Binding of Bexarotene to the Amyloid Precursor Protein

May 2, 2018 - Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer´s disea...
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Binding of Bexarotene to the Amyloid Precursor Protein Transmembrane Domain in Liposomes Alters its #-Helical Conformation but Inhibits #-Secretase Non-Selectively Frits Kamp, Holger A. Scheidt, Edith Winkler, Gabriele Basset, Hannes Heinel, James M. Hutchison, Loren M. LaPointe, Charles R. Sanders, Harald Steiner, and Daniel Huster ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00068 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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Binding of Bexarotene to the Amyloid Precursor Protein Transmembrane Domain in Liposomes Alters its α-Helical Conformation but Inhibits γ-Secretase Non-Selectively Frits Kamp,1,# Holger A. Scheidt,2,# Edith Winkler,1 Gabriele Basset,1Hannes Heinel,2 James M. Hutchison,3 Loren M. LaPointe,3 Charles R. Sanders,3 Harald Steiner,1,4,* Daniel Huster2,* 1

Biomedical Center - BMC, Metabolic Biochemistry, Ludwig-Maximilians–University

Munich, Germany 2

Institute for Medical Physics and Biophysics, Leipzig University, Härtelstr. 16-18, D-04107

Leipzig, Germany 3

Department of Biochemistry and Center for Structural Biology, Vanderbilt University,

Nashville, TN 37240, USA 4

German Center for Neurodegenerative Diseases (DZNE) – Munich, Feodor-Lynen-Str. 17,

D-81377 Munich, Germany, Germany #

These authors contributed equally

Correspondence:

Daniel Huster Phone: +49 (0) 341 97-17501 Fax: +49 (0) 341 97-15709 e-mail: [email protected]

Harald Steiner Phone: +49 (0) 89 4400 46535 Fax: +49 (0) 89 4400 46508 e-mail: [email protected]

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Abstract Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer´s disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aβ clearance mechanism. However, whether or not bexarotene induces removal of Aβ plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP Cterminal fragment C99 to release Aβ in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited the cleavage of Notch1, another major γ-secretase substrate, demonstrating that its inhibition of γ-secretase is not substrate specific and not mediated by acting via the cholesterol binding site of C99. Our data suggest that bexarotene is a pleiotropic molecule that interfere with Aβ metabolism through multiple mechanisms. Keywords: 2H NMR, order parameter, CD spectroscopy, γ-secretase cleavage, cholesterolprotein interaction, intramembrane proteolysis

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INTRODUCTION A hallmark of Alzheimer’s disease (AD) is the formation and deposition of amyloid plaques, which consist mainly of amyloid β (Aβ) peptides of different length in the brain.1-5 These deposits represent the end product of a complicated aggregation process, which starts from monomeric peptides derived from the amyloid precursor protein (APP), a type I membrane protein, by the sequential action of β- and γ-secretases.6 γ-Secretase cleavage occurs in the transmembrane domain (TMD) of APP C99, the C-terminal fragment (CTF) left by βsecretase cleavage and results in the release of Aβ peptides of various lengths (see Supplementary Fig. S1). The longer, neurotoxic, 42 residue form of Aβ is highly aggregation prone and represents the major Aβ species deposited in the brain.1-5 Amyloid fibril formation progresses via several transient intermediates such as oligomeric and protofibrillar states of Aβ to form the final mature fibrils.7,8 Although the role of Aβ in the disease is still not fully understood and has been challenged based on the failure of clinical trials, a number of approaches to interfere with the generation of Aβ and/or its aggregation process have been developed, which might eventually provide the basis for a therapy.2 One promising strategy is the use of small molecules that interfere with protein aggregation and the formation of amyloid structures.9-12 Among the plethora of lead molecules for pharmacological intervention is the aromatic anticancer drug bexarotene. While bexarotene was initially reported to rapidly remove Aβ plaque pathology by boosting apolipoprotein E (apoE)-mediated Aβ clearance in AD mouse models, rather contradictory results were later reported.13-18 Alternatively, it was shown that can suppress memory defects in AD mouse models by activating retinoid X receptors.19 Recently, it was found that bexarotene can suppress primary nucleation in Aβ aggregation20 suggesting that the drug might be a potent inhibitor of Aβ plaque formation.21 Alternatively, bexarotene might inhibit formation of Aβ by the secretases. Bexarotene is a lipophilic molecule that has been ascribed to have high structural similarity to the abundant membrane lipid cholesterol.22,23 Interestingly, mechanistic in vitro studies to understand the mode of action of bexarotene22,23 suggest that this molecule may compete with cholesterol for direct binding to a site on the juxtamembrane/transmembrane region of APP (see Supplementary Fig. S1).24,25 Furthermore, based on molecular modeling, monolayer experiments, and binding assays for bexarotene, it has been hypothesized that this molecule binds to extracellular Αβ peptides and inhibits the cholesterol-driven insertion of these peptides into the membrane, thereby preventing oligomerization and subsequent neurotoxic 3 ACS Paragon Plus Environment

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pore formation.22,23 These calculations suggest a similar binding mode for bexarotene and cholesterol to Aβ reflecting common structural features of the molecules. However, experimental data regarding binding of bexarotene to membrane-associated Aβ and C99 is missing. In the current study, we investigated in detail the binding of bexarotene to full-length C99 by NMR spectroscopy. Combined with circular dichroism data using a shorter transmembrane APP segment containing part of the cholesterol binding region (i.e. C9926-55, see Supplementary Fig. 1) we revealed a helix-stabilizing effect of bexarotene on the TMD of APP in its cholesterol binding region. As these effects could also be due to the indirect modification of the membrane properties by bexarotene, we compared the influence of bexarotene and cholesterol on membrane bilayer properties. It was observed that bexarotene did not induce the distinctive modifications of lipid membranes that are known for cholesterol. Taken together, these findings suggest a direct and specific interaction of bexarotene with the APP TMD. Consistent with this, cleavage of C99 by γ-secretase reconstituted in liposomes was inhibited in the presence of bexarotene. However, cleavage of Notch1, another major substrate lacking a cholesterol binding site, was also inhibited suggesting that bexarotene acts non-selectively on γ-secretase in model membranes. Interestingly, in cellular assays with HEK293 cells bexarotene lowered Aβ but did not inhibit γ-secretase. We conclude that bexarotene acts as a pleiotropic drug that can interfere with Aβ metabolism by multiple mechanisms.

RESULTS Bexarotene inserts into lipid membranes but does not influence lipid chain order and packing in the membrane. As bexarotene is a lipophilic molecule that was ascribed a high degree of structural similarity to the abundant membrane lipid cholesterol,22,23 its mode of action could be related to (i) its influence on lipid membrane structure and dynamics or (ii) direct interaction with either the C99 substrate or the γ-secretase. As it is known that very small changes in the molecular structure of cholesterol have a profound impact on its physicochemical profile in the membrane,26-31 we first compared the influence of bexarotene and cholesterol on lipid membranes. Cholesterol induces a very characteristic condensation of lipid chains that is highly specific for the molecule resulting in a much reduced cross-sectional area of the phospholipids in the membrane.30,32 Even seemingly small alterations in the cholesterol structure lead to an attenuation of lipid condensation.27,31,33 Given the highly specific interactions cholesterol undergoes with lipids, the proposed high 4 ACS Paragon Plus Environment

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similarity between bexarotene and cholesterol is questionable. Furthermore, from the comparison of the chemical structures of the two molecules displayed in Fig. 1 it is obvious that their structural similarity is limited. While cholesterol is characterized by a relatively flat sterol tetracyclic ring system featuring a smooth α-face and a C8 iso-branched lipid chain, bexarotene is completely lacking a flat ring structure and a lipid chain, which contributes significantly to phospholipid condensation by cholesterol.30 Nevertheless, both molecules are amphiphilic, featuring a carboxyl (bexarotene) or a hydroxyl group (cholesterol) on one end, leading to relatively similar octanol-water partition coefficients (logP) of 6.8 for bexarotene and 7.7 for cholesterol (calculated with Molinspiration, www.molinspiration.com). However, bexarotene has a much higher dipole moment of 6.23 D compared to cholesterol (2.01 D).

Figure 1. Chemical structures (A, B) and electrostatic surface potential (indicated by the gray clouds) determined from quantum chemical calculations done with Spartan (Wavefunction, Inc., Irvine, CA) (C, D) of bexarotene (A, C) and cholesterol (B, D). Both compounds feature an amphipathic character. Bexarotene has a higher polarity of the carboxyl head group compared to the hydroxyl group of cholesterol. Also, the hydrophobic moiety of bexarotene, which is thought to embed into the hydrophobic core of the membrane, shows some polarity, whereas the sterol part of cholesterol is very hydrophobic.

