Zip14 is a member of the SLC39A zinc transporter family, which is involved in zinc uptake by cells. Up-regulation of Zip14 by IL-6 appears to contribute to the hepatic zinc accumulation and hypozincemia of inflammation. At least three members of the SLC39A family transport other trace elements, such as iron and manganese, in addition to zinc. We analyzed the capability of Zip14 to mediate non-transferrin-bound iron (NTBI) uptake by overexpressing mouse Zip14 in HEK 293H cells and Sf9 insect cells. Zip14 was found to localize to the plasma membrane, and its overexpression increased the uptake of both 65 Zn and 59 Fe. Addition of bathophenanthroline sulfonate, a cell-impermeant ferrous iron chelator, inhibited Zip14-mediated iron uptake from ferric citrate, suggesting that iron is taken up by HEK cells as Fe 2؉ . Iron uptake by HEK and Sf9 cells expressing Zip14 was inhibited by zinc. Suppression of endogenous Zip14 expression by using Zip14 siRNA reduced the uptake of both iron and zinc by AML12 mouse hepatocytes. Zip14 siRNA treatment also decreased metallothionein mRNA levels, suggesting that compensatory mechanisms were not sufficient to restore intracellular zinc. Collectively, these results indicate that Zip14 can mediate the uptake of zinc and NTBI into cells and that it may play a role in zinc and iron metabolism in hepatocytes, where this transporter is abundantly expressed. Because NTBI is commonly found in plasma of patients with hemochromatosis and transfusional iron overload, Zip14-mediated NTBI uptake may contribute to the hepatic iron loading that characterizes these diseases.hemochromatosis ͉ iron transport ͉ liver ͉ zinc transport ͉ inflammation

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ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n . Collectively, these data suggest that ZIP14 may not only function during iron overload to take up NTBI, but also under normal or iron-deficient conditions when cells take up iron via endocytosis of transferrin. The aim of the present study was to determine how iron deficiency and overload affect the expression of ZIP14 and DMT1 in the liver, pancreas, and heart. The localization of ZIP14 in liver and pancreas was also determined. Knowledge of where ZIP14 is expressed in these organs and how ZIP14 and DMT1 are regulated in vivo by iron will help us to better assess the contribution of these transporters to tissue iron uptake. Design and Methods Animals, diets, and non-heme iron determinationRats were made iron-deficient, iron-adequate, or iron-loaded as described previously. 10 Briefly, weanling (21-day-old) male Sprague-Dawley rats were fed modified AIN-93G purified diets containing iron at 10 ppm (iron deficient, FeD), 50 ppm (iron adequate, FeA), or 18,916 ppm (iron overload, FeO) for 3 weeks. Male Zip14 (Slc39a14) knockout and wild-type control mice maintained on the 129+Ter/SvJcl x C57BL/6 background 11 were analyzed at 6 weeks of age. Male and female hypotransferrinemic (hpx) mice and wild-type controls maintained on the BALB/cJ background were analyzed at 16 weeks of age. Homozygous hpx mice were given a weekly intraperitoneal injection of human apo-transferrin (0.6-1.8 mg) (EMD Chemicals). All mice consumed a commercial rodent diet containing 240 ppm iron (Teklad 7912, Harlan Laboratories). At the end of the studies, animals were anesthetized with vaporized isoflurane and sacrificed by exsanguination via the descending aorta. Tissues were harvested, immediately frozen in liquid nitrogen, and stored at -80°C until use. Animal protocols were approved by the University of Florida Institutional Animal Care and Use Committee. Tissue non-heme iron concentrations were determined colorimetrically after acid digestion of tissues. 12 Generation of ZIP14 antibodyRabbit anti-ZIP14 antiserum was generated against peptide ENEQTEEGKPSAIEVC corresponding to amino acids 138-153 of rat ZIP14. Antibodies specific to the ZIP14 peptide immunogen were affinity purified by using a peptide-agarose column of SulfoLink coupling gel (Pierce). Sample preparation and western blot analysisThe preparation of samples and western blot analysis are described in the Online Supplementary Design and Methods. Transfection, immunoprecipitation, and enzymatic deglycosylationEffectene reagent (Qiagen) was used to transiently transfect HEK 293T cells with either empty pCMV-Sport2 (control) or pCMV-Sport6 containing rat ZIP14 cDNA. To immunoprecipitate ZIP14, anti-ZIP14 antibody (400 mg) was covalently linked to AminoLink Plus Coupling Resin (Thermo Scientific) and added to a Pierce Spin Column according to the manufacturer's protocol. Immunoprecipitations were performed using the CoImmunoprecipitation Kit (Pierce) according to the manufacturer's instructions. To assess protein glycosylati...

