Iron Redox Chemistry Promotes Antiparallel Oligomerization of α

1 hour ago - Brain metal dyshomeostasis and altered structural dynamics of the presynaptic protein α-synuclein (αS) are both implicated in the patho...
1 downloads 0 Views 483KB Size
Download PDF
Subscriber access provided by UNIV OF DURHAM

Iron Redox Chemistry Promotes Antiparallel Oligomerization of #-Synuclein Dinendra L. Abeyawardhane, Ricardo D. Fernández, Cody J. Murgas, Denver R. Heitger, Ashley K. Forney, Madeleine K. Crozier, and Heather R. Lucas J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02013 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Iron Redox Chemistry Promotes Antiparallel Oligomerization of αSynuclein. Dinendra L. Abeyawardhane, Ricardo D. Fernández, Cody J. Murgas, Denver R. Heitger, Ashley K. Forney, Madeleine K. Crozier, and Heather R. Lucas* Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284

Supporting Information Placeholder ABSTRACT: Brain metal dyshomeostasis and altered structural dynamics of the presynaptic protein α-synuclein (αS) are both implicated in the pathology of Parkinson’s disease (PD), yet a mechanistic understanding of disease progression in the context of αS structure and metal interactions remains elusive. In this Communication, we detail the influence of iron, a prevalent redox-active brain biometal, on the aggregation propensity and secondary structure of N-terminally acetylated αS (NAcαS), the physiologically relevant form in humans. We demonstrate that under aerobic conditions, Fe(II) commits NAcαS to a PD-relevant oligomeric assembly, verified by the oligomer-selective A11 antibody, that does not have any parallel β-sheet character but contains a substantial righttwisted antiparallel β-sheet component based on CD analyses and descriptive deconvolution of the secondary structure. This NAcαSFeII oligomer does not develop into the β-sheet fibrils that have become hallmarks of PD, even after extended incubation, as verified by TEM imaging and the fibril-specific OC antibody. Thioflavin T (ThT), a fluorescent probe for β-sheet fibril formation, also lacks coordination to this antiparallel conformer. We further show that this oligomeric state is not observed when O2 is excluded, indicating a role for iron(II) mediated O2 chemistry in locking this dynamic protein into a conformation that may have physiological or pathological implications.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder in which insoluble aggregates of α-synuclein (αS) protein and abnormal iron deposits accumulate within the substantia nigra pars compacta.1,2 Cerebral iron levels are known to increase with aging, yet are further enhanced in PD.3 It remains unknown whether heightened levels of cerebral iron and αS aggregates termed Lewy Bodies (LBs) are the cause or effect of PD;4 nevertheless, αS remains at the forefront of biochemical and clinical studies focused on PD since it is the principal structural component within LBs. Compartmental brain metal alterations have also prompted multiple studies that examine the effect of metals on αS folding;5 yet their precise roles, whether functional or dysfunctional, remain elusive. In recent years, human αS was conclusively defined as acetylated at the N-terminus, which would have dramatic effects on protein structure and dynamics.6,7 Indeed, NAcαS has been shown to affect membrane interactions through enhanced lipid coordination and to modulate potential protein partner interactions leading to altered endocytic trafficking.8-10 Recombinant NAcαS overexpression has also been shown to increase and redistribute iron accumulation in

