promotion of amyloid β protein misfolding and fibrillogenesis by a lipid oxidation product

doi:10.1016/j.jmb.2008.01.057 J. Mol. Biol. (2008) 377, 1236–1250

Available online at www.sciencedirect.com

Promotion of Amyloid β Protein Misfolding andFibrillogenesis by a Lipid Oxidation Product

Liu Liu, Hiroaki Komatsu, Ian V. J. Murray and Paul H. Axelsen⁎

Department of Pharmacology,University of Pennsylvania,Philadelphia, PA 19104, USA

Department of Biochemistryand Biophysics, University ofPennsylvania, Philadelphia,PA 19104, USA

Department of Medicine,University of Pennsylvania,Philadelphia, PA 19104, USA

Received 18 December 2007;received in revised form 13January 2008; accepted 21January 2008Available online30 January 2008

*Corresponding author. E-mail [email protected] address: I. V. J. Murray, D

Neuroscience and Experimental MeHealth Science Center, College StatiAbbreviations used: Aβ, amyloid

Alzheimer's disease; Aβ40, the 40-reAβ42, the 42-residue form of Aβ; amindicates a spectrum collected in D2Ononenal; HHE, 4-hydroxy-2-hexenaspectroscopy; PATIR-FTIR, polarizedinternal reflection IR spectroscopy; Ddimyristoyl-sn-glycero-3-phosphochRed; HFIP, hexafluoro-2-propanol; Pcross-linking of unmodified protein

0022-2836/$ - see front matter © 2008 E

Oxidatively damaged lipid membranes are known to promote theaggregation of amyloid β proteins and fibril formation. Oxidative damagetypically produces 4-hydroxy-2-nonenal when lipid membranes containω-6polyunsaturated fatty acyl chains, and this compound is known to modifythe three His residues in Aβ proteins by Michael addition. In this report, theability of 4-hydroxy-2-nonenal to reproduce the previously observedamyloidogenic effects of oxidative lipid damage on amyloid β proteins isdemonstrated and the mechanism by which it exerts these effects isexamined. Results indicate that 4-hydroxy-2-nonenal modifies the three Hisresidues in amyloid beta proteins, which increases their membrane affinityand causes them to adopt a conformation on membranes that is similar totheir conformation in a mature amyloid fibril. As a consequence, fibrilformation is accelerated at relatively low protein concentrations, and theability to seed the formation of fibrils by unmodified amyloid beta proteinsis enhanced. These in vitro findings linking oxidative stress to amyloid fibrilformation may be significant to the in vivo mechanism by which oxidativestress is linked to the formation of amyloid plaques in Alzheimer's disease.

© 2008 Elsevier Ltd. All rights reserved.

Keywords: Alzheimer's disease; internal reflection infrared spectroscopy;mass spectrometry; surface plasmon resonance; hydroxynonenal
Edited by J. Bowie
Introduction

Amyloid β (Aβ) proteins form fibrils and accu-mulate as dense “senile plaques” in the cortical braintissues of patients with Alzheimer's disease (AD).The 40-residue form of Aβ (Aβ40) along with itsvarious amino-terminal derivatives appears to pre-dominate in fully developed plaques.1 The 42-

ess:

epartment ofdicine, Texas A&Mon, TX 77840, USA.β proteins; AD,sidue form of Aβ;ide I′, the prime; HNE, 4-hydroxy-2-

l; IR, infraredattenuated totalMPC, 1,2-

oline; CR, CongoICUP, photoinduceds.

lsevier Ltd. All rights reserve

residue form (Aβ42), on the other hand, appears topredominate in early-stage diffuse plaques.2 There-fore, it has been proposed that Aβ42 serves tonucleate amyloid plaque formation.3–6 Neverthe-less, the fundamental reason amyloid plaques formin patients with sporadic AD remains elusive.Oxidative stress has been frequently implicated in

the pathogenesis of AD,7–16 although not directly inthe formation of amyloid plaques. Polyunsaturatedfatty acyl chains are abundant in lipid membranes ofthe brain17 and they are highly vulnerable tooxidative stress. When considering which com-pounds are likely to arise during lipid oxidation,interact with Aβ proteins, explain the effects ofoxidatively damaged membranes, and have a role inthe pathogenesis of AD, 4-hydroxy-2-nonenal (HNE)is an obvious candidate. HNE is derived from ω-6fatty acyl chains via nonenzymatic degradationpathways from lipid hydroperoxide intermediates.18

It is a highly reactive compound that tends to formMichael adducts with the His side chains in aprotein.19–26 HNE concentrations ranging from 8 to20μMhave been reported in normal humanplasma27

and cerebral ventricular fluid,28 along with evidenceof increased levels of HNE in AD.28–30 Spontaneous

d.

mailto:[email protected]

1237Hydroxynonenal and Membrane-Mediated Amyloidogenesis

formation of HNE–Aβ adducts24,31 and immunohis-tochemical evidence of HNE–His adducts in amyloidplaques32 have both been reported.In addition to HNE, lipid hydroperoxides also

degrade into isoprostanes—chemically stable eico-sanoids frequently used as biomarkers of oxidativestress33–35 The isoprostane 8,12-iso-iPF2α-VI appearsto be of value as a biomarker for AD, since levels inthe spinal fluid correlate to the severity of ADsymptoms,36 an increase in their production pre-cedes plaque formation in AD,11,37 and they appearto exhibit some specificity for AD over other types ofdementia.38 Because isoprostanes and HNE have acommon origin, these associations between isopros-tanes and AD also implicate HNE.A remarkable relationship between oxidatively

damaged lipids and amyloid fibril formation wasobserved several years ago in this laboratory usingpolarized attenuated total internal reflection Four-ier-transform infrared (PATIR-FTIR) spectroscopy.The initial observation was that oxidativelydamaged lipid membranes promoted Aβ aggrega-tion and fibrillogenesis.39 The fibrils that formed hada long axis oriented parallel with the membranesurface, and they are situated on the membranewithout penetrating the surface or laterally displa-cing membrane lipids. Subsequent studies demon-strated that oxidatively damaged membranesincreased the ability of Aβ42 to seed the formationof fibrils by Aβ40.40 The effect of oxidative damageon fibril formation was verified using lipid vesiclesand a Congo Red dye binding assay, and theformation of true amyloid fibrils under theseconditions was verified by electron microscopy.The membranes in these two original investiga-

tions contained arachidonoyl acyl chains that hadbeen subjected to copper-mediated oxidativedamage. This type of damage is known toproduce arachidonoyl hydroperoxides, HNE, andisoprostanes.41 Aggregated Aβ proteins accelerateoxidative lipid damage and increase the produc-tion of HNE from phospholipids containingarachidonoyl fatty acyl chains.42 The HNE thatis produced has been shown by mass spectro-metry sequencing to form covalent adducts withthe three His side chains of Aβ proteins (residues6, 13, and 14) and by PATIR-FTIR spectroscopy tocause fibril formation at exceptionally low con-centrations of protein.31 Therefore, all componentsof a chemical amplification mechanism have beenobserved in vitro in which aggregated Aβ proteinspromote oxidative damage and the products oflipid oxidation promote fibril formation underconditions where it would otherwise not occur. Ifthis mechanism operates in vivo, it is potentiallyof great significance to the pathogenesis of AD.The investigations described herein were aimed at

characterizing the behavior of Aβ proteins that havebeen specifically modified by HNE and gaininginsight into the mechanism bywhich HNEmodifica-tion promotes Aβ misfolding and fibril formation.Results indicate that HNEmodification increases theaffinity of Aβ proteins for neutral lipid membranes,

causing them to adopt a conformation on mem-branes that is similar to their conformation in a ma-ture amyloid fibril and creating nucleation sites forfibril formation by unmodified Aβ proteins.