Sialic acid binding is required for infectious cell surface receptor recognition by parvovirus minute virus of mice (MVM). We have utilized a glycan array consisting of ϳ180 different carbohydrate structures to identify the specific sialosides recognized by the prototype (MVMp) and immunosuppressive (MVMi) strains of MVM plus three virulent mutants of MVMp, MVMp-I362S, MVMp-K368R, and MVMp-I362S/K368R. All of the MVM capsids specifically bound to three structures with a terminal sialic acid-linked ␣2-3 to a common Gal␤1-4GlcNAc motif: Neu5Ac␣2-3Gal␤1-4GlcNAc␤1-4Gal␤1-4GlcNAc (3SiaLN-LN), Neu5Ac␣2-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc (3SiaLN-LN-LN), and Neu5Ac␣2-3Gal␤1-4(Fuc␣1-3)-GlcNAc␤1-3Gal␤1-4(Fuc␣1-3)GlcNAc␤1-3Gal␤1-4(Fuc␣1-3)GlcNAc (sLe x -Le x -Le x ). In addition, MVMi also recognized four multisialylated glycans with terminal ␣2-8 linkages: Neu5Ac␣2-8Neu5Ac␣2-8Neu5Ac␣ ((Sia) 3 ), Neu5Ac␣2-8Neu5Ac␣2-3Gal␤1-4Glc (GD3), Neu5Ac␣2-8Neu5Ac␣2-8Neu5Ac␣2-3Gal␤1-4Glc (GT3), and Neu5Ac␣2-8Neu5Ac␣2-3(GalNAc␤1-4)Gal␤1-4Glc (GD2). Interestingly, the virulent MVMp-K368R mutant also recognized GT3. Analysis of the relative binding affinities using a surface plasmon resonance biospecific interaction (BIAcore) assay showed the wild-type MVMp and MVMi capsids binding with higher affinity to selected glycans compared with the virulent MVMp mutants. The reduced affinity of the virulent MVMp mutants are consistent with previous in vitro cell binding assays that had shown weaker binding to permissive cells compared with wild-type MVMp. This study identifies the sialic acid structures recognized by MVM. It also provides rationale for the tropism of MVM for malignant transformed cells that contain sLe x motifs and the neurotropism of MVMi, which is likely mediated via interactions with multisialylated glycans known to be tumor cell markers. Finally, the observations further implicate a decreased binding affinity for sialic acid in the in vivo adaptation of MVMp to a virulent phenotype.Attachment to a cell surface receptor is an essential first step in the life cycle of many viruses. The initial viral attachment frequently determines the tissue tropism, and subsequent interactions determine the pathogenic outcome. In many virushost interactions, glycan chains often function as initial recognition molecules, with or without a protein component. Glycans are a major element of living systems, with Ͼ50% of all proteins being glycosylated. They are abundantly present in the outer cell surface and thus serve as convenient targets for viral attachment. The recognition of specific glycan motifs by viruses can be mediated by a specific viral surface protein component, such as the hemagglutinin of influenza virus (1), or specific regions of the viral capsid itself, as is proposed for members of the single-stranded DNA Parvoviridae family (2-8). The initial attachment of viruses to host cells is followed by an internalization process that often involves concerted actions of many factors, such as co-receptors and/or the endocytot...