We first compared the influence of bexarotene and cholesterol on membranes, which is conveniently investigated by 2H NMR to determine the phospholipid chain order. Mixtures of chain deuterated POPC-d31 with either cholesterol or bexarotene were prepared and solid-state 2

H NMR spectra acquired. From such data, order parameter plots along the saturated POPC

sn-1 chain are calculated, which exhibit a characteristic decrease from the upper chain segments towards the chain end (Fig. 2). As demonstrated in many experimental studies, addition of cholesterol leads to a significant increase in lipid chain order along the entire chain 5 ACS Paragon Plus Environment

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and much higher order parameters are measured.32,34,35 In striking contrast, the presence of bexarotene causes no decrease in lipid chain order, indicating no lipid condensation at all. Instead, a small decrease in order is observed in the presence of varying amounts of bexarotene. We repeated these measurements for three additional lipid systems, including (i) saturated DMPC-d54 membranes, (ii) a more physiological mixture of POPC-d31 (37 mol%), POPE (18 mol%), porcine brain sphingomyelin (12 mol%), and cholesterol (33 mol%), and (iii) a mixture of lipids extracted from bovine heart tissue and POPC-d31 (80/20, mol/mol). The obtained 2H NMR order parameter profiles are shown in Supplementary Fig. S2. In accordance with the data for pure POPC membranes, no significant lipid condensation was induced by bexarotene.

Figure 2. Bexarotene does not cause membrane condensation like cholesterol. 2H NMR lipid chain order parameter of POPC-d31 in the presence of 10 and 20 mol% bexarotene at 30°C. For comparison order parameter plots for a pure POPC-d31 membrane and for POPC-d31 membranes in the presence of 10 and 20 mol% cholesterol are given.

We calculated the hydrophobic membrane thickness from the experimentally determined 2

H NMR order parameters using the mean torque model.36 For pure POPC membranes, the

hydrophopic thickness of one membrane leaflet is 11.6 Å. Upon addition of cholesterol, this value increases to 12.5 Å in the presence of 10 mol% cholesterol and to 13.2 Å in the presence of 20 mol%. This 14% increase in membrane thickness induced by cholesterol is not observed when bexarotene is incorporated into the membrane. In the latter case, we calculated single leaflet hydrophobic thicknesses of 11.6 Å and 11.4 Å for POPC membranes in the presence of 10 or 20 mol% bexarotene, respectively. Thus, bexarotene does not share the 6 ACS Paragon Plus Environment

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typical chain condensation and concomitant membrane thickening effects caused by cholesterol. The same analysis was carried out for the three other lipid systems investigated. In the presence of bexarotene, only insignificant alterations (∆L ≤ 0.1 Å) of the thickness of one lipid leaflet were observed, which are reported in Supplementary Table S1.

Orientation of Bexarotene in Lipid Membranes. Cholesterol is known to adopt an upright membrane orientation with a slight tilt angle of ~15° relative to the membrane normal.37,38 In this orientation, the hydroxyl group faces the aqueous phase. Membrane embedded cholesterol molecules undergo axially symmetric reorientations around their long axis and are subject to fluctuations along the membrane normal with amplitudes exceeding 5 Å.27,39 To obtain insight about the membrane orientation and location of bexarotene, 1H MAS NMR measurements were performed. In the

1

H MAS NMR spectrum of

POPC/bexarotene membranes NMR signals of both molecular species can be observed (Fig. 3). While the ring protons of bexarotene are well separated from the aliphatic lipid signals, the resonances of its methyl groups partially superimpose with the POPC signals.

Figure 3. Bexarotene aligns along the normal of POPC membranes. 1H MAS NMR spectrum of a POPC membrane in the presence of 20 mol% bexarotene at 30°C and a MAS frequency of 8 kHz. The signals of bexarotene are assigned by numbers according to the chemical structure shown on the left; the molecular groups of POPC are assigned according to the structure shown on the right. The aromatic region of the NMR spectrum is enlarged.

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The aromatic rings of bexarotene induce chemical shift changes for the POPC signals, which is related to the distance between the aromatic ring and the individual molecular groups of POPC.40-42 A plot of the induced chemical shift vs. the molecular groups of POPC along the membrane normal illuminates the position of the ring in the lipid membrane (Fig. 4). The insertion of the aromatic rings of bexarotene in the acyl chain region of the membrane is evident.

Figure 4. Induced 1H NMR chemical shift changes of the POPC signals in membranes induced by the addition of 20 mol% bexarotene. The plot of the induced chemical shift vs. the POPC segments provides a distribution profile for the bexarotene aromatic rings in the membrane.40-42

Further insight into the membrane position and orientation of bexarotene was obtained by measuring the cross-relaxation rates between specific protons of bexarotene and POPC in 1H1

H NOESY NMR experiments. Cross-relaxation rates can be determined quantitatively and

represent the contact probability between interacting molecules in membranes.43 The quantitative determination of the bexarotene-POPC cross-relaxation rates can provide the distribution function of a respective bexarotene proton within the membrane.44 For all aromatic protons of bexarotene the cross-relaxation rates to most signals of POPC could be measured and plotted against the POPC structure (Fig. 5). All plots exhibit a quite broad distribution for each bexarotene segment in the membrane, which is a consequence of the high mobility and molecular disorder in lipid membranes. Nevertheless, an estimation of the location and orientation of bexarotene in the membrane is possible from these plots. While the protons H1 and H2 exhibit a maximum of their distribution function at the upper chain/glycerol region, these distribution functions are shifted downwards to the hydrophobic core of the membrane for the protons H5 of the CH2-group, in between the aromatic rings. 8 ACS Paragon Plus Environment

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This is even more pronounced for the H3 and H4 protons, which show a maximum in the acyl chain region. Therefore, for bexarotene a position with the polar acid group in the upper chain region oriented toward the membrane-water interface and the more hydrophobic non-aromatic ring buried deeper in the membrane appears to pertain. This orientation and localization, which is similar to cholesterol, is a result of the physical interaction within the membrane. Bexarotene is an amphiphilic molecule (see also Fig. 1) that orients with its polar carboxyl group towards the aqueous phase, while the hydrophobic part of the molecule is deeply buried in the membrane.

Figure 5. Amphipathic orientation of bexarotene in a POPC membrane. Cross-relaxation rates obtained from 1H-1H NOESY NMR experiments of protons 1 through 5 of bexarotene to the molecular groups of POPC, confirm the amphiphilic nature of bexarotene. On the right side a sketch of the membrane position and orientation of bexarotene relative to a POPC molecule in the membrane is given.

Considering all the data at once, bexarotene exhibits a similar membrane topology as cholesterol, but completely fails to condense the phospholipid chains, which is a characteristic feature of cholesterol. 9 ACS Paragon Plus Environment

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Bexarotene Binds Weakly to C99. Having established its orientation and the impact of bexarotene on phospholipid membranes, we investigated its direct interaction with C99. 15

N-Labeled C99 was reconstituted into DMPC/DHPC bicelles and 1H-15N TROSY NMR

spectra were acquired as a function of the bexarotene concentration in the bicelles. Figure 6A shows the NMR spectra of C99 in DMPC/DHPC bicelles as it was titrated with bexarotene up to 10 mol% (mol% relative to the lipids present in the sample; bulk concentration of 10 mM). The narrow dispersion of the NMR signals of C99 is typical for largely unstructured proteins with a transmembrane α-helix and confirms previous investigations.24 Upon addition of bexarotene, some NMR signals shift, suggesting interaction/binding with bexarotene. Prominent signals that change their position and can be confidently assigned include V689, F690, A701, and I702 (APP770 numbering, see Supplementary Fig S1). These sites are located near the reported cholesterol binding site of C99, although these particular residues were reported not to be critical for binding.24 Plots of changes in peak chemical shifts versus bexarotene concentrations revealed that some peaks, such as I702, exhibited a hyperbolic curve that can be fit by the model for 1:1 complex formation with a weak KD of 7 ± 3 mol% (Fig. 6B). Other peaks, such as F690 exhibited shifts that were linearly dependent on bexarotene concentration up through 10 mol% (plot not shown), suggesting these peaks change only as a result of non-specific specific interaction between C99 and bexarotene. A third set of peaks showed modest curvature in plots of chemical shift versus bexarotene concentration, but were not well fit by a 1:1 binding model (Fig. 6B). The behavior of these peaks is consistent with changes that reflect the combined impact both of weak binding and non-specific interactions. We conclude from these data that bexarotene may bind specifically to the N-terminal extracellular end of the C99 transmembrane segment and flanking interfacial sites, but that binding is weak and likely does not reflect a mode of binding that resembles that of cholesterol. Moreover, bexarotene also appears to impact C99 via nonspecific interactions.

