Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells
Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28...
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| Veröffentlicht in: | The Journal of nutrition 2021-11, Vol.151 (11), p.3329-3338 |
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| creator | Cao, Lei Pechan, Tibor Lee, Sanggil Cheng, Wen-Hsing |
| description | Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically.
We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells.
A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0–200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA.
Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P |
| doi_str_mv | 10.1093/jn/nxab290 |
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We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells.
A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0–200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA.
Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P < 0.05) protein levels of the long, but not the short, SELENOH variants in the presence of Na2SeO3 (100 nM). By contrast, SELENOH mRNA levels were increased by 53-fold upon FLAG-SELENOH transfection but were comparable with or without supplemental Se (100 nM). LC-MS/MS analyses of anti-FLAG immunoprecipitates designated both anti-FLAG bands as SELENOH and co-identified three 60S ribosomal and 9 other proteins. Overexpression of FLAG-SELENOH 1) reduced glutathione peroxidase 1 and thioredoxin reductase 1 expression at the protein rather than the mRNA level in the absence but not presence of supplemental Se (100 nM; P < 0.05); 2) increased mRNA levels of 3 heat shock proteins (HSP27, HSP70-1A, and HSP70-1B; P < 0.05); and 3) reduced senescence induced by H2O2 (20 μM, 4 hours; P < 0.05).
These cellular studies demonstrate a Se-independent, shortened SELENOH variant and suggest competition of overexpressed FLAG-SELENOH with 2 other selenoproteins for the expression at the protein but not the mRNA level in Se insufficiency.</description><identifier>ISSN: 0022-3166</identifier><identifier>EISSN: 1541-6100</identifier><identifier>DOI: 10.1093/jn/nxab290</identifier><identifier>PMID: 34510207</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Biochemical, Molecular, and Genetic Mechanisms ; Chromatography, Liquid ; DNA-Binding Proteins ; gene expression ; Glutathione Peroxidase ; HEK293 Cells ; Humans ; Hydrogen Peroxide ; isoform ; Protein Isoforms - genetics ; Proteomics ; RNA, Messenger - genetics ; Selenium ; selenoprotein H ; Selenoproteins - genetics ; Tandem Mass Spectrometry</subject><ispartof>The Journal of nutrition, 2021-11, Vol.151 (11), p.3329-3338</ispartof><rights>2021 American Society for Nutrition.</rights><rights>The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition. 2021</rights><rights>The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.</rights><rights>The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition. 2021</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c453t-7899b03254f96f281630729c8c60da0eca8e38bbf1a3e9d9f4a85dc0d7ba0aa13</citedby><cites>FETCH-LOGICAL-c453t-7899b03254f96f281630729c8c60da0eca8e38bbf1a3e9d9f4a85dc0d7ba0aa13</cites><orcidid>0000-0003-2720-5164</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34510207$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Cao, Lei</creatorcontrib><creatorcontrib>Pechan, Tibor</creatorcontrib><creatorcontrib>Lee, Sanggil</creatorcontrib><creatorcontrib>Cheng, Wen-Hsing</creatorcontrib><title>Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells</title><title>The Journal of nutrition</title><addtitle>J Nutr</addtitle><description>Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically.
We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells.
A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0–200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA.
Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P < 0.05) protein levels of the long, but not the short, SELENOH variants in the presence of Na2SeO3 (100 nM). By contrast, SELENOH mRNA levels were increased by 53-fold upon FLAG-SELENOH transfection but were comparable with or without supplemental Se (100 nM). LC-MS/MS analyses of anti-FLAG immunoprecipitates designated both anti-FLAG bands as SELENOH and co-identified three 60S ribosomal and 9 other proteins. Overexpression of FLAG-SELENOH 1) reduced glutathione peroxidase 1 and thioredoxin reductase 1 expression at the protein rather than the mRNA level in the absence but not presence of supplemental Se (100 nM; P < 0.05); 2) increased mRNA levels of 3 heat shock proteins (HSP27, HSP70-1A, and HSP70-1B; P < 0.05); and 3) reduced senescence induced by H2O2 (20 μM, 4 hours; P < 0.05).
