Article pubs.acs.org/Langmuir
Omega‑3 Fatty Acids Regulate the Interaction of the Alzheimer’s Aβ(25−35) Peptide with Lipid Membranes Giuseppe Vitiello,†,‡ Sara Di Marino,§ Anna Maria D’Ursi,§ and Gerardino D’Errico*,†,‡ †
Department of Chemical Science, University of Naples ‘‘Federico II’’, Complesso di Monte S. Angelo, Via Cinthia, I-80126 Naples, Italy ‡ CSGI (Consorzio per lo Sviluppo dei Sistemi a Grande Interfase), via della Lastruccia 3, 50019 Florence, Italy § Department of Pharmaceutical Science, University of Salerno, 84084 Fisciano, Italy S Supporting Information *
ABSTRACT: Polyunsaturated omega-3 fatty acids are increasingly proposed as dietary supplements able to reduce the risk of development or progression of the Alzheimer’s disease (AD). To date, the molecular mechanism through which these lipids act has not been yet univocally identified. In this work, we investigate whether omega-3 fatty acids could interfere with the fate of the Alzheimer-related amyloid peptide by tuning the microstructural and dynamical properties of the neuronal membrane. To this aim, the influence of the omega-3 lipid, 1,2didocosahexaenoyl-sn-glycero-3-phosphocholine [22:6(cis)PC] on the biophysical properties of lipid bilayers, and on their interaction with the amyloid peptide fragment Aβ(25−35) has been investigated by Electron Spin Resonance (ESR), using spin-labeled phospholipids. The results show that the peptide selectively interacts with bilayers enriched in cholesterol (Chol) and sphingomyelin (SM). [22:6(cis)PC] enhances the Aβ(25−35)/membrane interaction, favoring a deeper internalization of the peptide among the lipid acyl chains and, consequently, hindering its pathogenic self-aggregation. results7 and on the evidence that decreased levels of omega-3 fatty acids have been observed in brain tissue of people with AD, specifically in areas that mediate learning and memory.8 Nowadays, omega-3 fatty acids are increasingly proposed as dietary supplements that may reduce the risk of disease development or progression.9,10 The molecular interpretation of the omega-3 fatty acid involvement in the molecular processes underlying AD is still debated.11 Some studies have suggested that docosahexaenoic acid (DHA), the most abundant omega-3 fatty acid in the mammalian brain, could be broadly neuroprotective via multiple biochemical mechanisms that include DHA metabolites. DHA seems also to suppress several signal transduction pathways induced by Aβ, including two major kinases that phosphorylate the microtubule-associated protein tau and promote neurofibrillary tangle pathology.12 Furthermore, DHA is also protective against several risk factors for dementia, including head trauma, diabetes, and cardiovascular disease.13 In this work, we investigate whether omega-3 fatty acids could affect the Aβ peptides fate by an indirect mechanism (i.e., by tuning the neuronal membrane properties). Indeed, incorporation of omega-3 fatty acids into membranes increases
1. INTRODUCTION Alzheimer’s disease (AD) is the most common form of neurodegenerative disorder, affecting an increasing number of people every year. From a molecular viewpoint, AD is characterized by abnormal accumulations of extracellular amyloid plaques, composed by insoluble fibrils of β-amyloid (Aβ) peptides and intracellular neurofibrillary tangles throughout cortical and limbic brain regions.1 Aβ peptides result from the cleavage of the amyloid precursor protein (APP) and are mainly composed of 40 and 42 amino-acids [Aβ(1−40) and Aβ(1−42), respectively]. In accordance with the amyloid cascade hypothesis,2 the central event of plaque formation is the expulsion of the β-amyloid peptides from the neuronal membrane, which is followed by their self-aggregation into protofibrils and fibrils. Indeed, soluble Aβ oligomeric intermediates, rather than fully formed fibrils, are currently recognized as the predominant toxic species, capable of initiating pathogenic events.3,4 To date, a unique and clear strategy for AD therapy and/or prevention is not defined because many aspects of this pathology are still obscure. An innovative approach focuses on the protective action exerted by molecules naturally occurring in food and, at higher content, in dietary supplements.5 As an example, polyphenols have been recognized as effective neuroprotective agents.6 Possible effects of omega-3 fatty acids have also been hypothesized based on epidemiologic © 2013 American Chemical Society
