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.

Download full-text


Available from: Eiji Goto, Feb 04, 2015
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Frontiers in Plant Science 03/2014; 5:84. DOI:10.3389/fpls.2014.00084 · 3.95 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Journal of Experimental Medicine 02/2014; 211(3). DOI:10.1084/jem.20131424 · 12.52 Impact Factor
  • Source
    • "Anti-HOIP (SAB2102031), anti-HOIL-1L (NBP1-88301), anti-TRAF2 (558890), and anti-TRAF6 (H-274) antibodies were obtained from Sigma-Aldrich, Novus, BD Pharmingen, and Santa Cruz Biotechnology, respectively. Other antibodies and reagents were obtained as described previously [25]. HEK293T cells were grown in DMEM plus 10% fetal bovine serum, 100 IU ml−1 penicillin G and 100 µg ml−1 streptomycin. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS) is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING), resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.
    PLoS ONE 10/2013; 8(10):e76983. DOI:10.1371/journal.pone.0076983 · 3.23 Impact Factor
Show more