posted on 2019-10-30, 12:08authored byPeter Verwilst, Kyutae Kim, Kyoung Sunwoo, Hye-Ri Kim, Chulhun Kang, Jong Seung Kim
Endoplasmic
reticulum-thioflavin T (ER-ThT), a thioflavin
T-based fluorescent chemosensor, was developed to detect protein aggregates
in the endoplasmic reticulum (ER) and was applied to live cells under
various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive
denaturation of lysozyme and albumin, the intensity was increased
in a protein concentration-dependent way, following a nonfluorescent
lag phase. ER-ThT detects protein aggregates rather than
unfolded proteins in solution, and the protein aggregation can be
visualized in the presence of lipid membranes or native proteins.
Within live HeLa cells, ER-ThT is localized in the ER
and its fluorescence was dramatically increased upon ER stress induction
by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of
ER stress modulators (tauroursodeoxycholic acid, trimethylamine N-oxide, or 4-phenylbutyric acid), also known as chemical
chaperones, the fluorescence under Thapsigargin treatment was suppressed
to the level of the control group. Thus, ER-ThT is capable
of detecting the accumulation of protein aggregates under ER stress
in living cells and acts as an in vitro screening tool for ER stress
modulators, putative prodrugs against ER-related proteopathy. Overall,
the results strongly suggest that protein aggregation is intricately
involved in the activation of the unfolded protein response following
ER stress.