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Figure 6. Interaction of bexarotene with the residues of the cholesterol binding domain of C99. (A) 1H-15N TROSY NMR spectra of C99 (150 µM) in DMPC/DHPC bicelles (20%, i.e. 400 mM total lipid + detergent) at constant 15N-C99 and varying bexarotene (mol% relative to DMPC) at a temperature of 45°C and a pH of 5.8. Partial assignment is indicated. (B) Plot of proton chemical shifts as a function of bexarotene concentration. Dissociation constants were determined by fitting the chemical shifts to a binding model for one site specific binding without linear subtraction of non-specific interactions.

Bexarotene Stabilizes the Transmembrane Segment of APP. In light of the reported cholesterol binding site in C99,24 we reconstituted a 30 amino acid long peptide containing the TMD of APP, including the G700xxxG704 motif present in the cholesterolbinding domain24 (i.e. G29AIIG33 in Aβ numbering, see Supplementary Fig. S1) as well as the N-terminal and C-terminal lysine anchors (S26NKG AIIGLMVGGV VIATVIVITL VMLKKK55). This peptide, termed C9926-55, was reconstituted into POPC model membranes and examined in the absence and in the presence of cholesterol. Figure 7A displays the CD spectra of C9926-55 in the presence and absence of 10 mol % cholesterol. Both spectra display expected α-helical conformational character. The shape of the CD spectrum in the absence of 11 ACS Paragon Plus Environment

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cholesterol represents a conformation with a very high degree of helicity indicated by the stronger band at 218 nm compared to 212, and is similar to the spectrum of the model GWALP peptide (Supplementary Fig. S3), a peptide that has been reported to form an ideal TMD α-helix.45 In the presence of cholesterol an increase in the band at 218 nm is observed (i.e. θ218 is more negative), which usually indicates a higher degree of helicity. However, as demonstrated clearly in the detail of the CD spectrum (205-230 nm, Fig. 7B), cholesterol also causes a dramatic change in the shape of the spectrum. In the presence of cholesterol the θ218/θ212 ratio (θ218/θ212 = 1.01) is decreased compared to the ratio in the absence of cholesterol (θ218/θ212 = 1.05). For poly-alanine peptides with increasing residue numbers, changes in the CD spectrum resulting in increasing in θ218/θ212 ratios were interpreted as an increasing degree of helicity of longer poly-alanine peptides.46 Therefore, we interpret the lower θ218/θ212 ratio of the CD spectrum of C9926-55 in the cholesterol containing membrane (Fig. 7B) as a result of a decrease in helicity due to interaction of C9926-55 with cholesterol. Notably, when GWALP as well as a peptide containing the TMD of Notch1 (an alternative γ-secretase substrate), neither of which contain a cholesterol binding site,47 were reconstituted in a POPC membrane, the shape of the CD spectrum reflected a perfect α-helix, which was not altered by the presence of cholesterol as dramatically compared to the effect of cholesterol on the spectrum of C9926-55 (Supplementary Fig. S3). Thus, it is probably not the cholesterol induced lipid condensation (Fig. 2) but a direct interaction between cholesterol and C9926-55 causing its conformational change as detected by circular dicroism. Markedly, when we titrated bexarotene to cholesterol-containing vesicles, the shape of the CD spectra of C9926-55 approached that of the spectrum in the absence of cholesterol (Fig. 7C). The quantification of the ratio of θ218/θ212 at increasing bexarotene concentrations (Fig. 7D) shows that the effect of bexarotrene appeared to saturate not earlier than at 120 µM (5 mol% relative to lipid; measurements at higher concentrations became very inaccurate due to increased noise in the CD spectra). In these titrations, the peptide concentration was about 83 µM, i.e. the effect of bexarotene saturated approximately at stoichiometric bexarotene/C992655

ratios. The lipid concentration was 2.5 mM of which 10 mol% was cholesterol. Thus, the

bulk cholesterol-concentration was 250 µM, i.e. 3 times in excess over C9926-55 while the 10 mol% membrane concentration of cholesterol is 2 times the KD of cholesterol for full length C99,24 such that it is reasonable to assume that a majority of the binding sites of all C9926-55 molecules were occupied with cholesterol prior to addition of bexarotene. These observations suggest that bexarotene can compete with cholesterol at the cholesterol-binding site in the C9926-55. When cholesterol is replaced by bexarotene, the C9926-55 reverts to a high degree of 12 ACS Paragon Plus Environment

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helicity. Finally, the experiment of Fig. 7 was repeated at lower pH (pH 6.4) with model membranes composed of a lipid mixture (POPC/POPE/sphingomyelin, 3/1.5/1 molar ratio,78 which better reflects the environment of the cellular (early) endosomal compartment where cleavage of C99 by γ-secretase takes place and where the enzyme has optimal activity.79,80 The effect of bexarotene on the helicity of the TMD of APP was similar to the experiment with POPC (Supplementary Fig. S4).

Figure 7. Changes in the CD spectra of the C9926-55 peptide incorporated in a POPC/cholesterol (90/10) bilayer indicate a direct interaction of bexarotene with the C9926-55. (A) CD spectra of C9926-55 (83 µM) in LUV of POPC in the absence and in the presence of cholesterol. Lipid/protein molar ratio was 30. (B) Detail of the CD spectra displayed in panel (A). The altered shape of the CD spectrum of the peptide in large unilamellar POPC vesicles in the presence of cholesterol indicates a helix destabilization due to the interaction with cholesterol. (C) CD spectra of C9926-55 in POPC/cholesterol (90/10) measured at increasing bexarotene concentrations. (D) The increased ratio of ellipticity (θ) at 218/212 nm 13 ACS Paragon Plus Environment

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demonstrates a stabilization of the TMD helix due to the binding of bexarotene to the cholesterol binding site in the C9926-55. Data present the mean and SD of 2 experiments.

Bexarotene Non-Selectively Inhibits Processing of C99 by γ-Secretase in Liposomes, but not in Cells. To investigate if bexarotene directly affects γ-secretase activity, we used our previously described cell-free γ-secretase cleavage assay.48,49 In this assay, endogenous γ-secretase purified from HEK293 cells was reconstituted into POPC vesicles. Following addition of a C99-based substrate (C100-His6) and incubation at 37°C, Aβ and AICD cleavage products were analyzed by immunoblotting. As shown in Fig. 8A, bexarotene inhibited γ-secretase cleavage of C100-His6 in a dose-dependent manner. In order to test whether this inhibition is due to a specific binding of bexarotene to the C100-His6 substrate, we repeated the experiment with a Notch1-based substrate (N102-FmH).73 Strikingly, bexarotene also inhibited cleavage of the N102-FmH substrate by γ-secretase (Fig 8B). Thus, bexarotene-mediated γ-secretase inhibition is not substrate-specific. Inhibition of γ-secretase cleavage of C100-His6 is also observed in a modified in vitro assay using membranes derived from HEK cells (Supplementary Fig. S5), but to a lesser degree than in the in vitro assay using proteolipsomes (Fig. 8C). Finally, we tested the potential of bexarotene to inhibit substrate cleavage by γ-secretase in two HEK293 cell lines (Fig. 8C, D). In the first cell line, stably co-expressing the APP- and Notch1-based γ-secretase substrates C99-6myc and F-NEXT,81 no significant inhibition of the ε-site cleavage products AICD and NICD, and no accumulation of the substrates was observed in the presence of bexarotene, using the same concentrations as in the in vitro assays (Fig. 8A, B). Some inhibition was observed at the highest concentration at which however some cell toxicity was already observed. Interestingly, in another cell line that stably expresses the APPsw mutant,82 Aβ levels decreased in the presence of increasing bexarotene (Fig. 8D). However, again the latter was not due to inhibition of ε-cleavage by γ-secretase, since the substrates C99 and C83 did not accumulate in the presence of bexarotene. Apparently, at the tested concentrations bexarotene does not inhibit γ-secretase, but enhances the decay of secreted Aβ or its aggregation.

