An ortho-Iminoquinone Compound Reacts with Lysine Inhibiting Aggregation while Remodeling Mature Amyloid Fibrils

Article abstract of DOI:10.1021/acschemneuro.7b00017

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aβ_1-40 as model amyloidogenic proteins, we observed that IQ was able to react with lysine residues and reduce amyloid aggregation. We also observed that IQ reacted with free amines within the amyloid fibrils preventing their dissociation and seeding capacity.

Full text of DOI:10.1021/acschemneuro.7b00017

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Article
An ortho-Iminoquinone Compound Reacts with Lysine Inhibiting
Aggregation while Remodeling Mature Amyloid Fibrils.
Luiza Fernandes, Nathália Moraes, Fernanda Savacini Sagrillo, Augusto V. Magalhães, Marcela Cristina
de Moraes, Luciana Romão, Jeffery W. Kelly, Debora Foguel, Neil P. Grimster, and Fernando L Palhano
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017
Downloaded from http://pubs.acs.org on April 22, 2017
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An ortho-Iminoquinone Compound Reacts with Lysine Inhibiting
Aggregation while Remodeling Mature Amyloid Fibrils.
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Luiza Fernandes‡, Nathalia Moraes‡, Fernanda S. Sagrillo§, Augusto V. Magalhães‡, Marcela C.
de Moraes§, Luciana Romão#, Jeffery W. Kelly†, Debora Foguel‡, Neil P. Grimster† and
Fernando L. Palhano‡,*
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‡Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941ꢀ590, Brazil.
†Departments of Chemistry and Molecular and Experimental Medicine and the Skaggs Institute
for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla,
CA 92037, USA.
#Universidade Federal do Rio de Janeiro – Pólo de Xerém, Duque de Caxias, Rio de Janeiro
25245ꢀ390, Brazil.
§Department of Organic Chemistry, Chemistry Institute, Fluminense Federal University, Niteroi,
Rio de Janeiro, 24020ꢀ141, Brazil.
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ABSTRACT
Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer’s
and Parkinson’s diseases. It has been shown that lysine residues play a key role in the formation
of these aggregates. Thus the ability to disrupt aggregate formation by covalently modifying
lysine residues could lead to the discovery of therapeutically relevant antiꢀamyloidogenesis
compounds. Herein, we demonstrate that an orthoꢀiminoquinone (IQ) can be utilized to inhibit
amyloid aggregation. Using alphaꢀsynuclein and Aβ1ꢀ40 as model systems, we observed that IQ
was able to react with lysine residues and reduce amyloid aggregation. We also observed that IQ
reacts with free amines within the amyloid fibrils preventing their dissociation and seeding
capacity.
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Keywords: imine, amyloid aggregation, Parkinson’s disease, alphaꢀsynuclein, Alzheimer’s
disease, crossꢀlink.
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INTRODUCTION
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The aggregation of peptides and proteins, exacerbated by aging, is genetically and pathologically
linked to numerous neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s
disease, as well as the systemic amyloidoses1. As such, a wide range of normally soluble
intrinsically disordered peptides or folded proteins with wellꢀdefined structures can form
amyloid fibrils, either through mutation or environmental changes1. These amyloid fibrils consist
of multiple interacting filaments, which are at least two crossꢀβꢀsheets thick. To date, a very
limited number of therapies exist that can slow the progression of one of the known
amyloidoses2. Therefore, compounds capable of directly interacting with amyloidogenic proteins
and blocking their selfꢀassembly, or possessing the ability to remodel mature amyloid fibrils,
may help in addressing this unmet medical need2. Two compounds that fulfill these criteria are
the molecular tweezers CLR013,4 and epigallocatechinꢀ3ꢀgallate (EGCG) (Fig S1) 5ꢀ7. CLR01
binds with high affinity to lysine (Lys) residues of proteins, thereby affecting the aggregation
process of a broad range of amyloidogenic proteins3. The molecular mechanisms by which
EGCG blocks amyloid aggregation are not completely understood; however, some studies point
toward hydrophobic binding and Schiff base formation with Lys residues as important features
of the mechanism5,8. Moreover, Lys residues have been shown to be important in the aggregation
of several amyloidogenic proteins9.
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Recently, inspired by the amine oxidase enzymes10, Largeron and Fleury developed an oꢀ
iminoquinone (IQ) compound that catalyzes the oxidation of amines to imines under mild
conditions11 (Fig 1). To accomplish this transformation, however, it was necessary to first
oxidize IQ itself (step 1, Fig 1). This initial step can occur through aerobic oxidation, but with a
very slow rate of reaction (~ seven days)11. In contrast, the presence of metal salts, e.g., copper,
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enhances the rate of reaction ~50 fold. The authors demonstrated that under these conditions IQ
was capable of oxidizing several primary amines to imines, in methanolic solutions11. Due to the
ability of this transformation to function under polar protic conditions, we reasoned that it may
be possible to oxidize the Lys sideꢀchains of proteins under aqueous conditions, thereby
generating imines capable of forming interꢀ or intraꢀmolecular crossꢀlinks.
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Our data show that IQ blocks the aggregation of alphaꢀsynuclein and remodels mature amyloid
fibrils of alphaꢀsynuclein and Aβ1ꢀ40 into amorphous material. The molecular mechanisms by
which IQ affects the aggregation of fibrils is not fully elucidated, but here we demonstrate that
these effects are in part dependent of presence of lysine residues within the fibrils. The data
presented here open several new possible avenues of investigation in the amyloid field, and the
mechanisms described should therefore be considered during the design of amyloid inhibitors to
treat these devastating diseases.
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RESULTS AND DISCUSSION
To test the activity of IQ on amyloid aggregation, we first utilized the amyloidogenic protein
alphaꢀsynuclein (syn) as a model system. The aggregation of syn in Lewy bodies is a typical
pathological hallmark of Parkinson’s disease. Syn possesses fifteen lysine residues (Fig 2A).
