Polyubiquitination of proteins has a pivotal role in the regulation of numerous cellular functions such as protein degradation, DNA repair and cell signaling. As deregulation of these processes can result in pathological conditions such as inflammatory diseases, neurodegeneration or cancer, tight regulation of the ubiquitin system is of tremendous importance. Ubiquitination by E3 ubiquitin ligases can be counteracted by the activity of several deubiquitinating enzymes (DUBs). CYLD, A20 and OTULIN have been implicated as key DUBs in the negative regulation of NF-κB transcription factor-mediated gene expression upon stimulation of cytokine receptors, antigen receptors and pattern recognition receptors, by removing distinct types of polyubiquitin chains from specific NF-κB signaling proteins. In addition, they control TNF-induced cell death signaling leading to apoptosis and necroptosis via similar mechanisms. In the case of A20, also catalytic-independent mechanisms of action have been demonstrated to have an important role. CYLD, A20 and OTULIN have largely overlapping substrates, suggesting at least partially redundant functions. However, mice deficient in one of the three DUBs show significant phenotypic differences, indicating also non-redundant functions. Here we discuss the activity and polyubiquitin chain-type specificity of CYLD, A20 and OTULIN, their specific role in NF-κB signaling and cell death, the molecular mechanisms that regulate their activity, their role in immune homeostasis and the association of defects in their activity with inflammation, autoimmunity and cancer.

A20 (also known as TNFAIP3) is a cytoplasmic protein that plays a key role in the negative regulation of inflammation and immunity. Polymorphisms in the A20 gene locus have been identified as risk alleles for multiple human autoimmune diseases, and A20 has also been proposed to function as a tumor suppressor in several human B-cell lymphomas. A20 expression is strongly induced by multiple stimuli, including the proinflammatory cytokines TNF and IL-1, and microbial products that trigger pathogen recognition receptors, such as Toll-like receptors. A20 functions in a negative feedback loop, which mediates its inhibitory functions by downregulating key proinflammatory signaling pathways, including those controlling NF-κB-and IRF3-dependent gene expression. Activation of these transcription factors is controlled by both K48-and K63-polyubiquitination of upstream signaling proteins, respectively triggering proteasome-mediated degradation or interaction with other signaling proteins. A20 turns off NF-B and IRF3 activation by modulating both types of ubiquitination. Induction of K48-polyubiquitination by A20 involves its C-terminal zincfinger ubiquitin-binding domain, which may promote interaction with E3 ligases, such as Itch and RNF11 that are involved in mediating A20 inhibitory functions. A20 is thought to promote de-ubiquitination of K63-polyubiquitin chains either directly, due to its N-terminal deubiquitinase domain, or by disrupting the interaction between E3 and E2 enzymes that catalyze K63-polyubiquitination. A20 is subject to different mechanisms of regulation, including phosphorylation, proteolytic processing, and association with ubiquitin binding proteins. Here we review the expression and biological activities of A20, as well as the underlying molecular mechanisms.

Linear polyubiquitination of proteins has recently been implicated in NF-jB signalling and is mediated by the linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP and Sharpin. However, the mechanisms that regulate linear ubiquitination are still unknown. Here, we show that A20 is rapidly recruited to NEMO and LUBAC upon TNF stimulation and that A20 inhibits LUBAC-induced NF-jB activation via its C-terminal zinc-finger 7 (ZF7) domain. Expression of a polypeptide corresponding to only ZF7 was sufficient to inhibit TNF-induced NF-jB activation. Both A20 and ZF7 can form a complex with NEMO and LUBAC, and are able to prevent the TNF-induced binding of NEMO to LUBAC. Finally, we show that ZF7 preferentially binds linear polyubiquitin chains in vitro, indicating A20-ZF7 as a novel linear ubiquitin-binding domain (LUBID). We thus propose a model in which A20 inhibits TNF-and LUBACinduced NF-jB signalling by binding to linear polyubiquitin chains via its seventh zinc finger, which prevents the TNF-induced interaction between LUBAC and NEMO.