Absence Of Cu Research Articles

Tau-peptide fragments and their copper(II) complexes: Effects on Amyloid-β aggregation

Abstract Recent studies suggest that the interaction of Aβ and Tau may be significant in the pathogenesis of Alzheimer’s diseases (AD). In addition, the potential influence of copper on Tau-related pathology in AD has not been previously addresseded and the interaction between Tau protein, Aβ and copper has even more recently been associated with AD. While the copper(II) interaction with the Aβ peptide has exhaustively been studied, the few studies carried out on copper(II) complexes with peptide fragments from Tau protein have been focused on the pseudo-repeats of Tau protein in the microtubule-binding region. No data have been reported about the metal complexes with peptides derived from the N-terminal portion of Tau protein, outside the microtubule-binding domain, despite increased levels of peptide fragments from this region have been detected in the Cerebrospinal fluid (CSF) of AD patients. Here we examine the interaction of two peptides fragments, encompassing the 1–25 or 26–44 residues of the human Tau protein sequence, with Aβ as well as the Cu2+-binding features of these two naturally occurring peptides. The CD experiments showed that copper(II) differently affects the peptide conformation of the two ligands and provided also insight into the donor atoms involved in metal coordination. Stoichiometry of copper(II) complexes was obtained by means of High resolution ESI-MS. Finally, the influence of the studied peptide on Aβ’s fibrillogenesis, either in the presence or absence of Cu2+, was investigated by means of Th-T fluorescence coupled with turbidimetric measurements. The observed different effect on the in vitro Aβ’s aggregation, was correlated with the affinity of copper(II) with the two peptide ligands. The overall results indicate that copper(II) can bind these peptides using the histidine residue or amino group as anchoring sites and that copper(II) binding may have a possible involvement in AD.

Dynamic Stress Analysis Applied to the Underpotential Deposition of Copper Onto (111)-Textured Au

Wafer curvature and cantilever bending techniques have been used by the electrochemical community to examine stress development during electrochemical processing. Surface stress changes as low as 10−3 N/m can typically be resolved from cantilever electrodes immersed in solution and under potential control. Such resolution makes this measurement useful for examining virtually all aspects of electrochemistry; i.e., electrocapillarity, adsorption processes, underpotential deposition, electrodeposition, etc. Often these processes occur either simultaneously or in rapid succession and we are often limited to measuring the influence of the dominant process in the time-scale of the experiment. Similar to electrochemical impedance spectroscopy (EIS) where electrochemical processes with different characteristic time constants can be separated, dynamic stress analysis (DSA) allows us to study the dynamics of any particular stress-generating process and link the stress to specific electrochemical and surface phenomena. We have demonstrated the technique by examining the electrocapillarity (charge-induced stress) of both Pt and Au in HClO4 electrolyte.1,2 In this case the figure of merit is the stress-charge coefficient ( ς ) which captures the fundamental surface mechanics associated with charging the electrode surface and can be obtained from the following equation, ς = jωYsZe where Ze is the electrochemical impedance, Ys is the stress admittance (with units of N/(V-m), ω is the angular frequency, and j =√-1. In this talk, we use DSA to examine the underpotential deposition (upd) of Cu onto (111)-textured Au cantilever electrodes from 0.1 M H2SO4 containing 0.01 M CuSO4. Figure 1(a) shows the potentiodynamic scan and surface stress response of a Au cantilever electrode in the potential region covering both Cu upd and Au oxide formation. Cu upd clearly exhibits the two distinct electrochemical steps that have been well-described in the literature. A prominent feature of the surface stress response is the significant change in the double layer region. This can be attributed to both electrocapillarity and sulfate adsorption/desorption. The stress response in the Cu upd region suggests that the honeycomb structure has little impact on the surface stress as the response seems to naturally extend from the double layer region. However, completion of the Cu monolayer, which includes the re-adsorption of sulfate onto the Cu surface, causes a -0.5 N/m shift in the surface stress. Interestingly, the large tensile stress expected from the +12.8 % lattice misfit between Cu and Au is absent. This is in part due to the interfacial contributions to the stress as well as the stabilizing influence of the sulfate adlayer.3 DSA can provide additional information on the relationship between surface stress and charge density. Figure 1(b) shows the phase angle of the stress-charge coefficient ( ς ) in the potential range of interest. The steady-state potential was stepped in 10 mV increments with a superimposed AC signal of ±10 mV at 10 Hz. In the absence of Cu2+ the phase angle of the stress with respect to the charge remains constant at 180°; i.e., negative charge produces tensile stress. This is a normal electrocapillarity response for Au in sulfuric acid at these potentials. However, in the presence of Cu2+, the phase angle begins to increase in the earliest stages of Cu upd, suggesting that the stress response for the honeycomb structure does indeed have a unique response that can be separated from that of electrocapillarity. With proper use of an equivalent circuit that adequately describes the electrochemical impedance, one should be able to obtain unique stress-charge coefficients that capture both the electrocapillarity and Cu upd response as a function of potential.