Initially, we incubated monomeric syn (70 ꢀM) with agitation in the presence of thioflavin T
(ThT), a fluorogenic amyloid probe, and monitored aggregation by the increase in ThT
fluorescence intensity as a function of time (Fig 2B). As hypothesized, there is a dose dependent
reduction in the rate of syn aggregation in the presence of IQ (Fig 2B). When a ratio of 1:7 or
greater (syn:IQ) was used, complete inhibition of syn aggregation was observed. As a positive
control EGCG (Fig S1), a wellꢀknown inhibitor of syn aggregation6, was also studied. Here, syn
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aggregation was completely inhibited at a ratio of 1:5 syn:EGCG, indicating that EGCG was
slightly more effective at preventing aggregation when compared to IQ. It is also important to
note that all subsequent experiments with IQ were performed in the presence of zinc (Zn2+). As a
control, we observed no difference in the aggregation of syn monomers when incubated in the
presence or absence of zinc, over the time course of our experiments (Fig S3). When syn was
incubated with IQ at a ratio of 1:5 in the presence of 1mM zinc, we observed increased inhibitory
aggregation activity of IQ when compared to the absence of zinc (Fig S3). When a ratio of 1:10
(syn:IQ) was used, IQ efficiently blocked syn aggregation and this inhibition was not further
enhanced by the presence of zinc (Fig S3). In other words, zinc is important, but not essential,
for catalyzing the reaction between IQ and syn, especially when lower concentrations of IQ are
used.
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In an effort to further characterize the effects of IQ on syn aggregation, 70 ꢀM syn was
incubated in the presence of either IQ or EGCG (1:10 ꢀ syn:compound) for 60 h. The samples
were then fractionated by highꢀspeed centrifugation to yield soluble (S) and pellet (P) fractions.
Each fraction was subsequently treated with 8 M urea, sonicated for 30 min, boiled for 10 min
with SDS in order to solubilize any amyloids fibrils present12, and resolved by SDSꢀPAGE (Fig
2C and S4). In the absence of compound, the vast majority of the syn (90%) is found in the
pellet, consistent with amyloid formation (Fig 2C, lanes 1 and 2; Fig S4), whereas in the
presence of EGCG, only soluble species with different masses (monomer, dimer, trimer, etc.)
were detected (Fig 2C, lanes 3 and 4; Fig S4). These crossꢀlinked species are believed to derive
from Schiff base formation between the lysine of the protein and a quinone intermediate formed
via oxidation of EGCG6. In the presence of IQ (1:10 syn:IQ), approximately 80% of the syn was
found in the soluble fraction, thus demonstrating IQ’s ability to retard aggregate formation (Fig
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2C, lane 5; Fig S4). It should be noted, however, that syn was also observed in the pelleted
material, suggesting only partial inhibition of aggregation (Fig 2C, lane 6; Fig S4).
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To gain further insights into the species formed during aggregation in the presence of IQ, a filter
retardation assay (FRA) was employed (Fig 2D). Briefly, syn was incubated for 60 h in the
absence or presence of IQ, and then an aliquot of each was boiled with 2% SDS and applied to a
membrane (0.2 ꢀm pores), which allows the passage of soluble proteins, but traps larger
aggregated species. As expected, the sample incubated in the absence of IQ passed through the
membrane, because the aggregates formed in the incubation had been denatured through boiling
(Fig 2D). In contrast, when syn was incubated with IQ, part of the sample was retained in the
membrane. This suggests a degree of intermolecular crossꢀlinking amongst the fibrils which
renders them stable to boiling with SDS (Fig 2D). It is important to note that in our FRA
protocol the membrane was stained with Ponceau S, the sensitivity of which is far less than some
other detection methods, e.g., immune detection. This may, therefore, explain why we observed
no retained fibrils after SDS boiling while others have observed some SDS resistant fibrils13. To
study the secondary structure of the species formed during aggregation in the presence of IQ,
circular dichroism (CD) was employed. In the absence of IQ, syn aggregates displayed the well
documented βꢀsheet secondary structure. In contrast, a randomꢀcoil pattern, typical of syn
monomers, was observed when aggregation was performed in the presence of IQ (Fig 2E).
Finally, the seeding capacity of fibrils formed in the presence of IQ was investigated. As has
been previously demonstrated, fragments of the aggregates formed in the presence of EGCG do
not seed the formation of other aggregates (6 and Fig 2F, green). In contrast, syn fibrils formed in
the absence of any compound do seed aggregation (Fig 2F, black). Interestingly, sonication of
the aggregates formed in the presence of IQ displayed reduced seeding capacity (Fig 2F ꢀ
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compare blue with black curve). Collectively, these data suggest that IQ was able to reduce
amyloid aggregation of syn, resulting in soluble and insoluble species, and generating some
SDSꢀresistant aggregates with reduced seeding competence.
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In order to demonstrate that the effect of IQ on syn aggregation originates from the Lys residues,
aggregation assays were performed in the presence and absence of free lysine (Fig 3A).
Interestingly, when free lysine (150:1, Lys:IQ) was added to the aggregation of syn in the
presence of IQ, the ability of IQ to inhibit aggregation was almost completely blocked. This
result suggests that, at least in part, IQ exerts its effect on syn aggregation through reaction with
the lysine side chains. TEM analysis of the morphology of the syn aggregates formed in the
absence or presence of IQ demonstrated mature fibrils when aggregation was performed in the
absence of IQ (Fig. 3B), while amorphous, spherical aggregates were observed in the presence of
IQ (1:5, Fig. 3C). Interestingly, the typical fibril architecture was restored when free Lys was
present (Fig. 3D). The presence of free Lys also reduced the amount of SDSꢀresistant aggregates
produced upon addition of IQ (Fig 3E).
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In order to analyze whether IQ chemically modified syn, monomeric syn was incubated for 60 h
under aggregating conditions and subsequently analyzed by mass spectrometry (Fig 3FꢀI). In the
absence of IQ, a single peak corresponding to unmodified syn was observed (mass observed =
14,460 Da) (Fig 3F). However, when aggregation was performed in the presence of IQ, several
additional peaks were detected (Fig 3H and 3I). First, we noted the formation of oxidized syn,
with masses equivalent to the addition of one (+16), two (+32) or three (+48) oxygens. We also
observed the presence of masses corresponding to the addition of one (+164) or two (+328) IQ
molecules. Additional peaks for the oxidation (1ꢀ3 oxygens) of these adducts were also observed.
This analysis also confirmed that Zn2+ is important for the catalytic activity of IQ, since all peaks
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related to the adduct IQ:syn were less intense in its absence (compare Fig 3H with Fig 3I). Also,
Zn2+ alone caused no changes in the primary structure of syn (compare Fig 3F with 3G).
Moreover, free Lys blocked the formation of the IQ:syn adduct, while the oxidation of syn was
still observed (data not shown). It should be noted that fully oxidized syn cannot aggregate14.
