N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization

Many immune responses depend upon activation of NF-kappa B, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-kappa B by interacting with the NF-kappa B signaling essential modulator (NEMO, also known as I kappa B kinase gamma) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-kappa B activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1(-/-) mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-kappa B, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished. NF-kappa B signalling is critical to TLR mediated cytokine release in various immune responses. Here the authors show how N4BP1 inhibits NEMO signalling and subsequent NF-kappa B activation and how this pathway is negatively regulated by caspase-8 cleavage of N4BP1.