Summary Vectors based on the adeno-associated virus are attractive and versatile vehicles for in vivo gene transfer. The virus capsid is the primary interface with the cell that defines many pharmacological, immunological and molecular properties. Determinants of these interactions are often restricted to a limited number of capsid amino acids. In this study, a portfolio of novel AAV vectors was developed following a structure-function analysis of naturally occurring AAV capsid isolates. Singletons, which are particular residues on the AAV capsid that were variable in otherwise conserved amino acid positions were found to impact on vector's ability to be manufactured or to transduce. Data for those residues that mapped to monomer-monomer interface regions on the particle structure suggested a role in particle assembly. The change of singleton residues to the conserved amino acid resulted in the rescue of many isolates that were defective upon initial isolation. This led to the development of an AAV vector portfolio that encompasses 6 different clades and 3 other distinct AAV niches. Evaluation of the in vivo gene transfer efficiency of this portfolio following intravenous and intramuscular administration highlighted a clade-specific tropism. These studies further the design and selection of AAV capsids for gene therapy applications.

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The Cancer Genome Atlas (TCGA) and other large-scale genomic data pipelines have been integral to the current understanding of the molecular events underlying renal cell carcinoma (RCC). These data networks have focused mostly on primary RCC, which often demonstrates indolent behavior. However, metastatic disease is the major cause of mortality associated with RCC and data sets examining metastatic tumors are sparse. Therefore, a more comprehensive analysis of gene expression and DNA methylome profiling of metastatic RCC in addition to primary RCC and normal kidney was performed. Integrative analysis of the methylome and transcriptome identified over 30 RCC-specific genes whose mRNA expression inversely correlated with promoter methylation, including several known targets of hypoxia inducible factors. Notably, genes encoding several metabolism-related proteins were identified as differentially regulated via methylation including hexokinase 2, aldolase C, stearoyl-CoA desaturase, and estrogen-related receptor-γ (ESRRG), which has a known role in the regulation of nuclear-encoded mitochondrial metabolism genes. Several gene expression changes could portend prognosis in the TCGA cohort. Mechanistically, ESRRG loss occurs via DNA methylation and histone repressive silencing mediated by the polycomb repressor complex 2. Restoration of ESRRG in RCC lines suppresses migratory and invasive phenotypes independently of its canonical role in mitochondrial metabolism. Collectively, these data provide significant insight into the biology of aggressive RCC and demonstrate a novel role for DNA methylation in the promotion of HIF signaling and invasive phenotypes in renal cancer.

The transmembrane protein ferroportin (Fpn) is essential for iron efflux from the liver, spleen, and duodenum. Fpn is regulated predominantly by the circulating iron regulatory hormone hepcidin, which binds to cell surface Fpn, initiating its degradation. Accordingly, when hepcidin concentrations decrease, Fpn levels increase. A previous study found that Fpn levels were not elevated in copper-deficient (CuD) mice that had anemia, a condition normally associated with dramatic reductions in hepcidin. Lack of change in Fpn levels may be because CuD mice do not display reduced concentrations of plasma iron (holotransferrin), a modulator of hepcidin expression. Here, we examined Fpn protein levels and hepcidin expression in CuD rats, which exhibit reduced plasma iron concentrations along with anemia. We also examined hepcidin expression in anemic CuD mice with normal plasma iron levels. We found that CuD rats had higher liver and spleen Fpn levels and markedly lower hepatic hepcidin mRNA expression than did copper-adequate (CuA) rats. In contrast, hepcidin levels did not differ between CuD and CuA mice. To examine potential mediators of the reduced hepcidin expression in CuD rats, we measured levels of hepatic transferrin receptor 2 (TfR2), a putative iron sensor that links holotransferrin to hepcidin production, and transcript abundance of bone morphogenic protein 6 (BMP6), a key endogenous positive regulator of hepcidin production. Diminished hepcidin expression in CuD rats was associated with lower levels of TfR2, but not BMP6. Our data suggest that holotransferrin and TfR2, rather than anemia or BMP6, are signals for hepcidin synthesis during copper deficiency.