Divalent Metal Ion Transporter Research Articles

Structural characterization and molecular dynamics simulations of the caprine and bovine solute carrier family 11 A1 (SLC11A1).

Natural Resistance-Associated Macrophage Proteins are a family of transmembrane divalent metal ion transporters, with important implications in life of both bacteria and mammals. Among them, the Solute Carrier family 11 member A1 (SLC11A1) has been implicated with susceptibility to infection by Mycobacterium avium subspecies paratuberculosis (MAP), potentially causing Crohn's disease in humans and paratuberculosis (PTB) in ruminants. Our previous research had focused on sequencing the mRNA of the caprine slc11a1 gene and pinpointed polymorphisms that contribute to caprine SLC11A1's susceptibility to infection by MAP in PTB. Despite its importance, little is known on the structural/dynamic features of mammalian SLC11A1 that may influence its function under normal or pathological conditions at the protein level. In this work we studied the structural architecture of SLC11A1 in Capra hircus and Bos taurus through molecular modeling, molecular dynamics simulations in different, functionally relevant configurations, free energy calculations of protein-metal interactions and sequence conservation analysis. The results of this study propose a three dimensional structure for SLC11A1 with conserved sequence and structural features and provide hints for a potential mechanism through which divalent metal ion transport is conducted. Given the importance of SLC11A1 in susceptibility to PTB, this study provides a framework for further studies on the structure and dynamics of SLC11A1 in other organisms, to gain 3D structural insight into the macromolecular arrangements of SLC11A1 but also suggesting a potential mechanism which divalent metal ion transport is conducted.

Low DMT1 Expression Associates With Increased Oxidative Phosphorylation and Early Recurrence in Hepatocellular Carcinoma

BackgroundDespite a high rate of recurrences, long-term survival can be achieved after the resection of hepatocellular carcinoma (HCC) with effective local treatment. Discovery of adverse prognostic variables to identify patients with high risk of recurrence could improve the management of HCC. Accumulating evidence showing a link between carcinogenesis and increased expression of iron import proteins and intracellular iron prompted us to investigate a role of divalent metal-ion transporter-1 (DMT1) that binds and regulates a variety of divalent metals in HCC. Materials and methodsClinical and gene expression data from RNA seq in 369 HCC patients were obtained from The Cancer Genome Atlas. Disease-free survival was compared between DMT1 high- and low-expressing tumors, and gene set enrichment analysis was conducted. ResultsPatients with lower expression of DMT1 exhibited significantly worse disease-free survival compared with the DMT1 high group (P = 0.044), notably in advanced-stage patients (P = 0.008). DMT1 expression did not differ in etiologies, stages, and differentiation status of HCC. Interestingly, DMT1 expression levels inversely associated with cellular respiratory function in HCC. Furthermore, gene set enrichment analysis revealed that metabolism-related gene sets such as glycolysis, oxidative phosphorylation, and reactive oxygen species pathway were significantly enriched in the DMT1 low-expressing HCC. ConclusionsLow DMT1 expression associates with increased oxidative phosphorylation as well as glycolysis and identifies early recurrence in HCC patients after surgical treatment.

Homology modelling of human divalent metal transporter (DMT): Molecular docking and dynamic simulations for duodenal iron transport

Iron transport through the duodenum is regulated by carrier proteins, one of which is the ubiquitously distributed divalent metal transporter (DMT1) which is responsible for the uptake of iron across the apical surface of the duodenal enterocyte. The crystallographic structure of Staphylococcus capitis divalent metal ion transporter (ScaDMT1) was obtained and it was used as a template for the construction of a homology model of human divalent metal transporter (hDMT1). The binding site for hDMT1 was determined by using SiteMap as well as molecular docking studies on ScaDMT1. The differences in binding modes between ScaDMT1 and hDMT1 were noted for a set of 7 iron containing compounds, including ferrous sulphate. Diffusion of ferrous ion was observed during the course of molecular dynamic simulation which corresponded to the postulated mechanism of iron transport. Further, the dock scores correlated well with relative bioavailabilities of the iron compounds. The study confirmed the efficacy of the in silico model which could be used for future studies on the absorption of micronutrients.

