Copper Binding Ability Research Articles

A Water-Soluble Multifunctional Probe for Colorimetric Copper Sensing, Lysosome Labelling and Live-Cell Imaging.

We report a water-soluble fluorescence and colorimetric copper probe (LysoBC1); this system can also serve for lysosome labeling and for the dynamic tracking of Cu2+ in living cells. The sensing mechanism takes advantage of the synergic action by the following three components: i) a lysosome targeting unit, ii) the spirolactam ring-opening for the selective copper chelation and iii) the metal-mediated hydrolysis of the rhodamine moiety for fluorescence enhancement. In aqueous environment the molecule acts as a fluorescent reversible pH sensor and as colorimetric probe for Cu2+ at physiological pH; the hydrolysis of the copper targeting unit resulted in a 50-fold increase of the fluorescence intensity. Most importantly, in vitro cell analyses in undifferentiated (SH SY5Y) and differentiated (d-SH SY5Y) neuroblastoma cells, LysoBC1 is able to selectively accumulate into lysosome while the copper binding ability allowed us to monitor intracellular copper accumulation into lysosome.

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from 1H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

A Combined NMR and UV-Vis Approach to Evaluate Radical Scavenging Activity of Rosmarinic Acid and Other Polyphenols.

Oxidative stress results from an imbalance between reactive oxygen species (ROS) production and the body's ability to neutralize them. ROS are reactive molecules generated during cellular metabolism and play a crucial role in normal physiological processes. However, excessive ROS production can lead to oxidative damage, contributing to various diseases and aging. This study is focused on rosmarinic acid (RA), a hydroxycinnamic acid (HCA) derivative well known for its antioxidant activity. In addition, RA has also demonstrated prooxidant behavior under specific conditions involving high concentrations of transition metal ions such as iron and copper, high pH, and the presence of oxygen. In this study, we aim to clarify the underlying mechanisms and factors governing the antioxidant and prooxidant activities of RA, and to compare them with other HCA derivatives. UV-Vis, NMR, and EPR techniques were used to explore copper(II)'s binding ability of RA, caffeic acid, and p-coumaric acid. At the same time, UV-Vis and NMR methods were exploited to evaluate the polyphenols' free radical scavenging abilities towards ROS generated by the ascorbic acid-copper(II) system. All the data indicate that RA is the most effective polyphenol both in copper binding abilities and ROS protection.

Investigation of metal interactions with YrpE protein of Bacillus subtilis by a polyhistidine peptide model

The use of model peptides that can simulate the behaviour of a protein domain is a very successful analytical method to study the metal coordination sites in biological systems. Here we study zinc and copper binding ability of the sequence HTHEHSHDHSHAH, which serves as model for the metal interactions with YrpE, a putative metal-binding protein of the ZinT family identified in Bacillus subtilis. Compared to other ZinT proteins secreted by Gram-negative bacteria, the metal-coordination properties of YrpE N-terminal histidine-rich domain have not been yet characterized. Different independent analytical methods, aimed at providing information on the stability and structure of the formed species, have been employed, including potentiometric titrations, electrospray ionization mass spectrometry, UV–Vis spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopy. The obtained speciation models and equilibrium constants allowed to compare the metal-binding ability of the investigated polyhistidine sequence with that of other well-known histidine-rich peptides. Our thermodynamic results revealed that the YrpE domain HTHEHSHDHSHAH forms more stable metal complexes than other His-rich domains of similar ZinT proteins. Moreover, the studied peptide, containing the alternated (-XH-)n motif, proved to be even more effective than the His6-tag (widely used in immobilized metal ion affinity chromatography) in binding zinc ions.

Discovery and biosynthesis of tricyclic copper-binding ribosomal peptides containing histidine-to-butyrine crosslinks

Cyclic peptide natural products represent an important class of bioactive compounds and clinical drugs. Enzymatic side-chain macrocyclization of ribosomal peptides is a major strategy developed by nature to generate these chemotypes, as exemplified by the superfamily of ribosomally synthesized and post-translational modified peptides. Despite the diverse types of side-chain crosslinks in this superfamily, the participation of histidine residues is rare. Herein, we report the discovery and biosynthesis of bacteria-derived tricyclic lanthipeptide noursin, which is constrained by a tri amino acid labionin crosslink and an unprecedented histidine-to-butyrine crosslink, named histidinobutyrine. Noursin displays copper-binding ability that requires the histidinobutyrine crosslink and represents the first copper-binding lanthipeptide. A subgroup of lanthipeptide synthetases, named LanKCHbt, were identified to catalyze the formation of both the labionin and the histidinobutyrine crosslinks in precursor peptides and produce noursin-like compounds. The discovery of the histidinobutyrine-containing lanthipeptides expands the scope of post-translational modifications, structural diversity and bioactivity of ribosomally synthesized and post-translational modified peptides.

