SARS‐CoV‐2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets
The current work investigated SARS‐CoV‐2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC‐based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with th...
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| Veröffentlicht in: | FEBS letters 2021-12, Vol.595 (23), p.2872-2896 |
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| creator | Somasekharan, Syam Prakash Gleave, Martin |
| description | The current work investigated SARS‐CoV‐2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC‐based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress‐stimulated NCAP to undergo liquid–liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS‐CoV‐2 NCAP.
We have identified novel interactors of the SARS‐CoV‐2 nucleocapsid (NCAP) protein in human lung epithelial cancer cells. NCAP showed specific interaction with the stress granule (SG) proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following stress treatments. NCAP exhibited RNA‐dependent phase separation. The RNA intercalator mitoxantrone disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the NCAP. |
| doi_str_mv | 10.1002/1873-3468.14229 |
| format | Article |
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We have identified novel interactors of the SARS‐CoV‐2 nucleocapsid (NCAP) protein in human lung epithelial cancer cells. NCAP showed specific interaction with the stress granule (SG) proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following stress treatments. NCAP exhibited RNA‐dependent phase separation. The RNA intercalator mitoxantrone disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the NCAP.</description><identifier>ISSN: 0014-5793</identifier><identifier>EISSN: 1873-3468</identifier><identifier>DOI: 10.1002/1873-3468.14229</identifier><identifier>PMID: 34780058</identifier><language>eng</language><publisher>England: John Wiley and Sons Inc</publisher><subject>A549 Cells ; Adaptor Proteins, Signal Transducing - metabolism ; Coronavirus Nucleocapsid Proteins - chemistry ; Coronavirus Nucleocapsid Proteins - metabolism ; DNA Helicases - metabolism ; eIF-2 Kinase - metabolism ; G3BP1 ; Humans ; liquid–liquid phase separation ; mitoxantrone ; NCAP ; nucleocapsid ; Phosphoproteins - chemistry ; Phosphoproteins - metabolism ; Poly-ADP-Ribose Binding Proteins - metabolism ; Protein Binding ; RNA Helicases - metabolism ; RNA Recognition Motif Proteins - metabolism ; RNA-Binding Proteins - metabolism ; SARS‐CoV‐2 ; SILAC ; stress granules ; Stress Granules - chemistry ; Stress Granules - metabolism ; Ubiquitin Thiolesterase - metabolism ; viral factory ; viral infection</subject><ispartof>FEBS letters, 2021-12, Vol.595 (23), p.2872-2896</ispartof><rights>2021 The Authors. published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies</rights><rights>2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4689-a981aba18bcbd08940bf911d8b4a77c63d3c7050b4b75617a6a58fa752e084c53</citedby><cites>FETCH-LOGICAL-c4689-a981aba18bcbd08940bf911d8b4a77c63d3c7050b4b75617a6a58fa752e084c53</cites><orcidid>0000-0002-9006-8206</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2F1873-3468.14229$$EPDF$$P50$$Gwiley$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2F1873-3468.14229$$EHTML$$P50$$Gwiley$$Hfree_for_read</linktohtml><link.rule.ids>230,314,776,780,881,1411,1427,27903,27904,45553,45554,46387,46811</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34780058$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Somasekharan, Syam Prakash</creatorcontrib><creatorcontrib>Gleave, Martin</creatorcontrib><title>SARS‐CoV‐2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets</title><title>FEBS letters</title><addtitle>FEBS Lett</addtitle><description>The current work investigated SARS‐CoV‐2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC‐based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress‐stimulated NCAP to undergo liquid–liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS‐CoV‐2 NCAP.
We have identified novel interactors of the SARS‐CoV‐2 nucleocapsid (NCAP) protein in human lung epithelial cancer cells. NCAP showed specific interaction with the stress granule (SG) proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following stress treatments. NCAP exhibited RNA‐dependent phase separation. The RNA intercalator mitoxantrone disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the NCAP.