Molecular chirality mediated amyloid formation on phospholipid surfaces

One of the neuropathological features of Alzheimer's disease (AD) is the misfolding of amyloid-β to form amyloid aggregates, a process highly associated with biological membranes. However, how molecular chirality affects the amyloid formation on phospholipid surfaces has seldom been reported. Here, l- and d-aspartic acid-modified 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (l-/d-Asp–DPPE) is synthesized to construct chiral phospholipid bilayers. We discover that the l-Asp–DPPE liposomes slightly inhibit the Aβ(1–40) nucleation process but cannot affect the oligomer elongation process. By contrast, the d-Asp–DPPE liposomes strongly inhibit both nucleation and elongation of the peptide. Notably, l- and d-Asp–DPPE liposomes not only have good biocompatibility but can also rescue Aβ(1–40)-aggregation induced cytotoxicity with significant chiral discrimination, in which the cell viability is higher in the presence of d-Asp–DPPE liposomes. Mechanism analysis and molecular dynamics simulation clearly demonstrate that differential electrostatic interactions of Lys16 in Aβ(1–40) with l- or d-Asp on the phospholipid contribute to the remarkable chiral discrimination. This study provides a deeper understanding of the crucial amyloidosis process from the perspective of the chiral interface and reveals that the convergence of d-amino acids with the liposomes might be a feasible route for AD prevention.

Liposomes were obtained through a classical extrusion method. 5,6 L-Asp-DPPE (2 mg) mixed with DPPE (2 mg) was dissolved in a mixture of chloroform and methanol (v/v 2:1). Evaporation of the solvent under reduced pressure causes the residue to form a film at the bottom of the flask.
To prepare liposomes, the dried lipid film was allowed to rehydrate by adding 4 mL of PBS and heated to 70 °C under stirring for at least 1 h. The multilamellar vesicle suspension was then extruded through a mini-extruder (Avanti Polar Lipids Inc.). A polycarbonate membrane with 0.1 μm pore diameter was used and nineteen extrusion cycles were performed. The resulting clear unilamellar vesicle solution at a concentration of 1 mg·mL −1 was stored in a vial and used in fresh.
Similar method was used to prepare other unilamellar vesicles. Unless otherwise stated, the chiral liposomes used in this work (i.e., L-Asp-DPPE and D-Asp-DPPE) were prepared by mixing L-or D-Asp-DPPE and DPPE at a mass ratio of 1: 1.

ThT fluorescence spectroscopy experiment
To study the influence of various liposomes on nucleation phase of Aβ(1−40) peptide, a series of working solutions (100 µL) were prepared containing 50 µM Aβ(1−40) peptide, 50 µM ThT in PBS. One of various liposomes (i.e., L-and D-Asp-DPPE, DPPE) or amino acid small molecules (i.e., L-and D-Asp) (100 µL, 1 mg·mL −1 ) was then added to the pre-prepared solutions. The liposome was replaced by PBS in the blank experiment. Dynamic growth curves were monitored by a standard ThT fluorescence assay. Fluorescence data were record using a Synergy™ H1M Multifunctional Microplate Tester with a bottom-reading mode in 96-well flat bottom plates sealed with a platemax film. Plates were shaken for 2 s before reading fluorescent data every 10 min using an excitation wavelength of 445 nm and emission wavelength of 485 nm at 37 °C. Each S-7 experiment was run in triplicated in a 96-well plate.
To study the influence of the chiral liposomes on the fiber elongation phase, the mixtures of 50 µM Aβ(1−40) peptide and 50 µM ThT in PBS were incubated in a 37 °C water bath until the Aβ(1−40) has become oligomers. After that, the peptide solutions (100 µL) were diluted by 100 µL solution of chiral liposomes (1 mg·mL −1 ). Subsequently, dynamic fibrillation processes were monitored by ThT fluorescence assay as described above. 1% DMSO was used in the ThT binding assays.   where I vv is the intensity of fluorescence parallel to excitation plane, I vh is the intensity of fluorescence perpendicular to excitation plane, G is the grating correction coefficient, G = I hv /I hh .

Dynamic light scattering experiment
A Zetasizer Nano system was employed with an argon-neon laser (λ= 633 nm, θ= 173°). Newly extruded liposomes (i.e., L-and D-Asp-DPPE, DPPE) in PBS of 1 mg·mL −1 or samples (Aβ (1−40) alone, Aβ(1−40) with L-or D-Asp-DPPE liposomes added) incubated at 37 °C for 80 h were placed in a quartz cuvette (light path: 10 mm, volume: 3.5 mL). After waiting for 120 s (well dispersed and stabilized), the mean hydrodynamic diameter was obtained by an average of 12 measurements at 25 °C, and each data was collected in 10 s intervals. Due to the measure range of DLS is 2 nm−3 μm, the measured data beyond the range are for reference only. The preparation method of peptide solutions was the same as the ThT binding assay, 1% DMSO was used.

