SummaryUbiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes. LUBAC-generated linear (M1) ubiquitin is important for signaling via various immune receptors. We show here that the deubiquitinases CYLD and A20, but not OTULIN, are recruited to the TNFR1- and NOD2-associated signaling complexes (TNF-RSC and NOD2-SC), at which they cooperate to limit gene activation. Whereas CYLD recruitment depends on its interaction with LUBAC, but not on LUBAC’s M1-chain-forming capacity, A20 recruitment requires this activity. Intriguingly, CYLD and A20 exert opposing effects on M1 chain stability in the TNF-RSC and NOD2-SC. While CYLD cleaves M1 chains, and thereby sensitizes cells to TNF-induced death, A20 binding to them prevents their removal and, consequently, inhibits cell death. Thus, CYLD and A20 cooperatively restrict gene activation and regulate cell death via their respective activities on M1 chains. Hence, the interplay between LUBAC, M1-ubiquitin, CYLD, and A20 is central for physiological signaling through innate immune receptors.

LUBAC modulates signalling by various immune receptors. In TNF signalling, linear (also known as M1) ubiquitin enables full gene-activation and prevents cell death. However, the mechanisms underlying cell-death prevention remain ill-defined. We show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNFR1-signalling complex (TNFR1-SC). Whilst exerting only limited effects on TNF-induced gene-activation, TBK1/IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1/IKKε phosphorylate RIPK1 in the TNFR1-SC, thereby preventing RIPK1-kinase-activity-dependent cell death. This activity is essential in vivo , as it prevents TNF-induced lethal shock. Strikingly, NEMO/IKKγ, which mostly, but not exclusively, binds to the TNFR1-SC via M1-ubiquitin, mediates recruitment of the adaptors TANK and NAP1/AZI2 which are constitutively associated with TBK1/IKKε and TBK1, respectively. We here discover a previously unrecognised TBK1/IKKε-mediated cell-death checkpoint and uncover an essential survival function for NEMO by enabling recruitment and activation of these noncanonical IKKs to prevent TNF-induced cell death.

SummaryTumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology.

The Linear Ubiquitin chain Assembly Complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR11. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, due to deregulation of TNFR1-mediated cell death2–8. In humans, deficiency in the third LUBAC component, HOIL-1, causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9–11. By creating HOIL-1-deficient mice, we here show that HOIL-1 is, however, as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is LUBAC’s catalytically active component, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1-signalling complex (TNFR1-SC), thereby preventing aberrant cell death. Both, HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of Caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- embryos, yet only combined loss of Caspase-8 with MLKL results in viable HOIL-1-deficient mice. Interestingly, Ripk3-/-Caspase-8-/-Hoil-1-/- embryos die at late-gestation due to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both, HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they unveil that, when LUBAC and Caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in foetal haematopoiesis.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ubiquitin ligase which catalyses the generation of linear ubiquitin linkages de novo. LUBAC is a crucial component of various immune receptor signalling pathways. Here, we show that LUBAC forms part of the TRAIL‐R‐associated complex I as well as of the cytoplasmic TRAIL‐induced complex II. In both of these complexes, HOIP limits caspase‐8 activity and, consequently, apoptosis whilst being itself cleaved in a caspase‐8‐dependent manner. Yet, by limiting the formation of a RIPK1/RIPK3/MLKL‐containing complex, LUBAC also restricts TRAIL‐induced necroptosis. We identify RIPK1 and caspase‐8 as linearly ubiquitinated targets of LUBAC following TRAIL stimulation. Contrary to its role in preventing TRAIL‐induced RIPK1‐independent apoptosis, HOIP presence, but not its activity, is required for preventing necroptosis. By promoting recruitment of the IKK complex to complex I, LUBAC also promotes TRAIL‐induced activation of NF‐κB and, consequently, the production of cytokines, downstream of FADD, caspase‐8 and cIAP1/2. Hence, LUBAC controls the TRAIL signalling outcome from complex I and II, two platforms which both trigger cell death and gene activation.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 AbstractMarine environment has frequently afforded a variety of biologically active compounds with strong anticancer and cytotoxic properties. In the present study, the mechanism of action of Jaspine B, an anhydrophytosphingosine derivative isolated from the marine sponge Jaspis sp., was investigated. Jaspine B was able to doseand time-dependently decrease the viability of murine B16 and human SK-Mel28 melanoma cells. On these cells, Jaspine B treatment triggered cell death by typical apoptosis as illustrated by phosphatidylserine externalization, the release of cytochrome c and caspase processing. These effects were associated with increased intracellular ceramide levels owing to perturbed ceramide metabolism. Indeed, Jaspine B exposure strongly inhibited the activity of sphingomyelin synthase (SMS), an enzyme that converts de novo ceramide into the membrane lipid sphingomyelin.Moreover, whereas Jaspine B-induced cell death was enhanced in SMS1-depleted cells, it was strongly inhibited in cells that stably overexpress human SMS1. Finally, the cytotoxic effects of Jaspine B truncated analogs were also shown to be dependent on SMS activity.Altogether, Jaspine B is able to kill melanoma cells by acting on SMS activity and consequently on ceramide formation, and may represent a new class of cytotoxic compounds with potential applications in anticancer melanoma therapy.

Despite its name, signalling induced by the tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is versatile. Besides eliciting cell death by both apoptosis and necroptosis, TRAIL can also induce migration, proliferation, and cytokine production in cancerous and non-cancerous cells. Unravelling the mechanisms regulating the intricate balance between these different outputs could therefore facilitate our understanding of the role of TRAIL in tissue homeostasis, immunity, and cancer. Ubiquitination and its reversal, deubiquitination, are crucial modulators of immune receptor signalling. This review discusses recent progress on the orchestration of TRAIL signalling outcomes by ubiquitination of various components of the signalling complexes, our understanding of the molecular switches that decide between cell death and gene activation, and what remains to be discovered.

Ceramide can be converted into sphingomyelin by sphingomyelin synthases (SMS) 1 and 2. In this study, we show that in human leukemia Jurkat cells, which express mainly SMS1, Fas ligand (FasL) treatment inhibited SMS activity in a dose-and timedependent manner before nuclear fragmentation. The SMS inhibition elicited by FasL (1) was abrogated by benzyloxycarbonyl valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor; (2) did not occur in caspase-8-deficient cells and (3) was not affected in caspase-9-deficient cells. Western blot experiments showed SMS1 cleavage in a caspase-dependent manner upon FasL treatment. In a cell-free system, caspase-2, -7, -8 and -9, but not caspase-3 and -10, cleaved SMS1. In HeLa cells, SMS1 was Golgi localized and relocated throughout the cytoplasm in cells exhibiting an early apoptotic phenotype on FasL treatment. zVAD-fmk prevented FasL-induced SMS1 relocation. Thus, FasL-mediated SMS1 inhibition and relocation depend on caspase activation and likely represent proximal events in Fas signaling. FasL-induced ceramide production and cell death were enhanced in cells stably expressing an siRNA against SMS1. Conversely, in cells stably overexpressing SMS1, FasL neither increased ceramide generation nor efficiently induced cell death. Altogether, our data show that SMS1 is a novel caspase target that is functionally involved in the regulation of FasL-induced apoptosis.