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Abstract

In the light of conflicting reports on the ability of copper(II) complexes of amyloid beta (Aβ) peptides to form ternary complexes with small molecules co-present in the biological milieu, we performed a study of coordination equilibria in the system containing Cu(II) ions, the Aβ1–16 peptide, glutamic acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, HEPES) buffer. Using potentiometry, isothermal titration calorimetry (ITC), UV–visible spectroscopy and EPR, we concluded that glutamic acid was not able to form such a ternary complex, but can efficiently compete for the Cu(II) ion with the Aβ peptide at Glu concentrations relevant for the synaptic cleft. We also found that the literature constants for Cu(II) complexes with Glu were overestimated, but this effect was partially compensated by the formation of a ternary Cu(Glu)(HEPES) complex. Our results indicate that small molecules co-present with Cu(II) ions and Aβ peptides in the synaptic cleft are not very likely to enhance Cu(II)/Aβ interactions, but instead should be considered as a Cu(II) buffering system that may help prevent these interactions and participate in Cu(II) clearance from the synaptic cleft.

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... Interestingly, glutamate is released to the same synaptic clefts as Zn(II) ions [80]. Glutamate can compete for Cu(II) from Aβ 1−x [82] but not from Aβ 4−x because of a much higher affinity to Cu(II) of the latter one [66]. However, it can probably effectively compete for Zn(II) with the proposed here mixed 2:1:2 Cu(II)-Zn(II) (Aβ 4−x ) by substituting Glu11 from the peptide. ...
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Amyloid-β (Aβ) peptides are involved in Alzheimer’s disease (AD) development. The interactions of these peptides with copper and zinc ions also seem to be crucial for this pathology. Although Cu(II) and Zn(II) ions binding by Aβ peptides has been scrupulously investigated, surprisingly, this phenomenon has not been so thoroughly elucidated for N-truncated Aβ4−x—probably the most common version of this biomolecule. This negligence also applies to mixed Cu–Zn complexes. From the structural in silico analysis presented in this work, it appears that there are two possible mixed Cu–Zn(Aβ4−x) complexes with different stoichiometries and, consequently, distinct properties. The Cu–Zn(Aβ4−x) complex with 1:1:1 stoichiometry may have a neuroprotective superoxide dismutase-like activity. On the other hand, another mixed 2:1:2 Cu–Zn(Aβ4−x) complex is perhaps a seed for toxic oligomers. Hence, this work proposes a novel research direction for our better understanding of AD development. Graphical Abstract
... The resulting oxidative stress leads to the formation of toxic Ab oligomers and advanced glycation end products (AGEs) [49]. Oxidative processes or Ab copper complexes may affect neurotransmitters oxidation as well as copper homeostasis [50][51]. ...
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Some metal ions of the d-block are necessary for the proper growth, functioning and maintenance of the central nervous system. In particular, copper and zinc ions have emerged as effective non-structural intracellular mediator of cell signaling. The high zinc and copper levels in the synaptic cleft are associated with the shaping of synaptic plasticity and, thus, with learning and memory formation processes. Recent findings identify neurotrophins (NTs) as potential targets of metal ions in the kinase signaling networks of neuronal tissues. Neurotrophins are a class of proteins crucial for the growth and preservation of the central nervous system as they preside over processes/functions like neuroplasticity, memory and learning. A deficit in the expression and activity of these proteins is associated to many neurodegenerative pathologies as Alzheimer and Parkinson’s diseases. Knowing the coordination chemistry of metal ions to NTs is an essential step to identify the basis of NTs/metal ions physiology in learning and memory formation as well as the factors that trigger memory impairment in neurodegeneration, NTs conformations, NTs chemical properties and their biological functions. Unfortunately, characterizing the metal ion coordination of the whole protein is hardly feasible if not impossible. Thus, the design and synthesis of peptides able to mimic the binding and functional sites of proteins may be a viable strategy to explore protein activity and conformational features, metal ion binding as well as to design new molecules for neurodegenerative disorders.
