Interplay between Copper, Neprilysin, and N-Truncation of β-Amyloid

Communication pubs.acs.org/IC

Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Interplay between Copper, Neprilysin, and N‑Truncation of β‑Amyloid Mariusz Mital,†,‡ Wojciech Bal,‡ Tomasz Frączyk,‡,§ and Simon C. Drew*,∥ †

Florey Department of Neuroscience and Mental Health, The University of Melbourne, Melbourne, Victoria 3010, Australia Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland § Department of Immunology, Transplantology and Internal Medicine, Medical University of Warsaw, Warsaw, Poland ∥ Department of Medicine (Royal Melbourne Hospital), The University of Melbourne, Melbourne, Victoria 3010, Australia ‡

S Supporting Information *

These properties contrast with the metals hypothesis of AD and suggest a functional role for N-truncated Aβ in Cu2+ binding.12−15 An important family of Aβ-degrading enzymes is the Zndependent endopeptidases,1,16,17 which include insulin-degrading enzyme (IDE)18,19 and neprilysin (NEP)20,21 both of which can hydrolytically cleave the Glu3−Phe4 bond in vitro to generate an Aβ4−x fragment. Brain Aβ levels in aging and AD are inversely correlated with the NEP levels and activity.22−27 This raises the question of whether the widely speculated loss of homeostasis of transition-metal ions such as copper can be related, in part, to variations in the levels of Cu2+-binding Aβ4−x species. NEP is a membrane-bound enzyme with the active site located within its ectodomain, enabling it to hydrolyze extracellular peptide substrates at the plasma membrane.28,29 Elevated extracellular Cu2+ has been associated with a reduction of NEP protein levels. In a cell culture model, exposure to exogenous Cu2+ was reported to decrease NEP levels by modulating its proteosomal degradation, with a concomitant increase in secreted Aβ1−40.30 RNAi silencing of the orthologue of human copper uptake protein Ctr1 in a transgenic drosophila model of AD also resulted in reduced levels of NEP (NEP1 and NEP3) and IDE.31 However, inhibition of NEP itself by Cu2+-binding interaction is yet to be considered. Moreover, there exist discrepancies in the reported Aβ cleavage profile using NEP derived from nonhuman sources. Here, we sought to clarify the N-terminal cleavage profile using recombinant human NEP and modulation of its activity by Cu2+ and Zn2+ ions. To begin, the influence of Cu2+ on NEP activity was assessed using an internally quenched fluorescent substrate McaRPPGFSAFK(Dnp). The 2,4-dinitrophenol (Dnp) group quenches the fluorescence of the 7-methoxycoumarin (Mca)based fluorophore until NEP hydrolyzes the Gly−Phe or Ala− Phe bonds. For a nominal NEP concentration of 0.1 nM, the range of substrate (S) concentrations suitable for determination of the Michaelis−Menten constant (Km) was first established (Figure S1). Using this range, the influence of varying Cu2+ concentration on the initial rate of substrate cleavage (V) was determined (Figure S2), from which the inhibition constant (Ki) was derived by global nonlinear analysis (Figure 1a). Divalent

ABSTRACT: Sporadic Alzheimer’s disease (AD) is associated with an inefficient clearance of the β-amyloid (Aβ) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aβ levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aβ fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP (Ki = 1.0 μM), while proteolysis of Aβ yielded the soluble, Aβ4−9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aβ4−9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations (Ki = 20 μM) further suggests a mechanism for modulating NEP activity, Aβ4−9 production, and Cu2+ homeostasis.