Received: September 5, 2013 Revised: October 17, 2013 Published: October 21, 2013 14239
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their fluidity. This could reflect on the Aβ generation and selfaggregation in two ways. First, enhanced membrane fluidity favors the nonamyloidogenic pathway of APP processing, thus reducing the Aβ generation.14 Second, accumulating evidence indicates that neuronal membranes are deeply involved in the mechanism of Aβ self-aggregation and cytotoxicity. Particularly, lipid bilayers have been shown to play a pivotal role in the early stages of the peptides misfolding and self-aggregation, so that even slight changes of their properties, as determined by the lipid composition, could reflect in the process.15 In this scenario, this work is aimed at analyzing the influence of the omega-3 lipid 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine [22:6(cis)PC] on the biophysical properties of lipid membranes and on their interaction with a fragment of the Aβ peptide. As model of amyloid peptides, we used Aβ(25−35), the shortest Aβ fragment that exhibits large β-sheet aggregated structures.16 Aβ self-aggregation and membrane interaction could be affected by the length and sequence of the considered fragment17−20 and any generalization has to be handled with care. Nevertheless, Aβ(25−35) has been recently confirmed as a good tool to gain atomistic details of AD-related molecular processes.21 We have considered three lipid systems: POPC, POPC/Chol (75:25 wt/wt), and POPC/SM/Chol (1:1:1 wt/wt/wt). POPC is one of the most abundant diacylglycerol phospholipids in neuronal membranes. One-component POPC bilayers are often used in physicochemical studies of protein- or peptidebiomembrane interactions. Despite that their biologic relevance is questionable, they still constitute a sort of “reference” for more complex and biomimetic lipid bilayers. Chol is a major component of neuronal cell membranes, influencing their thickness and fluidity.22 A 25 wt %/wt represents the average content of Chol in these membranes. Last, POPC/SM/Chol lipid mixtures are considered representative of the lipid rafts naturally occurring in neuronal membranes, whose direct involvement in the generation, accumulation, and aggregation of Aβ peptides has been recently hypothesized.23 In all these three lipid systems, we have monitored the effect of the omega3 lipids by replacing 20% by weight of glycerophospholipids (POPC, SM) with 22:6(cis)PC. The main focus of this work is on the changes in lipid ordering and dynamics due to membrane-peptide interactions. These perturbations have been investigated by electron spin resonance (ESR), using phospholipids spin-labeled on the acyl chain. On the other hand, the secondary structure of the Aβ(25−35) peptide interacting with the lipid membranes was monitored by performing circular dichroism (CD) experiments.
reported.24 The peptide was characterized on a Finningan LCQ-Deca ion trap instrument equipped with an electrospray source (LCQ Deca Finnigan, San José, CA). The sample were directly infused in the ESI source by using a syringe pump at the flow rate of 5 μL/min. Data were analyzed with Xcalibur software. The samples purity was >98%. In order to ensure sample reproducibility and removal of aggregated states which can be present, dry peptide was pretreated with neat TFA (Fluka, St. Louis, MO) for 3 h, followed by a 10-fold dilution with Milli-Q water and lyophilization (Millipore, Billerica, MA). 2.3. Sample Preparation. Multi-lamellar vesicles (MLVs) of POPC, POPC/Chol 75:25 wt/wt, and POPC/SM/Chol 1:1:1 wt/wt/ wt were prepared mixing appropriate amounts of lipids, dissolved in dichloromethane−methanol mixtures (2:1 v/v, 10 mg/mL lipid concentration), in a round-bottom test tube. Total weight of the lipid for each sample was 0.2 mg. In the case of samples prepared for ESR experiments, spin-labeled phosphatidylcholines (5-PCSL or 14PCSL) were added to the lipid mixture (1 wt %/wt on the total lipid) by mixing appropriate amounts of a spin-label solution in ethanol (1 mg/mL) with the lipid organic mixture. A thin lipid film was produced by evaporating the solvents with dry nitrogen gas and final traces of solvents were removed by subjecting the sample to vacuum desiccation for at least 3 h. The samples were then hydrated with 20 μL of 10 mM phosphate buffer at pH = 7.4 and repeatedly vortexed, obtaining a MLV suspension. This suspension was transferred into a 25 μL glass capillary