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

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.

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. 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. 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. 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.