Nanopore Single-Molecule Analysis of Metal Ion–Chelator Chemical Reaction

Metal ions play critical roles in wide range of biochemical and physiological processes, but they can cause toxicity if excessive ingestion or misregulation. Chelating agents offer an efficient mean for metal ions intoxication and therapeutics of diseases. Studies on metal ion-chelator interactions are important for understanding the reaction mechanism and developing new specific metal chelator drugs. However, it remains a significant challenge to detect the metal ion-chelator interactions at the molecular level. Here, we report a label-free nanopore sensing approach that enables single-molecule investigation of the complexation process. We demonstrate that the chemical reaction between Cu2+ and carboxymethyl-β-cyclodextrin (CMβCD) in a nanoreactor is completely different from in the bulk solution. The formation constant (Kf = 4.70 × 104 M-1) increases 14 417-fold in the nanopore than that in the bulk solution (Kf = 3.26 M-1). The bioavailable CMβCD as a natural derivative with higher affinity for Cu2+ could be used in the safe medicinal removal of toxic metal. On the basis of the different ionic current signatures across an α-hemolysin (α-HL) mutant (M113N)7 nanopore lodged with a CMβCD adaptor in the presence and absence of Cu2+, the reversible molecular binding events to CMβCD can be in situ recorded and the single-molecule thermodynamic and kinetic information can be obtained. Interestly, we found that the Cu2+ binding leads to the increase of the channel current, rather than the blocking as usual nanopore experiment. The uncommon (on/off) characteristic could be very useful for fabricating the nanodevice. Furthermore, the unique nanopore sensor can provide a highly sensitive approach for detecting metal ions.

Structural and Dynamics Characterization of the MerR Family Metalloregulator CueR in its Repression and Activation States

CueR (Cu export regulator) is a metalloregulator protein that "senses" Cu(I) ions with very high affinity, thereby stimulating DNA binding and the transcription activation of two other metalloregulator proteins. The crystal structures of CueR when unbound or bound to DNA and a metal ion are very similar to each other, and the role of CueR and Cu(I) in initiating the transcription has not been fully understood yet. Using double electron-electron resonance (DEER) measurements and structure modeling, weinvestigate the conformational changes that CueR undergoes upon binding Cu(I) and DNA in solution. We observe three distinct conformations, corresponding to apo-CueR, DNA-bound CueR in the absence of Cu(I) (the "repression" state), and CueR-Cu(I)-DNA (the "activation" state). We propose a detailed structural mechanism underlying CueR's regulation of the transcription process. The mechanism explicitly shows the dependence of CueR activity on copper, thereby revealing the important negative feedback mechanism essential for regulating the intracellular copper concentration.

(Invited) New Glycofullerenes for Ebola Virus Infection

Multivalency is an important issue in the study of carbohydrate−protein interactions as well as for getting a better understanding of the virus infection processes. Thus, different chemical and bio-inspired scaffolds have been used in the literature in the search for new glycoconjugates bearing larger units of carbohydrates located in the appropriate spatial arrangement. At the molecular level, there are a wide variety of examples where multivalency drastically enhances the interactions between biomolecules in comparison with the monovalent binding. The search for high-affinity ligands for the study and understanding of the mechanisms involved in multivalent interactions has yielded a wide variety of artificial glycoconjugates which include, among others, glycopolymers, glycodendrimers, and glyconanoparticles. Recently, we have shown that hexakis-adducts of [60]fullerene endowed with 12, 24 or 36 mannoses, act as strong inhibitors for DC-SIGN in an Ebola infection assay model.1 Furthermore, a drastic increase in the inhibition process to the subnanomolar scale has been observed when the size and mannoses´ number are increased in the tridecafullerenes endowed with 120 mannose units decorating the periphery of the molecule.2 In this work, we report the synthesis and study of novel carbon nanostructures and carbohydrates for the multivalent presentation in an artificial Ebola virus infection model. These new carbon nanoforms have been chemically modified by the covalent attachment of glycofullerenes using the CuAAC “click chemistry” approach as well as in the absence of Cu in order to prevent the unfavorable metal effect.3 The efficiency to block DC-SIGN mediated viral infection by an artificial Ebola virus has been tested in a cellular experimental assay finding that, these systems are potent inhibitors of viral infection.