cellulo.11 To date, no work has explored the effect of αS N-acetylation on iron mediated fibrillization and oligomerization even though iron coordination sites have been established for the unacetylated protein.12 Moreover, previously reported experiments were not conducted in the absence of O2, despite the redox activity of iron, which can promote Fenton chemistry or other oxidative pathways. In this work, we demonstrate a distinct change in NAcαS folding and aggregation induced by iron(II) in the presence of O2 that is not observed under anaerobic conditions. Monomeric αS exists as an intrinsically disordered protein.13,14 It is notorious for aggregating into elongated β-sheet fibrils similar to those found in LBs. Intermediary oligomers, prefibrils, and/or off-pathway aggregates are among a multitude of other conformers that may have physiological or pathological relevance.15 To explore the effects of iron on the structure and aggregation propensity of NAcαS, monomeric protein was supplemented with stoichiometric iron(II) or iron(III). A 1:1 metal-protein binding ratio for NAcαS-FeIII and NAcαS-FeII was confirmed by conducting a metal depletion assay.16,17 Quantification of the protein-bound metal was confirmed by ICP-OES, and the residual unbound metal concentration was measured by ICP-MS and found to be negligible. Aggregation experiments were then carried out by agitating 1:1 metal:protein samples at 500-1000 rpm under aerobic and anaerobic conditions in 20 mM MOPS, 100 mM NaCl buffer at pH 7.

Figure 1. CD spectra following aggregation of NAcαS-FeII and NAcαS-FeIII, and comparison to unfolded monomeric NAcαS. In the absence of O2, NAcαS-FeIII and NAcαS-FeII similarly polymerize into parallel β-sheet aggregates as evidenced by CD spectroscopy based on the intense negative minima at ~218 nm (Figure 1). Under aerobic conditions, NAcαS-FeIII also exhibits a strong parallel β-sheet feature in its CD spectrum. Conversely, NAcαS-FeII does not exhibit a parallel β-sheet spectral transition in the presence of O2. Instead, a discrete and reproducible (n=40) change occurs from the spectrum associated with the disordered, random-coil monomer to a novel spectrum with a reduced and slightly blue-

ACS Paragon Plus Environment

Journal of the American Chemical Society shifted molar ellipticity minimum (Figure 1) for 1:1 metal/protein concentrations ranging from 5µM ‒ 75µM. This spectral shift is dependent on both O2 and the presence of FeII (Figure S1). Deconvolution of the CD spectra using the Beta Structure Selection (BeStSel)18 algorithm confirms that NAcαS-FeII adopts a discrete secondary structural fold under aerobic conditions that is distinct from both the parallel β-sheet fibrils observed in the anaerobic samples and the disordered NAcαS monomer. The aerobic NAcαSFeII spectrum is reminiscent of right-twisted antiparallel β-II proteins,19,20 such as trypsin inhibitor A.18,21 In fact, NAcαS-FeII has a 5-fold higher right-twisted antiparallel component than that of the monomeric NAcαS protein (Figure 2). Moreover, NAcαS-FeII incubated under aerobic conditions does not develop any parallel βstructure whatsoever. In contrast, similar proportions of parallel and antiparallel β-strands contribute to the anaerobic NAcαS-FeII and NAcαS-FeIII spectra with the parallel β-strands dominating the CD spectral features, underscoring a clear structural consequence of O2 exposure with respect to the NAcαS-FeII sample.

Figure 2. Normalized β-structure distribution of NAcαS samples as determined by deconvolution of the CD spectra with the BeStSel algorithm. Correlation with response to the A11 antioligomer antibody is noted for each sample. Unlike NAcαS-FeII samples that lack a parallel β-sheet component in the presence of O2, aerobic NAcαS-FeIII aggregates exhibit 40% β-strand structure with a parallel to antiparallel β-strand ratio of approximately 1:3 (Table S1). It is noteworthy that aerobic NAcαSFeIII exhibits a ~10% larger proportion of β-structure than the analogous anaerobic sample, likely due to the O2-induced enhancement in the antiparallel character (Figure S2), suggesting an autoreduction mechanism that subsequently results in FeII + O2 chemistry. In previous work, it has been suggested that αS behaves as a cellular ferrireductase.22 Hence, we employed X-band EPR spectroscopy to gain insight into the iron redox state. Double integral analyses of our NAcαS-FeIII samples demonstrate an increase in the iron(III) signal within the g=4.3 region23 following supplementation of O2-saturated buffer to anaerobic NAcαS-FeIII (Figure S3), providing evidence of a ferrous component in our NAcαS-FeIII samples.

