A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin
Hepcidin is a liver‐secreted small disulfide‐rich peptide that plays a key role in iron homeostasis by binding and mediating the internalization and degradation of the only iron efflux transporter so far known, ferroportin (Fpn). To study hepcidin–Fpn interactions, in the present study we establishe...
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| Veröffentlicht in: | The FEBS journal 2013-04, Vol.280 (8), p.1773-1781 |
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| creator | Song, Ge Jiang, Qian Xu, Ting Liu, Ya‐Li Xu, Zeng‐Guang Guo, Zhan‐Yun |
| description | Hepcidin is a liver‐secreted small disulfide‐rich peptide that plays a key role in iron homeostasis by binding and mediating the internalization and degradation of the only iron efflux transporter so far known, ferroportin (Fpn). To study hepcidin–Fpn interactions, in the present study we established a convenient luminescence assay for the quantitative measurement of hepcidin‐induced Fpn internalization by fusing a small nanoluciferase (NanoLuc, 171 amino acids) at the Fpn C‐terminus. Once the NanoLuc‐tagged Fpn was internalized, the measured luminescence was significantly decreased when assayed with the intact transiently transfected cells and an inducible expression system. Through the coexpression of a NanoLuc‐tagged Fpn and an enhanced green fluorescent protein (EGFP)‐tagged Fpn by the use of an inducible bidirectional promoter, we could measure the hepcidin‐induced Fpn internalization qualitatively and quantitatively on the basis of the fluorescence of the tagged EGFP and the luminescence of the tagged NanoLuc. Thus, our present study provides a novel and convenient assay for measuring the hepcidin–Fpn interaction qualitatively and quantitatively. Through coexpression of a NanoLuc‐tagged wild‐type Fpn and an EGFP‐tagged hepcidin‐insensitive mutant [C326S]Fpn, we demonstrated that the mutant Fpn had no effect on hepcidin‐induced internalization of wild‐type Fpn, suggesting that wild‐type Fpn and mutant Fpn are functionally independent.
Ferroportin‐hepcidin system plays a key role in iron homeostasis. A convenient luminescent assay of ferroportin internalization was established by fusing a small nanoluciferase at the ferroportin C‐terminus, providing a novel quantitative assay for ferroportin‐hepcidin interaction studies. Through coexpression of a nanoluciferase‐tagged ferroportin and an EGFP‐tagged ferroportin controlled by a bi‐directional promoter, hepcidin‐induced ferroportin internalization could be analyzed qualitatively and quantitatively. |
| doi_str_mv | 10.1111/febs.12192 |
| format | Article |
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Ferroportin‐hepcidin system plays a key role in iron homeostasis. A convenient luminescent assay of ferroportin internalization was established by fusing a small nanoluciferase at the ferroportin C‐terminus, providing a novel quantitative assay for ferroportin‐hepcidin interaction studies. Through coexpression of a nanoluciferase‐tagged ferroportin and an EGFP‐tagged ferroportin controlled by a bi‐directional promoter, hepcidin‐induced ferroportin internalization could be analyzed qualitatively and quantitatively.</description><identifier>ISSN: 1742-464X</identifier><identifier>EISSN: 1742-4658</identifier><identifier>DOI: 10.1111/febs.12192</identifier><identifier>PMID: 23413836</identifier><language>eng</language><publisher>England: Blackwell Publishing Ltd</publisher><subject>Antimicrobial Cationic Peptides - chemistry ; Bioassays ; Cation Transport Proteins - chemistry ; Cell Membrane Permeability ; ferroportin ; HEK293 Cells ; hepcidin ; Hepcidins ; Homeostasis ; Humans ; interaction ; internalization ; Iron ; Luminescence ; luminescence assay ; Luminescent Measurements ; Mutation ; Peptides ; Proteins ; Transfection</subject><ispartof>The FEBS journal, 2013-04, Vol.280 (8), p.1773-1781</ispartof><rights>2013 The Authors Journal compilation © 2013 FEBS</rights><rights>2013 The Authors Journal compilation © 2013 FEBS.