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. 2014 May 6;111(18):6720-5.
doi: 10.1073/pnas.1321187111. Epub 2014 Apr 21.

Emergence of the A20/ABIN-mediated inhibition of NF-κB signaling via modifying the ubiquitinated proteins in a basal chordate

Shaochun Yuan  1 , Xiangru Dong, Xin Tao, Liqun Xu, Jie Ruan, Jian Peng, Anlong Xu
Affiliations

Emergence of the A20/ABIN-mediated inhibition of NF-κB signaling via modifying the ubiquitinated proteins in a basal chordate

Shaochun Yuan et al. Proc Natl Acad Sci U S A. .

Abstract

In the past decade, ubiquitination has been well documented to have multifaceted roles in regulating NF-κB activation in mammals. However, its function, especially how deubiquitinating enzymes balance the NF-κB activation, remains largely elusive in invertebrates. Investigating bbtA20 and its binding proteins, bbt A20-binding inhibitor of NF-κB (bbtABIN1) and bbtABIN2, in Chinese amphioxus Branchiostoma belcheri tsingtauense, we found that bbtABIN2 can colocalize and compete with bbt TNF receptor-associated factor 6 to connect the K63-linked polyubiquitin chains, whereas bbtABIN1 physically links bbtA20 to bbt NF-κB essential modulator (bbtNEMO) to facilitate the K48-linked ubiquitination of bbtNEMO. Similar to human A20, bbtA20 is a dual enzyme that removes the K63-linked polyubiquitin chains and builds the K48-linked polyubiquitin chains on bbt receptor-interacting serine/threonine protein kinase 1b, leading to the inhibition of NF-κB signaling. Our study not only suggests that ubiquitination is an ancient strategy in regulating NF-κB activation but also provides the first evidence, to our knowledge, for ABINs/A20-mediated inhibition of NF-κB via modifying the ubiquitinated proteins in a basal chordate, adding information on the stepwise development of vertebrate innate immune signaling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Sequence and phylogenetic analyses of bbtA20 and bbtABINs. (A) Protein architecture of bbtA20 shows an N-terminal OTU domain and seven ZnFs at its C terminus. (B) Phylogenetic analysis of bbtA20. (C) Protein architecture shows that bbtABIN1 possesses all four AHD regions, whereas bbtABIN2 lacks AHD3. Both bbtABIN1 and bbtABIN2 contain a conserved UBAN domain. All A20 protein sequences were obtained from the GenBank database. Protein sequences were first aligned using ClustalX 1.83 and were manually corrected using GeneDoc. The neighbor-joining tree was obtained using MEGA4 with 1,000 bootstrap tests.
Fig. 2.
Fig. 2.
Expression patterns of bbtABIN2 and bbtA20. (A) Section in situ hybridization showed that transcripts of bbtABIN2 are abundant in intestine, hepatic cecum, and gonad. (B) Macroscopic view of the hybridization signals of bbtABIN2 in hepatic cecum and ovary. (C) Transcripts of bbtABIN2 are sparse in gill slits but abundant in testis. (D) Negative control of bbtABIN2 using the sense probe. g, gill slit; h, hepatic cecum; i, intestine; m, muscle; n, notochord; o, ovary; t, testis. Blue indicates strong hybridization, and dark brown indicates a weak signal. (E) RT-PCR analyses of expression patterns of bbtA20 after bacterial challenge in the presence or absence of the NF-κB inhibitor helenalin. Results were presented as “fold induction” of mRNA expression done in triplicate, using the 2-ΔΔCt method. Endogenous control for quantification was cytoplasmic β-actin. Values were considered significant at P < 0.05. (F) Sequence analysis indicated that all bbtABINs and bbtA20 possess several conserved κB-binding motifs in the promoter region. A reporter assay confirmed that the region containing two κB-binding sites upstream of the ATG of bbtA20 is essential for the binding to bbtRel. Hsp65 indicates the Homo sapiens p65. Reporter experiments were conducted in triplicate; vertical bars indicate mean ± SD. Data are representative of three independent experiments.
