Lys63/Met1-hybrid ubiquitin chains are commonly formed during the activation of innate immune signalling.

We have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitinchains containing both Lys63-linked and Met1-linked ubiquitin(Ub) oligomers, some of which are attached covalently to Interleukin ReceptorAssociated 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 ( NOD1pathway). 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 ubiquitinchain.