Taken together, these data suggest that part of IQ’s ability to prevent syn aggregation is due to
oxidation of the monomers (Fig 1A and Fig 3A) and part to the Lys adducts formed upon
reaction with IQ.
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Next, we were interested in evaluating the ability of IQ to remodel mature fibrils, as has been
reported for EGCG5,8. To this end, mature amyloid fibrils of syn were produced (see Fig S5 for
fibril characterization), and subsequently incubated with IQ (1:20) or EGCG (1:20). It is
important to note that EGCG displaces ThT from amyloid fibrils5 and this explains the reduction
in ThT florescence at time point 0 (Fig 4A). This effect was not observed upon incubation with
IQ when fibrils of syn (Fig 4A) or fibrils of Aβ1ꢀ40 (data not shown) were used. As can be seen in
Fig 4A, a marked reduction in ThT fluorescence after 24 h was observed for both EGCG and IQ.
The remodeling activity displayed by IQ was analyzed by filter retardation assay (Fig 4B). We
observed that amyloid fibrils incubated in the presence of either compound became increasingly
resistant to SDS/boiling over time (Fig 4B). While the EGCG fibril crossꢀlinking was faster
when compared with IQ (Fig 4B), we confirmed IQ’s remodeling of mature amyloid fibrils by
electron microscopy analysis (compare Fig 4C with Fig 4D) and also their reduced seeding
capacity (Fig 4G). We also observed that after incubation with IQ or EGCG the syn fibrils were
largely no longer recognized by the conformationꢀdependent antibody LOC (Fig 4F), an
antibody raised against mature amyloid fibrils15,16. Consistent with our hypothesis that IQ
remodels amyloid fibrils through interaction with Lys residues, we observed that IQ was
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ineffective at remodeling syn fibrils when the Lys side chains were blocked by vitamin B6 or
acetylation (Fig 5A), or in the presence of excess of free Lys (Fig 5B,5C). We, therefore,
concluded that IQ was able to remodel mature amyloid fibrils of syn into SDSꢀresistant
amorphous aggregates that were also seeding incompetent.
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In order to determine whether IQ was also effective at remodeling other amyloid fibrils, we
examined its effect on the aggregation of Aβ1ꢀ40 (Fig 6A), a peptide associated with Alzheimer’s
disease. The Aβ1ꢀ40 peptide was agitated for 6 d at 37 °C and the resulting amyloid fibrils were
characterized by ThT binding (Fig S6A), transmission electron microscopy (Fig S6B), and CD
spectroscopy (Fig S6C). We incubated Aβ1ꢀ40 mature fibrils with IQ (1:20) for 24 h and the
remodeling activity was analyzed by the filter retardation assay and ThT binding. The formation
of SDSꢀresistant aggregates (Fig 6B) and the reduction in ThT fluorescence (Fig 6C) in the
presence of IQ demonstrate that IQ also efficiently remodels mature Aβ1ꢀ40 amyloid fibrils.
We next wanted to investigate whether IQ or EGCG could remodel fibrils that lack lysine
residues. To investigate this we used mature amyloid fibrils from islet amyloid polypeptide
(IAPP), which lacks both Lys and methionine residues, and was synthesized with an acetylated
Nꢀterminus (Fig 7A and Fig S7)5,17. When IAPP fibrils were incubated with either IQ or EGCG,
neither compound was able to crossꢀlink the mature fibrils (Fig 7B), but EGCG was effective in
remodeling them, whereas IQ was not (Fig 7C). This result suggests that IQ activity is
completely dependent on the presence of free amines, and/or methionines, whereas EGCG is not.
The cytotoxicity of IQ was probed using human embryonic kidney cells (HEK293) (Fig S8).
Unlike EGCG, which was highly toxic to these cells in culture, IQ was innocuous up to 500 ꢁM.
In order to study the toxicity of IQ in a more physiologically relevant context, we cultured
primary murine dopaminergic neurons, the cells that degenerate in Parkinson’s disease patients18.
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These neuronal cells, which express the enzyme tyrosine hydroxylase, a marker for
dopaminergic neurons19 (Fig 8A), were incubated for 24 h with increasing concentrations of IQ
or EGCG in the presence of 100 ꢀM zinc. EGCG was much more toxic when compared with IQ
in neuronal cells (Fig 8B), as observed for HEK293 cells (Fig S8). Neuronal cells were less
robust than HEK cells, with the combination of IQ + zinc displaying some toxicity at 200 ꢀM IQ
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but more pronounced at 500 ꢀM IQ (compare Fig 8B with S8). The reduced cellular toxicity of
IQ represents an important advantage of IQ over EGCG in terms of its future use as an antiꢀ
amyloidogenic compound.
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Finally, the specificity of IQ was probed using competition assays. We incubated monomeric syn
in the presence of chicken egg albumin or eggꢀwhite lysozyme and analyzed the ability of IQ to
inhibit syn aggregation as well as the enzymatic activity of lysozyme. For the competition
experiments we used an equimolar concentration of lysines per protein. We observed that the
presence of albumin had no effect on syn aggregation, however the presence of lysozyme
changed the kinetics of syn aggregation, reducing the lag time (Fig 9A). When the same
experiment was performed in the presence of IQ (1:10, syn:IQ), IQ completely blocked syn
aggregation even when albumin or lysozyme were present (Fig 9A). These data were confirmed
by fractionation of samples followed by SDSꢀPAGE (Fig 9B), where it is evident that the
samples incubated with albumin or lysozyme exhibit the same profile when compared to the
sample incubated in the absence of these proteins, even when IQ was present. In order to
evaluate the enzymatic activity of lysozyme after IQ treatment, syn was incubated in the
presence or absence of lysozyme with or without IQ (1:10, syn:IQ) for 40 h and then added to a
culture of Micrococcus luteus, a known target of lysozyme20. Lysozyme hydrolyzes the betaꢀ
glycosidic linkage between Nꢀacetylmuramic acid and Nꢀacetyl glucosamine in the
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peptidoglycan of bacterial cell walls. Dead cells were quantified by labeling with an ethidium
homodimer probe (Fig 9C)20. We observed that the activity of lysozyme was the same in the
absence or in the presence of IQ (Fig 9D, Fig S9). With these experiments we conclude that IQ
prefers syn to lysozyme, suggesting some specificity. We speculate that the preference of IQ for
syn over lysozyme can be explained by the fact that syn is an intrinsically disordered protein
(IDP) with more lysines exposed to facilitate IQ attack.
