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

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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.

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David W Morgens

Department of Genetics, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.
Charlene Chan

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

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Andrew J Kane

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

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Nicholas R Weir

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

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"This ORCID iD identifies the author of this article:" 0000-0002-1797-849X
Amy Li

Department of Genetics, Stanford University, Stanford, United States

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Michael M Dubreuil

Department of Genetics, Stanford University, Stanford, United States

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C Kimberly Tsui

Department of Genetics, Stanford University, Stanford, United States

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Gaelen T Hess

Department of Genetics, Stanford University, Stanford, United States

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Adam Lavertu

Biomedical Informatics Training Program, Stanford University, Stanford, United States

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Kyuho Han

Department of Genetics, Stanford University, Stanford, United States

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Nicole Polyakov

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

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Jing Zhou

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

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Emma L Handy

Department of Chemistry, Brown University, Providence, United States

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Philip Alabi

Department of Chemistry, Brown University, Providence, United States

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Amanda Dombroski

Department of Chemistry, Brown University, Providence, United States

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David Yao

Department of Genetics, Stanford University, Stanford, United States

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Russ B Altman

Bioengineering, Genetics, and Medicine, Stanford University, Stanford, United States

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Jason K Sello

Department of Chemistry, Brown University, Providence, United States

For correspondence
jason_sello@brown.edu

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Vladimir Denic

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

For correspondence
vdenic@mcb.harvard.edu

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1982-7281
Michael C Bassik

Department of Genetics, Stanford University, Stanford, United States

For correspondence
bassik@stanford.edu

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The authors declare that no competing interests exist.

"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.

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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.