These cellular studies demonstrate a Se-independent, shortened SELENOH variant and suggest competition of overexpressed FLAG-SELENOH with 2 other selenoproteins for the expression at the protein but not the mRNA level in Se insufficiency.</description><subject>Biochemical, Molecular, and Genetic Mechanisms</subject><subject>Chromatography, Liquid</subject><subject>DNA-Binding Proteins</subject><subject>gene expression</subject><subject>Glutathione Peroxidase</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Hydrogen Peroxide</subject><subject>isoform</subject><subject>Protein Isoforms - genetics</subject><subject>Proteomics</subject><subject>RNA, Messenger - genetics</subject><subject>Selenium</subject><subject>selenoprotein H</subject><subject>Selenoproteins - genetics</subject><subject>Tandem Mass Spectrometry</subject><issn>0022-3166</issn><issn>1541-6100</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kcFu1DAQhi0EokvhwgMgX7gghY7tJBtfkNpVoSutugjK2XKcMfUqsSPbuyov02clZbdVK1Wc5jDf_400PyHvGXxmIMXJxp_4G91yCS_IjFUlK2oG8JLMADgvBKvrI_ImpQ0AsFI2r8mRKCsGHOYzcrvs0GdnndHZBU-DpT-xRx_GGDI6Ty_oMgUb4pCo9h1dDqM2-TlsvcOIN2PElP6JPP1-2J1tM70MmQ4_Lk_pCnfYpzsBp-t8jfGpKNEpwKW4ogvs-_SWvLK6T_juMI_Jr6_nV4uLYrX-tlycrgpTViIX80bKFgSvSitryxtWC5hzaRpTQ6cBjW5QNG1rmRYoO2lL3VSdgW7eatCaiWPyZe8dt-2AnZl-EnWvxugGHf-ooJ16uvHuWv0OOyVBlIKLSfBpLzAxpBTRPmQZqLuW1MarQ0sT_OHxtQf0vpYJ-LgHwnb8v6jcc9NLcecwqmQceoOdi2iy6oJ7LvYXR3SxDQ</recordid><startdate>20211101</startdate><enddate>20211101</enddate><creator>Cao, Lei</creator><creator>Pechan, Tibor</creator><creator>Lee, Sanggil</creator><creator>Cheng, Wen-Hsing</creator><general>Elsevier Inc</general><general>Oxford University Press</general><scope>6I.</scope><scope>AAFTH</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>5PM</scope><orcidid>https://orcid.org/0000-0003-2720-5164</orcidid></search><sort><creationdate>20211101</creationdate><title>Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells</title><author>Cao, Lei ; Pechan, Tibor ; Lee, Sanggil ; Cheng, Wen-Hsing</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c453t-7899b03254f96f281630729c8c60da0eca8e38bbf1a3e9d9f4a85dc0d7ba0aa13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Biochemical, Molecular, and Genetic Mechanisms</topic><topic>Chromatography, Liquid</topic><topic>DNA-Binding Proteins</topic><topic>gene expression</topic><topic>Glutathione Peroxidase</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Hydrogen Peroxide</topic><topic>isoform</topic><topic>Protein Isoforms - genetics</topic><topic>Proteomics</topic><topic>RNA, Messenger - genetics</topic><topic>Selenium</topic><topic>selenoprotein H</topic><topic>Selenoproteins - genetics</topic><topic>Tandem Mass Spectrometry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Cao, Lei</creatorcontrib><creatorcontrib>Pechan, Tibor</creatorcontrib><creatorcontrib>Lee, Sanggil</creatorcontrib><creatorcontrib>Cheng, Wen-Hsing</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of nutrition</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Cao, Lei</au><au>Pechan, Tibor</au><au>Lee, Sanggil</au><au>Cheng, Wen-Hsing</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells</atitle><jtitle>The Journal of nutrition</jtitle><addtitle>J Nutr</addtitle><date>2021-11-01</date><risdate>2021</risdate><volume>151</volume><issue>11</issue><spage>3329</spage><epage>3338</epage><pages>3329-3338</pages><issn>0022-3166</issn><eissn>1541-6100</eissn><abstract>Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically.
We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells.
A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0–200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA.
Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P < 0.05) protein levels of the long, but not the short, SELENOH variants in the presence of Na2SeO3 (100 nM). By contrast, SELENOH mRNA levels were increased by 53-fold upon FLAG-SELENOH transfection but were comparable with or without supplemental Se (100 nM). LC-MS/MS analyses of anti-FLAG immunoprecipitates designated both anti-FLAG bands as SELENOH and co-identified three 60S ribosomal and 9 other proteins. Overexpression of FLAG-SELENOH 1) reduced glutathione peroxidase 1 and thioredoxin reductase 1 expression at the protein rather than the mRNA level in the absence but not presence of supplemental Se (100 nM; P < 0.05); 2) increased mRNA levels of 3 heat shock proteins (HSP27, HSP70-1A, and HSP70-1B; P < 0.05); and 3) reduced senescence induced by H2O2 (20 μM, 4 hours; P < 0.05).
These cellular studies demonstrate a Se-independent, shortened SELENOH variant and suggest competition of overexpressed FLAG-SELENOH with 2 other selenoproteins for the expression at the protein but not the mRNA level in Se insufficiency.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>34510207</pmid><doi>10.1093/jn/nxab290</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0003-2720-5164</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Biochemical, Molecular, and Genetic Mechanisms Chromatography, Liquid DNA-Binding Proteins gene expression Glutathione Peroxidase HEK293 Cells Humans Hydrogen Peroxide isoform Protein Isoforms - genetics Proteomics RNA, Messenger - genetics Selenium selenoprotein H Selenoproteins - genetics Tandem Mass Spectrometry |
| title | Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells |
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