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Targeted protein destabilization reveals an estrogen-mediated ER stress response

Nature Chemical Biology volume 10, pages 957–962 (2014)Cite this article

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Abstract

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) of eukaryotic cells leads to an unfolded protein response (UPR) that either restores homeostasis or commits the cells to apoptosis. Tools traditionally used to study the UPR are proapoptotic and thus confound analysis of long-term cellular responses to ER stress. Here, we describe an ER-localized HaloTag (ERHT) protein that can be conditionally destabilized using a small-molecule hydrophobic tag (HyT36). Treatment of ERHT-expressing cells with HyT36 induces acute, resolvable ER stress that results in transient UPR activation without induction of apoptosis. Transcriptome analysis of late-stage responses to this UPR stimulus reveals a link between UPR activity and estrogen signaling.

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Figure 1: The ERHT system.
Figure 2: HyT36 treatment causes destabilization of the ERHT protein.
Figure 3: HyT36 treatment of ERHT cells induces a UPR response comparable to tunicamycin and thapsigargin.
Figure 4: Destabilization of the ERHT protein leads to acute ER stress.
Figure 5: The acute ER stress induced by HyT36 does not result in apoptosis.
Figure 6: Identification of estrogen signaling as a late-stage response to UPR activation.

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Acknowledgements

We wish to acknowledge financial support from the US National Institutes of Health (R01AI084140, R01CA083049 and T32GM067543) and the Department of Defense through the National Defense Science and Engineering Graduate Fellowship Program.

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Author notes

  1. Kanak Raina and Devin J Noblin: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Kanak Raina & Craig M Crews

  2. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Devin J Noblin, Yevgeniy V Serebrenik, Alison Adams & Craig M Crews

  3. Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Connie Zhao

  4. Department of Pharmacology, Yale University, New Haven, Connecticut, USA

    Craig M Crews

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  1. Kanak Raina
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Contributions

K.R. and D.J.N. contributed equally as first authors to this work. C.M.C. conceived the idea. K.R. generated the ERHT construct and cell line and performed the immunoprecipitation and immunoblotting studies. D.J.N., K.R., Y.V.S. and A.A. performed the qPCR experiments. D.J.N. analyzed the RNA-seq data. Y.V.S. and D.J.N. assessed compound cytotoxicity. C.Z. and Y.V.S. performed the ERAD experiments. D.J.N., K.R. and C.M.C. wrote the paper.

Corresponding author

Correspondence to Craig M Crews.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–23 and Supplementary Tables 1–3. (PDF 2195 kb)

Supplementary Data Set 1

Mean fold upregulation of genes from two independent mRNA-sequencing experiments: full gene list used for analysis using Ingenuity Pathway Analysis (IPA) is provided. (XLSX 1181 kb)

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Raina, K., Noblin, D., Serebrenik, Y. et al. Targeted protein destabilization reveals an estrogen-mediated ER stress response. Nat Chem Biol 10, 957–962 (2014). https://doi.org/10.1038/nchembio.1638

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