... Instead, we found that these two ligands only competed for Cu(II) at the physiological range of its concentrations. 122 These studies confirmed a pleiotropic and hardly predictable character of Cu(II) interactions with Aβ 1−x peptides. ...
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... Contribution of Glu to the equilibrium distributions of copper and zinc complexes is negligible, as demonstrated by results of calculations presented in Table S1 (ESI †), which were based on published stability constants. 9,[27][28][29] This excludes a dissociative mechanism, and hence suggests an associative one via a ternary complex [Glu-Cu(II)-Ab 4-16 ] (Fig. S13, ESI †). 29 In order to assess possible mechanisms, we replaced Zn 7 MT-3 with EDTA as a copper acceptor. ...
Article
Copper transfer from Cu(II)Amyloid-β4-16 to human Zn7-Metallothionein-3 can be accelerated by glutamate and by lowering the Zn-load of Metallothionein-3 with EDTA. Glutamate facilitates the Cu(II) release, Zn4-6-Metallothionein-3 react faster. These...
... The apparent dissociation constant of Aβ 1−x (x ≥ 16) at pH 7 is ∼0.1 nM (Alies et al., 2013;Young et al., 2014) and thus better described as "moderate, " since other metal-binding species with comparable (or higher) capacity to bind Cu 2+ are also present in the central nervous system (CNS). For example, glutamate also reaches transiently high local concentration during synaptic signaling (Danbolt, 2001), making it competitive at relevant physiological concentrations (Frączyk et al., 2016), while other neurotransmitters such as histamine (HA) have even higher Cu 2+ affinity than glutamate (Dawson et al., 1990). Aside from one study concluding Aβ 1−42 oligomers have an enhanced affinity (K d < 3 pM in HEPES pH 7.4; Jiang et al., 2013) that enables them to compete with human serum albumin (HSA), the latter has also been proposed as a major competitor with Aβ for copper ions within the CNS (Rózga and Bal, 2010), effectively competing for 99.9% of Cu 2+ (Perrone et al., 2010) and binding Cu + stronger than Aβ (Lu et al., 2015). ...
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The “therapeutic chelation” approach to treating Alzheimer’s disease (AD) evolved from the metals hypothesis, with the premise that small molecules can be designed to prevent transition metal-induced amyloid deposition and oxidative stress within the AD brain. Over more than 20 years, countless in vitro studies have been devoted to characterizing metal binding, its effect on Ab aggregation, ROS production, and in vitro toxicity. Despite a lack of evidence for any clinical benefit, the conjecture that therapeutic chelation is an effective approach for treating AD remains widespread. Here, the author plays the devil’s advocate, questioning the experimental evidence, the dogma, and the value of therapeutic chelation, with a major focus on copper ions.
Chapter
Electron paramagnetic resonance spectroscopy in conjunction with site-selective isotopic labelling provides a valuable tool for probing the local coordination environment of paramagnetic metal ions. Sometimes the metal ligands are inter-molecular in origin, such as during biological catalysis involving enzyme/metal-ion/substrate complexes or within oligomeric forms of peptides and proteins. Inter- and intra-molecular metal binding within peptide oligomers can be delineated by combining two fractions of peptide monomers containing the same amino acid sequence but having different isotopic content. Here, the principles of isotopic dilution in EPR spectroscopy are described and simulations provided to demonstrate its application to the identification of Cu²⁺-bridging of peptide oligomers.