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Figure 8. Bexarotene inhibits γ-secretase cleavage of C99 and Notch1-based substrates in vitro but not in cell-based assays using HEK293 cells. (A) Immunoblot analysis of C100-His6 substrate cleavage by γ-secretase in the presence of increasing concentrations of bexarotene. Cleavage products: Aβ, AICD (APP intracellular domain). Substrate concentration in the assay is 0.5 µM, while lipid concentration is 780 µM. (B) as in A, but for the N102-FmH substrate. Cleavage products: Nβ (Notch1 Aβ-like peptide), NICD (Notch1 intracellular domain). Quantification of γ-secretase activity compared to the vehicle control (mean ± S.E.; n = 4) is shown in the lower panels. (C) Immunoblot analysis of substrate cleavage in HEK293 cells stably co-expressing the APP- and Notch1-based γ-secretase substrates C996myc and F-NEXT. Whereas a γ-secretase inhibitor (GSI, 1 µM L-685,458) inhibits the formation of ε-cleavage products (AICD and NICD) compared to the control C, this is not observed for bexarotene. (D) Immunoblot analysis of substrate cleavage in HEK293/sw cells stably expressing the APPsw mutant. Bexarotene lowers secreted Aβ, but does not inhibit γsecretase as the APP-CTF substrates are not altered. C83: CTF substrate resulting from αsecretase shedding of full length (FL) APP.6

Discussion The role of cholesterol as a contributing factor to Alzheimer’s disease has been widely suspected and elevated neuronal cholesterol levels increase the formation of Aβ.50,51 However, the exact mechanistic link is still under debate.52 Some studies have suggested that cholesterol facilitates membrane pore formation by Aβ oligomers.22,53 Thus, alterations of the 15 ACS Paragon Plus Environment

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cholesterol content of the cells may modify the neurotoxic potential of these early Aβ aggregates.22 Furthermore, APP, from which Aβ peptides of varying lengths are derived, is a transmembrane cholesterol binding protein.24 Therefore, it seems plausible that any small molecule that targets the cholesterol binding site of APP to specifically reduce its cleavage would hold pharmacological potential. One such pharmacologically interesting molecule is bexarotene, which is currently in clinical trials as a drug against cancer and Alzheimer’s disease.54 It has been shown that bexarotene interacts with Aβ peptides, which was interpreted as a consequence of high structural similarity between cholesterol and bexarotene.22 However, considering the significant chemical differences between the two molecules (see Fig. 1), the suggested structural homology between cholesterol and bexarotene seems rather limited. This uncertainty is amplified by the fact that even very small alterations of the cholesterol tetracyclic ring system27,55-57 or of its methyl branched chain30,31,33,38 can completely abolish the highly specific impact cholesterol exerts on lipid membranes. It is therefore not expected that bexarotene would mimic the effects of cholesterol on lipid packing. This was demonstrated by comparing the ability of either molecule to condense lipid packing, a very characteristic molecular feature of cholesterol. Our data clearly show that bexarotene does not condense lipids in various membrane systems (Figs. 2 and S2). Although bexarotene does not seem to specifically interact with (saturated) lipid chains in the membrane as cholesterol does, it was seen to be efficiently incorporated into the bilayer, partitioning into the lipid water interface so as to expose its carboxylate carboxyl group toward the aqueous phase (Fig. 5). It is this carboxylate group that has been suggested to interact with the peptide backbone of residues 25 through 35 of Aβ at residue Gly25 (Gly696 in APP numbering).23 This site could be involved in hydrogen bonding and/or electrostatic interactions between bexarotene and C99. We found that the residues of C99 near those thought to interact with cholesterol24 also exhibited major 1H,15N-TROSY NMR peak shifts in response to titration with bexarotene. These include Phe690, Ala701, and and Ile702 (Fig. 6). The NMR peaks from the Gly700 and Gly704 sites thought be critical for cholesterol biding were, however, not impacted by bexarotene. This may suggest that the binding sites of bexarotene and cholesterol at the extracellular end of the APP transmembrane domain and flanking juxtamembrane segment are similarly located, but that their detailed interactions with C99 are different. Nevertheless, bexarotene interacts with the same segment on APP as cholesterol and it is tempting to suggest that these molecules are likely to compete for binding to C99. As Aβ peptides conserve a tandem GxxxG zipper motif after they are cleaved off C99 by γ-secretase, 16 ACS Paragon Plus Environment

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this would be in agreement with a recent molecular docking study which confirmed high affinity binding of bexarotene to Aβ peptides of varying length by occupying cholesterol binding sites,23 and supports the potential of bexarotene as an inhibitor of Aβ.20 Results from our NMR-monitored titration of C99 by bexarotene were consistent both with weak binding of bexarotene to C99 (KD on the order of 5-10 mol%) and to additional changes in the spectrum of C99 that arise from non-specific effects of the drug. KD for binding of bexarotene to C99 seems on the same order of that previously observed for cholesterol (ca. 5 mol%).24 We note also that because cholesterol binding to C99 appears to compete with homodimerization of C99,25 it is possible that bexarotene could impact homodimerization of C99. There are competing models for how C99 homodimerizes,58-63 which are not necessarily mutually exclusive—it is possible that APP and its derived C99 domain adopt different dimer modes depending on membrane lipid composition or other factors.64 We discovered that bexarotene binding leads to a stabilization of the α-helix structure of the C99-TMD (Fig. 7). Here we found evidence that bexarotene can compete with cholesterol at the cholesterol binding domain of the C99-TMD peptide. Thus, when C9926-55 is embedded in a pure POPC bilayer, its helicity is higher compared to its conformation in a POPC bilayer containing 10% cholesterol (Fig 7A, B). Apparently, cholesterol association with its partial binding site in C9926-55 leads to a destabilization of the α-helix. Subsequent replacement of cholesterol by bexarotene results in a dose-dependent stabilization of the helix, which saturates at approximately stoichiometric C9926-55/bexarotene ratios. Possibly, when bexarotene replaces cholesterol at its partial binding site in C9926-55, the more flexible structure of bexarotene compared to cholesterol allows the helix to adopt an energetically more favorable intrinsic conformation. The much stronger binding affinity for bexarotene found in the CD experiments (Fig. 7) involving C9926-55 compared to the NMR experiments (Fig. 6) on full length C99 might be due to different experimental conditions. The CD experiments were performed with detergent-free bilayers into which bexarotene might have partitioned more avidly than into the detergent-containing bicelles that were used in the NMR experiments. In addition, in the NMR experiments the large fraction of the bicelles that were devoid of C99 might have led to an overestimation of the KD. With these biophysical data on the membrane effect of bexarotene and the binding of bexarotene to C99 and its stabilizing effect on the TMD in hand, we hypothesized that bexarotene could effectively and selectively inhibit processing of C99 by γ-secretase. Indeed, bexarotene inhibited γ-secretase cleavage of C99 in a dose-dependent manner in our established cell-free γ-secretase cleavage assay48,49 with POPC proteoliposomes (Fig. 8A). 17 ACS Paragon Plus Environment

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Although no reliable KD for the interaction of bexarotene with C99 could be derived from our biophysical experiments, the interaction of bexarotene with C99 is suggested to be very strong, i.e. even stronger then previously observed for cholesterol (Fig. 7). Despite its presumably strong interaction with C99, in the in vitro assay, bexarotene could inhibit substrate

cleavage

only

significantly

at

relatively

high

concentrations,

i.e.

at

bexarotene/substrate molar ratios larger than 100. Moreover, our observation that cleavage of Notch1, which does not contain a cholesterol-binding site,47 can also be inhibited by bexarotene, suggests that the inhibition of γ-secretase substrate processing by bexarotene is not due to a specific interaction with the substrate, but due to some other mechanism. Hence, it seems unlikely that one could use bexarotene as a drug to specifically suppress Aβ production by γ-secretase without impeding cleavage of other crucial substrates, such as Notch1. Taken together, bexarotene decreases the processing of C99 by γ-secretase, in vitro, which is possibly due to a weak direct interaction of bexarotene with γ-secretase. Alternatively, as many studies have demonstrated that the lipid composition of the membrane in which γ-secretase is embedded directly and significantly influences its activity,65-68 bexarotene might also inhibit the proteolytic activity γ-secretase due to a yet to be defined modulation of biophysical properties of the host membrane. To further investigate the potential of bexarotene as an inhibitor of Aβ production by γ-secretase, we performed activity assays in membrane preparations of HEK293 cells (Fig. S5). Here, inhibition of processing of C99 by γ-secretase was inhibited but the effect was decreased compared to the in vitro assay. It should be noted that in the above-mentioned experiments extremely high concentrations of bexarotene were used (up to 160 µM). These concentrations would be too toxic to apply in vivo or in neurons where concentrations above 1 µM should be prevented19,69 and are beyond the concentrations which patients could ever receive. In our cellular assays with HEK293 cells no significant inhibition of γ-secretase could be observed up to 80 µM, above which bexarotene also became toxic. Interestingly, although γ-secretase was not inhibited by bexarotene in the cell-based assays, levels of secreted Aβ were reduced in HEK293/sw cells indicating that bexatorene exerted this effect by a distinct mechanism. In summary, although bexarotene may bind to the cholesterol binding site of the TMD of C99, our findings demonstrate that bexarotene cannot be effective as drug to reduce the production of toxic Aβ peptides by γ-secretase. However, as the cholesterol binding site of C99 is conserved in the released Aβ peptides, bound bexarotene might increase the helical content of secreted Aβ peptides. Recently, foldamer compounds, by increasing the helical 18 ACS Paragon Plus Environment

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content of Aβ, were shown to impede the formation of β-sheet rich Aβ amyloids.70 Similary, bexarotene, at low non-toxic concentrations, might impede the toxicy of Aβ by preventing its misfolding, oligomer formation and/or perturbation of cellular membranes.