A

lzheimer’s disease (AD) is characterized by the deposition within the brain and blood vessels of extracellular plaques containing aggregated β-amyloid (Aβ) and intracellular tangles of hyperphosphorylated τ protein.1 The Aβ peptide is cleaved from the amyloid precursor protein (APP) by β- and γ-secretases at the membranes of intracellular organelles and transport vesicles during APP trafficking to and from the plasma membrane.2 The peptide is a product of normal cellular metabolism, with neurotrophic3,4 and antimicrobial5,6 actions having been identified. The onset and progression of AD is believed to be causally linked to an inefficient clearance of Aβ from the central nervous system.7 A range of Aβ peptides with “ragged” N-termini are known, with the majority of species originally sequenced in AD plaque cores reported to begin at Phe4.8−10 The prevalence of Aβ4−x in AD brain was also reported more recently, with the additional finding that Aβ4−42 is at least as abundant as Aβ1−42 in the cortex of non-AD subjects.11 The amino-terminal Cu and Ni binding (ATCUN) motif of Aβ4−x isoforms binds Cu2+ with nearfemtomolar affinity (Kd = 30 fM at pH 7.4) via a {NH2F4, N− R5, N− H6, NImH6} coordination mode that efficiently outcompetes Aβ1−x for Cu2+ and generates negligible quantities of hydroxyl radicals in the presence of the cellular reductant ascorbate.12−14 © XXXX American Chemical Society

Received: February 13, 2018

A

DOI: 10.1021/acs.inorgchem.8b00391 Inorg. Chem. XXXX, XXX, XXX−XXX

Communication

Inorganic Chemistry

Figure 2. Approximate product concentrations during proteolysis of 0.6 mM Aβ1−16 by 15 nM recombinant human NEP in 20 mM HEPES and pH 7.4 and 37 °C. Inset: Routes of Aβ1−16 proteolysis, where the width of the arrows qualitatively indicates the relative rate of production; the Aβ4−16 fragment is a minor species and is not depicted. Corresponding data in the presence of 0.9 equiv of Cu2+ are shown in Figure S7.

Aβ1−16 to the ATCUN sites of Aβ4−9 and Aβ12−16 (Figures 3 and S6). The presence of substoichiometric Cu2+ increased the half-

Figure 1. Determination of the metal-NEP dissociation constant Ki for (a) noncompetitive Cu2+ inhibition and (b) “mixed model” Zn2+ inhibition of NEP, using the fluorogenic substrate (S) McaRPPGFSAFK(Dnp), 0.1 nM NEP, and 20 mM HEPES at pH 7.4 and 37 °C. Data are plotted as the mean ± standard deviation (n = 3). Solid lines and constants (mean ± SEM) were determined by a global nonlinear fitting (Supporting Information).

copper noncompetitively inhibited NEP with a Cu2+-NEP dissociation constant of Ki = 1.04 ± 0.07 μM, which is comparable with that previously determined for Cu2+ inhibition of angiotensin-converting enzyme.32 The addition of excess Zn2+ (100 μM) did not restore the loss of NEP activity caused by Cu2+. Rather, a high concentration of Zn2+ ions also had an inhibitory effect on the NEP activity (Figures 1b and S3). The apparent reduction in the activity was not an artifact of indirect Zn2+ or Cu2+ quenching of the substrate fluorescence (Figure S4). Quantitative analysis of the effect of Zn2+ required a mixed model of inhibition (see the Supporting Information), yielding a Zn2+-NEP dissociation constant of Ki = 19.5 ± 3.9 μM and some enhancement of Zn2+ binding to the enzyme when the substrate was also bound (α = 5.8 ± 4.7). The inhibition by Zn2+ has not previously been reported for NEP or other Zn-dependent endopeptidases. In this regard, it is noteworthy that 10 μM Aβ1−42 was reported to be resistant to degradation by human NEP in the presence of 100 μM ZnCl2,33 although inhibition of NEP by excess Zn2+ was not considered in that instance. Next, Aβ1−16 was incubated with human NEP and the cleavage products were analyzed by liquid chromatography-mass spectrometry (LC−MS; Figures 2 and S5 and Table S1). The initial cleavage event occurred between Gly9 and Tyr10, followed by subsequent hydrolysis of the Glu3−Phe4 and Glu11−Val12 bonds. The Glu11−Val12 bond of Aβ was previously proposed as a potential site for hydrolysis20 but this was not observed experimentally. The Aβ12−x fragment was identified during the first sequencing of brain tissue from AD subjects.9 Our observation of the Aβ10−11 and Aβ12−x fragments is, to our knowledge, the first reported for NEP. Proteolysis of Cu2+/Aβ1−16 (0.9:1) led to a concomitant transfer of Cu2+ from the low-affinity coordination modes of