Molecular Insights into a Novel Cu(I)-Sensitive ArsR/SmtB Family Repressor in Extremophile Acidithiobacillus caldus.

Acidithiobacillus caldus is a common bioleaching bacterium that is inevitably exposed to extreme copper stress in leachates. The ArsR/SmtB family of metalloregulatory repressors regulates homeostasis and resistance in bacteria by specifically responding to metals. Here, we characterized A. caldus Cu(I)-sensitive repressor (AcsR) and gained molecular insights into this new member of the ArsR/SmtB family. Transcriptional analysis indicated that the promoter (PIII) of acsR was highly active in Escherichia coli but inhibited upon AcsR binding to the PIII-acsR region. Size exclusion chromatography and circular dichroism spectra revealed that CuI-AcsR shared an identical assembly state with apo-AcsR, as a dimer with fewer α helices, more extended strands, and more β turns. Mutation of the cysteine site in AcsR did not affect its assembly state. Copper(I) titrations revealed that apo-AcsR bound two Cu(I) molecules per monomer in vitro with an average dissociation constant (KD) for bicinchoninic acid competition of 2.55 × 10-9 M. Site-directed mutation of putative Cu(I)-binding ligands in AcsR showed that replacing Cys64 with Ala reduces copper binding ability from two Cu(I) molecules per monomer to one, with an average KD of 6.05 × 10-9 M. Electrophoretic mobility shift assays revealed that apo-AcsR has high affinity for the 12-2-12 imperfect inverted repeats P2245 and P2270 in the acsR gene cluster and that Cu-loaded AcsR had lower affinity for DNA fragments than apo-AcsR. We developed a hypothetical working model of AcsR to better understand Cu resistance mechanisms in A. caldus. IMPORTANCE Copper (Cu) resistance among various microorganisms is attracting interest. The chemolithoautotrophic bacterium A. caldus, which can tolerate extreme copper stress (≥10 g/L Cu ions), is typically used to bioleach chalcopyrite (CuFeS2). Understanding of Cu resistance in A. caldus is limited due to scant investigation and the absence of efficient gene manipulation tools. Here, we characterized a new member of the ArsR/SmtB family of prokaryotic metalloregulatory transcriptional proteins that repress operons linked to stress-inducing concentrations of heavy metal ions. This protein can bind two Cu(I) molecules per monomer and negatively regulate its gene cluster. Members of the ArsR/SmtB family have not been investigated in A. caldus until now. The discovery of this novel protein enriches understanding of Cu homeostasis in A. caldus.

Multi-spectroscopic investigation of the molecular weight distribution and copper binding ability of dissolved organic matter in Dongping Lake, China

The properties and metal-binding abilities of dissolved organic matter (DOM) rely on its molecular weight (MW) structure. In this study, the spatial differences of DOM in compositions, MW structures, and binding mechanisms with copper (Cu2+) in Dongping Lake were investigated by applying excitation-emission matrix combining parallel factor analysis (EEM-PARAFAC), synchronous fluorescence (SF) spectra, two-dimensional correlation spectra (2D-COS), and Fourier transform infrared (FTIR) spectra. The EDOM for the entrance of the Dawen River and PDOM for the macrophyte-dominated region were divided from DOM of Dongping Lake based on hierarchical clustering analysis (HCA) and principal component analysis (PCA) and were size-fractioned into MW < 500 kDa and <100 kDa fractions. According to EEM-PARAFAC, Dongping Lake was dominated by tryptophan-like substances with MW < 500 kDa. The concentration of PDOM was higher than that of EDOM (p < 0.05). 2D-COS showed that protein-like components preceded humic-like components binding to Cu2+ regardless of sample type (215 nm > 285 nm > 310–360 nm). The Cu2+ binding capacity of DOM exhibited specific differences in space, components, and molecular weights. The humic-like component 1 (C1) and tryptophan-like component 4 (C4) of PDOM showed stronger binding abilities than those of EDOM. Endogenous tryptophan-like component 4 (C4) had a higher binding affinity for Cu2+ than humic-like components (logKa: C4 > C1 > C2) in PDOM irrespective of MW. Humic-like components with MW < 500 kDa displayed higher binding potentials for Cu2+. FTIR spectra showed that the main participants of DOM-Cu complexation included aromatic hydrocarbons, aliphatic groups, amide Ⅰ bands, and carboxyl functional groups. This study provides spatial-scale insights into the molecular weight structure of DOM in influencing the behavior, fate, and bioavailability of heavy metals in lakes.

