Chemical stresses fail to mimic the unfolded protein response resulting from luminal load with unfolded polypeptides

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.

Keywords: ER quality control; endoplasmic reticulum stress (ER stress); molecular chaperone; protein folding; protein misfolding; unfolded protein response (UPR).

Figure 1.

ER retention and BiP binding are requisite for UPR induction. A, panel of model proteins expressed by inducible HEK293 cells used in this study. B, Western blot (WB) showing the expression level of model proteins on induction with 100 ng/ml of tetracycline. Loadings were normalized based total protein concentration. C, BiP mRNA up-regulation upon 17 h induction of the different model proteins with 100 ng/ml of tetracycline. Mean ± S.E., n = 3, ***, p < 0.001, nonspecific (ns), p > 0.5, one-way ANOVA with Dunnett's test for multiple comparisons. D, same as C for HERP mRNA. E, BiP protein up-regulation upon 17 h induction of the different model proteins with 100 ng/ml of tetracycline quantified by Western blot. Mean ± S.E., n = 3, ***, p < 0.001, ns, p > 0.5, one-way ANOVA with Dunnett's test for multiple comparisons. F, same as D for HERP. G, Western blot showing tunable expression of BACE457-HA and progressive induction of UPR markers GRP94, BiP, and HERP. * indicates unspecific, cross-reactive band. In the quantification the blue line is calculated as the average fold-change of the three UPR markers ± S.E. H same as F for CD3δ, which does not induce UPR. I, BiP co-precipitation with unfolded proteins. * indicates anti-HA heavy chain. J, quantification of H. Mean ± S.E., n = 3, *, p < 0.05, BootsRatio (68). K, same as C for drug treatments. ****, p < 0.0001. BACE457 as reference. L, same as E for drug treatments. **, p < 0.01. BACE457 as reference. M same as F for drug treatments. **, p < 0.01. BACE457 as reference.

Figure 2.

Transcriptional responses to ER stress–inducing chemicals and unfolded proteins. A, schematic representation of the workflow for GEP (left) and LFQ-MS (right). B, distribution of fold-changes from GEP data. Numbers on top and bottom show the number of significantly up- and down-regulated transcripts (p < 0.05, fold-change >20%) in each condition, dashed line shows the ±20% fold-change. Shown are only values outside the 1–99 percentile range. C, histogram showing the number of significantly up-regulated UPR target genes. UPRome (red) are all UPR target genes as defined based on literature (see text). D, hierarchical clustering based on the 97 UPRome genes up-regulated by TM_high and shown as a heat map. Genes are ordered based on their dependence from the different UPR branches (colored lines on the right).

Figure 3.

Unfolded proteins induce a subset of chemically induced UPR genes. A, Venn diagrams showing the overlap of significantly up-regulated genes by TG_low and TM_low. B, same as A but considering UPRome genes only. Gene names are color-coded based on their dependence on the different UPR branches. C, overlap between UPRome genes induced by TG_low, TM_low, and unfolded proteins.

Figure 4.

Unfolded proteins do not affect cellular viability, in contrast to drugs. A, qPCR data for transcription PERK genes linked to apoptosis. Mean ± S.E., n = 3, nonspecific (ns), p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001, unpaired two-tailed t tests. B, percentage of living cells after 17 h induction of model proteins and drug treatments relative to mock, ns, p > 0.05, **, p < 0.01, one-way ANOVA with Dunnett's test for multiple comparisons. C, cell growth of BACE457-inducible cells, expressed as relative cell number, over 4 days with the respective treatment, ns, p > 0.05, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001, two-way ANOVA with Dunnett's test for multiple comparisons. N.B., TG_low and TM_high are highly significant (****) at all time points. D, same as C for CD3δ_QQQ expressing cells, ns, >0.05 two-way ANOVA with Dunnett's test for multiple comparisons.

Figure 5.

Unfolded proteins induce a subset of ER chaperones and folding enzymes. A, histograms showing UPRome coverage by MS with the respective percentage. B, correlation between mRNA log₂ fold-change (y axis) and protein log₂ fold-change (x axis) from GEP and LFQ-MS data, respectively, upon drug treatments for mRNA-protein pairs with a positive log₂ fold-change values. C, same as B for expression of unfolded proteins. D, interaction network (string-db.org/)³ (69) of identified core UPR genes induced by the expression of unfolded proteins. Color-code defines the dependence of different UPR branches and in italic their role and function.