Abstract
The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (HOIP) and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.
Highlights
Posttranslational modification of substrates with ubiquitin regulates a wide variety of biological functions
Purifications of the three linear ubiquitin chain assembly complex (LUBAC) components expressed individually in Escherichia coli consistently gave low yields and isolated proteins were co-purified with several contaminants; this was severe in purifications of full-length HOIL-1-interacting protein (HOIP) (Figure 1A)
Given that HOIP is destabilized in cells lacking Shank-associated RH domain-interacting protein (SHARPIN) or HOIL-1L (Gerlach et al, 2011; Ikeda et al, 2011; Tokunaga et al, 2011; Fujita et al, 2018; Peltzer et al, 2018), we conjectured that HOIP could be unstable when recombinantly expressed in the absence of its interaction partners
Summary
Posttranslational modification of substrates with ubiquitin (ubiquitination) regulates a wide variety of biological functions. Ubiquitin forms chains via its seven internal Lys residues (Lys, Lys, Lys, Lys, Lys, Lys, and Lys63) through an isopeptide bond, or via Met through a peptide bond (Komander and Rape, 2012). The different ubiquitin chain types contribute to determine the fate of the substrate and biological outcomes regulated. Ubiquitin can be conjugated through isopeptide bonds to Lys residues, thioester bonds formed with the side chain of Cys residues, or oxyester bonds formed with side chains of Ser and Thr residues (Carvalho et al, 2007; McClellan et al, 2019; McDowell and Philpott, 2013; Vosper et al, 2009; Wang et al, 2007; Williams et al, 2007).
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