and immediately sealed. In a different set of samples, 20% by weight of glycerophospholipids (POPC, SM) was replaced with 22:6(cis)PC. In preparing these samples, appropriate amounts of 22:6(cis)PC in chloroform (25 mg/ mL lipid concentration) were mixed with the other lipids before evaporating the organic solvents. Samples containing Aβ(25−35) were prepared by the same procedure, adding appropriate amounts of the peptide dissolved in HFIP to the lipid organic solutions. Lipid/Aβ(25−35) ratio was 1:0.5 wt/wt (which corresponds to about 20:1 mol/mol). Such lipid/ peptide ratio was chosen to highlight perturbation in lipid arrangement due to the peptide. Lau et al. have recently pointed out that the Aβ(25−35) positioning with respect to lipid bilayers could depend on the sample preparation procedure (i.e., it interacts with the headgroups if added after liposome preparation, while it inserts in the bilayer if inserted during liposome preparation).18 For this reason, we tested samples prepared through different procedures, finding negligible differences in the ESR spectra (data not shown). For CD experiments, large unilamellar vesicles (LUVs), obtained from MLVs by 9-fold extrusion through a polycarbonate membrane of 100 nm pore size, were preferred for their lower radiation scattering. The Aβ(25−35) concentration was set to 100 μM, and the same lipid/ peptide ratio used for ESR measurements was maintained. 2.4. ESR Spectroscopy. ESR spectra were recorded with a 9 GHz Bruker Elexys E-500 spectrometer (Bruker, Rheinstetten, Germany). The capillaries containing MLV suspensions to be investigated were placed in a standard 4 mm quartz sample tube containing light silicone oil for thermal stability. All the measurements were performed at 25 °C. Spectra were recorded using the following instrumental settings: sweep width, 100 G; resolution, 1024 points; time constant, 20.48 ms; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; incident power, 6.37 mW. Several scans, typically 16, were accumulated to improve the signal-to-noise ratio. 2.5. CD Measurements. CD measurements were performed on a JASCO J-810 spectropolarimeter (Jasco, Cremella, Italy) equipped with a thermostatted cell holder, using a quartz cell with a 1.0 mm path length. The spectra are an average of 4 consecutive scans from 260 to 190 nm with a bandwidth of 2.0 nm, a time constant of 8 s, and a scan rate of 10 nm/min. Each spectrum was corrected for the contribution of the buffer containing MLVs. The measurements were performed at 25 °C. For estimation of secondary structure content, CD spectra were analyzed using the CONTINN algorithm of the DICHROWEB online server.25
2. MATERIALS AND METHODS 2.1. Materials. Dichloromethane and methanol, HPLC-grade solvents, were obtained from Merck (Darmstadt, Germany), while 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was obtained from SigmaAldrich (St. Louis, MO). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM), and 1,2-didocosahexaenoyl-snglycero-3 phosphocholine (22:6(cis)PC) were obtained from Avanti Polar Lipids (Birmingham, AL). Spin-labeled phosphatidylcholines (1palmitoyl-2-stearoyl-(n-doxyl)-sn-glycero-3-phosphocholine, n-PCSL) with the nitroxide group in the positions 5 and 14 of the acyl chain were also obtained from Avanti Polar Lipids. Cholesterol (Chol) was obtained from Sigma. 2.2. Peptide Synthesis. The Aβ(25−35) peptide, GSNKGAIIGLM, was manually synthesized by conventional solid-phase chemistry using the Fmoc/tBu strategy and subsequently purified, as previously 14240
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3. RESULTS AND DISCUSSION In the present work, the role of the omega-3 lipid 22:6(cis)PC on the interaction of Aβ(25−35) with lipid bilayers was investigated by analyzing changes in ESR spectra of phosphatidylcholine spin-labeled at different positions, n, in the sn-2 chain (n-PCSL, n = 5 and 14) incorporated in the membranes. The well-assessed approach reported in the literature for membrane-interacting viral peptides,26,27 as well as for classical peripheral membrane proteins28,29 was followed. The 5-PCSL and 14-PCSL spectra in the investigated lipid/ peptide mixtures are shown in Figures 1 and 2, respectively. In
Figure 2. ESR spectra of 14-PCSL in bilayers of (A) POPC, (A′) POPC/22:6(cis)PC, (B) POPC/Chol, and (B′) POPC/Chol/22:6(cis)PC, (C) POPC/SM/CHOL and (C′) POPC/SM/CHOL/ 22:6(cis)PC in the absence (continuous lines) and presence (dotted lines) of Aβ(25−35).
