Structure and dimerization properties of the plant-specific copper chaperone CCH.

Copper chaperones of the ATX1 family are found in a wide range of organisms where these essential soluble carriers strictly control the transport of monovalent copper across the cytoplasm to various targets in diverse cellular compartments thereby preventing detrimental radical formation catalyzed by the free metal ion. Notably, the ATX1 family in plants contains two distinct forms of the cellular copper carrier. In addition to ATX1 having orthologs in other species, they also contain the copper chaperone CCH. The latter features an extra C-terminal extension whose function is still unknown. The secondary structure of this extension was predicted to be disordered in previous studies, although this has not been experimentally confirmed. Solution NMR studies on purified CCH presented in this study disclose that this region is intrinsically disordered regardless of the chaperone's copper loading state. Further biophysical analyses of the purified metallochaperone provide evidence that the C-terminal extension stabilizes chaperone dimerization in the copper-free and copper-bound states. A variant of CCH lacking the C-terminal extension, termed CCHΔ, shows weaker dimerization but similar copper binding. Computational studies further corroborate the stabilizing role of the C-terminal extension in chaperone dimerization and identify key residues that are vital to maintaining dimer stability.

Copper Chaperone for Superoxide Dismutase Subtypes as a Prognostic Marker in Luminal B Breast Cancer.

Background: Copper chaperone for superoxide dismutase (CCS) is an essential component of the oxidation-reduction system. In breast cancer cells, CCS expression is highly up-regulated, which contributes to cellular proliferation and migration. Breast cancer is a multifaceted disease with different tumor prognoses and responses to clinical treatments, which may be associated with multiple molecular subtypes of CCS. Methods: The CCS expression patterns in breast cancer were investigated by TNMplot, cBioPortal, and HPA network database. The correlation of CCS expression with clinicopathological parameters was analyzed using the UALCAN database. The Cancer Genome Atlas (TCGA) data set was used to analyze the Clinical characteristics of CCS in luminal B patients. The bc-GenExMiner database was used to analyze the effects of BReast-CAncer susceptibility gene (BRCA)1/2, TP53 mutation status, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER) expression on CCS expression. The survival curves and prognostic value of CCS in luminal B breast cancer were performed through Kaplan-Meier curves, univariate and multivariate Cox regression using the PrognoScan, bc-GenExMiner, and Clinical bioinformatics analysis platform. Results: We found that CCS expression was associated with patient age, race, ER, and PR status. We also discovered that BRCA1/2 mutations had an effect on CCS expression. The luminal B subtype had the highest CCS expression, which was linked to poor survival compared with other subtypes. In addition, Kaplan-Meier curve analysis showed that luminal B patients with high CCS mRNA expression showed a poor survival and the CCS gene is an independent predictor of outcome in patients with luminal B breast cancer by univariate and multivariate Cox regression. Conclusions: Our findings emphasize the significant expression of CCS in luminal B breast cancer and its potential as an autonomous prognostic determinant for this specific molecular subtype. These findings suggest that CCS holds promise as a prospective marker for the treatment of luminal B breast cancer.

MOFs Modulate Copper Trafficking in Tumor Cells for Bioorthogonal Therapy.

In situ drug synthesis using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has attracted considerable attention in tumor therapy because of its satisfactory effectiveness and reduced side-effects. However, the exogenous addition of copper catalysts can cause cytotoxicity and has hampered biomedical applications in vivo. Here, we design and synthesize a metal-organic framework (MOF) to mimic copper chaperone, which can selectively modulate copper trafficking for bioorthogonal synthesis with no need of exogenous addition of copper catalysts. Like copper chaperones, the prepared ZIF-8 copper chaperone mimics specifically bind copper ions through the formation of coordination bonds. Moreover, the copper is unloaded under the acidic environment due to the dissipation of the coordination interactions between metal ions and ligands. In this way, the cancer cell-targeted copper chaperone mimics can selectively transport copper ions into cells. Regulation of intracellular copper trafficking may inspire constructing bioorthogonal catalysis system with reduced metal cytotoxicity in live cells.