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For transthyretin related amyloidoses, a regulatory agency approved drug, tafamidis, is approved
to slow disease progression. Tafamidis is a small molecule that binds with high affinity and
specificity to the folded protein transthyretin, kinetically stabilizing the tetramer and preventing
protein aggregation21. Tafamidis is one of the few compounds that specifically inhibits one
amyloid disease, owing to the fact that it binds to a natively folded protein. For IDPs such as syn
and Aβ, the development of a specific drug is more challenging2. IDPs expose more of their
residues to the solvent compared to folded proteins which can be taken advantage of to achieve
selectivity.
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CONCLUSIONS
In conclusion, we believe that IQ can efficiently block amyloid aggregation and remodel mature
amyloid fibrils. The mechanism by which IQ operates is not totally elucidated; however, the
majority of its action appears to operate through reaction with free amines (Lys) present in the
protein/peptide target. It is still unclear if the IQ effect is mediated only by protein biding (Fig 1
step 4; Fig 3H and 3I) or also involves fibril crosslinking (Fig 1, step 6; Fig 4B and Fig 5). We
are currently attempting to elucidate which of the syn lysine residues are susceptible to IQ
reaction. We believe that the findings presented in this study can be utilized as starting points for
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medicinal chemistry efforts towards the development of compounds capable of reducing the
aggregation of amyloidogenic proteins or crosslinking amyloid to reduce dissociationꢀassociated
proteotoxicity.
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EXPERIMENTAL METHODS
IQ synthesis
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The IQ synthesis was performed as described previously, with some modifications22. Briefly, in a
dry 500 mL flask, polyphosphoric acid (110 g) was heated to about 50 °C. When it became less
sticky, acetic acid (0.46 g, 7.7 mmol) and 2ꢀnitroresorcinol (1.00 g, 6.5 mmol) were added and
the mixture was stirred for 2 h at 70ꢀ80 °C. The reaction was quenched by addition of cold water
(300 mL) and the reaction mixture was extracted with ethyl acetate (5 x 100 mL). The combined
organic layers were dried over anhydrous MgSO4 and the solvent was removed under reduced
pressure at 35 °C. Purification of the crude product was performed by column chromatography
(silica gel, hexane/EtOAc) affording the pure 1ꢀ(3ꢀAminoꢀ2,4ꢀdihydroxyphenyl)ꢀ1ꢀethanone in
72 % yield (0,92 g, 4.7 mmol). This compound (1.0 g, 5.0 mmol) was poured into the
hydrogenation flask, followed by the addition of Pt/C (0.1 g, 10 mol %), and the mixture was
subjected to hydrogenation at 28 atm (420 psi) at 50 ºC23. The reaction was monitored by TLC
until total conversion of the starting material was achieved. The catalyst was separated by
filtration, and the solvent was removed under vacuum. oꢀIminoquinone (0.8 g, 4.78 mmol, 95 %)
was obtained as a pale yellow solid of suitable purity for further use: mp 209ꢀ211 °C; 1H NMR
(500 MHz, DMSOꢀd6) δ 7.14 (d, J = 8.8 Hz, 1 H), 6.40 (d, J = 8.8 Hz, 1 H), 2.50 (s, 3 H); 13C
NMR (75 MHz, DMSOꢀd6) δ 204.0, 150.8, 123.8, 120.9, 113.2, 107.5, 26.5; MS (ESI+) m/z 168
[M + H]+.
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Preparation of alphaꢀsynuclein (syn)
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Syn was purified as described previously by acidification treatment with some modifications24.
To purify syn monomers, the lyophilized syn was resuspended in water immediately before each
experiment, filtered through a 0.22 ꢁm filter and then centrifuged through a Centricon with a 100
kDa cutꢀoff. The homogeneity of monomers was analyzed by analytical sizeꢀexclusion
chromatography performed using a SuperoseTM6 10/300 GL column equilibrated with phosphate
saline buffer (Fig S2A). Chromatograms were obtained by injecting samples of 10 ꢁL at a 0.5
mL/min flow rate. Elution was monitored by fluorescence (Ex = 275 nm, Em = 320 nm) and
absorbance at 280 nm. We also used dynamic light scattering in order to characterize the purified
syn. Dynamic lightꢀscattering (DLS) measurements were performed on a Brookhaven
Instruments Corp at 25 °C25. The hydrodynamic radium of 4 nm was calculated using the
diffusion coefficient and the Stokes–Einstein equation (Fig S2B). The identity of syn was
confirmed by mass spectrometry. The mature syn amyloid fibrils were obtained by incubation of
monomeric syn (140 ꢁM) in phosphate saline buffer (PBS) pH 7.4 with 0.02% NaN3 with
agitation (rotation at 24 rpm) at 25 °C for 6 d.
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Preparation of Aβ1ꢀ40
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Aβ1ꢀ40 fibrils were prepared as previously described12. Aβ1ꢀ40 was synthesized using a standard
Fmoc chemistry strategy for solid phase peptide synthesis. The resulting peptide was purified by
reversed phase C18 highꢀperformance liquid chromatography (RPꢀHPLC) and characterized by
matrixꢀassisted laser desorption ionization mass spectrometry using sinapinic acid as a matrix
and nanostructure initiator mass spectrometry (NIMS)ꢀTOF analysis was performed using a
VoyagerDE STR TOF (Applied Biosytems, Foster City, CA) mass spectrometer in reflectron
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mode. The fibrils were produced after 6 d aggregation at 37 °C in 50 mM phosphate buffer pH
7.4, 150 mM NaCl and 0.02% NaN3 with agitation (rotation at 24 rpm).
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Preparation of Islet Amyloid Polypeptide (IAPP)
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IAPPAc8ꢀ37 was synthesized with an acetylated Nꢀterminus (Ac) by GenScript. The purity was
greater than 95% as analyzed by RPꢀHPLC and the correct mass was confirmed by electrospray
ionization (ESI). Peptide stock solutions were prepared by dissolving a weighed amount of
peptide in 8 M guanidine HCl, 50 mM phosphate buffer pH 7.4 to obtain a final concentration of
20 mg/ml. The peptides were sonicated for 1 h and centrifuged (10 min at 16,000 g at 25 °C) to
remove insoluble material. The concentration of the stock solutions was determined by Bradford
assay. The peptide stock solutions were diluted (∼80 fold) in 50 mM phosphate buffer pH 7.4,
150 mM NaCl and 0.02% NaN3 to a final concentration of 0.25 mg/ml and 0.1 M guanidine HCl.
