A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

The Crossroads between Host Copper Metabolism and Influenza Infection.

Three main approaches are used to combat severe viral respiratory infections. The first is preemptive vaccination that blocks infection. Weakened or dead viral particles, as well as genetic constructs carrying viral proteins or information about them, are used as an antigen. However, the viral genome is very evolutionary labile and changes continuously. Second, chemical agents are used during infection and inhibit the function of a number of viral proteins. However, these drugs lose their effectiveness because the virus can rapidly acquire resistance to them. The third is the search for points in the host metabolism the effect on which would suppress the replication of the virus but would not have a significant effect on the metabolism of the host. Here, we consider the possibility of using the copper metabolic system as a target to reduce the severity of influenza infection. This is facilitated by the fact that, in mammals, copper status can be rapidly reduced by silver nanoparticles and restored after their cancellation.

Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis.

BACKGROUND & AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD. METHODS: We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy. RESULTS: After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.

In vivo effect of copper status on cisplatin-induced nephrotoxicity.

Cisplatin is a widely used antitumor agent; however, tumor resistance and severe side effects limit its use. It is well accepted that cisplatin toxicity can be modulated in vitro in cell cultures by copper salts. In the present work, mice with different blood serum copper status were treated with a single intraperitoneal cisplatin injection at a dose of 5 mg/kg, monitored for 3 days in metabolic cages and analyzed for renal function. Both copper-deficient and copper-overloaded mice displayed more severe early proteinuria and retarded platinum excretion than control mice. The effects of copper status on cisplatin-induced nephrotoxicity are discussed.

Chemical background of silver nanoparticles interfering with mammalian copper metabolism.

The rapidly increasing application of silver nanoparticles (AgNPs) boosts their release into the environment, which raises a reasonable alarm for ecologists and health specialists. This is manifested as increased research devoted to the influence of AgNPs on physiological and cellular processes in various model systems, including mammals. The topic of the present paper is the ability of silver to interfere with copper metabolism, the potential health effects of this interference, and the danger of low silver concentrations to humans. The chemical properties of ionic and nanoparticle silver, supporting the possibility of silver release by AgNPs in extracellular and intracellular compartments of mammals, are discussed. The possibility of justified use of silver for the treatment of some severe diseases, including tumors and viral infections, based on the specific molecular mechanisms of the decrease in copper status by silver ions released from AgNPs is also discussed.

Abnormalities in Copper Status Associated with an Elevated Risk of Parkinson's Phenotype Development.

In the last 15 years, among the many reasons given for the development of idiopathic forms of Parkinson's disease (PD), copper imbalance has been identified as a factor, and PD is often referred to as a copper-mediated disorder. More than 640 papers have been devoted to the relationship between PD and copper status in the blood, which include the following markers: total copper concentration, enzymatic ceruloplasmin (Cp) concentration, Cp protein level, and non-ceruloplasmin copper level. Most studies measure only one of these markers. Therefore, the existence of a correlation between copper status and the development of PD is still debated. Based on data from the published literature, meta-analysis, and our own research, it is clear that there is a connection between the development of PD symptoms and the number of copper atoms, which are weakly associated with the ceruloplasmin molecule. In this work, the link between the risk of developing PD and various inborn errors related to copper metabolism, leading to decreased levels of oxidase ceruloplasmin in the circulation and cerebrospinal fluid, is discussed.

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.

Influence of Silver Nanoparticles on the Growth of Ascitic and Solid Ehrlich Adenocarcinoma: Focus on Copper Metabolism.

The link between copper metabolism and tumor progression motivated us to use copper chelators for suppression of tumor growth. We assume that silver nanoparticles (AgNPs) can be used for lowering bioavailable copper. Our assumption is based on the ability of Ag(I) ions released by AgNPs in biological media and interfere with Cu(I) transport. Intervention of Ag(I) into copper metabolism leads to the replacement of copper by silver in ceruloplasmin and the decrease in bioavailable copper in the bloodstream. To check this assumption, mice with ascitic or solid Ehrlich adenocarcinoma (EAC) were treated with AgNPs using different protocols. Copper status indexes (copper concentration, ceruloplasmin protein level, and oxidase activity) were monitored to assess copper metabolism. The expression of copper-related genes was determined by real-time PCR in the liver and tumors, and copper and silver levels were measured by FAAS. Intraperitoneal AgNPs treatment beginning on the day of tumor inoculation enhanced mice survival, reduced the proliferation of ascitic EAC cells, and suppressed the activity of HIF1α, TNF-α and VEGFa genes. Topical treatment by the AgNPs, which was started together with the implantation of EAC cells in the thigh, also enhanced mice survival, decreased tumor growth, and repressed genes responsible for neovascularization. The advantages of silver-induced copper deficiency over copper chelators 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.