Figure 3. Transfer of 0.9 mM Cu2+ from 1 mM Aβ1−16 (Amax ∼ 620 nm) to the ATCUN motifs of the Aβ4−9 and Aβ12−16 (Amax ∼ 520 nm) cleavage products during hydrolysis by 25 nM NEP in 20 mM HEPES at pH 7.4 and 37 °C. Inset: Overlay of 0.9:1 Cu/Aβ1−16 after >24 h digestion, 0.9:1 Cu/Aβ4−9, and 0.9:1 Cu/Aβ12−16.

life of Aβ1−16 from 3 to 5 h (Figure S7). On the basis of the conditional binding constants of log cK = 10 and 8 for the respective binding of the first and subsequent Cu2+ ion to Aβ1−x,34 there should be 0.1 μM nonpeptide-bound Cu2+ available for inhibiting NEP at the Aβ concentration used in this assay. Because Ki = 1 μM (Figure 1a), Cu2+ inhibition of NEP only partially accounts for the observed decrease in the degradation rate. This indicates that N-terminal Cu2+ binding of Aβ impairs substrate recognition, an effect previously reported for Aβ degradation by IDE.19 To ascertain the relevance of the results obtained with Aβ1−16, we also assessed the degradation profile of Aβ1−40 using LC-MS/ MS. All major cleavages occurred prior to hydrophobic Phe, Tyr, Leu, Ile, and Val residues (Figures 4 and S8 and Table S2). In particular, the Aβ4−x and Aβ10−x truncations identified by Masters et al.8,9 were detected. As observed for the hydrolysis of Aβ1−16, cleavage of the Gly9−Tyr10 peptide bond was an early event, and this occurred concurrently with the hydrolysis of Leu17− Val18, Phe19−Phe20, and numerous cleavage events amoung residues 28−38. Truncation at Phe4 occurred more slowly, while hydrolysis of the Lys16−Leu17 peptide bond was even slower. Cleavage between Glu11 and Val12 detected during proteolysis B

DOI: 10.1021/acs.inorgchem.8b00391 Inorg. Chem. XXXX, XXX, XXX−XXX

Communication

Inorganic Chemistry

production of Aβ4−9 from Aβ1−40 by NEP, combined with its favorable properties (low molecular weight, high solubility, strong ATCUN binding, and negligible reactive oxygen species), suggests a possible function as a Cu2+ carrier. The phenomenon of Cu2+ binding to Aβ4−x is yet to be established in vivo. Indeed, an Aβ4−9 fragment might exist only transiently and would not be expected to accumulate in disease. Nevertheless, one can speculate that the high Cu2+-binding affinity of Aβ4−9 can protect NEP from noncompetitive Cu2+ inhibition that would otherwise impair the enzyme’s capacity to generate this fragment. On the other hand, it is unclear whether Ki = 1 μM for the Cu2+-NEP complex represents a physiologically relevant dissociation constant. High Cu2+ and Zn2+ concentrations during synaptic transmission have been reported;40−42 however, bulk measures of the concentration have limited meaning because of the small volume of a synaptic cleft, with binding dynamics being an important measure.43,44 If an efflux of excess intracellular Cu ions permitted transient synaptic binding to NEP, it is possible that inhibition may occur with attendant reduction in Aβ4−9 production. Thus, if Cu(Aβ4−9) is normally endocytosed or transfers the metal ion to Cu2+ importers, then transient NEP inhibition would result in a lower reuptake of Cu2+ in order to maintain an intra/extracellular balance. If sustained extracellular Cu2+ levels were to arise, then NEP activity could be chronically impaired, thereby preventing cellular uptake of Cu2+ ions. Finally, inhibition by high Zn2+ concentrations further suggests an additional mechanism of modulating NEP activity, Aβ4−9 production, and Cu2+ balance. Future investigations should examine a possible correlation between NEP activity, Aβ4−9, and cellular Cu2+ distribution. Such information could support development of AD therapeutics that avoid interfering with Aβ physiology.

