1

Neurotoxic, redox-competent Alzheimer’s β-amyloid is released from

lipid membrane by methionine oxidation

Kevin J. Barnham1*, Giuseppe D. Ciccotosto1, Anna K. Tickler2,3, Feda’ E. Ali2, Danielle

G. Smith1, Nicholas A. Williamson1, Yuen-Han Lam2, Darryl Carrington1, Deborah

Tew,1 Gulcan Kocak1, Irene Volitakis1, Frances Separovic2, Colin J. Barrow2, John D.

Wade3, Colin L. Masters1, Robert A. Cherny1, Cyril C. Curtain1,4, Ashley I. Bush1,5,

Roberto Cappai1

1Department of Pathology, The University of Melbourne, and The Mental Health

Research Institute of Victoria, Victoria, 3010, Australia

2School of Chemistry, The University of Melbourne, Victoria, 3010, Australia

3Howard Florey Institute of Medical Research, Victoria, 3010, Australia

4 School of Physics and Materials Engineering, Monash University, Victoria, 3168,

Australia

5Laboratory for Oxidation Biology, Genetics and Aging Research Unit and Department

of Psychiatry, Harvard Medical School, Massachusetts General Hospital East,

Charlestown, MA 02129 USA.

* Corresponding Author. Fax +61 3 99039655

E-mail: kbarnham@unimelb.edu.au

Running Title: Oxidized Aβ is toxic

Keywords: Aβ/amyloid/Alzheimer’s disease/ion channel/copper zinc/oxidation/peroxide

toxicity/membrane penetration/ sulfoxide/catalase.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 18, 2003 as Manuscript M305494200 by guest on June 25, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2

Summary

The amyloid β-peptide is toxic to neurons and it is believed that this toxicity plays a

central role in the progression of Alzheimer’s disease. The mechanism of this toxicity is

contentious. Here we report that an Aβ peptide with the sulfur atom of Met35 oxidized

to a sulfoxide (Met(O)Aβ) is toxic to neuronal cells and this toxicity is attenuated by the

metal chelator clioquinol and completely rescued by catalase implicating the same

toxicity mechanism. However, unlike the unoxidized peptide, Met(O)Aβ is unable to

penetrate lipid membranes to form ion channel-like structures and β-sheet formation is

inhibited, phenomena that are central to some theories for Aβ toxicity. Our results show

that, like the unoxidized peptide, Met(O)Aβ will coordinate Cu2+ and reduce the

oxidation state of the metal, and still produce H2O2. We hypothesize that Met(O)Aβ

production contributes to the elevation of soluble Aβ seen in the brain in AD.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

3

Introduction

Genetic evidence from early onset cases of Alzheimer’s disease indicates that

metabolism of the β-amyloid peptide (Aβ) is clearly linked to the disease (1). The β-

amyloid cascade theory states that excessive deposition of Aβ in the brain is an important

step in the pathogenesis of Alzheimer’s disease (2). While there is a poor correlation

between the density of deposits and the severity of the disease, a correlation is found

between the levels of soluble Aβ and synaptic damage and cognitive impairment (3).

There have been many reports of synthetic Aβ being toxic to neuronal cells (4,5).

However the precise mechanism(s) of action and the nature of the toxic Aβ species

remain to be identified (reviewed in (6)).

AD is associated with extensive oxidative damage to brain tissue (7-10). The

cause of the observed oxidative stress has been debated with a number of possible

mechanisms postulated. These include deficits in calcium regulation, brought about by

either the formation in cell membranes of calcium channels (11) or modulation of an

existing channel (12). An alternative model proposes the direct production of reactive

oxygen species (ROS) by Aβ (9,13). A common factor in most proposed mechanisms of

Aβ toxicity is the oligomerization of Aβ , whether as dimers or trimers (14,15) or larger

aggregates such as protofibrils (16) or the megadalton fully formed amyloid fibril

(17,18).

The generation of ROS usually requires the reaction of O2 with a redox metal ion

such as Cu2+ or Fe3+. When Aβ binds Cu2+ and Fe3+ extensive redox chemical reactions

take place, reducing the oxidation state of either metal and producing H2O2 from O2 in a

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

4

catalytic manner (19-22). Both zinc and copper are released into the synaptic cleft (300

uM for Zn, 30 uM Cu), and markedly elevated levels of Cu (400 µM), Zn (1 mM) and Fe

(1 mM) are found in amyloid deposits in AD-affected brains (23,24). The oxidative

stress observed in AD may be a consequence of the production of ROS by metal-bound

forms of Aβ . The observation that senile plaques isolated from AD brains generated

ROS in a manner dependent upon Cu and Fe (22,25) supports this hypothesis.

The ability of Aβ to coordinate and reduce Cu in vitro must result in the peptide being

oxidized. Indeed, mass spectrometry has shown that Cu complexes were able to

oxygenate Aβ , with the most likely site of oxidation being the sulfur atom of Met35 (26).

Aβ peptides with the sulfur of Met35 oxidized (hereafter referred to as Met(O)Aβ) have

been isolated from AD amyloid brain deposits (27,28). In addition, Dong et al. (29) have

recently shown in a Raman spectroscopic study of senile plaque cores isolated from

diseased brains that much of the Aβ in these deposits contained methionine sulfoxide and

that copper and zinc were coordinated to the histidine residues.

The importance of metal ions to the pathogenesis of AD is highlighted by our

observation that the amount of Aβ ?deposition in a transgenic mouse expressing

amyloidogenic human Aβ was substantially decreased by treating the mice with an orally

available metal chelator (clioquinol) capable of crossing the blood-brain barrier (30). The

significance of this result has been further highlighted by the successful conclusion of a

phase II clinical trial that showed that clioquinol inhibited cognitive decline in moderate

to severe AD patients (31). Recently, the importance of Zn2+ in plaque formation has

been emphasized by the finding that age and female sex related plaque formation in

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

5

APP2576 transgenic mice was greatly reduced upon the genetic ablation of the zinc

transporter 3 protein, which is required for zinc transport into synaptic vesicles (32).

An alternative, but not mutually exclusive, hypothesis to explain Aβ neurotoxicity

is based on Aβ interacting with membranes and/or membrane proteins. Given that the

Aβ peptide contains part of the putative transmembrane domain of the amyloid precursor

protein (APP), it is not surprising that the peptide interacts with cell membranes and

lipoproteins. Numerous reports have described the effects of Aβ on membranes and lipid

systems and their possible roles in Aβ neurotoxicity (33). Our previous studies (34,35)

indicated that the interaction between large unilamellar vesicles (LUVs) and Aβ in the

presence of Cu and Zn resulted in α-helical oligomeric channel-like structures being

formed.

