Abeta 4-x peptides as copper binding molecules resistant to chelating drugs, promoting the formation of amyloid oligomers.

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.

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.

Multiple intermediates were found in Cu( ii ) binding to Aβ 4–16 before the formation of a tight complex.

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.

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.

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.

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.

Aβ4-42 is a major species of Aβ peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind CuII with an affinity approximately 3000 times higher than the commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract CuII from Aβ1-40 when acting in its native Zn7MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7MT-3 against Aβ toxicity. In this work, we used the truncated model peptides Aβ1-16 and Aβ4-16 to demonstrate that the high-affinity CuII complex of Aβ4-16 is resistant to Zn7MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aβ4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aβ4-42 as a CuII scavenger in the synaptic cleft.

Ab4-42 is a major species of Ab peptide in the brains of both healthy individuals and those affected by Alzheimers disease. It has recently been demonstrated to bind Cu II with an affinity approximately 3000 times higher than the commonly studied Ab1-42 and Ab1-40 peptides, which are implicated in the pathogenesis of Alzheimers disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu II from Ab1-40 when acting in its native Zn 7 MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn 7 MT-3 against Ab toxicity. In this work, we used the truncated model peptides Ab1-16 and Ab4-16 to demonstrate that the high-affinity Cu II complex of Ab4-16 is resistant to Zn 7 MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Ab4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Ab4-42 as a Cu II scavenger in the synaptic cleft.

Alzheimer’s disease (AD) is the major cause of dementia and death in the elderly. While the etiology and pathological mechanisms of the AD are under debate, the oligomerization of amyloid-β-peptides (Aβ peptides) in the brain is its major pathological hallmark.1 Aβ1-40, Aβ1-42, and Aβ4-42 are major species of Aβ peptide in the brain both healthy individuals and those affected by the AD. It has recently been demonstrated that Aβ4-42 binds CuII with an affinity ca. 3000 times higher from commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in Alzheimer’s disease pathogenesis. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection was shown to extract CuII from Aβ1-40 acting in its native Zn7MT3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7MT3 against Aβ toxicity. In this study, using truncated model peptides Aβ1-16 and Aβ4-16, we demonstrated that the high-affinity CuII complex of Aβ4-16 is resistant to Zn7MT3 reactivity. This indicates that the analogous complex of the full-length peptide, Cu(Aβ4-42) will not yield copper to MT-3 in the brain. A reverse swap experiment, where apo-Aβ4-16 was added to the pre-formed (Cu/Zn)MT-3 complex did not yield CuII transfer from metallothionein to the peptide, but addition of CuII ions to a mixture of Zn7MT-3 and apo-Aβ4-16 resulted in a partition of copper between these biomolecules, with ca. 30% as Cu(Aβ4-16). We also demonstrated that physiological amounts of ascorbate or hydrogen peroxide did not influence the equilibrium of studied reactions.Our results support the concept of the physiological role of Aβ4-42 as a CuII scavenger in the synaptic cleft.