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A new fluorescent probe Aβ16wwa based upon the Aβ16 peptide has been developed with two orders of magnitude greater fluorescence intensity for sensitive detection of interactions with Cu(ii). In combination with the Cu(i) probe Ferene S, it is confirmed that the Aβ16 peptide binds either Cu(i) or Cu(ii) with comparable affinities at pH 7.4 (log K = -10.4; log K = -10.0). It follows from this property that the Cu-Aβ16 complex is a robust if slow catalyst for the aerial oxidation of ascorbate with H2O2 as primary product (initial rate, ∼0.63 min(-1) for Cu-Aβ16 versus >2.5 min(-1) for Cuaq(2+)). An integrated study of variants of this peptide identifies the major ligands and binding modes involved in its copper complexes in solution. The dependence of K upon pH is consistent with a two-coordinate Cu(i) site in which dynamic processes exchange Cu(i) between the three available pairs of imidazole sidechains provided by His6, His13 and His14. The N-terminal amine is not involved in Cu(i) binding but is a key ligand for Cu(ii). Acetylation of the N-terminus alters the redox thermodynamic gradient for the Cu centre and suppresses its catalytic activity considerably. The data indicate the presence of dynamic processes that exchange Cu(ii) between the three His ligands and backbone amide at physiological pH. His6 is identified as a key ligand for catalysis as its presence minimises the pre-organisation energy required for interchange of the two copper redox sites. These new thermodynamic data strengthen structural interpretations for the Cu-Aβ complexes and provide valuable insights into the molecular mechanism by which copper chemistry may induce oxidative stress in Alzheimer's disease.
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Amyloid-beta peptides (Abeta) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Abeta and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Abeta aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper-exchange reaction between Abeta and HSA or the tetrapeptide DAHK (N-terminal Cu-binding domain of HSA) and the consequence of this exchange on Abeta-induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu(II) from Abeta rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu-induced aggregation of Abeta, 3) HSA and DAHK suppressed the catalytic HO(.) production in vitro and ROS production in neuroblastoma cells generated by Cu-Abeta and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu-Abeta with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu(II)-HSA than Cu-DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu(II)-Abeta interaction. The repercussion of the competition between HSA and Abeta to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.
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Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Possible errors in the measurement of acid dissociation constants by potentiometric titration techniques have been considered, with particular references to nitrilotriacetic acid (NTA) and ethylenediaminetetraacetic acid (EDTA). Unknown junction potentials can arise when pH measurements are carried out using a glass electrode with saturated calomel reference electrode which have been previously calibrated with a standard buffer solution. The magnitude of the influence of these unknown potentials has been demonstrated and an experimental procedure recommended which gives meaningful results.
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A new computer program has been developed in which formation constants are determined by minimisation of an error-square sum based on measured electrode potentials. The program also permits refinement of any reactant concentration or standard electrode potential. The refinement is incorporated into a new procedure which can be used for model selection.
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Beyreuther K., Multhaup G., Simms G., Pottgiesser J., Schroder W., Martins R. N., and Masters C. L. (1986) Neurofibrillary tangles of alzheimer's disease and “aged” Down's syndrome contain the same protein as the amyloid of plaque cores and blood vessels.Discuss. Neurosci. 3, 68–79, 143–157.
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8-Hydroxyquinolines (8HQ) have found widespread application in chemistry and biology due to their ability to complex a range of transition metal ions. The family of 2-substituted 8HQs has been proposed for use in the treatment of Alzheimer's disease (AD). Most notably, the therapeutic PBT2 (Prana Biotechnology Ltd.) has been shown to act as an efficient metal chaperone, disaggregate metal-enriched amyloid plaques comprised of the Aβ peptide, inhibit Cu/Aβ redox chemistry, and reverse the AD phenotype in transgenic animal models. Yet surprisingly little is known about the molecular interactions at play. In this study, we show that the homologous ligand 2-[(dimethylamino)methyl]-8-hydroxyquinoline (HL) forms a CuL complex with a conditional (apparent) dissociation constant of 0.33 nM at pH 6.9 and is capable of forming ternary Cu(2+) complexes with neurotransmitters including histamine (HA), glutamic acid (Glu), and glycine (Gly), with glutathione disulfide (GSSG), and with histidine (His) side chains of proteins and peptides including the Aβ peptide. Our findings suggest a molecular basis for the strong metal chaperone activity of PBT2, its ability to attenuate Cu(2+)/Aβ interactions, and its potential to promote neuroprotective and neuroregenerative effects.