Synthetic Lethality Screening Identifies FDA-Approved Drugs that Overcome ATP7B-Mediated Tolerance of Tumor Cells to Cisplatin.

Tumor resistance to chemotherapy represents an important challenge in modern oncology. Although platinum (Pt)-based drugs have demonstrated excellent therapeutic potential, their effectiveness in a wide range of tumors is limited by the development of resistance mechanisms. One of these mechanisms includes increased cisplatin sequestration/efflux by the copper-transporting ATPase, ATP7B. However, targeting ATP7B to reduce Pt tolerance in tumors could represent a serious risk because suppression of ATP7B might compromise copper homeostasis, as happens in Wilson disease. To circumvent ATP7B-mediated Pt tolerance we employed a high-throughput screen (HTS) of an FDA/EMA-approved drug library to detect safe therapeutic molecules that promote cisplatin toxicity in the IGROV-CP20 ovarian carcinoma cells, whose resistance significantly relies on ATP7B. Using a synthetic lethality approach, we identified and validated three hits (Tranilast, Telmisartan, and Amphotericin B) that reduced cisplatin resistance. All three drugs induced Pt-mediated DNA damage and inhibited either expression or trafficking of ATP7B in a tumor-specific manner. Global transcriptome analyses showed that Tranilast and Amphotericin B affect expression of genes operating in several pathways that confer tolerance to cisplatin. In the case of Tranilast, these comprised key Pt-transporting proteins, including ATOX1, whose suppression affected ability of ATP7B to traffic in response to cisplatin. In summary, our findings reveal Tranilast, Telmisartan, and Amphotericin B as effective drugs that selectively promote cisplatin toxicity in Pt-resistant ovarian cancer cells and underscore the efficiency of HTS strategy for identification of biosafe compounds, which might be rapidly repurposed to overcome resistance of tumors to Pt-based chemotherapy.

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.

Silver Ions as a Tool for Understanding Different Aspects of Copper Metabolism.

In humans, copper is an important micronutrient because it is a cofactor of ubiquitous and brain-specific cuproenzymes, as well as a secondary messenger. Failure of the mechanisms supporting copper balance leads to the development of neurodegenerative, oncological, and other severe disorders, whose treatment requires a detailed understanding of copper metabolism. In the body, bioavailable copper exists in two stable oxidation states, Cu(I) and Cu(II), both of which are highly toxic. The toxicity of copper ions is usually overcome by coordinating them with a wide range of ligands. These include the active cuproenzyme centers, copper-binding protein motifs to ensure the safe delivery of copper to its physiological location, and participants in the Cu(I) ↔ Cu(II) redox cycle, in which cellular copper is stored. The use of modern experimental approaches has allowed the overall picture of copper turnover in the cells and the organism to be clarified. However, many aspects of this process remain poorly understood. Some of them can be found out using abiogenic silver ions (Ag(I)), which are isoelectronic to Cu(I). This review covers the physicochemical principles of the ability of Ag(I) to substitute for copper ions in transport proteins and cuproenzyme active sites, the effectiveness of using Ag(I) to study copper routes in the cells and the body, and the limitations associated with Ag(I) remaining stable in only one oxidation state. The use of Ag(I) to restrict copper transport to tumors and the consequences of large-scale use of silver nanoparticles for human health are also discussed.

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.

Copper Metabolism of Newborns Is Adapted to Milk Ceruloplasmin as a Nutritive Source of Copper: Overview of the Current Data.

