Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP

Linear ubiquitin chains are important regulators of cellular signaling pathways that control innate immunity and inflammation through NF-κB activation and protection against TNFα-induced apoptosis(1-5). They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multi-subunit E3 ligase(6). RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or “donor” ubiquitin(7). Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The C-terminal portion of HOIP adopts a novel fold that, together with a zinc finger, forms an ubiquitin-binding platform which orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3~ubiquitin thioester. The carboxy-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-kB pathway in vivo and explain the determinants of linear ubiquitin chain specificity by LUBAC

N4BP1 is dimerization-dependent linear ubiquitin reader regulating TNFR1 signalling through linear ubiquitin binding and Caspase-8-mediated processing

This article is a preprint and has not been certified by peer review.Data and material availability: All the plasmids generated in this study will be available upon request. All data is available in the main text or the supplementary materials online at: https://doi.org/10.1101/2021.11.02.466974 ..Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) was recently identified as a critical suppressor of proinflammatory cytokine production1, whose mode of action remained unknown. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerizationdependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure exclusive 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 and the resulting cleavage fragment of N4BP1, which retains the ability to bind linear ubiquitin, is rapidly degraded by the 26S proteasome, accelerating apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a unique linear ubiquitin reader that ensures 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)

N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling

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)

N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling

AbstractSignalling 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.</jats:p

RBR ligase–mediated ubiquitin transfer: a tale with many twists and turns

RBR ligases are an enigmatic class of E3 ubiquitin ligases that combine properties of RING and HECT-type E3s and undergo multilevel regulation through autoinhibition, post-translational modifications, multimerization and interaction with binding partners. Here, we summarize recent progress in RBR structures and function, which has uncovered commonalities in the mechanisms by which different family members transfer ubiquitin through a multistep process. However, these studies have also highlighted clear differences in the activity of different family members, suggesting that each RBR ligase has evolved specific properties to fit the biological process it regulates

Characterization of callase (β-1,3-d-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8–5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall

Navigating cross-cultural research: methodological and ethical considerations

Copyright © 2020 The Authors. The intensifying pace of research based on cross-cultural studies in the social sciences necessitates a discussion of the unique challenges of multi-sited research. Given an increasing demand for social scientists to expand their data collection beyond WEIRD (Western, educated, industrialized, rich and democratic) populations, there is an urgent need for transdisciplinary conversations on the logistical, scientific and ethical considerations inherent to this type of scholarship. As a group of social scientists engaged in cross-cultural research in psychology and anthropology, we hope to guide prospective cross-cultural researchers through some of the complex scientific and ethical challenges involved in such work: (a) study site selection, (b) community involvement and (c) culturally appropriate research methods. We aim to shed light on some of the difficult ethical quandaries of this type of research. Our recommendation emphasizes a community-centred approach, in which the desires of the community regarding research approach and methodology, community involvement, results communication and distribution, and data sharing are held in the highest regard by the researchers. We argue that such considerations are central to scientific rigour and the foundation of the study of human behaviour.Department of Human Behaviour, Ecology and Culture at the Max Planck Institute for Evolutionary Anthropology; French National Research Agency under the Investments for the Future (Investissements d'Avenir) programme (ANR-17-EURE-0010)

Progress Report on Target Development

The present document is the D08 deliverable report of work package 1 (Target Development) from the MEGAPIE TEST project of the 5th European Framework Program. Deliverable D08 is the progress report on the activities performed within WP 1. The due date of this deliverable was the 5th month after the start of the EU project. This coincided with a technical status meeting of the MEGAPIE Initiative, that was held in March 2002 in Bologna (Italy). The content of the present document reflects the status of the MEGAPIE target development at that stage. It gives an overview of the Target Design, the related Design Support activities and the progress of the work done for the safety assessment and licensing of the target