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Cu(II) binding to the amyloid-β peptide has been proposed to be a key event in the cascade leading to Alzheimer disease. As a direct consequence, the strength of the Cu(II) to Aβ interaction, i.e. the Cu(II) affinity of Aβ, is a very important parameter to determine. Because Aβ peptide contain one Tyr fluorophore in its sequence and because Cu(II) does quench Tyr fluorescence, fluorescence measurements appears to be a straightforward way to obtain this parameter. However, this proved to be wrong, mainly due to misinterpretation of fluorescence experiments in some previous studies that leads to a conflicting situation. In the present paper, we have investigated in details a large new set of fluorescence data that were analyzed with a new method taking into account the presence of two Cu(II) site and the inner-filter effect. This leads to re-interpretation of the published data and to the determination of a unified affinity value in the 1010 M-1 range.
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Copper coexists with amyloid-β (Aβ) peptides at a high concentration in the senile plaques of Alzheimer's disease (AD) patients and has been linked to oxidative damage associated with AD pathology. However, the origin of copper and the driving force behind its accumulation are unknown. We designed a sensitive fluorescent probe, Aβ(1-16)(Y10W), by substituting the tyrosine residue at position 10 in the hydrophilic domain of Aβ(1-42) with tryptophan. Upon mixing Cu(II), Aβ(1-16)(Y10W), and aliquots of Aβ(1-42) taken from samples incubated for different lengths of time, we found that the Cu(II) binding strength of aggregated Aβ(1-42) has been elevated by more than two orders of magnitude with respect to that of monomeric Aβ(1-42). Electron paramagnetic spectroscopic measurements revealed that the Aβ(1-42) aggregates, unlike their monomeric form, can seize copper from human serum albumin (HSA), an abundant copper-containing protein in brain and cerebrospinal fluid. The significantly elevated binding strength of the Aβ(1-42) aggregates can be rationalized by a Cu(II) coordination sphere constituted by three histidines from two adjacent Aβ(1-42) molecules. Our work demonstrates that the copper binding affinity by Aβ(1-42) is dependent on its aggregation state and provides new insight into how and why senile plaques accumulate copper in vivo.
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The potential importance of copper (Cu) in neurosecretion can be inferred from the demonstration that extracellular Cu modulates the secretory function of peptidergic neurons (in vitro studies) and from the presence of high Cu concentrations in nerve terminals and secretory vesicles, primarily within the soluble matrix of the latter. We have previously hypothesized that vesicular Cu is released from neurons undergoing exocytosis and that such extracellular Cu plays an important modulatory role in the central nervous system. To test this Cu release hypothesis, rat hypothalami were incubated under in vitro conditions for 1 or 2 hr with 20 nM radiolabeled Cu (67Cu), and then 67Cu release was stimulated by a depolarizing concentration (60 mM) of K+. K+ markedly (P < 0.001) stimulated 67Cu release in a Ca2+-dependent manner (stimulated release was 95 fmol/10 min/mg protein after 1 hr 67Cu loading and 160 after 2 hr). These amounts of released 67Cu account for about 10% of the total 67Cu taken up by the tissue. These results indicate that part of the 67Cu taken up by hypothalamic explants is directed into an intracellular compartment from where it can be released by a Ca2+-dependent mechanism, thus providing strong support to our hypothesis that release of copper is operative in situ in the brain.
Article
Amyloid-β peptides (Aβ) are key molecules in Alzheimer's disease (AD) pathology as they form amyloid plaques that are primary hallmarks of AD. There is increasing evidence demonstrating that the biometals zinc(ii) and copper(ii) interact with Aβ peptides and have an influence on their fibrillization and toxicity. Zinc and copper ions are abundantly present in the synaptic areas of the brain, and it is likely that the age-related dyshomeostasis of these biometals is associated with AD pathology. In this review we summarize the knowledge of the interactions of zinc and copper ions with Aβ peptides, their role in Aβ fibrillization and toxicity and provide a critical analysis of the conflicting results in the field. Copper ions entrapped in Aβ fibrils are electrochemically active and can generate ROS in the presence of hydrogen peroxide and reducing agents. This might provide a key for understanding the putative role of copper in Aβ toxicity and AD pathology.