described in the literature.30−32 S is a measure of the local orientational ordering of the labeled acyl chain with respect to the normal to the bilayer surface. aN′ is an index of the micropolarity experienced by the nitroxide. The values of these parameters for both the spin labels in all the considered systems are collected in Tables 1 and 2. An alternative approach to obtain S values relies on computer simulation of the line shape.33−37 To test the reliability of the values reported in the tables, we simulated some spectra (see the Supporting Information). A good agreement between the results obtained from the two methods was found. Below, we separately analyze the results obtained for the three lipid systems considered. First, POPC bilayers are considered. In this case, the spectrum of 14-PCSL presents a narrow three-line, quasi-isotropic line shape (see Figure 2A) very different from the 5-PCSL signal (Figure 1A). This indicates a marked flexibility increase in segmental chain mobility in going from the polar headgroups to the inner hydrophobic core, which is a characteristic hallmark of the disordered liquid-crystalline state (Ld) of fluid phospholipids bilayers. The inclusion of 22:6(cis)PC does not significantly affect the 14-PCSL spectrum (Figure 2, panel A′ vs panel A), while it slightly decreases the anisotropy of the 5-PCSL signal. This is also demonstrated by the S values reported in Table 1. These results indicate a rather limited impact of omega-3 lipids on the Ld bilayers, whose local ordering slightly decreases in the outer shell, while the inner core remains unperturbed. We analyze now the effects of the Aβ(25−35) addition to these lipid systems (dotted vs continuous line spectra). Only slight variations were observed in the spectra of both 5-PCSL and 14-PCSL in POPC and POPC/22:6(cis)PC bilayers, as also shown by the S values in Table 1. Thus, the results indicate that the Aβ(25−35) peptide does not solubilize in these bilayers, positioning in the external aqueous medium, where it can potentially self-aggregate. In this respect, no effect of the omega-3 lipid is observed.
Figure 1. ESR spectra of 5-PCSL in bilayers of (A) POPC, (A′) POPC/22:6(cis)PC, (B) POPC/Chol, (B′) POPC/Chol/22:6(cis)PC, (C) POPC/SM/Chol, and (C′) POPC/SM/Chol/22:6(cis)PC in the absence (continuous lines) and presence (dotted lines) of the Aβ(25− 35) peptide.
