Properties of recombinant extracellular N-terminal domain of human high-affinity copper transporter 1 (hNdCTR1) and its interactions with Cu(II) and Ag(I) ions.

High-affinity copper transporter 1 (CTR1) is a key link in the transfer of copper (Cu) from the extracellular environment to the cell. Violation in the control system of its expression, or mutations in this gene, cause a global copper imbalance. However, the mechanism of copper transfer via CTR1 remains unclear. It has been shown that transformed bacteria synthesizing the fused GB1-NdCTR become resistant to toxic silver ions. According to UV-Vis spectrophotometry and isothermal titration calorimetry, electrophoretically pure GB1-NdCTR specifically and reversibly binds copper and silver ions, and binding is associated with aggregation. Purified NdCTR1 forms SDS-resistant oligomers. The link between nontrivial properties of NdCTR1 and copper import mechanism from extracellular space, as well as potential chelating properties of NdCTR1, are discussed.

Anti-Influenza Effect of Nanosilver in a Mouse Model.

The present study assesses copper metabolism of the host organism as a target of antiviral strategy, basing on the "virocell" concept. Silver nanoparticles (AgNPs) were used as a specific active agent because they reduce the level of holo-ceruloplasmin, the main extracellular cuproenzyme. The mouse model of influenza virus A infection was used with two doses: 1 LD50 and 10 LD50. Three treatment regimens were used: Scheme 1-mice were pretreated 4 days before infection and then every day during infection development; Scheme 2-mice were pretreated four days before infection and on the day of virus infection; Scheme 3-virus infection and AgNP treatment started simultaneously, and mice were injected with AgNPs until the end of the experiment. The mice treated by Scheme 1 demonstrated significantly lower mortality, the protection index reached 60-70% at the end of the experiment, and mean lifespan was prolonged. In addition, the treatment of the animals with AgNPs resulted in normalization of the weight dynamics. Despite the amelioration of the infection, AgNP treatment did not influence influenza virus replication. The possibility of using nanosilver as an effective indirectly-acting antiviral drug is discussed.

Size-Dependent Bioactivity of Silver Nanoparticles: Antibacterial Properties, Influence on Copper Status in Mice, and Whole-Body Turnover.

PURPOSE: The ability of silver nanoparticles (AgNPs) of different sizes to influence copper metabolism in mice is assessed. MATERIALS AND METHODS: AgNPs with diameters of 10, 20, and 75 nm were fabricated through a chemical reduction of silver nitrate and characterized by UV/Vis spectrometry, transmission and scanning electronic microscopy, and laser diffractometry. To test their bioactivity, Escherichia coli cells, cultured A549 cells, and C57Bl/6 mice were used. The antibacterial activity of AgNPs was determined by inhibition of colony-forming ability, and cytotoxicity was tested using the MTT test (viability, %). Ceruloplasmin (Cp, the major mammalian extracellular copper-containing protein) concentration and enzymatic activity were measured using gel-assay analyses and WB, respectively. In vitro binding of AgNPs with serum proteins was monitored with UV/Vis spectroscopy. Metal concentrations were measured using atomic absorption spectrometry. RESULTS: The smallest AgNPs displayed the largest dose- and time-dependent antibacterial activity. All nanoparticles inhibited the metabolic activity of A549 cells in accordance with dose and time, but no correlation between cytotoxicity and nanoparticle size was found. Nanosilver was not uniformly distributed through the body of mice intraperitoneally treated with low AgNP concentrations. It was predominantly accumulated in liver. There, nanosilver was included in ceruloplasmin, and Ag-ceruloplasmin with low oxidase activity level was formed. Larger nanoparticles more effectively interfered with the copper metabolism of mice. Large AgNPs quickly induced a drop of blood serum oxidase activity to practically zero, but after cancellation of AgNP treatment, the activity was rapidly restored. A major fraction of the nanosilver was excreted in the bile with Cp. Nanosilver was bound by alpha-2-macroglobulin in vitro and in vivo, but silver did not substitute for the copper atoms of Cp in vitro. CONCLUSION: The data showed that even at low concentrations, AgNPs influence murine copper metabolism in size-dependent manner. This property negatively correlated with the antibacterial activity of AgNPs.