Met35 has been reported to play a key role in the toxicity of Aβ since Aβ peptides

with Met35 replaced by either norleucine or Cys are non-toxic and unable to induce

oxidative stress responses in cells (36). Aβ peptides with an oxidized methionine have

also been reported to be non-toxic (37). We have shown that when Met35 is either

missing, as in Aβ1-28, or sequestered within a lipid environment and, therefore, not

available as a co-factor, there is no metal reduction and hence no conditions for Fenton

chemistry (34). In addition to ameliorating the toxic behavior of Aβ, the oxidation of

Met35 also alters the physical properties of the peptide. Met(O)Aβ is more soluble in

aqueous solution and there is a disruption of the local helical structure when the peptide

is dissolved in sodium dodecyl sulfate micelles (38). The oxidation of Met35 also affects

the aggregation properties of the peptide leading to a different oligomerisation profile

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

6

with the formation of trimers and tetramers significantly attenuated (39) and inhibition of

fibril formation (40).

Given that the Met(O)Aβ peptide is abundant in the brains of AD patients (27-

29), we examined the peptide’s interactions with lipid vesicles and the ability of

Met(O)Aβ to produce hydrogen peroxide and correlated these results with the peptide’s

toxicity in neuronal cell cultures. In this study we show by solid-state NMR that when

Aβ binds and reduces Cu2+ to Cu+ the Met35 residue becomes oxidized. Our results

show that, like the native peptide the oxidized peptide will bind copper and produce H2O2

but the interactions of Met(O)Aβ with lipids are markedly altered. Importantly,

Met(O)Aβ is toxic to neuronal cell cultures, via a toxicity that is rescued by catalase and

the metal chelator clioquinol. This finding has profound implications for the

mechanism(s) of Aβ toxicity.

Experimental Procedures

Materials and Methods

The acidic phospholipid spin label 16NPS was synthesized according to Hubbell and

McConnell (41). The water-soluble spin label TEMPO choline chloride was obtained

from Molecular Probes Inc (Oregon). All spin probes were checked for purity and to

ensure that their number of spins/M were > 90% of theory (42).

Preparation of LUV.

Synthetic palmitoyl phosphatidyl choline (POPC) was purchased from Sigma, and

palmitoyl phosphatidyl serine (POPS) was purchased from Avanti Polar Lipids Inc.

Large unilamelar vesicles (LUV) were prepared by dissolving equal quantities of POPS

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

7

and POPC in chloroform, which was then evaporated off. The lipids were then dissolved

in 10mM phosphate buffer pH 6.8. Some glass beads were added and the mixture was

shaken for 1hr at 37°C. The solution was decanted from the glass beads and subjected to

5 freeze-thaw cycles in liquid nitrogen and a 37o waterbath. The lipids were then

extruded 11 times through a 0.2µm pore filter from Millipore, using an Avanti “mini-

extruder” apparatus. LUV were stored at 4°C and used within 48hrs of preparation as

described by Hope et al. (43). Peptides as a freeze-dried powder were added to the

desired concentration to a suspension of LUV in buffer and the mixture was vortexed

under N2 for 10 min. at 305K in polypropylene tubes. One hundred millimolar stock

solutions of analytical reagent grade CuCl2 were prepared and the desired amount was

added to the LUV after the peptide. For copper EPR measurements 99.99% pure 65Cu

(Cambridge Isotopes) was used. Metal concentrations were measured by ICP-MS and

peptide concentrations were determined by quantitative amino acid analysis. Uptake of

peptide by the LUV was estimated by density gradient centrifugation in Metrizamide™

(Nyegaard, Oslo, Norway)-Tris buffer (pH 7.0, 0.05 M) as described by Cornell et al.

(44) and then measuring the extinction at 210 nm of the sub-phase. The protein

concentration was estimated by using an extinction coefficient, E1cm1%, of 204 at 210 nm

(45). It was found that 10-15% of the added peptide was not taken up by the LUV.

Peptides not synthesized were obtained from the W.M. Keck Laboratory, Yale

University.

Peptide Synthesis

Synthesis of Met(O)Aβ40.

The peptide was prepared by continuous flow Fmoc-solid phase peptide synthesis (SPPS)

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

8

as previously described (46). Following global cleavage and deprotection in a mixture of

trifluoroacetic acid (TFA)/H2O/ethanedithiol (EDT)/triethylsilane (TIS) (94:2.5:2.5:1

v/v, 10 ml), the crude lyophilized Aβ40 was purified by semi-preparative RP-HPLC.

Met(O)Aβ40 was prepared from this by dissolving the crude peptide (21 mg ) in 0.1% aq

TFA solution (10 ml) and 1 ml aliquots were reacted with hydrogen peroxide (250µL) for

10 mins at room temperature (47) after which it these were immediately purified by RP-

HPLC and lyophilized. Analysis by matrix-assisted laser desorption ionisation time of

flight mass spectrometry (MALDI-TOF MS) [MH+ found: 4345, (expected 4345)], and

amino acid analysis showed good correlation with expected values.

Synthesis of Met(O)Aβ42

Met(O)Aβ42 was synthesized as described above with the exception that Met35 was

incorporated as the Nα-Fmoc-Met sulfoxide form. Following cleavage and sidechain

deprotection, the crude peptide was purified by RP-HPLC and characterized by MALDI-

TOF MS [MH+ found: 4530.4; expected 4530.1]. Amino acid analysis also showed good

correlation with expected values.

Peptide Characterization

RP-HPLC was performed on a Waters (Milford, USA) instrument controlled by

Millennium software equipped with PDA detection. The solvent system used throughout

this study was buffer A: 0.1% aq. TFA, buffer B: 0.1% TFA/CH3CN. Semi-preparative

RP-HPLC was performed using a Vydac polymer reversed phase column (10 x 250 mm,

8µm, 300Å) (Hesperia, USA), at a flow rate of 4 ml/min using a 10-60% linear gradient

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

9

of CH3CN over 30 mins. For MALDI-TOF MS analysis, samples were mixed with α-

cyano-4-hydroxycinnamic acid as matrix and examined using a Bruker BIFLEX

instrument (Bremen, Germany) in linear mode at 19.5 kV, or reflector mode at19.5 kV,

20 kV gradient reflector. Quantitative amino acid analysis was performed on Fmoc-

derivatized hydrolysates carried out on a GBC automatic analyzer (Melbourne, Australia)

equipped with an ODS HYPERSIL reversed phase column (Runcorn, UK) and an LC

1250 fluorescence detector.

