A20 ubiquitin binding, not its catalytic activity, is important in Rheumatoid Arthritis (RA)


A20, or tumor necrosis factor α-induced protein 3 (TNFAIP3), is a zinc finger ubiquitin editing anti-inflammatory enzyme that modulates immune responses and apoptosis. Mutations in A20 and A20 haploinsufficiency have been linked to several autoimmune and autoinflammatory diseases1-4, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and psoriatic arthritis5-7. Studies utilizing animal models have confirmed roles for A20 in the pathogenesis of autoinflammatory and RA-like diseases and helped to identify the mechanisms involved8, 9. The first identified mechanism by which A20 exerts its anti-inflammatory effect was through the inhibition of NF-κB activation10. Additional mechanisms by which the enzymatic function of A20 offers protection for autoinflammatory conditions have been reported, including inhibition of necroptosis by deubiquitinating receptor-interacting protein kinase 3 (RIPK3)11 (See Figure 1).

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammatory arthritis and a variety of systemic manifestations. The addition of immunosuppressant biologics to classic disease-modifying antirheumatic drugs (DMARDs) has improved the management of RA12. However, there is no cure, and the underlying mechanisms of disease pathogenesis remain to be fully elucidated. In recent years, two ubiquitin-binding domains, zinc finger 4 (ZnF4) and ZnF7, in the A20 protein have been shown to be essential for inhibiting inflammation-dependent arthritis9, 13. This newsletter will review evidence that unveiled the importance of A20 ubiquitin-binding in RA and related arthritic disorders.  

ZnF7 and ZnF4 ubiquitin-binding motifs mediate A20 anti-inflammatory roles


Though the loss of A20 results in severe inflammatory disease and premature death in animal models14, mice harboring inactivating mutations in the catalytic deubiquitinase (DUB) and E3 ubiquitin ligase domains develop normally and did not influence NF-κB signaling15-17. Thus, it is clear that something other than the catalytic activity of A20 is involved in the pathogenesis of inflammatory diseases.


To investigate the physiological role of the ZnF7 ubiquitin-binding domain in A20, Martens, et al., generated mutant mice bearing two inactivating point mutations in the ZnF7 domain of A2018. These mice were smaller than their wildtype counterparts and developed spontaneous inflammatory pathology characterized by splenomegaly, joint inflammation, swelling of paws, multi-tissue immune cell infiltration, elevated serum cytokine levels, and elevated myeloid, lymphoid, and natural killer (NK) cell populations in the spleen. Using embryonic fibroblasts isolated from these mice, they further demonstrated that the ZnF7 domain was critical for A20-mediated suppression of inflammatory signaling and cell death.


However, the phenotype observed in ZnF7 mutant mice was not as severe as that of A20-deficient mice. The addition of two inactivating point mutations in the ZnF4 ubiquitin-binding domain resulted in mice that mimicked complete loss of A20, with severely runted progeny that did not survive past weaning and developed severe multi-organ inflammation and liver cell apoptosis. Thus, the combined inactivation of these two ubiquitin-binding domains, ZnF7 and ZnF4, is essential for inflammatory disease development18. They further revealed that the ZnF7 domain was necessary to recruit A20 to the tumor necrosis factor receptor 1 (TNFR1) complex. Importantly, conditional tissue-specific ablation of or the expression of mutant A20 revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells to promote inflammatory phenotypes. 


Figure 1. Domain structure of A20. A20 consists of an N-terminal ovarian tumor (OTU) domain and 7C-terminal domain built up by seven zinc fingers (ZF1-ZF7).

A20 non-catalytic ubiquitin binding prevents psoriatic arthritis-like disease


Published just days later in the same journal, Nature Immunology, Razani et al. corroborated the importance of the ZnF7 and ZnF4 domains19. They compared mice harboring cysteine to alanine point mutations in either ZnF7 (A20ZNF7) or ZnF4 (A20ZNF4) with wild-type mice and those with mutant catalytic DUB domains. For at least the first four months of life, mice with mutant ZnF4 or catalytic domains developed normally, while those with mutant ZnF7 experienced progressive swelling of the digits starting at 6 weeks of age, with all digits swollen by 12 weeks of age. Additionally, the mutant ZnF7 mice developed arthritis in the distal interphalangeal joints and spontaneous immune activation, phenotypes that were not present in mice of the other genotypes. 


By crossing A20ZNF7 mice with Rag1-/- mice that lack B and T lymphocytes, they determined that A20ZNF7-driven arthritis requires lymphocytes as the A20ZNF7xRag1-/- developed normally. To determine whether B lymphocytes, T lymphocytes, or both contribute to this phenomenon, they further crossed A20ZNF7 mice with μMT mice that lack B cells, revealing that the development of arthritis in these mice phenocopied that of the immune-competent A20ZNF7 mice, demonstrating that T cells, but not B cells, are required for A20ZNF7-driven arthritis. The Martens group identified A20 in myeloid populations as essential for disease pathogenesis in their model18. Interestingly, the present publication corroborated that finding and demonstrated that myeloid differentiation primary response 88 (MyD88) signaling is required for A20ZNF7-driven arthritis development19. They further revealed that the ZnF7 motif of A20 was necessary to prevent prolonged IKK activity, expression of NF-κB-dependent genes, and the development of arthritis.

As with the previous paper, they then investigated the mutant ZnF7 and ZnF4 in combination, as the mutant ZnF7 alone did not recapitulate the severity of the complete deletion of A20. The combination was synergistic, with mice harboring mutant ZnF7 and ZnF4 developing much more severe disease. These two papers, published within days of one another, solidified the importance of these two A20 ubiquitin-binding domains in the prevention of inflammatory arthritis.     


A20 a potential therapeutic target for RA


As discussed in this newsletter, A20 deficiency or mutation of the ubiquitin-binding domains of A20 in myeloid cells results in RA-like polyarthritis in mice. A 2021 study revealed that patients with RA have increased circulating monocyte populations and that A20 levels were decreased in those circulating monocytes in RA patients compared to healthy participants, suggesting that A20 in monocytes may be playing a protective role against RA development20. With these findings in mind, researchers in China reported on a proof-of-concept study in mice to determine whether gene therapy delivery of A20 to overexpress the protective protein could improve the development of arthritis. Indeed, they observed an improvement in the clinical arthritis score, paw thickness and porosity, bone volume, and other measures of RA development in mice that received rAAV6-CMV-A20 compared to those receiving the control vector, rAAV6-CMV-EGFP21. Understanding the mechanisms by which the A20 protein influences disease pathogenesis is essential to effective RA therapy development, and the nearly simultaneous studies discussed in this newsletter underscore that the ubiquitin-binding function of A20, not its catalytic function, is imperative for protecting from arthritis development. 




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