Cu2+ Ions Modulate the Conductance Hysteresis of Lysenin Channels

Lysenin channels self-assembled into lipid membranes have unique biophysical features which distinguish them from other pore-forming toxins. Lysenin channels present hysteretic and asymmetrical voltage-induced gating in response to positive transmembrane voltages and reversible ligand-gating in the presence of multivalent ions. Previous work on lysenin channels reported no apparent dependency between the voltage and ligand gating mechanisms. Such a link is demonstrated by this work, where we show that Cu2+ ions, known to be very potent inhibitors of lysenin channels’ conductance, have a dramatic influence on the channel response to transmembrane voltages. Addition of Cu2+ ions (in the range 0-100 μM) to the solutions bathing a population of lysenin channels inserted into a planar lipid membrane induces the well-known conductance inhibition at any transmembrane voltage within the physiological range. However, the channels that remain open respond to positive voltages by closing. Unexpectedly, only minor changes of the midway voltage of activation and the open probability are encountered, which is indicative of a lack of strong ionic screening induced by Cu2+ ions. In the absence of Cu2+ ions, channel reopening, although it occurs at lower voltages than closing (indicative of hysteresis), is always complete at voltages lower than +10 mV. In the presence of Cu2+ ions, channel reopening is not complete until the voltages reach values as low as −40 mV. This channel arresting in the closed state is concentration dependent and manifests as dramatic changes in the opening and closing rate constants in the presence of Cu2+ ions. The presence of Cu2+ ions significantly accentuate the hysteretic behavior of lysenin channels, indicative of improved molecular memory capabilities. Given the link between hysteresis and memory, our work may open novel avenues for exploiting the voltage-controlled molecular memory for bioelectronics and biological interfacing.

Copper-promoted methylene C-H oxidation to a ketone derivative by O2.

The methylene group of the ligand 1,2-di(pyridin-2-yl)-ethanone oxime (dpeo) is slowly oxygenated by the O2 in air under ambient conditions when [Cu(dpeo)2](ClO4)2 is dissolved in ethanol or acetonitrile. An initial transient ketone product, 2-(hydroxyimino)-1,2-di(pyridine-2-yl)ethanone, (hidpe) was characterized in the heteroleptic copper(ii) complex [Cu(bpca)(hidpe)](ClO4). The co-ligand in this complex, N-(2'-pyridylcarbonyl)pyridine-2-carboximidate (bpca-), is derived from a copper-promoted Beckmann rearrangement of hidpe. In the presence of bromide only [Cu(bpca)Br] is isolated. When significant water is present in reaction mixtures copper complexes of dpeo, hidpe and bpca- are not recovered and [Cu(pic)2H2O] is isolated. This occurs since two equivalents of picolinate are ultimately generated from one equivalent of oxidized and hydrolysed dpeo. The copper-dependent O2 activation and consequent stoichiometric dpeo C-H oxidation is reminiscent of the previously observed catalysis of dpeo oxidation by Mn(ii) [C. Deville, S. K. Padamati, J. Sundberg, V. McKee, W. R. Browne, C. J. McKenzie, Angew. Chem., Int. Ed., 2016, 55, 545-549]. By contrast dpeo oxidation is not observed during complexation reactions with other late transition metal(ii) ions (M = Fe, Co, Ni, Zn) under aerobic conditions. In these cases bis and tris complexes of bidentate dpeo are isolated in good yields. It is interesting to note that dpeo is not oxidised by H2O2 in the absence of Cu or Mn, suggesting that metal-based oxidants capable of C-H activation are produced from the dpeo-Cu/Mn systems and specifically O2. The metastable copper complexes [Cu(dpeo)2](ClO4)2 and [Cu(bpca)(hidpe)](ClO4), along with [NiX2(dpeo)2] (X = Cl, Br), [Ni(dpeo)3](ClO4)2, [Co(dpeo)3](ClO4)3 and the mixed valence complex [FeIIIFe(dpeo-H)3(dpeo)3](PF6)4, have been structurally characterized.

Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation

BackgroundCopper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson’s Disease. Hence, residue specific interaction of Cu2+ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail. MethodsWe investigated the residue specific mapping of Cu2+ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu2+ were studied using fluorescence, CD, and AFM respectively. ResultsCopper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu2+ showed an enhanced rate of fibril formation with a pronounced effect for G51D. ConclusionCu2+ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu2+ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q. General SignificanceThese findings will help in the better understanding of Cu2+ catalyzed aggregation of synucleins.

Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport

Cu+-specific P-type ATPase membrane protein transporters regulate cellular copper levels. The lack of crystal structures in Cu+-binding states has limited our understanding of how ion entry and binding are achieved. Here, we characterize the molecular basis of Cu+ entry using molecular-dynamics simulations, structural modeling, and in vitro and in vivo functional assays. Protein structural rearrangements resulting in the exposure of positive charges to bulk solvent rather than to lipid phosphates indicate a direct molecular role of the putative docking platform in Cu+ delivery. Mutational analyses and simulations in the presence and absence of Cu+ predict that the ion-entry path involves two ion-binding sites: one transient Met148-Cys382 site and one intramembranous site formed by trigonal coordination to Cys384, Asn689, and Met717. The results reconcile earlier biochemical and x-ray absorption data and provide a molecular understanding of ion entry in Cu+-transporting P-type ATPases.

Enhancement of Solubility and Specific Activity of a Cu/Zn Superoxide Dismutase by Co-expression with a Copper Chaperone in Escherichia coli.

Background Human Cu/Zn superoxide dismutase (hSOD1) is an antioxidant enzyme with potential as a therapeutic agent. However, heterologous expression of hSOD1 has remained an issue due to Cu2+ insufficiency at protein active site, leading to low solubility and enzymatic activity. Objectives The effect of co-expressed human copper chaperone (hCCS) to enhance the solubility and enzymatic activity of hSOD1 in E. coli was investigated in the presence and absence of Cu2+. Materials and Methods pETDuet-1-hSOD1 and pETDuet-1-hCCS-hSOD1 were constructed and individually transformed into E. coli strain BL21(DE3). The recombinant hSOD1 was expressed and purified using immobilized metal affinity chromatography. The yield and specific activity of hSOD1 in all conditions were studied. Results Co-expression with hCCS increased hSOD1 solubility at 37°C, but this effect was not observed at 25°C. Notably, the specific activity of hSOD1 was enhanced by 1.5 fold and greater than 3 fold when co-expressed with hCCS at 25°C with and without Cu2+ supplement, respectively. However, the chaperone co-expression did not significantly increase the yield of hSOD1 comparable to the expression of hSOD1 alone. Conclusions This study is the first report demonstrating a potential use of hCCS for heterologous production of hSOD1 with high enzymatic activity.

Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1.

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67mgL-1h-1), higher nitrite accumulation (47.3∼99.8mgL-1), and higher N2O emission ratios (5∼283mgL-1/mgL-1). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05mgL-1) was found, and the absence of Cu resulted in massive N2O emission due to poor synthesis of N2O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5mgL-1)>Cu(II) (0.5∼5mgL-1)>Ni(II) (2∼10mgL-1)>Zn(II) (25∼50mgL-1). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ>nirS≈cnorB>napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

Cu(II) removal by Nostoc muscorum and its effect on biomass growth and nitrate uptake: A photobioreactor study

