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Abstract

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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... The Cu II ion is bound there via the N-terminal amine, the first two amides, and the imidazole of His3 (4N, Fig. 1), reaching nearly femtomolar Cu II affinities at pH 7.4. [12][13][14] Such strong binding suggests a potential role of ATCUN Cu II complexes in copper cellular import, which is different from the mechanism known in yeast, where Cu II is reduced to Cu I by a complimentary Cu reductase prior to its binding to yeast Ctr1. 15 The presence of a Cu II -specific binding site in the extracellular domain of hCtr1 may also facilitate the direct shuttling of Cu II ions from extracellular ligands, including Human Serum Albumin (HSA), one of the main Cu II carriers in the blood. ...
... 16 Studies on cell cultures showed that radioactive 64 Cu II loaded to albumin can be acquired by cells. 17 Furthermore, EPR experiments on spin-labelled hCtr1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] , and HSA indicated a close interaction between these molecules. 18 Considering Cu acquisition as a process involving hCtr1 and HSA, one can consider two non-exclusive possibilities (i) a direct Cu II transfer from HSA to hCtr1 or (ii) the reduction of Cu II prior to the binding to hCtr1. ...
... 8,20 Therefore, we decided to re-evaluate the Cu II affinity of the 14 amino acid peptide in the absence of interfering solution components. This hCtr [1][2][3][4][5][6][7][8][9][10][11][12][13][14] peptide is expected to perform as a better model of the extracellular N-terminus of hCtr1 than hCtr1 1-3 due to the presence of additional Cu-binding groups that are adjacent to the ATCUN site, including His5, His6, and Asp13 (see Fig. 1 for sequences). We also attempted to estimate the rate of Cu II transfer from HSA to hCTR1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] . ...
Article
Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 Cu(II) is bound to the model peptide hCtr1_1-14 via an ATCUN motif and such complexes are strong enough to collect Cu(II) from albumin, supporting the potential physiological role of Cu(II) binding to hCtr1.
... The kinetic traces in Figures 1 and 2 show that, as expected, [Cu-Aβ [4][5][6][7][8][9][10][11][12][13][14][15][16] is much less reactive than [Cu-Aβ 1−x ] in the oxidation of DA and MC under both saturating (3 mM- Figure 1) or subsaturating (0.3 mM- Figure 2) conditions. This effect is due to the stabilization of copper(II) trapped by the ATCUN motif, although it is clear from the residual reactivity that [Cu-Aβ [4][5][6][7][8][9][10][11][12][13][14][15][16] is not redox-inert. ...
... The kinetic traces in Figures 1 and 2 show that, as expected, [Cu-Aβ [4][5][6][7][8][9][10][11][12][13][14][15][16] is much less reactive than [Cu-Aβ 1−x ] in the oxidation of DA and MC under both saturating (3 mM- Figure 1) or subsaturating (0.3 mM- Figure 2) conditions. This effect is due to the stabilization of copper(II) trapped by the ATCUN motif, although it is clear from the residual reactivity that [Cu-Aβ [4][5][6][7][8][9][10][11][12][13][14][15][16] is not redox-inert. ...
... These observations indicate that the substrate binds to copper(II) in [Cu II -Aβ x−16 ] complexes and suggest that both Cu II -catechol and [Cu II -Aβ-catechol] species share the spectral feature at 300 nm. In the case of [Cu-Aβ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and free Cu II , the band intensifies with the progress of the reaction, with a parallel trend with product formation, while with [Cu-Aβ 4-16 ] the band initially present decreases with the progress of the reaction. Moreover, a similar decay of the 300 nm band can be also noticed in the reactions of copper(II) complexes with the longer Aβ x−28 fragments in the catechol oxidations ( Figure S5). ...
Article
Full-text available
The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-β peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aβ sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu–Aβ4−x] and [Cu–Aβ1−x] complexes toward dopamine and other catechols. The results show that the CuII–ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [CuII–Aβ–catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the CuI complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu–Aβ4−x reveal that the His-tandem is able to bind CuII ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of CuII bound to the secondary site.
... This site enables high affinity Cu(II) binding 46−49 with Aβ 4−16 binding Cu(II) with K d = 30 fM at pH 7.4. 50 Reduction of CuAβ 4−16 to Cu(I) appears possible, but relies on a dynamic equilibrium with the low affinity secondary Cu(II) binding site (K d = 0.19 μM at pH 7.4) 50 involving His13 and His14, 51 with electron transfer being too slow to observe a current response using conventional voltammetry. 50−52 The proteolysis of CuAβ 1−16 by NEP leads to a transfer of Cu(II) from Aβ 1−16 to Aβ 4−9 and Aβ 12−16 as these fragments are formed. ...
... This site enables high affinity Cu(II) binding 46−49 with Aβ 4−16 binding Cu(II) with K d = 30 fM at pH 7.4. 50 Reduction of CuAβ 4−16 to Cu(I) appears possible, but relies on a dynamic equilibrium with the low affinity secondary Cu(II) binding site (K d = 0.19 μM at pH 7.4) 50 involving His13 and His14, 51 with electron transfer being too slow to observe a current response using conventional voltammetry. 50−52 The proteolysis of CuAβ 1−16 by NEP leads to a transfer of Cu(II) from Aβ 1−16 to Aβ 4−9 and Aβ 12−16 as these fragments are formed. ...
... 53,54 We previously proposed that N-truncated Aβ 4−42 may play an important role in brain copper homeostasis, serving as a metallothionein-independent synaptic Cu(II) scavenger. 50,55,56 The soluble, low-molecular-weight ATCUN motifs generated by NEP from Aβ may augment or modulate this role, 17 because these peptides have similar Cu(II) binding properties but are not prone to aggregation like Aβ 4−42 , which also has affinity for membranes. 57−59 In this study, we therefore characterize in detail the Cu(II) binding properties of Aβ 4−9 and Aβ 12−16 , as well as the Aβ 11−16 and non-ATCUN pAβ 11−16 peptides representative of β′ cleavage products by BACE1. ...
Article
The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4-9 and a minor Aβ12-x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11-x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4-9), Glu-Val-His-His-Gln-Lys-am (Aβ11-16), Val-His-His-Gln-Lys-am (Aβ12-16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4-9), 9.5 fM (Aβ12-16), and 1.8 pM (Aβ11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4-9 and Aβ12-16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4-9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.
... The C K 7.4 value for this complex is 3.0 × 10 13 M −1 , which is about 3000 times more than that for the Aβ 1−x complexes. 181 By directly reacting Cu(Aβ 1−16 ) with apo-Aβ 4−16 , we showed that the Aβ 4−16 peptide takes up the Cu(II) ion from the Aβ 1−16 complex in a fashion similar to that of HSA, that is, instantly and completely (the reaction was completed within several seconds, during the sample mixing), in agreement with the gradient of stability constants and the known kinetic lability of the Cu(II) complex of Aβ 1−16 . 124 The redox properties of the Cu(Aβ 4−16 ) complex were examined in the same study using voltammetric methods, CV and DPV. ...
... This process overlapped with the irreversible oxidation of the Tyr10 phenolic ring to quinone derivatives. 181 Subsequent electrochemical studies were performed for truncated peptides Aβ 4−6 , Aβ 4−8 , and Aβ 4−10 and the modified Aβ 4−10(Y10F) peptide (all amidated Cterminally). All these peptides contained the Cu(II) binding Phe-Arg-His sequence but differed by the presence or absence of Tyr10. ...
... We also investigated the formation of hydroxyl radicals in the presence of Asc by Cu(II) complexes of Aβ 4−16 and Aβ 4−42 peptides at the 1:1 stoichiometry versus CuAβ 1−16 and CuAβ 1−42 as positive controls. 181 The radicals were detected with the APF test. 185 Expectedly, the complexes of Aβ 1−x peptide yielded significant amounts of such radicals, while the Aβ 4−16 and Aβ 4−42 complexes were completely inactive. ...
Article
As life expectancy increases, the number of people affected by progressive and irreversible dementia, Alzheimer’s Disease (AD), is predicted to grow. No drug designs seem to be working in humans, apparently because the origins of AD have not been identified. Invoking amyloid cascade, metal ions, and ROS production hypothesis of AD, herein we share our point of view on Cu(II) binding properties of Aβ4–x, the most prevalent N-truncated Aβ peptide, currently known as the main constituent of amyloid plaques. The capability of Aβ4–x to rapidly take over copper from previously tested Aβ1–x peptides and form highly stable complexes, redox unreactive and resistant to copper exchange reactions, prompted us to propose physiological roles for these peptides. We discuss the new findings on the reactivity of Cu(II)Aβ4–x with coexisting biomolecules in the context of synaptic cleft; we suggest that the role of Aβ4–x peptides is to quench Cu(II) toxicity in the brain and maintain neurotransmission.
... 9−12 Supported by reports on deranged copper metabolism in AD brains and colocalization of copper and aggregated Aβ peptides in amyloid plaques, these properties gave rise to a concept of Cu II −Aβ 1−x complexes as neurotoxic species in AD. 5,13,14 Remarkably, Aβ 4−42 and its C-terminally truncated analogs are Cu II chelators much more avid (3000 times at pH 7.4 for Aβ 4−16 vs Aβ 1−16 ) and specific than Aβ 1−x peptides. 15 This results from a specific character of their N-terminal sequence, Phe-Arg-His, belonging to the ATCUN/NTS family. 16 Furthermore, unlike Cu II −Aβ 1−x complexes, Cu II −Aβ 4−x did not generate ROS and could not be reduced electrochemically to Cu I species. ...
... 16 Furthermore, unlike Cu II −Aβ 1−x complexes, Cu II −Aβ 4−x did not generate ROS and could not be reduced electrochemically to Cu I species. 15 These findings suggest that Aβ 4−42 might actually serve as synaptic copper scavenger, helping restore the resting state of glutamatergic synapse, after the physiological Cu 2+ release during neurotransmission. 17,18 Digestion of Aβ peptides is considered as one of the major routes of their detoxification. ...
... It was not formed in the presence of Cu II (Aβ 4−16 ), because of the log K difference at pH 7.4 in favor of the latter, 10.37 vs 13.53. 15,35 The reaction rates increased with temperature ( Figure S5). The ESI-MS analysis of reaction products indicated the absence of covalent oxidative modification of Aβ 4−16 (Figures S6 and S7). ...
Article
Full-text available
Aβ4-42 is the major subspecies of Aβ peptides characterized by avid Cu(II) binding via the ATCUN/NTS motif. It is thought to be produced in vivo proteolytically by neprilysin, but in vitro experiments in the presence of Cu(II) ions indicated preferable formation of C-terminally truncated ATCUN/NTS species including CuIIAβ4-16, CuIIAβ4-9, and also CuIIAβ12-16, all with nearly femtomolar affinities at neutral pH. Such small complexes may serve as shuttles for copper clearance from extracellular brain spaces, on condition they could survive intracellular conditions upon crossing biological barriers. In order to ascertain such possibility, we studied the reactions of CuIIAβ4-16, CuIIAβ4-9, CuIIAβ12-16, and CuIIAβ1-16 with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found CuIIAβ4-16 and CuIIAβ4-9 to be strongly resistant to reduction and concomitant formation of Cu(I)-GSH complexes, with reaction times ∼10 h, while CuIIAβ12-16 was reduced within minutes and CuIIAβ1-16 within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the CuIIAβ complexes were reformed with no concomitant oxidative damage to peptides. These finding reinforce the concept of Aβ4-x peptides as physiological trafficking partners of brain copper.
... 20 In this work, we aimed to characterize Cu(II) coordination and electrochemical properties of resulting complexes, Aβ 5−x peptides, by using Aβ 5−16 as a suitable well-soluble model, analogously to Aβ 1−16 and Aβ 4−16 model peptides. 15,16 Because of the presence of two metal binding regions in this peptide, one at the N-terminus and another at the His13-His14 couple, we also used shorter peptides, Aβ 5−9 and Aβ 5−12 as simplified models. For a better understanding of the role of Tyr10 in the studied processes, we also used a modified Aβ 5−12 Y10F (Aβ 5−12F ) peptide in some experiments (see Scheme 1 for sequences). ...