Figure 4. TEM images of NAcαS-FeII and NAcαS-FeIII aggregates prepared under aerobic conditions. Fibrillization was also monitored by ThT analyses and TEM imaging. ThT is a fluorescent indicator of β-sheet rich structures present in LBs and other cross-β sheet deposits common among amyloids.24 Our work suggests that ThT interacts primarily with parallel β-sheet structure as it does not elicit a response from aerobic NAcαS-FeII (Figure 3, Figure S4), which lacks parallel β-strand components but does have approximately 20% antiparallel β-strand character. This observation may, in part, account for the false negative results that have been obtained in some ThT studies on amyloidogenic proteins.25-27 Similarly, ThT has previously been demonstrated to selectively bind to only parallel G-quadruplexes.28 TEM imaging of aggregates (Figure 4) confirmed that aerobic NAcαS-FeII aggregation results in soluble oligomers that lack the elongated fibrillar structure associated with aerobic NAcαS-FeIII aggregates and often observed for amyloidogenic proteins. Immunoblotting with the anti-fibril OC antibody29 corroborated the absence of fibrillar structure within aerobically aggregated NAcαSFeII (Figure S5). Extended aggregation of NAcαS-FeII did not result in formation of parallel β-sheet structure even after 10 days in the presence of O2, and the antiparallel structure became apparent during the early stages of incubation. Since NAcαS-FeII can aggregate to form parallel β-sheet structure under anaerobic conditions, our results demonstrate that under aerobic conditions, iron(II) mediated O2 chemistry locks this dynamic protein in a conformation that prevents a parallel β-sheet fold that would normally progress into fibrils.

400 350

Intensity / a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

300

NAc

S

NAc

S-FeII

NAc

III

Figure 5: Dot blot analysis with A11 anti-oligomer antibody.

S-Fe

250 200 150 100 50 0 460

480

500

520

540

560

580

600

Wavelength / nm

Figure 3. ThT assay following aerobic aggregation of NAcαS-FeII, and NAcαS-FeIII.

NAcαS,

In order to determine whether an oligomeric conformation associated with PD pathogenesis was present within iron-bound NAcαS samples, the anti-oligomer A11 polyclonal antibody was employed. A11 binds to soluble oligomeric epitopes common among amyloidogenic proteins, including αS, Aβ1-42 (Alzheimer’s disease), prion protein (Creutzfeldt-Jakob disease), and IAPP (type II diabetes),29,30 and studies have linked this quaternary motif to elevated toxicity.29 This anti-oligomer antibody is highly selective and does not bind to monomeric or fibrillar protein.31 Despite the marked differences between the aerobic CD spectra associated with NAcαS-FeIII and NAcαS-FeII, both aerobically prepared iron-bound species trigger a response from A11 when a dot blot is performed