Pea Ferritin Stability under Gastric pH Conditions Determines the Mechanism of Iron Uptake in Caco-2 Cells

BackgroundIron deficiency is an enduring global health problem that requires new remedial approaches. Iron absorption from soybean-derived ferritin, an ∼550-kDa iron storage protein, is comparable to bioavailable ferrous sulfate (FeSO4). However, the absorption of ferritin is reported to involve an endocytic mechanism, independent of divalent metal ion transporter 1 (DMT-1), the transporter for nonheme iron.ObjectiveOur overall aim was to examine the potential of purified ferritin from peas (Pisum sativum) as a food supplement by measuring its stability under gastric pH treatment and the mechanisms of iron uptake into Caco-2 cells.MethodsCaco-2 cells were treated with native or gastric pH–treated pea ferritin in combination with dietary modulators of nonheme iron uptake, small interfering RNA targeting DMT-1, or chemical inhibitors of endocytosis. Cellular ferritin formation, a surrogate measure of iron uptake, and internalization of pea ferritin with the use of specific antibodies were measured. The production of reactive oxygen species (ROS) in response to equimolar concentrations of native pea ferritin and FeSO4 was also compared.ResultsPea ferritin exposed to gastric pH treatment was degraded, and the released iron was transported into Caco-2 cells by DMT-1. Inhibitors of DMT-1 and nonheme iron absorption reduced iron uptake by 26–40%. Conversely, in the absence of gastric pH treatment, the iron uptake of native pea ferritin was unaffected by inhibitors of nonheme iron absorption, and the protein was observed to be internalized in Caco-2 cells. Chlorpromazine (clathrin-mediated endocytosis inhibitor) reduced the native pea ferritin content within cells by ∼30%, which confirmed that the native pea ferritin was transported into cells via a clathrin-mediated endocytic pathway. In addition, 60% less ROS production resulted from native pea ferritin in comparison to FeSO4.ConclusionWith consideration that nonheme dietary inhibitors display no effect on iron uptake and the low oxidative potential relative to FeSO4, intact pea ferritin appears to be a promising iron supplement.

Intestinal DMT1 Is Essential for Optimal Assimilation of Dietary Copper in Male and Female Mice with Iron-Deficiency Anemia

Divalent metal-ion transporter 1 (DMT1) may transport copper, but studies to date on this topic have been equivocal. Previously, an ex vivo experiment showed that intestinal copper transport was impaired in Dmt1-mutant Belgrade rats. In this study, we tested the hypothesis that intestinal DMT1 transports copper in vivo. Intestine-specific Dmt1 knockout (Dmt1int/int) mice and normal (control) littermates (Dmt1fl/fl) were used. In study 1, intestinal copper absorption was assessed in 7-wk-old mice of both sexes and genotypes by oral-intragastric gavage of 64Cu under normal and iron-deficiency anemia (IDA) conditions. In study 2, both sexes and genotypes of 8-wk-old mice were fed diets with adequate iron concentrations [72 parts per million (ppm)] plus adequate (9 ppm) or excessive (183 ppm) copper concentrations for 4 wk. Iron- and copper-related physiologic variables were subsequently assessed. Study 1 showed that intestinal copper transport was enhanced in normal (∼11% increase in males, 35% in females) and anemic (∼42% increase in males, 35% in females) Dmt1int/int mice. Study 2 showed that, with adequate copper intakes, serum ceruloplasmin (Cp) activity was decreased (by ∼29% in males and 20% in females) and spleens were enlarged (by 3-fold in both sexes) in Dmt1int/int mice. Higher dietary copper increased hepatic copper concentrations (by ∼3.3-fold in males and 1.5-fold in females), restored serum Cp activity, and mitigated the noted splenomegaly in Dmt1int/int mice. Copper homeostasis was disrupted in Dmt1int/int mice, particularly during IDA, despite the noted increases in intestinal copper transport. This was exemplified by the fact that extra dietary copper was required to restore serum Cp activity (a biomarker of copper status) and reduce the severity of the noted splenomegaly (which could reflect changes in erythropoietic demand) in Dmt1int/int mice. Collectively, these observations show that intestinal DMT1 is essential for the assimilation of sufficient quantities of dietary copper to maintain systemic copper homeostasis during IDA.