This study investigated the Cu(II) removal by Nostoc muscorum, a cyanobacterium isolated from a toxic metal polluted site in Meghalaya, using a batch photobioreactor with an aim to elucidate the removal mechanism and its effect on nitrate uptake by the cyanobacterium. Experiments were carried out using a batch photobioreactor by varying the initial Cu(II) concentration in the range of 5–30 mg L−1. Results obtained in this study showed that a maximum of 5.628 ± 0.05 g L−1 of biomass concentration could be obtained in the absence of Cu(II), whereas this value of biomass reduces to 4.3 ± 0.0057 g L−1 in presence of 5 mg L−1 of initial Cu(II) concentration, which further reduces to 2.51 ± 0.01 g L−1 for an initial Cu(II) concentration of 30 mg L−1. Besides, an increase in the initial Cu(II) concentration delayed the uptake of nitrate by the cyanobacterium. However, complete removal of Cu(II) was observed for all the four different initial Cu(II) concentration. The estimated value of nitrate uptake rate in the absence of Cu(II) was found to be 0.115 mg L−1 d−1, which was reduced to 0.029 mg L−1 d−1 due to 30 mg L−1 initial Cu(II) concentration in the medium. The experimental nitrate uptake rate values were fitted to three unstructured kinetic models reported in the literature for estimating the biokinetic constants. Among the three models, Han-levenspiel and Andrew models fitted the experimental data closely with a determination coefficient (R2) value of 0.942 and 0.921, respectively. The critical Cu(II) concentration obtained as per the Han-levenspiel model was found to be 0.0325 g L−1.

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability.

Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function.

Abstract 3914: Repurposing disulfiram for use as an anticancer drug: a story about metabolism and metal binding

Abstract Disulfiram (DSF) is an FDA approved agent for the treatment of alcoholism with over 60 years of clinical use. It was identified in several high-throughput screen to be effective against glioma tumor initiating cells; this activity is amplified more than 100-fold in the presence of copper (Cu2+). DSF is metabolized to diethyldithiocarbamate (DDC), an established copper chelating agent. One mechanism attributed to the copper DDC (Cu(DDC)2) complex is proteosome inhibition. Cu(DDC)2 is very poorly soluble in aqueous solution; making it difficult to purse development for any cancer indication. A novel liposomal formulation of Cu(DDC)2 is described here; which represents the first iv injectable Cu(DDC)2. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio) liposomes were prepared by extrusion methods in the presence of 300 mM unbuffered CuSO4 to ∼100 nm in size. DDC was added to these pre-formed liposomes at room temperature, unentrapped drug was removed using column chromatography. The therapeutic activity of this formulation was determined in vitro against a variety of cancer cell lines. The toxicity and pharmacokinetic (PK) behaviour of this formulation was determined in CD-1 mice. DDC was tested in the presence and absence of Cu2+ in vitro with U251, U87, MDA231-BR and A549 cell lines giving IC50 values greater than >10 μM in the absence and from 300- 900 nM in the presence of Cu2+. The liposomal Cu(DDC)2 formulation was as active as unencapsulated Cu(DDC)2 when tested in vitro. The maximum tolerated dose of liposomal Cu(DDC)2 was determined to be 8mg/kg given iv using a Q2D schedule for two weeks. The PK studies suggested that Cu(DDC)2 was rapidly released from circulating liposomes. Initial efficacy studies were completed using convection enhanced delivery of liposomal Cu(DDC)2 in the F98 rat glioma model. Treatment resulted in a statistically significant delay in tumour growth upon injection of 10 μL of 0.5 mg Cu(DDC)2 /mL solution. Further anti-tumor efficacy studies are being conducted in a subcutaneous leukemia xenograft model (MV-4-11). This work describes a novel formulation of Cu(DDC)2, the active agent generated when administering DSF and Cu to treat cancer. Citation Format: Moe Wehbe, Malathi Anantha, Ian Backstrom, Ada Leung, Kent Chen, Minghan Shi, Armaan Malhotra, Katarina Edwards, Léon Sanche, Marcel B. Bally. Repurposing disulfiram for use as an anticancer drug: a story about metabolism and metal binding. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3914.

