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. 2013 Feb 21;494(7437):371-4.
doi: 10.1038/nature11831. Epub 2013 Jan 20.

OTUD7B controls non-canonical NF-κB activation through deubiquitination of TRAF3

Hongbo Hu  1 , George C Brittain, Jae-Hoon Chang, Nahum Puebla-Osorio, Jin Jin, Anna Zal, Yichuan Xiao, Xuhong Cheng, Mikyoung Chang, Yang-Xin Fu, Tomasz Zal, Chengming Zhu, Shao-Cong Sun
Affiliations

OTUD7B controls non-canonical NF-κB activation through deubiquitination of TRAF3

Hongbo Hu et al. Nature. .

Abstract

The non-canonical NF-κB pathway forms a major arm of NF-κB signalling that mediates important biological functions, including lymphoid organogenesis, B-lymphocyte function, and cell growth and survival. Activation of the non-canonical NF-κB pathway involves degradation of an inhibitory protein, TNF receptor-associated factor 3 (TRAF3), but how this signalling event is controlled is still unknown. Here we have identified the deubiquitinase OTUD7B as a pivotal regulator of the non-canonical NF-κB pathway. OTUD7B deficiency in mice has no appreciable effect on canonical NF-κB activation but causes hyperactivation of non-canonical NF-κB. In response to non-canonical NF-κB stimuli, OTUD7B binds and deubiquitinates TRAF3, thereby inhibiting TRAF3 proteolysis and preventing aberrant non-canonical NF-κB activation. Consequently, the OTUD7B deficiency results in B-cell hyper-responsiveness to antigens, lymphoid follicular hyperplasia in the intestinal mucosa, and elevated host-defence ability against an intestinal bacterial pathogen, Citrobacter rodentium. These findings establish OTUD7B as a crucial regulator of signal-induced non-canonical NF-κB activation and indicate a mechanism of immune regulation that involves OTUD7B-mediated deubiquitination and stabilization of TRAF3.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Otud7b negatively regulates the noncanonical NF-κB pathway
a-c, EMSA of NF-κB and control DNA-binding factors (Oct-1, NF-Y) using nuclear extracts isolated from WT and Otud7b-KO primary MEFs (a) or B cells (b, c) stimulated with the indicated inducers. d-f, Immunoblot (IB) assays using cytoplasmic (CE) and nuclear (NE) extracts of WT and Otud7b-KO primary MEFs (d) or B cells (e, f) that were stimulated as indicated. Data are representative of three independent experiments.
Figure 2
Figure 2. Otud7b negatively regulates TRAF3 degradation by affecting TRAF3 ubiquitination
a, b, IB analyses of TRAF3 and NIK in WT and Otud7b-KO MEFs or B cells, stimulated as indicated. c, Endogenous TRAF3 was isolated by IP, under denaturing conditions, from the WT or Otud7b-KO B cells treated with anti-CD40 plus MG132. Total and K48-ubiquitination of TRAF3 were detected by IB using anti-ubiquitin and anti-K48-ubiquitin antibodies, respectively. d, HEK293 cells were transfected with the indicated expression vectors and subjected to TRAF3 ubiquitination (upper) and protein expression (lower) assays. e, TRAF3 was transfected with HA-ubiquitin or K48 ubiquitin in HEK293 cells. Purified TRAF3-ubiquitin conjugates were incubated with buffer control (C), GST, GST-Otud7b WT, or GST-Otud7bCH recombinant proteins followed by anti-HA IB. f, IB analysis of subcellular extracts from WT or Otud7b-KO MEFs, stably infected with vector, Otud7b, or Otud7bCH and stimulated as indicated. Data are representative of at least three independent experiments.
Figure 3
Figure 3. Otud7b inducibly interacts with TRAF3 and is recruited to the receptor complexes
a, M12 B cells (left) and WT MEFs (right) were stimulated and subjected to Otud7b/TRAF3 co-IP (upper) and direct IB (lower) assays. b, M12-hCD40 cells were stimulated with anti-human CD40. CD40 was isolated by IP, and its associated proteins identified by IB (left panel). Cell lysates were subjected to direct IB (right panel). c, WT and Otud7b-KO MEFs were stimulated and subjected to identification of LTβR-associated proteins (upper) and analysis of protein expression (lower). d, Otud7b-KO MEFs, stably infected with Otud7b WT or the indicated Otud7b mutants, were stimulated with anti-LTβR and subjected to co-IP assays to detect Otud7b-TRAF3 interaction (panel 1) and Otud7b recruitment to LTβR (panel 2) or direct IB assays (bottom two panels). Data are representative of two to three independent experiments.
Figure 4
Figure 4. Otud7b negatively regulates intestinal lymphoid homeostasis, anti-bacterial immunity, and B-cell responses
a, H&E picture (left) and summary graph (right) of WT and Otud7b-KO colon (n=4). Black arrows point to CLPs, and the squared area is enlarged in the 80X pictures. b, ELISA of IgA in the feces of WT and Otud7b-KO mice (μg IgA per gram of feces, n=13). c, Summary of CLP numbers in WT and Otud7b-KO mice (n=4) treated with PBS or LTβR-Ig. d, e, QPCR assays using RNAs prepared from colonic tissues (d) and anti-LTβR-stimulated MEFs (e). f, g, Survival (f) and body weight (g) of C. rodentium-infected WT or Otud7b-KO mice that were also injected with a control IgG or an anti-CD4 antibody on day 0, 5, and 10 (n=4). h, Proliferation assays of splenic B cells cultured without (NT) or with the indicated inducers in vitro. In vitro proliferation assays of splenic B cells. i, ELISA of serum NP-specific antibodies in NP-KLH-immunized Rag1-KO mice adoptively transferred with WT or Otud7b-KO B cells plus WT T cells. j, k, Mice were injected with BAFF (30mg/kg) i.v. every other day for 2 weeks and sacrificed on day 15 for flow cytometric analysis of B-cell frequency in splenocytes (j) and ELISA of the serum antibodies (k) (n=4). Data are representative of two-three independent experiments. Bar graphs are presented as mean±S.D. values. *p<0.05; **p<0.01; ***p<0.001.
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