Copper chaperones: personal escorts for metal ions

Copper serves as the essential cofactor for a number of enzymes involved in redox chemistry and virtually all organisms must accumulate trace levels of copper in order to survive. However, this metal can also be toxic and a number of effective methods for sequestering and detoxifying copper prevent the metal from freely circulating inside a cell. Copper metalloenzymes are therefore faced with the challenge of acquiring their precious metal cofactor in the absence of available copper. To overcome this dilemma, all eukaryotic organisms have evolved with a family of intracellular copper binding proteins that help reserve a bioavailable pool of copper for the metalloenzymes, escort the metal to appropriate targets, and directly transfer the copper ion. These proteins have been collectively called copper chaperones. The identification of such molecules has been made possible through molecular genetic studies in the bakers' yeast Saccharomyces cerevisiae. In this review, we highlight the findings that led to a new paradigm of intracellular trafficking of copper involving the action of copper chaperones. In particular, emphasis will be placed on the ATX1 and CCS copper chaperones that act to deliver copper to the secretory pathway and to Cu/Zn superoxide dismutase in the cytosol, respectively.     

Fluorescent Protein-Based Optical Biosensor for Copper Ion Quantitation

In the present study, spectroscopic determinations of copper ions using chimeric metal-binding green fluorescent protein (His6GFP) as an active indicator have been explored. Supplementation of copper ions to the GFP solution led to a remarkable decrease of fluorescent intensity corresponding to metal concentrations. For circumstances, rapid declining of fluorescence up to 60% was detected in the presence of 500 μM copper. This is in contrast to those observed in the case of zinc and calcium ions, in which approximately 10–20% of fluorescence was affected. Recovery of its original fluorescence up to 80% was mediated by the addition of ethylenediamine tetraacetic acid. More importantly, in the presence of metal ions, the emission wavelength maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed. This indicates that the chromophore’s ground state was possibly affected by the static quenching process. Results from circular dichroism measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly different from that of the control, where the majority of sharp positive band around 195–196 nm in combination with a broad negative deflection around 215–216 nm was obtained. Taken together, it can be presumed that copper ions exerted their static quenching on the fluorescence rather than structural or conformational alteration. However, notification has to be made that some peptide rearrangements may also occur in the presence of metal ions. Further studies were conducted to investigate the feasibility of using the His6GFP as a sensing unit for copper ions. The His6GFP was encapsulated in Sol-gel and immobilized onto the optical fiber connected with a fluorescence detecting device. The Sol-gel was doped into the metal solution where the quenching of fluorescence could be monitored in real time. The sensing unit provided a high sensitivity of detection in the range of 0.5 μM to 50 mM with high selectivity for copper ions. All these findings open up a high potential to apply the fluorescent protein-based bioanalytical tool for copper determination in the future.     

Copper complex-assisted DNA hybridization

2,2'-Bipyridine (bpy) or 1,10-phenanthroline (phen) metal-binding domains were covalently attached to oligonucleotides, and the influence of metal ions on the hybridization of the conjugates was investigated. Metal-binding domains were attached to oligonucleotides at 3'- and 5'-terminal positions, thus placing them in juxtaposed positions after hybridization to a common target strand. While the ligands alone had a positive effect (increased Tm) on hybrid stability, the duplex was further stabilized by the addition of copper(I) and/or copper(II) through the formation of a metal complex in which the two short sequences are linked through {Cu(bpy)2}, {Cu(phen)}, or {Cu(bpy)(phen)} domains. The increase in Tm, due to formation of the {Cu(bpy)2}, {Cu(phen)2}, {Cu(bpy)(phen)} motifs is reversed upon addition of EDTA, consistent with the stripping of copper from the ligands. The effect of metal complex formation on the duplex strength was shown to be highest if the two metal-coordinating ligand strands are placed as close to each other as possible.     