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A proposed key event in the pathogenesis of Alzheimer's disease (AD) is the formation of neurotoxic amyloid beta (Abeta) oligomers and amyloid plaques in specific brain regions that are affected by the disease. The main plaque component is the 42 amino acid isoform of Alphabeta (Abeta1-42), which is thought to initiate plaque formation and AD pathogenesis. Numerous isoforms of Abeta, e.g., Abeta1-42, Abeta1-40 and the 3-pyroglutamate derivate of Abeta3-42 (pGluAbeta3-42), have been detected in the brains of sporadic AD (SAD) and familial AD (FAD) subjects. However, the relative importance of these isoforms in the pathogenesis of AD is not fully understood. Here, we report a detailed study using immunoprecipitation in combination with mass spectrometric analysis to determine the Abeta isoform pattern in the cerebellum, cortex and hippocampus in AD, including subjects with a mutation in the presenilin (M146V) or amyloid precursor protein (KM670/671NL) genes, SAD subjects and non-demented controls. We show that the dominating Abeta isoforms in the three different brain regions analyzed from control, SAD, and FAD are Abeta1-42, pGluAbeta3-42, Abeta4-42 and Abeta1-40 of which Abeta1-42 and Abeta4-42 are the dominant isoforms in the hippocampus and the cortex in all groups analyzed, controls included. No prominent differences in Abeta isoform patterns between FAD and SAD patients were seen, underscoring the similarity in the amyloid pathology of these two disease entities.
Article
Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly population, above 65 years of age. Multiple lines of evidence confirm the central role of 40-42 residue Abeta peptides in the pathogenesis of AD, but exact mechanisms of Abeta toxicity remain unclear. Recently, evidence has accumulated in favor of small oligomers of the Abeta42 peptide as major toxic species. Metal ions, copper(II) in particular, have been implicated in molecular mechanisms of Abeta neurotoxicity, including oxidative damage of lipid membranes. While monomeric Abeta peptides are not neurotoxic, the deep understanding of their chemical properties is prerequisite for significant progress in Alzheimer research. Monomeric Abeta40 and Abeta42 form a specific mononuclear complex with Cu(II), recruiting donor atoms within their common 16 amino acid N-terminal sequence. The formation of this complex, the exact structure of which is debated, correlates with increased Abeta toxicity. Human serum albumin (HSA) is a versatile carrier protein present, among others, in blood and cerebrospinal fluid. It binds one Cu(II) ion with a high, picomolar affinity and one Abeta molecule with a moderate, micromolar affinity. In this perspective, we present a model of interactions, which make HSA a likely guardian against Cu/Abeta toxicity in extracellular brain compartments.
Article
Interactions of amyloid beta (Abeta) peptides with Cu(II) are believed to play a crucial role in the molecular mechanisms of neurotoxicity of Alzheimer's disease. There is, however, a serious disagreement regarding the strength of Cu(II) binding to these peptides. We used recombinant amyloid beta peptide 1-40 (Abeta40) to determine the stoichiometry and dissociation constants of Cu(II)-Abeta40 complexes using fluorescence spectroscopy. A single Cu(Abeta40) complex, characterized with the conditional dissociation constant K(d)(cond) = 57 +/- 5 nM was identified. This complex does not bind Hepes buffer molecules, as indicated by the total lack of relationship between K(d)(cond) values and Hepes concentration. The differences between this and other determinations of this constant and its relevance for the understanding of Cu(II) interaction with Abeta peptides are discussed.