ACS Paragon Plus Environment

Page 2 of 5

Page 3 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society (Figure 5) ‒ it is worth noting that oligomeric proteins do not remain associated under denaturing conditions, thwarting molecular weight determination by SDS-PAGE and western blot analyses. In stark contrast, the anaerobic NAcαS-FeIII and NAcαS-FeII aggregates are not detected by the anti-oligomer A11 antibody. Taken together, our results suggest that A11 binds to a specific antiparallel motif that is present within both iron(II) and iron(III) aerobically prepared samples despite their drastically different CD spectral characteristics, indicative of FeII/O2-promoted oligomer formation. It has been well established that copper ions can form a stable conformation with αS.32-39 X-ray structural data has been reported for copper(II)-bound to the non-acetylated protein,34 and solutionstate NMR data was used to confirm that copper(I) binds to the Nterminus of NAcαS.32 Thus, we additionally incubated NAcαS with stoichiometric Cu(I) and Cu(II) triflate under identical aerobic and anaerobic aggregation conditions, and none of these samples generated an A11 response (Figure S6). Hence, the quaternary structure induced by iron(II) coordination to NAcαS and subsequent aerobic aggregation is distinguishable from the other metal induced protein folds. Oligomeric aggregates that bind the A11 antibody have demonstrated elevated toxicity, suggesting a common structural type among amyloidogenic proteins relevant to various disease states.29 Our results suggest that such toxicity could be linked to the antiparallel right-twisted conformation of NAcαS that occurs following iron(II) coordination in the presence of O2. It is also possible that iron(II) coordination to NAcαS initiates the formation of a stable conformation of the protein with physiological relevance that becomes oligomer-locked upon exposure to a highly oxidizing environment. At this point, neither of these propositions can be ruled out; therefore, it is important to give consideration to iron(II) as a potential allosteric cofactor40 and not just an initiator of protein misfolding. Mechanistically, this oligomer-locked NAcαS-FeII/O2 conformation may prevent formation of parallel β-sheets by oxidizing key residues that promote fibrillar folds and/or by disrupting key hydrogen-bonding networks required for elongation (Scheme 1). For example, aerobic iron(II) can generate one electron reduced O2 to produce iron(III)-superoxide (i),41-43 which could facilitate protein oxidation via C-H activation44 or hydrogen atom abstraction45 that results in modified amino acid residues (ii). X-band EPR analysis of NAcαS-FeII after incubation under aerobic conditions indicated substantial conversion to the ferric protein (Figure S7), consistent with iron(II)-mediated reductive activation of O2.

DNPH forms a hydrazone (abs = 375 nm) upon reaction with aldehydes and ketones that result from NAcαS-FeII mediated radical chemistry under aerobic conditions. Based on this positive response in the DNPH assay, we suspected that the protein carbonyls formed from NAcαS-FeII/O2 mediated oxidation could promote the oligomer-locked structure that we have observed to be resistant to fibril formation. Schiff base formation between protein carbonyls and NAcαS lysine residues could generate covalent but reversible linkages (iii) that stabilize the observed soluble oligomeric conformation. To test this theory, we treated aggregate samples with sodium cyanoborohydride to reduce any imines to amines, rendering the covalent linkages irreversible (iv). Indeed, we observed a higher molecular weight band in the SDS-PAGE gel consistent with intermolecular crosslinking of NAcαS-FeII (Figure S8). We additionally sought to determine if other amino acid oxidation was occurring as a result of the FeII/O2 chemistry. Four tyrosyl residues are present within NAcαS that could become modified following FeII/O2 chemistry, leading to tyrosine hydroxylation, oxidation, and/or dityrosine formation.48 From the SDS-PAGE analysis of iron-bound species (NAcαS-FeIII, NAcαS-FeII) and protein alone, only a band indicative of monomeric protein was observed under these denaturing conditions (Figure S9); thus, no protein-protein crosslinking is evident. To investigate the existence of intramolecular dityrosine crosslinks, a monoclonal anti-dityrosine antibody that is selective for ortho-ortho-dityrosine coupling was employed, but the western blot did not detect the presence of any of this posttranslational modification (PTM) within the protein (Figure S9). Our results underscore a key role for both O2 and redox active iron in oligomerization. This is the first study to demonstrate direct oligomer formation via the sole addition of iron and serves as the first NAcαS fibrillization process investigated under controlled O2 environments. Although oligomeric αS has been reported, organic solvents such as DMSO or ethanol and/or established lyophilization techniques are commonly used to generate oligomers.49,50 Iron(II) can reductively activate O2 to produce iron-(di)oxygen species capable of oxidizing protein partners or NAcαS itself, which could affect native protein-protein and/or protein-membrane interactions as well as account for our demonstrated change in the NAcαS folding dynamics. The oligomer-locked NAcαS-FeII/O2 conformation that does not transition to parallel β-sheet fibrils yet exhibits A11 detectable structural features may pinpoint a previously unrecognized β-strand conformation of NAcαS, which could have physiological and/or pathological significance.

