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CuII Binding Properties of N‑Truncated Aβ Peptides: In Search of Biological Function Ewelina Stefaniak* and Wojciech Bal*

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Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland ABSTRACT: 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.

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INTRODUCTION: ALZHEIMER’S DISEASE Alzheimer’s Disease (AD) is a neurodegenerative disorder, manifesting itself as a progressive and irreversible dementia, leading inevitably to death. Clinical AD is preceded by the prodromal stage, when the massive death of neurons occurs in brain structures responsible for processes related to memory and emotions. AD affects tens of millions of people worldwide (current estimates are 47 million now, increasing to 75 million by 2030 and 131.5 million in 2050).1−3 The patient’s age is the main risk factor for AD. Thus, the disease is widespread in developed countries characterized by a long life expectancy. Nevertheless, as people tend to live longer in developing countries, AD spreads all over the world, causing suffering and enormous social and material costs, counted in the tens of billions of US dollars. The best preventive measure available currently is to maintain a healthy lifestyle, with a balanced diet and physical and mental exercise.4−6 Such an approach, which results in a statistically significant delay of AD onset, is aimed at maintaining as many healthy neurons and working synaptic connections as possible. While commendable, it is unfortunately not sufficient to combat AD, because the disease may be delayed or even slowed down but not cured. Despite many years of research and numerous clinical trials, an effective therapy that could cure or at least stop the development of AD has not emerged. Disappointing results of clinical trials bring the realization that our knowledge of fundamental brain biochemistry is not sufficient to grasp the source of AD. The ignorance about the fundamental process(es) that undergo activation or inhibition, triggering the cascade of events leading to AD hampers our efforts to design a therapy. Metal ion, in particular copper, toxicity has been invoked as a candidate for such a process by some but denied by others. A climax was a failure of the only clinical trial designed on a concept of toxicity of misplaced brain copper interacting with Aβ peptide, a molecule © XXXX American Chemical Society

considered to be the key brain toxin in AD. The purpose of this Viewpoint is to propose a switch of gears in thinking about copper and Aβ. Instead of forwarding another model of copper toxicity, we interpret results of recent chemical studies to propose the existence of a fundamental physiological process involving both copper and a variant Aβ peptide. We argue that considering this process will help avoid failures in the future. But first, we have to briefly review the known molecular aspects of AD and the roles assigned to Aβ peptides. There are two variants of AD, sporadic Alzheimer’s disease (SAD), which accounts for about 95% of all AD cases, and a much less frequent inherited form (familial Alzheimer’s disease, FAD). The symptoms of SAD appear later in life, after the age of 65, while the onset of FAD is observed much earlier, usually around the age of 40. In both cases, the disease develops slowly, over years, although it has a faster course in FAD.7 The biochemical markers of SAD precede the appearance of the first clinical symptoms by at least 10 years.8,9 Patients gradually lose their cognitive abilities, short- and long-term memory, proceed through psychiatric disorders from the spectrum of schizophrenia, and die. Genetic factors contribute to both forms of the disease, clearly dominating in FAD.10 FAD is usually due to rare and highly penetrant mutations in one of three genes: amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2),11 although mutations with incomplete penetrance have also been described.12 The more complex SAD form is likely determined by a combination of genetic and environmental factors. Despite a great amount of research on the molecular pathogenesis of AD, a full underSpecial Issue: Metals in Biology: From Metallomics to Trafficking Received: May 13, 2019

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DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

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Table 1. Sequences of the Most Important Aβ Family Peptides,a Formed upon the APP Protein Proteolysis and Post-translational Modifications49,53−57,59−61

a The numbering of amino acid residues and abbreviated peptide labels are based on the sequence of the Aβ1−42 peptide, except for the N-terminally extended peptides, which are numbered according to the APP sequence. M in italics means that the given peptide was detected with both native and methionine S-oxide forms; D in italics means that the occurrence of rearrangement of aspartyl to isoaspartyl residue was detected for the given peptide; pE denotes a pyroglutamyl residue; Amm denotes an ammonium ion adduct at the C-terminus. Peptides indicated as highly abundant in brain structures in numerous studies are marked bold.

standing of the etiology of the sporadic form is still out of reach. The presence of the epsilon4 allele in apolipoprotein E (APOE) is the only well-described genetic risk factor in SAD, and over

60% of all sporadic cases are not associated with APOE. The research on genetic causes of SAD is ongoing, with novel AD risk B

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

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death of neurons, preceded by a disturbance of their synaptic function, is caused by the formation of extracellular neurotoxic structures composed of Aβ peptides.24 Indeed, amyloid plaques, the deposits found in the brains of most AD patients, composed of Aβ fibrils, were observed postmortem in brains of the first diagnosed AD patients and until now are considered a hallmark of AD.25 A doubt has been cast on their actual role in AD pathology by more recent research, with soluble Aβ oligomers considered now to be key toxic species.26−28 The amyloid cascade hypothesis seems to explain the rapid course of the familial form of the disease. It also finds support in the fact that exposure of experimental animal brains to oligomerized Aβ peptide preparations elicits AD-like symptoms in a dose-dependent manner.29 A possibility of human to human transmission by contaminated drugs has also been raised.30 In the brain, the spread of aggregated Aβ matches the movement of a toxic agent in the extracellular space.31 Still, the amyloid cascade has been widely criticized as a key concept of the SAD etiology.32 This criticism is partly rooted in the fact that experimental therapies aimed at inhibiting Aβ generation or its aggregation, despite the apparent success on the biochemical level (reduction of Aβ peptide formation or elimination of Aβ deposits), have not stopped the disease. At best, a temporary slowdown was achieved.33 A recent failure of another phase 3 clinical trial using an antibody specific for soluble oligomers has been widely considered a decisive blow against this therapeutic approach.34 In fact, Aβ peptides turned out to be indispensable for brain physiology, and their removal or reduction also causes dementia.35 Having said that, there is no clarity about their physiological role.36 One possibility formulated for Aβ1−40/42 is the induction of long-term synaptic depression through conformational changes of N-methyl-D-aspartate (NMDA) receptors.37,38

factors emerging from genome-wide association studies (GWASs).11

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β-AMYLOID (Aβ) PEPTIDES AND AD Various possible cellular and molecular mechanisms of AD have been proposed, including astrogliosis, microglial cell proliferation, alterations in calcium signaling, oxidative stress, intraneuronal tau protein aggregation, infections, mitochondrial dysfunction, and lysosomal failure being most popular.13−15 However, it is beyond dispute that aggregation of β-amyloid peptides (Aβ) is the first measurable biochemical phenomenon in AD, occurring well before the onset of clinical symptoms.16 Aβ peptides are formed in the minor (10−20%) variant process of hydrolytic degradation of their precursor APP, a transmembrane protein with an only partially established function.17 The cleavage requires simultaneous or sequential action of two membrane-bound endoproteases (generally labeled β- and γ-secretase). The β-secretase cleaves APP to secrete the large product, sAPPβ, and CTFβ, a fragment of 99 amino acids, which remains membrane bound. Then, it is rapidly cleaved by γ-secretase to generate Aβ. Lack of precision of γsecretase leads to C-terminal heterogeneity of the resulting peptide population (Table 1).18,19 The major hydrolytic pathway involves one enzyme, α-secretase, and yields two membrane-bound protein fragments.20 Virtually all recognized variants of FAD are related to Aβ peptides (Table 2). There are Table 2. FAD Mutations in Aβ Peptides and Their Pathological Effects21,62−64 type of mutation

mutated position

A2V

A2V

English

H6R

Tottori

D7N

Taiwanese Flemish

D7H A21G

Arctic

E22G

Italian

E22K

Dutch

E22Q

Osaka

E22Δ

Iowa Piedmont

D23N L34V

reported effects enhanced production and accelerated aggregation production of larger oligomers; unchanged total Aβ levels; accelerated fibril formation with no increase in protofibril levels; enhanced elongation phase with no effects on nucleation production of more stable oligomers interference with amyloid core formation; slow fibrillation; increased secreted Aβ lower plasma levels of Aβ; enhanced protofibril formation interference with amyloid core formation; enhanced aggregation of stable oligomers increases aggregation without altering APP processing accelerated aggregation; decreased Aβ secretion; formation of intracellular aggregates increased rate of fibril formation hypothesized to enhance aggregation into βsheets

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Aβ PEPTIDES AND METAL IONS

Human Aβ peptides contain three histidine residues, which offer facile transition metal binding sites.39,40 Furthermore, amyloid plaques isolated from AD brains contained large amounts of copper, estimated at 25.0 ± 7.8 μg/g.41−43 These findings prompted the copper variant of the amyloid cascade hypothesis,44 where aggregation and oxidative stress, causing inflammation and neuronal damage are due to the formation of CuAβ complexes, which support the Cu(I)/Cu(II) redox pair.45,46 Large amounts of zinc, calcium, and iron accompany copper in amyloid plaques, but redox-silent zinc and calcium cannot be a direct source of oxidative stress. Iron might do that, but its direct interaction with Aβ peptides is weak to nonexistent, for both Fe(II) and Fe(III).47 Also, recent studies indicated that iron is present in the plaques in the form of magnetite microcrystals and metallic iron particles, so it is not related directly to Aβ peptides. The magnetite particles were initially thought to be a source of ROS, contributing to AD pathology, but later research indicated that such magnetite particles are redox-inert, implying that the amyloid plaque iron would not be a likely direct source of oxidative stress.48

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mutations within the Aβ sequence of APP that yield avidly aggregating peptides,21 and there are mutations within Aβ that significantly reduce the tendency to aggregate.22 Some mutations occur elsewhere in APP but enhance the cleavage producing Aβ. The third family of mutations comprises proteases that excrete Aβ peptides.23 These observations and numerous experimental animal studies prompted the amyloid cascade as a postulated mechanism of AD. In this concept, the

MUTATIONS AND N- AND C-TERMINAL MODIFICATIONS OF Aβ PEPTIDES Despite a collective name, Aβ is a diverse group of peptides. Compared within a given organism (and with a provision for mutations), they constitute a family of molecules with the same central amino acid sequences but differing by the presence of C

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

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not bring AD automatically, thus the Aβ clearance impairment does not seem to qualify as the sole explanation of SAD. Importantly for this Viewpoint, we did not find reports that considered specific clearance of Aβ variants, such as N-truncated species, except for a comment that a particular propensity of Aβ4−x species to aggregate may cause them to be preferentially retained.59,78 Proteolysis of Aβ in the brain is another process relevant for decreasing its burden. Neurons and other brain cells take up Aβ peptides as a necessary step in BBB clearance, and intracellular digestion of these peptides via the ubiquitin−proteasome system (UPS) and the lysosomes has been demonstrated, as reviewed.61,79 However, digestion of extracellular Aβ peptides by individual extracellular or external membrane-bound proteases has gained more attention as a possible culprit in AD and a facile target for intervention. In some cases, a relationship between the enzyme expression level and its activity or inhibition was correlated with the progression of disease in humans or experimental animals.61,80−83 Proteases listed in the context of Aβ degradation include insulin-degrading enzyme (IDE), neprilysin (NEP), matrix metalloproteinases (MMPs), especially MMP2, MMP7, and MMP9, angiotensin-converting enzyme (ACE), endothelin-converting enzymes (ECE1 and ECE2), and plasmin. Indeed, the catabolic activity of any individual protease is not likely to suffice to account for AD pathology, but rather the actions of anabolic and catabolic enzymes sum up to a dynamic Aβ equilibrium level, which may evolve with age as malfunctions of various metabolic pathways, including proteolysis and clearance, accrue.84 The Aβ toxicity is noted only in the brain, although the peptide is produced in other tissues as well.85−87 Human serum albumin (HSA), the main extracellular protein of body fluids, forms a molecular complex with Aβ1−40 with CK7.4 = 2 × 105 M−1, keeping the peptide in the largely α-helical conformation, which prevents its aggregation.88,89 Typical HSA concentration in blood serum is ca. 0.6 mM.90 In CSF, it is 3 μM, still being the major protein component.91,92 Consequently, ∼90% of Aβ in blood serum and ∼40% in CSF is bound to HSA.93,94 HSA is most likely absent from synaptic clefts and the vicinity of neurons in the intact brain, but HSA addition enhanced digestion of Aβ aggregates by microglia.95 The ability of Aβ peptides to interact with the cellular membrane has been associated with their toxicity in AD. The Aβ population residing in the membrane environment following their generation by APP proteolysis has been estimated at ca. 20%.96 There is a general belief that oligomers are key membrane-damaging species, due to their ability to form Ca2+permeable pores.26,97−102 However, some studies indicated that soluble Aβ monomers are more prone to insertion into membranes than oligomers and mature fibrils.103,104 Regarding the truncated species, experiments on primary cortical mouse neurons revealed enhancement of lipid peroxidation by Aβ3pE−42 compared with the Aβ1−42.105 On the other hand, a study implementing molecular dynamics simulations, atomic force microscopy, channel conductance measurements, calcium imaging, neuritic degeneration, and cell death assays indicated that nonamyloidogenic Aβ9−42 and Aβ17−42 peptides form ion channels, elicit single-channel conductances, and induce neuronal toxicity in a dose-dependent fashion by altering cell calcium homeostasis.106

longer or shorter sequences at both ends of the amino acid chain, mostly at the N-terminus.49 This heterogeneity is a result of inaccuracy of secretases and further hydrolytic and posttranslational modifications (PTMs). Two peptides, Aβ1−42 and Aβ1−40, are the primary β- and γ-secretase products, serving as platforms for truncations and PTMs. Table 1 presents their sequences together with the C-terminally extended Aβ (CTEAβ), formed by so-called ε-cleavage,50 and N-terminally extended Aβ (NTE-Aβ) variants with one or more additional APP residues.51,52 The information on the presence and abundance of Aβ peptides is based mainly on immunoprecipitation of peptides from tissue, combined with peptide analysis by mass spectrometry. Therefore, the effectiveness and selectivity of detection are highly dependent on the nature of the antibodies used, and the quantitative aspect of the data is prone to systematic errors. Not surprisingly, the results obtained in different laboratories may be significantly disparate. This methodology also has a limited ability to detect the shortest Aβ peptides, discussed in the final sections of this Viewpoint. Despite these limitations, the emerging consensus names three Aβ peptides most abundant in brains of both normal subjects and AD victims: Aβ4−42 ≈ Aβ1−42 > Aβ1−40 ≫ other Aβ peptides,53−56 although some authors found Aβ4−42 predominantly in AD brains.57,58 Nevertheless, the vast majority of biochemical studies and the entire molecular diagnostics of AD include only the “canonical” Aβ1−42 and Aβ1−40 peptides. The Aβ1−42/Aβ1−40 ratio in cerebrospinal fluid is currently the basic biochemical diagnostic marker for the prodromal AD stage in accordance with the amyloid cascade hypothesis.65,66 The Aβ4−42 peptide was detected as the major component of amyloid plaques already in the first sequencing studies around 1984.67,68 Mass spectrometry sequencing was not available then, requiring the samples to be processed for classical sequencing. The probable historical cause why Aβ4−42 has been ignored by mainstream AD research ever since was the mistaken belief that it was an artifactual product of pepsin digestion of Aβ1−42.69 One should also note that most antibodies used for detection of Aβ peptides recognize the central and C-terminal parts of the peptides. Without the MS analysis the N-truncated species would not be recognized. The methodical progress revived broader interest in truncated Aβ peptides. The Aβ4−42 peptide was demonstrated to aggregate more rapidly than Aβ1−42,59,70 which is a rationale for its apparent absence in the cerebrospinal fluid.71 Aβ4−42 was also more neurotoxic in vitro and in transgenic mice.58,72 This property, shared with other Ntruncated species, prominently Aβp3−42 and Aβp11−4273 is assigned to their propensity for fast formation of toxic oligomers.55,58 However, until 2015, this peptide did not attract the attention of the copper branch of AD research, fully focused on Aβ1−42, Aβ1−40, derivative sequences, and their mice analogs.74−76 Insufficient Aβ clearance across the blood−brain barrier (BBB) due to vascular damage is another postulated cause of AD. This idea relates AD to an unhealthy lifestyle (and thus prevention or delay of AD by healthy lifestyle)4−6 and explains the comorbidity of type 2 diabetes and cholesterol dysmetabolism with AD. Clearance mechanisms relevant for Aβ peptides include transport mediated by lipoprotein receptor-related protein 1 (LRP1), cerebral parenchymal cells, and choroid plexus secretion.77 On the other hand, the healthiest lifestyle cannot reverse AD, and the poor condition of vasculature does D

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Structures of component I (A), component II (B), and the redox active form (C) of Cu(II) complexes with Aβ1−16 and Aβ1−40 peptides (based upon refs 76, 113, and 130). Hisx and Hisy denote any combination of His6, His13, and His14.

