Enhanced expression and functional characterization of the human ferritin H- and L-chain genes in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Enhanced expression of the human ferritin H- and L-chain genes (hfH and hfL) was achieved in Saccharomyces cerevisiae by modifying the N-terminal region of the structural genes. The yeast episomal vector YEp352 with the galactokinase1 (GAL1) promoter was used to construct expression plasmids. The expression of each gene was examined using SDS-PAGE and Western blot analysis. Iron uptake was examined and the cellular iron concentration was increased in S. cerevisiae expressing hfH. When cultured cells were incubated with 14.3 mM Fe(2+), the recombinant yeast expressing hfH had a cellular iron concentration 1.5 times greater than that of the control strain. The relationship between the iron taken up by the cells and the expressed proteins was examined. Iron-binding H-chain ferritin (H-ferritin) was seen in the recombinant S. cerevisiae incubated with iron, while small amounts of iron-binding L-chain ferritin (L-ferritin) were observed. Combined, these observations demonstrate that human H-ferritin has a function in iron storage in S. cerevisiae, while L-ferritin does not.     

L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.     

Functional assembly of recombinant human ferritin subunits in Pichia pastoris

Ferritin is an iron storage protein found in most living organisms as a natural assembled macromolecule. For studying the functional ability of the ferritin assembly, human H- and L-ferritins were expressed and purified from Pichia pastoris strain GS115. The recombinant H- and L-ferritins showed a globular form with transmission electron microscopy. The rate of iron uptake for H-ferritin was significantly faster than that for the L-ferritin in vitro. By gel permeation chromatography analysis, recombinant ferritins were confirmed as multimeric subunits with high molecular weight and it was indicated that assembled subunits were able to store iron in vivo.     

Uptake and Distribution of Iron and Transferrin in the Adult Rat Brain

Brain uptake of iron-59 and iodine-125-labelled transferrin from blood in the adult rat has been investigated using graphical analysis to determine the blood-brain barrier permeability to these tracers in experiments that lasted between 5 min and 8 days. The blood-brain barrier permeability (K(in)) to 59Fe was 89 x 10(-5) ml/min/g compared to the value of 7 x 10(-5) ml/min/g for 125I-transferrin, which is similar to that of albumin, a plasma marker. The autoradiographic distribution of these tracers in brain was also studied to determine any regional variation in brain uptake after the tracers had been administered either systemically or applied in vitro. No regional uptake was seen for 125I-transferrin even after 24 h of circulation. In contrast, 59Fe showed selective regional uptake by the choroid plexus and extra-blood-brain barrier structures 4 h after administration. After 24 h of circulation, 59Fe distribution in brain was similar to the transferrin receptor distribution, as determined in vitro, but was unlike the distribution of nonhaem iron determined histochemically. The data suggest that brain iron uptake does not involve any significant transcytotic pathway of transferrin-bound iron into brain. It is proposed that the uptake of iron into brain involves the entry of iron-loaded transferrin to the cerebral capillaries, deposition of iron within the endothelial cells, followed by recycling of apotransferrin to the circulation. The deposited iron is then delivered to brain-derived transferrin for extracellular transport within the brain, and subsequently taken up via transferrin receptors on neurones and glia for usage or storage.     

Evaluation of blood-brain barrier thiamine efflux using the in situ rat brain perfusion method

Thiamine is an essential, positively charged (under physiologic conditions), water-soluble vitamin requiring transport into brain. Brain thiamine deficiency has been linked to neurodegenerative disease by subsequent impairment of thiamine-dependent enzymes used in brain glucose/energy metabolism. In this report, we evaluate brain uptake and efflux of [3H]thiamine using the in situ rat brain perfusion technique. To confirm brain distribution was not related to blood-brain barrier endothelial cell uptake, we compared parenchymal and cell distribution of [3H]thiamine using capillary depletion. Our work supports previous literature findings suggesting blood-brain barrier thiamine uptake is via a carrier-mediated transport mechanism, yet extends the literature by redefining the kinetics with more sensitive methodology. Significantly, [3H]thiamine brain accumulation was influenced by a considerable efflux rate. Evaluation of the efflux mechanism demonstrated increased stimulation by the presence of increased vascular thiamine. The influx transport mechanism and efflux rate were each comparable throughout brain regions despite documented differences in glucose and thiamine metabolism. The observation that [3H]thiamine blood-brain barrier influx and efflux is regionally homogenous may have significant relevance to neurodegenerative disease linked to thiamine deficiency.     

Receptor-mediated transcytosis of lactoferrin through the blood-brain barrier

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.     

Functional roles of the ferritin receptors of human liver, hepatoma, lymphoid and erythroid cells.

Ferritin receptors are present on the membranes of many normal and malignant cells. The binding specificity of these receptors for H and L subunits was examined using recombinant human ferritin homopolymers. At least two different types of ferritin receptors were found, one derived from normal rat, pig, and human liver which shows similar binding of H- and L-ferritin. The second receptor type, specific for the H-chain ferritin, has been identified on membranes of hepatic and other transformed cells, and of normal lymphoblasts and erythroid precursors. These two receptor types may have different metabolic functions: the hepatic receptor acting as a scavenger for circulating ferritin and possibly for iron exchange between hepatocytes and macrophages; the H-ferritin receptor having a regulatory role which is not directly related to iron metabolism. The expression of the H-ferritin receptor is closely related to the activation and proliferation state of the cells. Addition of H-ferritin to the culture medium of cells expressing the H-ferritin receptor resulted in inhibition of cell proliferation and of colony formation.     