Scheme 1. Putative NAcαS-FeII + O2 Reaction Mechanism

Supporting Information

ASSOCIATED CONTENT The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Experimental procedures, detailed data tables of BeStSel deconvolution of secondary structure, additional CD plots, ThT data, EPR analyses, DNPH assay, SDS-PAGE gels, and western blot assays.

AUTHOR INFORMATION Corresponding Author *[email protected]

Notes The authors declare no competing financial interests. By employing a spectrophotometric assay using 2,4-dinitrophenylhydrazine (DNPH),46,47 the existence of protein-carbonyls was confirmed with the quantity increasing over time (Figure S7).

ACKNOWLEDGMENT Research reported in this publication was supported by the VCU College of Humanities & Sciences. We additionally acknowledge

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the National Institute on Aging (L-13046) for the EPR spectrometer and the Altria Group, Inc., for research fellowship support of DRH (Altria graduate fellow). Mass spectroscopic analyses of NAcαS was performed by Kristina T. Nelson at the VCU Chemical and Proteomic Mass Spectrometry Core Facility. TEM imaging was performed by Judy Williamson at the VCU Microscopy Facility, supported, in part, by funding from NIH-NCI Cancer Center Support Grant P30 CA016059. The research laboratory of Nicholas P. Farrell is also acknowledged for their maintenance of the CD spectrometer and the research laboratory of T. Ashton Cropp for access to a gel imager. Finally, we would also like to express our appreciation to the United States Secret Service for donation of the Hitachi F-4500 fluorescence spectrophotometer and Hitachi U3310 UV/vis spectrophotometer to the Lucas lab.

