Met1-linked ubiquitin signalling in health and disease: inflammation, immunity, cancer, and beyond

Abstract

Post-translational modification of proteins with ubiquitin (ubiquitination) provides a rapid and versatile mechanism for regulating cellular signalling systems. Met1-linked (or 'linear') ubiquitin chains have emerged as a key regulatory signal that controls cell death, immune signalling, and other vital cellular functions. The molecular machinery that assembles, senses, and disassembles Met1-linked ubiquitin chains is highly specific. In recent years, the thorough biochemical and genetic characterisation of the enzymes and proteins of the Met1-linked ubiquitin signalling machinery has paved the way for substantial advances in our understanding of how Met1-linked ubiquitin chains control cell signalling and biology. Here, we review current knowledge and recent insights into the role of Met1-linked ubiquitin chains in cell signalling with an emphasis on their role in disease biology. Met1-linked ubiquitin has potent regulatory functions in immune signalling, NF-κB transcription factor activation, and cell death. Importantly, mounting evidence shows that dysregulation of Met1-linked ubiquitin signalling is associated with multiple human diseases, including immune disorders, cancer, and neurodegeneration. We discuss the latest evidence on the cellular function of Met1-linked ubiquitin in the context of its associated diseases and highlight new emerging roles of Met1-linked ubiquitin chains in cell signalling, including regulation of protein quality control and metabolism.

Fig. 1. Pathologies associated with Met1-linked ubiquitin.

In recent years, the understanding of the (patho)physiological importance of Met1-linked ubiquitin signalling has increased substantially. Dysregulation or defects in Met1-linked ubiquitin signalling is now recognised to be associated with severe human disease, including neurodegeneration, myopathy, immune disorders, and cancer.

Fig. 2. The Met1-Ub Machinery.

A The proteins involved in ‘writing’ (assembling), ‘reading’ (binding), and ‘erasing’ (disassembling) Met1-Ub signals. LUBAC, together with E1 and E2 enzymes, assembles Met1-Ub in an ATP-dependent process. The first Ub conjugated to a substrate (shown in grey) does not have a defined linkage as the lysine residue (K) is unique to the substrate. Several proteins can bind Met1-Ub signals, and these are involved in ‘interpreting’ the biochemical modification of the substrate and funnelling it into the correct cellular pathway or response. OTULIN and CYLD (in complex with SPATA2) are the best-described DUBs that disassemble Met1-Ub. OTULIN exclusively disassembles Met1-Ub whereas CYLD-SPATA2 is less specific and also disassembles Lys63-Ub and some other linkages in addition to Met1-Ub (this activity is not shown). B Domain architecture of LUBAC and associated Met1-Ub DUBs. Domain interactions are indicated by arrows. CAP-Gly, cytoskeleton-associated protein glycine-rich; IBR, in-between-RING; LDD, linear ubiquitin chain-determining domain; LTM, LUBAC-tethering motif; NZF, Npl4 zinc finger; OTU, ovarian tumour; PH, pleckstrin homology; PIM, peptide:N-glycanase/ubiquitin-associated UBA-containing or UBX-containing protein-interacting motif; PUB, peptide:N-glycanase/ubiquitin-associated UBA-containing or UBX-containing protein; RING, Really Interesting New Gene; UBA, ubiquitin-associated; UBL, ubiquitin-like; USP, Ubiquitin specific protease; Znf, Zinc finger.

Fig. 3. Principles of immune signalling regulation by Met1-Ub.

