Mads Gyrd-Hansen

Methionine 1‐linked ubiquitin (Met1‐Ub) is a non‐degradative protein modification that is generated solely by the Linear Ub Chain Assembly Complex (LUBAC) and hydrolysed by the deubiquitinases OTULIN and CYLD. The Met1‐Ub system has emerged as a critical regulator of immune receptor signalling, specially known for activating NF‐κB downstream of the TNF receptor superfamily. Studies from human patients and mouse models have shown that the Met1‐Ub system is also critical for the development, maintenance, and function of T cells. However, manipulation of the Met1‐Ub system in T cells has yielded discrepant results, and the molecular underpinnings underlying the regulation of T cells by the Met1‐Ub system remain unclear. This mini‐review summarises the current consensus and highlights the unresolved questions surrounding the role of the Met1‐Ub system in T cells.

Stimulator of interferon genes (STING) is a highly conserved signaling molecule that coordinates an antiviral innate immune response through activation of the nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF3) signaling pathways. In addition to an antiviral response, STING-mediated NF-κB signaling also leads to an inflammatory response, which can cause substantial pathology. However, the molecular mechanism whereby STING drives inflammation is poorly understood, and this has hindered the development of anti-inflammatory strategies targeting this pathway. Here, we define an evolutionarily conserved mechanism whereby STING activates NF-κB signaling via recruitment of inhibitor of NF-κB kinase subunit γ (IKKγ) in an M1-linked ubiquitin-dependent manner. We also show that the more recently evolved carboxyl-terminal tail of STING cooperates with the IKKγ-IKK complex for NF-κB–dependent expression of pro-inflammatory genes in mammalian cells through the kinases TBK1 (TANK-binding kinase 1)/IKKε. Thus, the expression of the key inflammatory genes CCL3 and CCL4 requires the formation of M1-linked ubiquitin and the activation of NF-κB, but we do not detect a role for IRF3 in STING-driven NF-κB signaling.

Ubiquitination is central to skin homeostasis, however the cutaneous ubiquitinome remains poorly characterised. We investigated ubiquitination architecture in human skin, discovering ubiquitin linkage type localisation within the hair follicle and epidermis. We profiled healthy skin through di-glycine remnant immunopurification proteomics, identifying 1465 sites across histone, S100 and keratin protein classes. Predictive upstream enzyme analysis ranked the most frequent E3 ligases and deubiquitinases as NEDD4, USP7 and CYLD. Analysis of CCS tumours, characterised 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 secretion, our study highlights the ubiquitin system as a broader determinant of tissue architecture.

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.

Tumor necrosis factor (TNF) orchestrates immune responses but can also drive inflammation-associated tissue damage. However, the mechanisms governing tissue tolerance to TNF remain poorly understood. Here, we reveal that TNF receptor 1 (TNFR1) abundance is regulated by two upstream open reading frames (uORFs) in the 5′ untranslated region of TNFRSF1A and demonstrate that this is a key determinant of TNF tolerance. uORF2 dominantly limits TNFR1 translation, and its disruption increases TNFR1 levels, leading to excessive TNF-induced gene activation and cell death in cell culture. By contrast, uORF1 dynamically regulates TNFR1 levels in response to inflammatory and stress signals. In mice, uORF2 protects against TNF-driven systemic inflammatory response syndrome and liver pathology. We additionally report that the translation of other immune receptor messenger RNAs, including TLR4 , IFNAR1 , and IFNGR2 , is also controlled by uORFs. Thus, regulation of TNFR1 levels and possibly of other immune receptors emerges as a mechanism safeguarding against excessive immune responses and tissue damage.

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 OTULIN’s 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 (OTULIN ^Y56A ) in the OTULIN PUB-interacting motif, which disrupts OTULIN-LUBAC interaction, are viable without spontaneous immunopathology. However, Otulin ^Y56A/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 LUBAC’s activity and increased Met1-Ub accumulation at the TNF receptor signalling complex. This stabilised the signalling complex even after dissociation from TNF, increased NF-κB 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 Otulin ^Y56A/Y56A mice exaggerated pathology without affecting bacterial burden. Collectively, we identify the physical association of OTULIN to LUBAC as a critical brake that restricts LUBAC’s function and Met1-Ub-dependent inflammatory signalling, thereby preserving tissue integrity and promoting disease tolerance during acute immune activation.