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

Many immune responses depend upon activation of NF-κB, a key transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation) domains in N4BP1 mediate the 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-κB 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 exhibit 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-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished. Examination of mRNA expression in N4bp1 KO and wild-type peritoneal macrophages from two mice of each genotype that were untreated or treated with R848 or LPS.