A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.
Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed an...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2022-06-01
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| Series: | PLoS Biology |
| Online Access: | https://doi.org/10.1371/journal.pbio.3001501 |
| _version_ | 1811288060965421056 |
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| author | Mengwen Zhang Jason M Berk Adrian B Mehrtash Jean Kanyo Mark Hochstrasser |
| author_facet | Mengwen Zhang Jason M Berk Adrian B Mehrtash Jean Kanyo Mark Hochstrasser |
| author_sort | Mengwen Zhang |
| collection | DOAJ |
| description | Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites. |
| first_indexed | 2024-04-13T03:29:24Z |
| format | Article |
| id | doaj.art-4d747f9c9c3347bbbb87cd87a87d79c8 |
| institution | Directory Open Access Journal |
| issn | 1544-9173 1545-7885 |
| language | English |
| last_indexed | 2024-04-13T03:29:24Z |
| publishDate | 2022-06-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Biology |
| spelling | doaj.art-4d747f9c9c3347bbbb87cd87a87d79c82022-12-22T03:04:32ZengPublic Library of Science (PLoS)PLoS Biology1544-91731545-78852022-06-01206e300150110.1371/journal.pbio.3001501A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.Mengwen ZhangJason M BerkAdrian B MehrtashJean KanyoMark HochstrasserProtein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.https://doi.org/10.1371/journal.pbio.3001501 |
| spellingShingle | Mengwen Zhang Jason M Berk Adrian B Mehrtash Jean Kanyo Mark Hochstrasser A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins. PLoS Biology |
| title | A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins. |
| title_full | A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins. |
| title_fullStr | A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins. |
| title_full_unstemmed | A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins. |
| title_short | A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins. |
| title_sort | versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins |
| url | https://doi.org/10.1371/journal.pbio.3001501 |
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