Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, Delaware 19716, United States.
ACS Chem Biol. 2023 Apr 21;18(4):837-847. doi: 10.1021/acschembio.2c00898. Epub 2023 Mar 27.
The important roles played by branched polyubiquitin chains were recently uncovered in proteasomal protein degradation, mitotic regulation, and NF-κB signaling. With the new realization of a wide presence of branched ubiquitin chains in mammalian cells, there is an urgent need of identifying the reader and eraser proteins of the various branched ubiquitin chains. In this work, we report the generation of noncleavable branched triubiquitin probes with combinations of K11-, K48-, and K63-linkages. Through a pulldown approach using the branched triUb probes, we identified human proteins that recognize branched triubiquitin structures including ubiquitin-binding proteins and deubiquitinases (DUBs). Proteomics analysis of the identified proteins enriched by the branched triubiquitin probes points to possible roles of branched ubiquitin chains in cellular processes including DNA damage response, autophagy, and receptor endocytosis. characterization of several identified UIM-containing proteins demonstrated their binding to branch triubiquitin chains with moderate to high affinities. Availability of this new class of branched triubiquitin probes will enable future investigation into the roles of branched polyubiquitin chains through identification of specific reader and eraser proteins, and the modes of branched ubiquitin chain recognition and processing using biochemical and biophysical methods.
支化多聚泛素链在蛋白酶体蛋白降解、有丝分裂调控和 NF-κB 信号转导中发挥着重要作用。随着支化泛素链在哺乳动物细胞中广泛存在的新认识，迫切需要鉴定各种支化泛素链的读取器和橡皮擦蛋白。在这项工作中，我们报告了具有 K11、K48 和 K63 连接的不可切割支化三泛素探针的生成。通过使用支化三 Ub 探针进行下拉方法，我们鉴定了识别支化三泛素结构的人类蛋白，包括泛素结合蛋白和去泛素酶 (DUBs)。通过支化三泛素探针富集的鉴定蛋白的蛋白质组学分析表明，支化泛素链可能在包括 DNA 损伤反应、自噬和受体内吞在内的细胞过程中发挥作用。对几种鉴定出的含 UIM 蛋白的特性分析表明，它们与分支三泛素链具有中等至高强度的结合亲和力。这种新类别的支化三泛素探针的可用性将使未来能够通过鉴定特定的读取器和橡皮擦蛋白以及使用生化和生物物理方法识别和处理支化泛素链的模式来研究支化多聚泛素链的作用。