Synthesis of Boc-13C-Met(O)-OH

2-tertbutylcarbonyloximino-2-phenylacetonitrile (BocON 1.8g, 7.3 mmol) was added to a

stirred solution of 13C labeled glycine (0.5g, 6.7 mmol) (Cambridge Isotope Laboratories,

Inc., Andover, MA) and triethylamine (1.4 ml, 10mmol) in a 1:1 mixture of dioxin and

water (8 ml). After stirring for 2 hours at room temperature water (10 ml) and ethyl

acetate (13 ml) were added and the aqueous phase extracted twice with additional ethyl

acetate. The organic layer was then acidified with 5% citric acid and the precipitate

washed with water and dried. (yield 85%, m.pt. 86.5-87.5oC)

A suspension of 13C labeled methionine (1g, 6.7mmol) (Cambridge Isotope Laboratories,

Inc.) was stirred in water (3.3 ml) and hydrogen peroxide (0.7 ml of 30% solution) added

slowly with cooling. After 1 hour at room temperature absolute ethanol was added and

the crystals filtered and air dried (yield 80%, m.pt. 252-254oC). 13C-Met(O) was then

reacted as above to give Boc-13C-Met(O) in 70% yield.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

10

Synthesis of Aβ39 with 13C labels at Cα of G9, 25, 33 and Cε of M35:

13C Gly9, 25, 33, 13C Met35-Aβ39 was synthesized on solid phase in an automated

manner using a model 430A peptide synthesizer (Applied Biosystems Japan Inc) using a

conventional t-Boc strategy and PAM resin. 13C amino acids were loaded into the

appropriate cartridges and the synthesis was performed using a method previously

described (48).

Solid-State NMR

Solid-State NMR experiments were conducted on a Varian Inova 300

spectrometer (Palo Alto, CA), operating at 75 MHz for 13C. Acquisition and processing

of data was performed using Varian software. The acquisition parameters used for 13C CP

(cross polarization) MAS (magic angle spinning) NMR (49) were: proton π/2 pulse 4 µs,

recycle delay 1 s, contact time 1 ms, sweep width 30 kHz, 4096 transients, and MAS

speed ~7000 Hz and processed using 50 Hz line broadening. Chemical shifts were

referenced to 0 ppm for an external tetramethylsilane sample

Spectra were obtained on 13C labeled Aβ39. To ascertain the effect of the of adding

copper to Aβ peptides the following protocol was used: 5 mg of the reduced form of

Aβ39 was dissolved in a small amount of trifluoroacetic acid (TFA) followed by addition

of 1 ml of H2O and then a small amount of dimethyl sulfoxide. 52 µl (1mmol) of 50 mM

Cu(Gly)2 solution was then added to the Aβ39 peptide and left at room temperature for 3

days. Spectra were acquired on the precipitate that was collected by centrifugation,

washed 3 times with MilliQ water and lyophilized overnight.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

11

EPR spectroscopy

Continuous-wave X-band EPR spectra were obtained using a Bruker ECS106

spectrometer equipped with a temperature controller and flow through cryostat. 65Cu2+

spectra were collected at 110K from samples contained in 4 mm i.d. “Suprasil” quartz

EPR tubes (Wilmad USA). In order to eliminate the possibility that any line broadening

observed in the spectra might be due to freezing-induced localized concentrations of

sample 10% glycerol was added to aqueous peptide buffer solutions. Labeled lipid

samples (25 µl) were contained in 0.8 mm i.d. quartz capillaries (Wilmad) and handled as

described by Gordon and Curtain (42) to ensure reproducibility. Other details, including

the use of the non-membrane penetrant spin label Tempo choline chloride as an internal

reference, have been given in Curtain et al (2001) (34).

CD Spectroscopy

CD spectra of peptides in the LUV were obtained using a CD spectropolarimeter model

62DS (AVIV, Lakewood, New Jersey, U.S.A.). Peptide concentrations were determined

using the molar extinction of the UV absorption from the tyrosine residue. The CD

spectrum was obtained for each solution both neat and after a 1:4 dilution with MilliQ®

water. CD spectra were obtained using a 1 mm-path length quartz cell, acquired at 297 K

in 0.5 nm steps over a 185 to 250 nm wavelength range.

Hydrogen Peroxide (H2O2) Assay H2O2 production by Aβ peptides was measured using a fluorimetric assay. 5 mM of the

fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCF) (Sigma) in Ar-purged DMSO

was deacetylated with 0.025 M NaOH (BDH) for 30 minutes, diluted to 1mM with

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

12

Dulbecco’s phosphate buffered saline (PBS) (Sigma) and neutralised to pH 7.5. Aβ

peptides (100 µM) preincubated with Cu(Gly)22+ (BDH) in a 1/2 molar ratio were

incubated in 96 well plates at 37°C for 1 hour with 5µM dopamine (Sigma), 100 µM

DCF and 100 mM horse radish peroxidase (HRP) (Sigma) in a final volume of 250 µL

with PBS. Known standards of H2O2 were used to quantitate the signal. Fluorescence was

measured in a Wallac Victor2 fluorimeter at 485 nm excitation and 535 nM emission.

The mean and standard deviations of triplicate samples were calculated and compared.

Neuronal cell assay

Aβ42 and Met(O)Aβ42 peptide stocks were prepared by initially weighing out peptide

and determining a theoretical 200 µM concentration by weight, dissolving the peptide in

20 mM NaOH, then rapidly diluting out in water and 10X PBS in a ratio of 2:7:1. All Aβ

peptide solutions were sonicated in a water bath for 5 min, centrifuged for 5 min at high

speed and the supernate used for the toxicity experiments. The final concentration of the

peptides were further quantified according to procedures outlined by Huang et al (1997).

Briefly, the absorbance value at OD214 was measured using a UV spectrophotometer for

the Aβ preparations and the protein concentration determined from BCA standard curves.

This method of Aβ preparation was found to be associated with increased solubility of

the peptides. Clioquinol was purchased from Sigma.