Figure 4. (a) Observed Aβ1−40 cleavage sites for human NEP. Major cleavage sites are indicated by long red arrows. (b) Approximate concentration of major products during proteolysis of 0.2 mM Aβ1−40 by 5 nM recombinant human NEP in 20 mM HEPES at pH 7.4 and 37 °C. Corresponding data in the presence of 0.9 equiv Cu2+ are shown in Figure S9. Additional minor fragments identified are listed in Table S2.

of Aβ1−16 could not be identified from the NEP digestion of Aβ1−40 possibly because N-truncation at Val12 requires prior formation of Aβ10−16, whose abundance remained relatively low. Alternatively, preferential hydrolysis of Glu11−Val12 may have been an artifact of an altered conformation of Aβ10−16 due to Cterminal amidation of the Aβ1−16 model peptide. Similar to Aβ1−16, Cu2+ binding to the N-terminus of Aβ1−40 impaired proteolysis of peptide bonds within this region (Figure S9). The cleavage profile obtained using human NEP differs in some respect from previous studies. Using NEP isolated from rabbit renal cortex, Howell et al. likewise detected major cleavage sites at Glu3−Phe4, Gly9−Tyr10, Phe19−Phe20, Ala30−Ile31, and Gly33−Leu34.20 However, with secreted recombinant rabbit NEP, Selkoe and co-workers observed hydrolysis of the Gly9− Tyr10 bond but alternative hydrolyses of the Ala2−Glu3, Arg5− His6, and Val12−His13 bonds.35 It is possible that the 0.05% (8 μM) bovine serum albumin (BSA) included in their buffer could have altered substrate recognition because of its known binding interaction with Aβ.36−38 Indeed, the affinity of Aβ1−40 for human serum albumin (Kd = 5 μM)38 suggests that most Aβ1−40 would be bound to BSA. It is noteworthy that the major Aβ4−x species produced from Aβ was the soluble Aβ4−9 fragment due to the faster kinetics of Gly9−Tyr10 hydrolysis. Despite uncertainty surrounding the propensity of Aβ1−x to access Cu2+ ions in vivo,15 it is clear that proteolysis by NEP leads to metal transfer from Aβ1−x to Aβ4−9. The present study showed that hydrolysis of the Glu3−Phe4 bond occurred only after cleavage at C-terminal locations, suggesting that Aβ4−x production might shift from soluble Aβ4−9 to insoluble Aβ4−40/42 if aggregation outweighed endoproteolysis by NEP and other proteases. Indeed, aggregation or membrane binding of Aβ in vivo may obscure efficient access to C-terminal cleavage sites. However, despite the fact that synthetic Aβ4−40/42 has an aggregation propensity that exceeds that of Aβ1−40/4239 and that aggregation was free to occur over a period exceeding 4 days, no Aβ4−40 was detected in the presence or absence of Cu2+. It is possible that while NEP can degrade monomeric and oligomeric forms of Aβ1−40,21 oligomeric Aβ4−40 was too insoluble under the conditions for LC-MS/MS. Alternatively, other endopeptidases such as IDE may cleave the Glu3−Phe4 bond more rapidly to generate Aβ4−40 before NEP can cleave the Gly9−Tyr10 bond. Notwithstanding, our data suggest that

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b00391.

