Ceruloplasmin-ferroportin system of iron traffic in vertebrates

Safe trafficking of iron across the cell membrane is a delicate process that requires specific protein carriers. While many proteins involved in iron uptake by cells are known, only one cellular iron export protein has been identified in mammals: ferroportin(SLC40A1). Ceruloplasmin is a multicopper enzyme endowed with ferroxidase activity that is found as a soluble isoform in plasma or as a membrane-associated isoform in specific cell types. According to the currently accepted view, ferrous iron transported out of the cell by ferroportin would be safely oxidized by ceruloplasmin to facilitate loading on transferrin. Therefore, the ceruloplasminferroportin system represents the main pathway for cellular iron egress and it is responsible for physiological regulation of cellular iron levels. The most recent findings regarding the structural and functional features of ceruloplasmin and ferroportin and their relationship will be described in this review.     

Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?

The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at 相似文献   

The active role of vitamin C in mammalian iron metabolism: Much more than just enhanced iron absorption!

Ascorbate is a cofactor in numerous metabolic reactions. Humans cannot synthesize ascorbate owing to inactivation of the gene encoding the enzyme l-gulono-γ-lactone oxidase, which is essential for ascorbate synthesis. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance nonheme iron absorption in the gut, ascorbate within mammalian systems can regulate cellular iron uptake and metabolism. Ascorbate modulates iron metabolism by stimulating ferritin synthesis, inhibiting lysosomal ferritin degradation, and decreasing cellular iron efflux. Furthermore, ascorbate cycling across the plasma membrane is responsible for ascorbate-stimulated iron uptake from low-molecular-weight iron–citrate complexes, which are prominent in the plasma of individuals with iron-overload disorders. Importantly, this iron-uptake pathway is of particular relevance to astrocyte brain iron metabolism and tissue iron loading in disorders such as hereditary hemochromatosis and β-thalassemia. Recent evidence also indicates that ascorbate is a novel modulator of the classical transferrin–iron uptake pathway, which provides almost all iron for cellular demands and erythropoiesis under physiological conditions. Ascorbate acts to stimulate transferrin-dependent iron uptake by an intracellular reductive mechanism, strongly suggesting that it may act to stimulate iron mobilization from the endosome. The ability of ascorbate to regulate transferrin iron uptake could help explain the metabolic defect that contributes to ascorbate-deficiency-induced anemia.     

Determination of the chelatable iron pool of single intact cells by laser scanning microscopy

We have previously established a method of detecting intracellular chelatable iron in viable cells based on digital fluorescence microscopy. To quantify cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the method to confocal laser scanning microscopy, which should allow the determination of the indicator concentration on the single-cell level. The fluorescent heavy-metal indicator phen green SK (PG SK), the fluorescence of which is quenched by iron, was loaded into cultured rat hepatocytes. The hepatocellular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transition metal chelator 2,2'-dipyridyl (2, 2'-DPD, 5 mM). We optimized the scanning parameters for quantitatively recording changes in fluorescence and determined individual intracellular PG SK concentrations from the unquenched cellular fluorescence (after 2,2'-DPD) compared with PG SK standards in a "cytosolic" medium. An ex situ calibration method based on laser scanning microscopy was set up to determine the concentration of cellular chelatable iron from the increase of PG SK fluorescence after addition of 2,2'-DPD (5 mM). As the stoichiometry of the PG SK:Fe(2+) complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from absolute changes in cellular fluorescence. Using this method, we found 2.5 +/- 2.2 microM chelatable iron in hepatocytes. This method makes it possible to determine the pool of chelatable iron in single vital cells independently of cellular differences (e.g., dye loading, cell volume) in heterogeneous cell populations.     

Duodenal cytochrome b: a novel ferrireductase in airway epithelial cells

Catalytically active iron in the lung causes oxidative stress and promotes microbial growth that can be limited by intracellular sequestration of iron within ferritin. Because cellular iron uptake requires membrane ferrireductase activity that in the gut can be provided by duodenal cytochrome b (Dcytb), we sought Dcytb in the lung to test the hypothesis that it contributes to epithelial iron regulation by reducing Fe(3+) for cellular iron transport. Dcytb expression was found in respiratory epithelium in vitro and in vivo and was responsive to iron concentration. Iron transport was measured in human bronchial epithelial (HBE) cells using inductively coupled plasma atomic emission spectroscopy and was demonstrated to be partially inhibited in the presence of Dcytb-blocking antibody, suggesting that Dcytb reduces Fe(3+) for cellular iron transport. A definite source of reducing equivalents for Dcytb was sought but not identified. We found no evidence that ascorbate was involved but did demonstrate that O(2)(-). production decreased when Dcytb function was blocked. The presence of Dcytb in airway epithelial cells and its regulation by iron therefore may contribute to pulmonary cytoprotection.     

