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SILAC-based quantitative proteomics using mass spectrometry quantifies endoplasmic reticulum stress in whole HeLa cells.


ABSTRACT: The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. To date, a comprehensive analysis of the UPR has not been possible because of technological limitations. Here, we employ stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics to quantify the response of over 6200 proteins to increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5?µg/ml) to those of thapsigargin (1?µM) and DTT (2?mM), both activating the UPR through different mechanisms. This systematic quantification of the proteome-wide expression changes that follow proteostatic stress is a resource for the scientific community, enabling the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified increased expression in 38 proteins not previously linked to the UPR, of which 15 likely remediate ER stress, and the remainder may contribute to pathological outcomes. Unexpectedly, there are few strongly downregulated proteins, despite expression of the pro-apoptotic transcription factor CHOP, suggesting that IRE1-dependent mRNA decay (RIDD) has a limited contribution to ER stress-mediated cell death in our system.

SUBMITTER: Itzhak DN 

PROVIDER: S-EPMC6899043 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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SILAC-based quantitative proteomics using mass spectrometry quantifies endoplasmic reticulum stress in whole HeLa cells.

Itzhak Daniel N DN   Sacco Francesca F   Nagaraj Nagarjuna N   Tyanova Stefka S   Mann Matthias M   Murgia Marta M  

Disease models & mechanisms 20191111 11


The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. To date, a comprehensive analysis of the UPR has not been possible because of technological limitations. Here, we employ stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics to quantify the response of over 6200 proteins to increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 µg/ml) to those of thapsigargin (1 µ  ...[more]

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