The Complexity of Being A20: From Biological Functions to Genetic Associations.

A20, encoded by TNFAIP3, is a critical negative regulator of immune activation. A20 is a ubiquitin editing enzyme with multiple domains, each of which mediates or stabilizes a key ubiquitin modification. A20 targets diverse proteins that are involved in pleiotropic immunologic pathways. The complexity of A20-mediated immunomodulation is illustrated by the varied effects of A20 deletion in different cell types and disease models. Clinically, the importance of A20 is highlighted by its extensive associations with human disease. A20 germline variants are associated with a wide range of inflammatory diseases, while somatic mutations promote development of B cell lymphomas. More recently, the discovery of A20 haploinsufficiency (HA20) has provided real world evidence for the role of A20 in immune cell function. Originally described as an autosomal dominant form of Behcet's disease, HA20 is now considered a complex inborn error of immunity with a broad spectrum of immunologic and clinical phenotypes.

A20/Nrdp1 interaction alters the inflammatory signaling profile by mediating K48- and K63-linked polyubiquitination of effectors MyD88 and TBK1.

A20 is a potent anti-inflammatory protein that mediates both inflammation and ubiquitination in mammals, but the related mechanisms are not clear. In this study, we performed mass spectrometry (MS) screening, gene ontology (GO) analysis, and coimmunoprecipitation (co-IP) in a lipopolysaccharide (LPS)-induced inflammatory cell model to identify novel A20-interacting proteins. We confirmed that the E3 ubiquitin ligase Nrdp1, also known as ring finger protein 41 (RNF41), interacted with A20 in LPS-stimulated cells. Further co-IP analysis demonstrated that when A20 was knocked out, degradation-inducing K48-linked ubiquitination of inflammatory effector MyD88 was decreased, but protein interaction-mediating K63-linked ubiquitination of another inflammatory effector TBK1 was increased. Moreover, western blot experiments showed that A20 inhibition induced an increase in levels of MyD88 and phosphorylation of downstream effector proteins as well as of TBK1 and a downstream effector, while Nrdp1 inhibition induced an increase in MyD88 but a decrease in TBK1 levels. When A20 and Nrdp1 were coinhibited, no further change in MyD88 was observed, but TBK1 levels were significantly decreased compared with those upon A20 inhibition alone. Gain- and loss-of-function analyses revealed that the ZnF4 domain of A20 is required for Nrdp1 polyubiquitination. Upon LPS stimulation, the inhibition of Nrdp1 alone increased the secretion of IL-6 and TNF-α but decreased IFN-ß secretion, as observed in other studies, suggesting that Nrdp1 preferentially promotes the production of IFN-ß. Taken together, these results demonstrated that A20/Nrdp1 interaction is important for A20 anti-inflammation, thus revealing a novel mechanism for the anti-inflammatory effects of A20.

A20 restores phorbol ester-induced differentiation of THP-1 cells in the absence of nuclear factor-κB activation.

A20, also referred to as tumor necrosis factor alpha (TNFα)-induced protein 3 (TNFAIP3), is an ubiquitin-editing enzyme whose expression is enhanced by NF-κB activation, and plays an important role in silencing NF-κB activity. Another well-known role for A20 is to protect cells from TNFα-induced apoptosis. Depletion of NF-κB in differentiating U937 monocytic leukemia cells is known to cause apoptotic cell death; however, much remains to be explored about the molecules that are expressed in an NF-κB-dependent manner and which support monocyte-macrophage differentiation. Using the monocytic cell line THP-1, and peripheral blood monocytes, we show here a sustained increase in A20 expression during monocyte-macrophage differentiation, which coincided with high NF-κB-dependent transcriptional activity. Depletion of NF-κB by stable expression of a super-repressor form of IκBα in THP-1 cells caused remarkable cell death during phorbol 12-myristate 13-acetate (PMA)-induced differentiation. A20 expression in these cells did not alter this NF-κB suppression, but was sufficient to protect the cells and restore the cell surface expression of a differentiation marker (CD11b) and phagocytic activity. Mutational analyses revealed that this A20 activity requires the carboxy-terminal zinc-finger domain, but not its deubiquitinase activity. Based on these findings, we conclude that A20, when ectopically expressed, can support both survival and differentiation of THP-1 cells in the absence of sustained NF-κB activity.

Intracelluar delivery of A20 protein inhibits TNFα-induced NF-κB activation.

A20 (also known as TNFAIP3) is a potent anti-inflammatory protein that suppresses many intracellular signaling pathways induced by inflammatory cytokines and bacterial and viral pathogens. The anti-inflammatory function of A20 depends on its modulation of or binding to polyubiquitin chains on key signaling proteins in the nuclear factor-κB (NF-κB) pathway. To test whether A20 can be used as therapeutic agent in these inflammatory diseases, we prepared a recombinant cell-penetrating form of A20 (TAT-A20) for intracellular delivery and examined its effect on tumor necrosis factor-α (TNFα)-induced NF-κB activation. We observed that TAT-A20 was effectively transduced into cells within 30 min, whereas A20 protein without TAT motive was not. TAT-A20 also inhibited NF-κB induction in fibroblasts stimulated with TNFα. These results suggest that increasing intracellular level of A20 can be an effective means to suppress NF-κB activation and treat inflammatory diseases.