Met1 (green) and Lys63 (blue) Ub linkages control signalling from a large range of innate and adaptive immune receptors. Generally, activation of immune receptors leads to the recruitment of adaptor proteins that become ubiquitinated with Met1-Ub and Lys63-Ub by different Ub ligases. Notably, Met1-Ub can be formed on pre-existing Lys63-Ub leading to the generation of hybrid Lys63/Met1-Ub chains. For canonical NF-κB activation triggered by TNF-R1, BCR or TCR, TLRs, IL-1R, or NOD1/2, engagement of these receptors leads to the formation of large, multi-protein receptor signalling complexes (RSCs) that contain adaptor proteins, such as TRADD and RIPK1 for TNF-R1, CARMA1-BCL10-MALT1 for BCR and TCR, MyD88 and IRAK1/4 for IL-1R and TLRs, and RIPK2 for NOD1/2, as well as Ub ligases, including cIAP1/2 for TNF-R1, XIAP for NOD2, and TRAF6 for IL-1R, TLRs, and BCR/TCR. LUBAC is recruited to these RSCs through binding to Lys63-Ub chains and conjugates Met1-Ub to existing Lys63-Ub to form hybrid Lys63/Met1-Ub chains or directly to adaptors in the RSCs. Lys63- and Met1-Ub modifications are recognised by the Ub-dependent kinase complexes TAK1-TAB2/3 and IKKα/β-NEMO, respectively. NEMO binding to Met1-Ub causes a conformational change in the IKK complex, leading to phosphorylation and activation NF-κB, which facilitates transcription of a plethora of genes encoding inflammatory mediators and pro-survival factors. For signalling triggered by TNF receptor-1 (TNF-R1), Met1-Ub also prevents the formation of cell death-inducing complexes that trigger either caspase-8-mediated apoptosis or RIPK3-MLKL-mediated necroptosis. Furthermore, LUBAC positively regulates the NLRP3 inflammasome where Met1-Ub conjugated to ASC promotes caspase-1 activation and IL-1β secretion. In contrast, LUBAC is a negative regulator of RIG-I signalling by conjugating Met1-Ub to TRIM25, which induces its proteasomal degradation and thereby inhibits IRF activation. OTULIN and CYLD disassembles Met1-Ub modifications in the TNF-R1- and NOD1/2-RSCs. It has not been reported which DUB(s) antagonises Met1-Ub in other immune signalling pathways. Lastly, OTULIN is required for LUBAC ‘maintenance’ by removing Met1-Ub autoubiquitination by HOIP. LUBAC autoubiquitination may contribute to signalling but can also lead to proteasomal degradation of HOIP, thereby destabilising LUBAC. ASC, apoptosis-associated speck-like protein containing a CARD; BCL10, B cell lymphoma 10; CARMA1, Caspase recruitment domain-containing membrane-associated guanylate kinase protein-1; Casp, caspase; FADD, Fas-associated protein with Death Domain; IRAK, interleukin-1 associated kinase; IRF, interferon regulatory factor; MALT1, Mucosa-associated lymphoid tissue lymphoma translocation protein 1; MLKL, Mixed Linkage Kinase Domain-Like; MyD88, myeloid differentiation primary response 88; PKC, protein kinase C; RIG-I, Retinoic acid-inducible gene I; RIPK, Receptor-interacting serine/threonine-protein kinase; TAB, TAK1- binding protein; TAK1, TGF-beta-activated kinase1; TANK, TRAF family member-associated NF-κB activator; TBK1, TANK-binding kinase 1; TRADD, Tumour necrosis factor receptor type 1-associated death domain protein; TRAF, TNF-receptor associated factor; TRIM25, Tripartite motif-containing protein 25.

Fig. 4. Pathogen interference with Met1-Ub signalling.

In response to bacterial and viral infections, Met1-Ub is assembled in activated immune receptor signalling complexes. In the receptor complexes, Met1-Ub stimulate the IKK complex and downstream NF-κB transcriptional activation. Met1-Ub can also be assembled on intracellular bacteria or their vacuoles (not shown), which promotes NF-κB activation and autophagosome-mediated killing of the bacteria (xenophagy). Some pathogens have evolved secreted effector proteins to specifically interfere with Met1-Ub signalling. Legionella pneumophila, the causative agent of Legionnaires’ disease, encodes the DUB RavD that specifically removes Met1-Ub signals to inhibit IKK activation and the NF-κB response and to possibly interfere with xenophagy of the invading Legionella. Shigella flexneri secretes the ubiquitin ligases IpaH1.4, IpaH2.5 and IpaH9.8 that promote the proteasomal degradation of LUBAC components or the Met1-Ub receptor NEMO, which prevents assembly or sensing of Met1-Ub for activation of the IKK complex and NF-κB. Porcine reproductive and respiratory syndrome virus (PRRSV) secretes NSP11 that hijacks endogenous host OTULIN to remove Met1-Ub from NEMO-containing signalling complexes and inhibit NF-κB activation. Epstein-Barr virus (EBV) encodes the membrane protein LMP1, which recruits LUBAC to ubiquitinate IRF7 and inhibit its transcriptional activity to limit the anti-viral interferon (IFN) response.