METHODS Materials. Bexarotene was purchased from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). The lipids 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as well as the sn-1 chain perdeuterated POPC-d31, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE), porcine brain sphingomyelin, bovine heart lipid extract, and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, Al). Expression and Purification of C99 for Reconstitution into Bicelles and NMR Studies. An expression construct of human C99 (residues 672-770 of full length APP) containing a C-terminal His6 purification tag24 was expressed in E. coli and purified using metal-ion affinity chromatography as described previously.24,71 Tagged C99 was expressed in BL21 DE3 cells using minimal medium containing 15NH4Cl. The 15N-labeled C99 from 8 g of cell pellet inclusion bodies was bound to 2 mL of Ni(II)-NTA resin. Resin was washed extensively with 0.2% SDS in TBS buffer pH 7.8, followed by on-column exchange to 1% DHPC with 10 mM imidazole pH 7.8, then to 2% isotropic q = 0.3 DMPC/DHPC bicelles with 10 mM imidazole, pH 7.8 (where q is the DMPC to DHPC mole ratio). C99 was then eluted from the column with elution buffer containing 2% DMPC/DHPC bicelles and 350 mM imidazole, pH 7.8. The eluate was collected and concentrated 10 times by centrifugal ultrafiltration using a 10 kDa cut-off filter. The concentrate was diluted in buffer containing DHPC at its CMC with 10 mM imidazole at pH 7.8 and concentrated again 10 times to reduce the final imidazole concentration. EDTA was added to the concentrated sample to 1 mM. The pH of the sample was then lowered to 5.8 with acetic acid and D2O was added to 10% for NMR field-locking purpose. Final samples contained C99 at a concentration of 150 µM, 20% DMPC/DHPC bicelles (total amphiphile concentration of ~400 mM, i.e. 91 mM DMPC and 304 mM DHPC), 35 mM imidazole, and 1 mM EDTA. A concentrated stock solution of 20 mol% bexarotene was prepared in 20% DMPC/DHPC bicelles at pH 5.8. A solvent mixture of 95% benzene and 5% ethanol was used to dissolve powdered bexarotene, lipids, and detergents before being lyophilized. Lyophilized material was reconstituted in 35 mM imidazole pH 5.8 with 1 mM EDTA and freeze-thaw cycles with sonication were used to 19 ACS Paragon Plus Environment

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solubilize the mixture. Additionally, a 20% bicelle stock without bexarotene was created in an identical manner. For titrations, these concentrated stock solutions were mixed with purified C99 as described above to yield bexarotene samples (where 10 mol% indicates a bexarotene:DMPC mole ratio of 1:9, i.e. 10 mol% corresponds to 91/9 = 10 mM bexarotene). Bexarotene was not soluble in C99-containing NMR samples at concentrations higher than 10 mol%. Samples were equilibrated for at least 8 hours before NMR spectroscopy. 1H-15NTROSY spectra were acquired for each sample at 45°C using a Bruker 800 MHz NMR spectrometer.

γ-Secretase Cleavage Assays. For cell-free γ-secretase cleavage assays of APP and Notch1, affinity purified C100-His672 and N102-FmH73 constructs were used. γ-Secretase cleavage assays were carried out using purified γ-secretase reconstituted into small unilamellar vesicles (SUV) composed of POPC, essentially as described.48,49 Alternatively, membranes isolated from HEK293 cells were used48. Here, cell homogenates were centrifuged for 30 min at 1000g and 4°C. The supernatant fraction was supplemented with 5 % (v/v) glycerol and centrifuged for 60 min at 130,000g, 4° C. The pellet containing the membrane fraction was washed and resuspended in PBS (4.5 dish/ml containing 1x protease inhibitors (Complete, Roche)48. After addition of CHAPSO to a final concentration of 0.25 % wt/vol, 0.1 dish/sample were mixed with 0.4 µM C100-His6 and the samples were incubated O/N at 37°C. In all assays, bexarotene was added prior to incubation at the indicated concentrations from stock solutions in DMSO. Following Tris-Tricine SDS-PAGE, cleavage products of C100-His6 were detected by immunoblotting using antibodies 2D874 (Aβ) and Pentahis (Qiagen) (AICD) and of N102-FmH using antibodies 5E9 (Nβ)75 and cleaved Notch1 (Val1744) (Cell Signalling) (NICD) and quantified as described.48,49 Cell-based γ-secretase assays were performed using HEK293 cells stably co-expressing C99-6myc and F-NEXT81 or expressing Swedish mutant APP (HEK293/sw),82 which were cultured as described.81,83 Bexarotene was added at the indicated concentrations from stock solutions in DMSO to confluent cells and media were conditioned for 4 h. L-685,45884 was used at 1 µM as a GSI control. C99-6myc, F-NEXT as well as AICD and NICD cleavage products were analyzed using antibody A14 against c-myc (Santa Cruz). Aβ and soluble sAPPα was analyzed from conditioned media of HEK293/sw cells by direct immunoblotting using antibody 2D874. Full length APP and APP CTFs including C99 in these cells were analyzed from cell lysates74 using antibody 6687.83 20 ACS Paragon Plus Environment

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Membrane Sample Preparation for Solid-State NMR. Chloroform solutions of bexarotene and the respective phospholipids were mixed to achieve the desired molar ratios. After evaporation of the solvent and re-dissolving in cyclohexane the samples were lyophilized overnight at high vacuum to yield a fluffy powder. The samples were hydrated with 40 wt% deuterium-depleted water for 2H NMR measurements or D2O for 1H NMR measurements. The samples were equilibrated by stirring and ten freeze-thaw cycles and transferred into 4 mm HR MAS rotors with spherical Kel-F inserts. 2

H NMR Measurements. 2H NMR spectra were acquired using a Bruker DRX300 NMR

spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) operating at a resonance frequency of 46.1 MHz for 2H using a double channel solids probe equipped with a 5 mm solenoid coil. The 2H NMR spectra were collected using a phase-cycled quadrupolar echo sequence. The two 90° pulses of ca. 3.3 µs were separated by a 50 µs delay. The relaxation delay was 1 s. All NMR spectra were measured at a temperature of 30°C. After de-Paking76 the smoothed order parameter profiles where calculated as described in detail in the literature.35 1

H MAS NMR Spectroscopy. 1H MAS NMR measurements were carried out on a

Bruker Avance III 600 MHz spectrometer using a 4 mm HR MAS probe at a MAS frequency of 8 kHz. A 2H lock was used for field stability. The 90° pulse length was 4 µs. All 1H NMR spectra were referenced with respect to the terminal methyl group of the POPC lipid chains at 0.885 ppm. All measurements were conducted at a temperature of 30°C. Two-dimensional 1H MAS NOESY spectra77 were accumulated at five mixing times (between 0.1 ms and 500 ms). 536 data points were acquired in the indirect dimension with a relaxation delay of 3.5 s. The volume of the respective diagonal and cross peaks was integrated using the Bruker Topspin 2.1 software package. NOE build-up curves were fit using Origin (OriginLab Cooperation, Northampton, MA) to the spin pair model yielding cross relaxation rates (σij) according to:44

Aij (t m ) =

A jj (0)

2

⋅(1 − exp(−2σ ij t m )) exp(−t m / Tij ) ,

(1)

where Aij(tm) represents the cross peak volume at mixing time tm and Ajj(0) is the diagonal peak volume at mixing time zero, while 1/Tij defines the rate of magnetization leakage towards the lattice.

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Circular Dichroism (CD) Spectroscopy. Wildtype C9926-55, a 30 amino acid long peptide comprising residues 697 to 726 of APP (using APP770 numbering, see Supplementary Fig. S1), i.e. S697NKG AIIGLMVGGV VIATVIVITL VMLKKK726, was purchased from Peptide Specialty Laboratories GmbH, Heidelberg, Germany). This peptide was incorporated into large unilamellar vesicles (LUV) composed of POPC at a lipid/protein molar ratio of 30:1 by co-mixing 500 µg peptide with 3.72 mg POPC in 1 ml hexafluoroisopropanol (HFIP). After evaporation of the HFIP, the mixture was dissolved in 1 ml cyclohexane and lyophilized. The resulting fluffy powder was dissolved in 977 µl buffer (10 mM sodium phosphate, pH 7.4). After 10 freeze-thaw cycles, LUVs were prepared through extrusion using a 100-nm polycarbonate membrane and a LipofastTM extruder device (Armatis GmbH, Weinheim, Germany). Alternatively, model membranes composed of a lipid mixture of POPC/POPE/sphingomyelin, 3/1.5/1 molar ratio were used. In control experiments TMDNotch1

(PAQLHFMYVA

AAA

FVLLFF

VGCGVLLSRK

RCD)

and

GWALP

(GGALWLALAL ALALALALWL AGA) peptides were incorporated with the same protocol. Spectra were recorded with a Jasco 810 spectropolarimeter. A cuvette with a 0.1-cm path length was filled with 200 µl of the LUV/ C9926-55 preparation in which the final peptide concentration was 83 µM and lipid concentration 2.5 mM. Bexarotene was added in aliquots from a 10 mM stock solution in ethanol.