Primary neuronal cultures

Cortical neuronal cultures were prepared as described previously (White et al, 2001) with

some modifications. Briefly, embryonic day 14 BL6Jx129sv mouse cortices were

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

13

removed, dissected free of meninges and dissociated in 0.025% (w/v) trypsin.

Dissociated cells were plated in poly-L-lysine coated sterile 48 well culture plates at a

density of 125,000 cells/cm2 in plating medium (MEM/10% FCS/5% HS). Cultures were

maintained at 37oC in 5% CO2 for 2 h before the plating medium was replaced with

Neurobasal growth medium containing B27 supplements. This method resulted in

cultures highly enriched for neurons. After 5-6 days in culture, the medium was replaced

with fresh Neurobasal medium supplemented with B27 lacking antioxidants for

treatment. For the treatment of neuronal cultures, fresh soluble Aß stock solutions were

diluted to a final concentration of 6 µM in Neurobasal medium. Inhibition of

neurotoxicity was challenged by using a specific hydrogen peroxide antagonist, catalase

(2000 unit/ml) or by using a Cu\Zn chelator, clioquinol (1 µM). The mixtures were then

added to neuronal cultures for 4 days and cell viability was then assayed.

Cell viability assays Cell survival was monitored by phase contrast microscopy and cell viability quantitated

using the MTS assay as described previously. Briefly, the medium was replaced with

fresh Neurobasal medium supplemented with B27 lacking antioxidants and 25 µL MTS

(Promega) was added to each well and incubated for 4 hr in at 37oC in 5% CO2. The

color change of each well was determined by measuring the absorbance at 490 nm using

a Wallac Victor Multireader and background readings of MTS incubated in cell-free

medium were subtracted from each value before calculations. The data was normalized

and calculated as a percentage of untreated vehicle control values.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

14

Immunoprecipitation of Cell culture Media and analysis by MALDI-MS

Cell culture conditioned media were removed after 4 days of treatment and analyzed by

immunoprecipitation for Aβ peptides. For immunoprecipitation, 200 µL of conditioned

media were added to 12.5 µL of prewashed, antibody coated (monoclonal antibody W02,

3 µg of mAb WO2/ 107 beads) magnetic microparticles (DynaBeads M-280, Dynal

Biotech Pty Ltd, Victoria, Australia). Samples were incubated under rotation for 16h at

4C. Beads were washed two times with PBS/0.1% bovine serum albumin. Peptides were

eluted from the dynabeads by the addition of 5 µL of 50% acetonitrile/ 0.1% TFA. 1 µL

was then added to 1µL of matrix (saturated sinapinic acid in 50% acetonitrile/ 0.1% TFA)

and the sample/matrix allowed to dry at room temperature for approximately 30 min. An

Applied Biosystems Voyager-DE STR MALDI-TOF mass spectrometer was used to

determine the mass values of the isolated peptides and compared to the original synthetic

peptide stocks.

Results

Solid-State NMR confirms Aβ plus Cu2+ oxidizes the sulfur of Met35

When Cu2+ was added to Aβ essentially all the peptide precipitates and very little

peptide remains in solution as has previously been reported (50,51). Subsequently, to

confirm that Met35 is being oxidized following Cu treatment a 13C CP MAS spectrum of

the unoxidized form of the Aβ39 peptide was obtained (Fig. 1(a)). The spectrum shows

two C resonances: one from the Cε of Met35 at 16 ppm and another at 44 ppm from the

Cαs Gly9, Gly25 and Gly33 residues. The chemical shift position of 44 ppm is consistent

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

15

with reported values for Cα resonances of glycine in polypeptides obtained under MAS

conditions (52). In Figure 1(b), the 13C CP MAS spectrum of Aβ plus Cu2+ shows a

change in chemical shift of the Met35 Cε peak from 16 ppm to ~40 ppm which is

consistent with the sulfur of Met35 of Aβ39 peptide being oxidized to the sulfoxide.

Oxidation of the sulfur of Met35 alters Aβ lipid interactions

To determine if the interaction between Aβ and Cu in a lipid environment is

altered by Met35 oxidation a series of EPR spectra of 16NPS in POPS/POPC LUV in pH

6.0, 0.05 M phosphate buffer were obtained after adding increasing mole fractions of Aβ

coordinated with 0.3 M Cu2+/M (Fig. 2). All the spectra were recorded at 300K. It can

be seen that the unoxidized, Aβ42 in the presence of Cu2+ gives the motionally-restricted

lipid component (marked with an arrow in Fig. 2(a)) which is characteristic of systems

with a rigid peptide segment inserted into the bilayer. In contrast, the Met(O)Aβ does not

give rise to this phenomena indicating that the peptide has not penetrated into the core of

the lipid bilayer (Fig 2b). However, most of the peptide (~90%) was still bound to the

LUV as determined by density gradient centrifugation in Metrizamide T Tris buffer.

In Fig. 3 the Cu2+ EPR spectra of the Cu2+/Aβ and Cu2+/Met(O)Aβ complexes at

pH 6.0 are compared. The spectra are characteristic of type 2 square planar coordination

of the Cu2+ (53) and are identical, indicating that the oxidation of Met35 does not alter

the copper coordination site of Aβ . Both samples were allowed to stand at 295K for a

series of time intervals (0-24 hr) then taken down to 110K and their spectra recorded. The

amount of Cu2+ visible in the spectra was determined by double integration and plotted in

Figure 4. This shows that the reduction of Cu2+ to the EPR silent Cu+ is much slower

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

16

with the oxidized peptide, confirming earlier observations (34) that the Met35 residue in

Aβ plays an important part in the redox behavior of the peptide in solution.

CD spectroscopy was used to determine if the oxidized Met35 altered the

secondary structure of the peptide in a lipid environment (Fig. 5). The Met(O)Aβ in a

LUV environment is random coil and there is no conformational change when Cu2+ is

added. This is consistent with the metal failing to promote peptide insertion into the

membrane. In contrast, the unoxidized peptide showed a β-sheet structure on the surface

that converted to an α-helical structure upon addition of Cu2+ or Zn2+, as previously

reported (34).

