Copper exposure effects on yeast mitochondrial proteome

Mitochondria play an important role on the entire cellular copper homeostatic mechanisms. Alteration of cellular copper levels may thus influence mitochondrial proteome and its investigation represents an important contribution to the general understanding of copper-related cellular effects. In these study we have performed an organelle targeted proteomic investigation focusing our attention on the effect of non-lethal 1mM copper concentration on Saccharomyces cerevisiae mitochondrial proteome. Functional copper effects on yeast mitochondrial proteome were evaluated by using both 2D electrophoresis (2-DE) and liquid chromatography coupled with tandem mass spectrometry. Proteomic data have been then analyzed by different unsupervised meta-analysis approaches that highlight the impairment of mitochondrial functions and the activation of oxidative stress response. Interestingly, our data have shown that stress response generated by 1mM copper treatment determines the activation of S. cerevisiae survival pathway. To investigate these findings we have treated yeast cells responsiveness to copper with hydrogen peroxide and observed a protective role of this metal. These results are suggestive of a copper role in the protection from oxidative stress possibly due to the activation of mechanisms involved in cellular survival and growth.     

Is there a role for copper in neurodegenerative diseases?

Copper is an essential metal in living organisms; thus, the maintenance of adequate copper levels is of vital importance and is highly regulated. Dysfunction of copper metabolism leading to its excess or deficiency results in severe ailments. Two examples of illnesses related to alterations in copper metabolism are Menkes and Wilson diseases. Several proteins are involved in the maintenance of copper homeostasis, including copper transporters and metal chaperones. In the last several years, the beta-amyloid-precursor protein (beta-APP) and the prion protein (PrP(C)), which are related to the neurodegenerative disorders Alzheimer and prion diseases respectively, have been associated with copper metabolism. Both proteins bind copper through copper-binding domains that also have been shown to reduce copper in vitro. Moreover, this ability to reduce copper is associated with a neuroprotective effect exerted by the copper-binding domain of both proteins against copper in vivo. In addition to a functional link between copper and beta-APP or PrP(C), evidence suggests that copper has a role in Alzheimer and prion diseases. Here, we review the evidence that supports both, the role of beta-APP and PrP(C), in copper metabolism and the putative role of copper in neurodegenerative diseases.     

The reactions of copper proteins with nitric oxide.

Nitric oxide (NO) can act as a ligand for copper atoms and may also engage in redox chemistry with the metal once bound. Furthermore NO posses an unpaired electron which can couple with the unpaired electron on Cu2+. These properties have been exploited to probe the active sites of copper-containing enzymes and proteins. We review these studies. In addition to the use as a spectroscopic probe for the active site we draw attention to the rapid reactions of NO at the copper sites in Cytochrome c oxidase (CcO) and laccase. These reactions in CcO occur in the ms time range, at low NO concentrations and in the presence of oxygen and may therefore be of physiological relevance to the control of respiration. Finally we speculate on the wider role that NO may play in regulation of an important group of Type 2 copper containing enzymes.     

Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Copper(I), copper(II) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus capsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron-sulphur centre of protein C with a secondary effect at the FAD centre when the copper(II):protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron-sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule. These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.     

XIAP Is a copper binding protein deregulated in Wilson's disease and other copper toxicosis disorders

X-linked inhibitor of apoptosis (XIAP), known primarily for its caspase inhibitory properties, has recently been shown to interact with and regulate the levels of COMMD1, a protein associated with a form of canine copper toxicosis. Here, we describe a role for XIAP in copper metabolism. We find that XIAP levels are greatly reduced by intracellular copper accumulation in Wilson's disease and other copper toxicosis disorders and in cells cultured under high copper conditions. Elevated copper levels result in a profound, reversible conformational change in XIAP due to the direct binding of copper to XIAP, which accelerates its degradation and significantly decreases its ability to inhibit caspase-3. This results in a lowering of the apoptotic threshold, sensitizing the cell to apoptosis. These data provide an unsuspected link between copper homeostasis and the regulation of cell death through XIAP and may contribute to the pathophysiology of copper toxicosis disorders.     

Copper transporters and chaperones: Their function on angiogenesis and cellular signalling

Copper, although known as a micronutrient, has a pivotal role in modulating the cellular metabolism. Many studies have reported the role of copper in angiogenesis. Copper chaperones are intracellular proteins that mediate copper trafficking to various cell organelles. However, the role and function of copper chaperones in relation to angiogenesis has to be further explored. The intracellular copper levels when in excess are deleterious and certain mutations of copper chaperones have been shown to induce cell death and influence various cellular metabolisms. The study of these chaperones will be helpful in understanding the players in the cascade of events in angiogenesis and their role in cellular metabolic pathways. In this review we have briefly listed the copper chaperones associated with angiogenic and metabolic signalling and their function.     

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two copper-transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNK and WND and their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles for MNK and WND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.     

Multiple forms of copper (II) co-ordination occur throughout the disordered N-terminal region of the prion protein at pH 7.4

Although the physiological function of the prion protein remains unknown, in vitro experiments suggest that the protein may bind copper (II) ions and play a role in copper transport or homoeostasis in vivo. The unstructured N-terminal region of the prion protein has been shown to bind up to six copper (II) ions, with each of these ions co-ordinated by a single histidine imidazole and nearby backbone amide nitrogen atoms. Individually, these sites have micromolar affinities, which is weaker than would be expected of a true cuproprotein. In the present study, we show that with subsaturating levels of copper, different forms of co-ordination will occur, which have higher affinity. We have investigated the copper-binding properties of two peptides representing the known copper-binding regions of the prion protein: residues 57-91, which contains four tandem repeats of the octapeptide GGGWGQPH, and residues 91-115. Using equilibrium dialysis and spectroscopic methods, we unambiguously demonstrate that the mode of copper co-ordination in both of these peptides depends on the number of copper ions bound and that, at low copper occupancy, copper ions are co-ordinated with sub-micromolar affinity by multiple histidine imidazole groups. At pH 7.4, three different modes of copper co-ordination are accessible within the octapeptide repeats and two within the peptide comprising residues 91-115. The highest affinity copper (II)-binding modes cause self-association of both peptides, suggesting a role for copper (II) in controlling prion protein self-association in vivo.     