Screening of Tropical Wood-Rotting Mushrooms for Copper Biosorption

Fruiting bodies (mushrooms) of nine nonedible macrofungi were screened for copper(II) uptake potential. The maximum uptake potentials (Q(infmax)s) derived from equilibrium studies indicated that all nine species exhibited higher Q(infmax)s at pH 4.0 than that of Filtrasorb-400, a generally used adsorbent for metal removal. Wide variation in Q(infmax) was observed among the species and ranged from 0.048 to 0.383 mmol per g of sorbent. The uptake capacity of Ganoderma lucidum, which exhibited the highest Q(infmax), was higher than those of other microbial biosorbents reported in the literature.     

Copper proteins immobilised on gold electrodes for (bio)analytical studies

Copper electrochemistry at modified gold electrodes was investigated with two different states of the metal ion: first bound in azurin from Pseudomonas aeruginosa and second introduced via metal ion uptake in metallothionein (MT) from rabbit liver. Azurin was immobilised on a mercaptosuccinic acid (MSA) layer self-assembled on gold. The redox behaviour in the adsorbed as well as in the covalently immobilised state was found to be quasi-reversible with a formal potential of +198 mV versus Ag/AgCl. The pH variation suggests an optimal pH range for efficient electrode communication in the neutral range. MT was fixed at electrochemically cleaned gold using the accessible cysteins of the protein. Copper was found to bind to the MT-modified gold electrode. The electrochemical behaviour of the bound copper was characterised in copper-free solution with a formal potential of +245 mV versus Ag/AgCl. Stability and potential use is discussed.     

Copper accumulation byAspergillus awamori

Aspergillus awamori accumulated Cu2+ from aqueous solutions. The level of copper uptake was dependent on the ambient metal concentration. The process consisted of two phases: a fast initial phase and a slower secondary phase. Chelation of these ions occurs by chemical, equilibrated and saturable mechanism, following the mathematical models of Langmuir and Freundlich, with better performance on the Langmuir model. Data transformation allowed us to calculate the kinetic constants of the sorption reaction.     

Explanatory Variables for per Capita Stocks and Flows of Copper and Zinc

A number of potential explanatory variables for the stocks and flows of copper and zinc in contemporary technological societies are co-analyzed with the tools of exploratory data analysis. A one-year analysis (circa 1994) is performed for 50 countries that comprise essentially all anthropogenic stocks and flows of the two metals. The results show that (1) The key explanatory variable for metal use is gross domestic product (GDP) per capita (purchasing power parity, PPP). By itself, GDP explains between one-third and one-half of the variance of per capita copper and zinc use. Other variables that were significantly correlated with copper and zinc use included stock of passenger cars and television sets (per 1, 000 people); two infrastructure variables, wired telephone connections, urban population, and value added inmanufacturing. The results do not provide evidence supporting the Kuznets curve hypothesis for these metals. (2) Metal use per capita can be estimated using multiple regression equations. For copper, the natural logarithm of use is related to the explanatory variables GDP (PPP), value added in manufacturing, and urban population. This model explains 80% of the variance among the different countries (r²= 0.79). The natural logarithm of zinc use is related to GDP (PPP) and value added in manufacturing with an r² of 0.75; (3) For both metals, rates of metal fabrication, use, net addition to stock, and discard in low-and high-income countries differ significantly from each other. Our statistical analyses thus provide a basis for estimating the potential development of metal use, net addition to stock, and discard, using data on explanatory variables that are available at the international level.     

Copper II complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin

A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (+/-0.3) x10(3) M(-1). It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.     

Copper resistance mechanisms in bacteria and fungi

Abstract: Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are related to reduced copper transport, enhanced effiux of cupric ions, or copper complexation by cell components. Copper tolerance in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.     