Article
Cu(2+) ions are found concentrated within senile plaques of Alzheimer's disease patients directly bound to amyloid-beta peptide (Abeta) and are linked to the neurotoxicity and self-association of Abeta. The affinity of Cu(2+) for monomeric Abeta is highly disputed, and there have been no reports of affinity of Cu(2+) for fibrillar Abeta. We therefore measured the affinity of Cu(2+) for both monomeric and fibrillar Abeta(1-42) using two independent methods: fluorescence quenching and circular dichroism. The binding curves were almost identical for both fibrillar and monomeric forms. Competition studies with free glycine, l-histidine, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10(-11) M, at pH 7.4. Previous studies of Cu-Abeta have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. Abeta fibers are able to bind a full stoichiometric complement of Cu(2+) ions with little change in their secondary structure and have coordination geometry identical to that of monomeric Abeta. Electron paramagnetic resonance studies (EPR) with Abeta His/Ala analogues suggest a dynamic view of the tetragonal Cu(2+) complex, with axial as well as equatorial coordination of imidazole nitrogens creating an ensemble of coordination geometries in exchange between each other. Furthermore, the N-terminal amino group is essential for the formation of high-pH complex II. The Abeta(1-28) fragment binds an additional Cu(2+) ion compared to full-length Abeta, with appreciable affinity. This second binding site is revealed in Abeta(1-42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu(2+) affinity for Abeta of 10(11) M(-1) supports a modified amyloid cascade hypothesis in which Cu(2+) is central to Abeta neurotoxicity.
Article
Numerous conflicting models have been proposed regarding the nature of the Cu(2+) coordination environment of the amyloid beta (Abeta) peptide, the causative agent of Alzheimer's disease. This study used multifrequency CW-EPR spectroscopy to directly resolve the superhyperfine interactions between Cu(2+) and the ligand nuclei of Abeta, thereby avoiding ambiguities associated with introducing point mutations. Using a library of Abeta16 analogues with site-specific (15)N-labeling at Asp1, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu(2+) coordination modes, {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H13)} (component Ia) and {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H14)} (component Ib), between pH 6-7. A third coordination mode (component II) was identified at pH 8.0, and simulation of the superhyperfine resonances indicated a 3N1O coordination sphere involving nitrogen ligation by His6, His13, and His14. No differences were observed upon (17)O-labeling of the phenolic oxygen of Tyr10, confirming it is not a key oxygen ligand in the physiological pH range. Hyperfine sublevel correlation (HYSCORE) spectroscopy, in conjunction with site-specific (15)N-labeling, provided additional support for the common role of His6 in components Ia and Ib, and for the assignment of a {O, N(epsilon)(H6), N(epsilon)(H13), N(epsilon)(H14)} coordination sphere to component II. HYSCORE studies of a peptide analogue with selective (13)C-labeling of Asp1 revealed (13)C cross-peaks characteristic of equatorial coordination by the carboxylate oxygen of Asp1 in component Ia/b coordination. The direct resolution of Cu(2+) ligand interactions, together with the key finding that component I is composed of two distinct coordination modes, provides valuable insight into a range of conflicting ligand assignments and highlights the complexity of Cu(2+)/Abeta interactions.
Article
A new suite of 10 programs concerned with equilibrium constants and solution equilibria is described. The suite includes data preparation programs, pretreatment programs, equilibrium constant refinement and post-run analysis. Data preparation is facilitated by a customized data editor. The pretreatment programs include manual trial and error data fitting, speciation diagrams, end-point determination, absorbance error determination, spectral baseline corrections, factor analysis and determination of molar absorbance spectra. Equilibrium constants can be determined from potentiometric data and/or spectrophotometric data. A new data structure is also described in which information on the model and on experimental measurements are kept in separate files.
Article
Membrane vesicles from rat cerebral cortex were prepared and the functional response of the GABAA receptor was followed by monitoring GABA-activated influx of the radiotracer 36Cl- ion. CuCl2 decreased GABA-activated 36Cl- influx into synaptosomal membrane vesicles. The effect of Cu2+ was concentration dependent (5-500 microM CuCl2) and occurred with saturating (1 mM) as well as low (30 microM) GABA concentrations. A similar inhibition of the responses to muscimol (30 microM) was also observed with 50 microM CuCl2. In addition, release of copper from cortical synaptosomes and median eminence was followed by atomic absorption technique. An increased release of copper into the extracellular space was observed upon depolarization with 50 mM K+. A minimal concentration of copper was estimated to be 100 microM in the synaptic cleft. These findings suggest that copper may play a role in regulating neuronal excitability.
