LUBAC PUB domain interactions restrict Met1-linked ubiquitination to prevent embryonic lethality and immune pathology

LUBAC PUB domain interactions restrict Met1-linked ubiquitination to prevent embryonic lethality and immune pathology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article LUBAC PUB domain interactions restrict Met1-linked ubiquitination to prevent embryonic lethality and immune pathology Mads Gyrd-Hansen, John Rizk, Frederik Timmermann, Wenxin Lyu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5519483/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Met1-linked ubiquitin (Met1-Ub) assembled by LUBAC crucially regulates immune receptor signalling. However, the regulation of LUBAC’s function remains widely debated. Here, we reveal that the interaction between LUBAC and PUB-interacting motif (PIM)-containing proteins, including deubiquitinases OTULIN and SPATA2-CYLD, safeguards the fidelity of Met1-Ub deposition to prevent embryonic lethality and immune pathology. Mutation of the PUB domain in the LUBAC subunit HOIP ablated PIM-interactions and led to excessive Met1-Ub on signalling components after immune receptor stimulation, yet impaired productive signalling and sensitised to TNF-induced cell death. In vivo, ablation of HOIP PIM-interactions caused embryonic lethality at midgestation and led to acute cytokine storm, immune dysregulation, and weight loss when induced in adult mice. Moreover, heterozygous ablation of HOIP PUB PIM-interactions, which did not cause spontaneous pathology, rendered mice remarkably sensitive to TNF-challenge. Thus, the PUB domain of HOIP is a critical protein-interaction interface regulating LUBAC’s function in receptor signalling and immune homeostasis. Biological sciences/Immunology/Inflammation/Acute inflammation Biological sciences/Cell biology/Post-translational modifications/Ubiquitylation Biological sciences/Immunology/Cell death and immune response LUBAC PUB domain Met1-linked ubiquitin OTULIN CYLD SPATA2 TNF immune receptor inflammation cell death immunology Full Text Additional Declarations There is NO Competing Interest. Breeding of mice and experiments involving mice were performed under breeding and experimental licenses approved by the Danish Animal Experiments Inspectorate. Supplementary Files RizkXTimmermannSupplinformationSFigsRev.pdf Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5519483","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":624750713,"identity":"1556afd9-f628-445d-9ec6-c38e03ec4b86","order_by":0,"name":"Mads 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