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.

中文翻译：

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在 TLR7 信号传导过程中鉴定酯连接的泛素化位点将泛素间连接的数量从 8 增加到 12

E3 连接酶 HOIL-1 在体外在泛素和蛋白质中的丝氨酸/苏氨酸残基之间形成酯键。在这里，我们利用 UbiSite 技术来鉴定在用 R848（TLR7/8 异二聚体的激活剂）刺激的巨噬细胞中经历 HOIL-1 催化泛素化的丝氨酸和苏氨酸残基。我们将泛素的 Thr12、Thr14、Ser20 和 Thr22 鉴定为与另一个泛素分子的 C 末端羧酸盐形成酯键的氨基酸残基。这使细胞中形成的泛素连锁类型的数量从 8 种增加到 12 种。我们还将 IRAK4 的 Ser175、IRAK2 的 Ser136、Thr163 和 Ser168 以及 MyD88 的 Thr141 鉴定为 HOIL-1 催化泛素化的进一步位点，以及这些蛋白质中也经历 R848 依赖性泛素化的赖氨酸残基。这些发现表明，附着在 myddosome 成分上的泛素链是由酯键和异肽键启动的。酯键形成发生在 IRAK2 和 IRAK4 的脯氨酸、丝氨酸、富含苏氨酸 (PST) 结构域和 MyD88 的中间结构域内。与IRAK2的Lys162、Thr163和Ser168相连的泛素分子分别与不同的IRAK2分子相连。