Direct three-dimensional visualization of membrane disruption by amyloid fibrils

Abstract

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from β 2 -microglobulin (β 2 m). Using cryoelectron tomography, we demonstrate that fragmented β 2 m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.T he failure of molecular chaperones to prevent the accumulation of misfolded proteins results in protein aggregation and amyloid formation, processes associated with severe human degenerative diseases (1, 2). Despite the attention focused on these problems during the century since these disorders were first identified (3-5) and advances in understanding the structure of the cross-β conformation of amyloid fibrils in atomic detail (6, 7), the basic pathological mechanisms of amyloidosis remain poorly understood and therapeutic intervention is lacking. The identity of the toxic species and the mechanisms of cytotoxicity remain major unsolved problems. In some systems, there is evidence suggesting that prefibrillar oligomers, rather than the fully formed fibrils, are the source of toxicity (8, 9). In these cases, cytotoxicity is thought to result from the formation of specific membrane pores (10, 11) although alternative models including membrane destabilization or membrane thinning have also been proposed (12-15). In other cases, toxicity may reside with the amyloid fibrils themselves. Evidence that toxicity correlates with fibrillar assemblies has been reported for yeast and mammalian prion proteins (16, 17), human lysozyme (18), Huntingtin exon 1, α-synuclein (19), and Amyloid-β (Aβ) (20, 21). Furthermore, Aβ plaques have been shown to form rapidly in vivo and to precede neuropathological changes in a mouse model (22). The end surfaces of fibrils (herein termed "fibril ends") are unusually reactive entities: they play a key role in catalyzing recruitment and conformational conversion of amyloid-forming proteins (23, 24) and provide the sites for templated...

Interactions of Lipid Membranes with Fibrillar Protein Aggregates

“…6.4). Our data agree with the results of Milanesi and colleagues indicating that fibrillar β 2 -microglobulin associates with the membrane surface, as follows from the fibrilinduced clustering of liposomes (Milanesi et al 2012). These studies suggest that protein fibers interact mainly with the surface of the lipid monolayer but cause little or no perturbation in its core.…”

pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers

“…Reduced fibril length can also contribute to disease by increasing the uptake of fibrils into intracellular compartments (8)(9)(10), or by enhancing transmission of aggregates between cells in prionlike processes (11). In addition, fibrils have been shown to interact with lipid bilayers, causing membrane deformations via interactions with fibril ends (9,12,13), or promoting the release of toxic oligomers by membrane-induced fibril disassembly (14). Together these data support a role of amyloid fibrils as direct or indirect agents of amyloid disease.…”

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits

“…They possibly involve hydrophobic interactions between the fatty acid tails and the relatively hydrophobic fibril core that is exposed at the fibril tip, as suggested by a recent structural model of Aβ(1-42) fibrils (42). Interactions of the tips of in vitro-formed β2-microglobulin fibrils were previously observed with artificial liposomes and found to perturb the structural integrity of their lipid bilayer structures (43), one possible toxic mechanism of amyloid fibrils (25,43). Interactions of the lateral fibril surface with the lipid bilayer are likely to involve greater contributions from polar or electrostatic groups and may underlie the binding of cross-β fibrils to lipid-enveloped viral particles in the course of viral infection enhancements (26).…”