MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, Scotland, U.K.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Biochem J. 2022 Dec 9;479(23):2419-2431. doi: 10.1042/BCJ20220510.
The E3 ligase HOIL-1 forms ester bonds in vitro between ubiquitin and serine/threonine residues in proteins. Here, we exploit UbiSite technology to identify serine and threonine residues undergoing HOIL-1 catalysed ubiquitylation in macrophages stimulated with R848, an activator of the TLR7/8 heterodimer. We identify Thr12, Thr14, Ser20 and Thr22 of ubiquitin as amino acid residues forming ester bonds with the C-terminal carboxylate of another ubiquitin molecule. This increases from 8 to 12 the number of ubiquitin linkage types that are formed in cells. We also identify Ser175 of IRAK4, Ser136, Thr163 and Ser168 of IRAK2 and Thr141 of MyD88 as further sites of HOIL-1-catalysed ubiquitylation together with lysine residues in these proteins that also undergo R848-dependent ubiquitylation. These findings establish that the ubiquitin chains attached to components of myddosomes are initiated by both ester and isopeptide bonds. Ester bond formation takes place within the proline, serine, threonine-rich (PST) domains of IRAK2 and IRAK4 and the intermediate domain of MyD88. The ubiquitin molecules attached to Lys162, Thr163 and Ser168 of IRAK2 are attached to different IRAK2 molecules.
E3 连接酶 HOIL-1 可在体外将泛素与蛋白质中的丝氨酸/苏氨酸残基形成酯键。在这里，我们利用 UbiSite 技术来鉴定 R848（TLR7/8 异二聚体的激活剂）刺激巨噬细胞中经 HOIL-1 催化的泛素化的丝氨酸和苏氨酸残基。我们鉴定出泛素中的 Thr12、Thr14、Ser20 和 Thr22 是与另一个泛素分子的 C 末端羧基形成酯键的氨基酸残基。这将细胞中形成的泛素连接类型的数量从 8 种增加到 12 种。我们还鉴定出 IRAK4 的 Ser175、IRAK2 的 Ser136、Thr163 和 Ser168 以及 MyD88 的 Thr141 是 HOIL-1 催化的泛素化的进一步位点，这些蛋白质中的赖氨酸残基也经历 R848 依赖性泛素化。这些发现确立了附着在 myddosome 成分上的泛素链是通过酯键和异肽键同时启动的。酯键的形成发生在 IRAK2 和 IRAK4 的脯氨酸、丝氨酸、苏氨酸丰富（PST）结构域和 MyD88 的中间结构域内。附着在 IRAK2 的 Lys162、Thr163 和 Ser168 上的泛素分子附着在不同的 IRAK2 分子上。
