TNFAIP3 (A20) is ubiquitin-editing enzyme with distinct peptidase and ligase domains. The amino-terminal domain of TNFAIP3 (A20), which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes K63-linked ubiquitin chains from adaptor proteins such as receptor interacting protein 1 (RIPK1) (Shembade N et al. 2010; Wertz IE et al. 2004; He KL&Ting AT 2002). The carboxy-terminal domain of TNFAIP3 (A20), composed of seven C2/C2 zinc fingers, functions as a ubiquitin ligase by polyubiquitinating target proteins with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation (Wertz IE et al. 2004). TNFAIP3 zinc fingers have been also shown to support TNFAIP3's binding to different ubiquitinated molecules (Wertz IE et al. 2004; Shembade N et al. 2010; Lu TT et al. 2013).

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).