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COPPER COMPLEXES OF Aβ1−x PEPTIDES The C-terminally amidated Aβ1−16 peptide has become the standard research model for studying the interaction of Cu(I)/ Cu(II) and Zn(II), as well as iron and other metal ions of biological importance, because it contains all residues able to bind metal ions (except Met35, which does not participate in known metal ion complexes of Aβ peptides). Good solubility (millimolar) in a wide pH range and maintenance of the monomeric state, which enables accurate physicochemical experiments are its two main advantages. For longer Cterminally extended model peptides, the physicochemical studies become difficult, due to their fast aggregation at concentrations required by most of these methods. The Aβ1−40 peptide aggregates spontaneously at concentrations above 10−15 μM.107 The critical aggregation concentration for the Aβ1−42 peptide, usually reported as 2 μM, may be much lower according to a recent study (∼90 nM).108 Numerous studies of many research groups, using Aβ1−16 (and Aβ1−28 to a lesser extent) led to a consensus, according to which at pH 7.4 Aβ1−16 forms two types of complexes (component I and component II, Figure 1A,B, respectively) in a dynamic equilibrium.39,76,109−114 Their main feature is the dynamic character of histidine coordination, with the three residues replacing each other while maintaining the overall composition of the first coordination sphere. In addition to the generally accepted view of component II coordination involving the Ala2 peptide nitrogen binding, it was alternatively proposed that all three histidine residues participate in the binding equally and simultaneously, complemented in the first coordination sphere of Cu(II) by the Ala2 carbonyl oxygen.40 In a number of studies, the ability of Aβ1−x to bind another Cu(II) ion was indicated. Its presence is evident upon the observation of Tyr10 fluorescence quenching in the course of Cu2+ titration of Aβ1−x peptides, but the resulting Cu(II)2 site has never been characterized accurately.115,116 In a very broad and technically accurate study, the Aβ1−16 peptide extended with a poly(ethylene glycol) chain (PEG) was employed.117 This approach was aimed at obtaining a quantitative characterization of the coordination process. Aβ1−16(PEG) was reported to bind up to four Cu(II) ions. A weak point of this approach is that PEG is a very strong modifier of peptide conformation, acting by water displacement, and thus Cu(II) binding to PEG-ylated Aβ1−16 cannot be directly compared to that of the native peptide.118 The kinetic lability of histidine residues around the Cu(II) ion makes the Cu(Aβ1−x) complex prone to ternary complex formation. Two general types of ternary complexes were reported. In one, Aβ1−x was the major partner of the ternary interaction, with small monodentate ligands, such as buffer components, able to bind to Cu(II) at high molar excess.119,120

In another, terdentate chelators, such as 2-(dimethylamino)methyl-8-hydroxyquinoline, occupied three sites around Cu(II), with Aβ1−x acting as a monodentate chelator through one of its histidine imidazole nitrogens.121 We checked whether glutamate, which may be co-present with Aβ peptides in glutamatergic synapses, might be a ternary partner for Aβ1−16 in Cu(II) binding. Instead, we found that these two ligands only competed for Cu(II) at the physiological range of its concentrations.122 These studies confirmed a pleiotropic and hardly predictable character of Cu(II) interactions with Aβ1−x peptides. Many efforts were undertaken to determine the stability constant for Cu(Aβ1−x) complexes, using a number of methodologies, including calorimetry, potentiometry, and spectroscopic techniques. Disparate and often controversial results were obtained, as reviewed.105,112 These controversies seem to have been solved by a couple of comprehensive studies from separate laboratories, which used fluorescence quenching for two slightly different peptide models (one containing native Tyr10116 and another employing a Trp substitution123). These two studies yielded virtually the same conditional binding constant for Cu(Aβ1−16) at pH 7.4 (CK7.4) as 1.1 × 1010 M−1. The corresponding CK7.4 value for Cu(Aβ1−40) was very similar, 2.7 × 1010 M−1.116 The binding is pH dependent; hence the binding constant determined around pH 7 is closer to 109 M−1.124,125 Redox properties of Cu(Aβ1−x) complexes have attracted a great deal of attention as a possible source of neuronal damage, as reviewed.126 These complexes require suitable reducing or oxidizing agents to generate reactive oxygen species (ROS) via the Cu(I)/Cu(II) redox pair. Ascorbate (Asc) has been investigated most frequently, due to its biological abundance. The overall brain Asc is in the millimolar range, with particular enrichment in neurons (about 10 mM).127,128 Generally considered to be neuroprotective, Asc is often a source of deleterious radical species when contacted with adventitious redox-active metal ions, for example, in carcinogenesis.129 In line with this feature, copper complexes of Aβ1−x peptides are moderately active catalysts of ROS formation in the presence of Asc, less active than free or “loosely bound” copper.130,131 In this context, one should note that buffers, such as Hepes, Aces, or Tris can coordinate Cu(II) and the real Cu2+ aqua ion concentrations are thus nanomolar and lower.132−134 Earlier studies were focused on the generation of hydroxyl radicals and H2O2.135 Recently the formation of superoxide radicals was also found.46,136 Strikingly, a reverse correlation was found between the ROS generating activity and the peptide length and degree of aggregation of Aβ1−x species (i.e., the monomeric CuAβ1−16 was the most active and the fibrillary CuAβ1−42 was the least active E

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry species).136,137 The ROS producing reversible Cu(I)/Cu(II) redox couple in CuAβ1−x complexes has been attributed to the structure shown in Figure 1C, whose contribution to equilibrium at pH 7.4 does not exceed 0.1% (thus explaining the relatively low activity).130,138,139 Aβ species with oxidized side chains of histidine, methionine, and tyrosine residues were detected in AD brains.140 The current focus is directed at the ability of hydroxyl radicals to oxidize the Tyr10 aromatic ring to yield dityrosine bridges (diTyr).141,142 Such diTyr bridged Aβ1−x peptide dimers have been detected in abundance in the brain regions most susceptible to AD pathology and are thought to be one of the causative (seeding) agents in the formation of toxic Aβ oligomers.143,144 The CK7.4 ≈ 1010 M−1 for Cu(II)Aβ1−x at pH 7.4 is rather low, compared to other extracellular Cu(II) carriers and enzymes in the human body. The most abundant of them, HSA, binds Cu(II) with CK7.4 = 1012 M−1145 and was demonstrated to withdraw Cu(II) from CuAβ1−x directly and completely.146 Metallothionein-3 (MT3), another copper-binding protein, was also demonstrated to decompose Cu(II)Aβ1−x complexes and to abolish their toxicity in neuronal cultures.147 MT3, which is the brain-specific metallothionein, can bind multiple Cu(I) ions with the average CK7.4 = 1016 M−1 and is also able to reduce Cu(II) to Cu(I) prior to the binding by one of its 20 cysteine residues.148 It is thought to protect the brain from copper oxidative toxicity, as Cu(I) ions bound to MT3 remain redoxinert. A number of chelating agents were designed in order to mimic the action of HSA and MT3 and defuse the copperrelated Aβ1−x toxicity, as comprehensively reviewed recently.149 Chelation of Cu(II) is usually addressed, but recently, targeting Cu(I) was proposed.150 These chelators, sometimes equipped with additional functionalities, usually perform well in vitro and even in animal studies. A related concept proposed that the introduction of AD-matched copper ionophores to the brain could prevent or halt the disease process by decomposing Cu(II) complexes of Aβ peptides in the extracellular space and eventually transferring recovered Cu(II) ions into cells.151 This concept resulted in the design and synthesis of a putative brainfriendly Cu(II) ionophore, 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2) by Prana Biotechnology.152 This compound gave promising results in experimental animals and a pilot clinical study but failed to improve the cognitive performance of patients in a larger phase II clinical trial. The failure of PBT2 was ascribed to the fact that instead of forming an ionophoric CuL2 complex, it preferentially formed CuLA ternary complexes where A represents the imidazole ring of any histidine residue of the Aβ1−x peptide.121 Similar results were obtained later for genuine PBT2.153 A thorough discussion and critique of attempts to use chelators or ionophores for treating AD have recently been published.69 The presence of Cu in amyloid plaques43 and copurification of copper with Aβ from AD brain tissues154 inspired the copper amyloid cascade hypothesis and prompted multiple studies of Cu(II)-dependent Aβ aggregation. Reproducibility of results among or even within different laboratories is a significant issue in these studies, due to a high sensitivity of the aggregation process to even small changes in the experimental protocol.155,156 However, a considerable number of reports indicated that Cu(II) ions push the aggregation pathway of Aβ1−40/42 away from fibrillar structures toward amorphous aggregates or oligomers, assumed to be the crucial neurotoxic forms.157 These aggregated forms displayed various toxicities, namely,

Cu(II) amorphous aggregates were deemed nontoxic,158,159 the more fibrillar Cu(II) aggregates as cytotoxic,160,161 and oligomers as highly cytotoxic.162−164 The crucial issue of relationship between Cu(II) complexation by Aβ1−x complexes and their membrane interactions remains to be solved. In the only structural study, SDS micelles were used as membrane model for the CuAβ1−40 complex. The Cu(II) ion was bound in the same way as in the absence of SDS, to the N-terminus of the peptide that was not structured by SDS, and retained its ROS generating ability.165 Acknowledging the fact that this model system stabilizes the monomeric state of Aβ peptide, it is interesting to see that there was no tendency noted for Aβ1−40 to leave the hydrophobic environment and aggregate with Cu(II). The studies summarized above reveal another weak spot in the copper amyloid cascade concept. The ROS production by monomeric CuAβ1−x complexes is similar to or higher than that of the aggregated species. This observation appears to be at odds with the lack of toxicity of monomeric Aβ1−x. One ought to ask whether oxidative stress in AD brains can really be caused by the relatively weak CuAβ1−x complex, so easy to silence by external chelators present in brain interstitial fluid and CSF,166 by way of ternary complex formation or even by simple competition, as was the case with glutamic acid?122

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COPPER COMPLEXES OF Aβ4−x PEPTIDES Upon encountering the analytical data that indicated the high abundance of the N-terminally truncated Aβ4−42 variant and, more broadly the existence of the Aβ4−x family of peptides (Table 1), we immediately realized that these peptides should have very high affinities for Cu(II) ions. This is due to the presence of the Phe-Arg-His amino acid sequence at their Nterminus, belonging to the ATCUN (amino-terminal Cu,Nibinding)167/NTS (N-terminal site)168/His-3169 motif, first discovered in serum albumins.170 This motif, shown in Figure 2, is formed by the tripeptide amino acid sequence of Xaa-Zaa-

Figure 2. Structure of the ATCUN/NTS complex formed by Aβ4−x peptides (based upon refs 181−183).

His, where the amino acid residue Xaa contains an unmodified amino group and the Xaa-Zaa peptide bond contains a dissociable hydrogen atom at the peptide nitrogen (i.e., Zaa ≠ Pro). In such situation, four consecutive nitrogen atoms, the Xaa N-terminal amine, the amide nitrogens of Zaa and histidine residues, and the N-1 nitrogen of the imidazole ring in the histidine side chain, form a square-planar coordination structure around the Cu(II) ion with a concomitant release of four hydrogen ions (4N complex). This structure has been documented by X-ray,171,172 EXAFS,172−174 and numerous spectroscopic studies, as reviewed comprehensively.167,169,175,176 The ATCUN/NTS structure binds several other metal ions in the analogous fashion. There are X-ray structures available for Ni(II),177 Pd(II), and Au(III) ions.178 F

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

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together with no, one (major species), or two local peptide nitrogens. The first of these species is identical with the redox active complex of Aβ1−x peptides. As expected, therefore, the Cu2Aβ4−16 complex at pH 7.4 yielded the irreversible oxidation wave of Cu(II) bound at ATCUN/NTS and the quasi-reversible reduction wave of Cu(II) bound at His13−His14 to a Cu(I) species.181 Spectroscopic and electrochemical parameters of the ATCUN/NTS complex were not affected by the presence of the second Cu(II) ion. Interestingly, however, Tyr10 fluorescence quenching resulting from the coordination of the first Cu(II) ion was practically identical for both Aβ1−16 and Aβ4−16. Together with the oxidation of Tyr10 by the Cu(III) complex in CuAβ4−16, this finding indicates that the N-terminal segment of CuAβ4−x has high conformational freedom, and, unlike CuAβ1−x, the structuring of the peptide by Cu(II) coordination is indeed limited to three N-terminal residues. In the next step of the studies, we checked whether the Cu(II) ion bound to Aβ4−x peptides is mobile, that is, it can be easily transferred to other molecules. Aβ peptides are thought to exhibit their neurotoxic properties primarily in the extracellular space and in the synaptic cleft in particular. Our first choice was HSA, which is the major Cu(II) scavenger in extracellular fluids, with a demonstrated superiority over Aβ1−x.146 The CK7.4 value for Aβ4−16 is higher, but HSA prevails in terms of biological concentration: 0.6 mM in blood90 and 3 μM in CSF91,92 vs nanomolar Aβ in blood and CSF.186−188 HSA should therefore prevent all Aβ, including Aβ4−x peptides from Cu(II) binding in the bulk of body fluids if given sufficient reaction time to reach chemical equilibrium. Metallothionein-3 (MT-3), as mentioned above, is one of the major proteins that bind extracellular copper in the brain. Metallothioneins are Zn(II) binding proteins, and MT-3 is released from cells as Zn7MT-3 complex.189 Zn7MT-3 was shown to protect neuronal cell cultures from the CuAβ1−40 toxicity.190 Background chemical studies indicated that this action likely resulted from the reductive swap of copper and zinc, yielding ZnAβ1−40 and Cu(I)-occupied MT-3. MT-3 itself was a reductant in this process, sacrificing some of its thiol groups with the formation of intramolecular disulfide bridges.147,191 As CK7.4 of Cu(I) binding to MT-3 is estimated at about 1016 −1 148 M , the transfer of copper from Aβ4−x to MT-3 should be possible. First experiments carried out by us have shown that this reaction did not take place even after several hours of incubation of CuAβ4−16 with Zn7MT-3 but could be forced by coincubation with very high, nonphysiological concentrations of Asc, yielding primarily Cu(I)4Zn(II)4MT-3 as a product of copper transfer. In a separate experiment, Cu(II) ions were added to a solution containing equimolar Aβ4−16 and Zn7MT-3. An apparent equilibrium was established rapidly, in which both molecules participated significantly in copper binding. The partition of copper was not related quantitatively to the ratio of affinity constants.192 The second Cu(II) ion, bound at the His13−His14 pair in Aβ4−16, was easily and promptly reduced and transferred to MT-3, in a fashion similar to that seen for CuAβ1−16 and CuAβ1−40 complexes.147,192 In a follow-up study, we further looked for conditions enabling copper transfer from the ATCUN/NTS site in Aβ4−16 to MT-3. Long (several hours) incubations of Cu(II)Aβ4−16 with Zn(II)7MT-3 in the presence of millimolar reduced glutathione (GSH) resulted in a partial transfer. The amino acid cysteine was much more effective, providing a complete transfer within minutes, by reducing Cu(II) to Cu(I) and shuttling Cu(I) to MT-3.193 While more effective than Asc, the concentrations of thiols needed for the