both figures, spectra A, B, and C refer to the POPC, POPC/ Chol (75:25 wt/wt), and POPC/SM/Chol (1:1:1 wt/wt/wt) bilayers, respectively. Spectra A′, B′, and C′ refer to the same lipid systems in which 20% by weight of glycerophospholipids (POPC, SM) has been replaced with 22:6(cis)PC. Finally, dotted lines refer to the spectra obtained in the presence of Aβ(25−35). 5-PCSL presents the nitroxide reporter group close to the hydrophilic lipid headgroup, while in 14-PCSL, the nitroxide group is positioned close to the terminal methyl group. Thus, a combined use of the two spin-labels permit the monitoring of both the outer and inner membrane regions, since the analysis of the spectra furnishes information about the local microstructure and polarity. Inspection of Figure 1 shows that the 5PCSL spectrum in all the investigated samples presents a clearly defined axially anisotropic line shape. Figure 2 shows that the 14-PCSL spectrum line shape is more sensitive to the lipid composition of the membrane and can vary from a narrow three-line, quasi-isotropic spectrum to a well-resolved anisotropic one. In order to quantitatively analyze the spectra, we evaluated the order parameter, S, and the isotropic hyperfine coupling constant, aN′ , using a spectra parametrization method 14241
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Table 1. Order parameter, S, of 5-PCSL and 14-PCSL Spin-Labels in Bilayers at Different Lipid Composition with and without the Aβ(25-35) Peptide S POPC
POPC/22:6(cis)PC
POPC/Chol
POPC/Chol/22:6(cis)PC
POPC/SM/Chol
POPC/SM/Chol/22:6(cis)PC
5-PCSL 14-PCSL
0.64 ± 0.01 0.14 ± 0.02
0.61 ± 0.01 0.14 ± 0.02
0.71 ± 0.01 0.40 ± 0.01
0.68 ± 0.01 0.20 ± 0.02
0.74 ± 0.01 0.38 ± 0.02
0.60 ± 0.01 0.13 ± 0.02
5-PCSL 14-PCSL
Aβ(25−35) 0.65 ± 0.01 0.15 ± 0.02
Aβ(25−35) 0.61 ± 0.01 0.16 ± 0.02
Aβ(25−35) 0.74 ± 0.02 0.33 ± 0.01
Aβ(25−35) 0.70 ± 0.01 0.27 ± 0.02
Aβ(25−35) 0.82 ± 0.01 0.44 ± 0.02
Aβ(25−35) 0.72 ± 0.01 0.18 ± 0.02
Table 2. Hyperfine Coupling Constant, aN′ , of 5-PCSL and 14-PCSL in Bilayers at Different Lipid Composition with and Without the Aβ(25-35) Peptide aN′ /G POPC
POPC/22:6(cis)PC
POPC/Chol
POPC/Chol/22:6(cis)PC
POPC/SM/Chol
POPC/SM/Chol/22:6(cis)PC
5-PCSL 14-PCSL
15.3 ± 0.1 14.0 ± 0.2
15.4 ± 0.1 13.9 ± 0.2
15.5 ± 0.1 14.1 ± 0.1
15.6 ± 0.1 13.9 ± 0.2
16.2 ± 0.2 13.6 ± 0.1
15.8 ± 0.2 14.1 ± 0.1
5-PCSL 14-PCSL
Aβ(25−35) 15.5 ± 0.1 14.0 ± 0.2
Aβ(25−35) 15.3 ± 0.1 13.8 ± 0.2
Aβ(25−35) 15.6 ± 0.2 13.7 ± 0.1
Aβ(25−35) 15.5 ± 0.1 13.9 ± 0.2
Aβ(25−35) 15.6 ± 0.2 14.3 ± 0.2
Aβ(25−35) 16.1 ± 0.2 13.9 ± 0.1
interplay between cholesterol and amyloid peptides in regulating the bilayer microstructure. Association of Aβ(25−35) with POPC/Chol/22:6(cis)PC bilayers causes an evident increase of the anisotropy of both 5PCSL and 14-PCSL spectra. In particular, the 14-PCSL spectrum, at high magnetic field, turns from a broad minimum to a well-defined double well line shape (Figure 2B′, continuous vs dotted line). This evidence, confirmed by the increased S, indicate an at least partial insertion of the peptide in the inner core of the bilayers. Finally, POPC/SM/Chol bilayers were studied. At the composition used in this work, this mixture forms bilayers in the Lo state,40 as detectable by the anisotropic line shape of both 5- and the 14-PCSL spectra (Figures 1C and 2C). Particularly, the 5-PCSL spectrum shows that this lipid mixture presents the most anisotropic behavior with respect to the other lipid bilayers considered in the present work. This is confirmed by the higher S value (see Table 1). This effect is to be ascribed to favorable SM−Chol interactions, which are fundamental in stabilizing lipid rafts.26 The addition of 22:6(cis)PC causes a strong decrease in the anisotropy of both spin-labels. The changes in the line shape are qualitatively similar to those observed for POPC/Chol, and quantitatively much higher, as confirmed by the dramatic decrease of the S values. These results indicate that omega-3 lipids are extremely effective in influencing microstructure of raftlike bilayers, increasing their fluidity and destabilizing the local order of phospholipid chains. This order decrease could also suggest the formation of domains with different lipid ordering. Significant changes were observed in both 5-PCSL and 14PCSL spectra for POPC/SM/Chol bilayers in the presence of Aβ(25−35), as also supported by S values reported in Table 1. The 5-PCSL spectrum presents a significant increase of the anisotropy, as confirmed by the evident increase in the S value (from 0.74 to 0.82). In addition, in the 14-PCSL spectrum, a second anisotropic component is observed (highlighted by the well-detectable shoulder peak at low field), indicating that Aβ(25−35) determines an increase of the lipids order packing. These results point to a capability of the peptide to partition