Zinc-mediated interaction of copper chaperones through their heavy-metal associated domains.

BACKGROUND: A copper chaperone CCS is a multi-domain protein that supplies a copper ion to Cu/Zn-superoxide dismutase (SOD1). Among the domains of CCS, the N-terminal domain (CCSdI) belongs to a heavy metal-associated (HMA) domain, in which a Cys-x-x-Cys (CxxC) motif binds a heavy metal ion. It has hence been expected that the HMA domain in CCS has a role in the metal trafficking; however, the CxxC motif in the domain is dispensable for supplying a copper ion to SOD1, leaving an open question on roles of CCSdI in CCS. METHODS: To evaluate protein-protein interactions of CCS through CCSdI, yeast two-hybrid assay, a pull-down assay using recombinant proteins, and the analysis with fluorescence resonance energy transfer were performed. RESULTS: We found that CCS specifically interacted with another copper chaperone HAH1, a HMA domain protein, through CCSdI. The interaction between CCSdI and HAH1 was not involved in the copper supply from CCS to SOD1 but was mediated by a zinc ion ligated with Cys residues of the CxxC motifs in CCSdI and HAH1. CONCLUSION: While physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCSdI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.

A pathological link between dysregulated copper binding in Cu/Zn-superoxide dismutase and amyotrophic lateral sclerosis.

Mutations in the gene coding Cu/Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (ALS), and its pathological hallmark includes abnormal accumulation of mutant SOD1 proteins in spinal motorneurons. Mutant SOD1 proteins are considered to be susceptible to misfolding, resulting in the accumulation as oligomers/aggregates. While it remains obscure how and why SOD1 becomes misfolded under pathological conditions in vivo, the failure to bind a copper and zinc ion in SOD1 in vitro leads to the significant destabilization of its natively folded structure. Therefore, genetic and pharmacological attempts to promote the metal binding in mutant SOD1 could serve as an effective treatment of ALS. Here, I briefly review the copper and zinc binding process of SOD1 in vivo and discuss a copper chaperone for SOD1 as a potential target for developing ALS therapeutics.

Effects of Fructose and Stress on Rat Renal Copper Metabolism and Antioxidant Enzymes Function.

The effects of a fructose-rich diet and chronic stress on copper metabolism in the kidneys are still understudied. We investigated whether fructose and/or chronic unpredictable stress modulate copper metabolism in a way that affects redox homeostasis, thus contributing to progression of metabolic disturbances in the kidney. We determined protein level of copper transporters, chaperones, and cuproenzymes including cytochrome c oxidase, as well as antioxidant enzymes function in the kidneys of male Wistar rats subjected to 20% liquid fructose supplementation and/or chronic stress. Liquid fructose supplementation increased level of copper chaperone of superoxide dismutase and decreased metallothionein level, while rendering the level of copper importer and copper chaperones involved in copper delivery to mitochondria and trans Golgi network unaffected. Stress had no effect on renal copper metabolism. The activity and expression of renal antioxidant enzymes remained unaltered in all experimental groups. In conclusion, fructose, independently of stress, decreased renal copper level, and modulated renal copper metabolism as to preserve vital cellular function including mitochondrial energy production and antioxidative defense, at the expense of intracellular copper storage.

Copper in the tumor microenvironment and tumor metastasis.

