Identification of ester-linked ubiquitylation sites during TLR7 signalling increases the number of inter-ubiquitin linkages from 8 to 12

Elisha H. McCrory, Vyacheslav Akimov, Philip Cohen*, Blagoy Blagoev*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

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.

Original languageEnglish
JournalThe Biochemical journal
Volume479
Issue number23
Pages (from-to)2419-2431
ISSN0264-6021
DOIs
Publication statusPublished - Dec 2022

Keywords

  • HOIL-1
  • innate immunity
  • Toll-like receptors
  • ubiquitin ligases
  • ubiquitins

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