A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.
A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.
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DOI：
10.1371/journal.pbio.3001501
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发表时间：
2022-06
期刊：
影响因子：
9.8
通讯作者：
中科院分区：
文献类型：
--
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蛋白质泛素化是一种重要的翻译后修饰，影响广泛的细胞过程。由于生物样品中泛素化种类的丰度低，已经在纯化和检测泛素化蛋白的方法上花费了相当大的努力。我们已经开发并表征了一种基于OtUBD的泛素检测和纯化的新工具，OtUBD是来自恙虫病东方体去泛素化酶（DUB）的高亲和力泛素结合结构域（UBD）。我们证明，OtUBD可用于纯化monoubiquitylated和polyubiquitylated底物从酵母和人类组织培养样品，并比较其性能与现有的方法。重要的是，我们发现的条件，无论是选择性纯化的共价泛素化的蛋白质或共分离的泛素化的蛋白质和它们的相互作用的蛋白质。作为这些新开发方法的原理证明，我们分析了芽殖酵母酿酒酵母的泛素组和泛素相关蛋白质组。将OtUBD亲和纯化与定量蛋白质组学相结合，我们确定了E3连接酶Bre1和Pib 1的潜在底物。OtUBD为泛素研究提供了一种通用、高效和经济的工具，与某些其他方法相比具有特定的优势，例如有效地检测单泛素化或泛素与非经典位点的连接。本研究提出了OtUBD，一种新的工具，来自细菌去泛素化酶，用于纯化和分析广泛的内源性泛素化蛋白，包括单泛素化，多泛素化，非赖氨酸泛素化和潜在的其他大分子。
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. This study presents OtUBD, a new tool derived from a bacterial deubiquitylase, for the purification and analysis of a broad range of endogenous ubiquitylated proteins, including monoubiquitylation, polyubiquitylation, non-lysine ubiquitylation and potentially other macromolecules.
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影响因子：
14.9
作者：
Gene Ontology Consortium
通讯作者：
Gene Ontology Consortium
影响因子：
16
作者：
Anindya, Roy;Ayguen, Ozan;Svejstrup, Jesper Q.
通讯作者：
Svejstrup, Jesper Q.
影响因子：
16
作者：
Elia AE;Boardman AP;Wang DC;Huttlin EL;Everley RA;Dephoure N;Zhou C;Koren I;Gygi SP;Elledge SJ
通讯作者：
Elledge SJ
DOI：
10.1038/nrd.2017.152
发表时间：
2018-01
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
Harrigan JA;Jacq X;Martin NM;Jackson SP
通讯作者：
Jackson SP
DOI：
10.1016/j.jbc.2021.100660
发表时间：
2021-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Cheng CL;Wong MK;Li Y;Hochstrasser M
通讯作者：
Hochstrasser M