Results

HNE and Aβ aggregation on lipid monolayermembranes

An effect of HNE on the formation of β structureby Aβ42 could be detected by PATIR-FTIR atconcentrations of HNE as low as 25 μM, concentra-tions of Aβ42 as low as 180 nM, and times as short as30 min (Fig. 1a). Under the conditions of theseexperiments, the amide I′ (the prime indicates aspectrum collected in D2O) maximum was∼1635 cm−1 in the absence of HNE. At a concentra-tion of 25 μM, HNE specifically increased the low-frequency amplitude and red-shifted the amide I′maximum to ∼1630 cm−1. Increasing the HNEconcentration to 125 μM increased the overallamplitude of the amide I′ absorption and furtherred-shifted the low-frequency component to∼1625 cm− 1—the absorption frequency that ischaracteristic of the β structure in mature Aβfibrils.40 In previously described experiments, pro-tein concentrations were somewhat higher (230–300 nM), and times were somewhat longer (90–125min).31 The current experiments better define thecurrent limits of our ability to detect an effect of HNEon β structure formation by Aβ42 and roughlydemonstrate that the effects of HNE are concentra-tion dependent.In control experiments, the use of 4-hydroxy-2-

hexenal (HHE) (a shorter-chain analogue of HNEderived from ω-3 fatty acyl chains) instead of HNEhad no effect on Aβ42 under the same conditions. Torule out the possibility that the results were affectedby preformed fibril seeds of Aβ42 present in thesamples at the time of injection, the experimentswere repeated with samples that had been filteredthrough a 10-kDa cutoff filter in alkaline buffer at4 °C immediately prior to injection (further describedin Experimental Procedures). The results obtainedusing these ultrafiltered samples were indistinguish-able from those obtained using unfiltered samples(results shown in Supplementary Material).Additional control experiments were performed

with mutant and scrambled forms of Aβ42.Compared to the 30-min amide I′ spectrum of180 nM, Aβ42 (shown in Fig. 1a), the spectrum after100 min exhibits a stronger low-frequency compo-nent at ∼1625 cm−1 (Fig. 2a), and it more closelyresembles previously published spectra of 300 nMAβ42 after 60 min and of mature amyloid fibrils.39

Therefore, Aβ42 does eventually develop a domi-nant ∼1625 cm−1 absorption characteristic of βstructure in mature amyloid fibrils in the absence ofHNE. However, Aβ42 forms this structure morequickly at lower concentrations in the presence ofHNE.

Fig. 1. The effects of HNE on the adsorption of Aβproteins to a DMPCmonolayer. The subphase consisted of2.5 mL of 30 mM Hepes–NaOD (pD 7.4) with either 0, 25,or 125 μM HNE. (a) PATIR-FTIR amide I′ spectra 30 minafter the addition of 2 μg of Aβ42 to yield ∼180 nM. (b)Integrated absorbance of the 1625 cm−super 1 amide I′component calculated from a simultaneous fit of spectrafrom multiple experiments using IRfit.43 At time 0, 125 ngof Aβ42 was added to yield a subphase concentration of∼11 nM. At 25-min intervals, four 500-ng aliquots of Aβ40were added (each aliquot increased the nominal Aβ40concentration by ∼46 nM). For 0 μM HNE, the datarepresent four independent experiments performed ondifferent days, and the error bars represent±standarddeviation. For 25 and 125 μM HNE, the data representthree independent experiments; one-sided error bars(either + or − standard deviation) are shown for clarity.Between 50 and 125 min, results with 0 μM HNE aresignificantly different from those with either concentrationof HNE, although results using the two differentconcentrations of HNE are not significantly different.

1238 Hydroxynonenal and Membrane-Mediated Amyloidogenesis

Because HNE is known to form Michael adductswith the three His residues of Aβ proteins,31 mutantforms of Aβ42 were examined to determine whichHis residues were necessary for HNE to exert itseffects. The spectra of H6A, H13A, and H14Amutants of Aβ42 at 180 nM after 100 min havelower overall spectral amplitudes than Aβ42, buttheir amide I′ spectra remain dominated by the∼1625 cm−1 low-frequency component (Fig. 2b–d).These results agree with an earlier report thatsubstitution of the His residues at positions 13 and14 yielded fibrils with normal structure.44 In con-trast, a 42-residue polypeptide with the same amino

acid composition as Aβ42, but a scrambled sequence(Aβscr), yielded a spectrum that was approximatelysymmetric and featureless (Fig. 2e). HNE greatlyincreased the amide I′ amplitude of Aβ42 butslightly decreased the amplitudes of all threemutants and Aβscr (Fig. 2f–j). The overall spectralshapewas not changed byHNE in any of these cases.An IRfit analysis43 was performed on the spectra

shown in Fig. 2 to quantify changes in the low-frequency components characteristic of β structure.In the absence of HNE, the low-frequency compo-nents for Aβscr and the three mutant forms wereslightly lower in amplitude than for Aβ42, althoughthe decrease was only statistically significant forAβscr (Fig. 3a). HNE markedly decreased theamplitude of the low-frequency component forAβscr and slightly decreased it for the three mutantforms, but markedly increased it for Aβ42 (Fig. 3b).Overall, the data in Figs. 2 and 3 indicate that thepromotion of fibril formation by HNE requires allthree His residues to be present and in correctsequence. HNE modification of Aβscr almost cer-tainly increases the overall hydrophobicity of thisprotein; the marked decrease in signal upon HNEmodification of Aβscr, compared to the markedincrease upon HNE modification of Aβ42, demon-strates that increased signal is not due solely to anincrease in hydrophobicity of Aβ42.In previously described experiments, nonspecifi-

