The Angiotensin Metabolite His-Leu Is a Strong Copper Chelator Forming Highly Redox Active Species.

His-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet-visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 µM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu.

Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster.

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

The Aggregation Pattern of Aß1-40 is Altered by the Presence of N-Truncated Aß4-40 and/or CuII in a Similar Way through Ionic Interactions.

Alzheimer's disease (AD) is one of the most common of the multifactorial diseases and is characterized by a range of abnormal molecular processes, such as the accumulation of extracellular plaques containing the amyloid-ß (Aß) peptides and dyshomeostasis of copper in the brain. In this study, we have investigated the effect of CuII on the aggregation of Aß1-40 and Aß4-40 , representing the two most prevalent families of Aß peptides, that is, the full length and N-truncated peptides. Both families are similarly abundant in healthy and AD brains. For either of the studied peptides, substoichiometric CuII concentrations accelerated aggregation, whereas superstoichiometric CuII inhibited fibril formation, likely by stabilizing the oligomers. The addition of either Aß4-40 or substoichiometric CuII affected the aggregation profile of Aß1-40 , by yielding shorter and thicker fibrils; amorphous aggregates were formed in the presence of a molar excess of CuII . The similarity of these two effects can be attributed to the increase in the positive charge on the Aß N terminus, caused both by CuII complexation and N truncation at position 4. Our findings provide a better understanding of the biological Aß aggregation process as these two Aß species and CuII coexist and interact under physiological conditions.

Copper Transporters? Glutathione Reactivity of Products of Cu-Aß Digestion by Neprilysin.

Aß4-42 is the major subspecies of Aß 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 CuIIAß4-16, CuIIAß4-9, and also CuIIAß12-16, 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 CuIIAß4-16, CuIIAß4-9, CuIIAß12-16, and CuIIAß1-16 with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found CuIIAß4-16 and CuIIAß4-9 to be strongly resistant to reduction and concomitant formation of Cu(I)-GSH complexes, with reaction times ∼10 h, while CuIIAß12-16 was reduced within minutes and CuIIAß1-16 within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the CuIIAß complexes were reformed with no concomitant oxidative damage to peptides. These finding reinforce the concept of Aß4-x peptides as physiological trafficking partners of brain copper.

Exploration of the Potential Role for Aß in Delivery of Extracellular Copper to Ctr1.

Amyloid beta (Aß) 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. Aß 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. Aß 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 Aß1-16 and Aß4-16 peptides as well-established non-aggregating models of major Aß 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 Aß peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aß1-16 fully and rapidly delivered Cu(II) to Ctr1-14 along the affinity gradient. Such delivery was only partial for the Aß4-16/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 Aß 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 Aß model peptides. These results are discussed in terms of the fundamental difference between the peptides' Cu(II) complexes (pleiotropic ensemble of open structures of Aß1-16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aß4-16) 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 Aß 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.

Peptide Bond Cleavage by Ni(II) Ions within the Nuclear Localization Signal Sequence.

Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr-Xaa-His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68-like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.

CuII Binding Properties of N-Truncated Aß Peptides: In Search of Biological Function.

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 Aß4-x, the most prevalent N-truncated Aß peptide, currently known as the main constituent of amyloid plaques. The capability of Aß4-x to rapidly take over copper from previously tested Aß1-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)Aß4-x with coexisting biomolecules in the context of synaptic cleft; we suggest that the role of Aß4-x peptides is to quench Cu(II) toxicity in the brain and maintain neurotransmission.

Using N-Terminal Coordination of Cu(II) and Ni(II) to Isolate the Coordination Environment of Cu(I) and Cu(II) Bound to His13 and His14 in Amyloid-ß(4-16).

The amyloid-ß (Aß) 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 Aß is variable, and different proportions of these variable-length Aß peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aß starting at position 4 (Aß4-x) has a His residue as the third amino acid (His6 using the formal Aß 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 Aß1-x peptides. In addition to the ATCUN site, Aß4-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 Aß. Using the ATCUN and bis-His motifs, the Aß4-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 Cu2Aß4-x state. The identification of two functionally different copper binding environments within the Aß4-x sequence may have important implications for this peptide in vivo.

N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications.

Peptides and proteins with N-terminal amino acid sequences NH2 -Xxx-His (XH) and NH2 -Xxx-Zzz-His (XZH) form well-established high-affinity CuII -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity CuII -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH2 -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64 Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong CuII binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application.

Structure and Affinity of Cu(I) Bound to Human Serum Albumin.