Atomic force microscopy experiment
AFM measurements were conducted on a NanoWizard Ultra Speed AFM (JPK, Germany) in a QI mode. The samples after ThT binding asssay were used for AFM study. Sample was prepared by dropping 10 μL of peptide solution or chiral liposomes on a freshly cleaved mica and allowed it to dry in the air. Then AFM images were acquired under ambient conditions.

Cell viability assay
Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay in N2a cells. N2a cells were cultured in high glucose DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin, in 5% CO 2 at 37 °C. Once the cells reached 95% confluence, they were split (using 0.25% trypsin-EDTA), seeded at a density of 5 × 10 3 cells/well in 100 μL complete medium in 96-well plates and cultured for 48 h. After that, the medium was removed, the cells were washed with PBS and 100 μL serum-free DMEM was added to the wells. To investigate the conditions. The reaction heat was recorded during the binding process, and subtracting the control experiment using software gave the corrected reaction heat for each injection. Then the data was fitted using a one-site model.

Molecular dynamics simulation
To mimic the experimental condition, two model membranes were composed of 200 L-or D-Asp-DPPE and DPPE phospholipids with a mixture ratio of 1:1. Then the systems were solvated with TIP3P water molecules. The monomer of Aβ(1−40) (PDB code 2LFM) 11 was used to construct the model of peptide binding to model membrane. Ten models of Aβ(1−40) binding to two heterogeneous bilayers were built. The charges of the systems were balanced to neutral using S-13 calculated using the Gaussian 09 program with the B3LYP functional under 6-311G* basis set. 12 The partial charges of the substrate molecules were derived using the RESP charge fitted with the antechamber module in Amber 16. The other parameters, including vdW, bond, angle and torsion terms, were obtained with the antechamber module. 13  The semi isotropic pressure coupling was employed using the Monte Carlo barostat 16 controlled the pressure at 1 bar with a coupling constant of 5 ps when the production run was performed. To maintain the stability of the lipid system, all simulations were performed above the experimental liquid-crystalline phase transition temperature (∼315 K for pure dipalmitoyllecithin). 17 The Langevin thermostat 18 was employed to couple the temperature of the systems around 323. 5 K 19 with a time constant of 1 ps. For each system, three independent MD simulations have been carried out and each simulation was performed for 1 μs, and totally run for 60 μs. The process of protein binding to membrane needs more simulation time to equilibrium, the last 500 ns of each simulation were used to analysis, and the analysis of interaction energies, native contacts etc. were performed by cpptraj module in Amber 16. 20 The positions of P atom of phospholipid molecules were statically analyzed to investigate the difference of L-and D-Asp-DPPE behaviors, when the Aβ(1−40) binding to it. The phospholipid bilayers were aligned and shifted to make the membrane molecules geometrical center on the origin of coordinate. All the phospholipid molecules were chosen for statically analysis. The z coordinates of P atom were applied to fit the P z surface. And the median filtering 21 was applied to smooth the surface. 500 frames after the system reached the equilibrium state were analyzed according to the steps mentioned above. And the mean z surface of the P surface distribution was got by following formula: where is the mean of . is the surface in frame of MD trajectory of equilibrium state. is the total number of the frame applied in static process. The illustration of fitting the surface of the equilibrium trajectory was shown in Fig. S17.

Binding site validation
First, fluorescence titration experiments were performed to calculate association constant (K a ) according to remarkable fluorescence quenching of fluorescein-labelled oligopeptide caused by the addition of PEA L-or D-Asp (Fig. S10 in ESI). The calculation results revealed that the K a of VHHQKLVFF with PEA D-Asp was 89920 L•mol −1 , which was larger than that with PEA L-Asp (K a : 56684 L•mol −1 ), validating the chiral discrimination observed by the 1 H-15 N HSQC NMR spectra. Remarkable differences between PEA L-and D-Asp were also detected when they interacted with VHHQ or LVFF, observed by the fluorescence (Fig. S10 in ESI) and 1 H NMR titration experiments ( Fig. S12 and S13 in ESI). However, their K a values only ranged from 968 to 5344 L•mol −1 , which were substantially smaller than that with VHHQKLVFF ( Fig. S11 in ESI).
These results indicated that the electrostatic interactions between the residue K16 in Aβ(1−40) and PEA L-or D-Asp were the source of the chiral effect for the amyloid fibrillation.
The binding preference of VHHQKLVFF for PEA D-Asp was further confirmed by isothermal titration calorimetry (ITC), which is a powerful technique to detect the thermodynamic parameters of interaction between molecules without label. 22 With the titration of PEA D-Asp in VHHQKLVFF solution, endothermic data were recorded, and the optimal fitting curve yielded a K a value of 3590 L•mol −1 (Fig. S14A and S14C in ESI). Under the same condition, when PEA L-Asp was added to VHHQKLVFF solution, the endathermic data were too small to give a reliable K a value ( Fig. S14B and S14D in ESI). Therefore, the binding affinity results indicated the stronger binding capacity of VHHQKLVFF towards PEA D-Asp than its enantiomer.     The P z surface in each frame of MD trajectory. (E) The mean smooth P z surface of trajectory in equilibrium state.