cally oxidized lipid membranes increased the abilityof Aβ42 to seed fibril formation by Aβ40.40 There-fore, the ability of HNE to increase the seedingactivity of Aβ42 was examined. When 1,2-dimy-ristoyl-sn-glycero-3-phosphocholine (DMPC) mem-branes were treated with 11 nM Aβ42 for 25 min,there was a small increase in amide I′ signal, but theintegrated amplitudes of the spectra at that pointwere not affected by 25 or 125 μMHNE. The additionof 46 nM Aβ40 after a 25-min pretreatment withAβ42 yielded a small increase in amide I′ signal,although subsequent additions up to 184 nM did notsignificantly increase it further (Fig. 1b). When thesame experiment was repeated with 25 or 125 μMHNE present at the beginning of the experiment, theaddition of 46 nM Aβ40 after 25 min caused amarked increase in the amide I′ signal that wassignificantly greater than the increase observed inthe absence of HNE.The concentration of Aβ42 in these experiments

was only 1/16 of what was used in the experimentsdepicted in Fig. 1a. This Aβ42 concentration isbelow the threshold needed to observe the HNEeffect illustrated in Fig. 1a, so the lack of an HNEeffect at 25 min is not surprising or inconsistent withother results. Nevertheless, HNE clearly had aneffect at this subthreshold concentration, since itcaused significantly greater amide I′ absorptionsupon subsequent injections of Aβ40.Taken together, the results in Figs. 1–3 demon-

strate that HNE induces Aβ42 to accumulate onlipid membranes in a fibril-like conformation morequickly, and at lower concentrations. HNE alsoincreases the ability of membrane-bound Aβ42 to

Fig. 2. PATIR-FTIR spectra illustrating the effects of HNE on the adsorption of Aβ42 variant proteins to a DMPCmonolayer. The protein concentration was 180 nM in all experiments, the HNE concentration was 125 μM in (f)–(j), andthe spectra were collected 100 min after injection of Aβ42 into the subphase. In each panel, experimental data arerepresented by black dots. Each set of identical experiments was fitted simultaneously using IRfit software (seeExperimental Procedures), although only one representative spectrum from each set is shown. Individual fittedcomponents are represented by thin lines, and their sum is represented by a thick line running through most of theexperimental points.


seed fibril formation by Aβ40. The effects of HNErequire all three His residues of Aβ42 to be present,and these effects could be detected within minutes ata near-physiological concentration of HNE and at11 nM Aβ42.

HNE and Aβ aggregation on lipid bilayer vesicles

The effect of HNE was also examined in DMPCvesicles with a Congo Red (CR) binding assay offibril formation. This approach is much less sensitiveand much slower than PATIR-FTIR spectroscopy,

requiring orders of magnitude more protein anddays instead of minutes to detect fibril formation.However, it provides a true bilayer membrane forthe reaction and uses a time-honored means ofdetecting amyloid fibril formation. Therefore, it isvaluable for examining the interaction betweenHNE and Aβ proteins at different concentrationsand under different conditions. A related dye,thioflavin T, used for quantifying fibril formationby fluorescence,45 was not suitable for these experi-ments because fluorescence of the dye is stronglyaffected by the presence of lipid vesicles.

Fig. 3. Quantitative analysis ofthe integrated absorbance of∼1625 cm−1 components illustratedin Fig. 2. The data for each protein ateach time point represents three tofour independent measurements,and the error bars represent±standard deviations. Results forAβscr are not included because thisprotein did not exhibit a 1625 cm−1

component. (a) Data collected in theabsence of HNE. There is a trendtowards slightly lower absorbancefor the His→Ala mutants, but thedifferences at any given time pointare not statistically significant. (b)Data collected in the presence of125 μM HNE. The absorbance ofAβ42 in the presence of HNE isgreater than in its absence andgreater than the absorbance ofHis→Ala mutants whether or notHNE was present.


When HNE was added to a suspension of DMPCvesicles, CR binding at 24 and 48 h increased forboth Aβ40 and Aβ42 (Fig. 4). A substantial amountof fibril formation occurred in the absence of HNE,as expected for the protein concentrationsused.4,46,47 CR binding declined after 2 days forAβ40, but not Aβ42. This decline with Aβ40 hasbeen observed by others48 and presumably repre-sents the precipitation of fibrils formed at therelatively high concentrations used in the Aβ40experiments.These experiments provide several types of valu-

able information. First, they constitute an indepen-dent corroboration of PATIR-FTIR spectroscopyresults showing that fibril formation is increasedwhen Aβ42 is treated with HNE (Fig. 3). Second,they demonstrate that HNE concentrations as low as60–330 nM (subphysiological) can exert a significantamyloidogenic effect, given a sufficiently longperiod of observation. Third, they indicate that aneffect of HNE can be detected at [HNE]/[protein]ratios ≪1 (with a long period of observation), incontrast to PATIR-FTIR experiments where the[HNE]/[protein] ratios are ≫1 (and only a rela-tively short period of observation is possible).Fourth, they demonstrate that HNE has similareffects on both Aβ40 and Aβ42.

Fig. 4. Congo Red binding to Aβ proteins incubatedwith saturated lipid vesicles (DMPC) in the presence andabsence of HNE. (a) [Aβ42]=2 μM, (b) [Aβ40]=11 μM. Inboth panels, [Aβ]/[DMPC]=1.0, and [HNE]/[DMPC]=0.03 in 10 mM phosphate, 150 mM NaCl, pH 7.4. Eachdata point represents an average and standard deviationof three to five experiments. Per the established protocolfor this assay, the apparent amount of Congo Red bindingat the beginning of the experiment is subtracted from allsubsequent results so that the result at time zero corres-ponds to a concentration of zero.

The fourth point is important because all of theother experiments described above were performedusing Aβ42. For various technical reasons, all of theexperiments described below required higher pro-tein concentrations than could be achieved withoutspontaneous fibril formation using Aβ42, so theywere performed with Aβ40.

Partitioning of HNE and Aβ40 to lipid membranes

HNE reacts spontaneously with side chains on Aβto form covalent adducts.24,31 However, when Aβproteins reacted with HNE in the preceding experi-ments, it was not clear whether HNE–Aβ adductsformed in the aqueous phase before associating withvesicle membranes, or only after HNE had parti-tioned to the vesicle membranes and encounteredAβ40 on the membrane surface. Therefore, thepartitioning behaviors of HNE and Aβ40 withrespect to DMPC vesicles were characterized.A dimensionless water–octanol partition coeffi-

cient for HNE has been calculated as 70.8.49

However, the coefficient between water and lipidbilayer membranes is likely to be different, and adimensionless coefficient is not particularly usefulfor systems involving membranes. A more usefulmodel was proposed by Schurtenberger et al.50 anddeveloped by Almog et al.51 in which the ratio of thetotal lipid concentration, [DMPC], to the lipid-bound HNE concentration, [HNE]v, is plotted versusthe aqueous HNE concentration, [HNE]a, to yield aline with slope 1/PHNE according to

½DMPC�½HNE�v

¼ 1PHNE

1½HNE�a

� 1 ð1Þ

The results of experiments with [DMPC] rangingfrom 2.3 to 12.9 mM, and HNE ranging from 5.0 to23.8 μM, yielded PHNE=20.5±0.9 M− 1 (see supple-mental information). Therefore, under conditions inwhich the CR binding experiments were conducted(11 μM DMPC, 0.33 μM HNE, Fig. 4), assumingthere are 9×104 DMPCmolecules per vesicle,52 therewas less than one molecule of HNE in each vesicle.The partitioning behavior of Aβ40 with respect to

