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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 18; pp. 5691 - 5696
• Main Authors: Tipping, Kevin W., Karamanos, Theodoros K., Jakhria, Toral, Iadanza, Matthew G., Goodchild, Sophia C., Tuma, Roman, Ranson, Neil A., Hewitt, Eric W., Radford, Sheena E.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 05.05.2015; National Acad Sciences
• Subjects: Amyloid - chemistry; Amyloidosis - metabolism; Benzothiazoles; beta 2-Microglobulin - chemistry; Biological Sciences; Endosomes - chemistry; Fluorescence; HSP70 Heat-Shock Proteins - chemistry; Humans; Hydrogen-Ion Concentration; Kinetics; Lysosomes - chemistry; Membranes; Molecules; Monocytes - metabolism; Muramidase - chemistry; NMR; Nuclear magnetic resonance; Protein Binding; Proteins; Recombinant Proteins - chemistry; Spectrometry, Fluorescence; Thiazoles - chemistry; Toxicity; oligomer; amyloid; membrane disruption; disassembly; fibrils
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1423174112

Significance Oligomers formed en route to amyloid fibrils are thought to be the perpetrators of toxicity in many amyloid disorders. How amyloid fibrils contribute to disease, however, is less clear. Here, using β ₂-micoglobulin (β ₂m) as a model system, we show that the stability of amyloid fibrils is highly pH-dependent, with mild acidification enhancing the formation of fibril-derived nonnative oligomers that disrupt membranes and alter cellular function. Enhancing fibril stability by incubation with the molecular chaperone, hsp70, or by cross-linking, protects against fibril-induced membrane disruption and cellular dysfunction. The results highlight the importance of pH in determining fibril stability and suggest that uptake of fibrils into acidic cellular compartments may contribute to amyloid disease by pH-induced molecular shedding of toxic species. Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β ₂-microglobulin (β ₂m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.