Copper (Cu), an essential micronutrient, plays an essential role in several physiological processes, including cell proliferation and angiogenesis; however, its dysregulation induces oxidative stress and inflammatory responses. Significant Cu accumulation is observed in several tumor tissues. The bioavailability of intracellular Cu is tightly controlled by Cu transporters, including Cu transporter 1 (CTR1) and Cu-transporting P-type ATPase α and ß (ATP7A and ATP7B), and Cu chaperones, including Cu chaperone for superoxide dismutase 1 (CCS) and antioxidant-1 (Atox-1). In several tumor tissues, these abnormalities that induce intra-cellular Cu accumulation are involved in tumor progression. In addition, functional disturbance in Cu-containing secretory enzymes, such as superoxide dismutase 3 (SOD3), and lysyl oxidase enzymes (LOX and LOXL1-4) with abnormal Cu dynamics plays a key role in tumor metastasis. For example, the loss of SOD3 in tumor tissues induces oxidative stress, which promotes neovascularization and epithelial-to-mesenchymal transition (EMT). LOX promotes collagen crosslinking, which functions in the metastatic niche formation. Accordingly, restricted Cu regulation may be a novel strategy for the inhibition of tumor metastasis. However, it is unclear how these Cu disturbances occur in tumor tissues and the exact molecular mechanisms underlying Cu secretory enzymes. In this review article, I discuss the role of Cu transporters, Cu chaperones, and Cu-containing secretory enzymes in tumor progression to better understand the role of Cu homeostasis in tumor tissues.

Restoration of the Immunogenicity of Tumor Cells for Enhanced Cancer Therapy via Nanoparticle-Mediated Copper Chaperone Inhibition.

Recent progress in studying copper-dependent targets and pathways in the context of tumor treatment has provided new insights into therapeutic strategies of leveraging copper-dependent disease vulnerabilities and pharmacological manipulation of intratumor copper transportation to improve chemotherapy. Here, we developed reactive oxygen species (ROS)-sensitive nanoparticles loaded with copper chaperone inhibitor DC_AC50 and cisplatin(IV) prodrug. The released DC_AC50 can promote a remarkable accumulation of intracellular cisplatin and copper through inhibition of the Atox1-ATPase pathways, thereby enhancing the chemotherapeutic effect of cisplatin and inducing significant ROS generation. Excessive ROS then elicits intense endoplasmic reticulin (ER) stress which facilitates the immunogenic cell death (ICD) spurring a sustained immune response. Our study suggests that nanoparticle-mediated copper chaperone inhibition via DC_AC50 can restore the immunogenicity of tumor cells for enhanced chemotherapy and cancer immunotherapy.

An Emerging Role of Defective Copper Metabolism in Heart Disease.

Copper is an essential trace metal element that significantly affects human physiology and pathology by regulating various important biological processes, including mitochondrial oxidative phosphorylation, iron mobilization, connective tissue crosslinking, antioxidant defense, melanin synthesis, blood clotting, and neuron peptide maturation. Increasing lines of evidence obtained from studies of cell culture, animals, and human genetics have demonstrated that dysregulation of copper metabolism causes heart disease, which is the leading cause of mortality in the US. Defects of copper homeostasis caused by perturbed regulation of copper chaperones or copper transporters or by copper deficiency resulted in various types of heart disease, including cardiac hypertrophy, heart failure, ischemic heart disease, and diabetes mellitus cardiomyopathy. This review aims to provide a timely summary of the effects of defective copper homeostasis on heart disease and discuss potential underlying molecular mechanisms.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

RANBP2 mutation causing autosomal dominant acute necrotizing encephalopathy attenuates its interaction with COX11.

PURPOSE: Autosomal dominant acute necrotizing encephalopathy (ADANE) is caused by missense mutations in the gene encoding Ran-binding protein 2 (RANBP2), a nuclear pore protein regulating mitochondrial localization and function. Previous studies have found that RANBP2 binds to COX11 and suppresses its inhibitory activity over hexokinase1. To further elucidate mitochondrial dysfunction in ADANE, we analyzed the interaction between mutated RANBP2 and COX11. METHODS: We extracted cDNA from a patient and constructed pGEX wild-type or mutant-type vectors including RANBP2 c.1754C>T, the commonest variant in ADANE. We transformed E. coli competent cells with the vectors and had them express GST-RANBP2 recombinant protein, and conducted a pull-down assay of RANBP2 and COX11. RESULTS: The amount of COX11 bound to mutated RANBP2 was significantly smaller than that bound to the wild-type RANBP2. CONCLUSION: Mutated RANBP2 had an attenuated binding ability to COX11. Whether this change indeed decreases ATP production remains to be further explored.

Expression of cardiac copper chaperone encoding genes and their correlation with cardiac function parameters in goats.

