Met1-linked ubiquitination in immune signalling

Abstract

N-terminal methionine-linked ubiquitin (Met1-Ub), or linear ubiquitin, has emerged as a central post-translational modification in innate immune signalling. The molecular machinery that assembles, senses and, more recently, disassembles Met1-Ub has been identified, and technical advances have enabled the identification of physiological substrates for Met1-Ub in response to activation of innate immune receptors. These discoveries have significantly advanced our understanding of how nondegradative ubiquitin modifications control proinflammatory responses mediated by nuclear factor-κB and mitogen-activated protein kinases. In this review, we discuss the current data on Met1-Ub function and regulation, and point to some of the questions that still remain unanswered.

Keywords: LUBAC; Met1-linked ubiquitin; NEMO; OTULIN; immune receptor signalling; inflammation; innate immunity.

Fig 1

The Met1-Ub machinery. Schematic model of the proteins and their domain organisation involved in Met1-Ub assembly (Writer), Met1-Ub sensing (Reader), and Met1-Ub disassembly (Eraser). Double arrows indicate interacting domains. LZ, leucine zipper; PH, pleckstrin homology; ZnF, zinc finger.

Fig 2

Assembly and disassembly of Met1-Ub. (A) Schematic model of Met1-Ub assembly by HOIP and the cofactors HOIL-1 and SHARPIN. HOIP is catalytically active when bound by HOIL-1 and/or SHARPIN. Cognate Ub-charged E2s (e.g. UbcH7) dock to RING1, which facilitates transfer of the Ub to RING2/CBR. The HOIP RING2-LDD/CBR orients the acceptor Ub, here shown conjugated to a Ub substrate (Step 1). The charged Ub is transferred from the E2 to Cys885 in RING2/CBR. The orientation of the acceptor Ub relative to the catalytic Cys in the CBR juxtaposes Met1 in the acceptor to the thioester bond between the donor Ub (G76) and Cys885 (Step 2). The donor Ub is subsequently conjugated to the α-amino group on Met1 of the acceptor to form a Met1 0linkage. This process can be repeated with a new Ub-charged E2 to extend the Met1-Ub chain further (Step 3). (B) Schematic model of how Met1 linkages are disassembled by OTULIN. Met1-Ub (here attached to a substrate) is bound by OTULIN's OTU. Glu16 of the proximal Ub inserts into the catalytic site of the OTU domain, which reorients the catalytic triad to enhance the hydrolytic activity of the enzyme, facilitating disassembly of the Met1 linkage. The Met1-Ub chains can be trimmed down to the Ub conjugated directly to a Lys in the substrate. This linkage is different from any Ub–Ub linkage, and is most likely less prone to hydrolysis by linkage-selective DUBs.

Fig 3

Model of innate immune signalling pathways controlled by Met-Ub. For NOD2 (left), RIPK2 is the central adaptor for assembly of the signalling complex and is the predominant target for ubiquitination. LUBAC is recruited through the Ub chains conjugated by XIAP, and LUBAC, in turn, conjugates Met1-Ub on existing Ub chains (or mono-Ub). The formation of Met1-Ub is essential for activation of NF-κB and MAPK pathways and for a productive transcriptional response. Conversely, OTULIN is needed to restrict accumulation of Met1-Ub that otherwise leads to an excessive transcriptional response. In IL-1β signalling (centre), MyD88 is the central adaptor for assembly of the signalling complex. In addition to MyD88, several other complex components are modified by Lys63-Ub. LUBAC extends the Lys63-Ub with Met1-Ub, thereby generating Lys63/Met1 mixed-linkage Ub chains. The specific Ub chains responsible for recruiting LUBAC have not been established. Notably, NEMO is predominantly modified by Lys63-Ub but binds Met1-Ub to enable productive signalling. In TNF signalling (right), cIAPs are responsible for recruiting LUBAC to the TNFR1 signalling complex. RIPK1 is a main target for ubiquitination, and is modified with Lys11-Ub, Lys63-Ub, and Met1-Ub. LUBAC conjugates Ub chains on RIPK1 and NEMO, and probably other complex components. This stabilises the signalling complex and enables optimal activation of NF-κB and MAPK pathways. In addition, Met1-Ub prevents the formation of cell death-inducing complexes that trigger either caspase-mediated apoptosis or RIPK3-mediated necroptosis. AP-1, activator protein-1; FADD, Fas-associated protein with Death Domain; ITCH, itchy E3 ubiquitin protein ligase; MLKL, Mixed Linkeage Kinase Domain-Like.