Case of Early-Onset Parkinson's Disease in a Heterozygous Mutation Carrier of the ATP7B Gene.

In this paper, we report a clinically proven case of Parkinson's disease (PD) with early onset in a patient who is a heterozygous mutation carrier of ATP7B (the Wilson's disease gene). The patient was observed from 2011 to 2018 in the Center for Neurodegenerative Diseases, Institute of Experimental Medicine (St. Petersburg, Russia). During this period, the patient displayed aggravation of PD clinical symptoms that were accompanied by a decrease in the ceruloplasmin concentration (from 0.33 to 0.27 g/L) and an increase in serum nonceruloplasmin copper, which are typical of the late stages of Wilson's disease. It was found that one of the alleles of exon 14 in the ATP7B gene, which partially codes of the nucleotide-binding domain (N-domain), carries a mutation not previously reported corresponding to Cys1079Gly substitution. Alignment of the ATP7B N-domain amino acid sequences of representative vertebrate species has shown that the Cys at 1079 position is conserved throughout the evolution. Molecular dynamic analysis of a polypeptide with Cys1079Gly substitution showed that the mutation causes profound conformational changes in the N-domain, which could potentially lead to impairment of its functions. The role of ATP7B gene mutations in PD development is discussed.

CRISP-R/Cas9 Mediated Deletion of Copper Transport Genes CTR1 and DMT1 in NSCLC Cell Line H1299. Biological and Pharmacological Consequences.

Copper, the highly toxic micronutrient, plays two essential roles: it is a catalytic and structural cofactor for Cu-dependent enzymes, and it acts as a secondary messenger. In the cells, copper is imported by CTR1 (high-affinity copper transporter 1), a transmembrane high-affinity copper importer, and DMT1 (divalent metal transporter). In cytosol, enzyme-specific chaperones receive copper from CTR1 C-terminus and deliver it to their apoenzymes. DMT1 cannot be a donor of catalytic copper because it does not have a cytosol domain which is required for copper transfer to the Cu-chaperons that assist the formation of cuproenzymes. Here, we assume that DMT1 can mediate copper way required for a regulatory copper pool. To verify this hypothesis, we used CRISPR/Cas9 to generate H1299 cell line with CTR1 or DMT1 single knockout (KO) and CTR1/DMT1 double knockout (DKO). To confirm KOs of the genes qRT-PCR were used. Two independent clones for each gene were selected for further studies. In CTR1 KO cells, expression of the DMT1 gene was significantly increased and vice versa. In subcellular compartments of the derived cells, copper concentration dropped, however, in nuclei basal level of copper did not change dramatically. CTR1 KO cells, but not DMT1 KO, demonstrated reduced sensitivity to cisplatin and silver ions, the agents that enter the cell through CTR1. Using single CTR1 and DMT1 KO, we were able to show that both, CTR1 and DMT1, provided the formation of vital intracellular cuproenzymes (SOD1, COX), but not secretory ceruloplasmin. The loss of CTR1 resulted in a decrease in the level of COMMD1, XIAP, and NF-κB. Differently, the DMT1 deficiency induced increase of the COMMD1, HIF1α, and XIAP levels. The possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed.

A low blood copper concentration is a co-morbidity burden factor in Parkinson's disease development.

