Abstract
The stress sensors ATF6, IRE1, and PERK monitor deviations from homeostatic conditions in the endoplasmic reticulum (ER), a protein biogenesis compartment of eukaryotic cells. Their activation elicits unfolded protein responses (UPR) to re-establish proteostasis. UPR have been extensively investigated in cells exposed to chemicals that activate ER stress sensors by perturbing calcium, N-glycans, or redox homeostasis. Cell responses to variations in luminal load with unfolded proteins are, in contrast, poorly characterized. Here, we compared gene and protein expression profiles in HEK293 cells challenged with ER stress-inducing drugs or expressing model polypeptides. Drug titration to limit up-regulation of the endogenous ER stress reporters heat shock protein family A (Hsp70) member 5 (BiP/HSPA5) and homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERP/HERPUD1) to levels comparable with luminal accumulation of unfolded proteins substantially reduced the amplitude of both transcriptional and translational responses. However, these drug-induced changes remained pleiotropic and failed to recapitulate responses to ER load with unfolded proteins. These required unfolded protein association with BiP and induced a much smaller subset of genes participating in a chaperone complex that binds unfolded peptide chains. In conclusion, UPR resulting from ER load with unfolded proteins proceed via a well-defined and fine-tuned pathway, whereas even mild chemical stresses caused by compounds often used to stimulate UPR induce cellular responses largely unrelated to the UPR or ER-mediated protein secretion.
Highlights
At endoplasmic reticulum (ER) stress magnitudes comparable with those elicited by low drug concentrations, cell responses to ER load with BACE457, BACE457⌬, and CD3␦QQQ were characterized by the induction of a more restricted number of unfolded protein responses (UPR) gene products, and by the fact that no activation of the PERK pathway was measurable in gene expression profiling (GEP) and quantitative RT-PCR (qPCR) (Figs. 2D and 4A)
GEP analyses led to the identification of a set of UPR gene transcripts, whose expression was induced by drugs, and a more restricted set that was induced by ER load with unfolded proteins (Figs. 2, C and D, and 3, and Table S4)
Activation of the UPR is a hallmark of the general perturbation of cellular homeostasis or organelle-specific challenges such as accumulation of unfolded proteins in the ER lumen [6]
Summary
The stress sensors ATF6, IRE1, and PERK monitor deviations from homeostatic conditions in the endoplasmic reticulum (ER), a protein biogenesis compartment of eukaryotic cells. Drug titration to limit up-regulation of the endogenous ER stress reporters heat shock protein family A (Hsp70) member 5 (BiP/HSPA5) and homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERP/HERPUD1) to levels comparable with luminal accumulation of unfolded proteins substantially reduced the amplitude of both transcriptional and translational responses. The UPR have been extensively studied by exposing cells to chemical stress inducers such as dithiothreitol (DTT), tunicamycin (TM), or thapsigargin (TG) [9], which perturb redox, N-glycans, and calcium homeostasis, respectively These studies led to characterizing the signaling cascades regulating transcription and translation of several hundred gene products involved in protein biogenesis, function of the secretory pathway, and cell death in yeast [10] and in mammalian cells [11,12,13,14,15,16,17]. As a crucial difference with chemical stresses of the same magnitude that modify expression of a variety of genes, compartmental load with unfolded proteins induces transcription and translation of a subset of chaperones engaged in a functional complex previously reported to bind unfolded polypeptide chains
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