The samples were incubated with agitation (rotation at 24 rpm) at 25 °C for 6 d. The fibrils were
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centrifuged (16,000 g for 10 min at 4 °C), the supernatant was removed and the pellet was stored
at ꢀ20 °C until use.
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EGCG and IQ preparation
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EGCG (Fig S1) obtained from SigmaꢀAldrich was prepared in fresh ultraꢀpure water and aliquots
of the stock solution (5 mM) were stored at ꢀ20 °C until use. Ten milligrams of IQ was dissolved
in 100% DMSO to a final concentration of 300 mM. The stock solution was diluted in ultra pure
water to a final concentration of 5 mM and stored protected from light at ꢀ20 °C until use.
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Thioflavin T (ThT) assay
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For kinetics experiments, the samples containing ThT (10 ꢁM) were incubated at 37 °C in a 96ꢀ
well plate (Costar # 3631) together with Teflon spheres, 0.3 cm diameter. Every 5 min, the plates
were shaken for 30 s, and fluorescence (excitation at 450 nm, emission at 477 nm) was
monitored using a SpectraMax Paradigm MultiꢀMode Microplate Reader. For steady state ThT
binding assays, the samples were diluted to 5 ꢁM in PBS pH 7.4 containing 20 ꢀM ThT and
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incubated at 25 °C for 0.5 h. Binding was monitored using a spectrofluorimeter to measure the
fluorescence increase (excitation at 450 nm and fluorescence emission at 470ꢀ520 nm) at 25 °C.
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Filter retardation (FR) assay
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The samples were incubated in the presence of 2% SDS and boiled or not for 10 min, then
applied to a nitrocellulose membrane using a DOT blot apparatus under vacuum26. For the FR
assay, the samples were stained using Ponceau.
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Circular dichroism
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Circular dichroism (CD) measurements were performed in a Jasco spectropolarimeter using a
0.01 mm pathꢀlength quartz cuvette. The buffer used for CD measurements was phosphate saline
buffer, pH 7.4. Data were averaged for three scans at a speed of 100 nm/min collected in 0.2 nm
steps at 25 °C. The baseline (buffer alone) was subtracted from the corresponding spectra.
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Fractionation of aggregates
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The samples were fractioned through highꢀspeed centrifugation (16,000 g for 30 min, 15 °C) to
yield a soluble and a pellet fraction and then each fraction was treated with 8 M urea, sonicated
for 30 min and boiled for 10 min with 2% SDS in order to solubilize any amyloids fibrils
present.
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Mass spectrometry
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The samples were diluted to 7.5 ꢁM in 3% acetonitrile and 0.1% formic acid and loaded onto a
Waters Nano Acquity system (Waters, Milford, MA). The sample injection volume was 2.0 ꢁl.
Proteins were desalted using a trap column (Waters Symmetry C18 180 ꢁm X 20 mm, 5 ꢁm) and
the LC was performed with a linear gradient (0–60%) of acetonitrile containing 0.1% formic acid
at a 1.0 ꢁL/min flow. Electrospray tandem mass spectra were recorded using a QꢀTof
quadrupole/orthogonal acceleration timeꢀofꢀflight spectrometer (Waters, Milford, MA)
interfaced to the Nano Acquity system capillary chromatograph. The ESI voltage was set at
3500 V, the source temperature was 80 °C, and the cone voltage was 30 V. The instrument
control and data acquisition were conducted by a MassLynx data system (Version 4.1, Waters),
and experiments were performed by scanning from a massꢀtoꢀcharge ratio (m/z) of 50–2000
using a scan time of 1 s, applied during the whole chromatographic process. All data were
processed manually in MassLynx and the average molecular weight was determined through
charge state deconvolution using the Maximum entropy Algorithm (MaxEnt 1, Waters, Milford,
MA).
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Seeding Experiments.
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A monomeric solution of protein (syn = 70 ꢁM) was incubated in the absence or presence of 5%
seeds in phosphate saline buffer pH 7.4 with agitation. The seeds were produced by 30 min
sonication. The samples containing ThT (10 ꢁM) were incubated at 37 °C in a 96ꢀwell plate, and
every 5 min the fluorescence (excitation at 450 nm, emission at 477 nm) was monitored.
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MTT Metabolic Assay.
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Primary cultures of dopaminergic neurons were prepared from the mesencephalon of 14ꢀdayꢀold
Swiss Mice embryos (E14), as previously described27. Pregnant Swiss mice were anesthetized
and decapitated; the brain structures of the 14ꢀdayꢀold embryos were removed and dissociated
cells were plated on coverslips treated with polyꢀLꢀornithine (1.5 mg/ml; SigmaꢀAldrich) in
serumꢀfree neurobasal medium. The cultures were incubated at 37 °C in a humidified 5% CO2
and 95% air chamber for 7 days. These neuronal cell cultures were assayed by
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immunocytochemistry with antiꢀtyrosine hydroxylase (1:100; Millipore) antibody and antiꢀ
MAP2 (1:100; Millipore). The cells were incubated with increasing concentrations of IQ or
EGCG in the presence of 0.1 M zinc sulfate for 24 h. HEK293 cells were cultured in DMEM
medium supplemented with 10% FBS, 100 U/mL gentamicin in a 5% CO2 humidified
environment at 37 °C. Cells were plated at a density of 10,000 cells/well on 96ꢀwell plates in 100
ꢁL of fresh medium. After 24 h, the compounds were added, and the cells were incubated for 2 d
at 37 °C. Cytotoxicity was measured utilizing the 3ꢀ4,5ꢀdimethylthiazolylꢀ2,5ꢀ
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diphenyltetrazolium bromide (MTT) assay. Absorbance values of formazan were determined at
570 nm after 0.5 h of incubation.
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Electron Microscopy
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Copper grids (carbonꢀ and formvarꢀcoated 400 mesh) (Electron Microscopy Sciences, Hatfield
PA) were glow discharged and 2 ꢁL of sample was applied for 3 min. Excess sample was
removed and the grids immediately incubated with 2 % uranyl acetate solution for 2 min. Excess
stain was removed and the grid allowed to dry thoroughly. Grids were then examined on a
Tecnai Spirit (FEI Company) microscopy at 120 kV.
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Chemical blocking of lysines of amyloid fibrils.