The Cu(II) binding constants for the ATCUN/NTS motif fall within the range of 1.0 × 1012 M−1 for HSA145 to 3.2 × 1014 M−1 for endostatin179 and even 4.6 × 1014 M−1 for hepcidin Nterminus.180 Hinted by the fact that even the weakest of these ligands, HSA, is able to remove Cu(II) from its complexes with Aβ1−x peptides promptly and smoothly,146 we hypothesized that Aβ4−x peptides will be preferably targeted by Cu(II) ions under biological conditions over Aβ1−x peptides or other truncated peptides without a His at position 3. With this rationale, we initiated a plan of systematic comparative research on Cu(II) complexation with Aβ4−x peptides vs Aβ1−x peptides and properties of the respective complexes. We started our studies of Cu(II) interactions by determining the stoichiometry, binding constants, and structure of complexes for the Aβ4−16 model peptide (amidated Cterminally). We used potentiometric titrations for quantitation of complex formation, accompanied by UV−vis, CD, and EPR spectroscopies for corroboration of the pH dependence of the complexation process and for structural analysis. We established that the first Cu(II) equivalent added to the peptide forms the ATCUN/NTS complex at the Phe-Arg-His sequence in a very wide range of pH. The CK7.4 value for this complex is 3.0 × 1013 M−1, which is about 3000 times more than that for the Aβ1−x complexes.181 By directly reacting Cu(Aβ1−16) with apo-Aβ4−16, we showed that the Aβ4−16 peptide takes up the Cu(II) ion from the Aβ1−16 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 Aβ1−16.124 The redox properties of the Cu(Aβ4−16) complex were examined in the same study using voltammetric methods, CV and DPV. The Cu(II) ion bound to the Phe-Arg-His sequence was not reduced on a glassy carbon electrode to Cu(I) in the potential range available in the aqueous solution, but the irreversible electrochemical oxidation of Cu(II) to Cu(III) was observed at a potential of about 1.0 V. This process overlapped with the irreversible oxidation of the Tyr10 phenolic ring to quinone derivatives.181 Subsequent electrochemical studies were performed for truncated peptides Aβ4−6, Aβ4−8, and Aβ4−10 and the modified Aβ4−10(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 demonstrated the electrochemical reversibility of the Cu(II)/Cu(III) redox pair in the absence of Tyr10 and its lack (irreversible oxidation) in its presence.184 This strict dependence indicates the existence of an electron transfer path between the ATCUN/NTS complex and the phenol ring of the Tyr10 residue. We also investigated the formation of hydroxyl radicals in the presence of Asc by Cu(II) complexes of Aβ4−16 and Aβ4−42 peptides at the 1:1 stoichiometry versus CuAβ1−16 and CuAβ1−42 as positive controls.181 The radicals were detected with the APF test.185 Expectedly, the complexes of Aβ1−x peptide yielded significant amounts of such radicals, while the Aβ4−16 and Aβ4−42 complexes were completely inactive. The Aβ4−16 peptide can also bind the second Cu(II) ion. The binding is sequential, with the second Cu(II) ion binding only upon the saturation of the ATCUN/NTS site, because its CK7.4 value (5.2 × 106 M−1) is more than a million times smaller than that of the primary site. The binding site of this ion is at the His13−His14 pair. At pH 7.4, three complex species coexist. In all of them, Cu(II) is bound to His13 and His14 imidazoles, G

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Some studies reported the effects of Cu(II) on the aggregation of truncated Aβ peptides, others followed the aggregation of Aβ4−x peptides, but the combination of Cu(II) and Aβ4−42 was not investigated in this respect. For example, stoichiometric Cu(II) accelerated the aggregation of Aβ14−23 by apparently bridging peptide molecules, but reduced aggregation of Aβ11−28 by forming a stoichiometric CuAβ11−28 complex.199,200 However, the biological relevance of CuAβ11−x is disputable for the low abundance of unmodified Aβ11−x species201 and a relatively weak Cu(II) complexation to the EVH(11−13) sequence.183 Studies on truncated Aβ peptides in the absence of Cu(II) ions showed different results depending on the type of truncation. The N-terminally truncated Aβ2−40 showed an increased aggregation propensity compared to Aβ1−40 and even acted as a seed for Aβ1−40 aggregation.202 Aβ4−x peptides (x = 40, 42) aggregated faster than the corresponding Aβ1−x peptides. Interestingly, this effect could be counteracted by a parallel Cterminal truncation, indicating cross-talk between the Aβ termini during aggregation.59 Our preliminary results, carried out according to analogous methodology,203 confirmed the very high ability of Aβ4−40 to form ThT-reactive fibrils relative to Aβ1−40, but this effect was partially eliminated by Cu(II) ions, roughly proportional to the Aβ4−40/Cu(II) ratio. This finding suggests that Cu(II) ions slightly elongate the oligomer formation phase (lag phase) and reduce fibril formation, probably by enhancing the oligomer stability. In addition, a significant difference in aggregate morphology in the presence of Cu(II) ions was observed. The Aβ1−40 fibril formation was largely inhibited, and only very short fibrils were seen, together with other more globular agglomerates. The Aβ4−40 fibrils formed in the presence of Cu(II) had altered, clog-like morphology. Overall, our results confirm the extreme importance of the N-terminus of Aβ peptides for their aggregation, irrespective of their participation in the amyloid structure, and indicate significance of the relatively small conformational alteration actuated by the Cu(II) complex.204

CuAβ4−16 decomposition were still nonphysiologically high. The next molecule tested by us for assistance in copper transfer was glutamate, the main neurotransmitter in the brain regions primarily affected by AD pathology.194 Glutamate is thus copresent in synaptic clefts together with Aβ peptides and MT-3 upon release from highly packed (100 mM) presynaptic vesicles.195 Peak concentration of glutamate in the synaptic cleft during neurotransmission is estimated at several millimolar.196 We observed an acceleration of copper transfer from Cu(II)Aβ4−16 to Zn(II)7MT-3 by glutamate. The transfer was also enhanced by lowering the Zn(II) load of MT-3 with EDTA.197 The affinity of glutamate for Cu(II) is so much lower than that of the other reactants that Cu(Glu) complexes could not participate in the equilibrium with any significance. The rate enhancement by removal of Zn(II) from Zn(II)7MT-3 was found to be equally unlikely. On the basis of kinetic arguments, we proposed that the transfer enhancement is due to a transient formation of a hypothetical ternary complex [Glu−Cu(II)− Aβ4−16], which could then deliver copper to metallothionein in an associative fashion. We find it very interesting that despite the low affinity of Glu for Cu(II) compared to Aβ4−16 and MT-3, such a small molecule can affect the kinetics of copper transfer. In another segment of our systematic research, we studied the interaction of Aβ4−16 with Cu(II) in the presence of NDHQ, the model hydroxyquinoline introduced in our previous work.121 In accord with the order of affinities, the equilibrium of the reaction is shifted toward CuAβ4−16 at sufficiently high peptide concentrations. However, the overall affinities of CuAβ4−16, Cu(NDHQ)2 and Cu(NDHQ)Aβ4−16 complexes are similar and the state of equilibrium strongly depends on relative reagent concentrations. In the ternary complex, three coordination sites are occupied by NDHQ, and the fourth position by an Aβ4−16 histidine imidazole (or any other imidazole ligand, Figure 3).198

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FORMATION OF Aβ4−x AND OTHER ATCUN PEPTIDES BY NEPRILYSIN We also examined the digestion of Aβ1−16 and Aβ1−40 peptides by neprilysin (NEP), the zinc protease listed above as one of the main enzymes of brain Aβ catabolism and a proposed source of Aβ4−x peptides.205,206 In our hands, NEP proved to be an excellent producer of ATCUN peptides indeed, of Aβ4−x and Aβ12−x types, but not really of long Aβ4−x species.182 This was because the elimination of the Tyr-Glu dipeptide (Aβ10−11) was among the fastest processes catalyzed by NEP in both Aβ1−x substrates. Hence, Aβ4−9 and Aβ12−16 peptides emerged as significant products (the latter also in Aβ1−40, due to the cleavage behind Lys16). In the presence of Cu(II), the CuAβ4−9 complex was formed quantitatively from the initial CuAβ1−x substrate. We found that the excess of Cu(II) and Zn(II) ions inhibited NEP, with Ki values of 1 and 20 μM, respectively.182 The micromolar Cu2+ concentrations are easily available in the synaptic cleft,207 and so Aβ4−9 has the potential to enhance its own production by sequestering Cu(II) released during neurotransmission, thereby preventing NEP inhibition.208 In the follow-up work, we described the Cu(II) binding properties of Aβ4−9, Aβ11−16, and Aβ12−16 peptides. Aβ11−16 and its pyroGlu variant were included following the report of a femtomolar affinity of CuAβ11−x complexes,209 remembering that the analytical data indicated the prevalence of pyroglutamylation of N-terminal Glu11 peptides in the brain.73 Aβ4−9 and Aβ12−16

Figure 3. Structures of transient ternary Cu(II) hydroxyquinoline/ imidazolate complexes.198

Aβ1−16 formed a similar structure. Interestingly, the equilibration of the reaction with Aβ1−16 occurred within the mixing time of the samples (seconds), but for Aβ4−16, it was slow (t1/2 ≈ 90 min at millimolar concentrations). Therefore, NDHQ could form ternary complexes with both types of Aβ peptides. Control experiments with imidazole as a generic ternary ligand for Cu(NDHQ)L complexes confirmed that such ternary complexes can yield substantial amounts of hydroxyl radicals in the APF test (although this activity was significantly quenched by sufficient amounts of Aβ4−16 by virtue of equilibrium shift).198 We concluded that terdentate hydroxyquinolines, such as NDHQ or PBT2, can indeed interfere with Cu(II) binding to both Aβ1−x and Aβ4−x peptides, but the outcome is rather harmful than beneficial, due to ROS generation by the resulting ternary complexes. H

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yielded strong Cu(II) complexes, CK7.4 = 1.5 × 1014 M−1 and 1.1 × 1014 M−1 at pH 7.4, respectively, that is, five and three times stronger than CuAβ4−16.183 Cu(Aβ11−16) and Cu(Aβp11−16) were 200 and 600 000 times weaker in terms of CK7.4. The time of equilibration of Cu(II) ions between the Aβ4−9 and Aβ12−16 sites was in the range of days, much slower than the six hours period of renewal of cerebrospinal fluid.210 Therefore such ATCUN complexes with oligopeptidic products of Aβ proteolysis should be considered as inert in the time scale of brain physiology. Their small size and good water solubility make them likely candidates for copper clearance across the BBB.183 Equally important in this respect is the high resistance of Aβ4−x peptides to reduction by GSH,173,192,197 which increases their chance of surviving the passage through endothelial cells constituting the BBB.

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SUMMARY AND OUTLOOK

We have shown that Aβ4−x peptides should be considered as primary Cu(II) binding agents in extracellular spaces of the brain, capable of rapidly taking over copper from previously tested Aβ1−x peptides and forming complexes that are highly stable, redox unreactive, and very resistant to Cu(II) release and Cu(II)/Cu(I) exchange.181,192,193 Altogether, these properties make Aβ4−42 a candidate molecule to help rapidly restore basal conditions in the glutamatergic synaptic cleft by cleansing it of excessive Cu2+ ions, thus enabling the next neurotransmitter release. We have to stress that the origin of our proposal of a physiological role for the Aβ4−42 peptide is purely molecular. It is chemistry in search of a biological function. We can only cite a weak indirect support for it by noting that endogenous antibodies were detected against Aβp3−x species and Aβ oligomers, but none were found against Aβ4−x peptides.58 Such function could have evolved recently; after all, the brain is the most divergent organ when humans are compared with other mammals or vertebrates in general. Nevertheless, a systematic analysis of the presence of histidine near the N-terminus in Aβ sequences together with the availability of appropriate proteases in other species could help ascertain our proposal in a broader evolutionary context. For example, the mouse Aβ sequence conserves His6, although the Arg5Gly replacement may alter the proteolytic production of mAβ4−x. His6 or His7 is also present in many, but not all lower animals.216 Chemical properties required for the proposed function of Aβ4−42 may be conveyed by essentially any ATCUN peptide, and at least one of the His13/ His14 couple is usually conserved. Human Aβ (Table 1) offers ATCUN peptides by cleavage at both positions 11 and 12. The former products are mostly pyroglutamylated but may not be so in other species. Aβ12−x peptides can have properties very similar to those of Aβ4−x. One should add that unlike Aβ1−x peptides, which form relatively strong Zn(II) complexes,217 Aβ4−x are extremely specific for Cu(II) and will not be modulated by Zn(II), Fe(II), or any other biological metal ion.169 The proposed Aβ4−x function should be compromised in patients carrying the English mutation His6Arg, as the mutated peptide loses its Cu(II) binding properties (Table 2). A deeper look at copper status and neurotransmission in these patients could help verify our concept. An alternative function could be assumed in Aβ5−x species carrying the Taiwanese Gly7His mutation. The proposed function of Aβ4−x may be easily disturbed by inappropriate pharmacological intervention and may be one of the reasons of failure of PBT2 in the clinical trial.198 Therefore, a thorough investigation of this function is very important for designing anti-Aβ therapies. The failure of all of them so far gives us a strong hint for the relevance of some Aβ species in the brain physiology. Our concept is very important for the idea of therapeutic chelation, because such chelator should be sufficiently weak to avoid interference with CuAβ4−x complexes, both the long ones presumably residing in synapses and the short ones, possibly participating in copper clearance.182,183 There is no doubt that Aβ4−42 apopeptide can be neurotoxic when in excess, even more neurotoxic than the Aβ1−x species, due to its rapid aggregation and the ability to seed the Aβ1−x coaggregates.56−58,218 Our preliminary experiments indicate that the Cu(II) binding may also have a modulatory effect here, by altering the morphology of aggregates to more amorphous species.204