膜铁转运蛋白1，铁调素的靶分子？

膜铁转运蛋白1是重要的跨膜铁输出分子,主要分布于十二指肠和单核巨噬系统的细胞膜上,参与机体的肠铁吸收和巨噬细胞对铁的再循环等过程。铁调素是调节机体铁代谢平衡的激素,机体通过肝脏分泌的铁调素对铁转运相关蛋白的表达进行调控,从而实现机体自身的铁稳态。最新研究显示,铁调素的靶分子可能是膜铁转运蛋白1,它通过直接的作用引起膜铁转运蛋白1的内化(internalization)、降解,从而调节其在细胞膜上的表达量,进而控制肠铁吸收和巨噬细胞对铁的再循环过程,以维持机体的铁稳态。     

Regulation of iron acquisition and iron distribution in mammals

Both cellular iron deficiency and excess have adverse consequences. To maintain iron homeostasis, complex mechanisms have evolved to regulate cellular and extracellular iron concentrations. Extracellular iron concentrations are controlled by a peptide hormone hepcidin, which inhibits the supply of iron into plasma. Hepcidin acts by binding to and inducing the degradation of the cellular iron exporter, ferroportin, found in sites of major iron flows: duodenal enterocytes involved in iron absorption, macrophages that recycle iron from senescent erythrocytes, and hepatocytes that store iron. Hepcidin synthesis is in turn controlled by iron concentrations, hypoxia, anemia and inflammatory cytokines. The molecular mechanisms that regulate hepcidin production are only beginning to be understood, but its dysregulation is involved in the pathogenesis of a spectrum of iron disorders. Deficiency of hepcidin is the unifying cause of hereditary hemochromatoses, and excessive cytokine-stimulated hepcidin production causes hypoferremia and contributes to anemia of inflammation.     

The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis

Ferroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpn(null/null) animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis.     

Effect of metabolic inhibitors on uptake of non-transferrin-bound iron by reticulocytes

The relationship between transferrin-free iron uptake and cellular metabolism was investigated using rabbit reticulocytes in which energy metabolism was altered by incubation with metabolic inhibitors (antimycin A, 2,4-dinitrophenol, NaCN, NaN3 and rotenone) or substrates. Measurements were made of cellular ATP concentration and the rate of uptake of Fe(II) from a sucrose solution buffered at pH 6.5. There was a highly significant correlation between the rate of iron uptake into cytosolic and stromal fractions of the cells and ATP levels. Iron transport into the cytosol showed saturation kinetics. The metabolic inhibitors all reduced the Vmax but had no effect on the Km values for this process. It is concluded that the uptake of transferrin-free iron by reticulocytes is dependent on the cellular concentration of ATP and that it crosses the cell membrane by an active, carrier-mediated transport process. Additional studies were performed using transferrin-bound iron. The metabolic inhibitors also reduced the uptake of this form of iron but the inhibition could be accounted for entirely by reduction in the rate of transferrin endocytosis.     

Effects of carboxylic acids on the uptake of non-transferrin-bound iron by astrocytes

The concentrations of non-transferrin-bound iron are elevated in the brain during pathological conditions such as stroke and Alzheimer's disease. Astrocytes are specialised for sequestering this iron, however little is known about the mechanisms involved. Carboxylates, such as citrate, have been reported to facilitate iron uptake by intestinal cells. Citrate binds iron and limits its redox activity. The presence of high citrate concentrations in the interstitial fluid of the brain suggests that citrate may be an important ligand for iron transport by astrocytes. This study investigates whether iron accumulation by cultured rat astrocytes is facilitated by citrate or other carboxylates. Contrary to expectations, citrate, tartrate and malate were found to block iron accumulation in a concentration-dependent manner; α-ketoglutarate had limited effects, while fumarate, succinate and glutarate had no effect. This blockade was not due to an inhibition of ferric reductase activity. Instead, it appeared to be related to the capacity of these carboxylates to bind iron, since phosphate, which also binds iron, diminished the capacity of citrate, tartrate and malate to block the cellular accumulation of iron. These findings raise the possibility that citrate may have therapeutic potential in the management of neurodegenerative conditions that involve cellular iron overload.     

Altered iron homeostasis involvement in arsenite-mediated cell transformation

Chronic exposure to low doses of arsenite causes transformation of human osteogenic sarcoma (HOS) cells. Although oxidative stress is considered important in arsenite-induced cell transformation, the molecular and cellular mechanisms by which arsenite transforms human cells are still unknown. In the present study, we investigated whether altered iron homeostasis, known to affect cellular oxidative stress, can contribute to the arsenite-mediated cell transformation. Using arsenite-induced HOS cell transformation as a model, it was found that total iron levels are significantly higher in transformed HOS cells in comparison to parental control HOS cells. Under normal iron metabolism conditions, iron homeostasis is tightly controlled by inverse regulation of ferritin and transferrin receptor (TfR) through iron regulatory proteins (IRP). Increased iron levels in arsenite transformed cells should theoretically lead to higher ferritin and lower TfR in these cells than in controls. However, the results showed that both ferritin and TfR are decreased, apparently through two different mechanisms. A lower ferritin level in cytoplasm was due to the decreased mRNA in the arsenite-transformed HOS cells, while the decline in TfR was due to a lowered IRP-binding activity. By challenging cells with iron, it was further established that arsenite-transformed HOS cells are less responsive to iron treatment than control HOS cells, which allows accumulation of iron in the transformed cells, as exemplified by significantly lower ferritin induction. On the other hand, caffeic acid phenethyl ester (CAPE), an antioxidant previously shown to suppress As-mediated cell transformation, prevents As-mediated ferritin depletion. In conclusion, our results suggest that altered iron homeostasis contributes to arsenite-induced oxidative stress and, thus, may be involved in arsenite-mediated cell transformation.     

