Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins
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 domai...
| Main Authors: | , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2019-11-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/48434 |
| _version_ | 1811252976219586560 |
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| author | David W Morgens Charlene Chan Andrew J Kane Nicholas R Weir Amy Li Michael M Dubreuil C Kimberly Tsui Gaelen T Hess Adam Lavertu Kyuho Han Nicole Polyakov Jing Zhou Emma L Handy Philip Alabi Amanda Dombroski David Yao Russ B Altman Jason K Sello Vladimir Denic Michael C Bassik |
| author_facet | David W Morgens Charlene Chan Andrew J Kane Nicholas R Weir Amy Li Michael M Dubreuil C Kimberly Tsui Gaelen T Hess Adam Lavertu Kyuho Han Nicole Polyakov Jing Zhou Emma L Handy Philip Alabi Amanda Dombroski David Yao Russ B Altman Jason K Sello Vladimir Denic Michael C Bassik |
| author_sort | David W Morgens |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T16:43:48Z |
| format | Article |
| id | doaj.art-f8b0b64eb9da49ff8b1b2ae4b6712fa1 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T16:43:48Z |
| publishDate | 2019-11-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-f8b0b64eb9da49ff8b1b2ae4b6712fa12022-12-22T03:24:41ZengeLife Sciences Publications LtdeLife2050-084X2019-11-01810.7554/eLife.48434Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteinsDavid W Morgens0Charlene Chan1Andrew J Kane2Nicholas R Weir3https://orcid.org/0000-0002-1797-849XAmy Li4Michael M Dubreuil5C Kimberly Tsui6Gaelen T Hess7Adam Lavertu8Kyuho Han9Nicole Polyakov10Jing Zhou11Emma L Handy12Philip Alabi13Amanda Dombroski14David Yao15Russ B Altman16Jason K Sello17Vladimir Denic18https://orcid.org/0000-0002-1982-7281Michael C Bassik19https://orcid.org/0000-0001-5185-8427Department of Genetics, Stanford University, Stanford, United StatesDepartment of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, United StatesDepartment of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, United StatesDepartment of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, United StatesDepartment of Genetics, Stanford University, Stanford, United StatesProgram in Cancer Biology, Stanford University, Stanford, United StatesDepartment of Genetics, Stanford University, Stanford, United StatesDepartment of Genetics, Stanford University, Stanford, United StatesBiomedical Informatics Training Program, Stanford University, Stanford, United StatesDepartment of Genetics, Stanford University, Stanford, United StatesDepartment of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, United StatesDepartment of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, United StatesDepartment of Chemistry, Brown University, Providence, United StatesDepartment of Chemistry, Brown University, Providence, United StatesDepartment of Chemistry, Brown University, Providence, United StatesDepartment of Genetics, Stanford University, Stanford, United StatesDepartment of Genetics, Stanford University, Stanford, United States; Bioengineering, Stanford University, Stanford, United StatesDepartment of Chemistry, Brown University, Providence, United StatesDepartment of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, United StatesDepartment of Genetics, Stanford University, Stanford, United States; Program in Cancer Biology, Stanford University, Stanford, United States; Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, United StatesThe 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.https://elifesciences.org/articles/48434CRISPRRetro-2TRC pathway |
| spellingShingle | David W Morgens Charlene Chan Andrew J Kane Nicholas R Weir Amy Li Michael M Dubreuil C Kimberly Tsui Gaelen T Hess Adam Lavertu Kyuho Han Nicole Polyakov Jing Zhou Emma L Handy Philip Alabi Amanda Dombroski David Yao Russ B Altman Jason K Sello Vladimir Denic Michael C Bassik Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins eLife CRISPR Retro-2 TRC pathway |
| title | Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins |
| title_full | Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins |
| title_fullStr | Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins |
| title_full_unstemmed | Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins |
| title_short | Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins |
| title_sort | retro 2 protects cells from ricin toxicity by inhibiting asna1 mediated er targeting and insertion of tail anchored proteins |
| topic | CRISPR Retro-2 TRC pathway |
| url | https://elifesciences.org/articles/48434 |
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