Structures of ferroportin in complex with its specific inhibitor vamifeport

A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe$^{2+}$ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter

A Model of the Cellular Iron Homeostasis Network Using Semi-Formal Methods for Parameter Space Exploration

This paper presents a novel framework for the modeling of biological networks. It makes use of recent tools analyzing the robust satisfaction of properties of (hybrid) dynamical systems. The main challenge of this approach as applied to biological systems is to get access to the relevant parameter sets despite gaps in the available knowledge. An initial estimate of useful parameters was sought by formalizing the known behavior of the biological network in the STL logic using the tool Breach. Then, once a set of parameter values consistent with known biological properties was found, we tried to locally expand it into the largest possible valid region. We applied this methodology in an effort to model and better understand the complex network regulating iron homeostasis in mammalian cells. This system plays an important role in many biological functions, including erythropoiesis, resistance against infections, and proliferation of cancer cells.Comment: In Proceedings HSB 2012, arXiv:1208.315

Membrane Transporters Involved in Iron Trafficking: Physiological and Pathological Aspects

Iron is an essential transition metal for its involvement in several crucial biological functions, the most notable being oxygen storage and transport. Due to its high reactivity and potential toxicity, intracellular and extracellular iron levels must be tightly regulated. This is achieved through transport systems that mediate cellular uptake and efflux both at the level of the plasma membrane and on the membranes of lysosomes, endosomes and mitochondria. Among these transport systems, the key players are ferroportin, the only known transporter mediating iron efflux from cells; DMT1, ZIP8 and ZIP14, which on the contrary, mediate iron influx into the cytoplasm, acting on the plasma membrane and on the membranes of lysosomes and endosomes; and mitoferrin, involved in iron transport into the mitochondria for heme synthesis and Fe-S cluster assembly. The focus of this review is to provide an updated view of the physiological role of these membrane proteins and of the pathologies that arise from defects of these transport systems

Characterizing Ferroportin Trafficking in Macrophages During Phagocytosis

Macrophages are important mediators of innate immunity and nutritional immunity via modulation of essential nutrients like iron during bacterial infection. Ferroportin (Fpn), an iron-exporting protein, is found on the plasma membrane of macrophages and, if not modulated during phagocytosis, would transport iron into phagosomes and supply phagocytosed bacteria with iron. Interestingly, the fate of Fpn during phagocytosis and bacterial infection remains unknown. We generated a Fpn-GFP fusion protein and, using fluorescence microscopy, demonstrated that, during phagocytosis in RAW264.7 macrophages, Fpn is removed from phagosomes containing IgG-coated beads or Staphylococcus aureus. Further, Fpn is present on Rab5-containing phagosomes but absent from PI(3)P- and LAMP1-positive phagosomes indicating Fpn removal occurs early during phagosome maturation. Co-localization analysis revealed that markers of cellular recycling pathways, Rab4 and transferrin receptor, do not co-localize with Fpn. Thus, our data support the conclusion that macrophages restrict Fpn residence on phagosomes presumably to prevent iron transport into phagosomes

Iron homeostasis in immune mononuclear cells : a potential role in a atherogenesis

Tese de doutoramento, Biologia (Biologia-Molecular), Universidade de Lisboa, Faculdade de Ciências, 2015Atherosclerosis is an inflammatory disease characterized by the formation of vessel wall plaques, initiated by oxidized LDL (oxLDL) accumulation and infiltration of immune cells. Iron accumulation in atherosclerotic plaques was proposed to constitute a risk factor in atherogenesis and immune cells could contribute to this risk. Herein, we studied the iron homeostasis of lymphocytes, monocytes and macrophages in a context of atherogenic conditions. Particularly, we investigated the expression and regulation of the iron exporter ferroportin-1 (Fpn1) and its potential functional partner ceruloplasmin (Cp). We demonstrated that lymphocytes, monocytes and macrophages express both a soluble Cp and a membrane GPI-Cp. Through oxidation of LDL, Cp expressed by immune cells could contribute to the reported association between high Cp levels and increased cardiovascular risk. We showed that GPI-Cp partially co-localizes with Fpn1 in lipid rafts of iron-treated macrophages and could participate in Fpn1-mediated iron export. However, we proposed that besides Cp, the β-amyloid precursor protein (APP) could be a functional partner of Fpn1 in macrophages that needs to be further studied. The effect of oxLDL on the iron homeostasis was investigated in macrophages. Despite oxLDL-driven Nrf2 activation and increased heme oxygenase-1 expression, Mox macrophages were not protected from foam cell formation like Mhem/M(Hb) macrophages, showing lipid accumulation, basal levels of Fpn1 at cell surface along with upregulation of interleukine-6 and hepcidin. Simultaneous exposure to oxLDL and LPS/IFNγ induced a Mox/M1 phenotype closer to M1 than Mox, with increased interleukine-6 and H-ferritin expression along with decreased Fpn1 expression. Our results suggest that macrophages exposed to a microenvironment rich in oxLDL and pro-inflammatory cytokines are prone to accumulate both lipids and iron while secreting high levels of pro-inflammatory cytokines and Cp, which will further promote the local inflammation and LDL oxidation. In summary, our results support the “iron hypothesis” in atherogenesis proposed by Sullivan.Fundação para a Ciência e a Tecnologia (FCT

