidin production is upregulated (C). Hepcidin binds to Fpn and rapidly triggers Fpn's internalization and abruptly stops free iron release.

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