■

Experimental methods and additional kinetic, chromatographic, and spectroscopic characterization (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wojciech Bal: 0000-0003-3780-083X Tomasz Frączyk: 0000-0003-2084-3446 Simon C. Drew: 0000-0002-1459-9865 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS S.C.D. was supported, in part, by a fellowship (FT110100199) administered by the Australian Research Council and a fellowship awarded by the faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne. M.M. received a graduate research scholarship administered by The University of Melbourne. W.B. was supported by National Science Center (Poland) OPUS Project 2014/15/B/ST5/05229. C

DOI: 10.1021/acs.inorgchem.8b00391 Inorg. Chem. XXXX, XXX, XXX−XXX

Communication

Inorganic Chemistry

■

activity in cerebral amyloid angiopathy. J. Biol. Chem. 2004, 279, 56004− 56013. (19) Grasso, G.; Pietropaolo, A.; Spoto, G.; Pappalardo, G.; Tundo, G. R.; Ciaccio, C.; Coletta, M.; Rizzarelli, E. Copper(I) and Copper(II) Inhibit Aβ Peptides Proteolysis by Insulin-Degrading Enzyme Differently: Implications for Metallostasis Alteration in Alzheimer’s Disease. Chem. - Eur. J. 2011, 17, 2752−2762. (20) Howell, S.; Nalbantoglu, J.; Crine, P. Neutral endopeptidase can hydrolyze beta-amyloid(1−40) but shows no effect on beta-amyloid precursor protein metabolism. Peptides 1995, 16, 647−652. (21) Kanemitsu, H.; Tomiyama, T.; Mori, H. Human neprilysin is capable of degrading amyloid beta peptide not only in the monomeric form but also the pathological oligomeric form. Neurosci. Lett. 2003, 350, 113−116. (22) Russo, R.; Borghi, R.; Markesbery, W.; Tabaton, M.; Piccini, A. Neprylisin decreases uniformly in Alzheimer’s disease and in normal aging. FEBS Lett. 2005, 579, 6027−6030. (23) Farris, W.; Schutz, S. G.; Cirrito, J. R.; Shankar, G. M.; Sun, X.; George, A.; Leissring, M. A.; Walsh, D. M.; Qiu, W. Q.; Holtzman, D. M.; Selkoe, D. J. Loss of neprilysin function promotes amyloid plaque formation and causes cerebral amyloid angiopathy. Am. J. Pathol. 2007, 171, 241−251. (24) Hellström-Lindahl, E.; Ravid, R.; Nordberg, A. Age-dependent decline of neprilysin in Alzheimer’s disease and normal brain: inverse correlation with A beta levels. Neurobiol. Aging 2008, 29, 210−221. (25) Yasojima, K.; Akiyama, H.; McGeer, E. G.; McGeer, P. L. Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide. Neurosci. Lett. 2001, 297, 97−100. (26) Zhou, L.; Wei, C.; Huang, W.; Bennett, D. A.; Dickson, D. W.; Wang, R.; Wang, D. Distinct subcellular patterns of neprilysin protein and activity in the brains of Alzheimer’s disease patients, transgenic mice and cultured human neuronal cells. Am. J. Transl. Res. 2013, 5, 608−621. (27) Mohajeri, M. H.; Wollmer, M. A.; Nitsch, R. M. Aβ42-induced Increase in Neprilysin Is Associated with Prevention of Amyloid Plaque Formation in Vivo. J. Biol. Chem. 2002, 277, 35460−35465. (28) Kerr, M. A.; Kenny, A. J. The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. Biochem. J. 1974, 137, 477−488. (29) 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, 392−396. (30) Li, M.; Sun, M.; Liu, Y.; Yu, J.; Yang, H.; Fan, D.; Chui, D. Copper downregulates neprilysin activity through modulation of neprilysin degradation. J. Alzheimer's Dis. 2010, 19, 161−169. (31) Lang, M.; Fan, Q.; Wang, L.; Zheng, Y.; Xiao, G.; Wang, X.; Wang, W.; Zhong, Y.; Zhou, B. Inhibition of human high affinity copper importer Ctr1 orthologous in the nervous system of Drosophila ameliorates Aβ42-induced alzheimer’s disease-like symptoms. Neurobiol. Aging 2013, 34, 2604−2612. (32) Gokhale, N. H.; Cowan, J. A. Inactivation of human angiotensin converting enzyme by copper peptide complexes containing ATCUN motifs. Chem. Commun. 2005, 5916−5918. (33) Crouch, P. J.; Tew, D. J.; Du, T.; Nguyen, D. N.; Caragounis, A.; Filiz, G.; Blake, R. E.; Trounce, I. A.; Soon, C. P.; Laughton, K.; Perez, K. A.; Li, Q. X.; Cherny, R. A.; Masters, C. L.; Barnham, K. J.; White, A. R. Restored degradation of the Alzheimer’s amyloid-β peptide by targeting amyloid formation. J. Neurochem. 2009, 108, 1198−1207. (34) 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, 1501−1508. (35) Leissring, M. A.; Lu, A.; Condron, M. M.; Teplow, D. B.; Stein, R. L.; Farris, W.; Selkoe, D. J. Kinetics of amyloid beta-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays. J. Biol. Chem. 2003, 278, 37314−37320. (36) Milojevic, J.; Melacini, G. Stoichiometry and Affinity of the Human Serum Albumin-Alzheimer’s Aβ Peptide Interactions. Biophys. J. 2011, 100, 183−192.