Acknowledgements We thank Drs. Masaysasu Okochi, Taisuke Tomita and Takeshi Iwatsubo for the kind gift of reagents, Brigitte Nuscher for technical assistance and Katherine LaClair for helpful comments on the manuscript. Dr. Alexander Vogel is acknowledged for support in the analysis of the order parameters.

Author Contributions DH, CRS, and HS designed the study, FK, HAS, EW, GB, HH, JMH, LMLP performed the experiments and analyzed the data, DH, FK, CRS, and HS wrote the paper, all authors made comments on the paper and discussed the results.

Funding Sources This study was supported by the DFG (FOR 2290, P4 and P8 (H.S. and D.H.), grant STE 847/6-1 (H.S.), by US NIH RO1AG056147 (C.R.S.) and T32 CA009582 (J.M.H.), and the VERUM Stiftung für Verhalten und Umwelt (F.K.). 22 ACS Paragon Plus Environment

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Conflict of Interest The authors declare no competing financial interest.

Supporting Information:

Peptide sequences (Fig. S1), 2H NMR order parameters of DMPC, POPC/POPE/SPM/Chol (37: 18:12:33 molar ratio), and bovine heart tissue lipids/POPC-d31 (2/1 molar ratio) (Fig. S2), CD spectra of C9926-55, GWALP and TMD-Notch1 peptides (Fig. S3), CD spectra of C9926-55, in POPC/POPE/BBSM (3/1.5/1 molar ratio) with 10 mol% cholesterol (Fig. S4), γ-secretase cleavage of C99 in membranes extracted from HEK cells (Fig. S5), and membrane leaflet thickness determined from the 2H NMR data (Table S1).

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15. Veeraraghavalu, K.; Zhang, C.; Miller, S.; Hefendehl, J. K.; Rajapaksha, T. W.; Ulrich, J.; Jucker, M.; Holtzman, D. M.; Tanzi, R. E.; Vassar, R.; Sisodia, S. S. Comment on "ApoEdirected therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models". Science. 2013, 340, 924-92f. 16. Tesseur, I.; Lo, A. C.; Roberfroid, A.; Dietvorst, S.; Van, B. B.; Borgers, M.; Gijsen, H.; Moechars, D.; Mercken, M.; Kemp, J.; D'Hooge, R.; De, S. B. Comment on "ApoEdirected therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models". Science. 2013, 340, 924-92e. 17. Price, A. R.; Xu, G.; Siemienski, Z. B.; Smithson, L. A.; Borchelt, D. R.; Golde, T. E.; Felsenstein, K. M. Comment on "ApoE-directed therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models". Science. 2013, 340, 924-92d. 18. Fitz, N. F.; Cronican, A. A.; Lefterov, I.; Koldamova, R. Comment on "ApoE-directed therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models". Science. 2013, 340, 924-92c. 19. Mounier,A.; Georgiev, D.; Nam, K. N.; Fitz, N. F., Castranio, E. L.; Wolfe, C. M.; Cronican, A. A.; Schug, J.; Lefterov, I.; Koldamova, R. Bexarotene-activated retinoid X receptors regulate neuronal differentiation and dendritic complexity. J. Neurosci. 2015, 35, 11862-11876. 20. Habchi, J.; Arosio, P.; Perni, M.; Costa, A. R.; Yagi-Utsumi, M.; Joshi, P.; Chia, S.; Cohen, S. I.; Muller, M. B.; Linse, S.; Nollen, E. A.; Dobson, C. M.; Knowles, T. P.; Vendruscolo, M. An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Abeta42 aggregates linked with Alzheimer's disease. Sci. Adv. 2016, 2, e1501244. 21. Huy, P. D. Q.; Thai, N. Q.; Bednarikova, Z.; Phuc, L. H.; Linh, H. Q.; Gazova, Z.; Li, M. S. Bexarotene does not clear amyloid beta plaques but delays fibril growth: molecular mechanisms. ACS Chem. Neurosci. 2017, 8, 1960-1969. 22. Di Scala, C.; Chahinian, H.; Yahi, N.; Garmy, N.; Fantini, J. Interaction of Alzheimer's beta-amyloid peptides with cholesterol: mechanistic insights into amyloid pore formation. Biochemistry. 2014, 53, 4489-4502. 23. Fantini, J.; Di, S. C.; Yahi, N.; Troadec, J. D.; Sadelli, K.; Chahinian, H.; Garmy, N. Bexarotene blocks calcium-permeable ion channels formed by neurotoxic Alzheimer's beta-amyloid peptides. ACS Chem. Neurosci. 2014, 5, 216-224. 24. Barrett, P. J.; Song, Y.; Van Horn, W. D.; Hustedt, E. J.; Schafer, J. M.; Hadziselimovic, A.; Beel, A. J.; Sanders, C. R. The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol. Science. 2012, 336, 1168-1171. 25. Song, Y.; Hustedt, E. J.; Brandon, S.; Sanders, C. R. Competition between homodimerization and cholesterol binding to the C99 domain of the amyloid precursor protein. Biochemistry. 2013, 52, 5051-5064. 26. Wang, J.; Megha; London, E. Relationship between sterol/steroid structure and participation in ordered lipid domains (lipid rafts): implications for lipid raft structure and function. Biochemistry 2004, 43, 1010-1018. 25 ACS Paragon Plus Environment

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27. Scheidt, H. A.; Müller, P.; Herrmann, A.; Huster, D. The potential of fluorescent and spinlabeled steroid analogs to mimic natural cholesterol. J. Biol. Chem. 2003, 278, 4556345569. 28. Rog, T.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen, M. Ordering effects of cholesterol and its analogues. Biochim. Biophys. Acta. 2009, 1788, 97-121. 29. Shaghaghi, M.; Chen, M. T.; Hsueh, Y. W.; Zuckermann, M. J.; Thewalt, J. L. Effect of sterol structure on the physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine membranes determined using 2H nuclear magnetic resonance. Langmuir. 2016, 32, 7654-7663. 30. Scheidt, H. A.; Meyer, T.; Nikolaus, J.; Baek, D. J.; Haralampiev, I.; Thomas, L.; Bittman, R.; Herrmann, A.; Müller, P.; Huster D. Cholesterol's aliphatic side chain structure modulates membrane properties. Angew. Chem. Int. Ed. 2013, 52, 12848-12851. 31. Meyer, T.; Baek, D. J.; Bittman, R.; Haralampiev, I.; Muller, P.; Herrmann, A.; Huster, D.; Scheidt, H. A. Membrane properties of cholesterol analogs with an unbranched aliphatic side chain. Chem. Phys. Lipids. 2014, 184, 1-6. 32. Vist, M. R.; Davis, J. H. Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry. Biochemistry 1990, 29, 451-464. 33. Huster, D.; Scheidt, H. A.; Arnold, K.; Herrmann, A.; Muller, P. Desmosterol may replace cholesterol in lipid membranes. Biophys. J. 2005, 88, 1838-1844. 34. Trouard, T. P.; Alam, T. M.; Zajicek, J.; Brown, M. F. Angular anisotropy of 2H NMR spectral densities in phospholipid-bilayers containing cholesterol. Chem. Phys. Lett. 1992, 189, 67-75. 35. Huster, D.; Arnold, K.; Gawrisch, K. Influence of docosahexaenoic acid and cholesterol on lateral lipid organization in phospholipid membranes. Biochemistry 1998, 37, 1729917308. 36. Petrache, H. I.; Dodd, S. W.; Brown, M. F. Area per lipid and acyl length distributions in fluid phosphatidylcholines determined by 2H NMR spectroscopy. Biophys. J. 2000, 79, 3172-3192. 37. Dufourc, E. J.; Parish, E. J.; Chitrakorn, S.; Smith, C. P. Structural and dynamical details of cholesterol-lipid interaction as revealed by deuterium NMR. Biochemistry 1984, 23, 6062-6071. 38. Vogel, A.; Scheidt, H. A.; Baek, D. J.; Bittman, R.; Huster, D. Structure and dynamics of the aliphatic cholesterol side chain in membranes as studied by 2H NMR spectroscopy and molecular dynamics simulation. Phys. Chem. Chem. Phys. 2016, 18, 3730-3738. 39. Endress, E.; Heller, H.; Casalta, H.; Brown, M. F.; Bayerl, T. M. Anisotropic Motion and Molecular Dynamics of Cholesterol, Lanosterol, and Ergosterol in Lecithin Bilayers Studied by Quasi-elastic Neutron Scattering. Biochemistry 2002, 41, 13078-13086.