Fig. 3.
Fig. 3.
K63-linked ubiquitinated bbtTRAF6 is the target of bbtABIN2. (A) Luciferase reporter assays showed that both bbtABIN1 and bbtABIN2 can attenuate the NF-κB activation mediated by bbtTRAF6 in a dose-dependent manner in HEK 293T cells. Reporter experiments were conducted in triplicate; vertical bars indicate mean ± SD. Data are representative of three independent experiments. (B) Vectors of bbtABIN2 used in this study. (C) Luciferase reporter assays showed that truncated mutants containing the AHD2 region of bbtABIN2 can inhibit the bbtTRAF6-mediated activation of NF-κB. FL2 indicates the WT bbtABIN2; 1–6 indicate bbtABIN2-1 to bbtABIN2-6. von indicates the blank control; vec indicates the pcDNA3.1 vector. (D) In vitro binding assays showed that bbtABIN2 can bind both K63-linked and K48-linked ubiquitin chains. WB, Western blot. (E) Confocal analysis showed that bbtABIN2, but not bbtABIN1, can colocalize with bbtTRAF6. Original magnification 400×. (F) Both WT bbtABIN2 and the truncated mutant bbtABIN2-4 can bind to K63-linked polyubiquitin chains by competing with bbtTRAF6, whereas bbtABIN2-3, which lacks the UBAN domain, cannot. The abbreviation bbtUb indicates the WT amphioxus ubiquitin, whereas bbtUbK63 indicates the amphioxus ubiquitin mutant with substitution of arginine for all lysine residues except the lysine at position 63. All of the co-IP (IP) assays were done at least twice.
Fig. 4.
Fig. 4.
BbtA20 was physically linked to bbtNEMO to facilitate the K48-linked ubiquitination of bbtNEMO. (A) Cotransfection of bbtNEMO with bbtA20 and bbtABIN1 results in the degradation of bbtNEMO dependent on the dose of bbtA20. (B) Cotransfection of bbtNEMO with bbtA20 and bbtABIN1, but not with bbtA20, alone or together with bbtABIN2, results in the degradation of bbtNEMO. (C) Vectors of bbtA20 used in this study. (D) In vivo ubiquitination showed that point mutations within bbtA20 ZnF4, but not within bbtA20 OTU, attenuate the degradation of bbtNEMO. (E) In vitro analyses showed that bbtA20 can function as an E3 to catalyze the K48-linked ubiquitination, which is dependent on its ZnFs. (F) Transfected WT and the ZnFs only of bbtA20 bind bbtABIN1 and btABIN2. (G) Co-IP assays showed that both bbtABIN1 and bbtABIN2, but not bbtA20, can directly interact with bbtNEMO. All co-IP experiments were done at least twice.
Fig. 5.
Fig. 5.
BbtA20 is a dual enzyme in removing the K63-linked ubiquitin chains and adding the K48-linked ubiquitination on bbtRIP1b. (A) Co-IP assays assessing the direct interaction between bbtRIP1b and bbtA20 and identifying both OTU and ZnFs of bbtA20 are responsible for the interaction. (B) Cotransfection of bbtA20 promotes bbtRIP1b degradation in HEK 293T cells. Such degradation could be inhibited by addition of MG132. Moreover, cotransfection of bbtA20 removes the K63-linked polyubiquitin chains from bbtRIP1b in a dose-dependent manner. (C) Cotransfection of WT bbtA20 or OTU mutant (bbtAM1), but not ZnF4 mutant (bbtAM2), results in the proteasome-dependent degradation of bbtRIP1b. (D) Cotransfection of WT bbtA20 or bbtAM2, but not bbtAM1, results in a low level of K63-linked ubiquitination, but not degradation, of bbtRIP1b in a dose-dependent manner. (E) In vitro ubiquitination assays showed that both WT bbtA20 and bbtA2 (the ZnFs only) can function as E3 to catalyze the K48-linked ubiquitination of hsRIP1b. (F) In vitro deubiquitination assays indicated that bbtA20 can deubiquitinate the K63-linked ubiquitinated bbtRIP1b. All co-IP experiments were done at least twice.
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