Linking molecular composition to proton and copper binding ability of fulvic acid: A theoretical modeling approach based on FT-ICR-MS analysis

Dissolved organic matter (DOM) is one of the most important ligands governing the geochemical cycling of metals in the environment, but recent studies with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) have shown enormous complexity and diversity of DOM composition. How the diverse molecular composition of DOM affects the reactivity of DOM with metals is still largely unknown, which precludes us from developing accurate geochemical models for the fate of metals in the environment. In this study, we combined FT-ICR-MS analysis and theoretical modeling approaches and specifically elucidated the link between molecular composition and the proton and Cu binding ability of DOM, using the Suwannee River fulvic acid (FA) as a model humic substance. Batch adsorption experiments were conducted to generate different extents of molecular fractionation of FA samples by ferrihydrite. FT-ICR-MS analyses were employed to investigate the changes of molecular composition while Cu titration and the Windermere Humic Aqueous Model (WHAM) were used to quantify the variations on the Cu binding capacities of FA samples. We developed a general theoretical modeling approach, which integrated a suite of theoretical modeling methods, including the Vienna Soil-Organic-Matter-Modeler (VSOMM), SPARC Performs Automated Reasoning in Chemistry (SPARC), and the linear free energy relationships (LFER), for molecular modeling based on FT-ICR-MS data. Based on the FT-ICR-MS results, we found that, despite of the complex molecular composition of FA, FA molecules can be divided into three representative groups and each group of molecules had distinct chemical properties. Interestingly, molecules within the same group had similar distributions of molecular properties. Based on the chemical properties of the three groups of FA molecules, we successfully constructed three molecular models of FA using VSOMM, and quantified the distributions of proton and Cu binding constants with SPARC and LFER. Those independently determined binding constants were comparable to the WHAM default proton and Cu binding constants, supporting the validity of our modeling approach. Our modeling results suggested that the molecular complexity of DOM may be simplified with representative groups of molecules based on their binding ability with metals in theoretical modeling. Our modeling approach based on FT-ICR-MS data shed light on developing mechanistical models for metal reactions with DOM based on the molecular data, which is helpful for predicting the geochemical cycling of carbon and metals in the environment.

Exploring the copper binding ability of Mets7 hCtr-1 protein domain and His7 derivative: An insight in Michael addition catalysis.

Mets7 is a methionine-rich motif present in hCtr-1 transporter that is involved in copper cellular trafficking. Its ability to bind Cu(I) was recently exploited to develop metallopeptide catalysts for Henry condensation. Here, the catalytic activity of Mets7-Cu(I) complex in Michael addition reactions has been evaluated. Furthermore, His7 peptide, in which Met residues have been substituted with His ones, was also prepared. This substitution allowed His7 to coordinate Cu (II), with the obtainment of a stable turn conformation as evicted by CD experiments. His7-Cu (II) proved also to be a better catalyst than Mets7-Cu(I) in the addition reaction. In particular, when the substrate was the (E)-1-phenyl-3-(pyridin-2-yl)prop-2-en-1-one, a conversion of 71% and a significative 58% of e.e. was observed.