Reassessment of the Transport Mechanism of the Human Zinc Transporter SLC39A2.

The human zinc transporter SLC39A2, also known as ZIP2, was shown to mediate zinc transport that could be inhibited at pH <7.0 and stimulated by HCO3-, suggesting a Zn2+/HCO3- cotransport mechanism [Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564]. In contrast, recent experiments in our laboratory indicated that the functional activity of ZIP2 increases at acidic pH [Franz, M. C., et al. (2014) J. Biomol. Screening 19, 909-916]. The study presented here was therefore designed to reexamine the findings about the pH dependence and to extend the functional characterization of ZIP2. Our current results show that ZIP2-mediated transport is modulated by extracellular pH but independent of the H+ driving force. Also, in our experiments, ZIP2-mediated transport is not modulated by extracellular HCO3-. Moreover, a high extracellular [K+], which induces depolarization, inhibited ZIP2-mediated transport, indicating that the transport mechanism is voltage-dependent. We also show that ZIP2 mediates the uptake of Cd2+ ( Km ∼ 1.57 μM) in a pH-dependent manner ( KH+ ∼ 66 nM). Cd2+ transport is inhibited by extracellular [Zn2+] (IC50 ∼ 0.32 μM), [Cu2+] (IC50 ∼ 1.81 μM), and to a lesser extent [Co2+], but not by [Mn2+] or [Ba2+]. Fe2+ is not transported by ZIP2. Accordingly, the substrate selectivity of ZIP2 decreases in the following order: Zn2+ > Cd2+ ≥ Cu2+ > Co2+. Altogether, we propose that ZIP2 is a facilitated divalent metal ion transporter that can be modulated by extracellular pH and membrane potential. Given that ZIP2 expression has been reported in acidic environments [Desouki, M. M., et al. (2007) Mol. Cancer 6, 37; Inoue, Y., et al. (2014) J. Biol. Chem. 289, 21451-21462; Tao, Y. T., et al. (2013) Mol. Biol. Rep. 40, 4979-4984], we suggest that the herein described H+-mediated regulatory mechanism might be important for determining the velocity and direction of the transport process.

Role of N‐glycosylation in the activity of divalent metal‐ion transporter‐1

Divalent metal‐ion transporter‐1 (DMT1) is critical for iron absorption from the gut lumen to the enterocytes and for iron utilization by red blood cell precursors. Rare mutations in DMT1 result in a severe anemia in human patients, and DMT1 upregulation is observed in iron‐overload disorders. DMT1 is described to be a glycoprotein [Gruenheid S et al (1999) J Exp Med 189, 831–841; Mackenzie B et al (2007) Biochem J 403, 59–69]. Glycosylation is the process by which sugars are attached to a nascent peptide. Glycosylation—common for membrane proteins—is important for cell signaling, attachment of proteins to the extra‐cellular matrix, protein folding and stability, trafficking to or from the plasma membrane, and protection from proteolysis. In the present study, we tested the hypothesis that N‐linked glycosylation of DMT1 is required for its iron‐transport activity. We used functional and biochemical assays in RNA‐injected Xenopus laevis oocytes expressing wildtype or mutant human DMT1 combined with DMT1 immunoblotting of oocyte membrane extracts. Treatment of oocyte membrane extracts with the bacterial glycosidase PNGase F produced a shift in the mobility of the DMT1 bands detected by immunoblotting, consistent with DMT1 glycosylation in oocytes. We used the NetNGlyc server to predict in silico N‐glycosylation sites in the 1A/IRE(+) isoform of DMT1: three sites—namely N62, N365, and N378—exceeded the probability threshold. Whereas N365 and N378 reside on the 4th extracellular loop, N62 resides within the N‐terminal intracellular domain and is followed by a proline residue, so glycosylation of N62 is therefore doubtful. We used site‐directed mutagenesis to substitute asparagine (N) with glutamine (Q) at each of the three putative N‐glycosylation sites, and generated double (N365/N378Q) and triple mutants. Mobility of N62Q did not differ from wildtype DMT1 (wtDMT1), whereas the N365Q and N378Q mutations each produced gel shifts. Each construct mediated 55Fe2+ uptake that roughly correlated with the level of expression of the protein as judged by GFP fluorescence or by immunoblotting. Unexpectedly, mutation of N365 and N378 resulted in an increase in the apparent affinity for Fe2+. K0.5 for Fe2+ (obtained by measuring iron‐evoked currents at pH 5.5) for N365Q (0.8 ± [SEM] 0.1 μM), and N378Q (0.4 ± 0.1 μM), differed from wtDMT1 (1.8 ± 0.1 μM), P &lt; 0.001. Our data reveal that DMT1 is glycosylated at N365 and N378. N‐linked glycosylation of DMT1 is not required for iron‐transport activity but may alter the apparent affinity for iron. Since certain commensal and pathogenic bacteria produce glysosidases, our findings raise the possibility that changes in the gut microbiota could modulate the activity of intestinal DMT1 in vivo.Support or Funding InformationNIH–NIDDK grants R01 DK080047 and R01 DK107309This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional analysis of SLC39A8 mutations and their implications for manganese deficiency and mitochondrial disorders

