Characterization of mouse embryonic cells deficient in the ctr1 high affinity copper transporter. Identification of a Ctr1-independent copper transport system

The trace metal copper is an essential cofactor for a number of enzymes that have critical roles in biological processes, but it is highly toxic when allowed to accumulate in excess of cellular needs. Consequently, homeostatic copper metabolism is maintained by molecules involved in copper uptake, distribution, excretion, and incorporation into copper-requiring enzymes. Previously, we reported that overexpression of the human or mouse Ctr1 copper transporter stimulates copper uptake in mammalian cells, and deletion of one Ctr1 allele in mice gives rise to tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. To investigate the physiological roles for mammalian Ctr1 protein in cellular copper metabolism, we characterized wild type, Ctr1 heterozygous, and Ctr1 homozygous knock-out cells isolated from embryos obtained by the inter-cross of Ctr1 heterozygous mice. Ctr1-deficient mouse embryonic cells are viable but exhibit significant defects in copper uptake and accumulation and in copper-dependent enzyme activities. Interestingly, Ctr1-deficient cells exhibit approximately 30% residual copper transport activity that is saturable, with a K(m) of approximately 10 microm, with biochemical features distinct from that of Ctr1. These observations demonstrate that, although Ctr1 is critical for both cellular copper uptake and embryonic development, mammals possess additional biochemically distinct functional copper transport activities.     

Ctr1 transports silver into mammalian cells

Silver is a non-essential, toxic metal. The use of silver as an antimicrobial agent in many applications and its presence as a contaminant in foods and air can lead to accumulation in tissues. Despite its widespread use, the systems involved in the uptake of silver into mammalian cells are presently unknown. Previous studies have shown that copper uptake at the plasma membrane by copper transporter 1 (Ctr1) is inhibited by an excess of silver, suggesting that Ctr1 may function in importing silver into cells. In this study we examined directly the role of Ctr1 in the accumulation of silver in mammalian cells using over-expression experiments and mouse embryonic fibroblast cells lacking Ctr1. COS-7 cells transfected to express a human Ctr1-green fluorescent protein (hCtr1-GFP) fusion protein hyper-accumulated silver when incubated in medium supplemented with low micromolar concentrations (2.5–10 μmol/L) of AgNO₃. An hCtr1-GFPM150L,M154L variant deficient for copper transport failed to stimulate accumulation of silver. Mouse embryonic fibroblast cells lacking Ctr1 showed approximately a 50% reduction in silver content when incubated in silver-supplemented medium compared to a wild-type isogenic cell line. Collectively, these data demonstrate that Ctr1 transports both copper and silver and suggest that Ctr1 is an important transport protein in the accumulation of silver in mammalian cells.     

Dynamical interplay between the human high-affinity copper transporter hCtr1 and its cognate metal ion

Abnormal cellular copper levels have been clearly implicated in genetic diseases, cancer, and neurodegeneration. Ctr1, a high-affinity copper transporter, is a homotrimeric integral membrane protein that provides the main route for cellular copper uptake. Together with a sophisticated copper transport system, Ctr1 regulates Cu(I) metabolism in eukaryotes. Despite its pivotal role in normal cell function, the molecular mechanism of copper uptake and transport via Ctr1 remains elusive. In this study, electron paramagnetic resonance (EPR), UV-visible spectroscopy, and all-atom simulations were employed to explore Cu(I) binding to full-length human Ctr1 (hCtr1), thereby elucidating how metal binding at multiple distinct sites affects the hCtr1 conformational dynamics. We demonstrate that each hCtr1 monomer binds up to five Cu(I) ions and that progressive Cu(I) binding triggers a marked structural rearrangement in the hCtr1 C-terminal region. The observed Cu(I)-induced conformational remodeling suggests that the C-terminal region may play a dual role, serving both as a channel gate and as a shuttle mediating the delivery of copper ions from the extracellular hCtr1 selectivity filter to intracellular metallochaperones. Our findings thus contribute to a more complete understanding of the mechanism of hCtr1-mediated Cu(I) uptake and provide a conceptual basis for developing mechanism-based therapeutics for treating pathological conditions linked to de-regulated copper metabolism.     

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.     

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.     