... The assignments of proton exchanging groups mainly contributing to given protonation constants are based on previous studies of analogous peptides. 16,29,30 The pK a values are typical for the respective groups and sufficiently well separated to make these assignments unambiguous. 31 The potentiometric titrations performed at various Cu(II)/ peptide ratios for Aβ 5−9 indicated the formation of complexes having solely a 1:1 copper-to-peptide stoichiometry and differing by the number of bound/released hydrogen ions ( Table 2). ...
... This complex is analogous to that observed before for Cu(II)-Aβ 4−16 and corresponds to an independent formation of the second Cu(II) ion at the His13 and His14 residues. 16 As expected, the binding of the second Cu(II) ion at His13/His14 prevented the entry of His13/His14 N im to the coordination sphere of the N-terminal 3N complex. ...
Article
Full-text available
The Aβ5–x peptides (x = 38, 40, 42) are minor Aβ species in normal brains but elevated upon the application of inhibitors of Aβ processing enzymes. They are interesting from the point of view of coordination chemistry for the presence of an Arg-His metal binding sequence at their N-terminus capable of forming a 3-nitrogen (3N) three-coordinate chelate system. Similar sequences in other bioactive peptides were shown to bind Cu(II) ions in biological systems. Therefore, we investigated Cu(II) complex formation and reactivity of a series of truncated Aβ5–x peptide models comprising the metal binding site: Aβ5–9, Aβ5–12, Aβ5–12Y10F, and Aβ5–16. Using CD and UV–vis spectroscopies and potentiometry, we found that all peptides coordinated the Cu(II) ion with substantial affinities higher than 3 × 1012 M–1 at pH 7.4 for Aβ5–9 and Aβ5–12. This affinity was elevated 3-fold in Aβ5–16 by the formation of the internal macrochelate with the fourth coordination site occupied by the imidazole nitrogen of the His13 or His14 residue. A much higher boost of affinity could be achieved in Aβ5–9 and Aβ5–12 by adding appropriate amounts of the external imidazole ligand. The 3N Cu-Aβ5–x complexes could be irreversibly reduced to Cu(I) at about −0.6 V vs Ag/AgCl and oxidized to Cu(III) at about 1.2 V vs Ag/AgCl. The internal or external imidazole coordination to the 3N core resulted in a slight destabilization of the Cu(I) state and stabilization of the Cu(III) state. Taken together these results indicate that Aβ5–x peptides, which bind Cu(II) ions much more strongly than Aβ1–x peptides and only slightly weaker than Aβ4–x peptides could interfere with Cu(II) handling by these peptides, adding to copper dyshomeostasis in Alzheimer brains.
... 37,38 The Aβ 4−16 peptide, a model of the N-terminally truncated Aβ 4−42 isoform, binds to Cu(II) with the relatively high affinity of C K 7.4 = 1.3 × 10 13 (K D = 30 fM) at pH 7.4: This is more than 3 orders of magnitude stronger than Aβ 1−16 binds Cu(II). 22 The dramatic increase in Cu(II) affinity for Aβ 4−16 compared to Aβ 1−16 is the result of a 4N amino-terminal Cu/Ni binding (ATCUN) site that is created upon truncation of the first three amino acids of full-length Aβ. The Aβ 4−16 peptide also contains a second Cu binding site located at the bis-His motif (His13/14) that accommodates either Cu(II) or Cu(I). ...
... 39 Cu(II) that is bound at the high-affinity Aβ 4−16 ATCUN site is redox silent, while Cu binding at the bis-His site is redoxactive. 22,39 The range of affinities and redox activity of Cu-Aβ complexes may allow Aβ to serve as a Cu-buffering system and as an environmentally sensitive prochelator that sequesters Cu(II) with high affinity under conditions of oxidative stress. Several lines of in vitro evidence support the proposal that Aβ, specifically the Aβ 4−x isoforms, may serve to sequester redoxactive Cu. ...
... For example, Aβ 4−16 instantly and completely sequesters Cu(II) from the Cu(II)Aβ 1−16 complex, thus preventing ROS production by Cu(II)Aβ 1−16 . 22 Further, Cu(II)Aβ 4−16 ablates the swapping of Cu/Zn when in competition with Zn 7 MT-3 (a brain protein responsible for controlling the balance of Cu and Zn metabolism). 21 While the Cu/Zn exchange was postulated as a key mechanism controlling the oxidative toxicity of CuAβ 1−x complexes by Zn 7 MT-3, 22 the resistance of Cu(II)Aβ 4−16 to this swap indicates that Aβ 4−x and MT-3 may play parallel roles in the synaptic copper clearance. ...
Article
Amyloid beta (Aβ) peptides are notorious for their involvement in Alzheimer’s disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood–brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aβ peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aβ peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aβ1–16 and Aβ4–16 peptides as well-established non-aggregating models of major Aβ species in healthy and AD brains, and the Ctr1–14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aβ peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aβ1–16 fully and rapidly delivered Cu(II) to Ctr1–14 along the affinity gradient. Such delivery was only partial for the Aβ4–16/Ctr1–14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aβ species released Cu(I) to Ctr1–14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aβ model peptides. These results are discussed in terms of the fundamental difference between the peptides’ Cu(II) complexes (pleiotropic ensemble of open structures of Aβ1–16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aβ4–16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aβ peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.
... Its ATCUN motif was found to bind Cu(II) much stronger than the Aβ1-x peptides (log K at pH 7.4 = 13.5 vs. 10.0). 40 The Cu(II) complexes of Aβ4-16 and other Aβ4-x analogues are redox-inert in the biologically relevant range of potentials (resistant to electrochemical reduction to Cu(I) species down to -0.6 V vs. NHE, oxidized to Cu(III) complexes only at 1.0 V). [52,53] In line with these observations, a very strong resistance of Cu(II)Aβ4-16 to reductively release copper to metallothionein was also reported. [54,55] Cu(II)Aβ4-16 was also kinetically inert in non-redox exchange reactions. ...
... The Aβ4-40 peptide has the N-terminal Asp-Ala-Glu sequence removed, hence its charge is roughly less negative by two units. Using the published pKa values for the C-terminally amidated Aβ4-16 peptide, [52] we found that this is indeed true, at pH 7.4 the Aβ4-40 N-terminus has an average charge of +0.75 (Scheme 3). The whole peptides are still charged negatively, because the 17-40 sequences have net charge of -2 (negative Glu22, Asp23 and C-terminus vs. positive Lys28). ...
... While details of these interactions can be explained only in the course of further experimental and theoretical structural studies, our results reinforce the concept of the catalytic role of the N-termini in the initial stages of Aβ aggregation, and underscore the relevance of electrostatic interactions. [26,27] [52,88,89] Color codes: Blue, single negative; Light blue, partial negative; Black, neutral; Pink, partial positive; Red, single positive; Dark red, double positive. ...
Article
Alzheimer’s Disease (AD) is one of the most common multifactorial diseases characterized by a range of abnormal molecular processes such as the accumulation of extracellular plaques containing the amyloid‐β (Aβ) peptides and dyshomeostasis of copper in the brain. Herein, we investigate the effect of Cu(II) ions on the aggregation of Aβ1‐40, and Aβ4‐40, representing two most prevalent families of Aβ peptides, full length and N‐truncated ones. Both families are similarly abundant in healthy and AD brains. In either of the studied peptides, substoichiometric Cu(II) concentrations accelerated aggregation, while superstoichiometric Cu(II) inhibited the fibril formation, likely by stabilizing oligomers. The addition of either Aβ4‐40 or substoichiometric Cu(II) ions affected the aggregation profile of Aβ1‐40, by yielding shorter and thicker fibrils. The similarity of these two effects can be attributed to the increase of positive charge at the Aβ N‐terminus, which is caused either by Cu(II) complexation or N‐truncation at position 4. Our findings provide a better understanding of the biological Aβ aggregation process as these two Aβ species and Cu(II) ions coexist and interact under physiological conditions.
... On the other hand, Cu(II) complexes of Aβ 4−x peptides are practically redox silent, as demonstrated in a ROS generation assay for Aβ 4−42 and its non-aggregating model Aβ 4−16 [16]. Moreover, the ascorbate activation ability of Cu(II)Aβ 4−16 is marginal compared to that of Cu(II)-Aβ 1−16 [17]. ...
... Moreover, the ascorbate activation ability of Cu(II)Aβ 4−16 is marginal compared to that of Cu(II)-Aβ 1−16 [17]. Furthermore, Cu(II)Aβ 4−x form high-affinity Cu(II) complexes (log K = 13.5 and 14.2 at pH 7.4, for x = 16 and 9, respectively [16,18]). Aβ 4−16 was also able to withdraw Cu(II) ions from Aβ 1−16 immediately and quantitatively [16], but, in contrast with Aβ 1−x peptides, strongly resisted copper transfer to metallothionein-3, except for under highly reducing conditions [19,20]. These properties prompted the concept that Aβ 4−42 may serve a physiological purpose in the maintenance of synaptic transmission as a Cu(II) scavenger, as reviewed recently [21]. ...
... Furthermore, Cu(II)Aβ 4−x form high-affinity Cu(II) complexes (log K = 13.5 and 14.2 at pH 7.4, for x = 16 and 9, respectively [16,18]). Aβ 4−16 was also able to withdraw Cu(II) ions from Aβ 1−16 immediately and quantitatively [16], but, in contrast with Aβ 1−x peptides, strongly resisted copper transfer to metallothionein-3, except for under highly reducing conditions [19,20]. These properties prompted the concept that Aβ 4−42 may serve a physiological purpose in the maintenance of synaptic transmission as a Cu(II) scavenger, as reviewed recently [21]. ...
Article
Full-text available
The Aβ 4−42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aβ 4−16 and Aβ 4−9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aβ 4−x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aβ 4−16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aβ 4−16 , controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1 H-13 C HSQC and 1 H-15 N HSQC NMR experiments, for natural abundance 13 C and 15 N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aβ 4−16 , Pd(II)-Aβ 4−16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aβ complexes.
... The redox chemistry of M-ATCUN derivatives is a useful index due to its wide variety of catalytic activities. So, intense interests in electrochemical studies on M-ATCUN derivatives are described in literature (Gonzalez et al., 2018;Neupane et al., 2013;Mital et al., 2015;Hureau et al., 2011;Wiloch et al., 2017). The redox potential of M n+ /M (nÀ1)+ in M-ATCUN derivatives highly depends on the nature of amino acid at periphery of ATCUN motif and geometry of metal-complexes. ...
... Human copper transporter (hCTR1-14aa) MDH 11.0 1.0 3 10 11 Ab 4-16 (Mital et al., 2015) RFH 13.5 3.2 x 10 13 HP2 N-term (Bal et al., 1997 (Bossu et al., 1977). The other analog, Co-ATCUN derivative, generally, shows Co II /Co III redox couple in their redox chemistry without geometry organization (both are sq. ...
... planar geometry). Such electrochemical studies are performed on variety of designed linear and cyclic ATCUN motifs Cowan, 2005, 2007;Neupane et al., 2013Neupane et al., , 2014Mital et al., 2015;Hureau et al., 2011). The redox chemistry of M-ATCUN derivative is influenced by several factors, such as geometry, stability of M III center, variable amino acid, positioning of amino acid in ATCUN motif, and stereochemical orientation of amino acid residues relative to the M-ATCUN equatorial plane. ...