DMPC vesicles was determined using a simplifiedversion of the protocol just described above forHNE. Lyophilized Aβ40 was dissolved in a DMPCvesicle suspension to yield final concentrations of1.5 μM for Aβ40 and 500 μM for DMPC. Thismixture was incubated for 90 min at roomtemperature and then centrifuged at 100,000g for2 h. The concentrations of Aβ40 and residualDMPC vesicles in the supernatant were determinedby bicinchoninic acid protein assay and phosphateassay, respectively. Results indicated that less than3% of DMPC remained in the supernatant, whilethe concentration of Aβ40 in the supernatant wasthe same as that of the mixture before centrifuga-tion. This result is consistent with the generalconclusion reached by many other laboratories,namely, that Aβ40 has little affinity for neutral lipidmembranes and that it only superficially adsorbs to


the surface of neutral lipid membranes.53 Takentogether, these results for HNE and Aβ40 partition-ing to lipid vesicles suggest that HNE and Aβ40only partition weakly to membranes and are mostlikely to react with each other in solution beforeassociating with a lipid membrane.

Partitioning of HNE-modified Aβ to lipidmembranes

The product of the reaction between HNE andAβ40 was difficult to characterize. Extreme insolu-bility and a strong tendency to aggregate (with bothHNE-modified and unmodified Aβ proteins) pre-cluded their purification. Therefore, two qualitativeand indirect assessments of partitioning behavior to

Fig. 5. MALDI-TOF mass spectra of HNE-treated Aβ1–16vesicles. Aβ40 was treated overnight with either 10-fold ounmodified 5 μM Aβ1–16. (b) Mass spectrum of Aβ1–16 preincumass units is apparent. (c) Mass spectrum of Aβ1–16 preincubat3×156 additional mass units are apparent. In (b) and (c) there amain adduct peaks due to dehydration of the HNE-modified H5 μM protein in running buffer at 30 μL/min for 300 s, followeAβ1–16 preincubated with 500 μM HNE exhibits increased abs

lipid membranes were sought using surface plas-mon resonance (SPR). The membranes in bothassessments used DMPC vesicles immobilized onan L1 chip.The first assessment examined the effect of HNE

on the interaction of Aβ1–16 with DMPC vesicles.Aβ1–16 contains all three of the His residues in Aβ40that are susceptible to modification by HNE, but incontrast to Aβ40, HNE-modified Aβ1–16 remainssoluble. The mass of Aβ1–16 was confirmed to be1955.0 by matrix-assisted laser desorption/ioniza-tion time-of-flight (MALDI-TOF) mass spectrometry(Fig. 5a). A 5 μM solution of Aβ1–16 was incubatedovernight with a 10-fold excess of HNE (50 μM) in10 mM Hepes with 150 mM NaCl at pH 7.4 and37 °C. MALDI-TOF mass spectrometry detected

and SPR sensorgrams of Aβ1–16 adsorption onto DMPCr 100-fold molar excess of HNE. (a) Mass spectrum ofbated with 50 μM HNE. A population with 156 additionaled with 500 μMHNE. Populations with 1×156, 2×156, andre prominent peaks at 18 and 2×18=36m/z lower than theis side chain (see the text). (d) SPR injections consisted of

d by a 500-s washout period with protein-free buffer. Onlyorption onto vesicles.


Aβ1–16 with one modified residue, but there waslittle or no signal representing Aβ1–16 moleculeswith two or three modified residues (Fig. 5b). When5 μM Aβ1–16 was treated with a 100-fold excess ofHNE (500 μM), however, Aβ1–16 with two and threemodified residues were readily apparent (Fig. 5c).Based on a previously published sequencing studyof HNE-modified Aβ40,31 it may be assumed thateach of the modified residues is His. The relativeabundance of Aβ1–16 modified by one, two, or threeHNE molecules cannot be determined in any singlemass spectrum, but it is reasonable to expect and toconclude from these spectra that a 100-fold excess ofHNE relative to Aβ1–16 yielded more polypeptideswith two or three HNE adducts than a 10-fold excessof HNE. Peaks 18 m/z lower than each of the majorpeaks are apparent, and these most likely representthe neutral loss of water from HNE-modified Hisside chains.22

When these preparations were examined by SPR,the responses generated by Aβ1–16 and Aβ1–16treated with 10-fold excess HNE were small andbarely distinguishable from a blank injection (Fig.5d). However, Aβ1–16 treated with 100-fold excessHNE yielded a response indicating significantlygreater affinity for the DMPC membrane. Theresponse persisted at a high level during washout,indicating a high degree of membrane affinity,but precluding quantitative determination of thisaffinity.Similar studies of HNE-modified Aβ40 could

not be performed by SPR because of insolubility.Therefore, experiments were performed in whichmembranes were pretreated with bovine serumalbumin (BSA) and HNE for a period that endedbefore a 1 μM solution of Aβ40 was injected. BSApretreatment was necessary because Aβ proteinsare sufficiently surface active that injected sampleswould otherwise be lost by nonspecific binding totubing and other interior surfaces. The period ofHNE pretreatment ended before Aβ40 injection toeliminate the possibility that HNE and Aβ40reacted before encountering each other in thevicinity of the lipid vesicles. When this precautionwas not taken, HNE–Aβ40 reaction products

formed, aggregated, and occluded flow throughthe system.When Aβ40 was injected over membranes pre-

treated with HNE, SPR signals indicating proteinadsorption were twice as large as when Aβ40 wasinjected over membranes that had not been pre-treated with HNE (Fig. 6). SPR signals on HNE-pretreated membranes did not reach a plateau in300 s, although a plateau level was nearly reached inthe same time interval when membranes were notpretreated with HNE. Most of the adsorbed Aβ40readily desorbed from the membrane regardless ofHNE pretreatment—behavior previously describedby others as being characteristic of monomeric,rather than aggregated, Aβ40 on a neutral lipidmembrane.54

HNE concentrations in the membrane were notat equilibrium during these experiments; the HNEthat partitioned into the membrane during pre-treatment began to leach out of the membraneimmediately after the HNE-containing buffer wasreplaced with an HNE-free buffer containingAβ40. Moreover, the production rates of variousreaction products between HNE and Aβ40 are notknown. Therefore, the SPR results are not suitablefor a quantitative assessment of affinity. However,the experiment mimics the circumstances of Aβ40approaching a membrane that is producing HNEthrough oxidative processes, and it clearly demon-strates in a qualitative way that HNE increases theaffinity of Aβ40 for lipid membranes.

