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

Elisha Honami McCrory, Vyacheslav Akimov, Philip Cohen (Lead / Corresponding author), Blagoy Blagoev (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)
40 Downloads (Pure)

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
Article numberBCJ20220510
Pages (from-to)2419-2431
Number of pages13
JournalBiochemical Journal
Volume479
Issue number23
Early online date21 Nov 2022
DOIs
Publication statusPublished - 7 Dec 2022

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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