Kelly Verhelst

ABINs: A20 binding inhibitors of NF-kappa B and apoptosis signaling.

ABINs have been described as three different proteins (ABIN-1, ABIN-2, ABIN-3) that bind the ubiquitin-editing nuclear factor-kappaB (NF-kappaB) inhibitor protein A20 and which show limited sequence homology. Overexpression of ABINs inhibits NF-kappaB activation by tumor necrosis factor (TNF) and several other stimuli. Similar to A20, ABIN-1 and ABIN-3 expression is NF-kappaB dependent, implicating a potential role for the A20/ABIN complex in the negative feedback regulation of NF-kappaB activation. Adenoviral gene transfer of ABIN-1 has been shown to reduce NF-kappaB activation in mouse liver and lungs. However, ABIN-1 as well as ABIN-2 deficient mice exhibit only slightly increased or normal NF-kappaB activation, respectively, possibly reflecting redundant NF-kappaB inhibitory activities of multiple ABINs. Other functions of ABINs might be non-redundant. For example, ABIN-1 shares with A20 the ability to inhibit TNF-induced apoptosis and as a result ABIN-1 deficient mice die during embryogenesis due to TNF-dependent fetal liver apoptosis. On the other hand, ABIN-2 is required for optimal TPL-2 dependent extracellularly regulated kinase activation in macrophages treated with TNF or Toll-like receptor ligands. ABINs have recently been shown to contain an ubiquitin-binding domain that is essential for their NF-kappaB inhibitory and anti-apoptotic activities. In this context, ABINs were proposed to function as adaptors between ubiquitinated proteins and other regulatory proteins. Alternatively, ABINs might disrupt signaling complexes by competing with other ubiquitin-binding proteins for the binding to specific ubiquitinated targets. Altogether, these findings implicate an important role for ABINs in the regulation of immunity and tissue homeostasis.

Expression, biological activities and mechanisms of action of A20 (TNFAIP3).

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-κB and IRF3 activation by modulating both types of ubiquitination. Induction of K48-polyubiquitination by A20 involves its C-terminal zinc-finger 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.

A20 inhibits LUBAC-mediated NF-κB activation by binding linear polyubiquitin chains via its zinc finger 7

Linear polyubiquitination of proteins has recently been implicated in NF‐κB 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‐κB 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‐κB 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 LUBAC‐induced NF‐κB signalling by binding to linear polyubiquitin chains via its seventh zinc finger, which prevents the TNF‐induced interaction between LUBAC and NEMO.

LIND/ABIN-3 Is a Novel Lipopolysaccharide-inducible Inhibitor of NF-κB Activation

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-κB (NF-κB). Although LPS-induced activation of NF-κB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-κB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-κB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-κB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-κB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKβ. NF-κB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-κB activity in the liver, thereby partially protecting mice against LPS/d-(+)-galactosamine-inducedmortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-κB activation.

Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.

The ubiquitin-editing enzyme A20 (tumor necrosis factor-α-induced protein 3) serves as a critical brake on nuclear factor κB (NF-κB) signaling. In humans, polymorphisms in or near the A20 gene are associated with several inflammatory disorders, including psoriasis. We show here that epidermis-specific A20-knockout mice (A20EKO) develop keratinocyte hyperproliferation, but no signs of skin inflammation, such as immune cell infiltration. However, A20EKO mice clearly developed ectodermal organ abnormalities, including disheveled hair, longer nails and sebocyte hyperplasia. This phenotype resembles that of mice overexpressing ectodysplasin-A1 (EDA-A1) or the ectodysplasin receptor (EDAR), suggesting that A20 negatively controls EDAR signaling. We found that A20 inhibited EDAR-induced NF-κB signaling independent from its de-ubiquitinating activity. In addition, A20 expression was induced by EDA-A1 in embryonic skin explants, in which its expression was confined to the hair placodes, known to be the site of EDAR expression. In summary, our data indicate that EDAR-induced NF-κB levels are controlled by A20, which functions as a negative feedback regulator, to assure proper skin homeostasis and epidermal appendage development.

IκB kinase ɛ (IKKɛ): A therapeutic target in inflammation and cancer

Abstract The innate immune system forms our first line of defense against invading pathogens and relies for a major part on the activation of two transcription factors, NF-κB and IRF3. Signaling pathways that activate these transcription factors are intertwined at the level of the canonical IκB kinases (IKKα, IKKβ) and non-canonical IKK-related kinases (IKKɛ, TBK1). Recently, significant progress has been made in understanding the function and mechanism of action of IKKɛ in immune signaling. In addition, IKKɛ impacts on cell proliferation and transformation, and is thereby also classified as an oncogene. Studies with IKKɛ knockout mice have illustrated a key role for IKKɛ in inflammatory and metabolic diseases. In this review we will highlight the mechanisms by which IKKɛ impacts on signaling pathways involved in disease development and discuss its potential as a novel therapeutic target.

Regulation of TNF-induced NF-κB activation by different cytoplasmic ubiquitination events

TNF is a multifunctional cytokine that plays a key role in innate immunity by inducing the expression of a variety of genes that are involved in an inflammatory response. TNF-induced NF-κB activation is one of the best studied signaling pathways in mammalian cells and has recently led to a revival of research in the biology of ubiquitin. Many NF-κB signaling proteins are modified by specific ubiquitin ligases with different types of ubiquitin chains that are recognized by other proteins and which determine the outcome of ubiquitination. In addition, specific de-ubiquitinases make the whole process reversible. This review summarizes recent findings that have shaped our current understanding on the role of cytoplasmic ubiquitination events in the regulation of TNF-induced NF-κB signaling.

TAX1BP1, a ubiquitin-binding adaptor protein in innate immunity and beyond

The innate immune system senses and protects against invading microorganisms and endogenous danger signals by triggering inflammatory and antimicrobial responses. However, dysregulation of these pathways, which involve the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) 3, can lead to severe inflammatory diseases. Tax1-binding protein 1 (TAX1BP1) plays a key role in the negative regulation of NF-κB and IRF3 signaling by acting in concert with the ubiquitin-editing enzyme A20. In addition to regulating A20 function in anti-inflammatory and antiviral signaling pathways, TAX1BP1 also coordinates its antiapoptotic activities. Moreover, TAX1BP1 can also function as a transcriptional coactivator for nuclear receptors and viral transactivators. In this review, we discuss these findings in light of the emerging role of TAX1BP1 as a ubiquitin-binding adaptor protein.

CYLD, A20 and OTULIN deubiquitinases in NF-κB signaling and cell death : so similar, yet so different

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.

The multifaceted role of the E3 ubiquitin ligase HOIL-1: beyond linear ubiquitination

Ubiquitination controls and fine‐tunes many signaling processes driving immunity, inflammation, and cancer. The E3 ubiquitin ligase HOIL‐1 (heme‐oxidized IRP2 ubiquitin ligase‐1) is increasingly implicated in different signaling pathways and plays a vital role in immune regulation. HOIL‐1 co operates with the E3 ubiquitin ligase HOIP (HOIL‐1 interacting protein) to modify specific nuclear factor‐κB (NF‐κB) signaling proteins with linear M1‐linked polyubiquitin chains. In addition, through its ability to also add K48‐linked polyubiquitin chains to specific substrates, HOIL‐1 has been linked with antiviral signaling, iron and xenobiotic metabolism, cell death, and cancer. HOIL‐1 deficiency in humans leads to myopathy, amylopectinosis, auto‐inflammation, and immunodeficiency associated with an increased frequency of bacterial infections. HOIL‐1‐deficient mice exhibit amylopectin‐like deposits in the myocardium, pathogen‐specific immunodeficiency, but minimal signs of hyper‐inflammation. This review summarizes current knowledge on the mechanism of action of HOIL‐1 and highlights recent advances regarding its role in health and disease.