CASPASE 8 initiates apoptosis downstream of TNF death receptors by undergoing autocleavage and processing the executioner CASPASE 31. However, the dominant function of CASPASE 8 is to transmit a pro-survival signal that suppresses programmed necrosis (or necroptosis) mediated by RIPK1 and RIPK32–6 during embryogenesis and hematopoiesis7–9. Suppression of necrotic cell death by CASPASE 8 requires its catalytic activity but not the autocleavage essential for apoptosis10, however, the key substrate processed by CASPASE 8 to block necrosis has been elusive. A key substrate must meet three criteria: (1) it must be essential for programmed necrosis; (2) it must be cleaved by CASPASE 8 in situations where CASPASE 8 is blocking necrosis; and (3) mutation of the CASPASE 8 processing site on the substrate should convert a pro-survival response to necrotic death without the need for CASPASE 8 inhibition. We now identify CYLD as a novel substrate for CASPASE 8 that satisfies these criteria. Upon TNF stimulation, CASPASE 8 cleaves CYLD to generate a survival signal. In contrast, loss of CASPASE 8 prevented CYLD degradation resulting in necrotic death. A CYLD substitution mutation at D215 that cannot be cleaved by CASPASE 8 switches cell survival to necrotic cell death in response to TNF.

On detecting viral RNAs, the RNA helicase retinoic acid-inducible gene I (RIG-I) activates the interferon regulatory factor 3 (IRF3) signalling pathway to induce type I interferon (IFN) gene transcription. How this antiviral signalling pathway might be negatively regulated is poorly understood. Microarray and bioinformatic analysis indicated that the expression of RIG-I and that of the tumour suppressor CYLD (cylindromatosis), a deubiquitinating enzyme that removes Lys 63-linked polyubiquitin chains, are closely correlated, suggesting a functional association between the two molecules. Ectopic expression of CYLD inhibits the IRF3 signalling pathway and IFN production triggered by RIG-I; conversely, CYLD knockdown enhances the response. CYLD removes polyubiquitin chains from RIG-I as well as from TANK binding kinase 1 (TBK1), the kinase that phosphorylates IRF3, coincident with an inhibition of the IRF3 signalling pathway. Furthermore, CYLD protein level is reduced in the presence of tumour necrosis factor and viral infection, concomitant with enhanced IFN production. These findings show that CYLD is a negative regulator of RIG-I-mediated innate antiviral response. Keywords: cylindromatosis; interferon; IRF3; RIG-I; ubiquitin EMBO reports (2008) 9, 930-936.

TNF receptor 1 (TNFR1) can trigger opposing responses within the same cell: a prosurvival response or a cell-death pathway [1, 2]. Cell survival requires NF-kappaB-mediated transcription of prosurvival genes [3-9]; apoptosis occurs if NF-kappaB signaling is blocked [5, 7-9]. Hence, activation of NF-kappaB acts as a cell-death switch during TNF signaling. This study demonstrates that the pathway includes another cell-death switch that is independent of NF-kappaB. We show that lysine 63-linked ubiquitination of RIP1 on lysine 377 inhibits TNF-induced apoptosis first through an NF-kappaB-independent mechanism and, subsequently, through an NF-kappaB-dependent mechanism. In contrast, in the absence of ubiquitination, RIP1 serves as a proapoptotic signaling molecule by engaging CASPASE-8. Therefore, RIP1 is a dual-function molecule that can be either prosurvival or prodeath depending on its ubiquitination state, and this serves as an NF-kappaB-independent cell-death switch early in TNF signaling. These results provide an explanation for the conflicting reports on the role of RIP1 in cell death; this role was previously suggested to be both prosurvival and prodeath [10-12]. Because TRAF2 is the E3 ligase for RIP1 [13], these observations provide an explanation for the NF-kappaB-independent antiapoptotic function previously described for TRAF2 [14-16].

TNF receptor 1 (TNFR1) ligation can result in cell survival or cell death. What determines which of the two opposing responses is triggered is not fully understood. The current model suggests that it is the activation of the NF-κB pathway and its induction of pro-survival genes, or the lack thereof, which determines the outcome. NF-κB essential modifier (NEMO)/IκB kinase gamma (IKKγ)-deficient cells are highly sensitive to apoptosis and since NEMO is essential for NF-κB activation, it has been assumed that this is due to the lack of NF-κB. This study demonstrates that this assumption was incorrect and that NEMO has another anti-apoptotic function that is independent of its role in the NF-κB pathway. NEMO prevents receptor interacting protein-1 (RIP1) from engaging CASPASE-8 prior to NF-κB-mediated induction of anti-apoptotic genes. Without NEMO, RIP1 associates with CASPASE-8 resulting in rapid tumor necrosis factor (TNF)-induced apoptosis. These results suggest that there are two cell death checkpoints following TNF stimulation: an early transcription-independent checkpoint whereby NEMO restrains RIP1 from activating the caspase cascade, followed by a later checkpoint dependent on NF-κB-mediated transcription of pro-survival genes.

TNF can trigger two opposing responses: cell survival and cell death. TNFR1 activates caspases that orchestrate apoptosis but some cell types switch to a necrotic death when treated with caspase inhibitors. Several genes that are required to orchestrate cell death by programmed necrosis have been identified, such as the kinase RIP1, but very little is known about the inhibitory signals that keep this necrotic cell death pathway in check. We demonstrate that T cells lacking the regulatory subunit of IKK, NFκB essential modifier (NEMO), are hypersensitive to programmed necrosis when stimulated with TNF in the presence of caspase inhibitors. Surprisingly, this pro-survival activity of NEMO is independent of NFκB-mediated gene transcription. Instead, NEMO inhibits necrosis by binding to ubiquitinated RIP1 to restrain RIP1 from engaging the necrotic death pathway. In the absence of NEMO, or if ubiquitination of RIP1 is blocked, necrosis ensues when caspases are blocked. These results indicate that recruitment of NEMO to ubiquitinated RIP1 is a key step in the TNFR1 signaling pathway that determines whether RIP1 triggers a necrotic death response.