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40. Scheidt, H. A.; Pampel, A.; Nissler, L.; Gebhardt, R.; Huster, D. Investigation of the membrane localization and distribution of flavonoids by high-resolution magic angle spinning NMR spectroscopy. Biochim. Biophys. Acta 2004, 1663, 97-107. 41. Stamm, H.; Jackel, H. Relative ring-current effects based on a new model for aromaticsolvent-induced shift. J. Am. Chem. Soc. 1989, 111, 6544-6550. 42. Scheidt, H. A.; Haralampiev, I.; Theisgen, S.; Schirbel, A.; Sbiera, S.; Huster, D.; Kroiss, M.; Muller, P. The adrenal specific toxicant mitotane directly interacts with lipid membranes and alters membrane properties depending on lipid composition. Mol. Cell Endocrinol. 2016, 428, 68-81. 43. Huster, D.; Arnold, K.; Gawrisch, K. Investigation of lipid organization in biological membranes by two-dimensional nuclear Overhauser enhancement spectroscopy. J. Phys. Chem. B 1999, 103, 243-251. 44. Scheidt, H. A.; Huster, D. The interaction of small molecules with phospholipid membranes studied by 1H NOESY NMR under magic-angle spinning. Acta Pharmacol. Sin. 2008, 29, 35-49. 45. de Planque, M. R.; Killian, J. A. Protein-lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring. Mol. Membr. Biol. 2003, 20, 271-284. 46. Heitmann, B.; Job, G. E.; Kennedy, R. J.; Walker, S. M.; Kemp, D. S. Water-solubilized, cap-stabilized, helical polyalanines: calibration standards for NMR and CD analyses. J. Am. Chem. Soc. 2005, 127, 1690-1704. 47. Deatherage, C. L.; Lu, Z.; Kroncke, B. M.; Ma, S.; Smith, J. A.; Voehler, M. W.; McFeeters, R. L.; Sanders, C. R. Structural and biochemical differences between the Notch and the amyloid precursor protein transmembrane domains. Sci. Adv. 2017, 3, e1602794. 48. Winkler, E.; Hobson, S.; Fukumori, A.; Dumpelfeld, B.; Luebbers, T.; Baumann, K.; Haass, C.; Hopf, C.; Steiner, H. Purification, pharmacological modulation, and biochemical characterization of interactors of endogenous human gamma-secretase. Biochemistry. 2009, 48, 1183-1197. 49. Winkler, E.; Kamp, F.; Scheuring, J.; Ebke, A.; Fukumori, A.; Steiner, H. Generation of Alzheimer disease-associated amyloid beta42/43 peptide by gamma-secretase can be inhibited directly by modulation of membrane thickness. J. Biol. Chem. 2012, 287, 2132621334. 50. Hartmann, T.; Kuchenbecker, J.; Grimm, M. O. Alzheimer's disease: the lipid connection. J. Neurochem. 2007, 103, 159-170. 51. Vetrivel, K. S.; Thinakaran, G. Membrane rafts in Alzheimer's disease beta-amyloid production. Biochim. Biophys. Acta. 2010, 1801, 860-867. 52. Wood, W. G.; Li, L.; Muller, W. E.; Eckert, G. P. Cholesterol as a causative factor in Alzheimer's disease: a debatable hypothesis. J. Neurochem. 2014, 129, 559-572.

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53. Di, S. C.; Troadec, J. D.; Lelievre, C.; Garmy, N.; Fantini, J.; Chahinian, H. Mechanism of cholesterol-assisted oligomeric channel formation by a short Alzheimer beta-amyloid peptide. J. Neurochem. 2014, 128, 186-195. 54. Cummings, J. L., Zhong, K., Kinney, J. W., Heaney, C., Moll-Tudla, J., Joshi, A. Doubleblind, placebo-controlled,proof-of-concept trial of bexarotene Xin moderate Alzheimer’s disease. Alzheimers Res. Ther. 2016, 8, 4. 55. Milles, S.; Meyer, T.; Scheidt, H. A.; Schwarzer, R.; Thomas, L.; Marek, M.; Szente, L.; Bittman, R.; Herrmann, A.; Gunther, P. T.; Huster, D.; Muller, P. Organization of fluorescent cholesterol analogs in lipid bilayers - lessons from cyclodextrin extraction. Biochim. Biophys. Acta. 2013, 1828, 1822-1828. 56. Wharton, S. A.; Green, C. Effect of sterol structure on the transfer of sterols and phospholipids from liposomes to erythrocytes in vitro. Biochim. Biophys. Acta 1982, 711, 398-402. 57. Child, P.; Kuksis, A. Critical role of ring structure in the differential uptake of cholesterol and plant sterols by membrane preparations in vitro. J. Lipid Res. 1983, 24, 1196-1209. 58. Kienlen-Campard, P.; Tasiaux, B.; Van, H. J.; Li, M.; Huysseune, S.; Sato, T.; Fei, J. Aimoto, S.; Courtoy, P. J.; Smith, S. O.; Constantinescu, S. N.; Octave, J. Amyloidogenic processing but not amyloid precursor protein (APP) intracellular terminal domain production requires a precisely oriented APP dimer assembled transmembrane GXXXG motifs. J. Biol. Chem. 2008, 283, 7733-7744.

Z.; N. Cby

59. Sato, T.; Tang, T. C.; Reubins, G.; Fei, J. Z.; Fujimoto, T.; Kienlen-Campard, P.; Constantinescu, S. N.; Octave, J. N.; Aimoto, S.; Smith, S. O. A helix-to-coil transition at the epsilon-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 1421-1426. 60. Chen, W.; Gamache, E.; Rosenman, D. J.; Xie, J.; Lopez, M. M.; Li, Y. M.; Wang, C. Familial Alzheimer's mutations within APPTM increase Abeta42 production by enhancing accessibility of epsilon-cleavage site. Nat. Commun. 2014, 5, 3037. 61. Miyashita, N.; Straub, J. E.; Thirumalai, D.; Sugita, Y. Transmembrane structures of amyloid precursor protein dimer predicted by replica-exchange molecular dynamics simulations. J. Am. Chem. Soc. 2009, 131, 3438-3439. 62. Decock, M.; El, H. L.; Stanga, S.; Dewachter, I.; Octave, J. N.; Smith, S. O.; Constantinescu, S. N.; Kienlen-Campard, P. Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing. FEBS Open. Bio. 2015, 5, 763-73. 63. Nadezhdin, K. D.; Bocharova, O. V.; Bocharov, E. V.; Arseniev, A. S. Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment. FEBS Lett. 2012, 586, 1687-1692. 64. Dominguez, L.; Foster, L.; Meredith, S. C.; Straub, J. E.; Thirumalai, D. Structural heterogeneity in transmembrane amyloid precursor protein homodimer is a consequence of environmental selection. J. Am. Chem. Soc. 2014, 136, 9619-9626. 28 ACS Paragon Plus Environment