A comprehensive study on sunlight driven photocatalytic hydrogen generation using low cost nanocrystalline Cu-Ti oxides

A series of Cu doped titania, CuxTi1−xO2−δ (x=0.0, 0.02, 0.06) and copper oxide-titanium oxide nanocomposites, xCuO-yTiO2 (x:y=1:9, 2:8, 5:5) were synthesized by sol-gel method and characterized by relevant techniques. The role of Cu ions in enhancement of photocatalytic evolution of H2 from H2O-methanol/glycerol mixtures in both sunlight and UV–visible irradiation over Cu/Ti oxides was investigated. X-ray absorption fine structure (XAFS), supported by X-ray diffraction and Raman studies, revealed that Cu substitution in TiO2 stabilized anatase lattice, lengthened Ti-O bonds, decreased the coordination number around Cu ions, and induced oxygen ion vacancies and distortion (σ) in lattice. Distorted structures are more open and flexible with improved charge carrier dynamics and favourable photocatalytic properties. Nanosized Cu-Ti-O powders with enhanced N2-BET surface area and microporosity exhibited improved photocatalytic properties. Reduction of CuO to photocatalytically more active Cu2O by photo-generated e- on the surface of composites was evident by the absence of Cu2+ peak in the XPS spectra of the composite sample after exposure to light. The most active formulations for sunlight assisted photocatalytic H2 generation were Cu0.02Ti0.98O2 for the doped samples, referred to as CuTi(2), and xCuO-yTiO2(x:y=2:8) for the composite samples. Performance of the most active, CuTi(2), was monitored in a up-scaled photoreactor in order to investigate the influence of illumination area, catalyst concentration, form of catalyst (powder/films) and different sacrificial agents on H2 yield. With an aim to identify practical materials for pilot plants, 6mg of CuTi(2) was dispersed on (30cm×0.7cm) ITO/PET films that exhibited enhanced efficiency (3.06%) as compared to same amount of CuTi(2) powder (1.41%). The results showed that the utilization of the CuTi(2) photocatalyst (without costly cocatalysts) with the proper selection of optimum operational conditions under sunlight in a up-scaled photoreactor, generated H2 yield of 1.167L/h/m2 with apparent quantum efficiency, AQE, of 7.5% and solar to fuel efficiency, SFE, of 3.9% for the photocatalytic hydrogen evolution reaction (HER). Our results suggest that with this efficiency, H2 at 1L/h would be evolved photocatalytically over 0.9m2 of CuTi(2) photocatalyst, exposed to sunlight. Inputs obtained from the present study will be useful for further scale up of sunlight driven photocatalytic hydrogen production over low cost and efficient Cu modified TiO2.

Multifunctional quinoline-triazole derivatives as potential modulators of amyloid-β peptide aggregation

Metal ion dyshomeostasis is hypothesized to play a role in the toxicity and aggregation of the amyloid beta (Aβ) peptide, contributing to Alzheimer's disease (AD) pathology. We report on the synthesis and metal complexation ability of three bidentate quinoline-triazole derivatives 3-(4-(quinolin-2-yl)-1H-1,2,3-triazol-1-yl)propan-1-ol (QOH), 4-(2-(4-(quinolin-2-yl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (QMorph), and 4-(2-(4-(quinolin-2-yl)-1H-1,2,3-triazol-1-yl)ethyl)thiomorpholine (QTMorph). We further study the utility of these ligands to modulate Aβ peptide aggregation processes in the presence and absence of Cu2+ ions. Ligand-peptide interactions were first investigated using both 2-D 1H–15N band-selective optimized flip angle short transient heteronuclear multiple quantum correlation (SOFAST-HMQC) NMR spectroscopy and molecular modeling techniques, indicating interactions with glutamic acid (E3) and several residues in the hydrophobic region of Aβ. Native gel electrophoresis with western blotting along with transmission electron microscopy provided information on the ability of each ligand to modulate Aβ aggregation. While the ligands alone did not modify Aβ peptide aggregation at the 24h timepoint, signifying relatively weak ligand-peptide interactions, the ligands did modify the aggregation profile of the peptide in the presence of stoichiometric and suprastoichiometric Cu. Interestingly, the thioether derivative QTMorph exhibited the most pronounced effect on peptide aggregation in the presence of Cu. Overall, the quinoline-triazole ligand series were shown to interact with the hydrophobic region of the Aβ peptide, and modulate the Cu-Aβ aggregation process.

Constant-pH MD Simulations Portray the Protonation and Structural Behavior of Four Decapeptides Designed to Coordinate Cu(2+).