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Syn amyloid fibrils (120 ꢁM) in 20 mM borate buffer pH 8.0 were incubated with 10 mM acetic
anhydride overnight at 25 °C. The acetylated fibrils were dialyzed overnight against phosphate
buffer pH 7.4. For pyridoxal 5'ꢀphosphate (PLP) modification, syn amyloid fibrils (120 ꢁM) in
20 mM borate buffer were incubated with 10 mM PLP for 2 h, then 20 mM sodium borohydride
was added for 1 h at 25 °C. The PLPꢀmodified fibrils were dialyzed overnight against phosphate
buffer pH 7.4. All reactions were performed protected from light.
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Dot blot
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LOC antibody was raised against mature amyloid fibrils derived from islet amyloid polypeptide.
This antibody can distinguish between amyloid fibrils and oligomeric and monomeric species15.
The samples were spotted (2 ꢁl) onto a nitrocellulose membrane. The membrane was blocked
using 1 vol PBS + 1 vol blocking solution (Odyssey) for 1 h. The membrane was incubated with
LOC antibody (1:1,000, Millipore) or synꢀ1 antibody (1:1,000, Sigma) (diluted in 1 vol TBST
(50 mM Tris pH 7.6, 0.9% NaCl, 0.1% Tween 20) +1 vol blocking solution for 1 h, washed 3
times with TBST and then incubated for 1 h with goat antiꢀrabbit secondary antibody conjugated
to IRDye 800 CW (1:5,000) for LOC or goat antiꢀmouse secondary antibody conjugated to
IRDye 680 CW (1:5,000) for synꢀ1 and developed/quantified using an Odyssey Infrared Imaging
System.
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Competition assay with lysozyme and albumin
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Monomeric syn (70 ꢁM) was incubated in the absence or in the presence of 52 ꢁM chicken egg
albumin (Uniprot ID: P01012, Sigma) or 175 ꢁM eggꢀwhite lysozyme (Uniprot ID: P00698,
Sigma) in order to have equimolar concentration of lysines for each protein (1 mM). The samples
were incubated at 37 °C in PBS in the absence or in the presence of 700 ꢁM IQ + 1 mM ZnSO4,
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as described in the “Thioflavin T (ThT) assay”. After 40 h aggregation, an aliquot of the samples
was processed as described in the “Fractionation of aggregates”. To assess the enzymatic activity
of lysozyme, we modified the previous method described by Shugar28. Micrococcus luteus was
grown overnight on Supplemented Tryptic Soy Broth (TSB) under aerobic conditions (200 rpm,
37 °C). The bacteria was centrifuged at 16,000 g 10 °C for 10 min, resuspended in 0.1M
potassium phosphate buffer pH 7.0 to give an OD450nm=2.0 and stored at 4 °C. One ꢁl of each
sample (syn, syn+IQ, syn+lysozyme or syn+lysozyme+IQ) was incubated with 5 ꢁl cells for 1 h
at 25 °C. One micromolar ethidium homodimer (Thermo Fisher) was added and the cells were
analyzed in EVOS Cell Imaging Fluorescence Microscopy under 40 X objective.
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Data Processing
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The error bars represent the S.D. of three independent measurements. For all experiments, the
graphics or images presented are representative of three independent experiments.
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ASSOCIATED CONTENT
Supporting Information
Additional figures S1 to S9. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
palhano@bioqmed.ufrj.br
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AUTHOR CONTRIBUTIONS
FLP and NPG conceived and coordinated the study. LF, NPG, JWK, DF and FLP wrote the
paper. LF, NM and FLP designed, performed and analyzed all experiments. NPG, NM, FSS and
MCM were responsible by the synthesis and characterization of orthoꢀiminoquinone. LR
provided assistance and contributed to the experiments with dopaminergic cells. AVM was
responsible for mass spectrometry experiments and analyses.
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FUNDING SOURCES
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This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq), the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do
Rio de Janeiro (FAPERJ) and the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES).
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGMENT
We thank Satiago Alonso and Ana Lucia O. Carvalho for technical assistance. We also thank Dr
Colleen Fearns for critical reading of the manuscript. We thank Dra Rossolina Zingali for the use
of her laboratory facilities. We thank Dr Marco Antônio Miguel for providing the Micrococcus
luteus strain.
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REFERENCES
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(1) Chiti, F., and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human
disease. Annu. Rev. Biochem. 75, 333–366.
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(2) Eisele, Y. S., Monteiro, C., Fearns, C., Encalada, S. E., Wiseman, R. L., Powers, E. T., and
Kelly, J. W. (2015) Targeting protein aggregation for the treatment of degenerative diseases. Nat
Rev Drug Discov 14, 759–780.
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(3) Sinha, S., Lopes, D. H. J., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P.,
Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P.ꢀY., Ehrmann, M., Benedek, G. B., Loo, J. A.,
Klärner, F.ꢀG., Schrader, T., Wang, C., and Bitan, G. (2011) LysineꢀSpecific Molecular
Tweezers Are BroadꢀSpectrum Inhibitors of Assembly and Toxicity of Amyloid Proteins. J. Am.
Chem. Soc. 133, 16958–16969.
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(4) Lump, E., Castellano, L. M., Meier, C., Seeliger, J., Erwin, N., Sperlich, B., Stürzel, C. M.,
Usmani, S., Hammond, R. M., Einem, von, J., Gerold, G., Kreppel, F., BravoꢀRodriguez, K.,
Pietschmann, T., Holmes, V. M., Palesch, D., Zirafi, O., Weissman, D., Sowislok, A., Wettig, B.,
Heid, C., Kirchhoff, F., Weil, T., Klärner, F.ꢀG., Schrader, T., Bitan, G., SanchezꢀGarcia, E.,
Winter, R., Shorter, J., and Münch, J. (2015) A molecular tweezer antagonizes seminal amyloids
and HIV infection. eLife 4, 10727.
(5) Palhano, F. L., Lee, J., Grimster, N. P., and Kelly, J. W. (2013) Toward the Molecular
Mechanism(s) by Which EGCG Treatment Remodels Mature Amyloid Fibrils. J. Am. Chem.
Soc. 135, 7503–7510.
(6) Ehrnhoefer, D. E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., Lurz, R., Engemann,
S., Pastore, A., and Wanker, E. E. (2008) EGCG redirects amyloidogenic polypeptides into
unstructured, offꢀpathway oligomers. Nat. Struct. Mol. Biol 15, 558–566.
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(7) Ehrnhoefer, D. E., Duennwald, M., Markovic, P., Wacker, J. L., Engemann, S., Roark, M.,
Legleiter, J., Marsh, J. L., Thompson, L. M., Lindquist, S., Muchowski, P. J., and Wanker, E. E.