Article
Full-text available
The designed “ATCUN” motif (amino-terminal copper and nickel binding site) is a replica of naturally occurring ATCUN site found in many proteins/peptides, and an attractive platform for multiple applications, which include nucleases, proteases, spectroscopic probes, imaging, and small molecule activation. ATCUN motifs are engineered at periphery by conjugation to recombinant proteins, peptides, fluorophores, or recognition domains through chemically or genetically, fulfilling the needs of various biological relevance and a wide range of practical usages. This chemistry has witnessed significant growth over the last few decades and several interesting ATCUN derivatives have been described. The redox role of the ATCUN moieties is also an important aspect to be considered. The redox potential of designed M-ATCUN derivatives is modulated by judicious choice of amino acid (including stereochemistry, charge, and position) that ultimately leads to the catalytic efficiency. In this context, a wide range of M-ATCUN derivatives have been designed purposefully for various redox- and non-redox-based applications, including spectroscopic probes, target-based catalytic metallodrugs, inhibition of amyloid-β toxicity, and telomere shortening, enzyme inactivation, biomolecules stitching or modification, next-generation antibiotic, and small molecule activation.
... The Aβ(5-16) peptides possess a His-2 motif in which Cu(II) ions bind to XH by three nitrogen atoms (3N) from the terminal amine, the first amide group and the imidazole ring. The emergence of the 3N binding mode results in higher stability of the complex for Cu(II)-Aβ(5-16) (K=9.55 × 10 12 M -1 at pH 7.4) compared to Cu(II)-Aβ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) complexes. [11] Cu(II) ions binding to Aβ(5-16) undergo both Cu(II)/Cu(III) oxidation and Cu(II)/Cu(I) reduction processes. ...
... [11,12] The process of enzymatic degradation of Aβ(1-x) at positions 4, 11 and 12 also leads to the production of a peptide family with very high affinities for Cu(II) ions (stability constant ~ 10^1 4 M -1 ). [13][14][15][16] Due to the presence of an XYH sequence at their N-terminus, called an amino-terminal copper and nickel motif (in short ATCUN), Cu(II) ions are coordinated in a square-planar geometry, which stabilises the Cu(II) and the Cu(II)/Cu(III) redox process is seen at a potential of ~0.8 V. [8,13] According to a recent report, Cu(II) bonded by Aβ(4-16) and ...
... [11,12] The process of enzymatic degradation of Aβ(1-x) at positions 4, 11 and 12 also leads to the production of a peptide family with very high affinities for Cu(II) ions (stability constant ~ 10^1 4 M -1 ). [13][14][15][16] Due to the presence of an XYH sequence at their N-terminus, called an amino-terminal copper and nickel motif (in short ATCUN), Cu(II) ions are coordinated in a square-planar geometry, which stabilises the Cu(II) and the Cu(II)/Cu(III) redox process is seen at a potential of ~0.8 V. [8,13] According to a recent report, Cu(II) bonded by Aβ(4-16) and ...
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Here we demonstrate a significant difference in redox behaviour of copper complexes with β-amyloids Aβ(11-x) and pAβ(11-x) which are models for important components of senile plaques. A small change in the peptide chain may enhance reactive oxygen species (ROS) formation which can severely damage nerve cells.
... [1][2][3] Previous studies demonstrated that each of them binds to Cu(II) ions, although the N-terminal modifications significantly impact their affinity and Cu(II)-binding sites. [4][5][6] The structures and properties of Cu(II)-Aβ complexes depend mostly on the localization of His residues in the peptide sequence. ...
... 12 The electrochemical Cu(II) oxidation has not been reported for this species at this pH. In contrast, Cu(II) in complex with Aβ 4-x , where His3 plays a dominant role in the metal ion coordination, could be oxidized to Cu(III), 5,11,13,14 but it is highly resistant to the reduction to Cu(I). 15 Interestingly, another form, Aβ 5-x , which contains His2, binds to Cu(II) in such a way that it enables both Cu(II) reduction and Cu(II) oxidation. ...
... The presence of Gly1 increased the Cu(II) oxidation potential (E Cu(II)/Cu(III) ) by 0.03-0.05 V ( Fig. 3 and Table 3) compared to the complex of the maternal RHD sequence of Aβ [5][6][7][8][9] (E Cu(II)/Cu(III) = 1.20 V). Conversely, the lowest E Cu(II)/Cu(III) of 1.18 V was determined for Cu(II)-RHG. ...
Article
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Copper(II) complexes of peptides with a histidine residue at the second position (His2 peptides) provide a unique profile of electrochemical behavior, offering signals of both Cu(II) reduction and Cu(II) oxidation. Furthermore, their structures with vacant positions in the equatorial coordination plane could facilitate interactions with other biomolecules. In this work, we designed a library of His2 peptides based on the sequence of Aβ5-9 (RHDSG), an amyloid beta peptide derivative. The changes in the Aβ5-9 sequence highly affect the Cu(II) oxidation signals, altered further by anionic species. As a result, Cu(II) complexes of Arg1 peptides without Asp residues were chosen as the most promising peptide-based molecular receptors for phosphates. The voltammetric data on Cu(II) oxidation for binary Cu(II)-His2 peptide complexes and ternary Cu(II)-His2 peptide/phosphate systems were also tested for His2 peptide recognition. We achieved a highly promising identification of subtle modifications in the peptide sequence. Thus, we introduce voltammetric measurement as a potential novel tool for peptide sequence recognition.
... To the best of our knowledge, no such data are published for Aβ 4−x . However, they can be critical because 1:1 complex Cu(Aβ 4−x ) is restricted to the first three N-terminal residues, and it excludes both His6 imidazole π nitrogen and N-terminal amine from Zn(II) coordination [66]. Therefore, zinc ions in mixed Cu(II)-Zn(II) complexes with Aβ 4−x may be coordinated in two Cu:Zn:Aβ stoichiometries: (i) 1:1:1, by His6 imidazole τ nitrogen, Glu11, His13, and His14 (analogously to that shown in Fig. 1A), and (ii) 2:1:2, by Glu11 and His14, from two adjacent Aβ monomers (as shown in Fig. 1B). ...
... When a copper ion is reduced in the SOD1 active site, His63 (bridging copper and zinc ions) detaches from the coordination sphere and the remaining three His residues bind Cu(I) in a trigonal planar geometry. Although ATCUN-motif-bound copper was found to be quite redox-inactive [66], it cannot be excluded that under some conditions, the Cu(II)/Cu(I) cycle is more pronounced even in such complex. Indeed, ATCUN-type peptide, GGH, forms a transient 2 N Cu(II) complex (copper ion coordinated by bidentate chelate provided by two nitrogen atoms from the peptide), presumably involved in Cu(II)/Cu(I) cycle [73]. ...
... 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 metal coordination properties of Ab(4-x) and Ab (11-x) fragments are dictated by their N-terminal sequence ( Fig. 10C and F), H 2 N-Xaa-Yaa-His-, which constitute an amino-terminal copper and nickel (ATCUN) motif, 298 as those found in several metal-binding proteins, such as human serum albumin (HSA), histatin-5 (Hst5) and Ctr1. The presence of a His residue in the third position enables a high-affinity Cu 2+ binding via the typical (H 2 N Xaa , N -Yaa , N -His , N Im His ) coordination sphere, yielding a highly stable (5,5,6)-membered chelate ring. ...
... CD and EPR studies show that the first site forms an ATCUN-type complex at the N-terminal sequence, with EPR parameters (g || : 2.183 and A || : 215 Â 10 −4 cm −1 ) that are consistent with Cu 2+ bound to the terminal NH 2 , two deprotonated backbone amides (Arg5 and His6), and the His imidazole group (Fig. 10F). 298 Furthermore, the best fit for the XANES and EXAFS spectra corresponds to a square pyramidal complex with an axial water ligand at 2.36 Å . 300 This high-affinity ATCUN motif is able to effectively compete with Ab(1-x) peptides for Cu 2+ ions, and it is a redox-inert copper-binding site because it harbors negligible hydroxyl radical production, as compared with the full-length Ab. ...
... 300 This high-affinity ATCUN motif is able to effectively compete with Ab(1-x) peptides for Cu 2+ ions, and it is a redox-inert copper-binding site because it harbors negligible hydroxyl radical production, as compared with the full-length Ab. 298 Moreover, the high-abundance of Ab in the brain supports a physiological role for Ab(4-x) species as a Cu 2+ scavenger that prevents ROS formation in the synaptic cleft. 301 Indeed, metallothionein-3 (MT-3), a protein involved in neuroprotection against antioxidant injuries, cannot compete for copper ions with the Ab(4-40/42) isoforms. ...
Chapter
d-block metal ions (Cu¹⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mn²⁺, and Zn²⁺) are essential for the brain; however, disruption of metal ion homeostasis is closely linked to neurodegenerative diseases. Interestingly, many of the proteins that play a key role in neurodegeneration can bind metal ions and, in some cases, impact metal homeostasis. This chapter reviews the role of d-block metal ions in different neurodegenerative diseases, including Prion, Alzheimer’s, Parkinson’s, and Huntington's diseases. For each pathology, the metal-binding properties of the proteins involved are discussed, attempting to link the bioinorganic chemistry of these proteins with the role of metal ions in function and disease. Therapeutic approaches that target metal-protein interactions for each disease are also discussed.
... They have limited solubility in water due to the hydrophobicity of the central and Cterminal Aβ segments, which may fold into a hairpin conformation upon aggregation (Abelein et al., 2014;Baronio et al., 2019). The charged N-terminal segment is hydrophilic 2013; Mital et al., 2015;Wärmländer et al., 2019;, as altered metal concentrations indicative of metal dyshomeostasis are a prominent feature in the brains and fluids of AD patients (Wang et al., 2015;Szabo et al., 2016), and because AD plaques contain elevated amounts of metal ions of e.g. Cu, Fe, and Zn (Beauchemin & Kisilevsky, 1998;Lovell et al., 1998;Miller et al., 2006). ...
... Interestingly, although the role of metal ions in AD pathogenesis remains debated (Duce et al., 2011;Modgil et al., 2014;Chin-Chan et al., 2015;Mital et al., 2015;Adlard & Bush, 2018;Huat et al., 2019;Wärmländer et al., 2019), monovalent ions of the alkali metal lithium [i.e., Li + ions] may provide beneficial effects to patients with neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) (Fornai et al., 2008;Morrison et al., 2013) or AD (Engel et al., 2008;Mauer et al., 2014;Sutherland & Duthie, 2015;Decker & Munoz-Torrero, 2016;Donix & Bauer, 2016;Morris & Berk, 2016;Kerr et al., 2018;Hampel et al., 2019;Kisby et al., 2019;Priebe & Kanzawa, 2020). Lithium salts are commonly used in psychiatric medication, even though it is not understood how the Li + ions affect the molecular mechanisms underlying the psychiatric disorders (Dell'Osso et al., 2016). ...
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Alzheimers disease (AD) is an incurable disease and the main cause of age-related dementia worldwide, despite decades of research. Treatment of AD with lithium (Li) has showed promising results, but the underlying mechanism is unclear. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils. The plaques contain also metal ions of e.g. Cu, Fe, and Zn, and such ions are known to interact with Aβ peptides and modulate their aggregation and toxicity. The interactions between Aβ peptides and Li ions have however not been well investigated. Here, we use a range of biophysical techniques to characterize in vitro interactions between Aβ peptides and Li ions. We show that Li ions display weak and non-specific interactions with Aβ peptides, and have minor effects on Aβ aggregation. These results indicate that possible beneficial effects of Li on AD pathology are not likely caused by direct interactions between Aβ peptides and Li ions.
... 35 Such a sequence beginning by a free N-terminal amine and having a His residue at the third position belongs to the broader family of ATCUN (Amino-Terminal Cu and Ni binding) motifs, and DAHK is the prototypical case. [36][37] Such ATCUN motifs have been reported to lessen Cu(Aβ) induced toxicity, and [38][39][40] some of the Ntruncated Aβ peptides (at positions 4 and 11) do themselves exhibit such motif. 26,[39][40][41][42][43][44][45][46] The GHK peptide and its Cu II complex have been extensively studied concerning their biological function. ...
