A single PDZ domain protein interacts with the menkes copper ATPase, ATP7A : a new protein implicated in copper homeostasis

The homeostatic regulation of essential elements such as copper requires many proteins whose activities are often mediated and tightly coordinated through protein-protein interactions. This regulation ensures that cells receive enough copper without intracellular concentrations reaching toxic levels. To date, only a small number of proteins implicated in copper homeostasis have been identified, and little is known of the protein-protein interactions required for this process. To identify other proteins important for copper homeostasis, while also elucidating the protein-protein interactions that are integral to the process, we have utilized a known copper protein, the copper ATPase ATP7A, as a bait in a yeast two-hybrid screen of a human cDNA library to search for interacting partners. One of the ATP7A-interacting proteins identified is a novel protein with a single PDZ domain. This protein was recently identified to interact with the plasma membrane calcium ATPase b-splice variants. We propose a change in name for this protein from PISP (plasma membrane calcium ATPase-interacting single-PDZ protein) to AIPP1 (ATPase-interacting PDZ protein) and suggest that it represents the protein that interacts with the class I PDZ binding motif identified at the ATP7A C terminus. The interaction in mammalian cells was confirmed and an additional splice variant of AIPP1 was identified. This study represents an essential step forward in identifying the proteins and elucidating the network of protein-protein interactions involved in maintaining copper homeostasis and validates the use of the yeast two-hybrid approach for this purpose.

Extraction of silver(i) from aqueous solutions in the absence and presence of copper(ii) with a methimazole-based ionic liquid

The ionic liquid (IL) 2-butylthiolonium bis(trifluoromethanesulfonyl)amide, [mimSBu][NTf₂], facilitates the efficient extraction of silver(i) from aqueous media via interaction with both the cation and anion components of the IL. Studies with a conventional aqueous-IL two phase system as well as microextraction of silver(i) by a thick IL film adhered to an electrode monitored in situ by cyclic voltammetry, established that [mimSBu][NTf₂] can extract electroactive silver(i) ions from an aqueous solution. The pH of the aqueous phase decreases upon addition of [mimSBu]⁺, which is attributed to partial release of the hydrogen attached to the N(3) nitrogen atom of the imidazolium ring. The presence of silver(i) further increase the acidity of the aqueous phase as a consequence of coordination with the IL cation component. Voltammetric and ¹H and ¹³C NMR techniques have been used to establish the nature of the silver(i) complexes extracted, and show that the form of interaction with the IL differs from that outlined previously for the extraction of copper(ii). Insights on the competition established when silver(i) is extracted in the presence of copper(ii) are provided. Finally, it is noted that metallic silver can be directly electrodeposited at the electrode surface after extraction of silver(i) into [mimSBu][NTf₂] and that back extraction of silver(i) into aqueous media is achieved by addition of an acidic aqueous solution.

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer.

Analysis of the current distribution in an aluminum electrolysis cell with copper collector-bar cathode assembly

Understanding the magneto-hydrodynamic forces generated due to the external magnetic field and current density distribution within the cell (current in cell linings) is important in the optimization of cell dynamics. It is well documented that these factors play a crucial role in establishing the metal-pad stability of the cell. Conventional cells use the cathode-collector-bar assembly to carry the current through molten aluminium, the cathode and the steel collector-bar to nearest external bus. The electrical conductivity of the steel is so poor relative to the molten aluminium that the outer third of the collector bar carries the maximum load, which in turn increases the horizontal components of the current within the cell. Previous studies have modelled improvement in the cell instability through external magnetic compensation by redistributing current in the cathode busbar. Very little to date has been published on work to improve the current distribution within the cell. In this work, the current distribution in an aluminium electrolysis cell with copper collector-bar was predicted using finite element modelling. A 2D cross-section of a commercial cell was used under steady conditions of electrical fields in anode, electrolyte, molten aluminium and copper cathode-assembly. Different shapes and sizes of the cathode assembly are also considered to optimise the distribution of current throughout the cathode lining. The findings indicated that the copper-bar of similar size to steel could save voltage up to 150 mV. There is a reduction of more than 70% in peak current density value due to the copper inserts. The predicted trends of current distribution show a good agreement with previously published data.