Coordination of platinum therapeutic agents to met-rich motifs of human copper transport protein1

Platinum therapeutic agents are widely used in the treatment of several forms of cancer. Various mechanisms for the transport of the drugs have been proposed including passive diffusion across the cellular membrane and active transport via proteins. The copper transport protein Ctr1 is responsible for high affinity copper uptake but has also been implicated in the transport of cisplatin into cells. Human hCtr1 contains two methionine-rich Mets motifs on its extracellular N-terminus that are potential platinum-binding sites: the first one encompasses residues 7-14 with amino acid sequence Met-Gly-Met-Ser-Tyr-Met-Asp-Ser and the second one spans residues 39-46 with sequence Met-Met-Met-Met-Pro-Met-Thr-Phe. In these studies, we use liquid chromatography and mass spectrometry to compare the binding interactions between cisplatin, carboplatin and oxaliplatin with synthetic peptides corresponding to hCtr1 Mets motifs. The interactions of cisplatin and carboplatin with Met-rich motifs that contain three or more methionines result in removal of the carrier ligands of both platinum complexes. In contrast, oxaliplatin retains its cyclohexyldiamine ligand upon platinum coordination to the peptide.     

Syntaxin 5 is required for copper homeostasis in Drosophila and mammals

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.     

Functional and molecular responses of suckling rat pups and human intestinal Caco-2 cells to copper treatment

Ctr1 and Atp7A are copper (Cu) transporters that may play a role in the regulation of intestinal Cu absorption; however, intestinal regulation of these transporters by Cu in vivo has not been well defined. In this study, we hypothesized that Cu supplementation would alter the expression of intestine Ctr1 and Atp7A in vivo and further documented effects of Cu exposure on Cu transport, Ctr1 and Atp7A levels and localization in enterocyte-like Caco-2 cells. Suckling rat pups were supplemented with Cu (0 and 25 microg Cu/day) for 10 days and small intestine Cu concentration, Ctr1, Atp7A and metallothionein (MT) gene expression were measured by Northern blot analysis. Caco-2 cells were treated with basal medium, or medium supplemented with 3 and 94 microM CuSO4 and 67Cu transport, Ctr1 and Atp7A levels and localization were determined. In rat pups, Cu supplementation increased intestinal Cu, Ctr1 and MT gene expression; however, Atp7A gene expression was not significantly affected. Caco-2 cells treated with 94 microM Cu had lower cellular Cu uptake and export compared to untreated cells. While Ctr1 and Atp7A gene and protein levels were unaffected, confocal microscopy indicated that Ctr1 was endocytosed and co-localized with transferrin in Cu treated cells. This study demonstrates the functional response of intestinal cells to Cu treatment and suggests that both Ctr1 and Atp7A may regulate Cu absorption.     

Ctr1 and its role in body copper homeostasis

Members of the Cu transporter (Ctr) family have been reported to be part of the copper uptake machinery in several organisms. Recently it has been suggested that human Ctr1 (hCtr1) may act as a copper transporter in several tissues including the intestine. hCtr1 is a 190 amino acid protein and is predicted to have three transmembrane-spanning domains and exist in the plasma membrane as a homo-trimer. Ctr1-transfected cell lines exhibit saturable, pH-dependent Cu(I) uptake indicating a role in copper transport. Recent studies with Ctr1 knockout mice have highlighted an essential function in mammalian embryonic development since homozygous mutants die in utero. Heterozygotes are indistinguishable from wild-type littermates but have a severely reduced brain copper content, suggesting that Ctr1 is a key component of the copper uptake pathway in the brain. However, its role in other tissues remains elusive.     

