Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Apr 25;3(4):1279-92.
doi: 10.1016/j.celrep.2013.03.024. Epub 2013 Apr 11.

Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments

Matthew D Shoulders  1 , Lisa M Ryno, Joseph C Genereux, James J Moresco, Patricia G Tu, Chunlei Wu, John R Yates 3rd, Andrew I Su, Jeffery W Kelly, R Luke Wiseman
Affiliations

Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments

Matthew D Shoulders et al. Cell Rep. .

Abstract

The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small-molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Orthogonal, Ligand-Dependent Control of XBP1s and ATF6 Transcriptional Activity
(A) Model illustrating the TMP-mediated, posttranslational regulation of DHFR.ATF6. (B) Immunoblot of nuclear (top) and postnuclear (bottom) fractions from HEK293T-REx cells expressing DHFR.YFP or DHFR.ATF6 treated 12 hr with TMP (10 μM). The immunoblot of matrin-3 shows the efficiency of the nuclear extraction. (C) qPCR analysis of Hyou1, HerpUD, and Erdj4 in HEK293T-REx cells expressing DHFR.YFP or DHFR.ATF6 following a 12 hr treatment with TMP (10 μM) or a 6 hr treatment with Tm (10 μg/ml). qPCR data are reported relative to vehicle-treated cells expressing DHFR.YFP. qPCR data are reported as the mean ± 95% confidence interval. (D) TMP dose dependence of HerpUD upregulation in HEK293T-REx cells expressing DHFR.ATF6 (12 hr treatments with TMP). qPCR data are reported as the mean ± 95% confidence interval. (E) qPCR analysis of the ATF6 target gene BiP in HepG2, Huh7, or primary fibroblast cells transiently transduced with DHFR.YFP- or DHFR.ATF6-expressing adenoviruses and treated for 12 hr with 100 μM TMP or vehicle. qPCR data are reported relative to the corresponding vehicle-treated cells. qPCR data are reported as the mean ± 95% confidence interval. (F) qPCR analysis of BiP and Erdj4 in HEK293DAX cells following a 12 hr activation of XBP1s (dox; 1 μg/ml), DHFR.ATF6 (TMP; 10 μM), or both. qPCR data are reported relative to vehicle-treated HEK293DYG cells. qPCR data are reported as the mean ± 95% confidence interval. See also Figure S1 and Table S4.
Figure 2
Figure 2. Transcriptional and Proteomic Profiling of Stress-Independent XBP1s and/or ATF6 Activation in HEK293DAX Cells
Data derived from Affymetrix whole-genome arrays and SILAC-MuDPIT whole-cell proteomic analyses of HEK293DAX cells following a 12 hr activation of XBP1s (dox; 1 μg/ml), DHFR.ATF6 (TMP; 10 μM), or both. Only genes with a FDR <0.05 are described (n = 3), unless otherwise indicated. (A) Plot depicting mRNA fold increase owing to XBP1s activation versus ATF6 activation. Dashed lines indicate a 1.9-fold filter to assign genes as selectively induced by XBP1s (red), ATF6 (blue), or lacking selectivity (purple). Only genes upregulated ≥1.5-fold are colored. (B) Plot depicting a ratio-of-ratios comparison to identify genes cooperatively induced by XBP1s and ATF6. Genes colored green are cooperatively induced ≥1.33-fold by the combined activation of XBP1s and ATF6 relative to activating either transcription factor individually. (C) qPCR analysis validating select genes cooperatively upregulated by the combination of XBP1s and ATF6 in HEK293DAX cells. qPCR data are reported relative to vehicle-treated HEK293DAX cells. Error bars indicate SE from biological replicates (n = 3). ***p < 0.005. (D–F) Plots of log2 fold change versus log FDR for all proteins identified in our SILAC-MuDPIT analysis following activation of (D) XBP1s, (E) ATF6, or (F) both. FDRs in (D) and (E) were calculated from ANOVA p values. FDRs in (F) were calculated using p values from t test distributions. (G–I) Plots of fold change in microarray experiments versus fold change in proteomics experiments for HEK293DAX cells following activation of (G) XBP1s, (H) ATF6, or (I) both. In each panel, the significance of the SILAC-MuDPIT quantification is indicated by shading. (J) Quantification of autoradiograms of media from HEK293DAX cells collected after a 4 hr chase in nonradioactive media. The metabolic-labeling protocol employed is shown. Error bars represent SE from biological replicates (n = 3). See also Tables S1 and S3.
Figure 3
Figure 3. Predictive Pathway Analysis for Stress-Independent XBP1s- and/or ATF6-Mediated Remodeling of the ER Proteostasis Network
Cartoon depicting the impact of activating XBP1s, ATF6, or both XBP1s and ATF6 on the composition of ER proteostasis pathways obtained by integrating transcriptional, proteomic, and biochemical results. XBP1s (red) and ATF6 (blue)-selective genes are genes where activating either XBP1s (but not ATF6) or ATF6 (but not XBP1s) independently results in >75% of the induction observed when both XBP1s and ATF6 are activated (“max induction”). Genes induced >75% of the max induction by activating XBP1s in isolation and induced >75% of the max induction by activating ATF6 in isolation (i.e., lacking selectivity) are colored purple. Genes cooperatively induced >1.33-fold upon activation of both XBP1s and ATF6 relative to the activation of either transcription factor alone are colored green. Plain type indicates results from array data. Italicized type indicates results from proteomics data. Underlined type indicates results confirmed at both the transcript and the protein levels. Thresholds for transcriptional analyses were set at a FDR of <0.05. Thresholds for proteomic analyses were set at a FDR of 0.1.