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of the total soluble protein of bacterial extract. The L-chains assemble to form a ferritin homopolymer with electrophoretic mobility, molecular weight, thermal stability, spectroscopic, and immunological properties analogous to natural ferritin from human liver (95% L-chain). This recombinant L-ferritin is able to incorporate and retain iron in solution at physiological pH values. At variance with the H-ferritin, the L form does not uptake iron at acidic pH values and does not show detectable ferroxidase activity. It is concluded that ferritin L-chain lacks the ferroxidase site present in the H-chain and that the two chains may have specialized functions in intracellular iron metabolism.     

Characterization and Distribution of Ferritin Binding Sites in the Adult Mouse Brain

Abstract : Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using ¹²⁵I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant ( K _D) of 4.65 × 10^-9 M and a binding site density ( B _max) of 17.9 fmol bound/μg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and whi     

Drug and gene delivery to the brain: the vascular route

Brain drug development of either small molecule or large molecule (recombinant proteins, gene medicines) neurotherapeutics has been limited, owing to the restrictive transport properties of the brain microvasculature, which forms the blood-brain barrier (BBB) in vivo. Widespread drug delivery to the brain, while not feasible via craniotomy and intracerebral injection, is possible if the drug is delivered to brain via the transvascular route through the BBB. Novel brain drug delivery and drug targeting strategies can be developed from an understanding of the molecular and cellular biology of the brain microvascular and BBB transport processes.     

Iron Uptake in Blood-Brain Barrier Endothelial Cells Cultured in Iron-Depleted and Iron-Enriched Media

Abstract: Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood-brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood-brain barrier endothelial cells were grown in either iron-enriched or iron-depleted medium. Iron-enriched cells showed a reduction in surface-bound and total transferrin receptor numbers compared with iron-depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron-enriched cultures was higher, whereas the transferrin receptor externalization rate in iron-enriched cultures was lower than the rate in iron-depleted cultures. Moreover, blood-brain barrier endothelial cells cultured in iron-enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood-brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.     

Quantification of Different Transferrin Receptor Pools in Primary Cultures of Porcine Blood-Brain Barrier Endothelial Cells

Abstract: Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood-brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood-brain barrier endothelial cells, we separately quantified surface-bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large "inactive" intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100-fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve-fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.     

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP^C) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP^Sc, nor the normal function of PrP^C is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP^C mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP^C increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP^C on ferritin iron content is enhanced by stimulating PrP^C endocytosis, and reversed by cross-linking PrP^C on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP^102L decreases ferritin iron content significantly relative to PrP^C expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP^C nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP^C in cellular iron uptake and transport to ferritin, and dysfunction of PrP^C as a significant contributing factor of brain iron imbalance in prion disorders.     

Metabolic capacity regulates iron homeostasis in endothelial cells

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.     

The Pivotal Role of Astrocytes in the Metabolism of Iron in the Brain

Iron is essential for the normal functioning of cells but since it is also capable of generating toxic reactive oxygen species, the metabolism of iron is tightly regulated. The present article advances the view that astrocytes are largely responsible for distributing iron in the brain. Capillary endothelial cells are separated from the neuropil by the endfeet of astrocytes, so astrocytes are ideally positioned to regulate the transport of iron to other brain cells and to protect them if iron breaches the blood-brain barrier. Astrocytes do not appear to have a high metabolic requirement for iron yet they possess transporters for transferrin, haemin and non-transferrin-bound iron. They store iron efficiently in ferritin and can export iron by a mechanism that involves ferroportin and ceruloplasmin. Since astrocytes are a common site of abnormal iron accumulation in ageing and neurodegenerative disorders, they may represent a new therapeutic target for the treatment of iron-mediated oxidative stress.     

Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1.

Interleukin-1 (IL-1 beta) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with L-ferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL-1 beta on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-1 beta. However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL-1 beta induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.     

Mutations of ferritin H chain C-terminus produced by nucleotide insertions have altered stability and functional properties

Ferritin is an iron storage protein made of 24 subunits. Previous mutational analyses showed that ferritin C-terminal region has a major role in protein stability and assembly but is only marginally involved in the mechanism of iron incorporation. However, it has recently been shown that patients who carry alterations of ferritin C-terminal sequence caused by nucleotide insertions show neurological disorders possibly related to altered protein functionality and cellular iron deregulation. To re-evaluate the role of this region, five mutants of mouse H-ferritin were produced by 2-nucleotide insertions that modified the last 6-29 residues and extended the sequence of 14 amino acids. The mutants were expressed in Escherichia coli and analysed for solubility, stability and capacity to incorporate iron. The alteration of the last 6-residue non-helical extension had no evident effect on the properties of ferritin, while solubility and capacity to assemble in ferritin shells decreased progressively with the extension of the modified region. The results also showed that the modification of even a part of the terminal E-helix abolished the capacity of ferritin to incorporate iron during expression in the cells, probably caused by conformational modification of the hydrophobic channels. The data support the hypothesis that the pathogenic mutations alter cellular iron homeostasis.     

Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity

Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data.