As the second lipid system, we consider the results obtained in POPC/Chol (75:25 wt/wt) bilayers. Inspection of Figures 1B and 2B show that both spin-labels present a clearly defined axially anisotropic spectrum, an evidence that, due to the high cholesterol content, the membrane is in the liquid-ordered state (Lo).38 The increased local ordering, due to Chol, appears evident in the S values reported in Table 1. Upon inclusion of 22:6(cis)PC, the 14-PCSL spectrum turns to an almost isotropic line shape (Figure 2, panel B′ vs panel B), as also confirmed by the dramatic S decrease. A slight anisotropy decrease is also observed for 5-PCSL (Figure 1, panel B′ vs panel B). These pieces of evidence indicate a strong increase in the bilayer fluidity due to the presence of omega-3 lipids, which effectively contrast the rigidifying effect of Chol, promoting the Lo → Ld transition. For POPC/Chol bilayers, association of Aβ peptides cause significant variations in the ESR spectra of both spin-labeled phospholipids. Particularly, the peptide causes a slight anisotropy increase for 5-PCSL (Figure 1B, dotted vs continuous line), as also detectable by the increase of the S value. This suggests that Aβ(25−35) interacts with the upper regions of the lipid acyl chains of POPC/Chol bilayers. Interestingly, an opposite effect is observed for 14-PCSL, whose spectrum becomes less anisotropic (Figure 2B, dotted vs continuous line), as evidenced by a noticeable S decrease. This evidence is not straightforward to understand. In a previous work conducted on DLPC/Chol bilayers, by using a spinlabeled analogue of Chol, we found that the peptide interaction with the bilayer interface causes a repositioning of cholesterol closer to the hydrophilic external layer, thus causing an increased disorder in the inner part of the bilayer.39 The results presented here seem to indicate a similar mechanism in POPC/ Chol bilayers interacting with Aβ(25−35). A deeper insertion of the full-length peptide Aβ(1−42) into the Chol-enriched phospholipid bilayer was proposed by Lau et al. based on 2H and 31P NMR data.17 While a different behavior could be ascribed to the different peptide length and sequence, it should also be highlighted that all these results support a strict 14242
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4. CONCLUSIONS Lipids play an important role in the development of the Alzheimer’s Disease, according to multiple mechanisms, some of which are still to be disclosed.41 Among others, lipid composition of neuronal membranes regulates their fluidity, which is associated with brain aging and play a central role in Alzheimer’s Disease.42 Polyunsaturated fatty acids (PUFAs) alter the packing of lipid acyl chains, potentially destabilizing the structural order of the bilayer. In the present work, we have investigated the extent of lipid bilayer perturbations due to the omega-3 lipid 22:6(cis)PC. Moreover, we have analyzed whether changes in bilayer properties induced by this PUFA affect the interaction of the amyloidogenic peptide Aβ(25−35). Our data show that the omega-3 lipid does not affect the structural properties of lipid bilayers in the Ld liquid crystalline state. In contrast, inclusion of 22:6(cis)PC in the bilayer formulation effectively reduce the acyl chain ordering of bilayers in the Lo state, resulting in a dramatic increase of the membrane fluidity. This is particularly interesting in the case of the POPC/SM/Chol bilayers, considered mimetic of lipid “raft” domains. The physical state of neuronal membranes is critical in the control of transfer of the neuronal information (i.e., neither too rigid nor too fluid for the ionic exchange between the inner and outer leaflets of the membrane). Thus, our results suggest that omega-3 lipids, modulating the properties of the membrane bilayer, could impact the formation of lipid rafts and the speed of signal transduction and neurotransmission. ESR results show that lipid composition critically affect Aβ(25−35) interaction with bilayers. Particularly, the peptide does not interact strongly with POPC bilayers, interacts with the interface of POPC/Chol