(2006) Green tea (ꢀ)ꢀepigallocatechinꢀgallate modulates early events in huntingtin misfolding and
reduces toxicity in Huntington's disease models. Hum. Mol. Gen 15, 2743–2751.
(8) Bieschke, J., Russ, J., Friedrich, R. P., Ehrnhoefer, D. E., Wobst, H., Neugebauer, K., and
Wanker, E. E. (2010) EGCG remodels mature ꢀsynuclein and amyloidꢀ fibrils and reduces
cellular toxicity. Proc Natl Acad Sci USA 107, 7710–7715.
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(9) Sinha, S., Lopes, D. H. J., and Bitan, G. (2012) A Key Role for Lysine Residues in Amyloid
βꢀProtein Folding, Assembly, and Toxicity. ACS Chem. Neurosci. 3, 473–481.
(10) Finney, J., Moon, H.ꢀJ., Ronnebaum, T., Lantz, M., and Mure, M. (2014) Archives of
Biochemistry and Biophysics. Arch. Biochem. Biophys. 546, 19–32.
9
10
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12
13
14
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19
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(11) Largeron, M., and Fleury, M.ꢀB. (2012) A Biologically Inspired Cu I/TopaquinoneꢀLike Coꢀ
Catalytic System for the Highly AtomꢀEconomical Aerobic Oxidation of Primary Amines to
Imines. Angew. Chem. Int. Ed. 51, 5409–5412.
(12) Murray, A. N., Palhano, F. L., Bieschke, J., and Kelly, J. W. (2013) Surface adsorption
considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes.
Protein Sci. 22, 1531–1541.
(13) Lam, H. T., Graber, M. C., Gentry, K. A., and Bieschke, J. (2016) Stabilization of αꢀ
Synuclein Fibril Clusters Prevents Fragmentation and Reduces Seeding Activity and Toxicity.
Biochemistry 55, 675–685.
(14) Hokenson, M. J., Uversky, V. N., Goers, J., Yamin, G., Munishkina, L. A., and Fink, A. L.
(2004) Role of individual methionines in the fibrillation of methionineꢀoxidized alphaꢀsynuclein.
Biochemistry 43, 4621–4633.
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(15) 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., and Glabe, C. G. (2007) Fibril
specific, conformation dependent antibodies recognize a generic epitope common to amyloid
fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol Neurodegener. 2, 18.
(16) Greiner, E. R., Kelly, J. W., and Palhano, F. L. (2014) Immunoprecipitation of Amyloid
Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid
Fibrils. PLoS ONE 9, e105433.
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(17) Cao, P., and Raleigh, D. P. (2012) Analysis of the Inhibition and Remodeling of Islet
Amyloid Polypeptide Amyloid Fibers by Flavanols. Biochemistry 51, 2670–2683.
(18) Rodrigues, T. M., JerónimoꢀSantos, A., Outeiro, T. F., Sebastião, A. M., and Diógenes, M.
J. (2014) Challenges and promises in the development of neurotrophic factorꢀbased therapies for
Parkinson's disease. Drugs Aging 31, 239–261.
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(19) St Martin, J. L., Klucken, J., Outeiro, T. F., Nguyen, P., KellerꢀMcGandy, C., Cantutiꢀ
Castelvetri, I., Grammatopoulos, T. N., Standaert, D. G., Hyman, B. T., and McLean, P. J.
(2007) Dopaminergic neuron loss and upꢀregulation of chaperone protein mRNA induced by
targeted overꢀexpression of alphaꢀsynuclein in mouse substantia nigra. J. Neurochem. 100,
1449–1457.
(20) Dutta, P., Ray, N., Roy, S., Dasgupta, A. K., Bouloussa, O., and Sarkar, A. (2011) Covalent
immobilization of active lysozyme on Si/glass surface using alkoxy Fischer carbene complex on
SAM. Org. Biomol. Chem. 9, 5123–5128.
(21) Bulawa, C. E., Connelly, S., Devit, M., Wang, L., Weigel, C., Fleming, J. A., Packman, J.,
Powers, E. T., Wiseman, R. L., Foss, T. R., Wilson, I. A., Kelly, J. W., and Labaudiniere, R.
(2012) Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid
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cascade. Proc Natl Acad Sci USA 109, 9629–9634.
(22) Xu, D., Chiaroni, A., Fleury, M.ꢀB., and Largeron, M. (2006) Electrochemically induced
cascade reaction for the assembly of libraries of biologically relevant 1,4ꢀbenzoxazine
derivatives. J. Org. Chem. 71, 6374–6381.
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(23) Facchinetti, V., Guimarães, F. A., Souza, M. V. N., Gomes, C. R. B., Souza, M. C. B. V.,
Wardell, J. L., Wardell, S. M. S. V., and Vasconcelos, T. R. A. (2015) Synthesis of Novel Ethyl
(substituted)phenyl 4 oxothiazolidin 3 yl) 1 ethyl 4 oxo 1,4 dihydroquinoline 3
Carboxylates as Potential Anticancer Agents. J. Heterocyclic Chem. 52, 1245–1252.
(24) CoelhoꢀCerqueira, E., CarmoꢀGonçalves, P., Pinheiro, A. S., Cortines, J., and Follmer, C.
(2013) αꢀSynuclein as an intrinsically disordered monomerꢀꢀfact or artefact? FEBS J. 280, 4915–
4927.
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(25) Dusa, A., Kaylor, J., Edridge, S., Bodner, N., Hong, D.ꢀP., and Fink, A. L. (2006)
Characterization of Oligomers during αꢀSynuclein Aggregation Using Intrinsic Tryptophan
Fluorescence. Biochemistry 45, 2752–2760.
(26) Wanker, E. E., Scherzinger, E., Heiser, V., Sittler, A., Eickhoff, H., and Lehrach, H. (1999)
Membrane filter assay for detection of amyloidꢀlike polyglutamineꢀcontaining protein
aggregates. Meth. Enzymol. 309, 375–386.
(27) Romão, L. F., Sousa, V. de O., Neto, V. M., and Gomes, F. C. A. (2008) Glutamate
activates GFAP gene promoter from cultured astrocytes through TGFꢀbeta1 pathways. J.
Neurochem. 106, 746–756.
(28) Shugar, D. (1952) The measurement of lysozyme activity and the ultraꢀviolet inactivation of
lysozyme. Biochim Biophys Acta 8, 302–309.