Hepcidin-mediated Iron Regulation in P19 Cells is Detectable by MRI

Magnetic resonance imaging (MRI) can be used to track cellular activities in the body using iron-based contrast agents. However, intrinsic cellular iron handling mechanisms may also influence the detection of magnetic resonance (MR) contrast. For instance, inflammation involves downregulation of iron export in macrophages by the hormone hepcidin, due to degradation of the iron export protein, ferroportin (Fpn). We examined the effect of hepcidin on iron regulation and MR transverse relaxation rates in multi-potent P19 cells, which display high iron export activity, similar to macrophages. In response to varying conditions of iron supplementation, our results showed similar Fpn expression in P19 cells as reported for M2 macrophages. Also, hepcidin treatment resulted in Fpn degradation in P19 cells, similar to the reported response of M1 macrophages. The correlation between total cellular iron content and MR transverse relaxation rates was significantly different between hepcidin and non-hepcidin treated P19 cells, providing a tool to non-invasively distinguish different macrophage phenotypes and potentially improve the monitoring of inflammatory cell activities

Gene-by-Environment Interaction of Hepatic Xenobiotic Transporter Polymorphisms and Nonalcoholic Steatohepatitis on Drug Disposition and Toxicity

Nonalcoholic steatohepatitis (NASH) is the advanced form of nonalcoholic fatty liver disease and is the hepatic manifestation of metabolic syndrome. The overall rate of NASH is on the rise globally, and currently approximately 1.5-6.45% of the population is afflicted with this disease(Z. M. Younossi et al., 2016). The progression of NASH is known to result in the dysregulation of many genes, including those that are responsible for ADME processes. These changes can alter the disposition of xenobiotics and result in adverse drug reactions (ADRs). Since ADRs are becoming increasingly frequent, with nearly 1 in 20 hospital patients experiencing ADRs in the United States(Bourgeois, Shannon, Valim, & Mandl, 2010), determining the factors that contribute to variations in drug response is increasingly important. Additionally, genetic polymorphisms are known to cause pharmacokinetic variations, and many of them have been extensively researched for their impacts on patient response. Less known, however, is how genetic polymorphisms and chronic liver disease interact, and how that interaction may increase patient risk for ADRs. As the goal of precision medicine is to provide the right dose to the right person at the right time, determining how factors like disease state can interact with genetic polymorphisms to alter pharmacokinetics can provide a better basis for individualized dosing. The purpose behind this study was therefore to utilize a methionine- and choline-deficient (MCD) rodent model to determine the gene-by-environment interactions of hepatic xenobiotic transporter polymorphisms and NASH on the disposition and toxicity of pharmaceutical compounds. To identify the impact of these gene-by-environment interactions, pharmacokinetic studies were designed to investigate the alterations to drug distribution that occurred with NASH in combination with disruptions of hepatic uptake and efflux transporters and to determine the underlying mechanism behind pharmacokinetic alterations during disease progression. The results in the MCD rodent model of NASH found a synergistic interaction between polymorphisms of both hepatic uptake and efflux transporters and NASH that lead to changes in plasma retention and biliary excretion of probe substrates. The comparison between biliary excretion of SN-38 in wild-type and Bcrp-/- rats demonstrated this synergistic interaction, as no alterations were found in biliary AUCs until the combination of the disease and genetic disruption, where biliary excretion significantly decreased. A similar interaction was demonstrated in a study investigating the gene dose of Oatp1b2 and NASH on pravastatin disposition, where the combination of full genetic Oatp1b2 knockout along with NASH was necessary to see dramatic increases in plasma AUC and tissue concentrations. Finally, in order to identify the underlying mechanism by which NASH alters pharmacokinetics, the disposition of sorafenib-glucuronide was determined in wild-type, Oatp1a/1b cluster knockouts, and Mrp2 knockouts, with and without MCD diet. By comparing the changes to plasma AUC and liver concentrations in these groups, the disruption to hepatocyte hopping that occurs during NASH was made quantifiable. In summation, these data identify synergistic gene-by-environment interactions of hepatic xenobiotic transporter polymorphisms and NASH, the alterations that these interactions cause to drug disposition, and the mechanism by which NASH causes pharmacokinetic changes through disruption of hepatocellular shuttling