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.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. By cross-linking TRAIL-Receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL has the capability to induce apoptosis in a wide variety of tumor cells while sparing vital normal cells. The discovery of this unique property among TNF superfamily members laid the foundation for testing the clinical potential of TRAIL-R-targeting therapies in the cancer clinic. To date, two of these therapeutic strategies have been tested clinically: (i) recombinant human TRAIL and (ii) antibodies directed against TRAIL-R1 or TRAIL-R2. Unfortunately, however, these TRAIL-R agonists have basically failed as most human tumors are resistant to apoptosis induction by them. It recently emerged that this is largely due to the poor agonistic activity of these agents. Consequently, novel TRAIL-R-targeting agents with increased bioactivity are currently being developed with the aim of rendering TRAIL-based therapies more active. This review summarizes these second-generation novel formulations of TRAIL and other TRAIL-R agonists, which exhibit enhanced cytotoxic capacity toward cancer cells, thereby providing the potential of being more effective when applied clinically than first-generation TRAIL-R agonists.

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.