(25) Rodrigues, T. M., JerónimoꢀSantos, A., Outeiro, T. F., Sebastião, A. M., and Diógenes, M.
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J. (2014) Challenges and promises in the development of neurotrophic factorꢀbased therapies for
Parkinson's disease. Drugs Aging 31, 239–261.
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(26) St Martin, J. L., Klucken, J., Outeiro, T. F., Nguyen, P., KellerꢀMcGandy, C., Cantutiꢀ
Castelvetri, I., Grammatopoulos, T. N., Standaert, D. G., Hyman, B. T., and McLean, P. J.
(2007) Dopaminergic neuron loss and upꢀregulation of chaperone protein mRNA induced by
targeted overꢀexpression of alphaꢀsynuclein in mouse substantia nigra. J. Neurochem. 100,
1449–1457.
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(27) Dutta, P., Ray, N., Roy, S., Dasgupta, A. K., Bouloussa, O., and Sarkar, A. (2011) Covalent
immobilization of active lysozyme on Si/glass surface using alkoxy Fischer carbene complex on
SAM. Org. Biomol. Chem. 9, 5123–5128.
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(28) Bulawa, C. E., Connelly, S., Devit, M., Wang, L., Weigel, C., Fleming, J. A., Packman, J.,
Powers, E. T., Wiseman, R. L., Foss, T. R., Wilson, I. A., Kelly, J. W., and Labaudiniere, R.
(2012) Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid
cascade. Proc Natl Acad Sci U S A. 109, 9629–9634.
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FIGURE CAPTIONS
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Figure 1. Catalytic oxidation of free amines from lysines into imines by oꢀiminoquinone (IQ).
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Figure 2. Effect of IQ on aggregation of syn. (A) Primary sequence of syn. (B) A kinetic
experiment of syn (70 ꢁM) aggregation, monitored by ThT fluorescence, was performed in the
absence or in the presence of EGCG or IQ. After 60 h aggregation, the samples were: (C)
fractionated by centrifugation and resolved by SDSꢀPAGE, (D) separated using a filter
retardation assay and the membrane was stained with Ponceau, and (E) analyzed by Circular
dichroism. (F) A monomeric solution of syn (70 ꢁM) was incubated in the absence or presence
of 5% seeds. The seeds were produced by sonication of final product of panel B.
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Figure 3. IQ acts upon syn aggregation through Lys modification and methionine oxidation. (A)
Syn monomers (70 ꢁM) were incubated in the absence or presence of IQ (350 ꢁM) and Lys (50
mM). ThT fluorescence was monitored over time. After aggregation, the morphology of the
samples was analyzed by TEM (B, C and D). After 60 h incubation, the samples were analyzed
by filter retardation assay (E) and mass spectrometry (F, G, H and I).
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Figure 4. IQ remodels mature amyloid fibrils of syn. (A) Mature fibrils of syn (15 ꢁM) were
incubated in the absence or in the presence of IQ (300 ꢁM) or EGCG (300 ꢁM) for 0 or 24 h and
the ThT fluorescence was measured. (B) A kinetic analysis by filter retardation assay of aliquots
of the samples described in panel A. TEM images of samples described in panel A after 24 h
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incubation (C, D and E). (F) Dot blot using the LOC antibody that recognizes a generic amyloid
epitope. Mature syn fibrils were incubated in the absence or in the presence of IQ or EGCG for
24 h then spotted onto nitrocellulose membrane and then probed with LOC or syn antibody. (G)
A monomeric solution of syn (70 ꢁM) was incubated in the absence or presence of 5% seeds.
The seeds were produced by sonication of final product of panel A after 24 h incubation.
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Figure 5. The amyloid remodeling activity of IQ requires free amines. (A) Syn mature amyloid
fibrils were incubated in the presence of pyridoxal 5'ꢀphosphate (VitB6) or acetic aldehyde (AA)
in order to block free amines. Chemically modified fibrils (15 ꢁM) were incubated in the absence
or presence of IQ (300 ꢁM) for 24 h and aggregation assessed using the FR assay stained with
Ponceau. Nonꢀmodified fibrils were used as control. (B and C) Mature fibrils of syn (15 ꢁM)
were incubated alone, in the presence of IQ (300 ꢁM), or in the presence of IQ (300 ꢁM) and lys
(50 mM) for 24 h and aggregation was assessed using (B) the FR assay stained with Ponceau or
(C) ThT fluorescence.
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Figure 6. IQ remodels mature amyloid fibrils of Aβ1ꢀ40. Mature fibrils of Aβ1ꢀ40 (A) (15 ꢁM)
were incubated in the absence or in the presence of IQ (300 ꢁM) or EGCG (300 ꢁM) for 24 h
and remodeling was assessed using (B) the filter retardation assay or (C) ThT fluorescence.
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Figure 7. The amyloid remodeling activity of IQ differs from EGCG. (A) IAPPAc8ꢀ24 mature
amyloid fibrils (15 ꢁM) were incubated in the absence or presence of IQ (300 ꢁM) or EGCG
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(300 ꢁM) for 24 h. An aliquot of these samples was boiled or not in 2% SDS and then applied to
FR membrane and stained with Ponceau (B) or ThT fluorescence was measured (C).
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Figure 8. Toxicity of IQ and EGCG in murine dopaminergic cells. (A) Primary dopaminergic
neurons characterized by the presence of tyrosine hydroxylase and microtubuleꢀassociated
protein 2 (MAP2) were incubated with 100 ꢁM zinc and different concentrations of IQ or EGCG
and after 24 h the viability was measured by MTT assay (B).
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Figure 9. IQ blocks the aggregation of syn without affecting the enzymatic activity of lysozyme.
(A) A kinetic experiment of syn (70 ꢁM) aggregation, monitored by ThT fluorescence, was
performed in the absence or in the presence of 700 ꢁM IQ (1:10). For the competition assay, syn
(70 ꢁM) was incubated in the presence of 52 ꢁM chicken egg albumin or 175 ꢁM eggꢀwhite
lysozyme. After 40 h aggregation, the samples were: (B) fractionated by centrifugation and
resolved by SDSꢀPAGE or (C and D) incubated in the presence of Micrococcus luteus to test for
the antimicrobial activity of lysozyme. After 1 h incubation, the Micrococcus luteus were stained
with ethidium homodimer and analyzed by fluorescence microscopy. Bacteria stained red (C)
were considered dead bacteria (D).
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