Copper-induced conformational changes in the N-terminal domain of the Wilson disease copper-transporting ATPase

The Wilson disease copper-transporting ATPase plays a critical role in the intracellular trafficking of copper. Mutations in this protein lead to the accumulation of a toxic level of copper in the liver, kidney, and brain followed by extensive tissue damage and death. The ATPase has a novel amino-terminal domain ( approximately 70 kDa) which contains six repeats of the copper binding motif GMTCXXC. We have expressed and characterized this domain with respect to the copper binding sites and the conformational consequences of copper binding. A detailed analysis of this domain by X-ray absorption spectroscopy (XAS) has revealed that each binding site ligates copper in the +1 oxidation state using two cysteine side chains with distorted linear geometry. Analysis of copper-induced conformational changes in the amino-terminal domain indicates that both secondary and tertiary structure changes take place upon copper binding. These copper-induced conformational changes could play an important role in the function and regulation of the ATPase in vivo. In addition to providing important insights on copper binding to the protein, these results suggest a possible mechanism of copper trafficking by the Wilson disease ATPase.     

Ctr1 and its role in body copper homeostasis

Members of the Cu transporter (Ctr) family have been reported to be part of the copper uptake machinery in several organisms. Recently it has been suggested that human Ctr1 (hCtr1) may act as a copper transporter in several tissues including the intestine. hCtr1 is a 190 amino acid protein and is predicted to have three transmembrane-spanning domains and exist in the plasma membrane as a homo-trimer. Ctr1-transfected cell lines exhibit saturable, pH-dependent Cu(I) uptake indicating a role in copper transport. Recent studies with Ctr1 knockout mice have highlighted an essential function in mammalian embryonic development since homozygous mutants die in utero. Heterozygotes are indistinguishable from wild-type littermates but have a severely reduced brain copper content, suggesting that Ctr1 is a key component of the copper uptake pathway in the brain. However, its role in other tissues remains elusive.     

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts

Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.     

Insights into prion protein function from atomistic simulations

Computer simulations are a powerful tool for studies of biological systems. They have often been used to study prion protein (PrP), a protein responsible for neurodegenerative diseases, which include “mad cow disease” in cattle and Creutzfeldt-Jacob disease in humans. An important aspect of the prion protein is its interaction with copper ion, which is thought to be relevant for PrP’s yet undetermined function and also potentially play a role in prion diseases. For studies of copper attachment to the prion protein, computer simulations have often been used to complement experimental data and to obtain binding structures of Cu-PrP complexes. This paper summarizes the results of recent ab initio calculations of copper-prion protein interactions focusing on the recently discovered concentration-dependent binding modes in the octarepeat region of this protein. In addition to determining the binding structures, computer simulations were also used to make predictions about PrP’s function and the role of copper in prion diseases. The results demonstrate the predictive power and applicability of ab initio simulations for studies of metal-biomolecular complexes.     

Overexpression and mechanistic characterization of blastula protease 10, a metalloprotease involved in sea urchin embryogenesis and development

Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated "tyrosine switch." It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid-related metalloenzyme involved in sea urchin embryogenesis.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.     

The importance of juveniles in structuring a littoral macrobenthic community

The relationships between individual dry weight and bodycontent of the essential metals copper and zinc and of therespiratory pigment haemocyanin have been investigated in fourBritish species of caridean decapod crustaceans, Palaemonelegans, Palaemon longirostris, Palaemonetesvarians and Crangon crangon. Also considered were thebody concentrations of soluble copper and copper associatedwith haemocyanin. These decapods regulate total bodyconcentrations of copper and zinc to approximately constantlevels which can vary interspecifically. An attempt is made tointerpret these regulated concentrations in terms of essentialrequirements for the metals in the physiological functioningof haemocyanin, the body concentration of which variesinterspecifically and intraspecifically with environmentalvariables.     

Glutaredoxin1 protects neuronal cells from copper-induced toxicity

Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P_1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.     

Aquatic pollution increases the relative success of invasive species

Although individual ecosystems vary greatly in the degree to which they have been invaded by exotic species, it has remained difficult to isolate mechanisms influencing invader success. One largely anecdotal observation is that polluted or degraded areas will accumulate more invaders than less-impacted sites. However, the role of abiotic factors alone in influencing invisibility has been difficult to isolate, often because the supply of potential invaders is confounded with conditions thought to increase vulnerability to invasion. Here, we conducted a field experiment to test how the assemblages of exotic versus native marine invertebrates changed during community assembly under different exposure levels of a common pollutant, copper. The experiment was conducted by deploying fouling panels in a Randomized Block Design in San Francisco Bay. Panels were periodically removed, placed into buckets with differing copper concentrations, and returned to the field after 3 days. This design allowed propagule availability to the plates to be statistically independent of short-term copper exposure. The results demonstrate that copper caused significant differences in community structure. Average native species richness was significantly affected by copper exposure, but average exotic richness was not. The total native species pool within treatments exhibited a greater than 40% decline within increasing copper, while the exotic species pool did not change significantly. These results confirm that anthropogenic alteration of abiotic factors influences invader success, indicating that management strategies to reduce invader impacts should include both efforts to improve environmental conditions as well as reduce invader supply.