... [36][37] Such ATCUN motifs have been reported to lessen Cu(Aβ) induced toxicity, and [38][39][40] some of the Ntruncated Aβ peptides (at positions 4 and 11) do themselves exhibit such motif. 26,[39][40][41][42][43][44][45][46] The GHK peptide and its Cu II complex have been extensively studied concerning their biological function. Some of the critical roles of Cu(H-1GHK) and GHK include stimulating wound healing and tissue regeneration. ...
... The charged N-terminal segment is hydrophilic and readily interacts with cationic molecules and metal ions Tiiman et al., 2016;Wallin et al., 2016;Wallin et al., 2017;Owen et al., 2019;, while the hydrophobic C-terminal segment can interact with membranes where Aβ may exert its toxicity (Österlund et al., 2018;Wärmländer et al., 2019). The interactions between Aβ and metal ions are of particular interest (Duce et al., 2011;Wärmländer et al., 2013;Mital et al., 2015;Wärmländer et al., 2019;, as altered metal concentrations indicative of metal dyshomeostasis are a prominent feature in the brains and fluids of AD patients (Wang et al., 2015;Szabo et al., 2016), and because AD plaques contain elevated amounts of metal ions of e.g. Cu, Fe, and Zn (Beauchemin & Kisilevsky, 1998;Lovell et al., 1998;Miller et al., 2006). ...
... Interestingly, although the role of metal ions in AD pathogenesis remains debated (Duce et al., 2011;Modgil et al., 2014;Chin-Chan et al., 2015;Mital et al., 2015;Adlard & Bush, 2018;Huat et al., 2019;Wärmländer et al., 2019), monovalent ions of the alkali metal lithium [i.e., Li + ions] may provide beneficial effects to patients with neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) (Fornai et al., 2008;Morrison et al., 2013) or AD (Engel et al., 2008;Mauer et al., 2014;Sutherland & Duthie, 2015;Decker & Munoz-Torrero, 2016;Donix & Bauer, 2016;Morris & Berk, 2016;Kerr et al., 2018;Hampel et al., 2019;Kisby et al., 2019;Priebe & Kanzawa, 2020). Lithium salts are commonly used in psychiatric medication, even though it is not understood how the Li + ions affect the molecular mechanisms underlying the psychiatric disorders (Dell'Osso et al., 2016). ...
Article
Full-text available
Alzheimer's disease (AD) is an incurable disease and the main cause of age-related dementia worldwide, despite decades of research. Treatment of AD with lithium (Li) has showed promising results, but the underlying mechanism is unclear. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils. The plaques contain also metal ions of e.g. Cu, Fe, and Zn, and such ions are known to interact with Aβ peptides and modulate their aggregation and toxicity. The interactions between Aβ peptides and Li+ ions have however not been well investigated. Here, we use a range of biophysical techniques to characterize in vitro interactions between Aβ peptides and Li+ ions. We show that Li+ ions display weak and non-specific interactions with Aβ peptides, and have minor effects on Aβ aggregation. These results indicate that possible beneficial effects of Li on AD pathology are not likely caused by direct interactions between Aβ peptides and Li+ ions.
... The examples of such sequences are: Asp-Ala-His in human serum albumin (HSA), [6] and Phe-Arg-His in N-truncated human amyloid beta (Aβ) peptide. [7] Both these sequences bind Cu(II) ions strongly, with femtomolar dissociation constants at pH 7.4. [6,7] Interaction of Cu(II) with proteins comprising such sequences is biologically relevant as, e. g., 10 -15 % of plasma copper is kept in the ATCUN motif of HSA. ...
... [7] Both these sequences bind Cu(II) ions strongly, with femtomolar dissociation constants at pH 7.4. [6,7] Interaction of Cu(II) with proteins comprising such sequences is biologically relevant as, e. g., 10 -15 % of plasma copper is kept in the ATCUN motif of HSA. [8] Interestingly, peptides/proteins with histidine residue in the second position of the amino acid sequence (His 2 ) also provide excellent coordination ligands for Cu(II), as was shown for Gly-His-Lys peptide, the wound healing factor. ...
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Link to Free Full Text: ............................................................................................................................................ https://onlinelibrary.wiley.com/share/author/J5YPUTRMDWJNMENJYC8K?target=10.1002/cbdv.202100043 ............................................................................................................................................ Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1, His2, and His3) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2, thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His1, His2, or His3. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.
... The so-called metal hypothesis proposes that this process contributes to disease progression and that the Aβ aggregation process can be prevented by "therapeutic chelation" [9,10]. A deep understanding of differences in Cu(II)-binding properties to various forms of Aβ [4][5][6], together with the knowledge about how they can interact with other copper-binding partners in the brain, is required to assess their relevance to metal homeostasis in healthy and AD brains [11] and to investigate potential chelation-based drugs. Here, complex type and coordination characteristics were analyzed for complex between Cu(II) and model peptide containing ATCUN motif (Aβ12-16-VHHQK-NH 2 ) with the help of ultraviolet-visible spectrometry (UV-Vis) and circular dichroism (CD) [12]. ...
Chapter
Dishomeostasis of Cu(II) ions in the human body is connected with several serious diseases such as Alzheimer’s disease or Wilson’s disease. Therefore, a deep understanding of Cu(II)-binding properties to metal ions carriers, together with the knowledge about how they can interact with other copper-binding partners, e.g., amyloid-β (Aβ), is required to assess their relevance to the brain metal homeostasis. Ultraviolet-visible spectrometry (UV-Vis) and circular dichroism (CD) were used to study the coordination characteristics of Cu(II) with peptide containing the amino-terminal (H2N–Xaa–Yaa–His–) copper-binding (ATCUN) motif (Aβ12–16—VHHQK-NH2) derived from Aβ peptide.
... Dyshomeostasis and miscompartmentalization of metal ions such as Cu(II) and Zn(II) are observed in the brains of AD patients and linked to neurodegeneration. 10 Moreover, metal ions can affect the aggregation and conformation of Ab by directly binding to the peptide. 9,10,15 Based on their physiological roles and reactivity with Ab, metal ions are considered an important part of AD pathogenesis. 16 Research regarding the pathological relationship between metal ions and Ab introduced the concept of metal-Ab as a pathogenic factor based on its potential toxicity and involvement in producing ROS. ...
Article
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Amyloid-β (Aβ) accumulation, metal ion dyshomeostasis, oxidative stress, and cholinergic deficit are four major characteristics of Alzheimer's disease (AD). Herein, we report the reactivities of 12 flavonoids against four pathogenic elements of AD: metal-free and metal-bound Aβ, free radicals, and acetylcholinesterase. A series of 12 flavonoids was selected based on the molecular structures that are responsible for multiple reactivities including hydroxyl substitution and transfer of the B ring from C2 to C3. Our experimental and computational studies reveal that the catechol moiety, the hydroxyl groups at C3 and C7, and the position of the B ring are important for instilling multiple functions in flavonoids. We establish a structure–activity relationship of flavonoids that should be useful for designing chemical reagents with multiple reactivities against the pathological factors of AD.
... Indeed, ascorbate concentration can be measured by UV-visible spectroscopy by monitoring absorbance at 265 nm (ε = 14,500 M −1 cm −1 ). This is a straightforward method, which compares well with fluorescence-based ROS detection method [14,52,[54][55][56]. In presence of copper, dioxygen, ascorbate and other species (such as Aβ peptide or potential drug candidates), the ascorbate consumption rate will provide information on ROS production rate, and thus on oxidative stress produced in this environment. ...
Article
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Abstract: We here report the synthesis of three new hybrid ligands built around the phenanthroline scaffold and encompassing two histidine-like moieties: phenHH, phenHGH and H’phenH’, where H correspond to histidine and H’ to histamine. These ligands were designed to capture Cu(I/II) from the amyloid-� peptide and to prevent the formation of reactive oxygen species produced by amyloid-� bound copper in presence of physiological reductant (e.g., ascorbate) and dioxygen. The amyloid-� peptide is a well-known key player in Alzheimer’s disease, a debilitating and devasting neurological disorder the mankind has to fight against. The Cu-A� complex does participate in the oxidative stress observed in the disease, due to the redox ability of the Cu(I/II) ions. The complete characterization of the copper complexes made with phenHH, phenHGH and H’phenH’ is reported, along with the ability of ligands to remove Cu from A�, and to prevent the formation of reactive oxygen species catalyzed by Cu and Cu-A�, including in presence of zinc, the second metal ions important in the etiology of Alzheimer’s disease. The importance of the reduced state of copper, Cu(I), in the prevention and arrest of ROS is mechanistically described with the help of cyclic voltammetry experiments.
... Together with promoting oxidative stress, Cu potentiates the aggregation and misfolding of Aβ. Cu 2+ ions bind with high affinity to Aβ peptides [75][76][77][78][79] [80]. This interaction increases the proportion of β-sheet and α-helix structures, which promotes Aβ aggregation [81]. ...
Article
Copper (Cu) and iron (Fe) are redox active metals essential for the regulation of cellular pathways that are fundamental for brain function, including neurotransmitter synthesis and release, neurotransmission, and protein turnover. Cu and Fe are tightly regulated by sophisticated homeostatic systems that tune the levels and localization of these redox active metals. The regulation of Cu and Fe necessitates their coordination to small organic molecules and metal chaperone proteins that restrict their reactions to specific protein centres, where Cu and Fe cycle between reduced (Fe2+, Cu+) and oxidised states (Fe3+, Cu2+). Perturbation of this regulation is evident in the brain affected by neurodegeneration. Here we review the evidence that links Cu and Fe dyshomeostasis to neurodegeneration as well as the promising preclinical and clinical studies reporting pharmacological intervention to remedy Cu and Fe abnormalities in the treatment of Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS).
... . Most notably, Ab exists in vivo in various modified forms, with emphasis on N-terminal modified peptides of the type Ab 4Àx . Since these modifications affect the primary copper binding site, they also affect the K d of copper and can reach much higher affinity than the full peptide and thus be functionally important [198][199][200][201][202][203]. ...
Article
In this perspective we list the many clinical, histopathological, genetic and chemical observations relating copper to Alzheimer’s disease (AD). We summarize how the coordination chemistry of the APP/Aβ system is centrally involved in neuronal copper transport at the synapses, and that genetic variations in the gene coding for the copper transporter ATP7B cause a subset of AD, which we call CuAD. Importantly, the distinction between loss of function and gain of toxic function breaks down in CuAD, because copper dyshomeostasis features both aspects directly. We argue that CuAD can be described by a single control variable, a critical, location-dependent copper dissociation constant, Kdc. Loss of functional copper from protein-bound pools reduces energy production and oxidative stress control and is characterized by a reduced pool of divalent Cu(II) with Kd < Kdc. Gain of redox-toxic function is described by more copper with Kd > Kdc. In the blood, the critical threshold is estimated to be Kdc ∼10⁻¹² M whereas at synapses it is argued to be Kdc ∼10⁻⁹ M. The synaptic threshold is close to the values of Kd for Cu(II)-binding to Aβ, prion protein, APP, and α-synuclein, implied in copper buffering at the synapses during glutamatergic transmission. The empirical support for and biochemical and pathological consequences of CuAD are discussed in detail.
... The accessibility of a Cu + site might determine the origin of the "ascorbate oxidase" activity and ROS production observed for both Cu(BSA) [24] and Cu(HSA). [17,25] Reduction of Cu(HSA) by ascorbate has recently been proposed as a possi- Figure 2. A) UV/Vis spectrum, B) CD spectrum, and C) species distribution plot of the pH-dependent Cu 2 + complexes formed by GGH (0.8 mm CuCl 2 , 0.95 mm GGH). The pH dependence of selected parameters from UV/Vis (685 nm) and CD (305, 580 nm) spectra is overlaid with the species distribution in (C). ...
