Abstract
We have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitin chains containing both Lys63-linked and Met1-linked ubiquitin (Ub) oligomers, some of which are attached covalently to Interleukin Receptor Associated kinase 1. Here we show that Lys63/Met1-Ub hybrids are also formed rapidly when the TNFR1/TRADD, TLR3/TRIF- and NOD1/RIP2-signaling networks are activated, some of which are attached covalently to Receptor-Interacting Protein 1 (TNFR1 pathway) or Receptor-Interacting Protein 2 (NOD1 pathway). These observations suggest that the formation of Lys63/Met1-Ub hybrids are of general significance for the regulation of innate immune signaling systems, and their potential roles in vivo are discussed. We also report that TNFα induces the attachment of Met1-linked Ub chains directly to TNF receptor 1, which do not seem to be attached covalently to Lys63-linked or other types of ubiquitin chain.
Highlights
The innate immune system is vital for defense against infection by microbial pathogens, especially in young children [1]
We stimulated THP1 cells with Tumour Necrosis Factor a (TNFa) and captured the Met1-linked ubiquitin (M1-Ub) chains and Lys63-linked ubiquitin (K63-Ub) chains formed after 10 min on Halo-NF-kB Essential Modifier (NEMO) beads or Halo-TAB2 beads (Fig. 1A)
Similar to our previous findings in the IL-1 signaling network [9], we found that the complete hydrolysis of K63-Ub chains induced the appearance of small Ub oligomers containing 4e7 ubiquitins, which co-migrated with M1-Ub standard oligomers (Fig. 1C, compare lanes 7e9 with lanes 5 and 6) and could be hydrolysed by Otulin (Fig. 1C, compare Lanes 10 and 11 with lanes 7 and 8)
Summary
The innate immune system is vital for defense against infection by microbial pathogens, especially in young children [1]. Most TLRs, as well as the IL-1R, initiate signal transduction by recruiting the adaptor protein MyD88, which is followed by the binding of IRAK4 to MyD88, and the association of other IRAK family members with IRAK4 to form an oligomeric complex, termed the Myddosome [2,3]. This leads within minutes to the interaction of IRAKs 1 and 2 with the E3 ubiquitin ligase TRAF6 [4,5] and to the formation of Lys63-linked ubiquitin and Met1-linked ubiquitin chains (K63-Ub, M1-Ub chains). The M1-Ub chains ( called linear Ub chains) are formed by the E3 ligase LUBAC [6], while K63-Ub chains can be formed by the action of TRAF6 in combination with the E2-conjugating complex Ubc13-Uev1a ( called UBE2N-UBE2V1) [7,8]
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