The cyclic decapeptide C-Asp, containing one Asp residue and three His residues, was designed by Fragoso et al. (Chem. Eur. J. 2013, 19, 2076) to bind Cu(2+) exclusively through the side chain groups and mimic copper coordination in metalloproteins. A variant of the cyclodecapeptide where Asp is substituted by Asn (C-Asn) has also been synthesized in addition to the linear ("open") counterparts of both forms (O-Asp and O-Asn), testing the importance of cyclization and the presence of Asp in Cu(2+) coordination (Chem. Eur. J. 2013, 19, 2076; Dalton Trans. 2013, 42, 6182). All peptides formed a major species at neutral pH that was able to coordinate Cu(2+) exclusively through the neutral imidazole groups and the Asp side chain, when present, with C-Asp being the most effective. A detailed description of the protonation behavior of each histidine could help understanding the coordination species being formed in the pH range and eventually further optimizing the peptide's design. However, the standard current methods (NMR titrations) are not very suited for proximal groups titrating in the same pH range. In this work, we used the stochastic titration constant-pH molecular dynamics method to calculate the protonation curves and pKa of each titrable residue in the four decapeptides, in the absence of Cu(2+) ions. The global protonation curves obtained in our simulations are in very good agreement with the existing potentiometric titration curves. The histidines are titrating very closely, and the Asp forms abundant salt bridges with the basic residues, displaying an unusually low pKa value. In addition, we could observe that the four peptides are very unstructured in the absence of copper, and not even the cyclic forms exhibit a significant β-sheet, unlike what could be expected from the presence of β-turn inducer units in this type of scaffold.

Effects of Trace Metal Concentrations on the Growth of the Coral Endosymbiont Symbiodinium kawagutii.

Symbiodinium is an indispensable endosymbiont in corals and the most important primary producer in coral reef ecosystems. During the past decades, coral bleaching attributed to the disruption of the symbiosis has frequently occurred resulting in reduction of coral reef coverage globally. Growth and proliferation of corals require some specific trace metals that are essential components of pertinent biochemical processes, such as in photosynthetic systems and electron transport chains. In addition, trace metals are vital in the survival of corals against oxidative stress because these metals serve as enzymatic cofactors in antioxidative defense mechanisms. The basic knowledge about trace metal requirements of Symbiodinium is lacking. Here we show that the requirement of Symbiodinium kawagutii for antioxidant-associated trace metals exhibits the following order: Fe >> Cu/Zn/Mn >> Ni. In growth media with Cu, Zn, Mn, and varying Fe concentrations, we observed that Cu, Zn, and Mn cellular quotas were inversely related to Fe concentrations. In the absence of Cu, Zn, and Mn, growth rates increased with increasing inorganic Fe concentrations up to 1250 pM, indicating the relatively high Fe requirement for Symbiodinium growth and potential functional complementarity of these metals. These results demonstrate the relative importance of trace metals to sustain Symbiodinium growth and a potential metal inter replacement strategy in Symbiodinium to ensure survival of coral reefs in an oligotrophic and stressful environment.

The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1

Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, KD, of ∼10−14 M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.

Inhibitory effect of human serum albumin on Cu-induced Aβ40 aggregation and toxicity

It has been suggested that the aggregation and cytotoxicity of amyloid-β (Aβ) peptide with transition-metal ions in neuronal cells is involved in the development and progression of Alzheimer's disease (AD). As the most abundant protein in blood plasma and in cerebrospinal fluid, human serum albumin (HSA) can bind Aβ in vivo and subsequently inhibit Aβ fibril growth. However, the roles of albumin in Cu-induced Aβ aggregation and toxicity, and its potential biological relevance to AD therapy, were not stressed enough. Here, we showed that HSA was capable of binding Cu (I) with much higher affinity than Aβ, competitively inhibiting the interaction of Aβ and Cu ions. In the presence of biological reducing agent ascorbate, HSA inhibited Cu (II)/Cu (I)-mediated Aβ40 aggregation, reactive oxygen species production, and neurotoxicity. However, in the absence of Cu (II)/Cu (I), HSA could not effectively inhibit Aβ40 aggregation and neurotoxicity at 24h (or less) incubation time, but decreased Aβ40 aggregation at much longer incubation (120h). Our data suggested that through competitively decreasing Cu–Aβ interaction, HSA could effectively inhibit Cu (II)/Cu (I)-induced Aβ40 aggregation and neurotoxicity, and play important roles in regulating redox balance as well as metal homeostasis in AD prevention and therapy.