Article
The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log c K Cu Cu ( HSA ) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.
... [71][72][73][74][75][76][77] The Cu 2+ ion binds Ab with high affinity where the metal-peptide complex displays some peroxidase activity and in the reduced state generates ROS by reacting with molecular oxygen (Scheme 1). [78][79][80][81][82][83][84][85] This can potentially introduce oxidative stress in the brain and lead to enhanced cell toxicity. In fact, both Ab and APP have Cu binding sites. ...
Article
Full-text available
The amyloid cascade theory attributes the neurodegeneration observed in Alzheimer’s disease (AD) to the deposition of amyloid β (Aβ) peptide into plaques and fibrils in the AD brain. The metal ion hypothesis which implicates several metal ions, viz. Zn, Cu and Fe in the AD pathology on account of their abnormal accumulation in the Aβ plaques along with an overall dyshomeostasis of these metals in the AD brain has been forwarded a while back. Metal ion chelators and ionophores, put forward as possible drug candidates for AD, are yet to succeed in clinical trials. Heme, which is widely distributed in the mammalian body as the prosthetic group of several important proteins and enzymes, has been thought to be associated with AD by virtue of its colocalization in the Aβ plaques along with similarity of several heme deficiency symptoms with those of AD and most importantly, due to its ability to bind Aβ. This feature article illustrates the active site environment of heme-Aβ which resembles those of peroxidases. It also discusses the peroxidase activity of heme-Aβ, its ability to effect oxidative degradation of neurotransmitters like serotonin and also the identification of the highly reactive high-valent intermediate, compound I. The effect of second sphere residues on formation and peroxidase activity of heme-Aβ along with the generation and decay of compound I is highlighted throughout the article. The reactivities of heme bound Aβ peptides give an alternate theory to understand the possible cause of this disease.
... The Cu II (P) complexes were also studied using cyclic voltammetry (CV) (Figures 2, S3, and S4). In the cathodic scan, no processes were observed for the Cu II complex made with one-His peptides; one irreversible anodic process was observed at about −1.0 V vs. SCE (−0.76 V vs. NHE) for the four Cu II complexes constructed using the double-His peptides, which was in line with previously reported data [39,42,50,56,59]. For the two-His-containing Cu II (GHHW) and Cu II (WHHG), an additional cathodic peak was detected at about -0.5 V vs. SCE (0.74 V vs. NHE) (Figures 2 and S3). ...
Article
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Amino-terminal CuII and NiII (ATCUN) binding sequences are widespread in the biological world. Here, we report on the study of eight ATCUN peptides aimed at targeting copper ions and stopping the associated formation of reactive oxygen species (ROS). This study was actually more focused on Cu(Aβ)-induced ROS production in which the Aβ peptide is the “villain” linked to Alzheimer’s disease. The full characterization of CuII binding to the ATCUN peptides, the CuII extraction from CuII(Aβ), and the ability of the peptides to prevent and/or stop ROS formation are described in the relevant biological conditions. We highlighted in this research that all the ATCUN motifs studied formed the same thermodynamic complex but that the addition of a second histidine in position 1 or 2 allowed for an improvement in the CuII uptake kinetics. This kinetic rate was directly related to the ability of the peptide to stop the CuII(Aβ)-induced production of ROS, with the most efficient motifs being HWHG and HGHW.
... This result underlines the potential of peptide ligands in the search for Cu-targeting therapeutic approaches in the context of AD. It is also interesting to note that for all the previous ATCUN peptides studied, only experiments where the peptide is added to Cu II (Aβ) and then the ROS production is triggered by addition of Asc have been reported [67][68][69][70][71][72][73]. Once formed, Cu II (ATCUN) complexes have a very low ability to form ROS, as shown here and previously [55,56,87]. ...
Article
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The progressive, neurodegenerative Alzheimer’s disease (AD) is the most widespread dementia. Due to the ageing of the population and the current lack of molecules able to prevent or stop the disease, AD will be even more impactful for society in the future. AD is a multifactorial disease, and, among other factors, metal ions have been regarded as potential therapeutic targets. This is the case for the redox-competent Cu ions involved in the production of reactive oxygen species (ROS) when bound to the Alzheimer-related Aβ peptide, a process that contributes to the overall oxidative stress and inflammation observed in AD. Here, we made use of peptide ligands to stop the Cu(Aβ)-induced ROS production and we showed why the AHH sequence is fully appropriate, while the two parents, AH and AAH, are not. The AHH peptide keeps its beneficial ability against Cu(Aβ)-induced ROS, even in the presence of ZnII-competing ions and other biologically relevant ions. The detailed kinetic mechanism by which AHH could exert its action against Cu(Aβ)-induced ROS is also proposed.
... (depending on the C-terminal extension). [85,86] Due to their abundance, high affinity and redox inertness, a role as Cu 2+ carrier or synaptic Cu 2+ scavenger has been postulated for such N-truncated species. [84] Indeed, it was lately reported that Cu 2+ -Aβ 4-x is only slowly reduced by physiological concentrations of GSH and hence it could shuttle Cu across the BBB. ...
Article
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Copper (Cu) is an essential micronutrient for most organisms and serves mainly as a redox-active catalytic centre in enzymes cycling between Cu+ and Cu2+. Membrane transporters and shuttles are involved to bring and insert Cu into these enzymes and to control tightly the copper metabolism, whose failure can lead to severe diseases. The main oxidation state intracellularly is Cu+, whereas Cu2+ is mainly found in extracellular fluids. A basic approach to investigate Cu metabolism in vivo and in cellulo contemplates the use of luminescent (mostly fluorescent) or magnetic resonance imaging (MRI)-active probes. Here, we focus on sensors of the Cu2+ state. First, Cu metabolism and speciation are revised, focusing on the main extracellular fluids (blood plasma, urine, cerebrospinal fluid, synaptic cleft, milk, saliva, sweat) and cell culture media, and highlighting the notion of exchangeable Cu2+ pool. Indeed, in contrast to bulk Cu measurements, sensors can only detect Cu2+ that is labile and thermodynamically accessible. Thus, the kinetics and thermodynamics of the exchangeable pools determine the quantity of Cu2+ that can be measured and influence the design of the sensor. The study of the best-known exchangeable Cu2+ pool in blood plasma, i.e. serum albumin, shows that a sensor might need a sub-femtomolar affinity for Cu2+ to compete with endogenous Cu2+ ligands. The selectivity of the probe for Cu2+ is also discussed, in particular against Zn2+, which is much more available than Cu2+ in the extracellular fluids (e.g. at least 106 times in the blood). Finally, the analysis of the literature on luminescent and MRI-active Cu2+ sensors applied in extracellular media show indeed how challenging such measurement is, and that none of the sensors reported convincingly and specifically detects Cu2+ in a biological system. Indeed, when considering all the sought parameters, i.e. thermodynamics and kinetics of the Cu2+-sensor, the specificity towards Cu2+, the reversibility, the sensitivity of the luminescent or MRI response and hence the required sensor concentration, it becomes clear that this is a huge challenge and that we stand just at the dawn of this field.
... Amino acid interactions with copper and iron may also play a crucial role in preventing oxidative damage and diseases that arise due to oxidative stress. Loss of metal homeostasis, mitochondrial malfunction, and the resulting oxidative stress is linked to neurodegenerative disease development, but the mechanistic details that cause this oxidative damage is poorly understood [8][9][10]. Labile copper and iron produce reactive oxygen species (ROS) such as hydroxyl radical that can damage nucleic acids, proteins, and lipids, and this oxidative damage is catalytic in cells (Fig. 1) [11][12][13]. ...
Article
Sulfur- and selenium-containing amino acids are of great biological importance, but their metal-binding properties with biologically-relevant metal ions are not well investigated. Stability constants of the methionine, selenomethionine, methylcysteine, and methylselenocysteine with Cu(II) and Fe(II) were determined by potentiometric titration. Stability constants of Cu(II) with these thio- and selenoether amino acids are in the range of 8.0–8.2 ([CuL] ⁺ ) and 14.5–14.7 (CuL 2 ) (L = amino acid). Fe(II) interactions with the same thio- and selenoether amino acids are much weaker, with stability constants between 3.5 and 3.8 ([FeL] ⁺ ) and −4.9 and −5.7 (FeL(OH)). Stability of Fe(II) with penicillamine, a thiol-containing amino acid, is much higher (FeL = 7.48(7) and [FeL] ²⁻ = 13.74(2)). For both copper and iron complexes, thio- and selenoether amino acid coordination occurs through the carboxylate and the amine groups as confirmed by infrared spectroscopy, with no stability afforded by thio- or selenoether coordination. The first single-crystal structure of Cu(II) with a selenium-containing amino acid, Cu(SeMet) 2 , also confirms binding through only the amine and carboxylate groups. The measured Cu(II)-amino-acid stability constants confirm that nearly 100% of the available Cu(II) can be coordinated by these amino acids at pH 7, but very little Fe(II) is bound under these conditions. The relative instability of Fe(II) complexes with thio- and selenoether amino acids is consistent with their inability to prevent metal-mediated oxidative DNA damage. In contrast, the stability constants of these amino acids with Cu(II) weakly correlate to their ability to inhibit DNA damage inhibition.
... Zinc is another essential trace element that plays a key role in neurotransmission and redox regulation (Grochowski et al., 2019). Amyloid beta plaques have high affinity to trace metals copper and zinc and have thus been found to contain high concentrations of these trace metals (Bush et al., 1994;Atwood et al., 1998;Lovell et al., 1998;Sayre et al., 2000;Suh et al., 2000;Cherny et al., 2001;Dong et al., 2003;Miller et al., 2006;Mital et al., 2015;Ejaz et al., 2020). For instance, a 339% increase in Zn and a 466% increase in Cu were found in amyloid beta plaques of AD patients in comparison to healthy subjects (Leskovjan et al., 2009). ...
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Alzheimer's Disease (AD), a progressive neurodegenerative disease characterized by the buildup of amyloid-beta (Aβ) plaques, is believed to be a disease of trace metal dyshomeostasis. Amyloid-beta is known to bind with high affinity to trace metals copper and zinc. This binding is believed to cause a conformational change in Aβ, transforming Aβ into a configuration more amenable to forming aggregations. Currently, the impact of Aβ-trace metal binding on trace metal homeostasis and the role of trace metals copper and zinc as deleterious or beneficial in AD remain elusive. Given that Alzheimer's Disease is the sixth leading cause of adult death in the U.S., elucidating the molecular interactions that characterize Alzheimer's Disease pathogenesis will allow for better treatment options. To that end, the model organism C. elegans is used in this study. C. elegans, a transparent nematode whose connectome has been fully established, is an amenable model to study AD phenomena using a multi-layered, interconnected approach. Aβ-producing and non-Aβ-producing C. elegans were individually supplemented with copper and zinc. On day 6 and day 9 after synchronization, the percent of worms paralyzed, concentration of copper, and concentration of zinc were measured in both groups of worms. This study demonstrates that dyshomeostasis of trace metals copper or zinc triggers further trace metal dyshomeostasis in Aβ-producing worms, while dyshomeostasis of copper or zinc triggers a return to equilibrium in non-Aβ-producing worms. This supports the characterization of Alzheimer's Disease as a disease of trace metal dyshomeostasis.
... Complexation to peptides is proposed to play important roles in Cu(II) physiology and toxicology. Cu(II) activates GHK, a wound healing factor 1,2 and α-factor, a yeast pheromone, 3,4 and is likely transported to neurons by neurokinin B. 5 It also elicits toxicity and probably gets detoxified by some variants of Aβ peptides 6,7 and protamine HP2 8−10 and likely participates in the antifungal action of histatins, salivary antimicrobial peptides. 11 A recent study indicated that more peptides with such properties remain to be identified in human proteome. ...
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The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH–jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)–peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.
