Mechanism of negative regulation of NF-κB by N4BP1

Abstract

Many immune responses depend upon activation of NF-κB, a key transcription factor in the elicitation of a cytokine response. Here we demonstrate that N4BP1 inhibited 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 mediated the interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhanced 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 caused activation of caspase 8, which cleaved N4BP1 distal to residues D424 and D490 and abolished its inhibitory effect. N4bp1-/- mice also exhibited 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.

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“…FunRich analysis demonstrated interactions among these proteins, with a focus on CREB1 (Figure -S12B). On the basis of published literature that implicated these four proteins in the regulation of the nuclear factor-κB (NF-κB) signaling pathway, [26][27][28][29] the protein-protein interaction (PPI) network and schematic diagram that contained key proteins (NEDD4, IKBKG, CACTIN, and OTUD7B) suggested that CREB1, DEAF1, and N4BP1 might negatively regulate NF-κB signaling (Figure S12C).…”