Multi-spectroscopic investigation on the spatial distribution and copper binding ability of sediment dissolved organic matter in Nansi Lake, China

Interaction between dissolved organic matter (DOM) and heavy metals have been widely studied, and the differences in DOM properties have a great impact on the capacity of DOM-metals binding. In this study, the samples were collected from the upper lake and lower lake in Nansi Lake, respectively. According to the ratio of the fluorescence intensity of component to the total fluorescence intensity (%Fmax) in the two areas, obvious differences were found in the properties of DOM. The content of humic-like substances showed a decreasing trend from the north to south in Nansi Lake. On this basis, samples from north and south ends of Nansi Lake were selected for fluorescence titration. All components were quenched with the increasing of Cu2+ addition. Combining the results of Synchronous fluorescence (SF) spectra with two-dimensional correlation spectroscopy (2D-COS), fluorescent quenching near 275 nm was quicker than the changes that occurred at the wavelength that after 275 nm, which suggested that the complexation occurred firstly for protein-like components as compared with humic-like components. The modified Stern-Volmer model suggested that protein-like components had larger effective quenching constants (logKa = 3.47 ± 0.13 for component C5 in the upper lake and logKa = 3.57 ± 0.19 for component C4 in the lower lake) than humic-like components and it is not affected by the region, indicating that protein-like substances might have a higher metal binding potential than humic-like substances in Nansi Lake. The binding constants and the binding sites suggested that humic-like components also had a nonnegligible binding potential in Nansi Lake although the protein-like components revealed a stronger binding ability in fluorescence quenching.

Complex formation processes and metal ion catalyzed oxidation of model peptides related to the metal binding site of the human prion protein

Interaction of copper(II) and nickel(II) ions with the Ac-PHAAAGTHSMKHM-NH2 tridecapeptide containing the His85, His96 and His111 binding sites of human prion protein has been studied by various techniques. pH-potentiometry, UV–Vis and circular dichroism spectroscopy were applied to study the stoichiometry, stability and structure of the copper(II) and nickel(II) complexes, while HPLC-MS and MS/MS were used for identifying the products of copper(II) catalyzed oxidation. The copper binding ability of shorter fragments, namely the nonapeptide Ac-PHAAAGTHS-NH2 and pentapeptide Ac-PHAAA-NH2 have also been studied. The tridecapeptide is able to bind three equivalent of copper(II) ion, since the histidine residues behave as independent metal binding sites. Nevertheless, the metal binding ability of histidine residue mimicking the octarepeat domain (His85) is decreased, while the other parts of the peptide mimicking the histidines outside the octarepeat domain bind the copper ions in comparable concentration. On the other hand, this peptide is able to coordinate only two equivalents of nickel ion on the domains outside the octarepeat region. Furthermore the His96 binding site is more effective for the nickel ions. Both histidine and methionine residues are sensitive for oxidation, the oxidation of these residues are proved, and in the case of the histidine residues follows the order His96 > His85 ≫ His111.

Exploring the Extended Biological Functions of the Human Copper Chaperone of Superoxide Dismutase 1

The human copper chaperone of SOD1 (designated as CCS) was discovered more than two decades ago. It is an important copper binding protein and a homolog of Saccharomyces cerevisiae LYS7. To date, no studies have systematically or specifically elaborated on the functional development of CCS. This review summarizes the essential information about CCS, such as its localization, 3D structure, and copper binding ability. An emphasis is placed on its interacting protein partners and its biological functions in vivo and in vitro. Three-dimensional structural analysis revealed that CCS is composed of three domains. Its primary molecular function is the delivery of copper to SOD1 and activation of SOD1. It has also been reported to bind to XIAP, Mia40, and X11α, and other proteins. Through these protein partners, CCS is implicated in several vital biological processes in vivo, such as copper homeostasis, apoptosis, angiogenesis and oxidative stress. This review is anticipated to assist scientists in systematically understanding the latest research developments of CCS for facilitating the development of new therapeutics targeting CCS in the future.