REFERENCES

(1) Masters, C. L.; Selkoe, D. J. Biochemistry of amyloid β-protein and amyloid deposits in Alzheimer disease. Cold Spring Harbor Perspect. Med. 2012, 2, a006262. (2) Haass, C.; Schlossmacher, M. G.; Hung, A. Y.; Vigo-Pelfrey, C.; Mellon, A.; Ostaszewski, B. L.; Lieberburg, I.; Koo, E. H.; Schenk, D.; Teplow, D. B.; Selkoe, D. J. Amyloid beta-peptide is produced by cultured cells during normal metabolism. Nature 1992, 359, 322−325. (3) Whitson, J. S.; Selkoe, D. J.; Cotman, C. W. Amyloid beta protein enhances the survival of hippocampal neurons in vitro. Science 1989, 243, 1488−1490. (4) Yankner, B. A.; Duffy, L. K.; Kirschner, D. A. Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science 1990, 250, 279−282. (5) Soscia, S. J.; Kirby, J. E.; Washicosky, K. J.; Tucker, S. M.; Ingelsson, M.; Hyman, B.; Burton, M. A.; Goldstein, L. E.; Duong, S.; Tanzi, R. E.; Moir, R. D. The Alzheimer’s disease-associated amyloid beta-protein is an antimicrobial peptide. PLoS One 2010, 5, e9505. (6) Kumar, D. K. V; Choi, S. H.; Washicosky, K. J.; Eimer, W. A.; Tucker, S.; Ghofrani, J.; Lefkowitz, A.; McColl, G.; Goldstein, L. E.; Tanzi, R. E.; Moir, R. D. Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease. Sci. Transl. Med. 2016, 8, 340ra72. (7) Mawuenyega, K. G.; Sigurdson, W.; Ovod, V.; Munsell, L.; Kasten, T.; Morris, J. C.; Yarasheski, K. E.; Bateman, R. J. Decreased clearance of CNS beta-amyloid in Alzheimer’s disease. Science 2010, 330, 1774. (8) 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, 4245−4249. (9) 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, 2757−2763. (10) Miller, D. L.; Papayannopoulos, I. A.; Styles, J.; Bobin, S. A.; Lin, Y. Y.; Biemann, K.; Iqbal, K. Arch. Peptide compositions of the cerebrovascular and senile plaque core amyloid deposits of Alzheimer’s disease. Arch. Biochem. Biophys. 1993, 301, 41−52. (11) Portelius, E.; Bogdanovic, N.; Gustavsson, M. K.; Volkmann, I.; Brinkmalm, G.; Zetterberg, H.; Winblad, B.; Blennow, K. Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer’s disease. Acta Neuropathol. 2010, 120, 185−193. (12) 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.; Drew, S. C. A Functional Role for Aβ in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding. Angew. Chem., Int. Ed. 2015, 54, 10460−10464. (13) 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, 7317−7319. (14) 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 Cu(II) Scavenger. Angew. Chem., Int. Ed. 2016, 55, 8235−8238. (15) Drew, S. C. The Case for Abandoning Therapeutic Chelation of Copper Ions in Alzheimer’s Disease. Front. Neurosci. 2017, 11, 317. (16) Jha, N. K.; Jha, S. K.; Kumar, D.; Kejriwal, N.; Sharma, R.; Ambasta, R. K.; Kumar, P. Impact of Insulin Degrading Enzyme and Neprilysin in Alzheimer’s Disease Biology: Characterization of Putative Cognates for Therapeutic Applications. J. Alzheimer's Dis. 2015, 48, 891−897. (17) Saido, T.; Leissring, M. A. Proteolytic degradation of amyloid βprotein. Cold Spring Harbor Perspect. Med. 2012, 2, a006379. (18) Morelli, L.; Llovera, R. E.; Mathov, I.; Lue, L. F.; Frangione, B.; Ghiso, J.; Castaño, E. M. Insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced D