Hydrogen Peroxide generation by Met(O)Aβ plus Cu2+

The ability of Aβ peptides incubated with Cu2+ to generate H2O2 has been

previously reported (20,22,54). The Aβ and Met(O)Aβ peptides were tested for their

ability to generate H2O2 in the presence of Cu2+ and a reducing substrate dopamine using

a fluorimetric assay with a DCF probe. The results shown in Figure 6 indicate that

Met(O)Aβ produced a similar amount of H2O2 as the Aβ . Therefore, Met35 is not

required for Aβ mediated H2O2 production. As a negative control the rat Aβ was also

assayed for its ability to generate H2O2, as has been previously reported (20), H2O2

production is inhibited.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

17

Neuronal cell assay

The neurotoxicity of the Met(O)Aβ peptide was tested to determine if, like the

unoxidised peptide, this peptide is toxic to neuronal cells. The results depicted in Fig. 7

show that Met(O)Aβ had a similar degree of toxicity on neuronal cultures to that of the

unoxidised Aβ . Importantly, the toxicity of Met(O)Aβ and Aβ was completely rescued

by the selective H2O2 scavenging enzyme, catalase (109 ± 6% and 95 ± 4% respectively,

p<0.05). Hence, the toxicity of Aβ and Met(O)Aβ could be attributed to catalytic H2O2

production. In addition, the metal chelator clioquinol is able to attenuate the toxicity of

both unoxidized Aβ and Met(O)Aβ . A result that highlights the importance metal ions

play in the production of the H2O2, as has been previously observed (20,22).

To confirm that the oxidized methionine was not reduced by the culturing

conditions, conditioned media was collected at the end of 4 days of treatment and

subjected to immunoprecipitation and the eluted peptide analyzed by mass spectrometry.

The spectra showed that the peptide mass profile of Met(O)Aβ was unchanged following

the treatment of the cells (data not shown).

Discussion

Oxidative damage has been identified as a possible cause of the

neurodegeneration associated with Alzheimer’s disease. Aβ can react with Cu2+ to

generate H2O2 with a reduction of the metal to Cu+ (19,22). Our solid-state NMR

experiments (Fig. 1) show that reduction of the metal is coupled with the oxidation of

Met35 to give the sulfoxide. One consequence of the redox chemical reactions associated

with oxidative damage is the oxidative modification of proteins with methionine, one of

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

18

the most readily oxidized amino acids. Of the various forms of Aβ peptide species

isolated from AD brains, peptides with Met(O) constitute a major component estimated

to comprise between 10-50% of total brain Aβ (27-29).

Oxidation of methionine residues, which converts a hydrophobic residue into a

more hydrophilic one, can markedly alter the properties of a protein. For example,

conformational transitions from a α-helix to β-sheet structure can occur (55). In the case

of Aβ , the oxidation of Met35 has been shown to alter the aggregation profile of the

peptide such that the formation of Aβ trimers and tetramers is attenuated (39) and fibril

formation inhibited (40). Our results are consistent with Met(O)Aβ markedly altering the

structural properties of Aβ . Met(O)Aβ is predominately random coil in the presence of a

lipid bilayer, whereas the unoxidised peptide adopts a β-sheet structure in this

environment (34,35). Moreover, the addition of Cu2+ or Zn2+ to Aβ will drive the

insertion of the unoxidized peptide into the membrane as an oligomeric complex that

adopts a channel-like α-helical structure in doing so. However, the addition of metal ions

to Met(O)Aβ does not result in such a structural transition and α-helical channel-like

structures are not observed (Fig. 2). Significantly, the oxidation of the sulfur of Met35

does not change the metal binding properties of the peptide (Fig. 3). This is consistent

with the structural transitions of Aβ being associated with the hydrophobic C-terminus

that also promotes higher-order oligomerisation. The inhibition of β-sheet formation on

the surface of the LUV following oxidation of Met35 suggests that these β-sheet

structures are the result of sequestering the Met residue into a hydrophobic environment.

Met(O), with a more hydrophilic character is not suited to this hydrophobic environment

and the formation of higher-order β-sheet oligomers is inhibited. Similarly, oxidation

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

19

inhibits the metal-mediated transition of Aβ to α-helical oligomeric channel-like

structures despite not changing the metal binding site (Fig. 5). This is probably due to the

more hydrophilic sulfoxide interfering with the hydrophobic interactions at either the

peptide/peptide interface or at the interface between the peptide and the hydrocarbon lipid

chains at the core of the lipid bilayer.

A critical role for Met35 in the pathogenic activity of Aβ has been suggested with

this residue being reported to be necessary for promoting neurotoxicity, aggregation and

the generation of ROS (36,37,56,57). However, given that metal binding occurs near the

N-terminus of Aβ, one would not expect oxidation of Met35 to have a large effect on the

metal binding ability. This is supported by the recent results of Dong et al. (29) who

used Raman spectroscopy to show the presence of Cu bound to the histidine residues of

Met(O)Aβ in diseased human tissue. This was confirmed by our EPR spectra (Fig. 3)

showing that the coordination sphere about the Cu was unaffected by Met(O). When Aβ

binds Cu2+ and reduces it to Cu+, Met35 is oxidized to the sulfoxide (Fig. 1), so it was

surprising that there was still significant reduction of the Cu, albeit slower, when Cu2+

was added to Met(O)Aβ (Fig. 4). Interestingly when Huang et al. (20) originally

reported that Aβ reduces Cu, they observed a fast (almost instantaneous) followed by a

slow phase over 24 hrs for the reduction of Cu2+ to Cu+. Our results suggest that the fast

stage occurs when Met35 is initially oxidized to Met(O)35 with the subsequent slow step

is due to the further slow oxidation of the sulfoxide to a sulfone. The further oxidation of

the sulfoxide to a sulfone highlights the strong oxidizing potential of the Aβ/Cu complex.

It has been previously reported (19) that the reduction of Cu2+ to Cu+ in the presence of

Aβ has a formal reduction potential that is highly positive (approximate to +550 mV

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

20

versus Ag/AgCl) suggesting that the Cu+/Aβ complex is very stable. Therefore, the

stability of this Cu+ adduct could drive the further oxidation of the Met sulfoxide to a

sulfone. This may also explain why Met(O)Aβ is able to generate as much peroxide as

the reduced form of the peptide in the presence of Cu2+ (Fig. 6). In the presence of an

exogenous reductant, such as dopamine, the Met(O)Aβ will readily reduce Cu2+ to Cu+

and produce H2O2. Our results demonstrate that, like the reduced peptide, Met(O)Aβ is

able to produce large quantities of H2O2 in the presence of a reducing substrate which are

highly abundant in brain. The strong oxidizing potential of Aβ/Cu2+ coupled with the

high affinity of Aβ for copper (51) and the rapid and complete reduction of Cu2+ to Cu+

by Aβ (34) explains why the reduced form of the peptide was not observed (Fig. 1) after

this reaction.