SLC39A8 encodes ZIP8, a divalent metal ion transporter. Mutations in the SLC39A8 gene are associated with congenital disorder of glycosylation type II and Leigh syndrome. Notably, affected patients with both disorders exhibited severe manganese (Mn) deficiency. The cellular function of human SLC39A8 (hSLC39A8) and the mechanisms by which mutations in this protein lead to human diseases are unclear. Herein, we show that hSLC39A8 mediates 54Mn uptake by the cells, and its expression is regulated by Mn. While expression of wild-type hSLC39A8 increased 54Mn uptake activity, disease-associated mutations abrogated the ability of the transporter to mediate Mn uptake into the cells, thereby providing a causal link to severe Mn deficiency. All mutants failed to localize on the cell surface and were retained within the endoplasmic reticulum. Interestingly, expression of hSLC39A8 mutants of both CDG type II and Leigh syndrome reduced mitochondrial 54Mn levels and activity of Mn-dependent mitochondrial superoxide dismutase MnSOD, and in turn increased oxidative stress. The expression of wild-type hSLC39A8, but not the disease-associated mutants, promoted mitochondrial functions. Moreover, loss of function analyses further corroborate hSLC39A8’s critical role in mediating Mn uptake and mitochondrial function. Our results provide a potential pathogenic mechanism of diseases that are associated with hSLC39A8 mutations.

Sorting Out the Role of α-Synuclein in Retromer-Mediated Endosomal Protein Sorting.

Retromer is a phylogenetically conserved, multisubunit coat complex that controls endosomal protein trafficking and sorting. Mutations in the retromer gene VPS35 cause late-onset Parkinson disease, suggesting that trafficking defects cause neurodegeneration. Sorting nexins assist retromer to guide cell surface proteins to their assigned destinations, and our interest here is sorting nexin 3 (Snx3). Snx3 binds to membranes via a phox homolog (PX) domain that binds phosphatidylinositol 3-phosphate (PI3P), and in human cells its cargo proteins are the transferrin and Wnt receptors and the divalent metal ion transporter, whereas in yeast the best characterized cargo is the iron permease Ftr1. We recently discovered that α-synuclein inhibits Snx3-retromer recycling of Ftr1 in an unexpected way: α-synuclein, which avidly binds to negatively charged lipids, blocks the association of Snx3 to early endosomes. Here, we discuss mechanisms by which α-synuclein can disrupt Snx3-retromer–mediated recycling.

