Biology subjects

Gyrd-Hansen, M.

Publications and source records attributed to Gyrd-Hansen, M..

7 recordsLinked to original sources

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↗

bioRxiv2025

Comparative ubiquitinomics of human skin reveals insulin receptor ubiquitination as a regulator of collagen secretion.

Ubiquitination is central to skin homeostasis and disease, however the cutaneous ubiquitinome remains poorly characterised. We investigated spatial patterns of ubiquitination in human skin, discovering specific ubiquitin linkages located within the hair follicle and epidermis. To create a comprehensive physiological profile of the ubiquitinated proteins within the skin, we profiled healthy skin and CYLD cutaneous syndrome (CCS) skin tumours through di-glycine remnant immunopurification proteomics, enabling identification of 1605 ubiquitin sites across 731 proteins. The healthy cutaneous ubiquitinome comprised substrate classes including keratins, S100 proteins and histones. Predictive upstream enzyme analysis ranked the most frequent E3 ligase as NEDD4, and the most frequent deubiquitinases included USP7 and CYLD. Analysis of the CCS tumor ubiquitinome, characterized by catalytically inactivated CYLD somatic mutations and prominent extracellular matrix (ECM) secretion, identified 251 differentially ubiquitinated sites compared to healthy skin. The CCS ubiquitinome was enriched for cell division and differentiation proteins, including predicted CYLD substrate insulin receptor (INSR). Secretome analysis of INSR knockdown CCS primary cells revealed reduced secretion of basement membrane proteins, particularly COL7A1. By defining how ubiquitination modulates pathophysiological ECM secretory output, our study highlights the ubiquitin post-translational system as a broader determinant of tissue architecture. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/691868v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@565e59org.highwire.dtl.DTLVardef@4c3084org.highwire.dtl.DTLVardef@52a43org.highwire.dtl.DTLVardef@11999d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

M1-linked Ubiquitination by LUBAC Regulates AMPK Activity and the Response to Energetic Stress

Methionine-1 (M1)-linked ubiquitin chains, assembled by the ubiquitin ligase LUBAC and cleaved by the deubiquitinase OTULIN, are critical regulators of inflammation and immune homeostasis. Genetic loss of either LUBAC or OTULIN causes autoinflammatory syndromes, which are associated with defects in glycogen and lipid metabolism. However, how LUBAC and OTULIN regulate metabolic signalling remains unknown. Here, we demonstrate that LUBAC promotes, while OTULIN restricts, activation of the key metabolic regulator AMP-activated protein kinase (AMPK) in cells, mice, and human samples. LUBAC and OTULIN interact with AMPK, control its M1-ubiquitination, and regulate its activation in response to glucose starvation and allosteric activation. During starvation, LUBAC deficiency impairs autophagy induction and hinders the shift from oxidative phosphorylation to glycolysis. Strikingly, LUBAC-deficient Drosophila have a strongly reduced survival rate after starvation. Our work identifies LUBAC and OTULIN as physiological regulators of AMPK, providing the first mechanism by which M1-linked ubiquitin chains regulate metabolic signalling.

cell biology↗

bioRxiv2023

Mitochondrial outer membrane integrity regulates a ubiquitin-dependent NF-κB inflammatory response

Mitochondria are often essential for apoptosis through mitochondrial outer membrane permeabilization (MOMP). This central event enables cytochrome c release leading to caspase activation and rapid cell death. Recently, MOMP has been shown to be inherently pro-inflammatory, for instance, by enabling mitochondrial DNA-dependent activation of cGAS-STING signalling. Alongside having emerging functions in health and disease, MOMP associated inflammation can also elicit anti-tumour immunity. Nonetheless, how MOMP triggers inflammation and how the cell counteracts this remains poorly defined. We find that upon MOMP, mitochondria are ubiquitylated in a promiscuous manner targeting proteins localised to both inner and outer mitochondrial membranes. Mitochondrial ubiquitylation serves to recruit the essential adaptor molecule, NEMO, leading to activation of pro-inflammatory NF-{kappa}B signalling. We find that disruption of mitochondrial outer membrane integrity through different means leads to engagement of a similar pro-inflammatory signalling platform. Thus, mitochondrial integrity directly controls inflammation, such that permeabilised mitochondria initiate NF-{kappa}B signalling. This event may be important for the various pathophysiological functions of MOMP-associated inflammation.

cell biology↗

bioRxiv2021

ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression

COVID-19 caused by the SARS-CoV-2 virus remains a threat to global health. The disease severity is mediated by cell death and inflammation, which regulate both the antiviral and the pathological innate immune responses. ZBP1, an interferon-induced cytosolic nucleic acid sensor, facilitates antiviral responses via RIPK3. Although ZBP1-mediated cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway that depends on ubiquitination and RIPK3s scaffolding ability independently of cell death. In human cells, ZBP1 associates with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. RIPK1 and ZBP1 are ubiquitinated to promote TAK1- and IKK-mediated inflammatory signaling. Additionally, RIPK1 recruits the p43/41-caspase-8-p43-FLIP heterodimer to suppress RIPK3 kinase activity, which otherwise promotes inflammatory signaling in a kinase activity-dependent manner. Lastly, we show that ZBP1 contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK1-RIPK3-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspase-8. Our results suggest the ZBP1 pathway contributes to inflammation in response to SARS-CoV-2 infection.

immunology↗

bioRxiv2020

Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling

Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 different stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.

cell biology↗