TIME SCALE OF Cu(II) COMPLEXATION, Aβ AGGREGATION, AND THE ISSUE OF RATES

In the earlier phase of copper/Aβ research, the main focus was on the static (equilibrium) description of coordination structures, mechanisms of redox catalysis, and slow processes of peptide/complex aggregation. This approach was necessary in order to grasp what kinds of objects are involved in the studied processes but is clearly not sufficient for explaining how they interact in the context of synaptic physiology, which is ruled by periodic and sporadic temporal events. The time scales in question range from the nearly millisecond periodicity of neurotransmitter pulses across the synaptic cleft through seconds to minutes of neuromodulator action.211−213 So far kinetic studies of the interaction of Cu(II) binding to Aβ peptides have been limited to Aβ1−x. In addition to establishing basal rate constants, they were aimed at finding out how Cu(II) coordination can be temporally related to nucleation of Aβ aggregation, according to a model involving formation of an (otherwise poorly documented) Cu(Aβ)2 species.124,164,214,215 A general conclusion from these studies was that Cu(II) related phenomena are fast, with initial complex formation essentially in the diffusion limit and with Cu(II) dependent Aβ dimers formed on a subsecond time scale. We analyzed these kinetic data in the framework of real synaptic geometry. Synaptic cleft volumes are extremely low, 2−20 aL. A direct consequence of this fact is that just a single molecule in the synaptic cleft yields a concentration of 83 nM (20 aL cleft) to 830 nM (2 aL cleft). Thus, micromolar concentrations are realized by as little as several molecules.208 This situation requires a stochastic approach, which we applied using the Chemical Master Equation formalism. Using a published comprehensive kinetic model,124 we quantitatively confirmed qualitative proposals formulated in chemical kinetic studies. We showed that left alone in solution and given time, even a few molecules of Aβ1−x peptides will eventually aggregate into an oligomer, but all Cu(II) related processes are much faster. Depending on the availability of Cu2+ ions, Aβ1−x aggregation may be enhanced (Cu2+ < Aβ1−x, facilitating Cu(Aβ)2) or suppressed (Cu2+ > Aβ1−x, facilitating CuAβ). The kinetic data for the formation of CuAβ4−x complexes are not yet available, but the ATCUN complexes are also formed at the diffusion limit.169 The key difference is that they are much more inert (due to higher stability) and thus less prone to interconvert to transient dimeric species. I

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Figure 4. Iceberg metaphor of Alzheimer’s Disease (AD), (copper) amyloid cascade and the proposed physiological role of Aβ4−x peptides. The visible tip of the iceberg represents the visible symptoms of AD. Just beneath the surface is the widely studied set of concepts on the toxic role of Aβ peptides in familial AD (FAD) and sporadic AD (SAD) via the amyloid cascade proposal and its copper variant. Interactions with molecules involved in brain copper metabolism are in a deeper layer, while the proposed specific physiological roles of Aβ4−x peptides in maintaining neurotransmission, copper clearance, and quenching copper toxicity are at the basis of the structure, hidden from the view of most AD researchers.

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OUTSTANDING QUESTIONS

An alternative mechanism could hypothetically be based on the direct entry of CuAβ4−42 to the cell across the membrane. Such mechanism has been postulated for neurokinin B, an ATCUN neuropeptide.221 We, however, find it doubtful, unless assisted by a yet unknown auxiliary process, because amphiphilic Aβ peptides have not been known to flip in phospholipid bilayers. Moreover, the uncontrolled entry of CuAβ4−42 into the cells and the eventual delivery of Cu(I) to GSH (which we demonstrated to occur slowly at a physiological GSH concentration)197 would result in severe neurotoxicity.222,223 However, it is conceivable that the smallest and very hydrophilic CuAβ4−x complexes, which are very sluggish to yield Cu(II) ions, could be taken up by epithelial cells and cleared to the bloodstream without shedding copper intracellularly. Such pathway would assume the suicidal function of Aβ4−42, requiring extensive proteolysis, and seems rather costly for routine synapse cleansing. The bottom line is that more information should be collected about how Aβ4−42 and CuAβ4−42 interact with cellular membranes before a viable hypothesis could be formulated. This issue is part of a broader question that in our opinion has not been convincingly resolved. What is the driving force for monomeric, α-helical Aβx−40/42 peptides with their high

The main unknown in our concept is what happens with Cu(II) bound to Aβ4−x peptides. The other issue to be resolved is whether copper may be toxic in conjunction with these peptides. The key process related to the former issue is the cellular copper uptake. In eukaryotic cells, the major route is provided by Ctr1, a transmembrane channel that transports Cu(I) ions.219 The human protein, hCtr1 has the ATCUN N-terminus, which can collect a Cu(II) ion from HSA, the major Cu(II) carrier in the bloodstream.174,220 The mechanism of Cu(II) reduction to Cu(I) in hCtr1 has not been resolved, but Asc is considered as the physiological reductant. The problem with Aβ4−x delivering Cu(II) to hCtr1 is that such transfer would have to occur upstream in the affinity gradient (CK7.4 = 1.0 × 1013 M−1 for hCtr11−14).174 We also showed that Asc could not reduce Cu(II) bound to Aβ4−16 fast enough to be physiologically relevant.192 Thus, an effective transfer of Cu(II) or Cu(I) from Aβ4−x to hCtr1 would require a catalyst. We demonstrated the existence of such catalysis already.197 Now the search is on for molecule(s) that could actuate it in synaptic conditions. J

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Inorganic Chemistry affinity to membranes, to jump out into the aqueous phase? As we showed in our stochastic calculations,208 the synaptic cleft volume is a sufficient factor to push them into oligomers, which then may approach the membranes as toxic, pore-forming species. All experimental data presented above indicate that Aβ4−x peptides quench rather than activate Cu(II) toxicity and have the ability to quench CuAβ1−x toxicity by rapid Cu(II) sequestration. This idea is presented in Figure 4 in the context of the mainstream understanding of the role of Aβ peptides in AD. The caveat is that we do not possess quantitative information about the abundance and temporal coincidence of Cu(II), Aβ4−x, and Aβ1−x peptides down to individual synapses. For example, at an excess of Cu(II), the Cu(II) ion bound at His13/His14 in Aβ4−x>14 peptides has all the deleterious properties of the Cu(II)/Cu(I) ROS producing system of Aβ1−x peptides, including the formation of diTyr crosslinks.192,224 The CK7.4 for Cu(II) binding at His13/His14 is 5 × 106 M−1, much weaker than that for Aβ1−x. Therefore, at a Cu2+ molar excess over Aβ4−x, Aβ1−x peptides may get their chance to bind copper and generate ROS. Novel probes, such as antibodies selective for Cu(II) complexes of individual Aβ species are needed to resolve this key issue, which we consider to be instrumental in designing any successful AD therapy.

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Wojciech Bal is a Professor and head of Department of Biophysics at the Institute of Biochemistry and Biophysics, PAS, in Warsaw (Poland). He investigates chemical and biochemical mechanisms underlying the transport of essential metal ions relevant for neurodegeneration, copper and zinc, and mechanisms of toxicity of environmental pollutants nickel, cadmium, and palladium.

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ACKNOWLEDGMENTS This work was supported by the PRELUDIUM Grant No. 2016/21/N/NZ1/02785 and ETIUDA Grant No. 2018/28/T/ NZ1/00452, National Science Centre in Poland to E.S.

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REFERENCES

(1) Alzheimer’s Disease International. World Alzheimer Report 2018 The state of the art of dementia research: New frontiers; 2018, https:// www.alz.co.uk/research/WorldAlzheimerReport2018.pdf. (2) Alzheimer’s Association. Alzheimer’s Disease Facts and Figures, 2018, https://www.alz.org/alzheimers-dementia/facts-figures. (3) Haaksma, M. L.; Vilela, L. R.; Marengoni, A.; Calderón-Larrañaga, A.; Leoutsakos, J.-M. S.; Olde Rikkert, M. G. M.; Melis, R. J. F. Comorbidity and Progression of Late Onset Alzheimer’s Disease: A Systematic Review. PLoS One 2017, 12 (5), No. e0177044. (4) Barnard, N. D.; Bush, A. I.; Ceccarelli, A.; Cooper, J.; de Jager, C. A.; Erickson, K. I.; Fraser, G.; Kesler, S.; Levin, S. M.; Lucey, B.; et al. Dietary and Lifestyle Guidelines for the Prevention of Alzheimer’s Disease. Neurobiol. Aging 2014, 35, S74−S78. (5) Kivipelto, M.; Håkansson, K. A Rare Success against Alzheimer’s. Sci. Am. 2017, 316 (4), 32−37. (6) Kivipelto, M.; Mangialasche, F.; Ngandu, T. Lifestyle Interventions to Prevent Cognitive Impairment, Dementia and Alzheimer Disease. Nat. Rev. Neurol. 2018, 14 (11), 653−666. (7) Panegyres, P. K.; Chen, H.-Y. Differences between Early and Late Onset Alzheimer’s Disease. Am. J. Neurodegener. Dis. 2013, 2 (4), 300− 306. (8) Ritchie, K.; Carrière, I.; Berr, C.; Amieva, H.; Dartigues, J.-F.; Ancelin, M.-L.; Ritchie, C. W. The Clinical Picture of Alzheimer’s Disease in the Decade Before Diagnosis. J. Clin. Psychiatry 2016, 77 (03), e305−e311. (9) Dorszewska, J.; Prendecki, M.; Oczkowska, A.; Dezor, M.; Kozubski, W. Molecular Basis of Familial and Sporadic Alzheimer’s Disease. Curr. Alzheimer Res. 2016, 13 (9), 952−963. (10) Nguyen, K. V. The Human β-Amyloid Precursor Protein: Biomolecular and Epigenetic Aspects. Biomol. Concepts 2015, 6 (1), 11−32. (11) Bertram, L.; Lill, C. M.; Tanzi, R. E. The Genetics of Alzheimer Disease: Back to the Future. Neuron 2010, 68 (2), 270−281. (12) Rossor, M. N.; Fox, N. C.; Beck, J.; Campbell, T. C.; Collinge, J. Incomplete Penetrance of Familial Alzheimer’s Disease in a Pedigree with a Novel Presenilin-1 Gene Mutation. Lancet 1996, 347 (9014), 1560.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Wojciech Bal: 0000-0003-3780-083X Notes

The authors declare no competing financial interest. Biographies

Ewelina Stefaniak is a Ph.D. student at the Institute of Biochemistry and Biophysics, PAS, in Warsaw (Poland). Her research focuses on inorganic chemistry in neurodegeneration of Alzheimer’s Disease. She uses biophysical techniques to investigate interactions between copper ions, amyloid β peptides, and biomolecules like MT-3, hCtr1, HSA, or GSH. K

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Inorganic Chemistry (13) Sanabria-Castro, A.; Alvarado-Echeverría, I.; Monge-Bonilla, C. Molecular Pathogenesis of Alzheimer’s Disease: An Update. Ann. Neurosci. 2017, 24 (1), 46−54. (14) Lin, M. T.; Beal, M. F. Alzheimer’s APP Mangles Mitochondria. Nat. Med. 2006, 12 (11), 1241−1243. (15) Nixon, R. A.; Cataldo, A. M. Lysosomal System Pathways: Genes to Neurodegeneration in Alzheimer’s Disease. J. Alzheimer's Dis. 2006, 9 (s3), 277−289. (16) Selkoe, D. J. Amyloid β Protein Precursor and the Pathogenesis of Alzheimer’s Disease. Cell 1989, 58 (4), 611−612. (17) Müller, U. C.; Deller, T.; Korte, M. Not Just Amyloid: Physiological Functions of the Amyloid Precursor Protein Family. Nat. Rev. Neurosci. 2017, 18 (5), 281−298. (18) Selkoe, D. J. Alzheimer’s Disease: Genes, Proteins, and Therapy. Physiol. Rev. 2001, 81 (2), 741−766. (19) Murphy, M. P.; LeVine, H., III Alzheimer̀s Disease and the βAmyloid Peptide. J. Alzheimer's Dis. 2010, 19 (1), 311. (20) Wang, Y. Q.; Qu, D. H.; Wang, K. Therapeutic Approaches to Alzheimer’s Disease through Stimulating of Non-Amyloidogenic Processing of Amyloid Precursor Protein. Eur. Rev. Med. Pharmacol Sci. 2016, 20 (11), 2389−2403. (21) Janssen, J. C.; Beck, J. A.; Campbell, T. A.; Dickinson, A.; Fox, N. C.; Harvey, R. J.; Houlden, H.; Rossor, M. N.; Collinge, J. Early Onset Familial Alzheimer’s Disease: Mutation Frequency in 31 Families. Neurology 2003, 60 (2), 235−239. (22) Panda, P. K.; Patil, A. S.; Patel, P.; Panchal, H. Mutation-Based Structural Modification and Dynamics Study of Amyloid Beta Peptide (1−42): An in-Silico-Based Analysis to Cognize the Mechanism of Aggregation. Genomics Data 2016, 7, 189−194. (23) Thordardottir, S.; Kinhult Ståhlbom, A.; Almkvist, O.; Thonberg, H.; Eriksdotter, M.; Zetterberg, H.; Blennow, K.; Graff, C. The Effects of Different Familial Alzheimer’s Disease Mutations on APP Processing in Vivo. Alzheimer's Res. Ther. 2017, 9 (1), 9. (24) Masters, C. L.; Selkoe, D. J. Biochemistry of Amyloid β-Protein and Amyloid Deposits in Alzheimer Disease. Cold Spring Harbor Perspect. Med. 2012, 2 (6), a006262. (25) De Strooper, B.; Karran, E. The Cellular Phase of Alzheimer’s Disease. Cell 2016, 164 (4), 603−615. (26) Sengupta, U.; Nilson, A. N.; Kayed, R. The Role of Amyloid-β Oligomers in Toxicity, Propagation, and Immunotherapy. EBioMedicine 2016, 6, 42−49. (27) Limbocker, R.; Chia, S.; Ruggeri, F. S.; Perni, M.; Cascella, R.; Heller, G. T.; Meisl, G.; Mannini, B.; Habchi, J.; Michaels, T. C. T.; et al. Trodusquemine Enhances Aβ42 Aggregation but Suppresses Its Toxicity by Displacing Oligomers from Cell Membranes. Nat. Commun. 2019, 10 (1), 225. (28) Tipping, K. W.; van Oosten-Hawle, P.; Hewitt, E. W.; Radford, S. E. Amyloid Fibres: Inert End-Stage Aggregates or Key Players in Disease? Trends Biochem. Sci. 2015, 40 (12), 719−727. (29) Morales, R.; Bravo-Alegria, J.; Duran-Aniotz, C.; Soto, C. Titration of Biologically Active Amyloid−β Seeds in a Transgenic Mouse Model of Alzheimer’s Disease. Sci. Rep. 2015, 5 (1), 9349. (30) Purro, S. A.; Farrow, M. A.; Linehan, J.; Nazari, T.; Thomas, D. X.; Chen, Z.; Mengel, D.; Saito, T.; Saido, T.; Rudge, P.; et al. Transmission of Amyloid-β Protein Pathology from Cadaveric Pituitary Growth Hormone. Nature 2018, 564 (7736), 415−419. (31) Mezias, C.; Raj, A. Analysis of Amyloid-β Pathology Spread in Mouse Models Suggests Spread Is Driven by Spatial Proximity, Not Connectivity. Front. Neurol. 2017, 8, 653. (32) Morris, G. P.; Clark, I. A.; Vissel, B. Inconsistencies and Controversies Surrounding the Amyloid Hypothesis of Alzheimer’s Disease. Acta Neuropathol. Commun. 2014, 2, 135. (33) Mehta, D.; Jackson, R.; Paul, G.; Shi, J.; Sabbagh, M. Why Do Trials for Alzheimer’s Disease Drugs Keep Failing? A Discontinued Drug Perspective for 2010−2015. Expert Opin. Invest. Drugs 2017, 26 (6), 735−739. (34) Servick, K. Another Major Drug Candidate Targeting the Brain Plaques of Alzheimer’s Disease Has Failed. What’s Left? Science 2019, DOI: 10.1126/science.aax4236.

