The Ubiquitin System and A20: Implications in Health and Disease

Abstract

Inflammation is triggered by stimulation of innate sensors that recognize pathogens, chemical and physical irritants, and damaged cells subsequently initiating a well-orchestrated adaptive immune response. Immune cell activation is a strictly regulated and self-resolving process supported by an array of negative feedback mechanisms to sustain tissue homeostasis. The disruption of these regulatory pathways forms the basis of chronic inflammatory diseases, including periodontitis. Ubiquitination, a covalent posttranslational modification of target proteins with ubiquitin, has a profound effect on the stability and activity of its substrates, thereby regulating the immune system at molecular and cellular levels. Through the cooperative actions of E3 ubiquitin ligases and deubiquitinases, ubiquitin modifications are implicated in several biological processes, including proteasomal degradation, transcriptional regulation, regulation of protein-protein interactions, endocytosis, autophagy, DNA repair, and cell cycle regulation. A20 (tumor necrosis factor α-induced protein 3 or TNFAIP3) is a ubiquitin-editing enzyme that mainly functions as an endogenous regulator of inflammation through termination of nuclear factor (NF)-κB activation as part of a negative feedback loop. A20 interacts with substrates that reside downstream of immune sensors, including Toll-like receptors, nucleotide-binding oligomerization domain-containing receptors, lymphocyte receptors, and cytokine receptors. Due to its pleiotropic functions as a ubiquitin binding protein, deubiquitinase and ubiquitin ligase, and its versatile role in various signaling pathways, aberrant A20 levels are associated with numerous conditions such as rheumatoid arthritis, diabetes, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, Sjögren syndrome, coronary artery disease, multiple sclerosis, cystic fibrosis, asthma, cancer, neurological disorders, and aging-related sequelae. Similarly, A20 has recently been implicated as an essential regulator of inflammation in the oral cavity. This review presents information on the ubiquitin system and regulation of NF-κB by ubiquitination using A20 as a representative molecule and highlights how the dysregulation of this system can lead to several immune pathologies, including oral cavity-related disorders mainly focusing on periodontitis.

Keywords: NF-κB; TLR; cytokines; inflammation; periodontitis; ubiquitination.

Figure 1.

The pathological host immune response in periodontitis. A dysbiotic oral microflora initiates the innate immune response by stimulating resident cells in the oral epithelium to produce mediators of inflammation. The secretion of these proinflammatory cytokines and chemokines drives the infiltration of various leukocytes of the innate and adaptive immune response into the compromised tissue. Among the first responders, neutrophils and macrophages act as professional phagocytes by engulfing and killing bacteria. Neutrophils attempt to kill invading agents by releasing potent effector molecules, such as reactive oxygen species (ROS). In addition to phagocytosis of microorganisms, macrophages clear apoptotic neutrophils in a process known as efferocytosis and activate lymphocyte-mediated adaptive immunity, bridging innate and adaptive responses. Dendritic cells uptake microbial antigenic material and present to lymphocytes in the lymphoid tissue, evoking the adaptive immune response. Failure of inflammation resolution now transitions the inflammatory response to a chronic state, altering bone homeostasis and leading to subsequent osteoclast activation and destruction of alveolar bone. Specifically, dendritic cells can interact with naive T-helper cells, driving their differentiation into several subsets, including Th1, Th2, Th17, and regulatory T cells (Treg). In addition to activating macrophages, T cells are important regulators of bone turnover through their elaborate production of proresorptive cytokines, including interleukin 17, which directly support osteoclastogenesis. Due to the importance of T-cell subsets, characteristics of prolonged adaptive immunity can differ depending on whichever class of T cells is present. In addition to prominent T-cell infiltration, B cells are also abundant in the chronic lesions. The dual functionality of B cells is highlighted by their protective ability in facilitating bacterial clearance and their destructive roles in promoting inflammation, bone resorption, and matrix dissolution. These observations suggest that the clinical progression from gingivitis to periodontitis is characterized by the shift to T- and B-cellular infiltrates. For a complete list of references used for this figure, please see the Appendix references for Figure 1 legend. DC, dendritic cell; Mφ, macrophage; PMN, polymorphonuclear neutrophil; ROS, reactive oxygen species.

Figure 2.

Individual ubiquitin linkage types and their associated biological roles. Ubiquitin, a small 76–amino acid protein, can be attached to a targeted substrate or a ubiquitin molecule that is already attached to a substrate, resulting in specific polyubiquitin linkage types. In a ubiquitin chain, ubiquitin moieties can be conjugated through one of their lysine resides (Lys11, Lys27, Lys6, Lys29, Lys33, Lys63, and Lys48) or the N-terminal methionine residue (Met1). Each chain is recognized by different ubiquitin-binding domains, targeting proteins in specific signaling pathways. Most extensively studied, Lys48- and Lys63-linked chains are the 2 most abundant chain types and regulate proteasomal degradation and a variety of proteolytic and nonproteolytic events, respectively. Recently, innovative technologies revealed the role of the remaining ubiquitin chain types in controlling cellular processes ranging from cell cycle control to cytokine signaling (Swatek and Komander 2016; Mendes et al. 2020).

Figure 3.

Regulation of NF-κB by ubiquitination and the deubiquitinases: A20, CYLD, and Cezanne. In the initiation of NF-κB signaling, ligand binding to a variety of immune sensors such as TNFR, IL-1R, TCR, and TLR results in the recruitment of TRAFs to the receptors. TRAF2 is recruited with a protein complex, which consists of multiple adaptor proteins, including TRADD, RIP1, and ubiquitin ligases: cIAP1 and cIAP2. cIAP promotes Lys63-linked polyubiquitination on both RIP1 and TRAF2 but also on themselves to generate a binding platform, allowing the subsequent attachment of further distal components. Polyubiquitinated RIP1 is recognized by the binding partners TAB1 and TAB2/TAB3. The Lys63-linked polyubiquitin chains also can bind the IKK regulatory subunit, NEMO. Due to the new proximity between the TAK1 and IKK, TAK1 phosphorylates IKKβ at 2 serine residues, resulting in the activation of IKK and rapid phosphorylation of IκBα. Subsequent ubiquitination of IκBα can now be carried out by E3 ligase, SCF-βTrCP. Although polyubiquitinated IκBα remains attached to NF-κB dimers, it is selectively degraded by the 26S proteasome, allowing the nuclear translocation of NF-κB, where it induces the activation of NF-κB through the transcription of NF-κB responsive genes. Conversely, once TRAF6 is activated, it functions as an E3 ubiquitin ligase and, together with the ubiquitin E2 complex, UBC13 and UEV1A, catalyzes the synthesis of Lys63-linked polyubiquitin chains onto itself as well as NEMO. Similar enzymatic events mentioned above follow, resulting in the ultimate degradation of IκBα by the 26S proteasome, allowing the nuclear translocation and activation of NF-κB–dependent genes (Rothschild et al. 2018). Lys63-linked polyubiquitin chains also are found to be bound to MALT1, which is a part of the CBM (CARMA1/BCL-10/MALT1) complex downstream of TCR, aiding in the activation of NF-κB signaling (Hu and Sun 2016). Deubiquitinating enzymes (A20, Cezanne, and CYLD) negatively regulate the NF-κB pathway by cleaving the Lys63-linked polyubiquitin chains from various target molecules, as denoted by black squares (Ma and Malynn 2012; Hu and Sun 2016; Rothschild et al. 2018). In the case of TNFR signaling, RIP1 can be subjected to the deubiquitinase function of all these enzymes, interfering with ubiquitin-mediated protein-protein interactions, thereby inhibiting downstream NF-κB activation. Furthermore, A20 and CYLD were also shown to inhibit NF-κB activation by the Lys63-linked deubiquitination and subsequent inactivation of TRAF6. Uniquely, A20 also can remove Lys63-linked polyubiquitin chains from MALT1 downstream of T- and B-cell antigen receptors. In addition, via its specialized E3 ligase activity, A20 adds Lys48-linked polyubiquitin chains to RIP1 and UBC13 for their subsequent degradation, as shown in pink squares (Martens and van Loo 2020). BCL-10, B-cell lymphoma 10; CARMA1, caspase recruitment domain-containing membrane-associated guanylate kinase protein 1; cIAP1 and 2, cellular inhibitor of apoptosis 1 and 2; IKK, IκB kinase complex; IL-1R, interleukin 1 receptor; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein; NEMO, NF-κB essential modulator; RIP1, receptor interacting protein 1; SCF-βTrCP, Skp1-Cull-F-box ligase containing the F-box protein βTrCP; TAB1,2,3, TAK1-binding protein 1,2,3; TAK1, TGFβ-activated kinase 1; TCR, T-cell receptor; TNFR, tumor necrosis factor receptor; TRADD, tumor necrosis factor receptor type 1–associated DEATH domain; TRAF, TNF receptor-associated factor; UBC13, ubiquitin-conjugating 13; UEV1A, ubiquitin-conjugating enzyme E2 variant 1A.

Figure 4.

A20 function and its contribution to cellular homeostasis and disease pathology. A20 is recognized as one of the central regulators of inflammation, apoptosis, autophagy, and necroptosis in a variety of innate and adaptive immune cells. Tightly regulated A20 levels are required to preserve tissue homeostasis and restrain inflammation following initial host response to tissue injury or infection. Aberrant A20 expression and activity may be caused by a variety of mechanisms, including genetics (single-nucleotide polymorphisms [SNPs]), epigenetics (DNA methylation, microRNAs [miRNAs]), and environmental or intrinsic stimuli (microbiome, danger signals, reactive oxygen species [ROS]). Impaired A20 activity for a prolonged period promotes an environment of sustained inflammation and delayed resolution and affects a variety of innate and adaptive immune responses, consequently leading to several pathologies in the oral cavity and at distant tissues. NET, neutrophil extracellular trap; SLE, systemic lupus erythematosus.