Size and morphology of toxic oligomers of amyloidogenic proteins: a case study of human stefin B

Abstract

Amyloid-induced toxicity is a well-known phenomenon but the molecular background remains unclear. One hypothesis relates toxicity to amyloid–membrane interactions, predicting that amyloid oligomers make pores into membranes. Therefore, the toxicity and membrane interaction of prefibrillar aggregates and individual oligomers of a non-pathological yet highly amyloidogenic protein human stefin B (cystatin B) was examined. By monitoring caspase-3 activity and by testing cell viability, we showed that the lag phase aggregates obtained at pH 5 and 3 were toxic to neuroblastoma cells. Of equal toxicity were the higher-order oligomers prepared at pH 7 by freeze–thaw cycles. The higher-order oligomers eluted on size-exclusion chromatography (SEC) as a broad peak comprising hexamers, octamers, 12- and 16-mers, well separated from monomers, dimers and tetramers. Only oligomers higher than the tetramers (Rh >3.5 nm) proved toxic, in contrast to dimers and tetramers. In accordance with data from SEC, dynamic light scattering and atomic force microscopy data indicate that the toxic oligomers have diameters larger than 4 nm. Critical pressure measurements showed that the toxic higher-order oligomers inserted more effectively into model lipid monolayers than dimers and tetramers. They also bound, similarly to prefibrillar aggregates, to the plasma membrane and became internalized. Taken together, our results confirm the importance of membrane interaction and perforation in the phenomenon of cytotoxicity.

Keywords:

• Amyloid fibrils
• toxic oligomers
• cystatins
• prefibrillar aggregates
• acidic phospholipid binding
• amyloid pores

Abbreviations
Aβ	=	amyloid β;
Rh	=	hydrodynamic radius;
SEC	=	size-exclusion chromatography;
DLS	=	dynamic light scattering;
AFM	=	atomic force microscopy;
TEM	=	transmission electron microscopy;
PBS buffer	=	phosphate-buffered saline.

Abbreviations
Aβ	=	amyloid β;
Rh	=	hydrodynamic radius;
SEC	=	size-exclusion chromatography;
DLS	=	dynamic light scattering;
AFM	=	atomic force microscopy;
TEM	=	transmission electron microscopy;
PBS buffer	=	phosphate-buffered saline.