The conformation of membrane-adsorbed Aβ40

To identify the effects of HNE on the amide I′spectrum of membrane-bound Aβ40, it wasnecessary to distinguish them from the effects ofprotein–protein interactions that ensued when theproteins aggregated and formed fibrils. Theseeffects were eliminated by mixing Aβ40 with alarge excess of DMPC in the form of vesicles—astrategy that substitutes protein–lipid interactionsfor protein–protein interactions. The amount oflipid was limited to a lipid/protein ratio of 200:1because the carbonyl ester stretching mode from

Fig. 6. SPR sensorgrams of theAβ40 adsorption onto DMPC vesi-cles. (dashed line) Pretreatment with90 μL of 100μMHNE followedby aninjection at time zero of 150 μL ofrunningbuffer. (dotted line) Pretreat-ment with 90 μL of running bufferfollowed by an injection at time zeroof 150 μL of 1 μM Aβ40. (solid line)Pretreatment with 90-μL injection of100 μM HNE followed by an injec-tion at time zero of 150 μL of 1 μMAβ40. The sensorgrams shown havehad blank injections subtracted andare representative of four indepen-dent sets of three injections.

Fig. 8. Effects of lipid vesicles and HNE on Aβ40conformation. (a) Internal reflection amide I′ infraredspectrum Aβ40 in HFIP after evaporation onto agermanium crystal, (b) 30 μM Aβ40 with 6 mM DMPCvesicles, (c) 30 μM Aβ40 with 6 mM DMPC vesicles and180 μM (6× relative to Aβ40) HNE (corresponding to lane4 in Fig. 6). (b and c) Infrared transmission spectra ofsamples incubated in 20 mM Hepes–D2O, pD 7.4, for 3 h,lyophilized, mixedwith KBr, and pressed into a pellet. The3-h incubation time corresponds to the incubation time ofsamples in Fig. 6.


the lipid obscured the amide I′ band of theprotein at higher ratios.A marked reduction in protein–protein interac-

tions was verified using the photoinduced cross-linking of unmodified proteins (PICUP) method tophoto-cross-linkAβ40 under various conditions.55,56

A 30 μM solution of Aβ40 incubated for 3 hcontained not only a large fraction of monomericAβ40, but also substantial amounts of severaloligomeric species between 7 and 20 kDa (Fig. 7,lane 2). Adding DMPC vesicles such that the[DMPC]/[Aβ40] concentration ratio was 200:1 leftonly traces of the oligomeric species (Fig. 7, lane 3).Adding HNE such that the [DMPC]/[HNE] ratioswere 33:1 or 10:1 produced dimers, trimers, and traceamounts of oligomer (Fig. 7, lanes 4 and 5). Never-theless, most of the protein remained monomeric.In principle, it is possible for HNE to cross-link

proteins apart from PICUP. This would occur viaa combination of Michael addition and Schiff baseformation. However, the amine groups in Aβ thatcould potentially form Schiff bases are mostlyprotonated at pH 7.4 and, thus, less likely to formSchiff bases. No evidence for HNE-mediatedcross-linking of Aβ was found by mass spectro-metry in prior studies (despite high reactantconcentrations).31 Our results in this respect areconsistent with numerous prior attempts to detectSchiff base formation between HNE and Lys invarious proteins.19

When Aβ40 was evaporated directly from a hexa-fluoro-2-propanol (HFIP) stock solution, its amide I′absorption was maximal at 1657 cm−1, roughly sym-metric, and featureless (Fig. 8a). These features indi-cate that it lacked secondary structure, particularlyβ-sheet structure.40,57 When DMPC vesicles wereadded at the same 200:1 [DMPC]/[Aβ40] ratio thatinhibited cross-linking in Fig. 7 (lane 3), the spectrumwas much broader, with numerous minor features(Fig. 8b). HNE (at a [DMPC]/[HNE] ratio of 33:1)caused a prominent and relatively narrow absorp-

Fig. 7. Effects of lipid vesicles and HNE on Aβ40aggregation. PICUP was performed on 30 μM Aβ40 in10 mM phosphate buffer, pH 7.4, with Ru(bpy)3 and APS,followed by SDS-PAGE and silver staining. Samples wereincubated for 3 h at room temperature before the additionof PICUP reagents. The gel is representative of threeexperiments. Lane 1,molecularweightmarkers. Lane 2, 1-sphotoactivation. Lane 3, 6 mM DMPC vesicles added([Aβ]/[DMPC]=200). Lane 4, 6 mM DMPC vesicles with180 μM (6× relative to Aβ40) HNE. Lane 5, 6 mM DMPCvesicles with 600 μM (20× relative to Aβ40) HNE.

tion at 1626.8 cm−1 to develop, along with a weakerbut clear absorption at 1684.8 cm−1 (Fig. 8c). Both ofthese features are characteristic ofmatureAβ proteinfibrils.58

Despite the resemblance of this spectrum to that ofamyloid fibrils, the photo-cross-linking results ofFig. 7 (especially lane 4) indicate that the spectrum ofFig. 8c arose mainly from monomeric protein (withlesser contributions from small oligomers). It shouldbe noted that all three samples used to generate Fig.8a–c were lyophilized, but the lack of strong βstructure features in Fig. 8a and b demonstrates thatlyophilization alone did not bring about an amyloid-like spectrum.The experiments that produced Figs. 4b and Fig.

8c both had a [DMPC]/[HNE] ratio of 33:1 (Table 1),although according to Eq. (1), the concentration ofHNE in each vesicle was slightly lower for Fig. 8cthan for Fig. 4b. The most striking differencebetween the circumstances of these two experimentsis that the HNE concentrations (aqueous andvesicle-associated) were orders of magnitude higher

Table 1. Comparative summary of the protein, lipid, and HNE concentration ratios in the experiments that producedFigs. 4b, 7 (lane 4), and 8c

Figure [DMPC]/[protein] [DMPC]/[HNE]a [DMPC]/[HNE]va [HNE]v/[protein]

a [HNE]a/[protein] Physical state

4b 1 33 ∼150 0.007 0.03 Fibril

7 (lane 4), 8c 200 33 ∼270 0.740 6 Predominantlymonomeric

a [HNE]v calculated using Eq. (1).


in the experiments that produced Fig. 8c. Thus,fibrils formed under conditions in which only asmall fraction of the Aβ40 protein could have beenmodified by HNE (Fig. 4b), whereas the protein waspredominantly monomeric when enough HNE waspresent for threefold modification of all the Aβ40present (Fig. 8c). These observations are consistentwith the suggestion that HNE-modified Aβ proteinsdo not merely have an increased tendency to self-associate, but instead accelerate fibril formation byunmodified Aβ proteins.