Parkinson's disease (PD) patients are often characterized by copper dyshomeostasis, which is responsible for ROS formation and fibrillogenesis. However, the relationships between copper metabolism and PD development are unclear. In this study in 50 patients with PD (pPD) and 50 age-matched healthy individuals, the serum total copper concentration, oxidase activity, ceruloplasmin and SOD3 protein concentrations were measured; and amount of copper atoms per ceruloplasmin molecule was calculated. These parameters were lower in pPD relatively to healthy volunteers. Decrease in concentrations of SOD3, ceruloplasmin, and copper but increase of interleukin-6 levels were associated with a risk of PD. Two consistent patterns were identified. First, a low serum copper concentration related with PD development and predominantly affected the non-motor symptoms of PD. There was no correlation between copper concentration and ceruloplasmin oxidase activity level (r = 0.27) in pPD. Second, Chelex 100 treatment revealed that pPD ceruloplasmin compared with ceruloplasmin of healthy individuals displayed smaller content of labile copper atoms. The presence or absence of these atoms had no effect on ceruloplasmin enzymatic activities. Our findings suggest that cuproenzyme deficiency, which is typical for PD, can be caused by violation of metabolic incorporation of the labile copper atoms into ceruloplasmin molecule.

The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo.

There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell's copper metabolism and its chelating properties are discussed.

Ceruloplasmin gene expression profile changes in the rat mammary gland during pregnancy, lactation and involution.

Copper metabolism disturbances in mammary gland (MG) cells have severe consequences in newborns. The mechanism that controls the balance of copper in the MG has not been thoroughly characterized. Four primary copper homeostasis genes in mammals: (1) ceruloplasmin (Cp) encoding multifunction multicopper blue (ferr)oxidase; (2) CTR1 encoding high affinity copper importer 1; and (3 and 4) two similar genes encoding Cu(I)/Cu(II)-ATPases P1 type (ATP7A and ATP7B) responsible for copper efflux from the cells and metallation of cuproenzymes formed in the Golgi complex are expressed in MG. This study aimed to characterize expression of these genes during pregnancy, lactation and forced involution in the rat MG. We found that Cp anchored to the plasma membrane and ATP7A were expressed during pregnancy and lactation. Soluble Cp and ATP7B were highly expressed in lactating MG decreasing to its ending. CTR1 activity increased during MG growth and reached its maximum at postpartum and then it decreased until the end of lactation. During early forced MG involution, Cp gene expression persisted; while a form of Cp that lacked exon 18 appeared. We suggest that Cp gene expressional changes at the transcriptional and posttranscriptional level reflect various physiological functions of Cp proteins during MG remodeling.

New silver nanoparticles induce apoptosis-like process in E. coli and interfere with mammalian copper metabolism.

Silver nanoparticles (SNPs) are new functional materials that are widely used in biomedical and industrial technologies. Two main features that make SNPs valuable are their strong antibacterial effects and low toxicity to eukaryotes. In this study, SNPs were synthesized using a modified method of reducing the metal ions to their atomic state followed by crystallization. SNPs were characterized by UV/vis spectroscopy, X-ray diffractometry, atomic force microscopy, and transmission electron microscopy (TEM). The SNPs were spherically shaped with an average linear dimension of 20 nm. In aqueous solution, the SNPs were beige-yellow in color, and they formed a black color in bacteria-rich growth media. The toxicity and bioavailability of the SNPs were tested using Escherichia coli cells and C57Bl/6 mice. Although the SNPs displayed bactericidal activity, an E. coli cell strain transformed with an expression plasmid carrying a human CTR1 ectodomain with three motives that bind Cu(II), Cu(I), and Ag(I) demonstrated increased resistance to treatment with SNPs. TEM showed that the SNPs were absorbed by the E. coli cell, and flow cytometry showed that the SNPs induced apoptosis-like death. In mice treated with SNPs (daily intraperitoneal injection of 10 µg SNPs/g body weight over 4 days), the ceruloplasmin (Cp) oxidase activity in the blood serum decreased. However, level of Cp gene expression, the relative contents of the Cp protein in the Golgi complex and in the serum did not change. Treatment with SNPs did not influence the activity of superoxide dismutase 1 in the liver and had no apparent toxic effects in mice. These findings expand the scope of application for the use of new SNPs. The data are discussed in a paradigm, in which the effects of SNPs are caused by the interference of silver ions with copper metabolism.