(35) Bishop, G. M.; Robinson, S. R. Physiological Roles of Amyloid-β and Implications for Its Removal in Alzheimer’s Disease. Drugs Aging 2004, 21 (10), 621−630. (36) Copani, A. The Underexplored Question of β-Amyloid Monomers. Eur. J. Pharmacol. 2017, 817 (May), 71−75. (37) Hsieh, H.; Boehm, J.; Sato, C.; Iwatsubo, T.; Tomita, T.; Sisodia, S.; Malinow, R. AMPAR Removal Underlies Abeta-Induced Synaptic Depression and Dendritic Spine Loss. Neuron 2006, 52 (5), 831−843. (38) Kessels, H. W.; Nabavi, S.; Malinow, R. Metabotropic NMDA Receptor Function Is Required for β-Amyloid-Induced Synaptic Depression. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (10), 4033−4038. (39) Drew, S. C.; Noble, C. J.; Masters, C. L.; Hanson, G. R.; Barnham, K. J. Pleomorphic Copper Coordination by Alzheimer’s Disease Amyloid-β Peptide. J. Am. Chem. Soc. 2009, 131 (3), 1195− 1207. (40) Drew, S. C.; Masters, C. L.; Barnham, K. J. Alanine-2 Carbonyl Is an Oxygen Ligand in Cu 2+ Coordination of Alzheimer’s Disease Amyloid-β Peptide − Relevance to N-Terminally Truncated Forms. J. Am. Chem. Soc. 2009, 131 (25), 8760−8761. (41) Miller, L. M.; Wang, Q.; Telivala, T. P.; Smith, R. J.; Lanzirotti, A.; Miklossy, J. Synchrotron-Based Infrared and X-Ray Imaging Shows Focalized Accumulation of Cu and Zn Co-Localized with β-Amyloid Deposits in Alzheimer’s Disease. J. Struct. Biol. 2006, 155 (1), 30−37. (42) Duce, J. A.; Bush, A. I.; Adlard, P. A. Role of Amyloid-β−Metal Interactions in Alzheimer’s Disease. Future Neurol. 2011, 6 (5), 641− 659. (43) Lovell, M. A.; Robertson, J. D.; Teesdale, W. J.; Campbell, J. L.; Markesbery, W. R. Copper, Iron and Zinc in Alzheimer’s Disease Senile Plaques. J. Neurol. Sci. 1998, 158 (1), 47−52. (44) Watt, A. D.; Villemagne, V. L.; Barnham, K. J. Metals, Membranes, and Amyloid-β Oligomers: Key Pieces in the Alzheimer’s Disease Puzzle? J. Alzheimer's Dis. 2012, 33 (s1), S283−S293. (45) Cheignon, C.; Tomas, M.; Bonnefont-Rousselot, D.; Faller, P.; Hureau, C.; Collin, F. Oxidative Stress and the Amyloid Beta Peptide in Alzheimer’s Disease. Redox Biol. 2018, 14, 450−464. (46) Reybier, K.; Ayala, S.; Alies, B.; Rodrigues, J. V.; Bustos Rodriguez, S.; La Penna, G.; Collin, F.; Gomes, C. M.; Hureau, C.; Faller, P. Free Superoxide Is an Intermediate in the Production of H2O2 by Copper(I)-Aβ Peptide and O2. Angew. Chem., Int. Ed. 2016, 55 (3), 1085−1089. (47) Moretz, R. C.; Iqbal, K.; Wisniewski, H. M. Microanalysis of Alzheimer Disease NFT and Plaques. Environ. Geochem. Health 1990, 12 (1−2), 15−16. (48) Gumpelmayer, M.; Nguyen, M.; Molnár, G.; Bousseksou, A.; Meunier, B.; Robert, A. Magnetite Fe3O4 Has No Intrinsic Peroxidase Activity, and Is Probably Not Involved in Alzheimer’s Oxidative Stress. Angew. Chem., Int. Ed. 2018, 57 (45), 14758−14763. (49) Wildburger, N. C.; Esparza, T. J.; Leduc, R. D.; Fellers, R. T.; Thomas, P. M.; Cairns, N. J.; Kelleher, N. L.; Bateman, R. J.; Brody, D. L. Diversity of Amyloid-Beta Proteoforms in the Alzheimer’s Disease Brain. Sci. Rep. 2017, 7, 9520. (50) Qi-Takahara, Y.; Morishima-Kawashima, M.; Tanimura, Y.; Dolios, G.; Hirotani, N.; Horikoshi, Y.; Kametani, F.; Maeda, M.; Saido, T. C.; Wang, R.; et al. Longer Forms of Amyloid β Protein: Implications for the Mechanism of Intramembrane Cleavage by γ-Secretase. J. Neurosci. 2005, 25 (2), 436−445. (51) Kaneko, N.; Nakamura, A.; Washimi, Y.; Kato, T.; Sakurai, T.; Arahata, Y.; Bundo, M.; Takeda, A.; Niida, S.; Ito, K.; et al. Novel Plasma Biomarker Surrogating Cerebral Amyloid Deposition. Proc. Jpn. Acad., Ser. B 2014, 90 (9), 353−364. (52) Szczepankiewicz, O.; Linse, B.; Meisl, G.; Thulin, E.; Frohm, B.; Sala Frigerio, C.; Colvin, M. T.; Jacavone, A. C.; Griffin, R. G.; Knowles, T.; et al. N-Terminal Extensions Retard Aβ42 Fibril Formation but Allow Cross-Seeding and Coaggregation with Aβ42. J. Am. Chem. Soc. 2015, 137 (46), 14673−14685. (53) Portelius, E.; Bogdanovic, N.; Gustavsson, M. K.; Volkmann, I.; et al. Mass Spectrometric Characterization of Brain Amyloid Beta Isoform Signatures in Familial and Sporadic Alzheimer’s Disease. Acta Neuropathol. 2010, 120, 185−193. L

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

Viewpoint

Inorganic Chemistry (54) Lewis, H.; Beher, D.; Cookson, N.; Oakley, A.; Piggott, M.; Morris, C. M.; Jaros, E.; Perry, R.; Ince, P.; Kenny, R. A.; et al. Quantification of Alzheimer Pathology in Ageing and Dementia: AgeRelated Accumulation of Amyloid-Beta(42) Peptide in Vascular Dementia. Neuropathol. Appl. Neurobiol. 2006, 32 (2), 103−118. (55) Bouter, Y.; Dietrich, K.; Wittnam, J. L.; Rezaei-Ghaleh, N.; Pillot, T.; Papot-Couturier, S.; Lefebvre, T.; Sprenger, F.; Wirths, O.; Zweckstetter, M.; et al. N-Truncated Amyloid β (Aβ) 4−42 Forms Stable Aggregates and Induces Acute and Long-Lasting Behavioral Deficits. Acta Neuropathol. 2013, 126 (2), 189−205. (56) Wirths, O.; Walter, S.; Kraus, I.; Klafki, H. W.; Stazi, M.; Oberstein, T. J.; Ghiso, J.; Wiltfang, J.; Bayer, T. A.; Weggen, S. NTruncated Aβ4−x Peptides in Sporadic Alzheimer’s Disease Cases and Transgenic Alzheimer Mouse Models. Alzheimer's Res. Ther. 2017, 9 (1), 80. (57) Antonios, G.; Saiepour, N.; Bouter, Y.; Richard, B. C.; Paetau, A.; Verkkoniemi-Ahola, A.; Lannfelt, L.; Ingelsson, M.; Kovacs, G. G.; Pillot, T.; et al. N-Truncated Abeta Starting with Position Four: Early Intraneuronal Accumulation and Rescue of Toxicity Using NT4X-167, a Novel Monoclonal Antibody. Acta Neuropathol. Commun. 2013, 1, 56. (58) Bayer, T. A.; Wirths, O. Focusing the Amyloid Cascade Hypothesis on N-Truncated Abeta Peptides as Drug Targets against Alzheimer’s Disease. Acta Neuropathol. 2014, 127 (6), 787−801. (59) Cabrera, E.; Mathews, P.; Mezhericher, E.; Beach, T. G.; Deng, J.; Neubert, T. A.; Rostagno, A.; Ghiso, J. Aβ Truncated Species: Implications for Brain Clearance Mechanisms and Amyloid Plaque Deposition. Biochim. Biophys. Acta, Mol. Basis Dis. 2018, 1864 (1), 208−225. (60) Kummer, M. P.; Heneka, M. T. Truncated and Modified Amyloid-Beta Species. Alzheimer's Res. Ther. 2014, 6 (3), 28. (61) Lanza, V.; Bellia, F.; Rizzarelli, E. An Inorganic Overview of Natural Aβ Fragments: Copper(II) and Zinc(II)-Mediated Pathways. Coord. Chem. Rev. 2018, 369, 1−14. (62) Irie, K.; Murakami, K.; Masuda, Y.; Morimoto, A.; Ohigashi, H.; Ohashi, R.; Takegoshi, K.; Nagao, M.; Shimizu, T.; Shirasawa, T. Structure of β-Amyloid Fibrils and Its Relevance to Their Neurotoxicity: Implications for the Pathogenesis of Alzheimer’s Disease. J. Biosci. Bioeng. 2005, 99 (5), 437−447. (63) Hayden, E. Y.; Teplow, D. B. Amyloid β-Protein Oligomers and Alzheimer’s Disease. Alzheimer's Res. Ther. 2013, 5 (6), 60. (64) Foroutanpay, B. V.; Kumar, J.; Kang, S. G.; Danaei, N.; Westaway, D.; Sim, V. L.; Kar, S. The Effects of N-Terminal Mutations on β-Amyloid Peptide Aggregation and Toxicity. Neuroscience 2018, 379, 177−188. (65) Janelidze, S.; Zetterberg, H.; Mattsson, N.; Palmqvist, S.; Vanderstichele, H.; Lindberg, O.; van Westen, D.; Stomrud, E.; Minthon, L.; Blennow, K.; et al. CSF Aβ42/Aβ40 and Aβ42/Aβ38 Ratios: Better Diagnostic Markers of Alzheimer Disease. Ann. Clin. Transl. Neurol. 2016, 3 (3), 154−165. (66) Niemantsverdriet, E.; Ottoy, J.; Somers, C.; De Roeck, E.; Struyfs, H.; Soetewey, F.; Verhaeghe, J.; Van den Bossche, T.; Van Mossevelde, S.; Goeman, J.; et al. The Cerebrospinal Fluid Aβ1−42/ Aβ1−40 Ratio Improves Concordance with Amyloid-PET for Diagnosing Alzheimer’s Disease in a Clinical Setting. J. Alzheimer's Dis. 2017, 60 (2), 561−576. (67) Masters, C. L.; Simms, G.; Weinman, N. A.; Multhaup, G.; McDonald, B. L.; Beyreuther, K. Amyloid Plaque Core Protein in Alzheimer Disease and Down Syndrome. Proc. Natl. Acad. Sci. U. S. A. 1985, 82 (12), 4245−4249. (68) Masters, C. L.; Multhaup, G.; Simms, G.; Pottgiesser, J.; Martins, R. N.; Beyreuther, K. Neuronal Origin of a Cerebral Amyloid: Neurofibrillary Tangles of Alzheimer’s Disease Contain the Same Protein as the Amyloid of Plaque Cores and Blood Vessels. EMBO J. 1985, 4 (11), 2757−2763. (69) Drew, S. C. The Case for Abandoning Therapeutic Chelation of Copper Ions in Alzheimer’s Disease. Front. Neurosci. 2017, 11, 317. (70) Dunys, J.; Valverde, A.; Checler, F. Are N- and C-Terminally Truncated Aβ Species Key Pathological Triggers in Alzheimer’s Disease? J. Biol. Chem. 2018, 293 (40), 15419−15428.