Copper and prion diseases

Transmissible spongiform encephalopathies are diseases of animals and humans that are also termed prion diseases. These diseases are linked together because a normal brain glycoprotein termed the prion protein is converted to a readily detectable protease-resistant isoform. There is now strong evidence to suggest that apart from this difference in resistance a major difference between the isoforms is that the normal prion protein binds copper and has an anti-oxidant function. Brains from Creutzfeldt-Jakob disease patients and brains from mice with experimental mouse scrapie have been shown to have changes in the levels of both copper and manganese. There is growing evidence that links prion diseases to disturbances of metal metabolism.     

Copper (II) interaction with proline-containing tetrapeptides

The Cu(II) interactions with four tetrapeptides: Ala-Ala-Ala-Ala, Ala-Ala-Ala-Pro, Ala-Ala-Pro-Ala, and Pro-Ala-Ala-Ala were studied by the absorption, circular dichroism, and electron paramagnetic resonance spectra. The results clearly show that proline residue is a specific structural factor in the formed complexes and, on the other hand, it is a break point in the metal ion coordination to the consecutive peptide bond nitrogens. The only position of proline residue ina peptide sequence that makes proline nitrogen available for the metal ion coordination is the N-terminal position. But even in this case (i.e., in the Cu(II) Pro-Ala-Ala-Ala system) proline plays a critical role in the creation of the specific structures in the complex formed in solution.     

Copper Resistance and Accumulation in the Zygomycete Mucor rouxii

Two copper-resistant (Cop^r) mutants, strains P1 and P3, were obtained from the dimorphic fungus Mucor rouxii. They were characterized as to their ability to take up copper in a growth medium supplemented with this metal ion. Detection of copper by linear sweep striping voltammetry in cell walls and in the cell wall-free fraction of disrupted cells revealed a higher content of the metal in both mutant fractions, as compared with those of the copper-sensitive (Cop^s) parental strain. Copper binding by M. rouxii growing cells was also studied through the use of a cytochemical method based on the compounds neocuproine (NCP) and sodium diethyldithiocarbamate (DTC). This method indicated that the P1 Cop^r strain accumulated more metal than the parental Cop^s strain, both on the cellular surface and in the intracellular milieu. Received: 30 January 1996 / Accepted: 7 March 1996     

Copper and the estradiol receptor

Copper is an essential element in living organisms and it appears to be involved in estrogen action. This study bears on the manner in which the metal may be linked to the mechanism of this action. Divalent copper was found to induce at 37 degrees C a several fold increase in estradiol binding to the receptors in rat uterine cytosols. An endogenous substance present in the uterine cytosol and separated from it by fractionation on a hydroxylapatite column was found to function as a potent inhibitor of the copper effect. This substance has been found so far also in human breast tissue and in some human breast tumors.     

Handling of Copper and Copper Oxide Nanoparticles by Astrocytes

Copper is an essential trace element for many important cellular functions. However, excess of copper can impair cellular functions by copper-induced oxidative stress. In brain, astrocytes are considered to play a prominent role in the copper homeostasis. In this short review we summarise the current knowledge on the molecular mechanisms which are involved in the handling of copper by astrocytes. Cultured astrocytes efficiently take up copper ions predominantly by the copper transporter Ctr1 and the divalent metal transporter DMT1. In addition, copper oxide nanoparticles are rapidly accumulated by astrocytes via endocytosis. Cultured astrocytes tolerate moderate increases in intracellular copper contents very well. However, if a given threshold of cellular copper content is exceeded after exposure to copper, accelerated production of reactive oxygen species and compromised cell viability are observed. Upon exposure to sub-toxic concentrations of copper ions or copper oxide nanoparticles, astrocytes increase their copper storage capacity by upregulating the cellular contents of glutathione and metallothioneins. In addition, cultured astrocytes have the capacity to export copper ions which is likely to involve the copper ATPase 7A. The ability of astrocytes to efficiently accumulate, store and export copper ions suggests that astrocytes have a key role in the distribution of copper in brain. Impairment of this astrocytic function may be involved in diseases which are connected with disturbances in brain copper metabolism.     