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65. Cordy, J. M.; Hooper, N. M.; Turner, A. J. The involvement of lipid rafts in Alzheimer's disease. Mol. Membr. Biol. 2006, 23, 111-122. 66. Osenkowski, P.; Ye, W.; Wang, R.; Wolfe, M. S.; Selkoe, D. J. Direct and potent regulation of gamma-secretase by its lipid microenvironment. J. Biol. Chem. 2008, 283, 22529-22540. 67. Holmes, O.; Paturi, S.; Ye, W.; Wolfe, M. S.; Selkoe, D. J. Effects of membrane lipids on the activity and processivity of purified gamma-secretase. Biochemistry. 2012, 51, 35653575. 68. Winkler, E.; Kamp, F.; Scheuring, J.; Ebke, A.; Fukumori, A.; Steiner, H. Generation of Alzheimer disease-associated amyloid beta42/43 peptide by gamma-secretase can be inhibited directly by modulation of membrane thickness. J. Biol. Chem. 2012, 287, 2132621334. 69. Tachibana, M.; Shinohara, M.; Yamazaki, Y.; Liu, C. C.; Rogers, J.; Bu, G.; Kanekiyo, T. Rexcuing effects of RXR agonists bexarotene on aging-related synapse loss depend on neuronal LRP1. Exp. Neurol. 2016, 277, 1-9. 70. Kumar, S.; Henning-Knechtel, A.; Chehade, I.; Magzoub, M.; Hamilton, A. D. Foldamermediated structural rearrangement attenuates Aβ oligomerization and cytotoxicity. J. Am., Chem. Soc. 2017, 139, 17098-17108. 71. Beel, A. J.; Mobley, C. K.; Kim, H. J.; Tian, F.; Hadziselimovic, A.; Jap, B.; Prestegard, J. H.; Sanders, C. R. Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor? Biochemistry. 2008, 47, 9428-9446. 72. Edbauer, D.; Winkler, E.; Regula, J. T.; Pesold, B.; Steiner, H.; Haass, C. Reconstitution of gamma-secretase activity. Nat. Cell Biol. 2003, 5, 486-488. 73. Takahashi, Y.; Hayashi, I.; Tominari, Y.; Rikimaru, K.; Morohashi, Y.; Kan, T.; Natsugari, H.; Fukuyama, T.; Tomita, T.; Iwatsubo, T. Sulindac sulfide is a noncompetitive gammasecretase inhibitor that preferentially reduces Abeta 42 generation. J. Biol. Chem. 2003, 278, 18664-18670. 74. Shirotani, K.; Tomioka, M.; Kremmer, E.; Haass, C.; Steiner, H. Pathological activity of familial Alzheimer's disease-associated mutant presenilin can be executed by six different gamma-secretase complexes. Neurobiol. Dis. 2007, 27, 102-107. 75. Okochi, M.; Fukumori, A.; Jiang, J.; Itoh, N.; Kimura, R.; Steiner, H.; Haass, C.; Tagami, S.; Takeda, M. Secretion of the Notch-1 Abeta-like peptide during Notch signaling. J. Biol. Chem. 2006, 281, 7890-7898. 76. McCabe, M. A.; Wassall, S. R. Fast-Fourier-transform DePaking. J. Magn. Reson. B 1995, 106, 80-82. 77. Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. Investigation of exchange processes by two-dimensional NMR spectroscopy. J. Chem. Phys. 1979, 71, 4546-4553.

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78. van Meer, G.; Voelker, D. R.; Feigenson, G. W. Membrane lipids: where they are and how they bahave. Nat. Rev. Cell Biol. 2008, 9, 112-124. 79. Kaether, C.; Schmitt, S.; Willem, M.; Haass, C. Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface. Traffic. 2006, 7, 408-415. 80. Xia, W.; Ostaszewski, B. L.; Kimberly, W. T.; Rahmati, T.; Moore, C. L.; Wolfe, M. S.; Selkoe, D. J. FAD mutations in presenilin-1 or amyloid precursor protein decrease the efficacy of a gamma-secretase inhibitor: evidence for direct infolvement of PS1 in the gamma-secretase cleavage complex. Neurobiol. Dis. 2000, 7, 673-681. 81. Ebke, A.; Luebbers, T.; Fukumori, A.; Shirotani, K.; Haass, C.; Baumann, K.; Steiner, H. Novel g-secretase enzyme modulators directly target presenilin protein. J. Biol. Chem. 2011, 286, 37181-38186. 82. Citron, M.; Oltersdorf, T.; Haass, C.; McConlogue, L.; Hung, A. Y.; Seubert, P.; VigoPelfrey, C.; Lieberburg, I.; Selkoe, D. J. Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production. Nature 1992, 360, 672-674. 83. Steiner, H.; Kostka, M.; Romig, H.; Basset, G.; Pesold, B.; Hardy, J.; Capell, A.; Meyn, L.; Grim. M. L.; Baumeister, R.; Fechteler, K.; Haass, C. Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases. Nat. Cell Biol. 2000, 2, 848-851. 84. Shearman, M. S.; Beher, D.; Clarke, E. E.; Lewis, H. D.; Harrison, T.; Hunt, P.; Nadin, A.; Smith, A. L.; Stevenson, G.; Castro, J. L. L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity. Biochemistry. 2000, 39, 8698-8704.

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A) H3C

C)

CH2

CH3

OH CH3

CH3

H3C

B)

O

D)

CH3 CH3 H

CH3

H H

H H

OH

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0.30

Order Parameter S

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

0.25 0.20 0.15 0.10 0.05 0.00

O

+ N

O

P

O-

pure POPC-d31 10 mol% cholesterol 20 mol% cholesterol 10mol% bexarotene 20 mol% bexarotene

Carbon Number n 2

4

6

8

10

12

14

16

O

O

O

H O O

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γ

γ

+ N

γ

β α

O

(CH2)n

O

O CH3

G-3

1

2 6

34

CH3

8 7

CH3

C-2 C-3 C-3

(CH2)n CH2-CH=

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x 30

O C-2 C-3

β

CH3

H 3C 8

H 3C

5

4

O

α

3

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G-1

5

2

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1

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1

O

G-1

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H2C

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G-2

OH

2

O-

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γ

CH=CH G-2

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

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6 7

CH2-CH=

(CH2)n

8

8

7

6 1

5

4

3

2

1

0

(CH2)n

H Chemical Shift / ppm CH3

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CH3

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0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002

= H H 3 ) n H C -3 -2 -1 -2 -3 α C CH 2 -2C H= C C G G G ( H C C O

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O H O

β

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O

O

0.000

O

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

Induced Chemical Shift / ppm

ACS Chemical Neuroscience

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+ N

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J

J

O G-3 OH

J

E

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D

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G

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G

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E

D

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G

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D

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=

-3

H

C

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3

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C

H

J

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O

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0.010 0.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.000

2

0.003

Cross Relaxation Rate / s-1

3

0.004

O

0.005

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Cross Relaxation Rate / s-1

C

H

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C-2 C-3

C-2 C-3

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3

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1

5

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Cross Relaxation Rate / s

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E

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D

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G

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G

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2

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2

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C

H

C

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0.003

H

0.001

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D

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2

0.003

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2

3

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2

1

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Cross Relaxation Rate / s-1

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Cross Relaxation Rate / s-1

+ N

O

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

ACS Chemical Neuroscience

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+ N

CH3

CH3

ACS Chemical Neuroscience

A)

0 mol% 2.5 mol% 5 mol% 7.5 mol% 10 mol%

Chemical Shift / ppm

110

115

V689 F690

A701

I702

15N

120

125 8.8

B) ∆ Chemical Shift / ppm

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

0.015

8.6

1H

8.4

8.2

8.0

Chemical Shift / ppm

7.8

A701 KD = 24 ± 19 mol% V689 KD = 12 ± 10 mol% I702 KD = 7± 3 mol%

0.010 0.005 0.000 0

2

4

6

8

10

[Bexarotene] / mol% of lipid

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C9926-55/POPC C9926-55/POPC/Chol (90/10)

50

B) -15

Ellipticity / mdeg

Ellipticity / mdeg

A) 100

0

-20 -25

more helical

-30 -35

-50

C)

λ / nm

-15

210

220

230

λ / nm

D)

C9926-55/POPC/Chol (90/10) 20 µM bexarotene 40 µM bexarotene 1.100 60 µM bexarotene 80 µM bexarotene 100 µM bexarotene 1.075 120 µM bexarotene

θ218 / θ212

-25 -30

helicity

1.050 1.025

-35 -40

-40

200 210 220 230 240 250 260

-20

Ellipticity / mdeg

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

ACS Chemical Neuroscience

210

220

λ / nm

230

1.000

0

20

40

60

80 100 120

[Bexarotene] / µM

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ACS Chemical Neuroscience

B) kDa

C100-His6

C)

[Bexarotene] / M 0 40 80 160

C

N102-FmH

16

36

[Bexarotene] / µM GSI 40 80 160 C99-6myc AICD

7

N

4

16

100 75

49

Actin

NICD

N NICD

D)

50

C 98 14 6 49 6

25 0

F-NEXT NICD

cell lysates

AICD

98

0 40 80 160 [Bexarotene] / M

ACS Paragon Plus Environment

media

A

-Secretase activity / %

-Secretase Activity / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16A) [Bexarotene] / M 17 40 80 160 kDa 0 18 19 20 7 21 4 22 23 24 25 7 26 27 28 100 A 29 AICD 30 75 31 32 50 33 34 25 35 36 0 0 40 80 160 37 [Bexarotene] / M 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

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[Bexarotene] / M GSI 40 80 160 APP FL C99 C83 Actin A

3 98

sAPP

Page 39 of 39

CH2

CH3

0.30 OH

H3C

CH3

CH3

O

CH3 CH3 H

CH3

H H

H H

OH

-Secretase Activity / %

H3C

Order Parameter S

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

ACS Chemical Neuroscience

0.25 0.20 0.15 0.10 0.05 0.00

2

4

6

8

10

12

Carbon Number n

14

16

100 75

A AICD

50 25 0

ACS Paragon Plus Environment

0 40 80 160 [Bexarotene] / M