(71) Bibl, M.; Gallus, M.; Welge, V.; Esselmann, H.; Wolf, S.; Rüther, E.; Wiltfang, J. Correction to: Cerebrospinal Fluid Amyloid-β 2−42 Is Decreased in Alzheimer’s, but Not in Frontotemporal Dementia. J. Neural Transm. 2018, 125 (10), 1515−1516. (72) Dietrich, K.; Bouter, Y.; Müller, M.; Bayer, T. A. Synaptic Alterations in Mouse Models for Alzheimer Disease-A Special Focus on N-Truncated Abeta 4−42. Molecules 2018, 23 (4), 718. (73) Schilling, S.; Lauber, T.; Schaupp, M.; Manhart, S.; Scheel, E.; Böhm, G.; Demuth, H.-U. On the Seeding and Oligomerization of PGlu-Amyloid Peptides (in Vitro). Biochemistry 2006, 45 (41), 12393− 12399. (74) Eury, H.; Bijani, C.; Faller, P.; Hureau, C. Copper(II) Coordination to Amyloid β: Murine versus Human Peptide. Angew. Chem., Int. Ed. 2011, 50 (4), 901−905. (75) Hureau, C.; Dorlet, P. Coordination of Redox Active Metal Ions to the Amyloid Precursor Protein and to Amyloid-β Peptides Involved in Alzheimer Disease. Part 2: Dependence of Cu(II) Binding Sites with Aβ Sequences. Coord. Chem. Rev. 2012, 256 (19−20), 2175−2187. (76) Drew, S. C.; Barnham, K. J. The Heterogeneous Nature of Cu2+interactions with Alzheimer’s Amyloid-β Peptide. Acc. Chem. Res. 2011, 44 (11), 1146−1155. (77) Cockerill, I.; Oliver, J.-A.; Xu, H.; Fu, B. M.; Zhu, D. Blood-Brain Barrier Integrity and Clearance of Amyloid-β from the BBB. In Molecular, Cellular, and Tissue Engineering of the Vascular System; Fu, B. M., Wright, N. T., Eds.; Springer International Publishing: Cham, 2018; pp 261−278. . (78) McIntee, F. L.; Giannoni, P.; Blais, S.; Sommer, G.; Neubert, T. A.; Rostagno, A.; Ghiso, J. In Vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer’s Aβ Protein. Front. Aging Neurosci. 2016, 8, 223. (79) Kanekiyo, T.; Bu, G. The Low-Density Lipoprotein ReceptorRelated Protein 1 and Amyloid-β Clearance in Alzheimer’s Disease. Front. Aging Neurosci. 2014, 6, 93. (80) Medoro, A.; Bartollino, S.; Mignogna, D.; Marziliano, N.; Porcile, C.; Nizzari, M.; Florio, T.; Pagano, A.; Raimo, G.; Intrieri, M.; et al. Proteases Upregulation in Sporadic Alzheimer’s Disease Brain. J. Alzheimer's Dis. 2019, 68 (3), 931−938. (81) Eckman, E. A.; Watson, M.; Marlow, L.; Sambamurti, K.; Eckman, C. B. Alzheimer’s Disease Beta-Amyloid Peptide Is Increased in Mice Deficient in Endothelin-Converting Enzyme. J. Biol. Chem. 2003, 278 (4), 2081−2084. (82) Farris, W.; Mansourian, S.; Chang, Y.; Lindsley, L.; Eckman, E. A.; Frosch, M. P.; Eckman, C. B.; Tanzi, R. E.; Selkoe, D. J.; Guenette, S. Insulin-Degrading Enzyme Regulates the Levels of Insulin, Amyloid Beta-Protein, and the Beta-Amyloid Precursor Protein Intracellular Domain in Vivo. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (7), 4162− 4167. (83) Zheng, C.; Geetha, T.; Gearing, M.; Ramesh Babu, J. Amyloid βAbrogated TrkA Ubiquitination in PC12 Cells Analogous to Alzheimer’s Disease. J. Neurochem. 2015, 133 (6), 919−925. (84) Paroni, G.; Bisceglia, P.; Seripa, D. Understanding the Amyloid Hypothesis in Alzheimer’s Disease. J. Alzheimer's Dis. 2019, 68 (2), 493−510. (85) Chen, M.; Inestrosa, N. C.; Ross, G. S.; Fernandez, H. L. Platelets Are the Primary Source of Amyloid Beta-Peptide in Human Blood. Biochem. Biophys. Res. Commun. 1995, 213 (1), 96−103. (86) Kucheryavykh, L. Y.; Dávila-Rodríguez, J.; Rivera-Aponte, D. E.; Zueva, L. V.; Washington, A. V.; Sanabria, P.; Inyushin, M. Y. Platelets Are Responsible for the Accumulation of β-Amyloid in Blood Clots inside and around Blood Vessels in Mouse Brain after Thrombosis. Brain Res. Bull. 2017, 128, 98−105. (87) Roher, A. E.; Esh, C. L.; Kokjohn, T. A.; Castaño, E. M.; Van Vickle, G. D.; Kalback, W. M.; Patton, R. L.; Luehrs, D. C.; Daugs, I. D.; Kuo, Y.-M.; et al. Amyloid Beta Peptides in Human Plasma and Tissues and Their Significance for Alzheimer’s Disease. Alzheimer's Dementia 2009, 5 (1), 18−29. (88) Rózga, M.; Kłoniecki, M.; Jabłonowska, A.; Dadlez, M.; Bal, W. The Binding Constant for Amyloid Aβ40 Peptide Interaction with M

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

Viewpoint

Inorganic Chemistry Human Serum Albumin. Biochem. Biophys. Res. Commun. 2007, 364 (3), 714−718. (89) Choi, T. S.; Lee, H. J.; Han, J. Y.; Lim, M. H.; Kim, H. I. Molecular Insights into Human Serum Albumin as a Receptor of Amyloid-β in the Extracellular Region. J. Am. Chem. Soc. 2017, 139 (43), 15437−15445. (90) Peters, T. Serum Albumin. Adv. Protein Chem. 1985, 37, 161− 245. (91) Licastro, F.; Morini, M. C.; Davis, L. J.; Biagi, R.; Prete, L.; Savorani, G. Studies of Blood Brain Barrier Permeability and of Intrathecal IgG Synthesis in Patients with Alzheimer’s Disease and Multi-Infarct Dementia. Adv. Biosci. (Oxford) 1993, 87, 283−283. (92) Christenson, R. H.; Behlmer, P.; Howard, J. F.; Winfield, J. B.; Silverman, L. M. Interpretation of Cerebrospinal Fluid Protein Assays in Various Neurologic Diseases. Clin. Chem. 1983, 29 (6), 1028−1030. (93) Milojevic, J.; Melacini, G. Stoichiometry and Affinity of the Human Serum Albumin-Alzheimer’s Aβ Peptide Interactions. Biophys. J. 2011, 100 (1), 183−192. (94) Stanyon, H. F.; Viles, J. H. Human Serum Albumin Can Regulate Amyloid-β Peptide Fiber Growth in the Brain Interstitium: Implications for Alzheimer Disease. J. Biol. Chem. 2012, 287 (33), 28163−28168. (95) Leinenga, G.; Götz, J. Scanning Ultrasound Removes Amyloid-β and Restores Memory in an Alzheimer’s Disease Mouse Model. Sci. Transl. Med. 2015, 7 (278), 278ra33−278ra33. (96) Janas, T.; Sapoń, K.; Stowell, M.; Janas, T. Selection of Membrane RNA Aptamers to Amyloid Beta Peptide: Implications for Exosome-Based Antioxidant Strategies. Int. J. Mol. Sci. 2019, 20 (2), 299. (97) Sciacca, M. F. M.; Kotler, S. A.; Brender, J. R.; Chen, J.; Lee, D.; Ramamoorthy, A. Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation. Biophys. J. 2012, 103 (4), 702−710. (98) Ramamoorthy, A.; Lim, M. H. H. Structural Characterization and Inhibition of Toxic Amyloid-β Oligomeric Intermediates. Biophys. J. 2013, 105 (2), 287−288. (99) Kotler, S. A.; Walsh, P.; Brender, J. R.; Ramamoorthy, A. Differences between Amyloid-β Aggregation in Solution and on the Membrane: Insights into Elucidation of the Mechanistic Details of Alzheimer’s Disease. Chem. Soc. Rev. 2014, 43 (19), 6692−6700. (100) Kayed, R.; Lasagna-Reeves, C. A. Molecular Mechanisms of Amyloid Oligomers Toxicity. J. Alzheimer's Dis. 2012, 33 (s1), S67− S78. (101) Evangelisti, E.; Cascella, R.; Becatti, M.; Marrazza, G.; Dobson, C. M.; Chiti, F.; Stefani, M.; Cecchi, C. Binding Affinity of Amyloid Oligomers to Cellular Membranes Is a Generic Indicator of Cellular Dysfunction in Protein Misfolding Diseases. Sci. Rep. 2016, 6, 32721. (102) Zhao, L. N.; Long, H.; Mu, Y.; Chew, L. Y. The Toxicity of Amyloid β Oligomers. Int. J. Mol. Sci. 2012, 13 (6), 7303−7327. (103) Zhang, Y.-J.; Shi, J.-M.; Bai, C.-J.; Wang, H.; Li, H.-Y.; Wu, Y.; Ji, S.-R. Intra-Membrane Oligomerization and Extra-Membrane Oligomerization of Amyloid-β Peptide Are Competing Processes as a Result of Distinct Patterns of Motif Interplay. J. Biol. Chem. 2012, 287 (1), 748− 756. (104) Niu, Z.; Zhang, Z.; Zhao, W.; Yang, J. Interactions between Amyloid β Peptide and Lipid Membranes. Biochim. Biophys. Acta, Biomembr. 2018, 1860 (9), 1663−1669. (105) Gunn, A. P.; Wong, B. X.; Johanssen, T.; Griffith, J. C.; Masters, C. L.; Bush, A. I.; Barnham, K. J.; Duce, J. A.; Cherny, R. A. Amyloid-β Peptide Aβ3pE-42 Induces Lipid Peroxidation, Membrane Permeabilization, and Calcium Influx in Neurons. J. Biol. Chem. 2016, 291 (12), 6134−6145. (106) Jang, H.; Arce, F. T.; Ramachandran, S.; Capone, R.; Azimova, R.; Kagan, B. L.; Nussinov, R.; Lal, R. Truncated Beta-Amyloid Peptide Channels Provide an Alternative Mechanism for Alzheimer’s Disease and Down Syndrome. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (14), 6538−6543.

(107) Rózga, M.; Kłoniecki, M.; Dadlez, M.; Bal, W. A Direct Determination of the Dissociation Constant for the Cu(II) Complex of Amyloid β 1−40 Peptide. Chem. Res. Toxicol. 2010, 23 (2), 336−340. (108) Novo, M.; Freire, S.; Al-Soufi, W. Critical Aggregation Concentration for the Formation of Early Amyloid-β (1−42) Oligomers. Sci. Rep. 2018, 8 (1), 1783. (109) Kowalik-Jankowska, T.; Ruta, M.; Wiśniewska, K.; Lankiewicz, L. Coordination Abilities of the 1−16 and 1−28 Fragments of BetaAmyloid Peptide towards Copper(II) Ions: A Combined Potentiometric and Spectroscopic Study. J. Inorg. Biochem. 2003, 95 (4), 270− 282. (110) Syme, C. D.; Nadal, R. C.; Rigby, S. E. J.; Viles, J. H. Copper Binding to the Amyloid-β (Aβ) Peptide Associated with Alzheimer’s Disease. J. Biol. Chem. 2004, 279 (18), 18169−18177. (111) Karr, J. W.; Kaupp, L. J.; Szalai, V. A. Amyloid-β Binds Cu2+ in a Mononuclear Metal Ion Binding Site. J. Am. Chem. Soc. 2004, 126 (41), 13534−13538. (112) Dorlet, P.; Gambarelli, S.; Faller, P.; Hureau, C. Pulse EPR Spectroscopy Reveals the Coordination Sphere of Copper(II) Ions in the 1−16 Amyloid-β Peptide: A Key Role of the First Two N-Terminus Residues. Angew. Chem., Int. Ed. 2009, 48 (49), 9273−9276. (113) Alies, B.; Eury, H.; Bijani, C.; Rechignat, L.; Faller, P.; Hureau, C. PH-Dependent Cu(II) Coordination to Amyloid-β Peptide: Impact of Sequence Alterations, Including the H6R and D7N Familial Mutations. Inorg. Chem. 2011, 50 (21), 11192−11201. (114) Atrián-Blasco, E.; Gonzalez, P.; Santoro, A.; Alies, B.; Faller, P.; Hureau, C. Cu and Zn Coordination to Amyloid Peptides: From Fascinating Chemistry to Debated Pathological Relevance. Coord. Chem. Rev. 2018, 375, 38−55. (115) Guilloreau, L.; Combalbert, S.; Sournia-Saquet, A.; Mazarguil, H.; Faller, P. Redox Chemistry of Copper−Amyloid-β: The Generation of Hydroxyl Radical in the Presence of Ascorbate Is Linked to RedoxPotentials and Aggregation State. ChemBioChem 2007, 8 (11), 1317− 1325. (116) Alies, B.; Renaglia, E.; Rózga, M.; Bal, W.; Faller, P.; Hureau, C. Cu(II) Affinity for the Alzheimer’s Peptide: Tyrosine Fluorescence Studies Revisited. Anal. Chem. 2013, 85 (3), 1501−1508. (117) Damante, C. A.; Ö sz, K.; Nagy, Z.; Pappalardo, G.; Grasso, G.; Impellizzeri, G.; Rizzarelli, E.; Sóvágó, I. The Metal Loading Ability of β-Amyloid N-Terminus: A Combined Potentiometric and Spectroscopic Study of Copper(II) Complexes with β-Amyloid(1−16), Its Short or Mutated Peptide Fragments, and Its Polyethylene Glycol (PEG)-Ylated Analogue. Inorg. Chem. 2008, 47 (20), 9669−9683. (118) Draper, S. R. E.; Lawrence, P. B.; Billings, W. M.; Xiao, Q.; Brown, N. P.; Bécar, N. A.; Matheson, D. J.; Stephens, A. R.; Price, J. L. Polyethylene Glycol Based Changes to β-Sheet Protein Conformational and Proteolytic Stability Depend on Conjugation Strategy and Location. Bioconjugate Chem. 2017, 28 (10), 2507−2513. (119) Rózga, M.; Protas, A. M.; Jabłonowska, A.; Dadlez, M.; Bal, W. The Cu(Ii) Complex of Aβ40 Peptide in Ammonium Acetate Solutions. Evidence for Ternary Species Formation. Chem. Commun. 2009, 0 (11), 1374−1376. (120) Bin, Y.; Jiang, Z.; Xiang, J. Side Effect of Tris on the Interaction of Amyloid β-Peptide with Cu2+: Evidence for Tris−Aβ−Cu2+ Ternary Complex Formation. Appl. Biochem. Biotechnol. 2015, 176 (1), 56−65. (121) Kenche, V. B.; Zawisza, I.; Masters, C. L.; Bal, W.; Barnham, K. J.; Drew, S. C. Mixed Ligand Cu 2+ Complexes of a Model Therapeutic with Alzheimer’s Amyloid-β Peptide and Monoamine Neurotransmitters. Inorg. Chem. 2013, 52 (8), 4303−4318. (122) Fraczyk, T.; Zawisza, I. A.; Goch, W.; Stefaniak, E.; Drew, S. C.; Bal, W. On the Ability of CuAβ1-Xpeptides to Form Ternary Complexes: Neurotransmitter Glutamate Is a Competitor While Not a Ternary Partner. J. Inorg. Biochem. 2016, 158, 5−10. (123) Young, T. R.; Kirchner, A.; Wedd, A. G.; Xiao, Z. An Integrated Study of the Affinities of the Aβ16 Peptide for Cu(I) and Cu(II): Implications for the Catalytic Production of Reactive Oxygen Species. Metallomics 2014, 6 (3), 505−517. N