In biological systems it would be expected that Met(O)Aβ would still bind and

reduce Cu and therefore have the capability to produce H2O2, resulting in conditions that

favor Fenton chemistry and subsequent hydroxyl radical formation. This is supported by

Met(O)Aβ being toxic to neuronal cells and this toxicity being rescued by catalase, and

attenuated by the metal chelator clioquinol (Fig. 7). Importantly, Cu bound Met(O)Aβ

exists in AD brains (29).

The ability of clioquinol to inhibit the toxicity of both Aβ and Met(O)Aβ is highly

significant in the light of recent findings showing that clioquinol is capable of markedly

inhibiting Aβ ?deposition in a transgenic mouse expressing amyloidogenic human Aβ

with a corresponding increase in general health and body weight parameters (30).

Furthermore, in a small phase II human clinical trial clioquinol was able to inhibit

cognitive decline in moderate to severe AD patients (31).

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

21

It has previously been reported that Met(O)Aβ is non-toxic to cells after 24 hrs

(37). In contrast we show that a longer incubation period of 96 hrs results in a significant

level of toxicity similar to unoxidised Aβ . Likewise, it was reported that Met(O)Aβ was

unable to reduce Cu2+ to Cu+ but again the assay only had a one hour incubation period

(37). We found that at one hour < 10% of the available Cu2+ was reduced in agreement

with these findings. However our data (Fig. 4) shows that reduction of Cu2+ to Cu+ still

does occur albeit more slowly. In our hands Met(O)Aβ was able to generate 2 µM H2O2

(Fig. 6). This result is consistent with the reported EPR spin trapping data by

Varadarajan et al. (37) and suggests Met(O)Aβ was able to generate radicals, which

would explain the damage to mitochondrial function that they also observed. The

reduction in mitochondrial function is recognized as an early step in the apoptotic

pathway and suggests that the toxicity of Met(O)Aβ observed by us would also have been

observed by Varadarajan et al. (37) if they had used a longer incubation period.

Our results suggest that both Aβ and Met(O)Aβ toxicity occurs via the same

mechanism of metal-dependent, H2O2 formation. Among the proposed mechanisms of

Aβ toxicity are fibril formation, ion channel formation, membrane depolarization, and

specific receptor mediated events. Many of these mechanisms are dependent on Aβ

aggregation. It should be noted that there are two methods of inducing aggregation of

Aβ , metal induced cross-linking which gives rise to amorphous aggregates and fibril

formation, usually induced by aging the Aβ solutions or lowering the pH (58). Oxidation

of the methionine inhibits fibril aggregation and not the metal-mediated aggregation. The

results presented here and elsewhere argue against fibrils as a toxic species as the

oxidation of the sulfur of methionine will modify the Aβ aggregation profile and inhibit

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

22

fibril formation. These changes in peptide structure also alter the peptide interactions

with membranes and inhibit membrane penetration. Yet these changes do not affect the

toxicity of the peptide.

The synaptic damage and cognitive impairment associated with AD correlates

with the soluble forms of Aβ, not the deposited forms (3). Intriguingly, Met(O)Aβ has

been found in AD brains in high concentrations, this peptide is highly soluble, is less

likely to be lipid associated and is toxic. Furthermore, there is a decrease in the enzyme

methionine sulfoxide reductase, that protects against oxidative modifications to

methionine residues, in AD brains (59) leading to the possibility of elevated Met(O)Aβ

generation. The release of Met(O)Aβ from membranes could allow the peptide to drift

into the synaptic cavity where it could coordinate synaptic Zn2+ and Cu2+ released upon

neurotransmission. This could promote the toxicity of the peptide as well as its

precipitation into amyloid, an event thought to commence at the synapse (60).

Acknowledgements

Supported in part by grants from the NH&MRC, NIA and Alzheimer’s Association (to

AIB), and Prana Biotechnology.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

23

References 1. Hardy, J. (1997) Trends Neurosci. 20, 154-159 2. Selkoe, D. J. (2001) Physiol Rev 81, 741-766 3. McLean, C. A., Cherny, R. A., Fraser, F. W., Fuller, S. J., Smith, M. J.,

Beyreuther, K., Bush, A. I., and Masters, C. L. (1999) Ann. Neurol. 46, 860-866 4. Yankner, B. A., Duffy, L. K., and Kirschner, D. A. (1990) Science 250, 279-282 5. Harkany, T., Hortobagyi, T., Sasvari, M., Konya, C., Penke, B., Luiten, P. G., and

Nyakas, C. (1999) Prog. Neuropsychopharmacol. Biol. Psychiatry 23, 963-1008 6. Harkany T, Abraham I, Konya C, Nyakas C, Zarandi M, Penke B, and P.G.M, L.

(2000) Rev Neurosci 11, 329-382 7. Martins, R. N., Harper, C. G., Stokes, G. B., and Masters, C. L. (1986) J.

Neurochem. 46, 1042-1045 8. Multhaup, G., Masters, C. L., and Beyreuther, K. (2000) Alzheimer's Rep 1, 147-

154 9. Behl, C., Davis, J. B., Lesley, R., and Schubert, D. (1994) Cell 77, 817-827 10. Butterfield, D. A., Drake, J., Pocernich, C., and Castegna, A. (2001) Trends Mol

Med 7, 548-554. 11. Arispe, N., Rojas, E., and Pollard, H. B. (1993) Proc Natl Acad Sci U S A 90,

567-571. 12. Mattson, M. P., Tomaselli, K. J., and Rydel, R. E. (1993) Brain Res 621, 35-49. 13. Bush, A. I. (2000) Curr. Opin. Chem. Biol. 4, 184-191 14. Roher, A. E., Chaney, M. O., Kuo, Y. M., Webster, S. D., Stine, W. B.,

Haverkamp, L. J., Woods, A. S., Cotter, R. J., Tuohy, J. M., Krafft, G. A., Bonnell, B. S., and Emmerling, M. R. (1996) J Biol Chem 271, 20631-20635

15. Walsh, D. W., Klyubin, I., Fadeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Nature 416, 535-539

16. Harper, J. D., Wong, S. S., Lieber, C. M., and Lansbury, P. T. (1999) Biochemistry 38, 8972-8980

17. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) J. Neurosci. 13, 1676-1687