REFERENCES 1. Duce, J. A.; Wong, B. X.; Durham, H.; Devedjian, J. C.; Smith, D. P.; Devos, D., Mol. Neurodegener. 2017, 12, 45. 2. Belaidi, A. A.; Bush, A. I., J. Neurochem. 2016, 139, 179-197. 3. Hare, D. J.; Double, K. L., Brain 2016, 139, 1026-35. 4. McDowall, J. S.; Brown, D. R., Metallomics 2016, 8, 385-97. 5. Carboni, E.; Lingor, P., Metallomics 2015, 7, 395-404. 6. Bartels, T.; Choi, J. G.; Selkoe, D. J., Nature 2011, 477, 107-10. 7. Fauvet, B.; Mbefo, M. K.; Fares, M. B.; Desobry, C.; Michael, S.; Ardah, M. T.; Tsika, E.; Coune, P.; Prudent, M.; Lion, N.; Eliezer, D.; Moore, D. J.; Schneider, B.; Aebischer, P.; El-Agnaf, O. M.; Masliah, E.; Lashuel, H. A., J. Biol. Chem. 2012, 287, 15345-64. 8. Bartels, T.; Kim, N. C.; Luth, E. S.; Selkoe, D. J., PLoS One 2014, 9, e103727. 9. Maltsev, A. S.; Ying, J.; Bax, A., Biochemistry 2012, 51, 500413. 10. Baksi, S.; Tripathi, A. K.; Singh, N., Free Radical Biol. Med. 2016, 97, 292-306. 11. Ortega, R.; Carmona, A.; Roudeau, S.; Perrin, L.; Dučić, T.; Carboni, E.; Bohic, S.; Cloetens, P.; Lingor, P., Mol. Neurobiol. 2016, 53, 1925-1934. 12. Binolfi, A.; Rasia, R. M.; Bertoncini, C. W.; Ceolin, M.; Zweckstetter, M.; Griesinger, C.; Jovin, T. M.; Fernández, C. O., J. Am. Chem. Soc. 2006, 128, 9893-901. 13. Cattani, J.; Subramaniam, V.; Drescher, M., Phys. Chem. Chem. Phys. 2017, 19, 18147-18151. 14. Theillet, F.-X.; Binolfi, A.; Bekei, B.; Martorana, A.; Rose, H. M.; Stuiver, M.; Verzini, S.; Lorenz, D.; van Rossum, M.; Goldfarb, D.; Selenko, P., Nature 2016, 530, 45-50. 15. Ono, K., Neurochem. Res. 2017, 42, 3362-3371. 16. Nakashige, T. G.; Zhang, B.; Krebs, C.; Nolan, E. M., Nat. Chem. Biol. 2015, 11, 765. 17. Nakashige, T. G.; Zygiel, E. M.; Drennan, C. L.; Nolan, E. M., J. Am. Chem. Soc. 2017, 139, 8828-8836. 18. Micsonai, A.; Wien, F.; Kernya, L.; Lee, Y. H.; Goto, Y.; Réfrégiers, M.; Kardos, J., Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E3095103. 19. Lees, J. G.; Miles, A. J.; Wien, F.; Wallace, B. A., Bioinformatics 2006, 22, 1955-62. 20. Whitmore, L.; Miles, A. J.; Mavridis, L.; Janes, R. W.; Wallace, B. A., Nucleic Acids Res. 2017, 45, D303-D307. 21. De Meester, P.; Brick, P.; Lloyd, L. F.; Blow, D. M.; Onesti, S., Acta Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54, 589-97. 22. McDowall, J. S.; Ntai, I.; Hake, J.; Whitley, P. R.; Mason, J. M.; Pudney, C. R.; Brown, D. R., Biochemistry 2017, 56, 2497-2505. 23. Gautam, R.; Akam, E. A.; Astashkin, A. V.; Loughrey, J. J.; Tomat, E., Chem. Commun., 2015, 51, 5104-7. 24. Younan, N. D.; Viles, J. H., Biochemistry 2015, 54, 4297-306. 25. Cloe, A. L.; Orgel, J. P.; Sachleben, J. R.; Tycko, R.; Meredith, S. C., Biochemistry 2011, 50, 2026-39. 26. Lin, Y.; Kardos, J.; Imai, M.; Ikenoue, T.; Kinoshita, M.; Sugiki, T.; Ishimori, K.; Goto, Y.; Lee, Y. H., Langmuir 2016, 32, 2010-22. 27. Gu, L.; Liu, C.; Stroud, J. C.; Ngo, S.; Jiang, L.; Guo, Z., J. Biol. Chem. 2014, 289, 27300-13.