Thesis
Alzheimer's disease is a neurodegenerative disease, affecting more than 30 million people all over the world. Nowadays, only symptomatic therapies exist, there is no cure yet. A dyshomeostasis of metal ions such as Cu and Zn ions in some areas of the brain is one of the different hypothesis about this disease. They would promote an accumulation of peptides, the Amyloid-ß (Aß) peptides, in the synaptic cleft. These aggregates would prevent the neuronal connections, triggering known symptoms of the disease, such as memory loss or cognitive impairments. Cu ions would also be responsible for an important oxidative stress, destroying the neuronal membranes for example. Cu ions are an important therapeutic target to cure the disease. Investigations are currently focusing on the development of new molecules, called chelators, in order to remove selectively Cu ions (over Zn ions), to regulate their concentrations and avoid the accumulation of the peptides. My research project focuses precisely on such kind of investigations. Different Cu(II) and Cu(I) chelators are studied, in the presence or not of Zn(II), in order to understand the different criteria to take into account for the development of good chelators. Different proof-of-concepts are developed in the first part. The kinetic aspect of the removal of Cu(II) from the Aß peptide by a chelator is studied with macrocyclic ligands Then, the redox state of Cu ions in the synaptic cleft staying unknown, two Cu(I) or Cu(I/II) chelators are proposed. The second part of the study takes into account the impact of Zn(II) in the Cu chelation. The thermodynamic part of the Cu(II) chelation in the presence of Zn(II) is evidenced with different chelators.
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The amyloid-β (Aβ) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer's disease. The N-terminal end of Aβ is variable, and different proportions of these variable-length Aβ peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aβ starting at position 4 (Aβ4-x) has a His residue as the third amino acid (His6 using the formal Aβ numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu(II). This motif is not present in the commonly studied Aβ1-x peptides. In addition to the ATCUN site, Aβ4-x contains an additional metal binding site located at the tandem His residues (bis-His at His13 and 14) which is also found in other isoforms of Aβ. Using the ATCUN and bis-His motifs, the Aβ4-x peptide is capable of binding multiple metal ions simultaneously. We confirm that Cu(II) bound to this particular ATCUN site is redox silent, but the second Cu(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu2Aβ4-x state. The identification of two functionally different copper binding environments within the Aβ4-x sequence may have important implications for this peptide in vivo.
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Alzheimer’s disease (AD) is a multifaceted disease that is characterized by increased oxidative stress, metal-ion dysregulation, and the formation of intracellular neurofibrillary tangles and extracellular amyloid-β (Aβ) aggregates. In this work we report the large affinity binding of the iron(III) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole complex FeL1 to the Aβ peptide (Kd ~ 10-7) and the ability of the bound FeL1 to act as a catalytic antioxidant in both the presence and absence of Cu(II) ions. Specific findings are that: a) an Aβ histidine residue binds axially to FeL1; b) that the resulting adduct is an efficient catalase; c) this interaction restricts the formation of high molecular weight peptide aggregates. UV-Vis and electron paramagnetic resonance (EPR) studies show that although the binding of FeL1 does not influence the Aβ-Cu(II) interaction (Kd ~ 10-10), bound FeL1 still acts as an antioxidant thereby significantly limiting reactive oxygen species (ROS) generation from Cu-Aβ. Overall, FeL1 is shown to bind to the Aβ peptide, and modulate peptide aggregation. In addition, FeL1 forms a ternary species with Aβ-Cu(II) and impedes ROS generation, thus showing the promise of discrete metal complexes to limit the toxicity pathways of the Aβ peptide.
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Metal ion interactions with weakly coordinating ligands, such as amino acids, are dependent on several factors, including metal ion availability, metal ion propensity for hydrolysis, ligand availability, and thermodynamic stability, as measured by stability constants. Metal ions in biological systems are often controlled by highly specific chaperone, transport, and storage proteins. Disruption in the homeostasis of redox active metal ions, such as Cu(I), Cu(II), Fe(II), and Fe(III), has been linked to increased oxidative damage and disease. Weakly binding ligands such as amino acids may play an active role in mitigating this metal-mediated damage, but a comprehensive understanding of the availability and thermodynamic likelihood of coordination must be understood to accurately predict complex formation in a competitive environment. This review presents an overview of amino acid stability constants with Cu(I), Cu(II), Fe(II), and Fe(III), the most common redox-active metal ions in biological systems. Specific attention is given to sulfur- and selenium-containing amino acids, since their interactions with Cu(I) and Fe(II) is of particular biological interest. This review also describes methods available for stability constant determination, with particular attention to specific difficulties encountered when working with weakly binding ligands and each of these four metal ions. Finally, the potential biological implications of these results are discussed based on reported stability constants as well as amino acid, copper, and iron bioavailability.
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It was shown that His3 of human copper transporter 1 (hCtr1) prompts the ATCUN-like Cu(II) coordination for model peptides of the hCtr1 N-terminus. Its high Cu(II) affinity is a potential driving force for the transfer of Cu(II) from extracellular Cu(II) carriers to hCtr1. Having a sequence similar to that of hCtr1, hCtr2 has been proposed as another human copper transporter. However, the N-terminal domain of hCtr2 is much shorter than that of hCtr1, with different copper binding motifs at its N-terminus. Employing a model peptide of the hCtr2 N-terminus, MAMHF-am, we demonstrated that His4 provides a unique pattern of Cu(II) complexes, involving Met sulfurs in their Cu(II) coordination sphere. The affinity of Cu(II) for MAMHF-am is a few orders of magnitude lower than that reported for the hCtr1 model peptides at the extracellular pH of 7.4, suggesting a maximal complementary role of Cu(II) binding to hCtr2 in the import of copper from the extracellular space to the cytoplasm. On the other hand, the ability of the hCtr2 model peptide to capture Cu(II) from amino acids and short peptides (potential degradation products of proteins) at pH 5.0 and the known predominant lysosomal localization of hCtr2 support an important potential role of the Cu(II)–hCtr2 interaction in the recovery of copper from lysosomes.
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Alzheimer’s disease (AD) is an irreversible, age-related progressive neurological disorder, and the most common type of dementia in aged people. Neuropathological lesions of AD are neurofibrillary tangles (NFTs), and senile plaques comprise the accumulated amyloid-beta (Aβ), loaded with metal ions including Cu, Fe, or Zn. Some reports have identified metal dyshomeostasis as a neurotoxic factor of AD, among which Cu ions seem to be a central cationic metal in the formation of plaque and soluble oligomers, and have an essential role in the AD pathology. Cu-Aβ complex catalyzes the generation of reactive oxygen species (ROS) and results in oxidative damage. Several studies have indicated that oxidative stress plays a crucial role in the pathogenesis of AD. The connection of copper levels in AD is still ambiguous, as some researches indicate a Cu deficiency, while others show its higher content in AD, and therefore there is a need to increase and decrease its levels in animal models, respectively, to study which one is the cause. For more than twenty years, many in vitro studies have been devoted to identifying metals’ roles in Aβ accumulation, oxidative damage, and neurotoxicity. Towards the end, a short review of the modern therapeutic approach in chelation therapy, with the main focus on Cu ions, is discussed. Despite the lack of strong proofs of clinical advantage so far, the conjecture that using a therapeutic metal chelator is an effective strategy for AD remains popular. However, some recent reports of genetic-regulating copper transporters in AD models have shed light on treating this refractory disease. This review aims to succinctly present a better understanding of Cu ions’ current status in several AD features, and some conflicting reports are present herein.
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The amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards Cu II ions and have been implicated in human copper physiology. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped‐flow and microsecond freeze‐hyperquenching (MHQ) techniques supported by steady‐state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate Cu II complexes formed by glycyl‐glycyl‐histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two‐nitrogen coordination, t ½ ∼100 ms at pH = 6.0 and the ability to maintain the Cu II /Cu I redox pair is the best candidate for the long‐sought reactive species in extracellular copper transport.
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ATCUN/NTS motifs participate in physiological CuII exchange. Using kinetic methods, spectroscopy, and electrochemistry, it was demonstrated that CuII binding to GGH, an ATCUN/NTS representative, proceeds via partially coordinated species. The 2N‐coordinated complex with t 1/2≈100 ms (pH 6.0) and CuII/CuI redox activity is the long‐sought reactive intermediate for extracellular copper delivery. Abstract The amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards CuII ions and have been implicated in human copper physiology. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped‐flow and microsecond freeze‐hyperquenching (MHQ) techniques supported by steady‐state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate CuII complexes formed by glycyl–glycyl–histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two‐nitrogen coordination, t 1/2≈100 ms at pH 6.0 and the ability to maintain the CuII/CuI redox pair is the best candidate for the long‐sought reactive species in extracellular copper transport.
Article
Although Alzheimer's disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The "amyloid cascade hypothesis" proposes that the amyloid β (Aβ) peptide is central to disease pathology, which is supported by elevated Aβ levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The "metals hypothesis" proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aβ, and metal ion chelators have been shown to reverse this reaction in vitro. 8-Hydroxyqinoline-based chelators showed early promise as anti-Alzheimer's drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aβ(1-42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered ∼83% of the Cu(II) from Aβ(1-42), whereas PBT2 sequestered only ∼59% of the Cu(II) from Aβ(1-42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aβ(1-42) species in solution, leaving a single Cu(II)Aβ(1-42) species. It follows that the Cu(II) site in this Cu(II)Aβ(1-42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aβ(1-42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aβ(1-42).
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The in vitro Cu(Aβ 1‐x ) induced ROS production has been extensively and thoroughly studied. Conversely, the ability of N‐truncated isoforms of Aβ to alter the Cu‐induced ROS production has been overlooked even though they are main constituents of amyloid plaques found in the human brain. N‐truncated peptides at the positions 4 and 11 (Aβ 4‐x and Aβ 11‐x ) contain an amino‐terminal copper and nickel (ATCUN) binding motif (NH 2 ‐Xxx‐Zzz‐His) that confer them different coordination sites and higher affinities for Cu(II) compared to the Aβ peptide. It has further been proposed that the role of Aβ 4−x peptide is to quench Cu(II) toxicity in the brain. However, the role of Cu(I) coordination has never been investigated so far. In contrast to Cu(II), the Cu(I) coordination is expected to be the same for N‐truncated and N‐intact peptides. Here, we report in‐depth spectroscopic characterizations (Cu(II) and Cu(I)) complexes of the Aβ 4‐16 and Aβ 11‐16 N‐truncated peptides and ROS production studies with copper (Cu(II) and Cu(I)) complexes of the Aβ 4‐16 and Aβ 11‐16 N‐truncated peptides. Our findings show that the N‐truncated peptides do produce ROS when Cu(I) in present in the medium, although to a lesser extent than the unmodified counterpart. In addition, when used as a drug‐candidate molecule ( id est , in the presence of Aβ 1‐16 ), the N‐truncated peptides are not able to fully preclude Cu(Aβ 1‐16 ) induced ROS production.
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Alzheimer’s disease is the most common neurodegenerative disease in the world and oxidative stress is a major factor in its pathogenesis. It is known that copper(II) ions forming complexes with peptides from the β-amyloid (Aβ) group can facilitate the production of reactive oxygen species. A very common amyloid in AD brain plaques Aβ(11-42) form very stable Cu(II)-complexes that suppress ROS formation. However, when glutamic acid in Aβ(11-42) undergoes dehydration form cyclic pyroglutamate, resulting in a new derivative pAβ(11-42), the Cu(II) stabilisation is much weaker. Here, we investigate, for the first time, the redox chemistry of pAβ(11-16)-Cu‑(II) complexes as a model system for pAβ(11-42). We show that the weaker Cu(II) affinity for the pyroglutamate-modified peptide leads to Cu(I)/Cu(II) oxidation at potentials associated with increased ROS production. Our study also shows a significant difference in the redox properties of the complex if phosphate ions are present in the electrolyte, underlining the importance of proper choice of buffer solutions. These results can be crucial for an increased understanding of AD pathogenesis.