DOI: 10.1021/acs.inorgchem.8b00391 Inorg. Chem. XXXX, XXX, XXX−XXX

Communication

Inorganic Chemistry (37) Milojevic, J.; Costa, M.; Ortiz, A. M.; Jorquera, J. I.; Melacini, G. In vitro amyloid-β binding and inhibition of amyloid-β self-association by therapeutic albumin. J. Alzheimer's Dis. 2014, 38, 753−765. (38) Rózga, M.; Kłoniecki, M.; Jabłonowska, A.; Dadlez, M.; Bal, W. The binding constant for amyloid Abeta40 peptide interaction with human serum albumin. Biochem. Biophys. Res. Commun. 2007, 364, 714− 718. (39) Bouter, Y.; Dietrich, K.; Wittnam, J. L.; Rezaei-Ghaleh, N.; Pillot, T.; Papot-Couturier, S.; Lefebvre, T.; Sprenger, F.; Wirths, O.; Zweckstetter, M.; Bayer, T. A. N-truncated amyloid β (Aβ) 4−42 forms stable aggregates and induces acute and long-lasting behavioral deficits. Acta Neuropathol. 2013, 126, 189−205. (40) Hartter, D. E.; Barnea, A. Evidence for release of copper in the brain: depolarization-induced release of newly taken-up 67copper. Synapse 1988, 2, 412−415. (41) Kardos, J.; Kovács, I.; Hajós, F.; Kálmán, M.; Simonyi, M. Nerve endings from rat brain tissue release copper upon depolarization. A possible role in regulating neuronal excitability. Neurosci. Lett. 1989, 103, 139−144. (42) Assaf, S. Y.; Chung, S. H. Release of endogenous Zn2+ from brain tissue during activity. Nature 1984, 308, 734−736. (43) 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, e0170749. (44) Vergnano, A. M.; Rebola, N.; Savtchenko, L. P.; Pinheiro, P. S.; Casado, M.; Kieffer, B. L.; Rusakov, D. A.; Mulle, C.; Paoletti, P. Zinc dynamics and action at excitatory synapses. Neuron 2014, 82, 1101− 1114.

E

DOI: 10.1021/acs.inorgchem.8b00391 Inorg. Chem. XXXX, XXX, XXX−XXX