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

TY - JOUR

T1 - SILAC-based quantitative mass spectrometry-based proteomics quantifies endoplasmic reticulum stress in whole HeLa cells

AU - Itzhak, Daniel N

AU - Sacco, Francesca

AU - Nagaraj, Nagarjuna

AU - Tyanova, Stefka

AU - Mann, Matthias

AU - Murgia, Marta

N1 - © 2019. Published by The Company of Biologists Ltd.

PY - 2019/10/18

Y1 - 2019/10/18

N2 - The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. So far, a comprehensive analysis has been missing due to technological limitations. Here we employ Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics to quantify over 6200 proteins at increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 ug/ml) to those of thapsigargin (1 µM) and DTT (2mM), both activating the UPR through different mechanisms. The systematic quantification of the proteome-wide expression changes following proteostatic stress is a resource for the scientific community, which enables the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified 38 proteins not previously linked to the UPR, whose expression increases, 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.

AB - The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. So far, a comprehensive analysis has been missing due to technological limitations. Here we employ Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics to quantify over 6200 proteins at increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 ug/ml) to those of thapsigargin (1 µM) and DTT (2mM), both activating the UPR through different mechanisms. The systematic quantification of the proteome-wide expression changes following proteostatic stress is a resource for the scientific community, which enables the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified 38 proteins not previously linked to the UPR, whose expression increases, 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.

U2 - 10.1242/dmm.040741

DO - 10.1242/dmm.040741

M3 - Journal article

C2 - 31628211

SN - 1754-8403

JO - Disease models & mechanisms

JF - Disease models & mechanisms

ER -