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Preeclampsia is a blood pressure disorder associated with significant proteinuria. Hypertensive women have increased levels of neurokinin B (NKB) and Cu(II) ions in blood plasma during pregnancy. NKB bears the ATCUN/NTS N-terminal motif empowering strong Cu(II) binding in a characteristic four-nitrogen (4N) square-planar motif, but an alternative Cu(II)NKB2 geometry was proposed earlier. We studied the coordination of DMHD-NH2, representing the NKB ATCUN/NTS motif, to Cu(II) by potentiometry, electronic absorption and circular dichroism spectroscopy in water and SDS micellar solutions. NKB was studied in SDS micelles. The experiments were aided by density functional theory (DFT) calculations. We found that under all investigated conditions NKB formed solely 1 : 1 complexes. In the absence of SDS, the 4N complex at physiological pH 7.4 has a very low dissociation constant of 3.5 fM, but the interaction with SDS weakened the binding nearly thousand-fold. This interaction may serve as a molecular switch for specific Cu(II) delivery to membrane receptors by NKB. Furthermore, the calculations based on clinical data indicate a potential toxic role of Cu(II)NKB in preeclampsia.
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In this review we give an updated outlook of the reactivity of catecholamines, particularly dopamine, and the redox effects produced by their interaction with copper(II) and dioxygen, with emphasis to the extensive studies carried out by our group. The interaction between copper(II) ions and neuronal proteins and peptides can contribute to neurodegeneration because in many cases the peptide fragments contain high affinity binding sites and the resulting complexes exhibit increased redox reactivity. It has become apparent in recent years that the redox reactivity of Cu-peptide complexes can be substantially improved by catecholamines, which are redox reactive molecules by themselves but also relatively good ligands for copper ions. Therefore, the toxic effects of copper dyshomeostasis will be particularly harmful in the brain areas producing and releasing catecholamines, i.e. the axon terminals of the substantia nigra and locus coeruleus. These are the brain regions which become affected in the early stages of Parkinson and Alzheimer’s disease, indicating that copper neurotoxicity may contribute to the outset of the diseases. Copper-β-amyloid and copper-prion complexes exhibit the highest redox activity induced by catecholamines; their reactivity is modulated by interaction with membranes, which tend to depress the reactivity unless the peptides interact with each other strengthening the binding of copper(II).
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Electrochemical biosensors have been adopted into a wide range of applications in the study of biometal-protein interactions in neurodegenerative diseases. Transition metals such as zinc, copper, and iron that are significant to biological functions have been shown to have strong implications in the progressive neural degeneration in Alzheimer's disease (AD), Parkinson's disease (PD), and prion protein diseases. This review presents a summative examination of the progress made in the design, fabrication, and applications of electrochemical biosensors in recent literature at understanding the metal-protein interactions in neurodegenerative diseases. The focus will be drawn on disease-causing biomarkers such as amyloid-β (Aβ) and tau proteins for AD, α-synuclein (α-syn) for PD, and prion proteins (PrP). Topics such as the use of electrochemical biosensing in monitoring biometal-induced conformational changes, elucidation of complexation motifs, production of reactive oxygen species (ROS) as well as the influence on downstream biomolecular interactions will be discussed. Major results and important concepts presented in these studies will be summarized in the hope to spark inspiration for the next generation of electrochemical sensors.
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Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by progressive and irreversible damage to the brain. One of the hallmarks of the disease is the presence of both soluble and insoluble aggregates of the amyloid beta (Aβ) peptide in the brain, and these aggregates are considered central to disease progression. Thus, the development of small molecules capable of modulating Aβ peptide aggregation may provide critical insight into the pathophysiology of AD. In this work we investigate how photoactivation of three distorted Ru(ii) polypyridyl complexes (Ru1-3) alters the aggregation profile of the Aβ peptide. Photoactivation of Ru1-3 results in the loss of a 6,6'-dimethyl-2,2'-bipyridyl (6,6'-dmb) ligand, affording cis-exchangeable coordination sites for binding to the Aβ peptide. Both Ru1 and Ru2 contain an extended planar imidazo[4,5-f][1,10]phenanthroline ligand, as compared to a 2,2'-bipyridine ligand for Ru3, and we show that the presence of the phenanthroline ligand promotes covalent binding to Aβ peptide His residues, and in addition, leads to a pronounced effect on peptide aggregation immediately after photoactivation. Interestingly, all three complexes resulted in a similar aggregate size distribution at 24 h, forming insoluble amorphous aggregates as compared to significant fibril formation for peptide alone. Photoactivation of Ru1-3 in the presence of pre-formed Aβ1-42 fibrils results in a change to amorphous aggregate morphology, with Ru1 and Ru2 forming large amorphous aggregates immediately after activation. Our results show that photoactivation of Ru1-3 in the presence of either monomeric or fibrillar Aβ1-42 results in the formation of large amorphous aggregates as a common endpoint, with Ru complexes incorporating the extended phenanthroline ligand accelerating this process and thereby limiting the formation of oligomeric species in the initial stages of the aggregation process that are reported to show considerable toxicity.
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ATCUN (amino terminal Cu(II) and Ni(II) binding) motifs chelate Cu(II) ions strongly. However, the impact of the phosphorylation of neighboring residues on such complexation has not been elucidated. The copper(II) dissociation constants of original and phosphorylated peptides from human histatin-1 and human serum albumin were compared using spectroscopic methods. Phosphorylation markedly weakened Cu(II) binding. Thus, these results indicate that phosphorylation may be a vital mechanism governing metal ion binding.
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Cu(II)-peptide complexes are intensely studied as models for biological peptides and proteins and for their direct importance in copper homeostasis and dyshomeostasis in human diseases. In particular, high-affinity ATCUN/NTS (amino-terminal copper and nickel/N-terminal site) motifs present in proteins and peptides are considered as Cu(II) transport agents for copper delivery to cells. The information on the affinities and structures of such complexes derived from steady-state methods appears to be insufficient to resolve the mechanisms of copper trafficking, while kinetic studies have recently shown promise in explaining them. Stopped-flow experiments of Cu(II) complexation to ATCUN/NTS peptides revealed the presence of reaction steps with rates much slower than the diffusion limit due to the formation of novel intermediate species. Herein, the state of the field in Cu(II)-peptide kinetics is reviewed in the context of physiological data, leading to novel ideas in copper biology, together with the discussion of current methodological issues.
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Two amide group containing pyridine derivatives, N-(pyridin-2-ylmethyl)picolinamide (PMPA) and N-(pyridin-2-ylmethyl)-2-((pyridin-2-ylmethyl)amino)acetamide (DPMGA), have been investigated as potential metallophores in the therapy of Alzheimer's disease. Their complex formation with Cu(II) and Zn(II) were characterized in details. Unexpectedly not only the Cu(II) but also the Zn(II) was able to induce deprotonation of the amide-NH, however, it occurred only at higher pH or at higher metal ion concentrations than the biological conditions. At μM concentration level mono complexes (MLH−1) dominate with both ligands. Direct fluorescence and reactive oxygen species (ROS) producing measurements prove that both ligands are able to remove Cu(II) from its amyloid-β complexes (CuAβ). Correlation was also established between the conditional stability constant of the Cu(II) complexes with different ligands and their ability of inhibition of ROS production by CuAβ.
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We used a series of modified/substituted GGH analogues to investigate the kinetics of Cu(II) binding to ACTUN peptides. Rules for rate modulation by 1st and 2nd sphere interactions were established,...
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N-truncated Aβ4-42 is highly abundant in Alzheimer disease (AD) brain and was the first Aβ peptide discovered in AD plaques. However, a possible role in AD aetiology has largely been neglected. In the present report, we demonstrate that Aβ4-42 rapidly forms aggregates possessing a high aggregation propensity in terms of monomer consumption and oligomer formation. Short-term treatment of primary cortical neurons indicated that Aβ4-42 is as toxic as pyroglutamate Aβ3-42 and Aβ1-42. In line with these findings, treatment of wildtype mice using intraventricular Aβ injection induced significant working memory deficits with Aβ4-42, pyroglutamate Aβ3-42 and Aβ1-42. Transgenic mice expressing Aβ4-42 (Tg4-42 transgenic line) developed a massive CA1 pyramidal neuron loss in the hippocampus. The hippocampus-specific expression of Aβ4-42 correlates well with age-dependent spatial reference memory deficits assessed by the Morris water maze test. Our findings indicate that N-truncated Aβ4-42 triggers acute and long-lasting behavioral deficits comparable to AD typical memory dysfunction.
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Progressive cerebral deposition of the amyloid β-protein (Aβ) in brain regions serving memory and cognition is an invariant and defining feature of Alzheimer disease. A highly similar but less robust process accompanies brain aging in many nondemented humans, lower primates, and some other mammals. The discovery of Aβ as the subunit of the amyloid fibrils in meningocerebral blood vessels and parenchymal plaques has led to innumerable studies of its biochemistry and potential cytotoxic properties. Here we will review the discovery of Aβ, numerous aspects of its complex biochemistry, and current attempts to understand how a range of Aβ assemblies, including soluble oligomers and insoluble fibrils, may precipitate and promote neuronal and glial alterations that underlie the development of dementia. Although the role of Aβ as a key molecular factor in the etiology of Alzheimer disease remains controversial, clinical trials of amyloid-lowering agents, reviewed elsewhere in this book, are poised to resolve the question of its pathogenic primacy.
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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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The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.
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Circular Dichroism (CD) spectroscopy in the visible region (Vis-CD) is a powerful technique to study metal-protein interactions. It can resolve individual d–d electronic transitions as separate bands and is particularly sensitive to the chiral environment of the transition metals. Modern quantum chemical methods enable CD spectra calculations from which, along with direct comparison with the experimental CD data, the conformations and the stereochemistry of the metal-protein complexes can be assigned. However, a clear understanding of the observed spectra and the molecular configuration is largely lacking. In this study, we compare the experimental and computed Vis-CD spectra of Cu2+-loaded model peptides in square-planar complexes. We find that the spectra can readily discriminate the coordination pattern of Cu2+ bound exclusively to main-chain amides from that involving both main-chain amides and a side-chain (i.e. histidine side chain). Based on the results, we develop a set of empirical rules that relates the appearance of particular Vis-CD spectral features to the conformation of the complex. These rules can be used to gain insight into coordination geometries of other Cu2+ or Ni2+-protein complexes.This article is protected by copyright. All rights reserved.
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Conspectus The interaction of d-block metal ions (Cu, Zn, Fe, etc.) with intrinsically disordered proteins (IDPs) has gained interest, partly due to their proposed roles in several diseases, mainly neurodegenerative. A prominent member of IDPs is the peptide amyloid-β (Aβ) that aggregates into metal-enriched amyloid plaques, a hallmark of Alzheimer's disease, in which Cu and Zn are bound to Aβ. IDPs are a class of proteins and peptides that lack a unique 3D structure when the protein is isolated. This disordered structure impacts their interaction with metal ions compared with structured metalloproteins. Metalloproteins either have a preorganized metal binding site or fold upon metal binding, resulting in defined 3D structure with a well-defined metal site. In contrast, for Aβ and likely most of the other IDPs, the affinity for Cu(I/II) and Zn(II) is weaker and the interaction is flexible with different coordination sites present. Coordination of Cu(I/II) with Aβ is very dynamic including fast Cu-exchange reactions (milliseconds or less) that are intrapeptidic between different sites as well as interpeptidic. This highly dynamic metal-IDP interaction has a strong impact on reactivity and potential biological role: (i) Due to the low affinity compared with classical metalloproteins, IDPs likely bind metals only at special places or under special conditions. For Aβ, this is likely in the neurons that expel Zn or Cu into the synapse and upon metal dysregulation occurring in Alzheimer's disease. (ii) Amino acid substitutions (mutations) on noncoordinating residues can change drastically the coordination sphere. (iii) Considering the Cu/Zn-Aβ aberrant interaction, therapeutic strategies can be based on removal of Cu/Zn or precluding their binding to the peptide. The latter is very difficult due to the multitude of metal-binding sites, but the fast koff facilitates removal. (iv) The high flexibility of the Cu-Aβ complex results in different conformations with different redox activity. Only some conformations are able to produce reactive oxygen species. (v) Other, more specific catalysis (like enzymes) is very unlikely for Cu/Zn-Aβ. (vi) The Cu/Zn exchange reactions with Aβ are faster than the aggregation process and can hence have a strong impact on this process. In conclusion, the coordination chemistry is fundamentally different for most of IDPs compared with the classical, structured metalloproteins or with (bio)-inorganic complexes. The dynamics is a key parameter to understand this interaction and its potential biological impact.