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

Viewpoint

Inorganic Chemistry (124) Branch, T.; Girvan, P.; Barahona, M.; Ying, L. Introduction of a Fluorescent Probe to Amyloid-β to Reveal Kinetic Insights into Its Interactions with Copper(II). Angew. Chem., Int. Ed. 2015, 54 (4), 1227−1230. (125) Conte-Daban, A.; Borghesani, V.; Sayen, S.; Guillon, E.; Journaux, Y.; Gontard, G.; Lisnard, L.; Hureau, C. Link between Affinity and Cu(II) Binding Sites to Amyloid-β Peptides Evaluated by a New Water-Soluble UV-Visible Ratiometric Dye with a Moderate Cu(II) Affinity. Anal. Chem. 2017, 89 (3), 2155−2162. (126) Hureau, C. Coordination of Redox Active Metal Ions to the Amyloid Precursor Protein and to Amyloid-β Peptides Involved in Alzheimer Disease. Part 1: An Overview. Coord. Chem. Rev. 2012, 256 (19−20), 2164−2174. (127) Rice, M.; Russo-Menna, I. Differential Compartmentalization of Brain Ascorbate and Glutathione between Neurons and Glia. Neuroscience 1997, 82 (4), 1213−1223. (128) Rice, M. E. Ascorbate Compartmentalization in the CNS. Neurotoxic. Res. 1999, 1 (2), 81−90. (129) Bal, W.; Protas, A. M.; Kasprzak, K. S. Genotoxicity of Metal Ions: Chemical Insights. Metal Ions in Toxicology: Effects, Interactions, Interdependencies; Royal Society of Chemistry, 2010; pp 319−373.. (130) Cheignon, C.; Jones, M.; Atrián-Blasco, E.; Kieffer, I.; Faller, P.; Collin, F.; Hureau, C. Identification of Key Structural Features of the Elusive Cu−Aβ Complex That Generates ROS in Alzheimer’s Disease. Chem. Sci. 2017, 8 (7), 5107−5118. (131) Zhang, W.; Liu, Y.; Hureau, C.; Robert, A.; Meunier, B. N4Tetradentate Chelators Efficiently Regulate Copper Homeostasis and Prevent ROS Production Induced by Copper-Amyloid-Β1−16. Chem. Eur. J. 2018, 24 (31), 7825−7829. (132) Sokołowska, M.; Bal, W. Cu(II) Complexation by “NonCoordinating” N-2-Hydroxyethylpiperazine-N’-2-Ethanesulfonic Acid (HEPES Buffer). J. Inorg. Biochem. 2005, 99 (8), 1653−1660. (133) Zawisza, I.; Rózga, M.; Poznański, J.; Bal, W. Cu(II) Complex Formation by ACES Buffer. J. Inorg. Biochem. 2013, 129, 58−61. (134) Nagaj, J.; Stokowa-Sołtys, K.; Kurowska, E.; Frączyk, T.; Jeżowska-Bojczuk, M.; Bal, W. Revised Coordination Model and Stability Constants of Cu(II) Complexes of Tris Buffer. Inorg. Chem. 2013, 52 (24), 13927−13933. (135) Huang, X.; Cuajungco, M. P.; Atwood, C. S.; Hartshorn, M. A.; Tyndall, J. D.; Hanson, G. R.; Stokes, K. C.; Leopold, M.; Multhaup, G.; Goldstein, L. E.; et al. Cu(II) Potentiation of Alzheimer Abeta Neurotoxicity. Correlation with Cell-Free Hydrogen Peroxide Production and Metal Reduction. J. Biol. Chem. 1999, 274 (52), 37111−37116. (136) Huang, H.; Lou, X.; Hu, B.; Zhou, Z.; Chen, J.; Tian, Y. A Comprehensive Study on the Generation of Reactive Oxygen Species in Cu-Aβ-Catalyzed Redox Processes. Free Radical Biol. Med. 2019, 135, 125−131. (137) Pedersen, J. T.; Chen, S. W.; Borg, C. B.; Ness, S.; Bahl, J. M.; Heegaard, N. H. H.; Dobson, C. M.; Hemmingsen, L.; Cremades, N.; Teilum, K. Amyloid-β and α-Synuclein Decrease the Level of MetalCatalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing. J. Am. Chem. Soc. 2016, 138 (12), 3966−3969. (138) Arrigoni, F.; Prosdocimi, T.; Mollica, L.; De Gioia, L.; Zampella, G.; Bertini, L. Copper Reduction and Dioxygen Activation in Cu− Amyloid Beta Peptide Complexes: Insight from Molecular Modelling. Metallomics 2018, 10 (11), 1618−1630. (139) Atrián-Blasco, E.; Del Barrio, M.; Faller, P.; Hureau, C. Ascorbate Oxidation by Cu(Amyloid-β) Complexes: Determination of the Intrinsic Rate as a Function of Alterations in the Peptide Sequence Revealing Key Residues for Reactive Oxygen Species Production. Anal. Chem. 2018, 90 (9), 5909−5915. (140) Näslund, J.; Schierhorn, A.; Hellman, U.; Lannfelt, L.; Roses, A. D.; Tjernberg, L. O.; Silberring, J.; Gandy, S. E.; Winblad, B.; Greengard, P.; et al. Relative Abundance of Alzheimer A Beta Amyloid Peptide Variants in Alzheimer Disease and Normal Aging. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (18), 8378−8382. (141) Barnham, K. J.; Haeffner, F.; Ciccotosto, G. D.; Curtain, C. C.; Tew, D.; Mavros, C.; Beyreuther, K.; Carrington, D.; Masters, C. L.;

Cherny, R. A.; et al. Tyrosine Gated Electron Transfer Is Key to the Toxic Mechanism of Alzheimer’s Disease β-Amyloid. FASEB J. 2004, 18 (12), 1427−1429. (142) Gu, M.; Bode, D. C.; Viles, J. H. Copper Redox Cycling Inhibits Aβ Fibre Formation and Promotes Fibre Fragmentation, While Generating a Dityrosine Aβ Dimer. Sci. Rep. 2018, 8 (1), 16190. (143) Hensley, K.; Maidt, M. L.; Yu, Z.; Sang, H.; Markesbery, W. R.; Floyd, R. A. Electrochemical Analysis of Protein Nitrotyrosine and Dityrosine in the Alzheimer Brain Indicates Region-Specific Accumulation. J. Neurosci. 1998, 18 (20), 8126−8132. (144) Vázquez de la Torre, A.; Gay, M.; Vilaprinyó-Pascual, S.; Mazzucato, R.; Serra-Batiste, M.; Vilaseca, M.; Carulla, N. Direct Evidence of the Presence of Cross-Linked Aβ Dimers in the Brains of Alzheimer’s Disease Patients. Anal. Chem. 2018, 90 (7), 4552−4560. (145) Rózga, M.; Sokołowska, M.; Protas, A. M.; Bal, W. Human Serum Albumin Coordinates Cu(II) at Its N-Terminal Binding Site with 1 pM Affinity. JBIC, J. Biol. Inorg. Chem. 2007, 12 (6), 913−918. (146) Perrone, L.; Mothes, E.; Vignes, M.; Mockel, A.; Figueroa, C.; Miquel, M.-C.; Maddelein, M.-L.; Faller, P. Copper Transfer from CuAβ to Human Serum Albumin Inhibits Aggregation, Radical Production and Reduces Aβ Toxicity. ChemBioChem 2010, 11 (1), 110−118. (147) Meloni, G.; Sonois, V.; Delaine, T.; Guilloreau, L.; Gillet, A.; Teissié, J.; Faller, P.; Vašaḱ , M. Metal Swap between Zn7-Metallothionein-3 and Amyloid-β−Cu Protects against Amyloid-β Toxicity. Nat. Chem. Biol. 2008, 4 (6), 366−372. (148) Artells, E.; Palacios, Ò .; Capdevila, M.; Atrian, S. In Vivo -Folded Metal-Metallothionein 3 Complexes Reveal the Cu-Thionein Rather than Zn-Thionein Character of This Brain-Specific Mammalian Metallothionein. FEBS J. 2014, 281 (6), 1659−1678. (149) Savelieff, M. G.; Nam, G.; Kang, J.; Lee, H. J.; Lee, M.; Lim, M. H. Development of Multifunctional Molecules as Potential Therapeutic Candidates for Alzheimer’s Disease, Parkinson’s Disease, and Amyotrophic Lateral Sclerosis in the Last Decade. Chem. Rev. 2019, 119 (2), 1221−1322. (150) Atrián-Blasco, E.; Cerrada, E.; Faller, P.; Laguna, M.; Hureau, C. Role of PTA in the Prevention of Cu(Amyloid-β) Induced ROS Formation and Amyloid-β Oligomerisation in the Presence of Zn. Metallomics 2019, 11, 1154. (151) Adlard, P. A.; Bush, A. I. Metal Chaperones: A Holistic Approach to the Treatment of Alzheimer’s Disease. Front. psychiatry 2012, 3, 15. (152) Adlard, P. A.; Bush, A. I. Metals and Alzheimer’s Disease: How Far Have We Come in the Clinic? J. Alzheimer's Dis. 2018, 62 (3), 1369−1379. (153) Nguyen, M.; Vendier, L.; Stigliani, J.-L.; Meunier, B.; Robert, A. Structures of the Copper and Zinc Complexes of PBT2, a Chelating Agent Evaluated as Potential Drug for Neurodegenerative Diseases. Eur. J. Inorg. Chem. 2017, 2017 (3), 600−608. (154) Opazo, C.; Huang, X.; Cherny, R. A.; Moir, R. D.; Roher, A. E.; White, A. R.; Cappai, R.; Masters, C. L.; Tanzi, R. E.; Inestrosa, N. C.; et al. Metalloenzyme-like Activity of Alzheimer’s Disease β-Amyloid. J. Biol. Chem. 2002, 277 (43), 40302−40308. (155) Teplow, D. B. On the Subject of Rigor in the Study of Amyloid β-Protein Assembly. Alzheimer's Res. Ther. 2013, 5 (4), 39. (156) Stewart, K. L.; Radford, S. E. Amyloid Plaques beyond Aβ: A Survey of the Diverse Modulators of Amyloid Aggregation. Biophys. Rev. 2017, 9, 405−419. (157) Hane, F.; Leonenko, Z. Effect of Metals on Kinetic Pathways of Amyloid-β Aggregation. Biomolecules 2014, 4 (1), 101−116. (158) Yoshiike, Y.; Tanemura, K.; Murayama, O.; Akagi, T.; Murayama, M.; Sato, S.; Sun, X.; Tanaka, N.; Takashima, A. New Insights on How Metals Disrupt Amyloid Beta-Aggregation and Their Effects on Amyloid-Beta Cytotoxicity. J. Biol. Chem. 2001, 276 (34), 32293−32299. (159) Tõugu, V.; Karafin, A.; Zovo, K.; Chung, R. S.; Howells, C.; West, A. K.; Palumaa, P. Zn(II)- and Cu(II)-Induced Non-Fibrillar Aggregates of Amyloid-β (1−42) Peptide Are Transformed to Amyloid O

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Histidine)] Occurs via NiIII: X-Ray Crystal Structure of [NiII(GlycylGlycyl-α-Hydroxy- D, L-Histamine)]·3H2O. J. Chem. Soc., Chem. Commun. 1994, 1889−1890. (178) Best, S. L.; Chattopadhyay, T. K.; Djuran, M. I.; Palmer, R. A.; Sadler, P. J.; Sóvágó, I.; Varnagy, K. Gold(III) and Palladium(II) Complexes of Glycylglycyl-L-Histidine: Crystal Structures of [AuIII(Gly-Gly-L-His-H−2)]Cl·H2O and [PdII(Gly-Gly-L-His-H−2)]· 1.5H2O and HisεNH Deprotonation. J. Chem. Soc., Dalton Trans. 1997, 2587−2596. (179) Kolozsi, A.; Jancsó, A.; Nagy, N. V.; Gajda, T. N-Terminal Fragment of the Anti-Angiogenic Human Endostatin Binds Copper(II) with Very High Affinity. J. Inorg. Biochem. 2009, 103 (7), 940−947. (180) Płonka, D.; Bal, W. The N-Terminus of Hepcidin Is a Strong and Potentially Biologically Relevant Cu(II) Chelator. Inorg. Chim. Acta 2018, 472, 76−81. (181) Mital, M.; Wezynfeld, N. E.; Frączyk, T.; Wiloch, M. Z.; Wawrzyniak, U. E.; Bonna, A.; Tumpach, C.; Barnham, K. J.; Haigh, C. L.; Bal, W.; et al. A Functional Role for Aβ in Metal Homeostasis? NTruncation and High-Affinity Copper Binding. Angew. Chem., Int. Ed. 2015, 54 (36), 10460−10464. (182) Mital, M.; Bal, W.; Frączyk, T.; Drew, S. C. Interplay between Copper, Neprilysin, and N-Truncation of β-Amyloid. Inorg. Chem. 2018, 57 (11), 6193−6197. (183) Bossak-Ahmad, K.; Mital, M.; Płonka, D.; Drew, S. C.; Bal, W. Oligopeptides Generated by Neprilysin Degradation of β-Amyloid Have the Highest Cu(II) Affinity in the Whole Aβ Family. Inorg. Chem. 2019, 58 (1), 932−943. (184) Wiloch, M. Z.; Wawrzyniak, U. E.; Ufnalska, I.; Bonna, A.; Bal, W.; Drew, S. C.; Wróblewski, W. Tuning the Redox Properties of Copper(II) Complexes with Amyloid-β Peptides. J. Electrochem. Soc. 2016, 163 (13), G196−G199. (185) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species. J. Biol. Chem. 2003, 278 (5), 3170−3175. (186) Ruiz, A.; Pesini, P.; Espinosa, A.; Pérez-Grijalba, V.; Valero, S.; Sotolongo-Grau, O.; Alegret, M.; Monleón, I.; Lafuente, A.; Buendía, M.; et al. Blood Amyloid Beta Levels in Healthy, Mild Cognitive Impairment and Alzheimer’s Disease Individuals: Replication of Diastolic Blood Pressure Correlations and Analysis of Critical Covariates. PLoS One 2013, 8 (11), No. e81334. (187) de Rojas, I.; Romero, J.; Rodríguez-Gomez, O.; Pesini, P.; Sanabria, A.; Pérez-Cordon, A.; Abdelnour, C.; Hernández, I.; Rosende-Roca, M.; Mauleón, A.; et al. Correlations between Plasma and PET Beta-Amyloid Levels in Individuals with Subjective Cognitive Decline: The Fundació ACE Healthy Brain Initiative (FACEHBI). Alzheimer's Res. Ther. 2018, 10 (1), 119. (188) Nakamura, A.; Kaneko, N.; Villemagne, V. L.; Kato, T.; Doecke, J.; Doré, V.; Fowler, C.; Li, Q.-X.; Martins, R.; Rowe, C.; et al. High Performance Plasma Amyloid-β Biomarkers for Alzheimer’s Disease. Nature 2018, 554 (7691), 249−254. (189) Vašaḱ , M.; Meloni, G. Mammalian Metallothionein-3: New Functional and Structural Insights. Int. J. Mol. Sci. 2017, 18 (6), 1117. (190) Irie, Y.; Keung, W. M. Metallothionein-III Antagonizes the Neurotoxic and Neurotrophic Effects of Amyloid β Peptides. Biochem. Biophys. Res. Commun. 2001, 282 (2), 416−420. (191) Meloni, G.; Polanski, T.; Braun, O.; Vašaḱ , M. Effects of Zn2+, Ca2+, and Mg2+ on the Structure of Zn7Metallothionein-3: Evidence for an Additional Zinc Binding Site. Biochemistry 2009, 48 (24), 5700− 5707. (192) Wezynfeld, N. E.; Stefaniak, E.; Stachucy, K.; Drozd, A.; Płonka, D.; Drew, S. C.; Krężel, A.; Bal, W. Resistance of Cu(Aβ4−16) to Copper Capture by Metallothionein-3 Supports a Function for the Aβ4−42 Peptide as a Synaptic CuIIScavenger. Angew. Chem., Int. Ed. 2016, 55 (29), 8235−8238. (193) Santoro, A.; Wezynfeld, N. E.; Vašaḱ , M.; Bal, W.; Faller, P. Cysteine and Glutathione Trigger the Cu−Zn Swap between Cu(II)Amyloid-β4−16 Peptide and Zn7-Metallothionein-3. Chem. Commun. 2017, 53 (85), 11634−11637.