Tissue-specific interplay between copper uptake and efflux in Drosophila

The vinegar fly Drosophila melanogaster is proving to be an excellent system to study the in vivo regulation of the essential metal copper. The Ctr1A/B and DmATP7 copper transport proteins have well-established roles in Drosophila copper uptake and efflux, respectively. Both Ctr1A and DmATP7 are essential genes, whereas Ctr1B mutants are viable but die in excess or depleted copper conditions. Less is known about the tissue-specific requirements for these three genes and how they interact to maintain copper homeostasis in different cell types. Here, we use targeted overexpression and suppression of each gene to examine these questions in vivo. We find that in the epidermal cells that form the adult thoracic and abdominal cuticle, Ctr1A plays a major role in copper uptake, whereas Ctr1B plays only a minor supporting role and DmATP7, as previously shown, is essential for transfer of copper to the trans-Golgi network. We also find that the copper chaperone dSco1 appears necessary for supplying the mitochondria with copper in these tissues. In contrast, in the developing Drosophila eye, DmATP7 appears to be non-essential unless copper levels in these cells are artificially elevated. Again, Ctr1A is the main copper uptake gene in the eye, but when ectopically expressed, Ctr1B has greater phenotypic effects than Ctr1A. Furthermore, Ctr1A and Ctr1B show a dramatic synergistic interaction, indicating their relationship is more complicated than a simply additive one and that they may in fact act cooperatively for optimal copper import.     

Copper-Dependent Trafficking of the Ctr4-Ctr5 Copper Transporting Complex

Background

In Schizosaccharomyces pombe, copper uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr4 and Ctr5 molecules at the cell surface.

Methodology/Principal Findings

We have developed a bimolecular fluorescence complementation (BiFC) assay to analyze protein-protein interactions in vivo in S. pombe. The assay is based on the observation that N- and C-terminal subfragments of the Venus fluorescent protein can reconstitute a functional fluorophore only when they are brought into tight contact. Wild-type copies of the ctr4⁺ and ctr5⁺ genes were inserted downstream of and in-frame with the nonfluorescent C-terminal (VC) and N-terminal (VN) coding fragments of Venus, respectively. Co-expression of Ctr4-VC and Ctr5-VN fusion proteins allowed their detection at the plasma membrane of copper-limited cells. Similarly, cells co-expressing Ctr4-VN and Ctr4-VC in the presence of Ctr5-Myc₁₂ displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co-expressed in the presence of Ctr4-Flag₂ failed to be visualized at the plasma membrane, suggesting a requirement for a combination of two Ctr4 molecules with one Ctr5 molecule. We found that plasma membrane-located Ctr4-VC-Ctr5-VN fluorescent complexes were internalized when the cells were exposed to high levels of copper. The copper-induced internalization of Ctr4-VC-Ctr5-VN complexes was not dependent on de novo protein synthesis. When cells were transferred back from high to low copper levels, there was reappearance of the BiFC fluorescent signal at the plasma membrane.

Significance

These findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability.     

Copper transporter Ctr1 contributes to enhancement of the sensitivity of cisplatin in esophageal squamous cell carcinoma

Increasing evidence has demonstrated that Ctr1 plays a crucial role in the regulation of cisplatin uptake in a variety of tumors. The purpose of this study was to investigate its role in mediating cisplatin sensitivity in ESCC cells. Immunohistochemistry (IHC), In situ hybridization (ISH) and semi-quantitative RT-PCR were used to detect Ctr1 expressions in ESCC tissues. qRT-PCR and Western blot was performed to investigate the levels of Ctr1 mRNA and protein in ESCC cells. CCK-8, Flow cytometry and Transwell chamber assay were carried out to examine cell proliferation, apoptosis, migration and invasion abilities in ESCC cells. We found that ESCC tissues and cells had higher Ctr1 level than normal tissues and Het-1A cell. Ctr1 expression was correlated with histological grade, invasion depth, TNM staging and lymph node metastasis in ESCC patients. Ctr1 depletion reduced the suppressive role of proliferation, migration and invasion as well as the inductive role of cell apoptosis and Caspase-3 activity evoked by cisplatin, whereas Ctr1 upregulation combined with cisplatin exerted the synergistic role in regulation of proliferation, apoptosis, Caspase-3 activity, migration and invasion in ESCC. In conclusion, Ctr1 is implicated in ESCC development and progression and its expression may be a novel predictor for assessment of cisplatin sensitivity in ESCC.     

Fluorimetric Analysis of Copper Transport Mechanisms in the B104 Neuroblastoma Cell Model: A Contribution from Cellular Prion Protein to Copper Supplying

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP^C) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP^C proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP^C-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP^C in mediating copper uptake by clathrin-dependent endocytosis.