Human MRCKα is regulated by cellular iron levels and interferes with transferrin iron uptake

Myotonic dystrophy kinase-related Cdc42-binding kinase α (MRCKα, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3′-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKα protein expression is also regulated by iron levels; MRCKα colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKα expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKα takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKα activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.     

The effects of ascorbic acid on the intracellular metabolism of iron and ferritin

An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron.     

A Golgi-localized MATE transporter mediates iron homoeostasis under osmotic stress in Arabidopsis

Iron is an essential micronutrient that acts as a cofactor in a wide variety of pivotal metabolic processes, such as the electron transport chain of respiration, photosynthesis and redox reactions, in plants. However, its overload exceeding the cellular capacity of iron binding and storage is potentially toxic to plant cells by causing oxidative stress and cell death. Consequently, plants have developed versatile mechanisms to maintain iron homoeostasis. Organismal iron content is tightly regulated at the steps of uptake, translocation and compartmentalization. Whereas iron uptake is fairly well understood at the cellular and organismal levels, intracellular and intercellular transport is only poorly understood. In the present study, we show that a MATE (multidrug and toxic compound extrusion) transporter, designated BCD1 (BUSH-AND-CHLOROTIC-DWARF 1), contributes to iron homoeostasis during stress responses and senescence in Arabidopsis. The BCD1 gene is induced by excessive iron, but repressed by iron deficiency. It is also induced by cellular and tissue damage occurring under osmotic stress. The activation-tagged mutant bcd1-1D exhibits leaf chlorosis, a typical symptom of iron deficiency. The chlorotic lesion of the mutant was partially recovered by iron feeding. Whereas the bcd1-1D mutant accumulated a lower amount of iron, the iron level was elevated in the knockout mutant bcd1-1. The BCD1 protein is localized to the Golgi complex. We propose that the BCD1 transporter plays a role in sustaining iron homoeostasis by reallocating excess iron released from stress-induced cellular damage.     

Microbial ferric iron reductases

Almost all organisms require iron for enzymes involved in essential cellular reactions. Aerobic microbes living at neutral or alkaline pH encounter poor iron availability due to the insolubility of ferric iron. Assimilatory ferric reductases are essential components of the iron assimilatory pathway that generate the more soluble ferrous iron, which is then incorporated into cellular proteins. Dissimilatory ferric reductases are essential terminal reductases of the iron respiratory pathway in iron-reducing bacteria. While our understanding of dissimilatory ferric reductases is still limited, it is clear that these enzymes are distinct from the assimilatory-type ferric reductases. Research over the last 10 years has revealed that most bacterial assimilatory ferric reductases are flavin reductases, which can serve several physiological roles. This article reviews the physiological function and structure of assimilatory and dissimilatory ferric reductases present in the Bacteria, Archaea and Yeast. Ferric reductases do not form a single family, but appear to be distinct enzymes suggesting that several independent strategies for iron reduction may have evolved.     

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.     

Iron, oxidative stress and the example of solar ultraviolet A radiation

Iron has outstanding biological importance as it is required for a wide variety of essential cellular processes and, as such, is a vital nutrient. The element holds this central position by virtue of its facile redox chemistry and the high affinity of both redox states (iron II and iron III) for oxygen. These same properties also render iron toxic when its redox-active chelatable 'labile' form exceeds the normal binding capacity of the cell. Indeed, in contrast to iron bound to proteins, the intracellular labile iron (LI) can be potentially toxic especially in the presence of reactive oxygen species (ROS), as it can lead to catalytic formation of oxygen-derived free radicals such as hydroxyl radical that ultimately overwhelm the cellular antioxidant defense mechanisms and lead to cell damage. While intracellular iron homeostasis and body iron balance are tightly regulated to minimise the presence of potentially toxic LI, under conditions of oxidative stress and certain pathologies, iron homeostasis is severely altered. This alteration manifests itself in several ways, one of which is an increase in the intracellular level of potentially harmful LI. For example acute exposure of skin cells to ultraviolet A (UVA, 320-400 nm), the oxidising component of sunlight provokes an immediate increase in the available pool of intracellular LI that appears to play a key role in the increased susceptibility of skin cells to UVA-mediated oxidative membrane damage and necrotic cell death. The main purpose of this overview is to bring together some of the new findings related to intracellular LI distribution and trafficking under physiological and patho-physiological conditions as well as to discuss mechanisms and consequences of oxidant-induced alterations in the intracellular pool of LI, as exemplified by UVA radiation.