18. Lorenzo, A., and Yankner, B. A. (1994) Proc Natl Acad Sci U S A 91, 12243-12247.

19. Huang, X., Cuajungco, M. P., Atwood, C. S., Hartshorn, M. A., Tyndall, J. D., Hanson, G. R., Stokes, K. C., Leopold, M., Multhaup, G., Goldstein, L. E., Scarpa, R. C., Saunders, A. J., Lim, J., Moir, R. D., Glabe, C., Bowden, E. F., Masters, C. L., Fairlie, D. P., Tanzi, R. E., and Bush, A. I. (1999) J. Biol. Chem. 274, 37111-37116

20. Huang, X., Atwood, C. S., Hartshorn, M. A., Multhaup, G., Goldstein, L. E., Scarpa, R. C., Cuajungco, M. P., Gray, D. N., Lim, J., Moir, R. D., Tanzi, R. E., and Bush, A. I. (1999) Biochemistry 38, 7609-7616

21. Cuajungco, M. P., Goldstein, L. E., Nunomura, A., Smith, M. A., Lim, J. T., Atwood, C. S., Huang, X., Farrag, Y. W., Perry, G., and Bush, A. I. (2000) J. Biol. Chem. 275, 19439-19442

22. Opazo, C., Huang, X., Cherny, R. A., Moir, R. D., Roher, A. E., White, A. R., Cappai, R., Masters, C. L., Tanzi, R. E., Inestrosa, N. C., and Bush, A. I. (2002) J Biol Chem 277, 40302-40308

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

24

23. Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., and Markesbery, W. R. (1998) J. Neurol. Sci. 158, 47-52

24. Smith, M. A., Harris, P. L., Sayre, L. M., and Perry, G. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9866-9868

25. Sayre, L. M., Perry, G., Harris, P. L., Liu, Y., Schubert, K. A., and Smith, M. A. (2000) J. Neurochem. 74, 270-279

26. Nishino, S., and Nishida, Y. (2001) Inorg. Chem. Commun. 4, 86-89 27. Naslund, J., Schierhorn, A., Hellman, U., Lannfelt, L., Roses, A. D., Tjernberg, L.

O., Silberring, J., Gandy, S. E., Winblad, B., Greengard, P., and et al. (1994) Proc Natl Acad Sci U S A 91, 8378-8382.

28. Kuo, Y. M., Kokjohn, T. A., Beach, T. G., Sue, L. I., Brune, D., Lopez, J. C., Kalback, W. M., Abramowski, D., Sturchler-Pierrat, C., Staufenbiel, M., and Roher, A. E. (2001) J Biol Chem 276, 12991-12998.

29. Dong, J., Atwood, C. S., Anderson, V. E., Siedlak, S. L., Smith, M. A., Perry, G., and Carey, P. R. (2003) Biochemistry 42, 2768-2773

30. Cherny, R. A., Atwood, C. S., Xilinas, M. E., Gray, D. N., Jones, W. D., McLean, C. A., Barnham, K. J., Volitakis, I., Fraser, F. W., Kim, Y., Huang, X., Goldstein, L. E., Moir, R. D., Lim, J. T., Beyreuther, K., Zheng, H., Tanzi, R. E., Masters, C. L., and Bush, A. I. (2001) Neuron 30, 665-676.

31. Ritchie, C. W., Bush, A. I., Mackinnon, A., Macfarlane, S., Mastwyk, M., MacGregor, L., Kiers, L., Cherny, R. A., Li, Q.-X., Tammer, A., Carrington, D., Mavros, C., Volitakis, I., Xilinas, M., Ames, D., Davis, S., Beyreuther, K., Tanzi, R. E., and Masters, C. L. (2003) Arch. Neurol., in press

32. Lee, J. Y., Cole, T. B., Palmiter, R. D., Suh, S. W., and Koh, J. Y. (2002) Proc Natl Acad Sci U S A 99, 7705-7710.

33. Kourie, J. I. (2001) Cell Mol Neurobiol 21, 173-213. 34. Curtain, C. C., Ali, F., Volitakis, I., Cherny, R. A., Norton, R. S., Beyreuther, K.,

Barrow, C. J., Masters, C. L., Bush, A. I., and Barnham, K. J. (2001) J Biol Chem 276, 20466-20473.

35. Curtain, C. C., Ali, F. E., Smith, D. G., Bush, A. I., Masters, C. L., and Barnham, K. J. (2003) J Biol Chem 278, 2977-2982

36. Varadarajan, S., Yatin, S., Kanski, J., Jahanshahi, F., and Butterfield, D. A. (1999) Brain Res Bull 50, 133-141.

37. Varadarajan, S., Kanski, J., Aksenova, M., Lauderback, C., and Butterfield, D. A. (2001) J Am Chem Soc 123, 5625-5631.

38. Watson, A. A., Fairlie, D. P., and Craik, D. J. (1998) Biochemistry 37, 12700-12706.

39. Palmblad, M., Westlind-Danielsson, A., and Bergquist, J. (2002) J Biol Chem 277, 19506-19510.

40. Hou, L., Kang, I., Marchant, R. E., and Zagorski, M. G. (2002) J Biol Chem 277, 40173-40176

41. Hubbell, W. L., and McConnell, H. M. (1971) J. Am. Chem. Soc. 93, 314-326 42. Gordon, L. M., and Curtain, C. C. (1988) Electron spin resonance analysis of

model and biological membranes. Advances in Membrane Fluidity 1: Methods for Studying Membrane Fluidity. (Aloia, R. C., Curtain, C. C., and Gordon, L. M., Eds.), 1, Alan R. Liss, New York

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

25

43. Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Biochim. Biophys. Acta 812, 55-56

44. Cornell, B., Separovic, F., Baldassi, A., and Smith, R. (1988) Biophys. J. 53, 67-76

45. Tombs, M. P., Souter, F., and Maclagan, N. F. (1959) Biochem. J 73, 167-171 46. Tickler, A. K., Barrow, C. J., and Wade, J. D. (2001) J Pept. Sci. 7, 488-494. 47. Atherton, E., Sheppard, R. C., and Wade, J. D. (1983) J. Chem. Soc. Chem

Commun., 1060-1062 48. Barrow, C. J., Yasuda, A., Kenny, P. T., and Zagorski, M. G. (1992) J Mol Biol