Fig. 5. Clinical manifestations and cell type-specific reactions in LUBAC deficiency and ORAS.

(A) Comparison of clinical manifestations of LUBAC deficiency and ORAS. Black text indicates overlapping manifestations, the green text indicates manifestations specific to LUBAC deficiency, and blue text indicates manifestations specific to ORAS. (B) Cell type-specific reactions to IL-1β or TNF stimulation. In LUBAC deficiency, patient-derived B cells and fibroblasts from HOIP- or HOIL-1-mutated patients show blunted NF-κB responses and decreased cytokine production. In contrast, patient monocytes have a hyperinflammatory transcriptional profile and hyper-secrete IL-6 in response to IL-1β stimulation. Decreased immune signalling in fibroblasts and B cells likely contribute to immunodeficiency in LUBAC deficiency patients, while the inflammatory profile of monocytes likely contributes to the paradoxical hyperinflammation. OTULIN-deficient lymphocytes (from mice) or patient-derived fibroblasts degrade and downregulate LUBAC. In these cells, TNF-induced NF-κB activity and cytokine production is decreased, and cells are sensitised to cell death. In contrast, OTULIN-deficient myeloid cells (primary murine or human cell lines) retain LUBAC expression and show increased basal and TNF-induced NF-κB activity, but the cells are still sensitive to TNF-induced cell death. TNF hyper-signalling and cytokine secretion from OTULIN-deficient myeloid cells combined with TNF-induced cell death of multiple cell types may in part explain the TNF-dependent inflammation and pathogenesis of ORAS. The molecular mechanism underlying these cell type-specific differences are unknown.

Fig. 6. Roles of the Met1-Ub machinery in cancer.

Examples of the involvement of the Met1-Ub machinery in cancer. Enhanced LUBAC activity, either as a consequence of HOIP mutations or overexpression, leads to constitutive NF-κB activation in ABC-DLBCL, which promotes B cell survival and proliferation as well as protects against DNA damage-induced cell death during B cell activation. In combination, this leads to sustained B cell survival and accumulation of somatic mutations, which contributes to lymphoma development. LUBAC activity and lymphoma cell survival can be inhibited by the N-Q622L peptide, which targets the HOIP-HOIL-1 interaction [132]. In LSCC, LUBAC overexpression leads to enhanced NF-κB activation, which confers tumour resistance to the chemotherapeutic agent cisplatin. Inhibition of LUBAC using the small-molecule LUBAC inhibitor gliotoxin reduces NF-κB activation and LSCC cell survival and sensitises tumours to cisplatin treatment [136]. OTULIN deficiency causes HCC in mice. The associated Met1-Ub accumulation causes aberrant mTOR activation, dysmetabolism, and increased RIPK1-mediated apoptosis and compensatory proliferation. Inhibition of mTOR by rapamycin leads to reduced tumour formation in mice [140]. In breast cancer patients, high OTULIN expression is associated with aggressive tumour subtypes and correlates with poor survival. Increased expression of OTULIN leads to removal of Met1-Ub from β-catenin, which stabilises the protein and allows Wnt/β-catenin-driven oncogenic signalling that promotes breast cancer cell survival, tumour growth, chemotherapy resistance, and metastasis in mice [142]. ABC-DLBCL, activated B cell diffuse large B cell lymphoma; HCC, hepatocellular carcinoma; LSCC, lung squamous cell carcinoma; mTOR, mammalian target, or rapamycin.

Fig. 7. Cellular functions of Met1-Ub.

In addition to the established functions of Met1-Ub, on which we have witnessed remarkable progress towards understanding their mechanistic underpinnings, recent studies have indicated new and emerging functions of Met1-Ub in the regulation of cellular signalling, function, and homeostasis.