Comparison of the Adsorption Capabilities of Myriophyllum spicatum and Ceratophyllum demersum for Zinc,Copper and Lead

Industrial wastewaters contain various heavy metal components and therefore threaten aquatic bodies. Heavy metals can be adsorbed by living or non‐living biomass. Submerged aquatic plants can be used for the removal of heavy metals. This paper exhibits the comparison of the adsorption properties of two aquatic plants Myriophyllum spicatum and Ceratophyllum demersum for lead, zinc, and copper. The data obtained from batch studies conformed well to the Langmuir Model. Maximum adsorption capacities (q_max) were obtained for both plant species and each metal. The maximum adsorption capacities (q_max) achieved with M. spicatum were 10.37 mg/g for Cu²⁺, and 15.59 mg/g for Zn²⁺ as well as 46.49 mg/g for Pb²⁺ and with C. demersum they were 6.17 mg/g for Cu²⁺, 13.98 mg/g for Zn²⁺ and 44.8 mg/g for Pb²⁺. It was found that M. spicatum has a better adsorption capacity than C. demersum for each metal tested. Gibbs free energy and the specific surface area based on the q_max values were also determined for each metal.     

Copper and zinc mixtures induce shifts in microbial communities and reduce leaf litter decomposition in streams

1. To assess the impact of metal mixtures on microbial decomposition of leaf litter, we exposed leaves previously immersed in a stream to environmentally realistic concentrations of copper (Cu) and zinc (Zn) (three levels), alone and in all possible combinations. The response of the microbial community was monitored after 10, 25 and 40 days of metal exposure by examining leaf mass loss, fungal and bacterial biomass, fungal reproduction and fungal and bacterial diversity.
2. Analysis of microbial diversity, assessed by denaturing gradient gel electrophoresis and identification of fungal spores, indicated that metal exposure altered the structure of fungal and bacterial communities on decomposing leaves.
3. Exposure to metal mixtures or to the highest Cu concentration significantly reduced leaf decomposition rates and fungal reproduction, but not fungal biomass. Bacterial biomass was strongly inhibited by all metal treatments.
4. The effects of Cu and Zn mixtures on microbial decomposition of leaf litter were mostly additive, because observed effects did not differ from those expected as the sum of single metal effects. However, antagonistic effects on bacterial biomass were found in all metal combinations and on fungal reproduction in metal combinations with the highest Cu concentrations, particularly at longer exposure times.     

Copper(II) complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin

A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (±0.3) ×10³ M⁻¹. It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.     

Copper resistance determinants in bacteria.

Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.     

Copper oxide nanoparticles induce autophagic cell death in a549 cells

Metal oxide nanoparticles (NPs) are among the most highly produced nanomaterials, and have many diverse functions in catalysis, environmental remediation, as sensors, and in the production of personal care products. In this study, the toxicity of several widely used metal oxide NPs such as copper oxide, silica, titanium oxide and ferric oxide NPs, were evaluated In vitro. We exposed A549, H1650 and CNE-2Z cell lines to metal oxide NPs, and found CuO NPs to be the most toxic, SiO2 mild toxic, while the other metal oxide NPs had little effect on cell viability. Furthermore, the autophagic biomarker LC3-II significantly increased in A549 cells treated with CuO NPs, and the use of the autophagy inhibitors wortmannin and 3-methyladenin significantly improved cell survival. These results indicate that the cytoxicity of CuO NPs may involve the autophagic pathway in A549 cells.     

Copper amine oxidases: Current state of knowledge

Summary A brief review on the structure nd functions of the copper-pyrroloquinoline quinone amine oxidases is presented. Data concerning the metal and organic cofactors are reported, giving special emphasis to their structural relationship with the protein molecule and mechanistic properties. Information on the functional role of these enzymes with respect to polyamine metabolism are also given.