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Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk
BACKGROUND: Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). METHODS: We adapted a method to analyze carbamylated peptides by...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10506243/ https://www.ncbi.nlm.nih.gov/pubmed/37723562 http://dx.doi.org/10.1186/s12964-023-01257-3 |
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author | You, Youngki Tsai, Chia-Feng Patel, Rishi Sarkar, Soumyadeep Clair, Geremy Zhou, Mowei Liu, Tao Metz, Thomas O. Das, Chittaranjan Nakayasu, Ernesto S. |
author_facet | You, Youngki Tsai, Chia-Feng Patel, Rishi Sarkar, Soumyadeep Clair, Geremy Zhou, Mowei Liu, Tao Metz, Thomas O. Das, Chittaranjan Nakayasu, Ernesto S. |
author_sort | You, Youngki |
collection | PubMed |
description | BACKGROUND: Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). METHODS: We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. RESULTS: By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. CONCLUSIONS: Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-023-01257-3. |
format | Online Article Text |
id | pubmed-10506243 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-105062432023-09-19 Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk You, Youngki Tsai, Chia-Feng Patel, Rishi Sarkar, Soumyadeep Clair, Geremy Zhou, Mowei Liu, Tao Metz, Thomas O. Das, Chittaranjan Nakayasu, Ernesto S. Cell Commun Signal Methodology BACKGROUND: Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). METHODS: We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. RESULTS: By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. CONCLUSIONS: Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-023-01257-3. BioMed Central 2023-09-18 /pmc/articles/PMC10506243/ /pubmed/37723562 http://dx.doi.org/10.1186/s12964-023-01257-3 Text en © Battelle Memorial Institute 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology You, Youngki Tsai, Chia-Feng Patel, Rishi Sarkar, Soumyadeep Clair, Geremy Zhou, Mowei Liu, Tao Metz, Thomas O. Das, Chittaranjan Nakayasu, Ernesto S. Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk |
title | Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk |
title_full | Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk |
title_fullStr | Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk |
title_full_unstemmed | Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk |
title_short | Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk |
title_sort | analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10506243/ https://www.ncbi.nlm.nih.gov/pubmed/37723562 http://dx.doi.org/10.1186/s12964-023-01257-3 |
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