Copper deficiency (CuD) is a common cause of oxidative cardiac tissue damage in ruminants. The expression of copper chaperone (Cu-Ch) encoding genes enables an in-depth understanding of copper-associated disorders, but no previous studies have been undertaken to highlight Cu-Ch disturbances in heart tissue in ruminants due to CuD. The current study aimed to investigate the Cu-Ch mRNA expression in the heart of goats after experimental CuD and highlight their relationship with the cardiac measurements. Eleven male goats were enrolled in this study and divided into the control group (n = 4) and CuD group (n = 7), which received copper-reducing dietary regimes for 7 months. Heart function was evaluated by electrocardiography and echocardiography, and at the end of the experiment, all animals were sacrificed and the cardiac tissues were collected for histopathology and quantitative mRNA expression by real-time PCR. In the treatment group, cardiac measurements revealed increased preload and the existence of cardiac dilatation, and significant cardiac tissue damage by histopathology. Also, the relative mRNA expression of Cu-Ch encoding genes; ATP7A, CTr1, LOX, COX17, as well as ceruloplasmin (CP), troponin I3 (TNNI3), glutathione peroxidase (GPX1), and matrix metalloprotease inhibitor (MMPI1) genes were significantly down-regulated in CuD group. There was a significant correlation between investigated genes and some cardiac function measurements; meanwhile, a significant inverse correlation was observed between histopathological score and ATP7B, CTr1, LOX, and COX17. In conclusion, this study revealed that CuD induces cardiac dilatation and alters the mRNA expression of Cu-Ch genes, in addition to TNNI3, GPX1, and MMPI1 that are considered key factors in clinically undetectable CuD-induced cardiac damage in goats which necessitate further studies for feasibility as biomarkers.

The Copper Chaperone NosL Forms a Heterometal Site for Cu Delivery to Nitrous Oxide Reductase.

The final step of denitrification is the reduction of nitrous oxide (N2 O) to N2 , mediated by Cu-dependent nitrous oxide reductase (N2 OR). Its metal centers, CuA and CuZ , are assembled through sequential provision of twelve CuI ions by a metallochaperone that forms part of a nos gene cluster encoding the enzyme and its accessory factors. The chaperone is the nosL gene product, an 18 kDa lipoprotein predicted to reside in the outer membrane of Gram-negative bacteria. In order to better understand the assembly of N2 OR, we have produced NosL from Shewanella denitrificans and determined the structure of the metal-loaded chaperone by X-ray crystallography. The protein assembled a heterodinuclear metal site consisting of ZnII and CuI , as evidenced by anomalous X-ray scattering. While only CuI is delivered to the enzyme, the stabilizing presence of ZnII is essential for the functionality and structural integrity of the chaperone.

High fat suppresses SOD1 activity by reducing copper chaperone for SOD1 associated with neurodegeneration and memory decline.

High fat consumption leads to reactive oxygen species (ROS) which is associated with age-progressive neurological disorders. Cu/Zn superoxide dismutase (SOD1) is a critical enzyme against ROS. However, the relationship between SOD1 and the high-fat-induced ROS and neurodegeneration is poorly known. Here we showed that, upon treatment with a saturated fatty acid palmitic acid (PA), the SOD1 activity was decreased in mouse neuronal HT-22 cell line accompanied by elevation of ROS, but not in mouse microglial BV-2 cell line. We further showed that PA decreased the levels of copper chaperone for SOD1 (CCS) in HT-22 cells, which promoted the nuclear import of SOD1 and decreased its activity. We demonstrated that the reduction of CCS is involved in the PA-induced decrease of SOD1 activity and elevation of ROS. In addition, compared with the adult mice fed with a standard diet, the high-fat-diet adult mice presented an increase of plasma free fatty acids, reduction of hippocampal SOD1 activity and CCS, mitochondrial degeneration and long-term memory decline. Taken together, our findings suggest that the high-fat-induced lower CCS level is essential for SOD1 suppression which may be associated with neurodegeneration and cognitive decline.

