Amyloid-like aggregates of the yeast prion protein ure2 enter vertebrate cells by specific endocytotic pathways and induce apoptosis.
BACKGROUND
A 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.


RESULTS
Here 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
The results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases.
Zhang
Chen
C
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Jackson
Antony P
AP
Zhang
Zai-Rong
ZR
Han
Yan
Y
Yu
Shun
S
He
Rong-Qiao
RQ
Perrett
Sarah
S
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002477:Cells / Q000166:cytology* / Q000378:metabolism*; D002645:Chickens; D004705:Endocytosis; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae; D002118:Calcium
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