The Reactome pathway shows that upon TNF receptor 1 (TNFR1) stimulation, the E3 ubiquitin ligases such as baculoviral IAP repeat-containing protein (BIRC2/3 or cIAP1/2) mediate polyubiquitination of RIPK1 generating K63-linked chains. TNFAIP3 removes these K63-linked polyubiquitin chains, preventing the interaction of RIPK1 with NFkappaB essential modulator (NEMO) (Wertz IE et al. 2004). Subsequently, TNFAIP3 facilitates addition of K48-linked polyubiquitin chains to RIP1, targeting it for proteasomal degradation (Wertz IE et al. 2004). In this way, TNFAIP3 restricts TNF-induced NFkappaB signaling by sequential deubiquitination and ubiquitin-mediated degradation of RIPK1.

TNFAIP3 (A20) is thought to limit NFkappaB activation, however the inhibitory mechanisms for TNFAIP3 are not fully understood and are partially contradicting. A study with knockin mice expressing DUB-inactive Tnfaip3 C103A mutant reported that the deubiquitinase (DUB) activity was dispensable for LPS- or TNF-stimulated NFkappaB signaling (De A et al 2014). In contrast to Tnfaip3 knockout mice that develop perinatal lethality, the knockin Tnfaip3 C103A mice were normal and did not have an inflammatory phenotype (De A et al 2014). These finding are in agreement with an earlier study reporting that the Tnfaip3 C103A knockin mice were grossly normal for at least 4 months and contained a normal number of lymphocytes (Lu TT et al. 2013). In addition, bone marrow-derived macrophage cells (BMDM) from the knockin mice showed normal LPS- and TNF-induced NFkappaB activation and downstream gene expression, comparable to cells from wild-type mice (De A et al 2014). The study suggests that the deubiquitinase activity of TNFAIP3 in general is not obligate for most of the regulatory functions of TNFAIP3 and alternative mechanisms might be involved in TNFAIP3-mediated NFkappaB regulation (De A et al 2014). Indeed, there is biochemical evidence for different mechanisms that may contribute to TNFAIP3 inhibitory effects on TNFR1 pro-inflammatory signaling in a cell type-dependent manner:

• Deubiquitination of K63-linked ubiquitin chains from RIPK1 by the OTU domain and adding of K48-linked polyubiquitin chains via its forth zinc finger (ZnF4) domain (Wertz IE et al. 2004).
• Triggering degradation of E2 enzymes such as ubiquitin conjugating enzyme Ubc13 by adding K48-linked polyubiquitin chains and disruption of interactions between E2 and E3 (BIRC2,3 or TRAF2) enzymes in the TNFR1 pathway. This event was impaired with DUB-inactive TNFAIP3 (C103A, within the OTU domain) suggesting that there might be a crosstalk with DUB-dependent mechanisms (Shembade N et al. 2010).
• A non-catalytic blockage of TAK1-mediated IKK activation by TNFAIP3 binding to polyubiquitin chain via its seventh zinc finger (ZnF7) and forming a complex involving specific NEMO:TNFAIP3 interaction (Skaug B et al. 2011).
• A non-catalytic inhibition of TNF- and LUBAC-induced NFkappaB signalling by binding to linear polyubiquitin chains via its ZnF7, which prevents the LUBAC:NEMO interaction (Verhelst K et al. 2012; Tokunaga F et al. 2012).
• Attenuation of TNFR1 signaling complex formation (He KL & Ting AT 2002).