225, 1075-1093. 49. Pines, A., Gibby, M. G., and Waugh, J. S. (1973) J. Chem. Phys. 59, 569-590 50. Atwood, C. S., Moir, R. D., Huang, X., Scarpa, R. C., Bacarra, N. M., Romano,

D. M., Hartshorn, M. A., Tanzi, R. E., and Bush, A. I. (1998) J. Biol. Chem. 273, 12817-12826

51. Atwood, C. S., Scarpa, R. C., Huang, X., Moir, R. D., Jones, W. D., Fairlie, D. P., Tanzi, R. E., and Bush, A. I. (2000) J. Neurochem. 75, 1219-1233

52. Saito, H. (1986) Magn. Res. Chem. 24, 835-852 53. Boas, J. F., Pilbrow, J. R., and Smith, T. D. (1978) Biological Magnetic

Resonance. Biological Magnetic Resonance (Berliner, L., and Reuben, J., Eds.), 1, Plenum Press, New York

54. Tabner, B. J., Turnbull, S., El-Agnaf, O. M., and Allsop, D. (2002) Free Radic Biol Med 32, 1076-1083

55. Dado, G. P., and Gellman, S. H. (1993) J. Am. Chem. Soc. 115, 12609-12610. 56. Schoneich, C. (2002) Arch Biochem Biophys 397, 370-376. 57. Yatin, S. M., Varadarajan, S., Link, C. D., and Butterfield, D. A. (1999)

Neurobiol. Aging 20, 325-330; discussion 339-342 58. Yoshiike, Y., Tanemura, K., Murayama, O., Akagi, T., Murayama, M., Sato, S.,

Sun, X., Tanaka, N., and Takashima, A. (2001) J Biol Chem 276, 32293-32299. 59. Gabbita, S. P., Aksenov, M. Y., Lovell, M. A., and Markesbery, W. R. (1999) J

Neurochem 73, 1660-1666 60. Terry, R. D. (1996) J Neuropathol Exp Neurol 55, 1023-1025

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

26

Figure Captions

Figure 1. 13C CP MAS spectrum of (a) reduced form of Aβ39, with the Cα of Gly9,

Gly25, Gly33 and Cε of Met35 13C labeled and (b) oxidized form of Aβ1-39 following the

addition of one equivalent of Cu(Gly)22+.

Figure 2. (a) EPR spectra of 16NPS in POPS/POPC LUV. Control spectrum is of vesicles

without peptide added. Numbers at right hand end of other spectra indicate mole fraction

of Aβ , unoxidized methionine, added/mole lipid. A 0.3 M Cu2+/M was added to the

peptide immediately before it was added to the LUV. All samples were adjusted to pH

6.0. Spectra were recorded at 300K. Instrument conditions were; microwave frequency

9.7679 GHz, modulation frequency 100 KHz, modulation amplitude 3.2 Gauss,

microwave power 6 mW, sweep time 400 sec, and time constant 0.5 sec. Scan width was

100 Gauss, mid-field 3,300 Gauss. The arrow marks the motionally-restricted lipid

component. (b) shows spectra taken under identical conditions except that Met(O)Aβ was

added to the system.

Figure 3. EPR spectra taken at 110K of (a), Cu2+- Aβ at pH 6.0 and (b) Met(O)Aβ at the

same pH. (Cu2+/peptide 0.3 mole/1 mole). Instrument settings were; microwave

frequency 9.7660 GHz, modulation frequency 100 KHz, modulation amplitude 1.0

Gauss, microwave power 2 mW, sweep time 83.886 s, time constant 10.240 ms. Spectra

were averaged over 16 sweeps.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

27

Figure 4. Time course of reduction of Cu2+. Proportion of Cu2+ was obtained by double-

integrating the EPR spectra of the peptide- Cu2+ complexes that had been recorded after

the samples had been held at 295K. (a) Αβ l , (b) Met(O)Aβ n

Figure 5. CD spectra of Met(O)Aβ42 (a) in the presence of negatively charged LUV.

The spectrum is indicative of a peptide undergoing conformational exchange; unlike the

reduced peptide that adopts a β-sheet structure under similar conditions (34). Following

addition of Cu2+(b), there is no change in the secondary structure of the peptide. Spectra

were recorded at 298K, pH 6.9.

Figure 6. Plot of H2O2 production by Aβ peptides (100 µM) in the presence two

equivalents of Cu2+ and the dopamine substrate, as determined by DCF assay. Met(O)Aβ

produces similar quantities of H2O2 in the presence of Cu2+ as the reduced peptide. H2O2

standards were used to quantitate the signal. By way of comparison the H2O2 production

by rat Aβ peptide is included as a negative control.

Figure 7. Aβ and Met(O)Aβ toxicity in primary cell culture. Primary cortical neurons

were grown at low density (125,000 cells/cm2) for 5 days and exposed to 6µM Aβ for an

additional 4 days. No differences in cell viability (measured by MTS assay) was

observed between Aβ or Met(O)Aβ (filled bars) following treatment of the cultures. The

addition of 1 µM Clioquinol (open bars) or 2000 U/ml catalase (grey bars) inhibited the

toxicity of both Aβ42 and Met(O)Aβ42; *, P<0.001; versus Vehicle; #, P<0.05; versus

peptide alone; ##, P<0.001; versus peptide alone. At least 3 samples were done per

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

28

group. Each sample was done in triplicate.

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

29

Figure 1

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

30

Figure2

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

31

Figure 3

2250 2750 3250

Gauss

(a)

(b)

Figure 4

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

32

Figure 5

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

33

Figure 6

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

34

Figure 7

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from

Masters, Robert A. Cherny, Cyril C. Curtain, Ashley I. Bush and Roberto CappaiKocak, Irene Volitakis, Frances Separovic, Colin J. Barrow, John D. Wade, Colin L.

Smith, Nicholas A. Williamson, Yuen-Han Lam, Darryl Carrington, Deborah Tew, Gulcan Kevin J. Barnham, Giuseppe D. Ciccotosto, Anna K. Tickler, Feda’ E. Ali, Danielle G.

by methionine oxidationNeurotoxic, redox-competent Alzheimer's b-amyloid is released from lipid membrane

published online August 18, 2003J. Biol. Chem. 

  10.1074/jbc.M305494200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on June 25, 2018http://w

ww

.jbc.org/D

ownloaded from