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Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dualacting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motifcontaining derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN–anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN–sh-buforin peptides. Our findings indicate that the addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents.
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The review describes the state of the art in the field of stability constant determination for Cu(II), Cu(I) and Zn(II) complexes of proteins and peptides involved in neurodegenerative diseases, α-synuclein (aS), prion protein (PrP), amyloid precursor protein (APP) and amyloid β peptides (Aβ). The methodologies and results are critically analyzed and recommendations are formulated about possible systematic errors in these studies. The possibility of formation of ternary complexes with titration competitors is discussed.
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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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The direct electrochemical behaviour of peptide methionine sulfoxide reductase A (MsrA) adsorbed on glassy carbon and boron doped diamond electrodes surface, was studied over a wide pH range by cyclic and differential pulse voltammetry. MsrA oxidation mechanism occurs in three consecutive, pH dependent steps, corresponding to the oxidation of tyrosine, tryptophan and histidine amino acid residues. At the glassy carbon electrode, the first step corresponds to the oxidation of tyrosine and tryptophan residues and occurs for the same potential. The advantage of boron doped diamond electrode was to enable the separation of tyrosine and tryptophan oxidation peaks. On the second step occurs the histidine oxidation, and on the third, at higher potentials, the second tryptophan oxidation. MsrA adsorbs on the hydrophobic carbon electrode surface preferentially through the three hydrophobic domains, C1, C2 and C3, which contain the tyrosine, tryptophan and histidine residues, and tryptophan exists only in these regions, and undergo electrochemical oxidation.
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Circular dichroism and electron spin resonance spectroscopy are used to investigate the second specific metal binding site on human, bovine and porcine albumins. Ni(II), Zn(II) and Cd(II) can displace Cu(II) from the second Cu(II) site but not from the first strong site of human and bovine albumins (the N-terminal site). The second Cu(II) binds more strongly than the other metal ions to the second site of all three proteins, except Zn(II) binding to porcine albumin which is ca. 10 × stronger than Cu(II). The second Cu(II) site appears to be a tetragonal {2N,4O} site.
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The Gly-His-Lys (GHK) peptide and the Asp-Ala-His-Lys (DAHK) sequences are naturally occurring high-affinity copper(II) chelators found in the blood plasma and are hence of biological interest. A structural study of the copper complexes of these peptides was conducted in the solid state and in solution by determining their X-ray structures, and by using a large range of spectroscopies, including EPR and HYSCORE (hyperfine sub-level correlation), X-ray absorption and 1H and 13C NMR spectroscopy. The results indicate that the structures of [Cu II(DAHK)] in the solid state and in solution are similar and confirm the equatorial coordination sphere of NH 2, two amidyl N and one imidazole N. Additionally, a water molecule is bound apically to Cu II as revealed by the X-ray structure. As reported previously in the literature, [Cu II(GHK)], which exhibits a dimeric structure in the solid state, forms a monomeric complex in solution with three nitrogen ligands: NH 2, amidyl and imidazole. The fourth equatorial site is occupied by a labile oxygen atom from a carboxylate ligand in the solid state. We probe that fourth position and study ternary complexes of [Cu II(GHK)] with glycine or histidine. The Cu II exchange reaction between different DAHK peptides is very slow, in contrast to [Cu II(GHK)], in which the fast exchange was attributed to the presence of a [Cu II(GHK) 2] complex. The redox properties of [Cu II(GHK)] and [Cu II(DAHK)] were investigated by cyclic voltammetry and by measuring the ascorbate oxidation in the presence of molecular oxygen. The measurements indicate that both Cu II complexes are inert under moderate redox potentials. In contrast to [Cu II(DAHK)], [Cu II(GHK)] could be reduced to Cu I around -0.62 V (versus AgCl/Ag) with subsequent release of the Cu ion. These complete analyses of structure and redox activity of those complexes gave new insights with biological impact and can serve as models for other more complicated Cu II-peptide interactions.
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Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive and memory impairment. Within the brain, senile plaques, which comprise extracellular deposits of the amyloid-β peptide (Aβ), are the most common pathological feature of AD. A high concentration of Cu2+ is found within these plaques, which are also areas under oxidative stress. Laboratory work has shown that in vitro Aβ will react with Cu2+ to induce peptide aggregation and the production of reactive oxygen species. As such, this interaction offers a possible explanation for two of the defining pathological features observed in the AD brain: the presence of amyloid plaques, which consist largely of insoluble Aβ aggregates, and the abundant oxidative stress therein. Researchers have accordingly put forth the “metals hypothesis” of AD, which postulates that compounds designed to inhibit Cu2+/Aβ interactions and redistribute Cu2+ may offer therapeutic potential for treating AD.
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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.
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Interaction of Cu ions with the amyloid-beta (Abeta) peptide is linked to the development of Alzheimer's disease; hence, determining the coordination of Cu(I) and Cu(II) ions to Abeta and the pathway of the Cu(I)(Abeta)/Cu(II)(Abeta) redox conversion is of great interest. In the present report, we use the room temperature X-ray absorption near edge structure to show that the binding sites of the Cu(I) and Cu(II) complexes are similar to those previously determined from frozen-solution studies. More precisely, the Cu(I) is coordinated by the imidazole groups of two histidine residues in a linear fashion. However, an NMR study unravels the involvement of all three histidine residues in the Cu(I) binding due to dynamical exchange between several set of ligands. The presence of an equilibrium is also responsible for the complex redox process observed by cyclic voltammetry and evidenced by a concentration-dependent electrochemical response.
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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
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
The potential of Ni(II) and Cu(II) complexes with Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide (HP2(1-15)), a peptide modeling the N-terminal amino acid sequence of human protamine HP2, to mediate oxidative DNA damage was studied by measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) generation from 2'-deoxyguanosine (dG) and calf thymus DNA and by formation of double-strand breaks in calf thymus DNA. The concentrations of reagents were 0.1 mM dG and the metal-HP2(1-15) complex, 1 mM H2O2, 1.5 mM DNA (per phosphate group), 100 mM phosphate buffer, pH 7.4, ambient O2. Samples were incubated at 37 degrees C for 16-24 h. The Cu(II)-HP2(1-15) complex was found to be an effective promoter of the formation of 8-oxo-dG from both dG and DNA with ambient O2 (approximately 13- and 3-fold increase versus the oxidant alone, respectively) and H2O2 (approximately 25-fold increase in either case). The Ni(II)-HP2(1-15) complex was ineffective with O2 versus 8-oxo-dG production from both substrates but markedly enhanced the attack of H2O2 on dG and DNA (approximately 5-fold increase of 8-oxo-dG production in either case). Both Cu(II)- and Ni(II)-HP2(1-15) equally promoted double-strand scission by H2O2 in calf thymus DNA. The promotion by the complexes of dG and DNA oxidation with H2O2 was accompanied by oxidative damage to the complexes themselves, consisting of decreasing contents of their His (to approximately 50% of control in either complex) and especially Tyr (down to 48% of control in Cu(II)- and 19% in Ni(II)-HP2[1-15]) residues, as well as appearance of aspartic acid, the known oxidation product of His residues in peptides (up to 22% vs Gly for Cu(II)- and 10% for Ni(II)-HP2(1-15)). The above results provide a novel chemical mechanism of Cu(II) and Ni(II) toxicity and may have wide implications for reproductive and transgenerational effects of metal exposure.
Article
A potentiometric and spectroscopic (UV/vis and CD) study of Cu(II) and Ni(II) binding to the N-terminal pentadecapeptide of human protamine HP2 (HP2(1-15)) was performed. The results indicate that the N-terminal tripeptide motif Arg-Thr-His is the exclusive binding site for both metal ions at a metal to HP2(1-15) molar ratio not higher than 1. The very high value of protonation-corrected stability constant (log *K) for Ni(II)-HP2(1-15) complex, -19.29, indicates that HP2 has the potential to sequester Ni(II) from other peptide and protein carriers, including albumin. The same is likely for Cu(II) (log *K = -13.13). The CD spectra of Cu(II) and Ni(II) complexes of HP2(1-15) indicate that the N-terminal metal binding affects the overall conformation of the peptide that, in turn, may alter interaction of HP2 with DNA. These results imply HP2 as a likely target for the toxic metals Ni(II) and Cu(II).
Article
A comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, respectively) and short peptide analogues was performed using potentiometry and spectroscopic techniques. It was found that while qualitative aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quantitative parameters (stability and rate constants) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modelling of Cu(II) and Ni(II) transport by albumins.
Article
GSH and L-His are abundant biomolecules and likely biological ligands for Zn(II) under certain conditions. Potentiometric titrations provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favour of L-His (ca. 1 log unit of stability constant). The solution structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, determined by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calculations of competitivity of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(II) carrier.
Article
Copper is implicated in the in vitro formation and toxicity of Alzheimer's disease amyloid plaques containing the beta-amyloid (Abeta) peptide (Bush, A. I., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11934). By low temperature electron paramagnetic resonance (EPR) spectroscopy, the importance of the N-terminus in creating the Cu(2+) binding site in native Abeta has been examined. Peptides that contain the proposed binding site for Cu(2+)-three histidines (H6, H13, and H14) and a tyrosine (Y10)-but lack one to three N-terminal amino acids, do not bind Cu(2+) in the same coordination environment as the native peptide. EPR spectra of soluble Abeta with stoichiometric amounts of Cu(2+) show type 2 Cu(2+) EPR spectra for all peptides. The ligand donor atoms to Cu(2+) are 3N1O when Cu(2+) is bound to any of the Abetapeptides (Abeta16, Abeta28, Abeta40, and Abeta42) that contain the first 16 amino acids of full-length Abeta. When a Y10F mutant of Abeta is used, the coordination environment for Cu(2+) remains 3N1O and Cu(2+) EPR spectra of this mutant are identical to the wild-type spectra. Isotopic labeling experiments show that water is not the O-atom donor to Cu(2+) in Abeta fibrils or in the Y10F mutant. Further, we find that Cu(2+) cannot be removed from Cu(2+)-containing fibrils by washing with buffer, but that Cu(2+) binds to fibrils initially assembled without Cu(2+) in the same coordination environment as in fibrils assembled with Cu(2+). Together, these results indicate (1) that the O-atom donor ligand to Cu(2+) in Abeta is not tyrosine, (2) that the native Cu(2+) binding site in Abeta is sensitive to small changes at the N-terminus, and (3) that Cu(2+) binds to Abetafibrils in a manner that permits exchange of Cu(2+) into and out of the fibrillar architecture.
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
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.
Cold Spring Harbor Perspect
  • C L Masters
  • D J Elkoe
C. L. Masters,D.J.S elkoe, Cold Spring Harbor Perspect. Med. 2012, 2,a006262.
Weinheim www.angewandte.org These are not the final page numbers
  • Wiley-Vch Verlag Gmbh
  • Co
  • Ü Ü Communications Kgaa
  • M Amyloids
  • N E Mital
  • T Wezynfeld
  • ˛ Fra
  • M Z Czyk
  • U E Wiloch
  • A Wawrzyniak
  • C Bonna
  • K J Tumpach
  • C L Barnham
  • W Haigh