The binding abilities of homodetic cyclic His-peptides toward copper ions

Abstract The biochemistry of copper is important and worthy to be broadly studied. In biological systems copper is usually bound by enzymes, proteins or peptides and the investigation of the occurring interactions is still a hot topic. In this overview we focus on the copper binding abilities of homodetic cyclic peptides (CPs) containing histidine moieties. This group of ligands is characterized by a cyclic ring composed only of amino acids. Several applications of cyclic peptides should be viable because of their special structures, stability and resistance against enzymatic degradation. The cyclic structure promotes the anchoring of metal ions by the amino acid side chains. We discuss the impact of peptide cyclization and ring size on the binding abilities toward copper(II) ions. We also compare the coordinating properties of ligands containing a different number of His residues with the (HX) n − motif, where X = Gly, Lys, Arg, Asn or Pro, based on the stability constants of nN Im (n = 2–4) complexes created by His 4 -cyclopeptides. In addition, we compare the sustainability and stability of nN Im (n = 1–4) complexes depending on the Asp residue in the peptide sequence and we also consider the aspects of forming dinuclear copper(II) complexes by cyclopeptides with two separated Pro residues in the sequence. The di-Cu(II)-complexes are favored in the basic pH range. So far, cyclopeptides found use in pharmaceutical chemistry, biochemistry, and life sciences, but also as ionophores and nano-materials. We advocate here to use their metal ion complexes because they enrich the spectrum of available activities of metallopeptides or change the properties of these ligands.

Biomphalaria glabrata Metallothionein: Lacking Metal Specificity of the Protein and Missing Gene Upregulation Suggest Metal Sequestration by Exchange Instead of through Selective Binding.

The wild-type metallothionein (MT) of the freshwater snail Biomphalaria glabrata and a natural allelic mutant of it in which a lysine residue was replaced by an asparagine residue, were recombinantly expressed and analyzed for their metal-binding features with respect to Cd2+, Zn2+ and Cu+, applying spectroscopic and mass-spectrometric methods. In addition, the upregulation of the Biomphalaria glabrata MT gene was assessed by quantitative real-time detection PCR. The two recombinant proteins revealed to be very similar in most of their metal binding features. They lacked a clear metal-binding preference for any of the three metal ions assayed—which, to this degree, is clearly unprecedented in the world of Gastropoda MTs. There were, however, slight differences in copper-binding abilities between the two allelic variants. Overall, the missing metal specificity of the two recombinant MTs goes hand in hand with lacking upregulation of the respective MT gene. This suggests that in vivo, the Biomphalaria glabrata MT may be more important for metal replacement reactions through a constitutively abundant form, rather than for metal sequestration by high binding specificity. There are indications that the MT of Biomphalaria glabrata may share its unspecific features with MTs from other freshwater snails of the Hygrophila family.

Cyclodextrins 3-Functionalized with 8-Hydroxyquinolines: Copper-Binding Ability and Inhibition of Synuclein Aggregation.

Neurodegenerative diseases such as Parkinson's and Alzheimer's diseases are multifactorial disorders related to protein aggregation, metal dyshomeostasis, and oxidative stress. To advance understanding in this area and to contribute to therapeutic development, many efforts have been directed at devising suitable agents that can target metal ions associated with relevant biomolecules such as α-synuclein. This paper presents a new cyclodextrin-8-hydroxyquinoline conjugate and discusses the properties of four cyclodextrins 3-functionalized with 8-hydroxyquinoline as copper(II) chelators and inhibitors of copper-induced synuclein aggregation. The encouraging results establish the potential of cyclodextrin-8-hydroxyquinoline conjugates as chelators for the control of copper toxicity.

Influence of effluent organic matter on copper speciation and bioavailability in rivers under strong urban pressure.

This study focuses on spatiotemporal variations in the type of dissolved organic matter (DOM) and copper binding ability both upstream and downstream of Paris. It also compares the relative influence of both natural DOM upstream of Paris and effluent dissolved organic matter (EfDOM) output from a wastewater treatment plant (WWTP) on trace metal speciation and bioavailability in aquatic systems. In addition to the typical high- and low-affinity binding sites, a third family of very high-affinity binding sites has been highlighted for EfDOM. In receiving waters downstream of Paris during low-flow periods, the percentage of high- and very high-affinity sites originating from EfDOM reaches nearly 60 %. According to the speciation computation, the free copper concentration upstream of Paris exceeds the downstream Paris concentration by a factor of 2 to 4. As regards copper bioavailability, the highest EC50tot values were observed for EfDOM and downstream DOM, with a very low aromaticity and low UV absorbance. This finding suggests that specific ultraviolet absorbance (SUVA) is unlikely to be useful in assessing metal speciation and toxicity in aquatic systems subject to strong urban pressures. These results also highlight that the copper speciation computation for surface water exposed to considerable human pressures should include not only the humic and/or fulvic part of dissolved organic carbon but more hydrophilic fractions as well, originating for example from EfDOM.