Article
We have purified and characterized the cerebral amyloid protein that forms the plaque core in Alzheimer disease and in aged individuals with Down syndrome. The protein consists of multimeric aggregates of a polypeptide of about 40 residues (4 kDa). The amino acid composition, molecular mass, and NH2-terminal sequence of this amyloid protein are almost identical to those described for the amyloid deposited in the congophilic angiopathy of Alzheimer disease and Down syndrome, but the plaque core proteins have ragged NH2 termini. The shared 4-kDa subunit indicates a common origin for the amyloids of the plaque core and of the congophilic angiopathy. There are superficial resemblances between the solubility characteristics of the plaque core and some of the properties of scrapie infectivity, but there are no similarities in amino acid sequences between the plaque core and scrapie polypeptides.
Article
Brain tissue has a remarkable ability to accumulate glutamate. This ability is due to glutamate transporter proteins present in the plasma membranes of both glial cells and neurons. The transporter proteins represent the only (significant) mechanism for removal of glutamate from the extracellular fluid and their importance for the long-term maintenance of low and non-toxic concentrations of glutamate is now well documented. In addition to this simple, but essential glutamate removal role, the glutamate transporters appear to have more sophisticated functions in the modulation of neurotransmission. They may modify the time course of synaptic events, the extent and pattern of activation and desensitization of receptors outside the synaptic cleft and at neighboring synapses (intersynaptic cross-talk). Further, the glutamate transporters provide glutamate for synthesis of e.g. GABA, glutathione and protein, and for energy production. They also play roles in peripheral organs and tissues (e.g. bone, heart, intestine, kidneys, pancreas and placenta). Glutamate uptake appears to be modulated on virtually all possible levels, i.e. DNA transcription, mRNA splicing and degradation, protein synthesis and targeting, and actual amino acid transport activity and associated ion channel activities. A variety of soluble compounds (e.g. glutamate, cytokines and growth factors) influence glutamate transporter expression and activities. Neither the normal functioning of glutamatergic synapses nor the pathogenesis of major neurological diseases (e.g. cerebral ischemia, hypoglycemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia) as well as non-neurological diseases (e.g. osteoporosis) can be properly understood unless more is learned about these transporter proteins. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity.
Article
Cu is thought to play an important role in the pathogenesis of several neurodegenerative diseases, such as Wilson's, Alzheimer's, and probably in prion protein diseases like Creutzfeld-Jakob's disease. Until now, no method existed to determine the concentration of this cation in vivo. Here, we present two possible approaches combined with a critical comparison of the results. The successful use of fluorescent ligands for the determination of Ca2+-concentrations in recent years encouraged us to seek a fluorophore which specifically reacts to Cu2+ and to characterize it for our purposes. We found that the emission of TSPP (tetrakis-(4-sulfophenyl)porphine) at an emission wavelength of 645 nm is in vitro highly specific to Cu2+ (apparent dissociation constant Kd=0.43 +/- 0.07 microM at pH 7.4). It does not react with the most common divalent cations in the brain, Ca2+ and Mg2+, unlike most of the other dyes examined. In addition, Zn2+ quenches TSPP fluorescence at a different emission wavelength (605 nm) with a Kd of 50 +/- 2.5 microM (pH 7.0). With these findings, we applied the measurement of Cu with TSPP to a biological system, showing for the first time in vivo that there is release of copper by synaptosomes upon depolarisation. Our findings were validated with a completely independent analytical approach based on ICP-MS (inductively-coupled-plasma mass-spectrometry).
Article
The combined potentiometric and spectroscopic studies of interactions of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) with Cu(II) demonstrated that this popular buffer, commonly labelled as "non-coordinating" forms a CuL+ complex, with the logbeta(CuL) value of 3.22. This complex undergoes alkaline hydrolysis above pH 6, resulting in Cu(OH)2 precipitation. However, the presence of HEPES at a typical concentration of 100 mM at pH 7.4 elevates the apparent binding constant, being determined for a complex of another ligand, by a factor of 80. HEPES does not form ternary complexes with aminoacids Ala, Trp, and His, but may do so with other bioligands, such as nucleotides. Therefore, HEPES can still be recommended for Cu(II) studies in place of other common buffers, such as Tris and phosphate, but appropriate corrections and precautions should be applied in quantitative experiments.
