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Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag

The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosyl...

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Autores principales: Overath, Thorsten, Kuckelkorn, Ulrike, Henklein, Petra, Strehl, Britta, Bonar, David, Kloss, Alexander, Siele, Dagmar, Kloetzel, Peter-Michael, Janek, Katharina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Biochemistry and Molecular Biology 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412975/
https://www.ncbi.nlm.nih.gov/pubmed/22556278
http://dx.doi.org/10.1074/mcp.M111.015966
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author Overath, Thorsten
Kuckelkorn, Ulrike
Henklein, Petra
Strehl, Britta
Bonar, David
Kloss, Alexander
Siele, Dagmar
Kloetzel, Peter-Michael
Janek, Katharina
author_facet Overath, Thorsten
Kuckelkorn, Ulrike
Henklein, Petra
Strehl, Britta
Bonar, David
Kloss, Alexander
Siele, Dagmar
Kloetzel, Peter-Michael
Janek, Katharina
author_sort Overath, Thorsten
collection PubMed
description The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted β-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either “light” biotin-cystamine or deuterated “heavy” biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.
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spelling pubmed-34129752012-08-10 Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag Overath, Thorsten Kuckelkorn, Ulrike Henklein, Petra Strehl, Britta Bonar, David Kloss, Alexander Siele, Dagmar Kloetzel, Peter-Michael Janek, Katharina Mol Cell Proteomics Research The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted β-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either “light” biotin-cystamine or deuterated “heavy” biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site. The American Society for Biochemistry and Molecular Biology 2012-08 2012-05-03 /pmc/articles/PMC3412975/ /pubmed/22556278 http://dx.doi.org/10.1074/mcp.M111.015966 Text en © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
spellingShingle Research
Overath, Thorsten
Kuckelkorn, Ulrike
Henklein, Petra
Strehl, Britta
Bonar, David
Kloss, Alexander
Siele, Dagmar
Kloetzel, Peter-Michael
Janek, Katharina
Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag
title Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag
title_full Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag
title_fullStr Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag
title_full_unstemmed Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag
title_short Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag
title_sort mapping of o-glcnac sites of 20 s proteasome subunits and hsp90 by a novel biotin-cystamine tag
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412975/
https://www.ncbi.nlm.nih.gov/pubmed/22556278
http://dx.doi.org/10.1074/mcp.M111.015966
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