Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins

Abstract

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

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All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. David W Morgens

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Charlene Chan

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Andrew J Kane

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Nicholas R Weir

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1797-849X
  5. Amy Li

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Michael M Dubreuil

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. C Kimberly Tsui

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Gaelen T Hess

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Adam Lavertu

    Biomedical Informatics Training Program, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Kyuho Han

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Nicole Polyakov

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Jing Zhou

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Emma L Handy

    Department of Chemistry, Brown University, Providence, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Philip Alabi

    Department of Chemistry, Brown University, Providence, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Amanda Dombroski

    Department of Chemistry, Brown University, Providence, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. David Yao

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Russ B Altman

    Bioengineering, Genetics, and Medicine, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Jason K Sello

    Department of Chemistry, Brown University, Providence, United States
    For correspondence
    jason_sello@brown.edu
    Competing interests
    The authors declare that no competing interests exist.
  19. Vladimir Denic

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    For correspondence
    vdenic@mcb.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1982-7281
  20. Michael C Bassik

    Department of Genetics, Stanford University, Stanford, United States
    For correspondence
    bassik@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5185-8427

Funding

National Institutes of Health (1DP2HD084069-01)

  • Michael C Bassik

National Human Genome Research Institute (T32 HG000044)

  • David W Morgens

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ramanujan S Hegde, MRC Laboratory of Molecular Biology, United Kingdom

Publication history

  1. Received: May 13, 2019
  2. Accepted: October 28, 2019
  3. Accepted Manuscript published: November 1, 2019 (version 1)
  4. Version of Record published: November 15, 2019 (version 2)

Copyright

© 2019, Morgens et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. David W Morgens
  2. Charlene Chan
  3. Andrew J Kane
  4. Nicholas R Weir
  5. Amy Li
  6. Michael M Dubreuil
  7. C Kimberly Tsui
  8. Gaelen T Hess
  9. Adam Lavertu
  10. Kyuho Han
  11. Nicole Polyakov
  12. Jing Zhou
  13. Emma L Handy
  14. Philip Alabi
  15. Amanda Dombroski
  16. David Yao
  17. Russ B Altman
  18. Jason K Sello
  19. Vladimir Denic
  20. Michael C Bassik
(2019)
Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins
eLife 8:e48434.
https://doi.org/10.7554/eLife.48434

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