Copper, which can potentially be a highly toxic agent, is an essential nutrient due to its role as a cofactor for cuproenzymes and its participation in signaling pathways. In mammals, the liver is a central organ that controls copper turnover throughout the body, including copper absorption, distribution, and excretion. In ontogenesis, there are two types of copper metabolism, embryonic and adult, which maintain the balance of copper in each of these periods of life, respectively. In the liver cells, these types of metabolism are characterized by the specific expression patterns and activity levels of the genes encoding ceruloplasmin, which is the main extracellular ferroxidase and copper transporter, and the proteins mediating ceruloplasmin metalation. In newborns, the molecular genetic mechanisms responsible for copper homeostasis and the ontogenetic switch from embryonic to adult copper metabolism are highly adapted to milk ceruloplasmin as a dietary source of copper. In the mammary gland cells, the level of ceruloplasmin gene expression and the alternative splicing of its pre-mRNA govern the amount of ceruloplasmin in the milk, and thus, the amount of copper absorbed by a newborn is controlled. In newborns, the absorption, distribution, and accumulation of copper are adapted to milk ceruloplasmin. If newborns are not breast-fed in the early stages of postnatal development, they do not have this natural control ensuring alimentary copper balance in the body. Although there is still much to be learned about the neonatal consequences of having an imbalance of copper in the mother/newborn system, the time to pay attention to this problem has arrived because the neonatal misbalance of copper may provoke the development of copper-related disorders.

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 role of subcutaneous adipose tissue in supporting the copper balance in rats with a chronic deficiency in holo-ceruloplasmin.

We have previously shown that (1) an acute deficiency in blood serum holo-ceruloplasmin (Cp) developed in rats that were fed fodder containing silver ions (Ag-fodder) for one month and (2) the deficiency in holo-Cp was compensated by non-hepatic holo-Cp synthesis in rats that were chronically fed Ag-fodder for 6 months (Ag-rats). The purpose of the present study is to identify the organ(s) that compensate for the hepatic holo-Cp deficiency in the circulation. This study was performed on rats that were fed Ag-fodder (40 mg Ag·kg-1 body mass daily) for 6 months. The relative expression levels of the genes responsible for copper status were measured by RT-PCR. The in vitro synthesis and secretion of [14C]Cp were analyzed using a metabolic labeling approach. Oxidase activity was determined using a gel assay with o-dianisidine. Copper status and some hematological indexes were measured. Differential centrifugation, immunoblotting, immunoelectrophoresis, and atomic absorption spectrometry were included in the investigation. In the Ag-rats, silver accumulation was tissue-specific. Skeletal muscles and internal (IAT) and subcutaneous (SAT) adipose tissues did not accumulate silver significantly. In SAT, the mRNAs for the soluble and glycosylphosphatidylinositol-anchored ceruloplasmin isoforms were expressed, and their relative levels were increased two-fold in the Ag-rats. In parallel, the levels of the genes responsible for Cp metallation (Ctr1 and Atp7a/b) increased correspondingly. In the SAT of the Ag-rats, Cp oxidase activity was observed in the Golgi complex and plasma membrane. Moreover, full-length [14C]Cp polypeptides were released into the medium by slices of SAT. The possibilities that SAT is part of a system that controls the copper balance in mammals, and it plays a significant role in supporting copper homeostasis throughout the body are discussed.

Biogenetic and morphofunctional heterogeneity of mitochondria: the case of synaptic mitochondria.

The mitochondria of different cells are different in their morphological and biochemical properties. These organelles generate free radicals during activity, leading inevitably to mitochondrial DNA damage. It is not clear how this problem is addressed in long-lived cells, such as neurons. We propose the hypothesis that mitochondria within the same cell also differ in lifespan and ability to divide. According to our suggestion, cells have a pool of 'stem' mitochondria with low metabolic activity and a pool of 'differentiated' mitochondria with significantly shorter lifespans and high metabolic activity. We consider synaptic mitochondria as a possible example of 'differentiated' mitochondria. They are significantly smaller than mitochondria from the cell body, and they are different in key enzyme activity levels, proteome, and lipidome. Synaptic mitochondria are more sensitive to different damaging factors. It has been established that neurons have a sorting mechanism that sends mitochondria with high membrane potential to presynaptic endings. This review describes the properties of synaptic mitochondria and their role in the regulation of synaptic transmission.

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.

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.