Fiil, B. K. | Search bioRxiv

bioRxiv2026

Development and Characterisation of a Versatile Single-Domain Antibody Specific for M1-linked Ubiquitin Chains

Ubiquitin signalling is mediated by structurally distinct polyubiquitin chains that encode discrete cellular functions. Progress in deciphering this ubiquitin code, particularly for the less abundant atypical chain types, has been hindered by limited availability of versatile chain type-specific affinity reagents. Here, we demonstrate that human single-domain antibodies (sdAbs) provide a versatile scaffold for the generation of ubiquitin linkage-specific binders. Using phage display and synthetic human sdAb libraries, we identified 2A6, an sdAb that specifically recognises methionine-1 (M1)-linked ubiquitin chains. To our knowledge, 2A6 represents the first reported sdAb with specificity for a defined homotypic ubiquitin chain linkage. 2A6 bound M1-linked ubiquitin chains with nanomolar affinity and was specific for M1-linked chains at the level of both diubiquitin and long polyubiquitin chains. AlphaFold3 modelling, supported by saturation mutagenesis, predicted that 2A6 recognises the proximal and distal ubiquitin moieties together with the region near the M1 linkage. Functionally, 2A6 enabled specific detection and enrichment of M1-linked ubiquitin across multiple applications, including ELISA, immunoblotting, immunoprecipitation under semi-denaturing conditions, substrate ubiquitination analysis, and immunofluorescence microscopy. The sdAb can be readily produced in E. coli from a single expression plasmid, providing a tractable, cost-effective and versatile reagent for investigating M1-linked ubiquitin signalling. Our work establishes sdAbs as a versatile scaffold for ubiquitin linkage-specific affinity reagents, providing a framework for the development of analogous binders specifically targeting additional ubiquitin linkages or architectures.

biochemistry↗

bioRxiv2026

The binding of OTULIN restrains LUBAC activity to prevent TNF-driven immunopathology

Met1-linked ubiquitin chains (Met1-Ub), synthesised by the linear ubiquitin chain assembly complex (LUBAC) and disassembled by the deubiquitinase OTULIN, critically regulate inflammatory signalling. Although OTULINs activity is essential to prevent TNF-driven autoinflammatory pathology and embryonic lethality, the regulatory significance of its direct interaction with LUBAC remains unclear. Here, we reveal that mice harbouring a point mutation (OTULINY56A) in the OTULIN PUB-interacting motif, which disrupts OTULIN-LUBAC interaction, are viable without spontaneous immunopathology. However, OtulinY56A/Y56A mice exhibited hypersensitivity to TNF-induced toxicity, which was not prevented by inhibiting RIPK1 kinase-mediated cell death. Mechanistically, disruption of the OTULIN-LUBAC interaction led to Met1-linked autoubiquitination, which enhanced LUBACs activity and increased Met1-Ub accumulation at the TNF receptor signalling complex. This stabilised the signalling complex even after dissociation from TNF, increased NF-{kappa}B signalling and, contrary to loss of OTULIN or its activity, protected cells from TNF-induced apoptosis. During systemic Listeria monocytogenes infection, the increased response to TNF in OtulinY56A/Y56A mice exaggerated pathology without affecting bacterial burden. Collectively, we identify the physical association of OTULIN to LUBAC as a critical brake that restricts LUBACs function and Met1-Ub-dependent inflammatory signalling, thereby preserving tissue integrity and promoting disease tolerance during acute immune activation.

molecular biology↗