Fibrils, Both Spontaneously and under the Influence of Metal Chelators. J. Neurochem. 2009, 110 (6), 1784−1795. (160) Sarell, C. J.; Wilkinson, S. R.; Viles, J. H. Substoichiometric Levels of Cu 2 Ions Accelerate the Kinetics of Fiber Formation and Promote Cell Toxicity of Amyloid-from Alzheimer Disease. J. Biol. Chem. 2010, 285, 41533. (161) Kepp, K. P. Alzheimer’s Disease: How Metal Ions Define βAmyloid Function. Coord. Chem. Rev. 2017, 351 (May), 127−159. (162) Sharma, A. K.; Pavlova, S. T.; Kim, J.; Kim, J.; Mirica, L. M. The Effect of Cu2+ and Zn2+ on the Aβ42 Peptide Aggregation and Cellular Toxicity. Metallomics 2013, 5 (11), 1529. (163) Matheou, C. J.; Younan, N. D.; Viles, J. H. Cu 2+ Accentuates Distinct Misfolding of Aβ (1−40) and Aβ (1−42) Peptides, and Potentiates Membrane Disruption. Biochem. J. 2015, 466 (2), 233−242. (164) Pedersen, J. T.; Teilum, K.; Heegaard, N. H. H.; Østergaard, J.; Adolph, H.-W.; Hemmingsen, L. Rapid Formation of a Preoligomeric Peptide-Metal-Peptide Complex Following Copper(II) Binding to Amyloid β Peptides. Angew. Chem., Int. Ed. 2011, 50 (11), 2532−2535. (165) Tiiman, A.; Luo, J.; Wallin, C.; Olsson, L.; Lindgren, J.; Jarvet, J.; Per, R.; Sholts, S. B.; Rahimipour, S.; Abrahams, J. P.; et al. Specific Binding of Cu(II) Ions to Amyloid-Beta Peptides Bound to Aggregation-Inhibiting Molecules or SDS Micelles Creates Complexes That Generate Radical Oxygen Species. J. Alzheimer's Dis. 2016, 54 (3), 971−982. (166) Dolgodilina, E.; Imobersteg, S.; Laczko, E.; Welt, T.; Verrey, F.; Makrides, V. Brain Interstitial Fluid Glutamine Homeostasis Is Controlled by Blood-Brain Barrier SLC7A5/LAT1 Amino Acid Transporter. J. Cereb. Blood Flow Metab. 2016, 36 (11), 1929−1941. (167) Harford, C.; Sarkar, B. Amino Terminal Cu(II)- and Ni(II)Binding (ATCUN) Motif of Proteins and Peptides: Metal Binding, DNA Cleavage, and Other Properties. Acc. Chem. Res. 1997, 30 (3), 123−130. (168) Bal, W.; Christodoulou, J.; Sadler, P. J.; Tucker, A. Multi-Metal Binding Site of Serum Albumin. J. Inorg. Biochem. 1998, 70 (1), 33−39. (169) Gonzalez, P.; Bossak, K.; Stefaniak, E.; Hureau, C.; Raibaut, L.; Bal, W.; Faller, P. N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, XxxHis) and Their Derivatives: Chemistry, Biology and Medicinal Applications. Chem. - Eur. J. 2018, 24 (32), 8029−8041. (170) Shearer, W. T.; Bradshaw, R. A.; Gurd, F. R.; Peters, T. The Amino Acid Sequence and Copper(II)-Binding Properties of Peptide (1−24) of Bovine Serum Albumin. J. Biol. Chem. 1967, 242 (23), 5451−5459. (171) Camerman, N.; Camerman, A.; Sarkar, B. Molecular Design to Mimic the Copper(II) Transport Site of Human Albumin. The Crystal and Molecular Structure of Copper(II) − Glycylglycyl- L -Histidine- N -Methyl Amide Monoaquo Complex. Can. J. Chem. 1976, 54 (8), 1309−1316. (172) Hureau, C.; Eury, H.; Guillot, R.; Bijani, C.; Sayen, S.; Solari, P.L.; Guillon, E.; Faller, P.; Dorlet, P. X-Ray and Solution Structures of CuIIGHK and CuIIDAHK Complexes: Influence on Their Redox Properties. Chem. - Eur. J. 2011, 17 (36), 10151−10160. (173) Streltsov, V. A.; Ekanayake, R. S. K.; Drew, S. C.; Chantler, C. T.; Best, S. P. Structural Insight into Redox Dynamics of Copper Bound N-Truncated Amyloid-β Peptides from in Situ X-Ray Absorption Spectroscopy. Inorg. Chem. 2018, 57 (18), 11422−11435. (174) Stefaniak, E.; Płonka, D.; Drew, S. C.; Bossak-Ahmad, K.; Haas, K. L.; Pushie, M. J.; Faller, P.; Wezynfeld, N. E.; Bal, W. The NTerminal 14-Mer Model Peptide of Human Ctr1 Can Collect Cu(II) from Albumin. Implications for Copper Uptake by Ctr1. Metallomics 2018, 10 (12), 1723−1727. (175) Sigel, H.; Martin, R. B. Coordinating Properties of the Amide Bond. Stability and Structure of Metal Ion Complexes of Peptides and Related Ligands. Chem. Rev. 1982, 82 (4), 385−426. (176) Kozłowski, H.; Bal, W.; Dyba, M.; Kowalik-Jankowska, T. Specific Structure−Stability Relations in Metallopeptides. Coord. Chem. Rev. 1999, 184 (1), 319−346. (177) Bal, W.; Djuran, M. I.; Margerum, D. W.; Gray, E. T.; Mazid, M. A.; Tom, R. T.; Nieboer, E.; Sadler, P. J. Dioxygen-Induced Decarboxylation and Hydroxylation of [NiII (Glycyl-Glycyl- LP

DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX

Viewpoint

Inorganic Chemistry (194) Wang, R.; Reddy, P. H. Role of Glutamate and NMDA Receptors in Alzheimer’s Disease. J. Alzheimer's Dis. 2017, 57 (4), 1041−1048. (195) Danbolt, N. C. Glutamate Uptake. Prog. Neurobiol. 2001, 65 (1), 1−105. (196) Budisantoso, T.; Harada, H.; Kamasawa, N.; Fukazawa, Y.; Shigemoto, R.; Matsui, K. Evaluation of Glutamate Concentration Transient in the Synaptic Cleft of the Rat Calyx of Held. J. Physiol. 2013, 591 (1), 219−239. (197) Santoro, A.; Wezynfeld, N.; Stefaniak, E.; Pomorski, A.; Płonka, D.; Krezel, A.; Bal, W.; Faller, P. Cu Transfer from Amyloid-β4−16 to Metallothionein-3: The Role of Neurotransmitter Glutamate and Metallothionein-3 Zn(II)-Load States. Chem. Commun. 2018, 54, 12634−12637. (198) Mital, M.; Zawisza, I. A.; Wiloch, M. Z.; Wawrzyniak, U. E.; Kenche, V.; Wróblewski, W.; Bal, W.; Drew, S. C. Copper Exchange and Redox Activity of a Prototypical 8-Hydroxyquinoline: Implications for Therapeutic Chelation. Inorg. Chem. 2016, 55 (15), 7317−7319. (199) Faller, P. Copper and Zinc Binding to Amyloid-β: Coordination, Dynamics, Aggregation, Reactivity and Metal-Ion Transfer. ChemBioChem 2009, 10 (18), 2837−2845. (200) Grasso, G.; Santoro, A. M.; Lanza, V.; Sbardella, D.; Tundo, G. R.; Ciaccio, C.; Marini, S.; Coletta, M.; Milardi, D. The Double Faced Role of Copper in Aβ Homeostasis: A Survey on the Interrelationship between Metal Dyshomeostasis, UPS Functioning and Autophagy in Neurodegeneration. Coord. Chem. Rev. 2017, 347, 1−22. (201) Liu, K.; Solano, I.; Mann, D.; Lemere, C.; Mercken, M.; Trojanowski, J. Q.; Lee, V. M.-Y. Characterization of Aβ11−40/42 Peptide Deposition in Alzheimer’s Disease and Young Down’s Syndrome Brains: Implication of N-Terminally Truncated Aβ Species in the Pathogenesis of Alzheimer’s Disease. Acta Neuropathol. 2006, 112 (2), 163−174. (202) Schönherr, C.; Bien, J.; Isbert, S.; Wichert, R.; Prox, J.; Altmeppen, H.; Kumar, S.; Walter, J.; Lichtenthaler, S. F.; Weggen, S.; et al. Generation of Aggregation Prone N-Terminally Truncated Amyloid β Peptides by Meprin β Depends on the Sequence Specificity at the Cleavage Site. Mol. Neurodegener. 2016, 11, 19. (203) Alies, B.; Solari, P.-L.; Hureau, C.; Faller, P. Dynamics of Zn(II) Binding as a Key Feature in the Formation of Amyloid Fibrils by Aβ11− 28. Inorg. Chem. 2012, 51 (1), 701−708. (204) Stefaniak, E.; Atrián-Blasco, E.; Hureau, C.; Bal, W. Can Copper Ions Modulate Self-Assembly of Amyloid-β? Presented at the AsBIC9 Conference, Singapore, 2018. (205) Kanemitsu, H.; Tomiyama, T.; Mori, H. Human Neprilysin Is Capable of Degrading Amyloid β Peptide Not Only in the Monomeric Form but Also the Pathological Oligomeric Form. Neurosci. Lett. 2003, 350 (2), 113−116. (206) Oefner, C.; Roques, B. P.; Fournie-Zaluski, M.-C.; Dale, G. E. Structural Analysis of Neprilysin with Various Specific and Potent Inhibitors. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60 (2), 392−396. (207) Millhauser, G. L. Copper and the Prion Protein: Methods, Structures, Function, and Disease. Annu. Rev. Phys. Chem. 2007, 58, 299−320. (208) Goch, W.; Bal, W. Numerical Simulations Reveal Randomness of Cu(II) Induced Aβ Peptide Dimerization under Conditions Present in Glutamatergic Synapses. PLoS One 2017, 12 (1), e0170749. (209) Barritt, J. D.; Viles, J. H. Truncated Amyloid-β(11−40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly. J. Biol. Chem. 2015, 290 (46), 27791− 27802. (210) Simon, M. J.; Iliff, J. J. Regulation of Cerebrospinal Fluid (CSF) Flow in Neurodegenerative, Neurovascular and Neuroinflammatory Disease. Biochim. Biophys. Acta, Mol. Basis Dis. 2016, 1862 (3), 442− 451. (211) Marder, E. Neuromodulation of Neuronal Circuits: Back to the Future. Neuron 2012, 76 (1), 1−11. (212) Valtcheva, S.; Venance, L. Control of Long-Term Plasticity by Glutamate Transporters. Front. Synaptic Neurosci. 2019, 11, 10.

(213) Helassa, N.; Dürst, C. D.; Coates, C.; Kerruth, S.; Arif, U.; Schulze, C.; Wiegert, J. S.; Geeves, M.; Oertner, T. G.; Török, K. Ultrafast Glutamate Sensors Resolve High-Frequency Release at Schaffer Collateral Synapses. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (21), 5594−5599. (214) Pedersen, J. T.; Borg, C. B.; Michaels, T. C. T.; Knowles, T. P. J.; Faller, P.; Teilum, K.; Hemmingsen, L. Aggregation-Prone Amyloid-β· CuII Species Formed on the Millisecond Timescale under Mildly Acidic Conditions. ChemBioChem 2015, 16 (9), 1293−1297. (215) Bin, Y.; Chen, S.; Xiang, J. pH-Dependent Kinetics of Copper Ions Binding to Amyloid-β Peptide. J. Inorg. Biochem. 2013, 119, 21− 27. (216) Tharp, W. G.; Sarkar, I. Origins of Amyloid-β. BMC Genomics 2013, 14 (1), 290. (217) Zawisza, I.; Rózga, M.; Bal, W. Affinity of Copper and Zinc Ions to Proteins and Peptides Related to Neurodegenerative Conditions (Aβ, APP, α-Synuclein, PrP). Coord. Chem. Rev. 2012, 256 (19−20), 2297−2307. (218) McLaurin, J.; Cecal, R.; Kierstead, M. E.; Tian, X.; Phinney, A. L.; Manea, M.; French, J. E.; Lambermon, M. H. L.; Darabie, A. A.; Brown, M. E.; et al. Therapeutically Effective Antibodies against Amyloid-β Peptide Target Amyloid-β Residues 4−10 and Inhibit Cytotoxicity and Fibrillogenesis. Nat. Med. 2002, 8 (11), 1263−1269. (219) De Feo, C. J.; Aller, S. G.; Siluvai, G. S.; Blackburn, N. J.; Unger, V. M. Three-Dimensional Structure of the Human Copper Transporter HCTR1. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (11), 4237−4242. (220) Bal, W.; Sokołowska, M.; Kurowska, E.; Faller, P. Binding of Transition Metal Ions to Albumin: Sites, Affinities and Rates. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830 (12), 5444−5455. (221) Russino, D.; McDonald, E.; Hejazi, L.; Hanson, G. R.; Jones, C. E. The Tachykinin Peptide Neurokinin B Binds Copper Forming an Unusual [CuII(NKB)2] Complex and Inhibits Copper Uptake into 1321N1 Astrocytoma Cells. ACS Chem. Neurosci. 2013, 4 (10), 1371− 1381. (222) Morgan, M. T.; Nguyen, L. A. H.; Hancock, H. L.; Fahrni, C. J. Glutathione Limits Aquacopper(I) to Sub-Femtomolar Concentrations through Cooperative Assembly of a Tetranuclear Cluster. J. Biol. Chem. 2017, 292 (52), 21558−21567. (223) Aliaga, M. E.; López-Alarcón, C.; Bridi, R.; Speisky, H. RedoxImplications Associated with the Formation of Complexes between Copper Ions and Reduced or Oxidized Glutathione. J. Inorg. Biochem. 2016, 154, 78−88. (224) Mital, M. N-Truncation of Aβ Peptides and Cu(II) Binding: Affinity, Structures, Reactivity. Ph.D. thesis, Melbourne/Warsaw, 2018.

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DOI: 10.1021/acs.inorgchem.9b01399 Inorg. Chem. XXXX, XXX, XXX−XXX