Human serum albumin (HSA) is a major Cu carrier in human blood and in cerebrospinal fluid. A major assumption is that Cu bound to HSA is in the Cu(II) oxidation state; thus, interactions between HSA and Cu(II) have been intensely investigated for over four decades. HSA has been reported previously to support the reduction of Cu(II) to the Cu(I) oxidation state in the presence of the weak reductant, ascorbate; however, the interactions between HSA and Cu(I) have not been explicitly investigated. Here, we characterize both the apparent affinity of HSA for Cu(I) using solution competition experiments and the coordination structure of Cu(I) bound to HSA using X-ray absorption spectroscopy and in silico modeling. We find that HSA binds to Cu(I) at pH 7.4 with an apparent conditional affinity of KCu(I):HSA = 1014.0 using digonal coordination in a structure that is similar to the bis-His coordination modes characterized for amyloid beta (Aß) and the prion protein. This high affinity and familiar Cu(I) coordination structure suggests that Cu(I) interaction with HSA in human extracellular fluids is unappreciated in the current scientific literature.

Resistance of Cu(Aß4-16) to Copper Capture by Metallothionein-3 Supports a Function for the Aß4-42 Peptide as a Synaptic Cu(II) Scavenger.

Aß4-42 is a major species of Aß peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu(II) with an affinity approximately 3000 times higher than the commonly studied Aß1-42 and Aß1-40 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 Cu(II) from Aß1-40 when acting in its native Zn7 MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7 MT-3 against Aß toxicity. In this work, we used the truncated model peptides Aß1-16 and Aß4-16 to demonstrate that the high-affinity Cu(II) complex of Aß4-16 is resistant to Zn7 MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aß4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aß4-42 as a Cu(II) scavenger in the synaptic cleft.

Interplay between Copper, Phosphatidylserine, and α-Synuclein Suggests a Link between Copper Homeostasis and Synaptic Vesicle Cycling.

Copper homeostasis is critical to the functioning of the brain, and its breakdown is linked with many brain diseases. Copper is also known to interact with the negatively charged lipid, phosphatidylserine (PS), as well as α-synuclein, an aggregation-prone protein enriched in the synapse, which plays a role in synaptic vesicle docking and fusion. However, the interplay between copper, PS lipid, and α-synuclein is not known. Herein, we report a detailed and predominantly kinetic study of the interactions among these three components pertinent to copper homeostasis and neurotransmission. We found that synaptic vesicle-mimicking small unilamellar vesicles (SUVs) can sequester any excess free Cu2+ within milliseconds, and bound Cu2+ on SUVs can be reduced to Cu+ by GSH at a nearly constant rate under physiological conditions. Moreover, we revealed that SUV-bound Cu2+ does not affect the binding between wild-type α-synuclein and SUVs but affect that between N-terminal acetylated α-synuclein and SUVs. In contrast, Cu2+ can effectively displace both types of α-synuclein from the vesicles. Our results suggest that synaptic vesicles may mediate copper transfer in the brain, while copper could participate in synaptic vesicle docking to the plasma membrane via its regulation of the interaction between α-synuclein and synaptic vesicle.

Hierarchical binding of copperII to N-truncated Aß4-16 peptide.

N-Truncated Aß4-42 displays a high binding affinity with CuII. A mechanistic scheme of the interactions between Aß4-42 and CuII has been proposed using a fluorescence approach. The timescales of different conversion steps were determined. This kinetic mechanism indicates the potential synaptic functions of Aß4-42 during neurotransmission.

The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences.

Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M-1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M-1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function.

Cu transfer from amyloid-ß4-16 to metallothionein-3: the role of the neurotransmitter glutamate and metallothionein-3 Zn(ii)-load states.

Copper transfer from Cu(ii)amyloid-ß4-16 to human Zn7-metallothionein-3 can be accelerated by glutamate and by lowering the Zn-load of metallothionein-3 with EDTA. Glutamate facilitates the Cu(ii) release, and Zn4-6-metallothionein-3 react more rapidly. These mechanisms are additive, proving the intricate and interconnected network of zinc and copper trafficking between biomolecules.

The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1.

Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiological role of CuII binding to hCtr1.

On the ability of CuAß1-x peptides to form ternary complexes: Neurotransmitter glutamate is a competitor while not a ternary partner.

In the light of conflicting reports on the ability of copper(II) complexes of amyloid beta (Aß) peptides to form ternary complexes with small molecules co-present in the biological milieu, we performed a study of coordination equilibria in the system containing Cu(II) ions, the Aß1-16 peptide, glutamic acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, HEPES) buffer. Using potentiometry, isothermal titration calorimetry (ITC), UV-visible spectroscopy and EPR, we concluded that glutamic acid was not able to form such a ternary complex, but can efficiently compete for the Cu(II) ion with the Aß peptide at Glu concentrations relevant for the synaptic cleft. We also found that the literature constants for Cu(II) complexes with Glu were overestimated, but this effect was partially compensated by the formation of a ternary Cu(Glu)(HEPES) complex. Our results indicate that small molecules co-present with Cu(II) ions and Aß peptides in the synaptic cleft are not very likely to enhance Cu(II)/Aß interactions, but instead should be considered as a Cu(II) buffering system that may help prevent these interactions and participate in Cu(II) clearance from the synaptic cleft.