Copper levels in buccal cells of vineyard workers engaged in various activities

Objectives: Copper-based compounds have been used as agricultural fungicides for many years. Their use in Australia is escalating with increase in the scale of planting and associated pest problems. The objective of this study was to identify viticulture activities associated with high exposure to foliage sprays. It would be determined if occupational exposure of vineyard workers to copper-based sprays was associated with raised body copper levels through analysis of saliva and buccal cells.

Methods: The activities of six vineyard workers from four vineyards in the Yarra Valley Victoria, Australia, were monitored over a period of 2 years. During this period, workers carried out seasonal activities, including fungicide spraying, canopy management, and tractor operation. Saliva and buccal cells from workers were collected and analysed for copper levels that were then correlated with the different types of vineyard activity.

Results: The buccal cells of vineyard workers exposed to copper through seasonal activities including fungicide spraying, canopy management, and tractor operation contained copper levels of 0.87, 1.24, and 0.95 ng Cu per 106 cells, respectively. This was up to 10-fold higher than the copper levels in buccal cells from the control subjects (0.1 ng Cu per 106). Copper levels in buccal cells from workers participating in other viticulture activities such as shoot thinning, bunch counting, and disbudding were not significantly different from those of control subjects. The levels of copper in saliva samples of both workers undertaking any vineyard activity and control subjects were below the level of detection.

Conclusions: Seasonal activities undertaken in vineyards that involved direct contact with copper, in particular canopy management, fungicidal spraying, and tractor operation were associated with high copper levels in buccal cells of workers. This indicates that copper derived from copper-based fungicidal compounds is accumulated within body cells. The lack of detectable copper levels in saliva suggests that the route of transport of copper into buccal cells is not through saliva. The results indicate potential adverse health risks associated with use of copper fungicide. Recommendations are made in relation to the precautions that should be taken in relation to use of copper sprays and to validate buccal cells as an indicator of body copper status.

Lack of ceruloplasmin expression alters aspects of copper transport to the fetus and newborn, as determined in mice

Copper transport and accumulation were studied in virgin and lactating C57BL/6 mice, with and without expression of ceruloplasmin (Cp), to assess the importance of Cp to these processes. One hour after i.p. injection of tracer ⁶⁴Cu, liver and kidney accounted for 80% of the radioactivity, and mammary gland 1%, while in lactating Cp+/+ mice 2–4 days post partum, uptake by mammary gland was 9-fold higher and that of liver and other organs was decreased, with ⁶⁴Cu rapidly appearing in milk. Parallel studies in Cp−/− mice (siblings from same colony) gave virtually identical results. However, their milk contained less ⁶⁴Cu, and actual copper contents determined by furnace atomic absorption were less than half those for milk from normal dams. Liver copper concentrations of pups born to Cp−/− dams also were half those of pups from wild type dams. Copper in pup brains was unaffected; but iron concentrations were reduced. We conclude that absence of Cp, while not affecting entry of exchangeable copper from the blood into the mammary gland, does have a significant effect on the availability of this metal to the newborn through the milk and in the form of stores accumulating in gestation.

Electrostatic forces induce poly(vinyl alcohol)-protected copper nanoparticles to form copper/poly(vinyl alcohol) nanocables via electrospinning

Copper/poly(vinyl alcohol) (PVA) nanocables have been successfully obtained by electrospinning a PVA-protected copper nanoparticle solution. The molar ratio of copper ions to PVA (in terms of VA repeating units) plays an important role in the formation of copper/PVA nanocables. The average diameter of the copper cores and PVA shells is about 100 and 400 nm, respectively. The structures of the copper/PVA nanocables are characterized by transmission electron microscopy (TEM) and their formation is confirmed by scanning electron microscopy (SEM).