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A Chemical Method for Labeling Lysine Methyltransferase Substrates
Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates ha...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
WILEY-VCH Verlag
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056122/ https://www.ncbi.nlm.nih.gov/pubmed/21243721 http://dx.doi.org/10.1002/cbic.201000433 |
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author | Binda, Olivier Boyce, Michael Rush, Jason S Palaniappan, Krishnan K Bertozzi, Carolyn R Gozani, Or |
author_facet | Binda, Olivier Boyce, Michael Rush, Jason S Palaniappan, Krishnan K Bertozzi, Carolyn R Gozani, Or |
author_sort | Binda, Olivier |
collection | PubMed |
description | Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts. |
format | Text |
id | pubmed-3056122 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | WILEY-VCH Verlag |
record_format | MEDLINE/PubMed |
spelling | pubmed-30561222012-01-24 A Chemical Method for Labeling Lysine Methyltransferase Substrates Binda, Olivier Boyce, Michael Rush, Jason S Palaniappan, Krishnan K Bertozzi, Carolyn R Gozani, Or Chembiochem Full Papers Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts. WILEY-VCH Verlag 2011-01-24 2010-11-17 /pmc/articles/PMC3056122/ /pubmed/21243721 http://dx.doi.org/10.1002/cbic.201000433 Text en Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://creativecommons.org/licenses/by/2.5/ Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. |
spellingShingle | Full Papers Binda, Olivier Boyce, Michael Rush, Jason S Palaniappan, Krishnan K Bertozzi, Carolyn R Gozani, Or A Chemical Method for Labeling Lysine Methyltransferase Substrates |
title | A Chemical Method for Labeling Lysine Methyltransferase Substrates |
title_full | A Chemical Method for Labeling Lysine Methyltransferase Substrates |
title_fullStr | A Chemical Method for Labeling Lysine Methyltransferase Substrates |
title_full_unstemmed | A Chemical Method for Labeling Lysine Methyltransferase Substrates |
title_short | A Chemical Method for Labeling Lysine Methyltransferase Substrates |
title_sort | chemical method for labeling lysine methyltransferase substrates |
topic | Full Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056122/ https://www.ncbi.nlm.nih.gov/pubmed/21243721 http://dx.doi.org/10.1002/cbic.201000433 |
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