Current understanding of iron homeostasis

Iron is an essential trace element, but it is also toxic in excess, and thus mammals have developed elegant mechanisms for keeping both cellular and whole-body iron concentrations within the optimal physiologic range. In the diet, iron is either sequestered within heme or in various nonheme forms. Although the absorption of heme iron is poorly understood, nonheme iron is transported across the apical membrane of the intestinal enterocyte by divalent metal-ion transporter 1 (DMT1) and is exported into the circulation via ferroportin 1 (FPN1). Newly absorbed iron binds to plasma transferrin and is distributed around the body to sites of utilization with the erythroid marrow having particularly high iron requirements. Iron-loaded transferrin binds to transferrin receptor 1 on the surface of most body cells, and after endocytosis of the complex, iron enters the cytoplasm via DMT1 in the endosomal membrane. This iron can be used for metabolic functions, stored within cytosolic ferritin, or exported from the cell via FPN1. Cellular iron concentrations are modulated by the iron regulatory proteins (IRPs) IRP1 and IRP2. At the whole-body level, dietary iron absorption and iron export from the tissues into the plasma are regulated by the liver-derived peptide hepcidin. When tissue iron demands are high, hepcidin concentrations are low and vice versa. Too little or too much iron can have important clinical consequences. Most iron deficiency reflects an inadequate supply of iron in the diet, whereas iron excess is usually associated with hereditary disorders. These disorders include various forms of hemochromatosis, which are characterized by inadequate hepcidin production and, thus, increased dietary iron intake, and iron-loading anemias whereby both increased iron absorption and transfusion therapy contribute to the iron overload. Despite major recent advances, much remains to be learned about iron physiology and pathophysiology.

Development of iron homeostasis in infants and young children

Healthy, term, breastfed infants usually have adequate iron stores that, together with the small amount of iron that is contributed by breast milk, make them iron sufficient until ≥6 mo of age. The appropriate concentration of iron in infant formula to achieve iron sufficiency is more controversial. Infants who are fed formula with varying concentrations of iron generally achieve sufficiency with iron concentrations of 2 mg/L (i.e., with iron status that is similar to that of breastfed infants at 6 mo of age). Regardless of the feeding choice, infants' capacity to regulate iron homeostasis is important but less well understood than the regulation of iron absorption in adults, which is inverse to iron status and strongly upregulated or downregulated. Infants who were given daily iron drops compared with a placebo from 4 to 6 mo of age had similar increases in hemoglobin concentrations. In addition, isotope studies have shown no difference in iron absorption between infants with high or low hemoglobin concentrations at 6 mo of age. Together, these findings suggest a lack of homeostatic regulation of iron homeostasis in young infants. However, at 9 mo of age, homeostatic regulatory capacity has developed although, to our knowledge, its extent is not known. Studies in suckling rat pups showed similar results with no capacity to regulate iron homeostasis at 10 d of age when fully nursing, but such capacity occurred at 20 d of age when pups were partially weaned. The major iron transporters in the small intestine divalent metal-ion transporter 1 (DMT1) and ferroportin were not affected by pup iron status at 10 d of age but were strongly affected by iron status at 20 d of age. Thus, mechanisms that regulate iron homeostasis are developed at the time of weaning. Overall, studies in human infants and experimental animals suggest that iron homeostasis is absent or limited early in infancy largely because of a lack of regulation of the iron transporters DMT1 and ferroportin.

Identification of eight copper (Cu) uptake related genes from yellow catfish Pelteobagrus fulvidraco, and their tissue expression and transcriptional responses to dietborne Cu exposure

The present working hypothesis is that absorption of dietary Cu is related to mRNA expressions of genes involved in Cu uptake and transport of the intestine in fish. To this end, the full-length cDNA sequences of eight Cu uptake related genes, including two isoforms of copper transporter genes (ctr1 and ctr2), three copper chaperone genes (atox1, ccs and cox17), two Cu-ATPase genes (atp7a and atp7b) and divalent metal ion transporter 1 (dmt1), were cloned and characterized in yellow catfish P. fulvidraco, respectively. Their mRNA tissue expression and transcriptional responses to dietborne Cu exposure were investigated. Compared to the corresponding members of mammals, all of these members in P. fulvidraco shared the similar conserved domain structures. Their mRNAs were expressed in a wide range of tissues (including liver, muscle, spleen, brain, gill, intestine, heart and kidney), but at variable levels. In anterior intestine, mRNA levels of ctr1, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. The mRNA levels of ctr2 and mt were the highest for excess dietary Cu group and showed no significant differences between other two treatments. Atox1 mRNA levels were the highest for Cu-deficient group and showed no significant differences between other two treatments. The mRNA levels of ccs were the highest for Cu-deficient group, followed by Cu-excess group and the lowest for adequate-Cu group. In contrast, atp7b mRNA levels were the highest for Cu-excess group and the lowest for adequate Cu group. In the mid-intestine, mRNA levels of ctr1, ctr2, atox1, ccs, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. Atp7b mRNA levels were the lowest for adequate Cu group and showed no significant differences between other two treatments. Mt mRNA levels were the lowest for adequate Cu group and highest for Cu-excess group. For the first time, our study cloned and characterized ctr1, ctr2, atox1, ccs, cox17, atp7a, atp7b and dmt1 genes in P. fulvidraco and determined their tissue-specific expression, and transcriptional responses in the anterior and mid-intestine of yellow catfish under dietborne Cu exposure, which shed new light on the Cu uptake system and help to understand the molecular mechanisms of Cu homeostasis in fish.