</rights><rights>Copyright © 2013 Federation of European Biochemical Societies</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3932-800f3ace22903f716c6eca1437055ccaa766c0f6d178b417ad87cb3d01d2a5693</citedby><cites>FETCH-LOGICAL-c3932-800f3ace22903f716c6eca1437055ccaa766c0f6d178b417ad87cb3d01d2a5693</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Ffebs.12192$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Ffebs.12192$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,1427,27901,27902,45550,45551,46384,46808</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23413836$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Song, Ge</creatorcontrib><creatorcontrib>Jiang, Qian</creatorcontrib><creatorcontrib>Xu, Ting</creatorcontrib><creatorcontrib>Liu, Ya‐Li</creatorcontrib><creatorcontrib>Xu, Zeng‐Guang</creatorcontrib><creatorcontrib>Guo, Zhan‐Yun</creatorcontrib><title>A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin</title><title>The FEBS journal</title><addtitle>FEBS J</addtitle><description>Hepcidin is a liver‐secreted small disulfide‐rich peptide that plays a key role in iron homeostasis by binding and mediating the internalization and degradation of the only iron efflux transporter so far known, ferroportin (Fpn). To study hepcidin–Fpn interactions, in the present study we established a convenient luminescence assay for the quantitative measurement of hepcidin‐induced Fpn internalization by fusing a small nanoluciferase (NanoLuc, 171 amino acids) at the Fpn C‐terminus. Once the NanoLuc‐tagged Fpn was internalized, the measured luminescence was significantly decreased when assayed with the intact transiently transfected cells and an inducible expression system. Through the coexpression of a NanoLuc‐tagged Fpn and an enhanced green fluorescent protein (EGFP)‐tagged Fpn by the use of an inducible bidirectional promoter, we could measure the hepcidin‐induced Fpn internalization qualitatively and quantitatively on the basis of the fluorescence of the tagged EGFP and the luminescence of the tagged NanoLuc. Thus, our present study provides a novel and convenient assay for measuring the hepcidin–Fpn interaction qualitatively and quantitatively. Through coexpression of a NanoLuc‐tagged wild‐type Fpn and an EGFP‐tagged hepcidin‐insensitive mutant [C326S]Fpn, we demonstrated that the mutant Fpn had no effect on hepcidin‐induced internalization of wild‐type Fpn, suggesting that wild‐type Fpn and mutant Fpn are functionally independent.
Ferroportin‐hepcidin system plays a key role in iron homeostasis. A convenient luminescent assay of ferroportin internalization was established by fusing a small nanoluciferase at the ferroportin C‐terminus, providing a novel quantitative assay for ferroportin‐hepcidin interaction studies. Through coexpression of a nanoluciferase‐tagged ferroportin and an EGFP‐tagged ferroportin controlled by a bi‐directional promoter, hepcidin‐induced ferroportin internalization could be analyzed qualitatively and quantitatively.</description><subject>Antimicrobial Cationic Peptides - chemistry</subject><subject>Bioassays</subject><subject>Cation Transport Proteins - chemistry</subject><subject>Cell Membrane Permeability</subject><subject>ferroportin</subject><subject>HEK293 Cells</subject><subject>hepcidin</subject><subject>Hepcidins</subject><subject>Homeostasis</subject><subject>Humans</subject><subject>interaction</subject><subject>internalization</subject><subject>Iron</subject><subject>Luminescence</subject><subject>luminescence assay</subject><subject>Luminescent Measurements</subject><subject>Mutation</subject><subject>Peptides</subject><subject>Proteins</subject><subject>Transfection</subject><issn>1742-464X</issn><issn>1742-4658</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp90E9LwzAYBvAgipvTix9AAl5E2MyfNm2Pc2wqCB5U8Fay9C3L6JKZpI766e3WuYMHc3kD748H3gehS0pGtH13Jcz9iDKasSPUp0nEhpGI0-PDP_rooTPvl4TwOMqyU9RjPKI85aKPYIyVNV9gNJiAq3qlDXgFRgGW3ssG2xKX4JxdWxe0wdoEcEZW-lsGbQ0OFvtQFw3WwXdLqXaLjQ4LvIC10oU25-iklJWHi_0coPfZ9G3yOHx-eXiajJ-HimecDVNCSi4VMJYRXiZUKAFK0ognJI6VkjIRQpFSFDRJ5xFNZJEmas4LQgsmY5HxAbrpctfOftbgQ77S7TVVJQ3Y2ueUM5GwLN3R6z90aevtZTsVx4ILQVp12ynlrPcOynzt9Eq6Jqck35afb8vPd-W3-GofWc9XUBzob9stoB3Y6Aqaf6Ly2fT-tQv9AWGxkC4</recordid><startdate>201304</startdate><enddate>201304</enddate><creator>Song, Ge</creator><creator>Jiang, Qian</creator><creator>Xu, Ting</creator><creator>Liu, Ya‐Li</creator><creator>Xu, Zeng‐Guang</creator><creator>Guo, Zhan‐Yun</creator><general>Blackwell Publishing