Growth kinetics of amyloid-like fibrils: An integrated atomistic simulation and continuum theory approach.

Amyloid fibrils have long been associated with many neurodegenerative diseases. The conventional picture of the formation and proliferation of fibrils from unfolded proteins comprises primary and secondary nucleation of oligomers followed by elongation and fragmentation thereof. In this work, we first employ extensive all-atom molecular dynamics (MD) simulations of short peptides to investigate the governing processes of fibril growth at the molecular scale. We observe that the peptides in the bulk solution can bind onto and subsequently diffuse along the fibril surface, which leads to fibril elongation via either bulk- or surface-mediated docking mechanisms. Then, to guide the quantitative interpretation of these observations and to provide a more comprehensive picture of the growth kinetics of single fibrils, a continuum model which incorporates the key processes observed in the MD simulations is formulated. The model is employed to investigate how relevant physical parameters affect the kinetics of fibril growth and identify distinct growth regimes. In particular, it is shown that fibrils which strongly bind peptides may undergo a transient exponential growth phase in which the entire fibril surface effectively acts as a sink for peptides. We also demonstrate how the relevant model parameters can be estimated from the MD trajectories. Our results provide compelling evidence that the overall fibril growth rates are determined by both bulk and surface peptide fluxes, thereby contributing to a more fundamental understanding of the growth kinetics of amyloid-like fibrils.

Nucleation and Growth of Amyloid Fibrils.

The formation of amyloid fibrils is a complex phenomenon that remains poorly understood at the atomic scale. Herein, we perform extended unbiased all-atom simulations in explicit solvent of a short amphipathic peptide to shed light on the three mechanisms accounting for fibril formation, namely, nucleation via primary and secondary mechanisms, and fibril growth. We find that primary nucleation takes place via the formation of an intermediate state made of two laminated ß-sheets oriented perpendicular to each other. The amyloid fibril spine subsequently emerges from the rotation of these ß-sheets to account for peptides that are parallel to each other and perpendicular to the main axis of the fibril. Growth of this spine, in turn, takes place via a dock-and-lock mechanism. We find that peptides dock onto the fibril tip either from bulk solution or after diffusing on the fibril surface. The latter docking pathway contributes significantly to populate the fibril tip with peptides. We also find that side chain interactions drive the motion of peptides in the lock phase during growth, enabling them to adopt the structure imposed by the fibril tip with atomic fidelity. Conversely, the docked peptide becomes trapped in a local free energy minimum when docked-conformations are sampled randomly. Our simulations also highlight the role played by nonpolar fibril surface patches in catalyzing and orienting the formation of small cross-ß structures. More broadly, our simulations provide important new insights into the pathways and interactions accounting for primary and secondary nucleation as well as the growth of amyloid fibrils.

Atomic Insights into Amyloid-Induced Membrane Damage.

Amphipathic peptides can cause biological membranes to leak either by dissolving their lipid content via a detergent-like mechanism or by forming pores on the membrane surface. These modes of membrane damage have been related to the toxicity of amyloid peptides and to the activity of antimicrobial peptides. Here, we perform the first all-atom simulations in which membrane-bound amphipathic peptides self-assemble into ß-sheets that subsequently either form stable pores inside the bilayer or drag lipids out of the membrane surface. An analysis of these simulations shows that the acyl tail of lipids interact strongly with non-polar side chains of peptides deposited on the membrane. These strong interactions enable lipids to be dragged out of the bilayer by oligomeric structures accounting for detergent-like damage. They also disturb the orientation of lipid tails in the vicinity of peptides. These distortions are minimized around pore structures. We also show that membrane-bound ß-sheets become twisted with one of their extremities partially penetrating the lipid bilayer. This allows peptides on opposite leaflets to interact and form a long transmembrane ß-sheet, which initiates poration. In simulations, where peptides are deposited on a single leaflet, the twist in ß-sheets allows them to penetrate the membrane and form pores. In addition, our simulations show that fibril-like structures produce little damage to lipid membranes, as non-polar side chains in these structures are unavailable to interact with the acyl tail of lipids.

Binding Mechanisms of Amyloid-like Peptides to Lipid Bilayers and Effects of Divalent Cations.

In several neurodegenerative diseases, cell toxicity can emerge from damage produced by amyloid aggregates to lipid membranes. The details accounting for this damage are poorly understood including how individual amyloid peptides interact with phospholipid membranes before aggregation. Here, we use all-atom molecular dynamics simulations to investigate the molecular mechanisms accounting for amyloid-membrane interactions and the role played by calcium ions in this interaction. Model peptides known to self-assemble into amyloid fibrils and bilayer made from zwitterionic and anionic lipids are used in this study. We find that both electrostatic and hydrophobic interactions contribute to peptide-bilayer binding. In particular, the attraction of peptides to lipid bilayers is dominated by electrostatic interactions between positive residues and negative phosphate moieties of lipid head groups. This attraction is stronger for anionic bilayers than for zwitterionic ones. Hydrophobicity drives the burial of nonpolar residues into the interior of the bilayer producing strong binding in our simulations. Moreover, we observe that the attraction of peptides to the bilayer is significantly reduced in the presence of calcium ions. This is due to the binding of calcium ions to negative phosphate moieties of lipid head groups, which leaves phospholipid bilayers with a net positive charge. Strong binding of the peptide to the membrane occurs less frequently in the presence of calcium ions and involves the formation of a "Ca2+ bridge".

Role of Cholesterol on Binding of Amyloid Fibrils to Lipid Bilayers.

Molecular dynamics simulations are used to provide insights into the molecular mechanisms accounting for binding of amyloid fibrils to lipid bilayers and to study the effect of cholesterol in this process. We show that electrostatic interactions play an important role in fibril-bilayer binding and cholesterol modulates this interaction. In particular, the interaction between positive residues and lipid head groups becomes more favorable in the presence of cholesterol. Consistent with experiments, we find that cholesterol enhances fibril-membrane binding.

Regulation of Endothelial Cell Adherence and Elastic Modulus by Substrate Stiffness.

Although substrate stiffness has been previously reported to affect various cellular aspects, such as morphology, migration, viability, growth, and cytoskeletal structure, its influence on cell adherence has not been well examined. Here, we prepared three soft, medium, and hard polyacrylamide (PAAM) substrates and utilized AFM to study substrate elasticity and also the adhesion and mechanical properties of endothelial cells in response to changing substrate stiffness. Maximum detachment force and cell stiffness were increased with increasing substrate stiffness. Maximum detachment force values were 0.28 ± 0.14, 0.94 ± 0.27, and 1.99 ± 0.59 nN while Young's moduli of cells were 218.85 ± 38.73, 385.58 ± 131.67, and 933.20 ± 428.92 Pa for soft, medium, and hard substrates, respectively. Human umbilical vein endothelial cells (HUVECs) showed round to more spread shapes on soft to hard substrates, with the most organized and elongated actin structure on the hard hydrogel. Our results confirm the importance of substrate stiffness in regulating cell mechanics and adhesion for a successful cell therapy.