Peptides derived from the copper-binding region of lysyl oxidase exhibit antiangiogeneic properties by inhibiting enzyme activity: an in vitro study.

Despite the rigorous research on abnormal angiogenesis, there is a persistent need for the development of new and efficient therapies against angiogenesis-related diseases. The role of Lysyl oxidase (LOX) in angiogenesis and cancer has been established in prior studies. Copper is known to induce the synthesis of LOX, and hence regulates its activity. Hypoxia-induced metastasis is dependent on LOX expression and activity. It has been believed that the inhibition of LOX would be a therapeutic strategy to inhibit angiogenesis. To explore this, we designed peptides (M peptides) from the copper-binding region of LOX and hypothesized them to modulate LOX. The peptides were characterized, and their copper-binding ability was confirmed by mass spectrometry. The M peptides were found to reduce the levels of intracellular copper when the cells were co-treated with copper. The peptides showed promising effect on aortic LOX, recombinant human LOX and LOX produced by human umbilical vein endothelial cells (HUVECs). The study also explores the effect of these peptides on copper and hypoxia-stimulated angiogenic response in HUVECs. It was found that the M peptides inhibited copper/hypoxia-induced LOX activity and inhibited stimulated HUVEC tube formation and migration. This clearly indicated the potential of M peptides in inhibiting angiogenesis, highlighting their role in the formulation of drugs for the same.

Identification of copper-binding proteins in soybean seeds by immobilized metal affinity chromatography and mass spectrometry

Metalloproteomics is defined as the structural and functional characterization of putative metal-binding proteins on a genome-wide scale. In this study, we carried out a systematic screen for copper-binding proteins in soybean seeds through the combined use of immobilized metal affinity chromatography and two-dimensional gel electrophoresis. A total of 32 protein spots displaying copper-binding ability were unambiguously identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis. About 78% of these identified proteins contain the possible copper-binding motifs, namely, H-(X)n-H (n = 0–5, 7, and 12), H-(X)3-C, H-(X)6-M, M-(X)7-H, and C-(X)n-C (n = 2–4). Available functional information suggested that the majority of the identified proteins are involved in storage, defense response, redox homeostasis, carbohydrate metabolism, and protein biosynthesis. Accordingly, the methodology reported here has the potential utility in additional metalloproteomic screening.

Dysregulation of intracellular copper homeostasis is common to transgenic mice expressing human mutant superoxide dismutase-1s regardless of their copper-binding abilities

Over 170 mutations in superoxide dismutase-1 (SOD1) have been linked to amyotrophic lateral sclerosis (ALS). The properties of SOD1 mutants differ considerably including copper-binding abilities. Nevertheless, they cause the same disease phenotype, suggesting a common neurotoxic pathway. We have previously reported that copper homeostasis is disturbed in spinal cords of SOD1G93A mice. However, it is unknown whether copper dyshomeostasis is induced by other SOD1 mutants. Using the additional mouse strains SOD1G127insTGGG, SOD1G85R, and SOD1D90A, which express SOD1 mutants with different copper-binding abilities, we show that copper dyshomeostasis is common to SOD1 mutants. The SOD1 mutants shifted the copper trafficking systems toward copper accumulation in spinal cords of the mice. Copper contents bound to the SOD1 active site varied considerably between SOD1 mutants. Still, copper bound to other ligands in the spinal cord were markedly increased in all. Zinc was also increased, whereas there were no changes in magnesium, calcium, aluminum, manganese and iron. Further support for a role of copper dyshomeostasis in ALS was gained from results of pharmacological intervention. Ammonium tetrathiomolybdate (TTM), a copper chelating agent, prolonged survival and slowed the disease progression of SOD1G93A mice, even when the treatment was started after the disease onset. TTM markedly attenuated pathology, including the loss of motor neurons and axons, and atrophy of skeletal muscles. Additionally, TTM decreased amounts of SOD1 aggregates. We propose that pharmacological agents that are capable of modulating copper dyshomeostasis, such as TTM, might be beneficial for the treatment of ALS caused by SOD1 mutations.

A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

BackgroundCopper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated.MethodsPlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy.ResultsSix P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein.ConclusionPlasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds.