Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation

1] Laboratory of Molecular Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan [2] Department of Biophysics and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
The EMBO Journal (Impact Factor: 10.43). 08/2012; 31(19):3856-70. DOI: 10.1038/emboj.2012.241
Source: PubMed


LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.

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Available from: Eiji Goto, Feb 04, 2015
    • "Alternative mechanisms might be important for the biological function of A20. For example, the seventh zinc-finger motif (ZnF7) of A20 was recently proposed to be involved in direct inhibition of IKK by a non-catalytic mechanism [22] , perhaps by suppressing LUBACmediated NF-jB activation by binding to linear ubiquitin chains [23,24]. This hypothesis will need to be explored in greater depth in the future.Figure 5. Biochemical analysis of NF-jB activation in wild-type and homozygous BMDMs. "
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    ABSTRACT: A20 has been suggested to limit NF-κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 is a ubiquitin-editing enzyme that removes K63-linked ubiquitin chains from adaptor proteins, such as RIP1, and then conjugates them to K48-linked polyubiquitin chains to trigger proteasomal degradation. To determine the role of the deubiquitinase function of A20 in downregulating NF-κB signaling, we have generated a knock-in mouse that lacks the deubiquitinase function of A20 (A20-OTU mice). These mice are normal and have no signs of inflammation, have normal proportions of B, T, dendritic, and myeloid cells, respond normally to LPS and TNF, and undergo normal NF-κB activation. Our results thus indicate that the deubiquitinase activity of A20 is dispensable for normal NF-κB signaling.
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    • "Additional UB-binding domain(s), such as the multiple NZFs of TRABID, are involved in targeting to substrate sites and enhance cleavage activity with longer UB chains (Licchesi et al., 2012). Furthermore, the different C-terminal linkage-specific UB chain-binding motifs (ZnF1- 7) in H. sapiens A20 are crucial for its recruitment by distinct signaling proteins and complexes (Bosanac et al., 2010; Skaug et al., 2011; Tokunaga et al., 2012; Verhelst et al., 2012; Lu et al., 2013). "
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    ABSTRACT: The reverse reaction of ubiquitylation is catalyzed by different classes of deubiquitylation enzymes (DUBs), including ovarian tumor domain (OTU)-containing DUBs; experiments using Homo sapiens proteins have demonstrated that OTU DUBs modulate various cellular processes. With the exception of OTLD1, plant OTU DUBs have not been characterized. We identified 12 Arabidopsis thaliana OTU loci and analyzed 11 of the encoded proteins in vitro to determine their preferences for the ubiquitin (UB) chains of M1, K48, and K63 linkages as well as the UB-/RUB-/SUMO-GST fusions. The A. thaliana OTU DUBs were shown to be cysteine proteases and classified into four groups with distinct linkage preferences: OTU1 (M1=K48>K63), OTU3/4/7/10 (K63>K48>M1), OTU2/9 (K48=K63), and OTU5/11/12/OTLD1 (inactive). Five active OTU DUBs (OTU3/4/7/9/10) also cleaved RUB fusion. OTU1/3/4 cleaved M1 UB chains, suggesting a possible role for M1 chains in plant cellular signaling. The different substrate specificities of the various A. thaliana OTU DUBs indicate the involvement of distinct structural elements; for example, the OTU1 oxyanion residue D89 is essential for cleaving isopeptide bond-linked chains but dispensable for M1 chains. UB-binding activities were detected only for OTU2 and OTLD1, with distinct linkage preferences. These differences in biochemical properties support the involvement of A. thaliana OTU DUBs in different functions. Moreover, based on the established phylogenetic tree, plant- and H. sapiens-specific clades exist, which suggests that the encoded proteins have taxa-specific functions. We also detected five OTU clades that are conserved across species, which suggests that the orthologs in different species within each clade are involved in conserved cellular processes, such as ERAD and DNA damage responses. However, different linkage preferences have been detected among potential cross-species OTU orthologs, indicating functional and mechanistic differentiation.
    Full-text · Article · Mar 2014 · Frontiers in Plant Science
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    • "These results, together with the frequent co-occurrence of MYD88 and TNFAIP3 mutations in ABC-DLBCL (Ngo et al., 2011), raised the possibility that MYD88 L265P might have an intrinsically self-limiting effect on B cells by inducing a negative feedback loop through induction of TNFAIP3 (also designated A20). TNFAIP3 mRNA and protein are rapidly induced by NF-B and form a critical negative feedback loop to diminish NF-B activity by adding K48- linked ubiquitin chains, removing K63-linked ubiquitin chains, and inhibiting the addition of linear ubiquitin chains to key molecules in the NF-B signaling axis (Harhaj and Dixit, 2012; Tokunaga et al., 2012; Verhelst et al., 2012). "
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    ABSTRACT: MYD88(L265P) has recently been discovered as an extraordinarily frequent somatic mutation in benign monoclonal IgM gammopathy, Waldenström's macroglobulinemia, and diffuse large B cell lymphoma. In this study, we analyze the consequences for antigen-activated primary B cells of acquiring MYD88(L265P). The mutation induced rapid B cell division in the absence of exogenous TLR ligands and was inhibited by Unc93b1(3d) mutation and chloroquine or TLR9 deficiency, indicating continued dependence on upstream TLR9 activation. Proliferation and NF-κB activation induced by MYD88(L265P) were nevertheless rapidly countered by the induction of TNFAIP3, an NF-κB inhibitor frequently inactivated in MYD88(L265P)-bearing lymphomas, and extinguished by Bim-dependent apoptosis. MYD88(L265P) caused self-reactive B cells to accumulate in vivo only when apoptosis was opposed by Bcl2 overexpression. These results reveal checkpoints that fortify TLR responses against aberrant B cell proliferation in response to ubiquitous TLR and BCR self-ligands and suggest that tolerance failure requires the accumulation of multiple somatic mutations.
    Full-text · Article · Feb 2014 · Journal of Experimental Medicine
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