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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Biochemistry and Molecular Biology
2012
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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. |
format | Online Article Text |
id | pubmed-3412975 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | The American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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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