Abstract
BackgroundA number of amyloid diseases involve deposition of extracellular protein aggregates, which are implicated in mechanisms of cell damage and death. However, the mechanisms involved remain poorly understood.Methodology/Principal FindingsHere we use the yeast prion protein Ure2 as a generic model to investigate how amyloid-like protein aggregates can enter mammalian cells and convey cytotoxicity. The effect of three different states of Ure2 protein (native dimer, protofibrils and mature fibrils) was tested on four mammalian cell lines (SH-SY5Y, MES23.5, HEK-293 and HeLa) when added extracellularly to the medium. Immunofluorescence using a polyclonal antibody against Ure2 showed that all three protein states could enter the four cell lines. In each case, protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that the structural differences between the three protein states lead to their different effects upon cells. Protofibrils of Ure2 increased membrane conductivity, altered calcium homeostasis, and ultimately induced apoptosis. The use of standard inhibitors suggested uptake into mammalian cells might occur via receptor-mediated endocytosis. In order to investigate this further, we used the chicken DT40 B cell line DKOR, which allows conditional expression of clathrin. Uptake into the DKOR cell-line was reduced when clathrin expression was repressed suggesting similarities between the mechanism of PrP uptake and the mechanism observed here for Ure2.Conclusions/SignificanceThe results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases.
Highlights
Amyloid diseases are characterized by misfolding and aggregation of normal soluble peptide into b-sheet-rich oligomeric structures and amyloid fibrils in specific tissues [1]
Protofibrils of Ure2 significantly inhibited the growth of four cell lines in a dose-dependent manner
Mature fibrils were defined as the longer, thicker fibrils that are abundant in the final plateau phase of fibril grown (Fig. 1A), with a height of 12–15 nm measured by atomic force microscopy (AFM) (Fig. 1D)
Summary
Amyloid diseases are characterized by misfolding and aggregation of normal soluble peptide into b-sheet-rich oligomeric structures and amyloid fibrils in specific tissues [1]. Proteins that are not associated with disease can in some cases be induced under extreme conditions to transform into amyloidlike aggregates; the cytotoxic properties of the amyloid-like aggregates formed from non-disease-related proteins [4] are closely similar to those of disease-associated proteins such as the Ab peptides, a-synuclein, and transthyretin [5,6,7,8,9]. This suggests that the cytotoxic properties may be determined by common structural features of certain types of aggregates, rather than by specific amino acid sequences.
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