A20 at the Crossroads of Cell Death, Inflammation, and Autoimmunity

Abstract

A20 is a potent anti-inflammatory protein, acting by inhibiting nuclear factor κB (NF-κB) signaling and inflammatory gene expression and/or by preventing cell death. Mutations in the A20/TNFAIP3 gene have been associated with a plethora of inflammatory and autoimmune pathologies in humans and in mice. Although the anti-inflammatory role of A20 is well accepted, fundamental mechanistic questions regarding its mode of action remain unclear. Here, we review new findings that further clarify the molecular and cellular mechanisms by which A20 controls inflammatory signaling and cell death, and discuss new evidence for its involvement in inflammatory and autoimmune disease development.

Figure 1.

Tumor necrosis factor (TNF)-induced nuclear factor κB (NF-κB) signaling, apoptosis, and necroptosis. Binding of TNF to TNF receptor 1 (TNFR1) induces receptor trimerization that allows the recruitment of TNF receptor-associated death domain protein (TRADD) and receptor-interacting protein 1 (RIPK1). TRADD recruits TRAF2/5 and the E3 ubiquitin (Ub) ligases cIAP1 and cIAP2. cIAP1 and cIAP2 conjugate RIPK1 as well as themselves with K63-linked polyubiquitin chains, which serve as a platform for the recruitment of the LUBAC complex, consisting of HOIP, HOIL1, and SHARPIN. Linear Ub chain assembly complex (LUBAC) conjugates several components of the TNFR1 complex, including NEMO and RIPK1, with linear Ub chains (M1). The TAB2/3–TAK1 complex binds K63-linked chains on RIPK1, whereas the IKK complex is recruited via NEMO binding to M1-linked polyubiquitin. This allows TAK1 to phosphorylate and activate the IKK complex. On activation, IKK2 phosphorylates the inhibitor of κB α (IκBα), targeting IκBα for ubiquitination with K48-linked chains and proteasomal degradation. This releases the p50/p65 NF-κB dimer, which translocates to the nucleus where it induces the expression of NF-κB response genes. A20 is recruited to the TNFR1 signaling complex via M1-linked Ub, which binds to ZnF7. In addition, A20 has been shown to act as a deubiquitinating enzyme (DUB) that removes K63-linked polyubiquitin from different target proteins, including RIPK1 and NEMO. Furthermore, A20 has been shown to target RIPK1 and TNFR1 for proteasomal degradation through its ZnF4 E3 ligase activity. Binding of A20 to M1-linked polyubiquitin prevents downstream signaling by competing with other Ub-binding proteins and by preventing the degradation of M1-linked chains by CYLD (not shown). Loss of M1-linked chains destabilizes complex I and results in the formation of a cytosolic death-inducing signaling complex (DISC), consisting of TRADD, Fas-associated death domain (FADD), and caspase 8 (complex IIa) or RIPK1, FADD, and caspase-8 (complex IIb). Homodimerization and activation of caspase-8 leads to the activation of downstream executioner caspases 3 and 7 (not shown) and apoptotic cell death. If caspase-8 activity is compromised, RIPK1 associates with RIPK3 (the necrosome), which is activated by autophosphorylation. The activated necrosome recruits and activates MLKL inducing necroptotic cell death.

Figure 2.

Domain structure of the A20 protein. The amino terminus of A20 contains an ovarian tumor (OTU) domain, which has deubiquitinating enzyme (DUB) activity relying on the catalytic residue Cys103. The carboxy-terminal part of A20 contains seven zinc finger (ZnF) domains. The fourth ZnF domain has K63-linked polyubiquitin-binding affinity and possesses E3 ubiquitin (Ub) ligase activity, whereas the seventh ZnF domain has strong binding affinity for linear (M1) Ub chains. IKK2-mediated phosphorylation of A20 at Ser381 enhances the DUB activity of A20 toward K63-linked polyubiquitin. MALT1 cleaves A20 at Ala439 in human, or between the third and fourth ZnF domains in mouse. Mutations that were introduced to generate OTU (C103A), ZnF4 (C609A, C612A), or ZnF7 (C764A, C767A) domain-specific mutant mice are depicted by red stars. Both the OTU and ZnF4 domains have been shown to bind to E2 enzymes Ubc13 and UbcH5c.

Figure 3.

Schematic representation of the TNFAIP3 gene indicating mutations causing A20 haploinsufficiency (HA20). Exons (1–9) are represented as rectangles. Exons encoding the ovarian tumor (OTU) domain are depicted in green and exons encoding the ZnF domains are depicted in orange; noncoding exons are shown in gray. HA20 mutations depicted in the figure are also listed in Table 1.