A putative nuclear copper chaperone promotes plant immunity in Arabidopsis.

Copper is essential for many metabolic processes but must be sequestrated by copper chaperones. It is well known that plant copper chaperones regulate various physiological processes. However, the functions of copper chaperones in the plant nucleus remain largely unknown. Here, we identified a putative copper chaperone induced by pathogens (CCP) in Arabidopsis thaliana. CCP harbors a classical MXCXXC copper-binding site (CBS) at its N-terminus and a nuclear localization signal (NLS) at its C-terminus. CCP mainly formed nuclear speckles in the plant nucleus, which requires the NLS and CBS domains. Overexpression of CCP induced PR1 expression and enhanced resistance against Pseudomonas syringae pv. tomato DC3000 compared with Col-0 plants. Conversely, two CRISPR/Cas9-mediated ccp mutants were impaired in plant immunity. Further biochemical analyses revealed that CCP interacted with the transcription factor TGA2 in vivo and in vitro. Moreover, CCP recruits TGA2 to the PR1 promoter sequences in vivo, which induces defense gene expression and plant immunity. Collectively, our results have identified a putative nuclear copper chaperone required for plant immunity and provided evidence for a potential function of copper in the salicylic pathway.

Oxidative stress and apoptotic effects of copper and cadmium in the zebrafish liver cell line ZFL.

Copper (Cu) and cadmium (Cd) are widely used in industrial activities, resulting in Cu and Cd contamination in aquatic systems worldwide. Although Cu plays an essential role in many biological functions, an excessive amount of the metal causes cytotoxicity. In contrast, Cd is a non-essential metal that usually co-exists with Cu. Together, they cause oxidative stress in cells, leading to cell damage. These metal ions are also believed to cause cell apoptosis. In this study, we used a zebrafish liver cell line, ZFL, to study combined Cu and Cd cytotoxicity. Although Cd is more toxic than Cu, both were found to regulate the expression of oxidative stress related genes, and neither significantly altered the activity of oxidative stress related enzymes. Co-exposure tests with the antioxidant N-acetyl-l-cysteine and the Cu chelator bathocuproinedisulfonic acid disodium salt demonstrated that Cd toxicity was due to the oxidative stress caused by Cu, and that Cu at a low concentration could in fact exert an antioxidant effect against the oxidative stress in ZFL. Excessive Cu concentration triggered the expression of initiator caspases (caspase 8 and caspase 9) but suppressed that of an executioner caspase (caspase 3), halting apoptosis. Cd could only trigger the expression of initiator caspases; it could not halt apoptosis. However, a low concentration of Cu reduced the mitochondrial superoxide level, suppressing the Cd-induced apoptotic effects in ZFL.

Novel insights into the transfer routes of the essential copper cofactor to the ethylene plant hormone receptor family.

The plant hormone ethylene is a key regulator of growth, development and stress adaptation at all stages of the plant life cycle. Signal perception and response to the plant hormone are mediated by a family of receptor kinases localized at the ER-Golgi network which gain their high affinity and specificity for the chemically simple ethylene molecule by an essential copper cofactor bound at their transmembrane domain. Transfer of this cofactor from the plant plasma membrane to the ER-localized receptors requires secured cellular transport of the reactive transition metal. In a recent study, we disclosed the transport proteins involved in the copper transfer to the receptors and identified that cytoplasmic chaperones of the ATX1-family and a membrane-bound P-type ATPase are involved in copper routing. Strictly speaking, our data show that receptors can acquire their copper load by different routes and adopt the metal ion from the plasma membrane either by sequential transfer from soluble chaperones of the ATX1-family via the ER-bound copper-transporting ATPase RAN1 or by direct transfer from the soluble chaperones. Here, we have studied the properties of the soluble plant copper chaperone isoforms, ATX1 and CCH, in more detail. Our data support different cellular functions of these isoforms on copper mobilization.

PCuAC domains from methane-oxidizing bacteria use a histidine brace to bind copper.

Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is copper-dependent. In addition to pMMO, many other copper proteins are encoded in the genomes of methanotrophs, including proteins that contain periplasmic copper-Achaperone (PCuAC) domains. Using bioinformatics analyses, we identified three distinct classes of PCuAC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCuAC domains from other types of bacteria bind a single Cu(I) ion via an HXnMX21/22HXM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCuAC domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCuAC domain. Crystal structures of the PmoF1 and PmoF2 PCuAC domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HX10H motif. This binding site is distinct from those of previously characterized PCuAC domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCuAC family reveals previously unappreciated diversity, including sequences that contain both the HXnMX21/22HXM and HX10H motifs, and sequences that lack either set of copper-binding ligands. These findings provide the first characterization of an additional class of copper proteins from methanotrophs, further expand the PCuAC family, and afford new insight into the biological significance of histidine brace-mediated copper coordination.

Copper-zinc superoxide dismutase (Sod1) activation terminates interaction between its copper chaperone (Ccs) and the cytosolic metal-binding domain of the copper importer Ctr1.

Copper-zinc superoxide dismutase (Sod1) is a critical antioxidant enzyme that rids the cell of reactive oxygen through the redox cycling of a catalytic copper ion provided by its copper chaperone (Ccs). Ccs must first acquire this copper ion, directly or indirectly, from the influx copper transporter, Ctr1. The three proteins of this transport pathway ensure careful trafficking of copper ions from cell entry to target delivery, but the intricacies remain undefined. Biochemical examination of each step in the pathway determined that the activation of the target (Sod1) regulates the Ccs·Ctr1 interaction. Ccs stably interacts with the cytosolic C-terminal tail of Ctr1 (Ctr1c) in a copper-dependent manner. This interaction becomes tripartite upon the addition of an engineered immature form of Sod1 creating a stable Cu(I)-Ctr1c·Ccs·Sod1 heterotrimer in solution. This heterotrimer can also be made by the addition of a preformed Sod1·Ccs heterodimer to Cu(I)-Ctr1c, suggestive of multiple routes to the same destination. Only complete Sod1 activation (i.e. active site copper delivery and intra-subunit disulfide bond formation) breaks the Sod1·Ccs·Ctr1c complex. The results provide a new and extended view of the Sod1 activation pathway(s) originating at cellular copper import.

The effect of maternal iron deficiency on zinc and copper levels and on genes of zinc and copper metabolism during pregnancy in the rat.

Fe deficiency is relatively common in pregnancy and has both short- and long-term consequences. However, little is known about the effect on the metabolism of other micronutrients. A total of fifty-four female rats were fed control (50 mg Fe/kg) or Fe-deficient diets (7·5 mg/kg) before and during pregnancy. Maternal liver, placenta and fetal liver were collected at day 21 of pregnancy for Cu and Zn analysis and to measure expression of the major genes of Cu and Zn metabolism. Cu levels increased in the maternal liver (P=0·002) and placenta (P=0·018) of Fe-deficient rats. Zn increased (P<0·0001) and Cu decreased (P=0·006) in the fetal liver. Hepatic expression of the Cu chaperones antioxidant 1 Cu chaperone (P=0·042) and cytochrome c oxidase Cu chaperone (COX17, P=0·020) decreased in the Fe-deficient dams, while the expression of the genes of Zn metabolism was unaltered. In the placenta, Fe deficiency reduced the expression of the chaperone for superoxide dismutase 1, Cu chaperone for superoxide dismutase (P=0·030), ceruloplasmin (P=0·042) and Zn transport genes, ZRT/IRT-like protein 4 (ZIP4, P=0·047) and Zn transporter 1 (ZnT1, P=0·012). In fetal liver, Fe deficiency increased COX17 (P=0·020), ZRT/IRT-like protein 14 (P=0·036) and ZnT1 (P=0·0003) and decreased ZIP4 (P=0·004). The results demonstrate that Fe deficiency during pregnancy has opposite effects on Cu and Zn levels in the fetal liver. This may, in turn, alter metabolism of these nutrients, with consequences for development in the fetus and the neonate.