Ltd</general><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>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>201304</creationdate><title>A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin</title><author>Song, Ge ; Jiang, Qian ; Xu, Ting ; Liu, Ya‐Li ; Xu, Zeng‐Guang ; Guo, Zhan‐Yun</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3932-800f3ace22903f716c6eca1437055ccaa766c0f6d178b417ad87cb3d01d2a5693</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Antimicrobial Cationic Peptides - chemistry</topic><topic>Bioassays</topic><topic>Cation Transport Proteins - chemistry</topic><topic>Cell Membrane Permeability</topic><topic>ferroportin</topic><topic>HEK293 Cells</topic><topic>hepcidin</topic><topic>Hepcidins</topic><topic>Homeostasis</topic><topic>Humans</topic><topic>interaction</topic><topic>internalization</topic><topic>Iron</topic><topic>Luminescence</topic><topic>luminescence assay</topic><topic>Luminescent Measurements</topic><topic>Mutation</topic><topic>Peptides</topic><topic>Proteins</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Song, Ge</creatorcontrib><creatorcontrib>Jiang, Qian</creatorcontrib><creatorcontrib>Xu, Ting</creatorcontrib><creatorcontrib>Liu, Ya‐Li</creatorcontrib><creatorcontrib>Xu, Zeng‐Guang</creatorcontrib><creatorcontrib>Guo, Zhan‐Yun</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The FEBS journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Song, Ge</au><au>Jiang, Qian</au><au>Xu, Ting</au><au>Liu, Ya‐Li</au><au>Xu, Zeng‐Guang</au><au>Guo, Zhan‐Yun</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin</atitle><jtitle>The FEBS journal</jtitle><addtitle>FEBS J</addtitle><date>2013-04</date><risdate>2013</risdate><volume>280</volume><issue>8</issue><spage>1773</spage><epage>1781</epage><pages>1773-1781</pages><issn>1742-464X</issn><eissn>1742-4658</eissn><abstract>Hepcidin is a liver‐secreted small disulfide‐rich peptide that plays a key role in iron homeostasis by binding and mediating the internalization and degradation of the only iron efflux transporter so far known, ferroportin (Fpn). To study hepcidin–Fpn interactions, in the present study we established a convenient luminescence assay for the quantitative measurement of hepcidin‐induced Fpn internalization by fusing a small nanoluciferase (NanoLuc, 171 amino acids) at the Fpn C‐terminus. Once the NanoLuc‐tagged Fpn was internalized, the measured luminescence was significantly decreased when assayed with the intact transiently transfected cells and an inducible expression system. Through the coexpression of a NanoLuc‐tagged Fpn and an enhanced green fluorescent protein (EGFP)‐tagged Fpn by the use of an inducible bidirectional promoter, we could measure the hepcidin‐induced Fpn internalization qualitatively and quantitatively on the basis of the fluorescence of the tagged EGFP and the luminescence of the tagged NanoLuc. Thus, our present study provides a novel and convenient assay for measuring the hepcidin–Fpn interaction qualitatively and quantitatively. Through coexpression of a NanoLuc‐tagged wild‐type Fpn and an EGFP‐tagged hepcidin‐insensitive mutant [C326S]Fpn, we demonstrated that the mutant Fpn had no effect on hepcidin‐induced internalization of wild‐type Fpn, suggesting that wild‐type Fpn and mutant Fpn are functionally independent.
Ferroportin‐hepcidin system plays a key role in iron homeostasis. A convenient luminescent assay of ferroportin internalization was established by fusing a small nanoluciferase at the ferroportin C‐terminus, providing a novel quantitative assay for ferroportin‐hepcidin interaction studies. Through coexpression of a nanoluciferase‐tagged ferroportin and an EGFP‐tagged ferroportin controlled by a bi‐directional promoter, hepcidin‐induced ferroportin internalization could be analyzed qualitatively and quantitatively.</abstract><cop>England</cop><pub>Blackwell Publishing Ltd</pub><pmid>23413836</pmid><doi>10.1111/febs.12192</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Antimicrobial Cationic Peptides - chemistry Bioassays Cation Transport Proteins - chemistry Cell Membrane Permeability ferroportin HEK293 Cells hepcidin Hepcidins Homeostasis Humans interaction internalization Iron Luminescence luminescence assay Luminescent Measurements Mutation Peptides Proteins Transfection |
| title | A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin |
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