Data availability;
Coordinates of the structure of N4BP1-CUE and the N4BP1-CUE/Ub complex have been deposited in the PDB-Dev Protein Data Bank (https://pdb-dev.wwpdb.org) under accession codes PDBDEV_00000076 and PDBDEV_00000093, respectively. Chemical shift data have been deposited in the Biological Magnetic Resonance Data Bank (https://bmrb.io) with BMRB entry ID 50688. The model of dimeric N4BP1 in complex with linear Ub2 is available in ModelArchive (modelarchive.org) with the accession code ma-2x3cw. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [76] with the dataset identifier PXD024355. All the plasmids generated in this study will be available upon request. All data is available in the main text or the supplementary materials. All original western blot images are available in the Supplemental Material.Supplementary information
is available at: https://www.nature.com/articles/s41420-024-01913-8#Sec40 .Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.This work was supported by the Francis Crick Institute through provision of access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029). KK was supported by the UPStream grant (EU, FP7, ITN project 290257). BS is supported by the UK Medical Research Council (MR/X036944/1)