Genome-Wide Identification and Expression Analysis of NRAMP Family Genes in Soybean (Glycine Max L.).

The NRAMP (natural resistance-associated macrophage protein) family of genes has been widely characterized in organisms ranging from bacteria to yeast, plants, mice, and humans. This gene family plays vital roles in divalent metal ion transport across cellular membranes. As yet, comprehensive analysis of NRAMP family genes has not been reported for soybean. In this study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes, along with their gene structures, phylogenetic relationships, and transmembrane domains. Expression analysis suggests that GmNRAMP genes function in numerous tissues and development stages. Moreover, soybean NRAMP genes were differentially regulated by deficiencies of N, P, K, Fe, and S, along with toxicities of Fe, Cu, Cd, and Mn. These results indicate that GmNRAMP genes function in many nutrient stress pathways, and might be involved in crosstalk among nutrient stress pathways. Subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of selected soybean NRMAP proteins. Protein-protein interaction analysis found that the networks of three GmNRAMP proteins which putatively interact with nodulin-like proteins, almost distinct from the network that is common to the other 10 soybean NRAMP proteins. Subsequent qRT-PCR results confirmed that these three GmNRMAP genes exhibited enhanced expression in soybean nodules, suggesting potential functions in the transport of Fe or other metal ions in soybean nodules. Overall, the systematic analysis of the GmNRAMP gene family reported herein provides valuable information for further studies on the biological roles of GmNRAMPs in divalent metal ion transport in various soybean tissues under numerous nutrient stresses and soybean-rhizobia symbiosis.

Heavy metal levels in patients with ineffective erythropoiesis

ObjectivesIron is taken into enterocytes at the duodenum via apical divalent metal-ion transporter 1 protein. Besides iron, divalent metal-ion transporter 1 also transports other divalent metals. We aimed to investigate blood heavy metal levels in patients with ineffective erythropoiesis. MethodsBlood levels of heavy metals including Pb, Al, Cd, Cr, Co, Cu, and Zn were measured in patients with thalassemia major (TM), thalassemia intermedia (TI), congenital dyserythropoietic anemia (CDA), and age- and sex-matched healthy controls. ResultsBlood samples were obtained from 68 patients (51 patients with TM, 8 with TI, 9 with CDA), and a control group that included 65 volunteers. Patients with TM were found to have lower Al, Pb, and Zn, and higher Cd levels compared with the control group. The patients treated with deferasirox were further analyzed and Pb and Zn levels were found lower compared with the control group. DiscussionPatients with TM had tendency to have elevated levels of plasma cadmium; however, the median level was not at a toxic level. Increased metal-ion transporter 1 activity may cause heavy metal accumulation, but deferasirox chelation may be protective against heavy metals besides iron.