Discussion

Aβ proteins are difficult to study because of theirlow solubility and tendency to aggregate. Whenthey are modified by HNE, they become even moredifficult, necessitating special techniques that cancharacterize HNE-modified Aβ proteins as they areformed and under the particular conditions in whichthey are formed. One such technique is PATIR-FTIRspectroscopy, which has been used previously todemonstrate that oxidatively damaged lipid mem-branes promote fibril formation by Aβ4239 andtransform Aβ42 into a more effective seed for fibrilformation by Aβ40.40 Some of the investigationsdescribed herein also employed PATIR-FTIR spec-troscopy to show that HNE alone can reproduce thefibrillogenic effects of oxidatively damaged mem-branes on Aβ40 and Aβ42. In addition, the currentresults also characterize the effects of HNE in fourways. First, the effects of HNE require the presenceof all three His residues. Second, HNE modificationenhances the affinity of the protein for neutral lipidmembranes. Third, HNE alters the conformation ofAβ proteins in a way that resembles their confor-mation in amyloid fibrils. Fourth, HNE promotesfibril formation even when only a minority of Aβproteins could have been modified by HNE. Thesefindings are significant because they suggest aspecific mechanistic link between oxidative stressand amajor histopathological feature of AD, namely,amyloid fibrils.Aβ proteins are generally regarded as having little

or no specific affinity for neutral lipid membranesand no enhanced tendency to form fibrils in theirpresence.53 Therefore, the fibrils that did form onordinary lipid membranes in Figs. 1–4 and 8b wouldmost likely have formed spontaneously in solutionwithout membranes present. Most investigatorsreport that Aβ proteins have a greater affinity for

membranes containing cholesterol, anionic lipids, organgliosides and that Aβ proteins exhibit a greatertendency to assume secondary structure (α or β) inthe presence of such membranes.53 However, theselipids are common in the brain, as is unaggregatedAβ protein in the extracellular space. Therefore, anaffinity between Aβ and normal lipid membranecomponents does not account for pathologicalamyloid formation. We are prompted to focus onthe consequences of oxidative stress because of itswell-known association with AD and becauseseveral lipid species abundant in brain tissue arehighly vulnerable to oxidative damage.Another reason to focus on the consequences of

oxidative stress is that Aβ proteins bind copper ionsand actually promote oxidative lipid damage.42

They increase the production of HNE via arachi-donate oxidation, and the HNE produced formscovalent Michael adducts with the three Hisresidues present in human Aβ proteins.24,31 Mate-rial reacting with anti-HNE-His antibody has beenidentified in senile plaques, but the identity of themodified protein is not clear.32,59 HNE-modified Aβproteins appear to inhibit the proteasome,60,61 andHNE accelerates fibril formation by Aβ40 insolution at relatively high concentrations.15,62 Thedata in Figs. 1–4 demonstrate that HNE acceleratesfibril formation on lipid membranes by Aβ40 andAβ42 at relatively low concentrations,46,47 suggest-ing that covalent modification of Aβ proteins bylipid oxidation products may cause Aβ proteins tobecome pathological and form fibrils in humanbrain tissue.At the concentrations of HNE and Aβ used to

obtain the data shown in Fig. 4, only a minority ofAβmolecules in each amyloid fibril could have beenmodified by HNE. Therefore, it is unlikely that HNEpromotes fibril formation by simply concentratingHNE-modified proteins on the membrane sur-face.14,15 The fibrils that formed under our experi-mental conditions were long and straight,40 whereasthe fibrils that formed when 100 μM Aβ40 wastreated with an equimolar concentration of HNEhad a curved morphology.62 It seems likely thatHNE promotes fibril formation by increasing thehydrophobicity of a small number of Aβ proteins,causing them to partition to a membrane andbecome nucleation sites for the aggregation ofunmodified Aβ into fibrils. These nucleation sitesmay act as templates that increase the thermody-namic stability of monomers in a fibrillar conforma-tion, but do not ultimately become part of the fibril.


Alternatively, they may act as seeds that reduce akinetic barrier to adopting a fibrillar conformationand become part of the fibril. The present data donot help distinguish between these possibilities. Ineither case, the role of increased hydrophobicity inmediating the effect of HNE is supported by thefinding that HHE has no discernable effect. Com-pared to HNE, HHE has the same reactive func-tional groups, but it is less hydrophobic because itsaliphatic chain is shorter by three methylene groups.Increased hydrophobicity may help explain how

HNE modification increases the affinity of Aβ forneutral lipid membranes (Fig. 6), but increasedhydrophobicity also makes the physical propertiesof HNE-modified Aβ difficult to characterize.Therefore, some of our studies were performed inthe presence of a high concentration of neutral lipidmembranes. These conditions substituted mem-brane–protein interactions for hydrophobic pro-tein–protein interactions. The photo-cross-linkingdata in Fig. 7 demonstrate that this strategy wassuccessful; lipid vesicles reduced the amount ofoligomeric unmodified Aβ to negligible levelsfollowing a 1-s photo-cross-linking event, andmonomeric species remained dominant even afterHNE exposure. Therefore, most of the protein ismonomeric under the conditions in which thespectra in Fig. 8b and c were recorded.These cross-linking data indicate that the amide I′

spectrum in Fig. 8c most likely corresponds to that ofmonomeric Aβ40. With this information, the spec-trum is remarkable in that it exhibits featuresusually interpreted as indicating a β-sheet confor-mation. It is difficult to imagine how a singlepolypeptide chain might be stabilized in a parallelβ-sheet conformation, although a single chain maywell form a hairpin and an intramolecular antipar-allel β sheet. Recent theoretical studies of β-sheetvibrational modes suggests that the positions of themost prominent infrared absorption bands arisingfrom a β sheet are determined by intrastrandcoupling, rather than interstrand coupling.63,64

Thus, features of the amide I′ spectrum in Fig. 8csuggesting β-sheet structure do not necessarilyimply that the structure is oligomeric.It is not entirely clearwhether the reaction between

HNE and Aβ occurs at the membrane, or whether itoccurs in bulk solution followed by adsorption of theHNE–Aβ adduct onto the membrane. On one hand,the protein and lipid concentrations that yielded theresults shown in Figs. 1–4 and the partitioningbehavior of HNE with respect to DMPC vesiclesindicate that HNE–Aβ adduct formation was morelikely to occur in solution. On the other hand, the SPRresults shown in Fig. 6 suggest that HNE remainingin the membrane after washout was responsible forincreased Aβ affinity for the membrane. Never-theless, significant encounters between reactants onthe membrane surface may occur, and significantencounters apart from the membrane cannot beruled out, because the concentration of HNE outsideof the membrane cannot be reduced to zero. HNEdoes promote Aβ fibril formation in the absence of

lipid membranes, but the reaction requires muchhigher concentrations of HNE and Aβ over muchlonger periods,62 and it yields a distinctivelydifferent fibril morphology than we observe in thepresence of membranes.40In summary, the results reported herein indicate

that modification by HNE promotes membraneassociation and fibril formation by Aβ. The mechan-ism involves stabilizing monomeric Aβ on themembrane in a conformation that at least partiallyresembles its conformation in a mature amyloidfibril, and stimulating fibril formation by unaggre-gated Aβ. These findings are important in thecontext of AD pathogenesis because they outline aspecific biophysical mechanism that may linkoxidative stress to one of the major histopathologicalfeatures of AD.