Figure 4
Figure 4. XBP1s and/or ATF6 Activation Differentially Influences the Degradation of NHK-A1AT and NHK-A1ATQQQ
(A) Representative autoradiogram of [35S]-labeled NHK-A1AT immunopurified from transfected HEK293DAX cells following a 15 hr preactivation of XBP1s (dox; 1 μg/ml), DHFR.ATF6 (TMP; 10 μM), or both. The metabolic-labeling protocol employed is shown. (B) Quantification of autoradiograms in (A) monitoring the degradation of [35S]-labeled NHK-A1AT. The fraction of NHK-A1AT remaining was calculated by normalizing the recovered [35S] signal to the total amount of labeling observed at 0 hr. Error bars represent SE from biological replicates (n = 18). (C) Bar graph depicting the normalized fraction of NHK-A1AT remaining at 3 hr calculated as in (B). (D) Representative autoradiogram of [35S]-labeled NHK-A1ATQQQ immunopurified from transfected HEK293DAX cells following a 15 hr preactivation of XBP1s (dox; 1 μg/ml), DHFR.ATF6 (TMP; 10 μM), or both. The metabolic-labeling protocol employed is shown. (E) Quantification of autoradiograms in (D) monitoring the degradation of [35S]-labeled NHK-A1ATQQQ. The fraction of NHK-A1ATQQQ remaining was calculated as in (B). Error bars represent SE from biological replicates (n = 6). (F) Bar graph depicting the normalized fraction of NHK-A1ATQQQ remaining at 4.5 hr calculated as in (B). *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S3.
Figure 5
Figure 5. ATF6 Activation Selectively Attenuates the Secretion of Amyloidogenic TTR
(A) Autoradiogram of [35S]-labeled FTTTRA25T immunopurified from media and lysates collected from transfected HEK293DAX cells following a 15 hr preactivation of XBP1s (dox; 1 μg/ml), DHFR.ATF6 (TMP; 10 μM), or both. The metabolic-labeling protocol employed is shown. (B) Quantification of autoradiograms as shown in (A). Fraction secreted was calculated as previously described by Sekijima et al. (2005). Error bars represent SE from biological replicates (n = 4). (C) Graph depicting the normalized fraction secreted of [35S]-labeled FTTTRWT (white bars) or FTTTRA25T (orange bars) at 4 hr following a 15 hr preactivation of DHFR.ATF6 (TMP; 10 μM) in HEK293DAX cells. Error bars represent SE from biological replicates (n = 8 for FTTTRA25T, and n = 9 for FTTTRWT). (D) Graph depicting the total [35S]-labeled FTTTRA25T remaining in HEK293DAX cells (combined media and lysate protein levels as in A). The fraction remaining was calculated as reported previously by Sekijima et al. (2005). Error bars represent SE from biological replicates (n = 8). (E) Graph depicting the normalized fraction secreted of [35S]-labeled FTTTRA25T (orange bars) at 4 hr following preactivation of DHFR.ATF6 (TMP; 10 μM; 15 hr) inthe presence or absence of tafamidis (10 μM; 15 hr) in HEK293DAX cells. Error bars represent SE from biological replicates (n = 4). (F) Bar graph depicting the normalized fraction secreted of FTTTRA25T and endogenous TTRWT at 4 hr following a 13 hr pretreatment with TMP (100 μM) in HepG2 cells stably expressing DHFR.ATF6. Error bars represent SE from biological replicates (n = 4). (G) Bar graph depicting the normalized fraction secreted of [35S]-labeled FTTTRD18G at 4 hr following a 15 hr pretreatment with TMP (10 μM) from HEK293DAX cellstransfected with both FTTTRD18G and TTRWT. Error bars represent SE from biological replicates (n = 4). (H) Immunoblot of α-FLAG M1FTTTRA25T immunoisolations from DSP-crosslinked lysates prepared from HEK293DAX cells expressing FTTTRA25T following 15 hr activation of XBP1s (dox; 1 μg/ml), DHFR.ATF6 (TMP; 10 μM), or both. HEK293DAX cells expressing GFP are shown as a negative control (Mock). The KDEL immunoblot shows BiP. *p < 0.05, **p < 0.01, ***p < 0.005. See also Figure S4.
See this image and copyright information in PMC

References

    1. Adachi Y, Yamamoto K, Okada T, Yoshida H, Harada A, Mori K. ATF6 is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum. Cell Struct Funct. 2008;33:75–89. - PubMed
    1. Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science. 2008;319:916–919. - PubMed
    1. Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AGL, Wandless TJ. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell. 2006;126:995–1004. - PMC - PubMed
    1. Braakman I, Bulleid NJ. Protein folding and modification in the mammalian endoplasmic reticulum. Annu Rev Biochem. 2011;80:71–99. - PubMed
    1. Brodsky JL, Skach WR. Protein folding and quality control in the endoplasmic reticulum: recent lessons from yeast and mammalian cell systems. Curr Opin Cell Biol. 2011;23:464–475. - PMC - PubMed

Publication types

MeSH terms

Associated data