A novel proton transfer mechanism in the SLC11 family of divalent metal ion transporters

In humans, the H+-coupled Fe2+ transporter DMT1 (SLC11A2) is essential for proper maintenance of iron homeostasis. While X-ray diffraction has recently unveiled the structure of the bacterial homologue ScaDMT as a LeuT-fold transporter, the exact mechanism of H+-cotransport has remained elusive. Here, we used a combination of molecular dynamics simulations, in silico pKa calculations and site-directed mutagenesis, followed by rigorous functional analysis, to discover two previously uncharacterized functionally relevant residues in hDMT1 that contribute to H+-coupling. E193 plays a central role in proton binding, thereby affecting transport properties and electrogenicity, while N472 likely coordinates the metal ion, securing an optimally “closed” state of the protein. Our molecular dynamics simulations provide insight into how H+-translocation through E193 is allosterically linked to intracellular gating, establishing a novel transport mechanism distinct from that of other H+-coupled transporters.

Hepatic metal ion transporter ZIP8 regulates manganese homeostasis and manganese-dependent enzyme activity.

Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.

The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells.

The relationship between iron and β-cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in β-cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human β-cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZRT/IRT-like protein 14 and 8 (ZIP14 and ZIP8) and divalent metal-ion transporter-1 (DMT1) to iron uptake by human β-cells. Iron was provided to the cells as nontransferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by βlox5 cells, a human β-cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in βlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to β-cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1β. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by β-cells and suggest differential regulation of ZIP14 in primary human islets compared with other cell types such as hepatocytes.

Intestinal brush-border Na+/H+ exchanger-3 drives H+-coupled iron absorption in the mouse.

Divalent metal-ion transporter-1 (DMT1), the principal mechanism by which nonheme iron is taken up at the intestinal brush border, is energized by the H(+)-electrochemical potential gradient. The provenance of the H(+) gradient in vivo is unknown, so we have explored a role for brush-border Na(+)/H(+) exchanger (NHE) isoforms by examining iron homeostasis and intestinal iron handling in mice lacking NHE2 or NHE3. We observed modestly depleted liver iron stores in NHE2-null (NHE2(-/-)) mice stressed on a low-iron diet but no change in hematological or blood iron variables or the expression of genes associated with iron metabolism compared with wild-type mice. Ablation of NHE3 strongly depleted liver iron stores, regardless of diet. We observed decreases in blood iron variables but no overt anemia in NHE3-null (NHE3(-/-)) mice on a low-iron diet. Intestinal expression of DMT1, the apical surface ferrireductase cytochrome b reductase-1, and the basolateral iron exporter ferroportin was upregulated in NHE3(-/-) mice, and expression of liver Hamp1 (hepcidin) was suppressed compared with wild-type mice. Absorption of (59)Fe from an oral dose was substantially impaired in NHE3(-/-) compared with wild-type mice. Our data point to an important role for NHE3 in generating the H(+) gradient that drives DMT1-mediated iron uptake at the intestinal brush border.

Regulation of the divalent metal ion transporter via membrane budding.

The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the divalent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins.

Five metal elements homeostasis-related genes in Synechogobius hasta: Molecular characterization, tissue expression and transcriptional response to Cu and Fe exposure

Two isoforms of Cu transporter (CTR1 and CTR2) and metallothionein (MT1 and MT2), and divalent metal ion transporter 1 (DMT1) were cloned and characterized in Synechogobius hasta, respectively. The protein sequences of S. hasta CTRs possessed two methionine-rich regions (MxM and MxxxM) and three transmembrane regions. At the C-terminus, CTR1 contained a sequence of conserved cysteine and histidine residues (HCH), while CTR2 did not contain the conserved sequence. The protein sequence of S. hasta DMT1 possessed all the characteristic features of DMT1, including twelve conserved hydrophobic cores of transmembrane domains. The protein sequences of S. hasta MTs were highly conserved in the total number of cysteine residues and their locations. mRNA of the five genes were expressed in a wide range of tissues but the levels were relatively higher in the liver. Cu exposure tended to up-regulate the mRNA expressions of CTR2, DMT1, MT1 and MT2. However, Fe down-regulated the Cu-induced increase of CTR2 and DMT1 mRNA levels. For the first time, our study cloned and characterized CTR1, CTR2, DMT1, MT1 and MT2 genes in S. hasta and determined their tissue-specific expression, and also the transcriptional change by Cu and Fe exposure, which shed new light on the CuFe relationship and help to understand the basic mechanisms of Cu and Fe homeostasis in fish.