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.
Bibliographical noteFunding Information:
This work was also supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (CRI project no. 2018R1A3B1052702, J.S.K., 2017R1A2A2A05069805, C.K.) and the Basic Science Research Program (2017R1D1A1B03032561, P.V.) funded by the Ministry of Education as well as the Korea Research Fellowship Program funded by the Ministry of Science and ICT through the National Research Foundation of Korea (2016H1D3A1938052, P.V.).
© 2019 American Chemical Society.
- ER stress
- chemical chaperones
- protein aggregation
ASJC Scopus subject areas
- Process Chemistry and Technology
- Fluid Flow and Transfer Processes