Experimental Procedures

Materials

DMPC was obtained from Avanti Polar Lipids (Alaba-ster, AL) in powder form, stored at −20 °C, and usedwithout further purification. D2O of 99% purity, CR, KBr,HFIP, ruthenium-tris(2,2′-bipyridyl) dichloride (Ru(byp)3)Cl2, ammonium persulfate (APS), n-octylglucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfo-nate (Chaps), were purchased from Sigma (St. Louis,MO). β-Mercaptoethanol and polypeptide SDS-PAGEmolecular weight standard were purchased from BioRad(Hercules, CA). Tricine sample buffer, SDS runningbuffer, 1 mm 10–20% Tris–Tricine gradient gels andSilverXpress silver staining kit were obtained fromInvitrogen (Carlsbad, CA). Pioneer L1 sensor chips werepurchased from Pharmacia (Uppsala, Sweden) and storedat 4 °C.Lyophilized Aβ1–16, Aβ40, and Aβ42 of greater than

95% purity were purchased from rPeptide (Bogart, GA)and stored at −20 °C. Variant forms of Aβ42 with H6A,H13A, or H14A mutations and a polypeptide with thesame amino acid composition as Aβ42 but with ascrambled sequence (KVKGLIDGAH IGDLVYEFMDSNSAIFREGV GAGHVHVAQV EF, Aβscr) were obtainedfrom the same source. A 0.5 mg/mL solution of eachprotein in HFIP was made and stored at −20 °C asdescribed by Stine et al.65 Aliquots of these stock solutionswere removed by Hamilton gas-tight syringe andlyophilized immediately prior to use. HNE and HHE ata concentration of 10 mg/mL in ethanol were purchasedfrom Cayman Chemical Co. (Ann Arbor, MI) and storedat −80 °C. N-Methyl-2-phenylindole in acetonitrile,methanesulfonic acid, and 1,1,3,3-tetramethoxypropanein Tris–HCl (TMOP) standard for HNE concentrationmeasurement assay were purchased from Calbiochem (LaJolla, CA) and stored at 4 °C. Water was purified throughan Elix and MilliQ A10 water purification system(Millipore, Bedford, MA) to 18 MΩ cm and b3 ppborganic carbon content.

MALDI-TOF mass spectrometry

Samples were dissolved in 30 μL of a 4-hydroxy-α-cyanocinnamic acid (CHCA) solution (5 mg CHCA in a1 mL solution of 50% acetonitrile with 0.3% trifluoroacetic


acid). Analysis was performed on a Voyager DE massspectrometer (Applied Biosystems/MDS Sciex, FosterCity, CA).

Lipid vesicle preparation

Ten millligrams of DMPC was dissolved in 200 μLcyclohexane in which one drop of methanol was addedto increase the solubility. This solution was lyophilizedovernight to remove all solvent, producing a whitepowder that is easily hydrated. This powder wasresuspended in 30 mM Hepes at pH 7.4 or 20 mMHepes in D2O at pD 7.4 for infrared spectroscopy, bath-sonicated for 10 min, and extruded through 100-nmpolycarbonate membranes to produce unilamellar vesi-cles. Lipid concentrations were determined by phosphateanalysis.66 Lipid vesicle stock suspensions including10 μg/mL cycloheximide to inhibit bacterial growthwere stored under argon for up to 2 weeks at 4 °C.Immediately prior to use, aliquots from these stockswere diluted by buffer solutions. HNE/DMPC vesiclemixtures with the HNE/DMPC molar ratio of 3% weremade immediately before every experiment by addingcertain amounts of HNE stock solution in ethanol toDMPC vesicle suspensions. All experiments were per-formed at room temperature (approximately 21 °C), i.e.,below the phase-transition temperature of our bilayermembranes.

Congo Red binding assay

A CR binding assay was employed to quantify fibrilformation. CR is a histologic dye that alters its absorbancespectrum upon binding to fibrillar amyloid.67,68 Lyophi-lized Aβ40 or Aβ42 proteins were redissolved in HNE/DMPC vesicle mixtures in phosphate-buffered saline(10 mM phosphate, 150 mM NaCl, pH 7.4) to yield aprotein/lipid molar ratio of 1:1. The final concentrations ofAβ40 or Aβ42 were 11 and 2 μM, respectively. Aβ proteinswere also dissolved in DMPC vesicle suspensions, with noHNE present, at the same protein and DMPC vesicleconcentrations as in the aggregation mixtures containingHNE. These mixtures were allowed to aggregate at roomtemperature with continuous gentle agitation on arotating table for multiday intervals. Aliquots (110 μL) ofthese mixtures were mixed with 30 μL of 10 μM CongoRed in phosphate-buffered saline and incubated at roomtemperature for 15 min before spectrometric measure-ments. Fibril formation was assayed by measuring theratio of sample absorptions at 541 and 403 nm, which arethe wavelengths of maximum difference and of anisosbestic point for fibril-bound CR and unbound CR,respectively.67,68

Cross-linking of Aβ40

PICUP was performed according to the protocol ofBitan et al.55 and Bitan and Teplow.56 Irradiation wasaccomplished using a 150-W lamp positioned 5 cm fromthe bottom of the reaction tube. The irradiation time was1 s and was controlled using a photographic shutter.Freshly lyophilized Aβ40 was dissolved in either

phosphate buffer (10 mM sodium phosphate, pH 7.4), aDMPC vesicle suspension, or a HNE/DMPC vesiclemixture and incubated at room temperature for 3 h. Onemicroliter of 1 mM Ru(bpy)3Cl2 and 1 μL of 20 mM APSin phosphate buffer were added to 18 μL of Aβ40

solutions and then irradiated. Cross-linking reactionswere quenched immediately with 10 μL of tricine samplebuffer (Invitrogen) containing 5% β-mercaptoethanol.Samples were analyzed by electrophoresis using 1 mm,10–20% Tris–Tricine gradient gels (Invitrogen). Proteinbands were visualized by silver staining (SilverXpress,Invitrogen). Gels were air-dried, following a 20-minincubation in gel-drying solution (40% EtOH, 5%glycerol, and 55% H2O).