</description><subject>A549 Cells</subject><subject>Adaptor Proteins, Signal Transducing - metabolism</subject><subject>Coronavirus Nucleocapsid Proteins - chemistry</subject><subject>Coronavirus Nucleocapsid Proteins - metabolism</subject><subject>DNA Helicases - metabolism</subject><subject>eIF-2 Kinase - metabolism</subject><subject>G3BP1</subject><subject>Humans</subject><subject>liquid–liquid phase separation</subject><subject>mitoxantrone</subject><subject>NCAP</subject><subject>nucleocapsid</subject><subject>Phosphoproteins - chemistry</subject><subject>Phosphoproteins - metabolism</subject><subject>Poly-ADP-Ribose Binding Proteins - metabolism</subject><subject>Protein Binding</subject><subject>RNA Helicases - metabolism</subject><subject>RNA Recognition Motif Proteins - metabolism</subject><subject>RNA-Binding Proteins - metabolism</subject><subject>SARS‐CoV‐2</subject><subject>SILAC</subject><subject>stress granules</subject><subject>Stress Granules - chemistry</subject><subject>Stress Granules - metabolism</subject><subject>Ubiquitin Thiolesterase - metabolism</subject><subject>viral factory</subject><subject>viral infection</subject><issn>0014-5793</issn><issn>1873-3468</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>EIF</sourceid><recordid>eNqFkUFvFCEYhonR2LV69mY4epkWZmBgLiZ109omTUxa9Uq-YZhdDDNMgbHpzaR_oL_RXyLr1I09eYHw8vAAeRF6S8kRJaQ8plJURcVqeURZWTbP0GqfPEcrQigruGiqA_Qqxu8kryVtXqKDiglJCJcrdH99cnX96-fD2n_LY4nHWTvjNUzRdngKPhk7YjsmE0CniG9t2mI7DPPog9nMDpIPEcPY4ZiCiRFvAoyzM0s2bSEaHM0EAVLOkse9DwN29mbO-i74yZkUX6MXPbho3jzOh-jr2emX9Xlx-fnTxfrkstD5O00BjaTQApWtbjsiG0bavqG0ky0DIXRddZUWhJOWtYLXVEANXPYgeGmIZJpXh-jD4p3mdjCdNmMK4NQU7ADhTnmw6unOaLdq438oWfOSM5IF7x8Fwd_MJiY12KiNczAaP0dV8kZIWlJaZfR4QXXwMQbT76-hRO2qU7ui1K4o9ae6fOLdv6_b83-7ykC9ALfWmbv_-dTZ6cdyMf8GLk6pJg</recordid><startdate>202112</startdate><enddate>202112</enddate><creator>Somasekharan, Syam Prakash</creator><creator>Gleave, Martin</creator><general>John Wiley and Sons Inc</general><scope>24P</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-9006-8206</orcidid></search><sort><creationdate>202112</creationdate><title>SARS‐CoV‐2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets</title><author>Somasekharan, Syam Prakash ; Gleave, Martin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4689-a981aba18bcbd08940bf911d8b4a77c63d3c7050b4b75617a6a58fa752e084c53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>A549 Cells</topic><topic>Adaptor Proteins, Signal Transducing - metabolism</topic><topic>Coronavirus Nucleocapsid Proteins - chemistry</topic><topic>Coronavirus Nucleocapsid Proteins - metabolism</topic><topic>DNA Helicases - metabolism</topic><topic>eIF-2 Kinase - metabolism</topic><topic>G3BP1</topic><topic>Humans</topic><topic>liquid–liquid phase separation</topic><topic>mitoxantrone</topic><topic>NCAP</topic><topic>nucleocapsid</topic><topic>Phosphoproteins - chemistry</topic><topic>Phosphoproteins - metabolism</topic><topic>Poly-ADP-Ribose Binding Proteins - metabolism</topic><topic>Protein Binding</topic><topic>RNA Helicases - metabolism</topic><topic>RNA Recognition Motif Proteins - metabolism</topic><topic>RNA-Binding Proteins - metabolism</topic><topic>SARS‐CoV‐2</topic><topic>SILAC</topic><topic>stress granules</topic><topic>Stress Granules - chemistry</topic><topic>Stress Granules - metabolism</topic><topic>Ubiquitin Thiolesterase - metabolism</topic><topic>viral factory</topic><topic>viral infection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Somasekharan, Syam Prakash</creatorcontrib><creatorcontrib>Gleave, Martin</creatorcontrib><collection>Wiley-Blackwell Open Access Titles</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>FEBS letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Somasekharan, Syam Prakash</au><au>Gleave, Martin</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>SARS‐CoV‐2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets</atitle><jtitle>FEBS letters</jtitle><addtitle>FEBS Lett</addtitle><date>2021-12</date><risdate>2021</risdate><volume>595</volume><issue>23</issue><spage>2872</spage><epage>2896</epage><pages>2872-2896</pages><issn>0014-5793</issn><eissn>1873-3468</eissn><abstract>The current work investigated SARS‐CoV‐2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC‐based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress‐stimulated NCAP to undergo liquid–liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS‐CoV‐2 NCAP.
We have identified novel interactors of the SARS‐CoV‐2 nucleocapsid (NCAP) protein in human lung epithelial cancer cells. NCAP showed specific interaction with the stress granule (SG) proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following stress treatments. NCAP exhibited RNA‐dependent phase separation. The RNA intercalator mitoxantrone disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the NCAP.</abstract><cop>England</cop><pub>John Wiley and Sons Inc</pub><pmid>34780058</pmid><doi>10.1002/1873-3468.14229</doi><tpages>25</tpages><orcidid>https://orcid.org/0000-0002-9006-8206</orcidid><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Wiley Free Content; Alma/SFX Local Collection |
| subjects | A549 Cells Adaptor Proteins, Signal Transducing - metabolism Coronavirus Nucleocapsid Proteins - chemistry Coronavirus Nucleocapsid Proteins - metabolism DNA Helicases - metabolism eIF-2 Kinase - metabolism G3BP1 Humans liquid–liquid phase separation mitoxantrone NCAP nucleocapsid Phosphoproteins - chemistry Phosphoproteins - metabolism Poly-ADP-Ribose Binding Proteins - metabolism Protein Binding RNA Helicases - metabolism RNA Recognition Motif Proteins - metabolism RNA-Binding Proteins - metabolism SARS‐CoV‐2 SILAC stress granules Stress Granules - chemistry Stress Granules - metabolism Ubiquitin Thiolesterase - metabolism viral factory viral infection |
| title | SARS‐CoV‐2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets |
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