Extended Data Figure 1: A model for A20 OTU and A20 ZnF4 regulation of TNF- and LPS-activated signalling.

a, Left complex. Upon TNF binding TNFR1 forms a trimer, thereby promoting recruitment of the adaptor protein TRADD and the RIP1 kinase (RIPK1). TRADD recruits TRAF2, TRAF5 and the ubiquitin ligases cIAP1 and cIAP2. The cIAP proteins promote K63-linked ubiquitination of signalling proteins including RIPK1, cIAP1/2 (autoubiquitination), and possibly TNFR1. K63 ubiquitination of cIAP1/2 subsequently recruits the LUBAC complex that promotes linear polyubiquitination of signalling proteins including RIPK1 and TNFR1. K63 ubiquitin chains on RIPK1 promote recruitment of the TAK1/TAB2/3 complex, whereas linear ubiquitin chains on RIPK1 promote IκK kinase complex recruitment via NEMO. Kinase complex recruitment promotes their subsequent activation and propagation of downstream JNK, p38 (via MKK3 and MKK4) and NFκB signalling pathways. We propose that A20 is recruited to the active TNFR1 signalling complex via ZnF7 binding to linear ubiquitin chains. The A20 OTU domain catalytic C103 is essential for attenuating TNF-activated signalling by removing K63 polyubiquitin chains from RIPK1 and other proteins including TNFR1, thereby promoting the dissociation of the active signalling complex. The A20 ZnF4 motif, that depends on C609/C612 for structural integrity and Y599/F600 for ubiquitin binding, is likely to collaborate with other proteins (not shown) to further downregulate TNF signalling by directing K48 polyubiquitination and subsequent degradation of proximal complex proteins, including RIPK1 and TNFR1. Right complex. LPS binding activates TLR4 and promotes the assembly of proximal signalling complexes via the adaptors TRIF and TRAM (not shown) or Mal and MyD88. Recruitment and activation of the proximal kinases IRAK4 and IRAK1, the ubiquitin ligase Pellino (not shown), and the LUBAC complex promote K63 and linear polyubiquitination of signalling proteins. As with TNFR1 signalling, this scaffolding-type ubiquitination promotes recruitment of TAK1/TAB2/3 and IκK kinase complexes, their subsequent activation, and propagation of downstream JNK, p38 and NFκB signalling pathways. A20 is probably recruited to the LPS-activated signalling complex via ZnF7 binding to linear ubiquitin chains. The A20 OTU domain catalytic C103 is essential for attenuating LPS-activated signalling by removing K63 polyubiquitin chains from TRAF6, and possibly other proximal signalling proteins. Although the structural integrity and the ubiquitin-binding function of A20 ZnF4 is dispensable for proper attenuation of TLR4 signalling, A20 ZnF4 could have a redundant function with another protein. b, In A20 OTU(C103A) cells removal of K63 ubiquitin chains on proximal signalling components is compromised, thus proteins are hyperubiquitinated with K63-linked chains. With sufficient linear ubiquitination, the infrastructure of the signalling complex is sustained, caspase recruitment to TNFR1 is prohibited, and pro-survival signalling is enhanced. c, In A20 OUT(C103A) cells with deficient linear ubiquitination, removal of K63 ubiquitin chains is still compromised; however, decreased linear chains favours enhanced association of hyperubiquitinated RIPK1 with FADD and caspase 8, the proximal components of the pro-death complex. Enhanced caspase 8 recruitment and activation in turn activates downstream effector caspases (such as caspase 3 and 7), culminating in cell death.