PATIR-FTIR spectroscopy

Spectra were recorded from lipid monolayers preparedin a Langmuir trough using instrumentation and methodsthat have been previously described.39,40,43,69–71 Thesubphase buffer in the trough consisted of 2.5 mL of30 mM Hepes–NaOD buffer in D2O at pD 7.4. HNE inHepes buffer was added where indicated. Monolayerswere created by applying approximately 4 nmol of DMPCdissolved in a mixture of n-hexane/ethanol (9:1 byvolume) to the surface of the subphase. The monolayerwas compressed to a surface pressure of 20 dyne/cm andapplied onto a silane-treated germanium internal reflec-tion crystal.Aliquots of lyophilized protein (0.125 μg of Aβ40 or

0.5 μg of Aβ42) were resuspended in 5 μL of 30 mMNa2DPO4–NaOD buffer, pD 11.9, per the method ofFezoui et al.48 and injected into the continuously stirredsubphase solution. This manner of preparing Aβ proteinshas been shown to produce solutions of low molecularweight Aβ with excellent solubility and fibrillogenesischaracteristics.48 To rule out the possibility that preformedseeds were being injected into the subphase, selectedexperiments were repeated with samples that had beenultrafiltered through a 10-kDa cutoff filter (Microcon YM-10) at 4 °C and 20,000g immediately prior to use.An enclosure around the Langmuir trough is filled with

argon to eliminate water vapor interference and avoidspontaneous air oxidation of lipids at the air–waterinterface. Spectra were collected in rapid-scanning modeas 1024 co-added interferograms using a Bio-Rad FTS-60A spectrometer, a liquid-nitrogen-cooled MCT detector,a resolution of 2 cm−1, scanning speed of 20 kHz,triangular apodization, and one level of zero filling. Thesubtraction of the gently sloping water vapor bands wascarried out to improve the background. All spectroscopicstudies were performed at 21 °C. Integrated componentareas were calculated over the range 1600 to 1710 cm−1

using IRfit software.43 A spectrum of Aβ40 withoutDMPC vesicles was obtained by internal reflection from3 μL of 0.5 mg/mL Aβ40 in HFIP evaporated onto agermanium internal reflection crystal.Transmission IR spectra were collected from KBr

pellets. Lyophilized Aβ40 was dissolved in 2 mM HClwith 40% HFIP, incubated 1 h, and lyophilized overnightto exchange the trifluoroacetic acid counterions withchloride and eliminate the strong band around 1673 cm−1

of trifluoroacetate salt that overlaps the amide I spectra.72

HFIP (40%) was introduced to inhibit Aβ40 aggregationunder low pH. Protein obtained was redissolved in 1%NH3·H2O in D2O and lyophilized overnight, since theHCl salt form of Aβ40 is known to self-aggregate.73

Protein powder obtained was resuspended in DMPCvesicle suspensions with and without the presence ofHNE to yield final protein and lipid vesicle concentra-tions of 30 μM and 6 mM, respectively. Protein/lipidvesicle mixtures in 20 mM Hepes buffer in D2O at pD 7.4were incubated at room temperature for 3 h, and then150-μL aliquots of the mixtures were flash-frozen in


liquid nitrogen and lyophilized overnight. The powderobtained was mixed with 150 mg KBr and pressed into apellet.Spectra were collected in rapid-scanning mode as 1024

co-added interferograms using a Bio-Rad FTS-60A spec-trometer, a liquid-nitrogen-cooled MCT detector, a resolu-tion of 2 cm−1, scanning speed of 20 kHz, and anundersampling ratio of 2. The instrument was continu-ously purged with dry CO2-free air. Spectra wereprocessed with one level of zero filling and triangularapodization, but no smoothing, deconvolution, vaporsubtraction, or nonlevel baseline correction. DMPCexhibits an IR absorption band at 1736 cm−1 due to itsester carbonyl group. This band partially overlappedprotein amide I′ bands when DMPC concentrations werehigh. Therefore, an IR spectrum of DMPC without proteinwas also collected and subtracted from spectra of theprotein/lipid mixtures.

Partition coefficients

DMPC lipid vesicle suspensions were mixed withvarying concentrations of HNE, equilibrated at 21 °C for18 h under argon, and centrifuged at 100,000g for 2 h topellet the vesicles. The supernatants were assayed forphosphate to determine residual DMPC concentrations,and HNE concentrations were determined by colorimetricassay.74 Three volumes of 10 mM N-methyl-2-phenylin-dole in acetonitrile was mixed with 1 volume of 25 μMFeCl3 in methanol to yield a chromogenic reagent. TMOP(200 μL) standard solutions or samples were mixed with650 μL of this reagent and 150 μL methanesulfonic acidand incubated at 45 °C for 1 h to yield a stablechromophore composed of one molecule of HNE andtwo molecules of N-methyl-2-phenylindole. HNE concen-trations were assayed by monitoring absorption of thechromophore at 586 nm.

Surface plasmon resonance

SPR experiments were performed on a PharmaciaBIAcore2000 biosensor system (Biacore AB, Uppsala,Sweden) with an L1 chip. The L1 chip contains lipophilicalkyl chain anchors immobilized to a carboxymethyldex-tran surface. Lipid vesicles are captured intact by thelipophilic anchors, retaining spherical shape and bilayernature.75 All experiments were performed at 25 °C, usingthe Hepes running buffer (10 mM Hepes, 150 mM NaCl,pH 7.4). The flow rate was 5 μL/min unless otherwisenoted. The surface of the chip was cleaned by 15 μL of10 mM Chaps, followed by washing with running bufferat 40 μL/min for 3 min. Eighty-five microliters of 1.2 mMDMPC vesicle suspensions was injected to populate thechip surface with vesicles, followed by a 3-min injectionof 100 μg/mL BSA to block possible uncovered non-specific binding sites. The adherent vesicles were thenwashed with 1 M NaCl for 3 min to establish a stablebaseline. Between experiments, the chip surface wasregenerated by sequential washing with 15 μL of 1 MNaCl, 20 μL of 10 mM Chaps, 20 μL of 0.5% n-octylglucoside, 10 μL of 10 mM NaOH, and 10 μL of10 mM glycine at pH 1.5.Two types of experiments were performed: (1) A 5 μM

solution of Aβ1–16 in 10 mMHepes, 150 mMNaCl, pH 7.4,was incubated with either 0, 50, or 500 μMHNE overnight(16–20 h) at 37 °C. These solutions, along with buffer andHNE-only control solutions, were then injected directlyinto the SPR instrument at a rate of 30 μL/min. (2) In the

second type of experiment, a 100 μM HNE solution wasprepared by diluting a 10 mg/mL (64.1 mM) ethanol stocksolution into running buffer, and a 1 μM Aβ40 solutionwas prepared by dissolving lyophilized Aβ40 powder intorunning buffer. Three different injection sequences werefollowed, all at a rate of 30 μL/min: (a) 90 μL of 100 μMHNE solution followed by 150 μL of 1 μMAβ40, (b) 90 μLrunning buffer followed by 150 μL of 1 μM Aβ40, and (c)90 μL of 100 μM HNE followed by 150 μL of runningbuffer. Injection sequence (a) was designed to avoid areaction between HNE and Aβ40 before they reachedimmobilized lipid vesicles. If these materials were co-injected, the reaction products were insoluble andoccluded the fluidic pathways. Injection sequences (b)and (c) were control experiments to characterize theinstrument response to Aβ40 without HNE, and HNEwithout Aβ40. The start time of the first injection of eachexperiment and the average absolute response signal priorto the first injection were adjusted to zero.

Acknowledgements

This study was supported by grants from theNational Institute of Aging, the AmericanHealth Assistance Foundation, and the Alzhei-mer's Association.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.01.057

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promotion of amyloid β protein misfolding and fibrillogenesis by a lipid oxidation product

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