Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

The aggregation of amyloid β peptides (Aβ) into amyloid fibrils is implicated in the pathology of Alzheimer’s disease. In light of the increasing number of proteins reported to retard Aβ fibril formation, we investigated the influence of small hydrophilic model proteins of different charge on Aβ aggregation kinetics and their interaction with Aβ. We followed the amyloid fibril formation of Aβ40 and Aβ42 using thioflavin T fluorescence in the presence of six charge variants of calbindin D_9k and single-chain monellin. The formation of fibrils was verified with transmission electron microscopy. We observe retardation of the aggregation process from proteins with net charge +8, +2, −2, and −4, whereas no effect is observed for proteins with net charge of −6 and −8. The single-chain monellin mutant with the highest net charge, scMN+8, has the largest retarding effect on the amyloid fibril formation process, which is noticeably delayed at as low as a 0.01:1 scMN+8 to Aβ40 molar ratio. scMN+8 is also the mutant with the fastest association to Aβ40 as detected by surface plasmon resonance, although all retarding variants of calbindin D_9k and single-chain monellin bind to Aβ40

Co-aggregation of α-synuclein with DOPC:DOPS 7∶3 (a, c) and DOPC (d, f) at L/P = 18 (molar ratio).

<p>Left: Lipid concentration derived from quantitative phosphorous analysis of aggregated and non-aggregated lipids after co-aggregation with 34 µM α-synuclein (a, d). Right: Polarization transfer solid state NMR on lamellar phase lipids (b, e) and lipids co-aggregated with α-synuclein (c, f). Stars indicate peaks originating from buffer molecules. Spectra are normalized to equal intensity for DP of C₁₈.</p

Relative intensities from INEPT and CP experiments for DOPC:DOPS 7∶3 in lamellar phase (top) and co-aggregated with α-synuclein (lipid/protein ratio, L/P, 1/1; bottom) (DOPS is used in representation).

<p>Brackets indicate the unresolved group of peaks from C_4–7 together with C_12–15. I_INEPT/I_CP depends on both the correlation time τ_c and the order parameter |S_CH| for the bond vector in the molecular segment. Detailed interpretation is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235.s005" target="_blank">Table S1</a>.</p

Schematic illustration of amyloid fibril formation in the presence of a lipid membrane.

<p>In the majority of published studies, the processes illustrated in (A) are investigated, including studies of the aggregation process (monomeric protein, via transient oligomeric aggregates to amyloid fibrils), and studies of how proteins in different aggregation stages adsorbs to and modify lipid membranes. In the present study, we focus on another aspect of the amyloid formation process, that is co-aggregation and the possibility of lipid uptake from the membrane into the fibrillar aggregates (B).</p

PT ssNMR spectra (DP black, CP blue, INEPT red) of DOPC:DOPS 7∶3 (top) and DOPC:DOPS 7∶3 co-aggregated with α-synuclein (bottom) at L/P = 18 (molar ratio).

<p>Numbers refer to assignments of the acyl chain from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone-0077235-g002" target="_blank">figure 2</a>. Spectra are scaled to equal DP intensities at 30–31 ppm.</p

Dynamic regimes and resulting intensities from polarisation transfer solid-state NMR experiments [30].

<p>Dynamic regimes and resulting intensities from polarisation transfer solid-state NMR experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235-Nowacka2" target="_blank">[30]</a>.</p

Main acyl chain region of the PT ssNMR spectra (a–d) and cryo-TEM images (e–h), scale bars 200 nm) of α-synuclein fibrils co-aggregated with different amounts of DOPC:DOPS 7∶3 vesicles.

<p>Inserted boxed peaks are INEPT and CP signals for the unresolved peaks from C_4–7 and C_12–15 baseline adjusted for background protein CP signal. Spectra are scaled to give equal protein CP intensity at 20 ppm. Filled dark spots in the cryo-TEM images are frost defects and are not part of the experimental system. More representative images can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235.s003" target="_blank">Figure S3</a>.</p

Cryo-TEM images of α-synuclein fibrils formed after co-aggregation with different amounts of DOPC vesicles.

<p>Inserted boxed peaks are INEPT and CP signals for the unresolved peaks from C_4–7 and C_12–15 adjusted for protein CP signal by baseline correction. Spectra are scaled to give equal protein CP intensity at 20 ppm. Filled dark spots in cryo-TEM images are technique dependent frost defects and are not part of the experimental system. More representative images can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235.s004" target="_blank">Figure S4</a>.</p

Schematic representation of lipid-protein co-aggregation at different L/P ratios.

<p>α-synuclein alone aggregates to fibrils that form bundles. When aggregation takes place in the presence of lipid vesicles (L/P = 1), fibrillar co-aggregates composed of protein and lipids form. These aggregates arrange into mesh-like tangles. At higher L/P ratios, the excess lipid vesicles adsorb to the fibrils, and due to strong interaction with the surface, the vesicles are deformed to non-spherical shape.</p

Adsorption of α‑Synuclein to Supported Lipid Bilayers: Positioning and Role of Electrostatics

An amyloid form of the protein α-synuclein is the major component of the intraneuronal inclusions called Lewy bodies, which are the neuropathological hallmark of Parkinson’s disease (PD). α-Synuclein is known to associate with anionic lipid membranes, and interactions between aggregating α-synuclein and cellular membranes are thought to be important for PD pathology. We have studied the molecular determinants for adsorption of monomeric α-synuclein to planar model lipid membranes composed of zwitterionic phosphatidylcholine alone or in a mixture with anionic phosphatidylserine (relevant for plasma membranes) or anionic cardiolipin (relevant for mitochondrial membranes). We studied the adsorption of the protein to supported bilayers, the position of the protein within and outside the bilayer, and structural changes in the model membranes using two complementary techniquesquartz crystal microbalance with dissipation monitoring, and neutron reflectometry. We found that the interaction and adsorbed conformation depend on membrane charge, protein charge, and electrostatic screening. The results imply that α-synuclein adsorbs in the headgroup region of anionic lipid bilayers with extensions into the bulk but does not penetrate deeply into or across the hydrophobic acyl chain region. The adsorption to anionic bilayers leads to a small perturbation of the acyl chain packing that is independent of anionic headgroup identity. We also explored the effect of changing the area per headgroup in the lipid bilayer by comparing model systems with different degrees of acyl chain saturation. An increase in area per lipid headgroup leads to an increase in the level of α-synuclein adsorption with a reduced water content in the acyl chain layer. In conclusion, the association of α-synuclein to membranes and its adsorbed conformation are of electrostatic origin, combined with van der Waals interactions, but with a very weak correlation to the molecular structure of the anionic lipid headgroup. The perturbation of the acyl chain packing upon monomeric protein adsorption favors association with unsaturated phospholipids preferentially found in the neuronal membrane