Article
Clinicopathological observations suggest there is considerable overlap between vascular dementia (VaD) and Alzheimer's disease (AD). We used immunochemical methods to compare quantities of amyloid-beta (Abeta) peptides in post mortem brain samples from VaD, AD subjects and nondemented ageing controls. Total Abeta peptides extracted from temporal and frontal cortices were quantified using a previously characterized sensitive homogenous time-resolved fluorescence (HTRF) assay. The HTRF assays and immunocapture mass spectrometric analyses revealed that the Abeta(42) species were by far the predominant form of extractable peptide compared with Abeta(40) peptide in VaD brains. The strong signal intensity for the peak representing Abeta(4-42) peptide confirmed that these N-terminally truncated species are relatively abundant. Absolute quantification by HTRF assay showed that the mean amount of total Abeta(42) recovered from VaD samples was approximately 50% of that in AD, and twice that in the age-matched controls. Linear correlation analysis further revealed an increased accumulation with age of both Abeta peptides in brains of VaD subjects and controls. Interestingly, VaD patients surviving beyond 80 years of age exhibited comparable Abeta(42) concentrations with those in AD in the temporal cortex. Our findings suggest that brain Abeta accumulates increasingly with age in VaD subjects more so than in elderly without cerebrovascular disease and support the notion that they acquire Alzheimer-like pathology in older age.
Article
This review covers copper homeostasis and its eventual link with copper involvement in different neurological disorders. Copper plays a dual role in producing reactive oxygen species: damaging the cells and eliciting antioxidant activity. Abnormal binding of metal ions to proteins or losing control over the copper homeostasis can result in disorders including neurodegenerative diseases. In addition to creating oxidative stress, copper, together with other metal ions influences protein aggregation processes that are critical in most of the neurodegenerative diseases.
Article
The conditional stability constant at pH 7.4 for Cu(II) binding at the N-terminal site (NTS) of human serum albumin (HSA) was determined directly by competitive UV–vis spectroscopy titrations using nitrilotriacetic acid (NTA) as the competitor in 100 mM NaCl and 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes). The log K NTSc value of 12.0 ± 0.1 was determined for HSA dissolved in 100 mM NaCl. A false log log K NTSc value of 11.4 ± 0.1 was obtained in the 100 mM Hepes buffer, owing to the formation of a ternary Cu(NTA)(Hepes) complex. The impact of the picomolar affinity of HSA for Cu(II) on the availability of these ions in neurodegenerative disorders is briefly discussed.
Article
There has been steadily growing interest in the participation of metal ions (especially, zinc, copper, and iron) in neurobiological processes, such as the regulation of synaptic transmission. Recent descriptions of the release of zinc and copper in the cortical glutamatergic synapse, and influencing the response of the NMDA receptor underscore the relevance of understanding the inorganic milieu of the synapse to neuroscience. Additionally, major neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease, are characterized by elevated tissue iron, and miscompartmentalization of copper and zinc (e.g. accumulation in amyloid). Increasingly sophisticated medicinal chemistry approaches, which correct these metal abnormalities without causing systemic disturbance of these essential minerals, are being tested. These small molecules show promise of being disease-modifying.
Control experiments showed that Glu did not interact with HEPES and Aβ 1-16 in the absence of Cu(II) ions (Figure S6 in the Supplementary Information file) For the
  • Hepes Samples In
samples in HEPES. Control experiments showed that Glu did not interact with HEPES and Aβ 1-16 in the absence of Cu(II) ions (Figure S6 in the Supplementary Information file). For the
Human serum albumin coordinates
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M. Rózga, M. Sokołowska, A. M. Protas, W. Bal; Human serum albumin coordinates
Studies of blood brain barrier permeability and of intrathecal IgG synthesis in patients with Alzheimer's disease and multi-infarct dementia
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