28. Zhao, D.; Dong, X.; Jiang, N.; Zhang, D.; Liu, C., Nucleic Acids Res. 2014, 42, 11612-21. 29. OC anti-fibril antibody: Kayed, R.; Head, E.; Sarsoza, F.; Saing, T.; Cotman, C. W.; Necula, M.; Margol, L.; Wu, J.; Breydo, L.; Thompson, J. L.; Rasool, S.; Gurlo, T.; Butler, P.; Glabe, C. G. Mol. Neurodegener. 2007, 2, 18.; A11 anti-oligomer antibody: Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G., Science 2003, 300, 486-9. 30. Larson, M. E.; Greimel, S. J.; Amar, F.; LaCroix, M.; Boyle, G.; Sherman, M. A.; Schley, H.; Miel, C.; Schneider, J. A.; Kayed, R.; Benfenati, F.; Lee, M. K.; Bennett, D. A.; Lesné, S. E., Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E4648-E4657. 31. de Oliveira, G. A.; Marques, M. A.; Cruzeiro-Silva, C.; Cordeiro, Y.; Schuabb, C.; Moraes, A. H.; Winter, R.; Oschkinat, H.; Foguel, D.; Freitas, M. S.; Silva, J. L., Sci. Rep. 2016, 6, 37990. 32. Miotto, M. C.; Valiente-Gabioud, A. A.; Rossetti, G.; Zweckstetter, M.; Carloni, P.; Selenko, P.; Griesinger, C.; Binolfi, A.; Fernández, C. O., J. Am. Chem. Soc. 2015, 137, 6444-7. 33. Lucas, H. R.; Lee, J. C., J. Inorg. Biochem. 2010, 104, 245-9. 34. Lucas, H. R.; Debeer, S.; Hong, M. S.; Lee, J. C., J. Am. Chem. Soc. 2010, 132, 6636-7. 35. Lucas, H. R.; Lee, J. C., Metallomics 2011, 3, 280-3. 36. Lucas, H. R.; Lee, J. C. In Brain Diseases and Metalloproteins; Brown, D. R., Ed.; Pan Stanford Publishing: Singapore, 2012; pp 295-326. 37. Rasia, R. M.; Bertoncini, C. W.; Marsh, D.; Hoyer, W.; Cherny, D.; Zweckstetter, M.; Griesinger, C.; Jovin, T. M.; Fernández, C. O., Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4294-9. 38. Moriarty, G. M.; Minetti, C. A.; Remeta, D. P.; Baum, J., Biochemistry 2014, 53, 2815-7. 39. Mason, R. J.; Paskins, A. R.; Dalton, C. F.; Smith, D. P., Biochemistry 2016, 55, 4737-41. 40. Chang, C. J., Acc. Chem. Res. 2017, 50, 535-538. 41. Solomon, E. I.; Goudarzi, S.; Sutherlin, K. D., Biochemistry 2016, 55, 6363-6374. 42. Sahu, S.; Goldberg, D. P., J. Am. Chem. Soc. 2016, 138, 1141011428. 43. Mukherjee, A.; Cranswick, M. A.; Chakrabarti, M.; Paine, T. K.; Fujisawa, K.; Münck, E.; Que, L., Inorg. Chem. 2010, 49, 3618-28. 44. Lee, Y. M.; Hong, S.; Morimoto, Y.; Shin, W.; Fukuzumi, S.; Nam, W., J. Am. Chem. Soc. 2010, 132, 10668-70. 45. Zhu, H.; Peck, S. C.; Bonnot, F.; van der Donk, W. A.; Klinman, J. P., J. Am. Chem. Soc. 2015, 137, 10448-51. 46. Levine, R. L.; Wehr, N.; Williams, J. A.; Stadtman, E. R.; Shacter, E., In Stress Response: Methods and Protocols, Walker, J. M.; Keyse, S. M., Eds.; Humana Press: Totowa, NJ, 2000; pp 15-24. 47. Ugur, Z.; Coffey, C. M.; Gronert, S., Anal. Bioanal. Chem. 2012, 404, 1399-1411. 48. Ali, F. E.; Barnham, K. J.; Barrow, C. J.; Separovic, F., J. Inorg. Biochem. 2004, 98, 173-84. 49. Chen, S. W.; Drakulic, S.; Deas, E.; Ouberai, M.; Aprile, F. A.; Arranz, R.; Ness, S.; Roodveldt, C.; Guilliams, T.; De-Genst, E. J.; Klenerman, D.; Wood, N. W.; Knowles, T. P.; Alfonso, C.; Rivas, G.; Abramov, A. Y.; Valpuesta, J. M.; Dobson, C. M.; Cremades, N., Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E1994-2003. 50. Kostka, M.; Högen, T.; Danzer, K. M.; Levin, J.; Habeck, M.; Wirth, A.; Wagner, R.; Glabe, C. G.; Finger, S.; Heinzelmann, U.; Garidel, P.; Duan, W.; Ross, C. A.; Kretzschmar, H.; Giese, A., J. Biol. Chem. 2008, 283, 10992-1003.

ACS Paragon Plus Environment

Page 4 of 5

Page 5 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

FOR TABLE OF CONTENTS ONLY:

ACS Paragon Plus Environment