membranes, and is at least partially internalized in the POPC/SM/Chol bilayers. From these results, one could conclude that Aβ(25−35) interacts only with bilayers in the Lo state, while it is excluded from those in the Ld state. This interpretation is not confirmed by the analysis of the effects of the inclusion of 22:6(cis)PC in the bilayer formulation. Indeed, the omega-3 lipid promotes the Lo → Ld transition but, at the same time, it strongly increases the bilayer propensity to interact with Aβ(25−35), favoring its internalization in the membrane. Thus, our results suggest that the presence of specific lipids (e.g., Chol and SM) could drive the peptide/membrane interaction, independent of the physical state of the lipid bilayer. Omega-3 lipids enhance this interaction, favoring the peptide insertion into the membrane. These conclusions point to a possible “protective role” of the omega-3 lipid which inhibits the peptide release from the membrane and its subsequent fibrillization. Also it may favor peptide insertion if released from plaques or other aggregates (e.g., oligomers and protofibrils). Finally, this work highlights the regulatory role of PUFAs in biological processes involving lipid membranes, such as amyloid peptides self-aggregation, further encouraging future chemical and biomedical investigation on the optimal strategy for their exploitation in the prevention and treatment of Alzheimer’s disease.
between the bilayer interface and its interior, effectively perturbing the whole lipid profile. It is to be noted that the SM−Chol interaction hinders the Chol repositioning due to the peptide observed in POPC/Chol bilayers. Addition of the peptide to the 22:6(cis)PC-containing POPC/SM/Chol bilayers, results in a dramatic increase of the 5-PCSL spectrum anisotropy (Figure 1C′, dotted vs continuous line), as also confirmed by the S increase which is the highest variation due to the peptide observed among the considered lipid systems. At the same time, the 14-PCSL spectrum is detectably broadened (see also the evident S increase). Thus, in the presence of omega-3 lipids, POPC/SM/ Chol bilayers present an enhanced ability to host the peptide. Table 2 shows that, for all considered lipid systems, a′N values are only marginally affected by both 22:6(cis)PC and Aβ(25− 35), suggesting that insertion of the omega-3 lipid and/or interaction with the peptide do not cause evident changes in the bilayer hydration. The CD spectra of Aβ(25−35) in buffer and in liposome suspensions are shown in Figure 3. Actually, in the case of short
Figure 3. CD spectra of Aβ(25−35) in PBS pH 7.4 buffer, recorded in the absence and presence of LUVs with different composition.
and flexible peptides, a refined quantitative interpretation of CD spectra is limited by the preponderance of intrinsically disordered secondary structures adopting a wide range of dihedral angles and by the non-negligible effects of the termini. Here, only some indications on possible conformational preferences are presented. In all systems, the data indicate the prevalence of random coil−turn conformations with limited amounts of β-structures, as also confirmed by the quantitative interpretation of CD spectra, indicating that an average of 50% of the peptides are unordered. The data also give a clear indication of the prevalence of soluble vs insoluble peptide forms. Particularly, inspection of Figure 3 shows that in the presence of liposomes, the absolute values of the mean residue elipsicity increase. This effect suggests that lipid bilayers contrast the incipient formation of CD-silent peptide aggregates. At the same time, a reduction of β-structure conformations is observed (from around 15% to 5%). Interestingly, the maximum effect is observed in 22:6(cis)PCcontaining POPC/SM/Chol bilayers.
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ASSOCIATED CONTENT
S Supporting Information *
Simulations of 14-PCSL ESR spectra in POPC, POPC/Chol, and POPC/Chol/22:6(cis)PC in the absence and presence of the Aβ(25−35) peptide. Methods used for spectra simulation. 14243
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Table of parameters obtained from spectra simulation. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by MIUR (PRIN 2010-2011, Grant 2010NRREPL) and by Università di Napoli Federico IICompagnia di S.Paolo (Progetto FARO-III tornata).
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REFERENCES
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