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SHARPIN regulates signaling from the tumor necrosis factor (TNF) superfamily and pattern-recognition receptors. An inactivating Sharpin mutation in mice causes TNF-mediated dermatitis. Blocking cell death prevents the phenotype, implicating TNFR1-induced cell death in causing the skin disease. However, the source of TNF that drives dermatitis is unknown. Immune cells are a potent source of TNF in vivo and feature prominently in the skin pathology; however, T cells, B cells, and eosinophils are dispensable for the skin phenotype. We use targeted in vivo cell ablation, immune profiling, and extensive imaging to identify immune populations driving dermatitis. We find that systemic depletion of Langerin + cells significantly reduces disease severity. This is enhanced in mice that lack Langerhans cells (LCs) from soon after birth. Reconstitution of LC-depleted Sharpin mutant mice with TNF-deficient LCs prevents dermatitis, implicating LCs as a potential cellular source of pathogenic TNF and highlighting a T cell-independent role in driving skin inflammation., Competing Interests: Declaration of interests The authors claim no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions., Competing Interests: Declaration of interests The authors declare that D.S.S., J.P., A.W., I.Y.K., M.R., J.P.C., K.C.D., S.H., H.A., M.D., Y.D., L.M., M.D., A.S.H., S.E.N., J.R.G., M.J.H., E.D.H., A.S., J.S., M.P., R.F., and J.E.V. are employees or former employees of the Walter and Eliza Hall Medical Institute, which receives milestone payments from Genentech and AbbVie for the development of ABT-199 for cancer therapy. J.E.V. sits on the advisory board of Avammune Therapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Intrinsic apoptosis resulting from BAX/BAK-mediated mitochondrial membrane damage is regarded as immunologically silent. We show here that in macrophages, BAX/BAK activation results in inhibitor of apoptosis (IAP) protein degradation to promote caspase-8-mediated activation of IL-1β. Furthermore, BAX/BAK signaling induces a parallel pathway to NLRP3 inflammasome-mediated caspase-1-dependent IL-1β maturation that requires potassium efflux. Remarkably, following BAX/BAK activation, the apoptotic executioner caspases, caspase-3 and -7, act upstream of both caspase-8 and NLRP3-induced IL-1β maturation and secretion. Conversely, the pyroptotic cell death effectors gasdermin D and gasdermin E are not essential for BAX/BAK-induced IL-1β release. These findings highlight that innate immune cells undergoing BAX/BAK-mediated apoptosis have the capacity to generate pro-inflammatory signals and provide an explanation as to why IL-1β activation is often associated with cellular stress, such as during chemotherapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Immune infiltration has been correlated with survival of patients with colorectal cancer. In this issue, Ziegler et al. reveal complex and unexpected connections between loss of STAT3, mitophagy, and the induction of an adaptive immune response that limits the growth of colorectal carcinoma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1's cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling in vivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

After exiting the thymus, Foxp3 + regulatory T (Treg) cells undergo further differentiation in the periphery, resulting in the generation of mature, fully suppressive effector (e)Treg cells in a process dependent on TCR signaling and the transcription factor IRF4. Here, we show that tumor necrosis factor receptor superfamily (TNFRSF) signaling plays a crucial role in the development and maintenance of eTreg cells. TNFRSF signaling activated the NF-κB transcription factor RelA, which was required to maintain eTreg cells in lymphoid and non-lymphoid tissues, including RORγt + Treg cells in the small intestine. In response to TNFRSF signaling, RelA regulated basic cellular processes, including cell survival and proliferation, but was dispensable for IRF4 expression or DNA binding, indicating that both pathways operated independently. Importantly, mutations in the RelA binding partner NF-κB1 compromised eTreg cells in humans, suggesting that the TNFRSF-NF-κB axis was required in a non-redundant manner to maintain eTreg cells in mice and humans., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

X-linked Inhibitor of Apoptosis (XIAP) deficiency predisposes people to pathogen-associated hyperinflammation. Upon XIAP loss, Toll-like receptor (TLR) ligation triggers RIPK3-caspase-8-mediated IL-1β activation and death in myeloid cells. How XIAP suppresses these events remains unclear. Here, we show that TLR-MyD88 causes the proteasomal degradation of the related IAP, cIAP1, and its adaptor, TRAF2, by inducing TNF and TNF Receptor 2 (TNFR2) signaling. Genetically, we define that myeloid-specific cIAP1 loss promotes TLR-induced RIPK3-caspase-8 and IL-1β activity in the absence of XIAP. Importantly, deletion of TNFR2 in XIAP-deficient cells limited TLR-MyD88-induced cIAP1-TRAF2 degradation, cell death, and IL-1β activation. In contrast to TLR-MyD88, TLR-TRIF-induced interferon (IFN)β inhibited cIAP1 loss and consequent cell death. These data reveal how, upon XIAP deficiency, a TLR-TNF-TNFR2 axis drives cIAP1-TRAF2 degradation to allow TLR or TNFR1 activation of RIPK3-caspase-8 and IL-1β. This mechanism may explain why XIAP-deficient patients can exhibit symptoms reminiscent of patients with activating inflammasome mutations., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exosomes represent an attractive vehicle for the delivery of biomolecules. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here we report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. We demonstrate that labeling of a target protein, Cre recombinase, with a WW tag leads to recognition by the L-domain-containing protein Ndfip1, resulting in ubiquitination and loading into exosomes. Our results show that Ndfip1 expression acts as a molecular switch for exosomal packaging of WW-Cre that can be suppressed using the exosome inhibitor GW4869. When taken up by floxed reporter cells, exosomes containing WW-Cre were capable of inducing DNA recombination, indicating functional delivery of the protein to recipient cells. Engineered exosomes were administered to the brain of transgenic reporter mice using the nasal route to test for intracellular protein delivery in vivo. This resulted in the transport of engineered exosomes predominantly to recipient neurons in a number of brain regions, including the olfactory bulb, cortex, striatum, hippocampus, and cerebellum. The ability to engineer exosomes to deliver biologically active proteins across the blood-brain barrier represents an important step for the development of therapeutics to treat brain diseases., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Henry and Martin (2017) and Hartwig et al. (2017) provide more insights into the non-apoptotic function of the FADD/caspase-8 duo in TRAIL signaling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease, and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice compared to Casp8(-/-)Ripk3(-/-) or Fadd(-/-)Ripk3(-/-) mice, respectively. These results demonstrate that MLKL is an essential effector of aberrant necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. It appears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity, and excess cytokine production that occur when FADD or Caspase-8-mediated apoptosis is abrogated., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Smac-mimetics are compounds that target cellular Inhibitor of APoptosis (cIAP) proteins and induce tumor cell death. In this issue of Cancer Cell, Yang and colleagues (2016) identify cIAPs at the CARD11-MALT1-BCL10 complex in the ABC subtype of diffuse large B cell lymphoma (DLBCL), making this malignancy a prime target for these drugs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Necroptosis is a form of regulated cell death implicated in a range of inflammatory diseases. In this issue of Cell Chemical Biology, Li et al. (2016) describe a novel inhibitor of necroptosis that targets the chaperone protein HSP90., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1) prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF) and lymphotoxin-α (LT-α), and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIP's catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Interleukin-1β (IL-1β) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1β activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1β that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1β by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1β and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling., (Copyright © 2010 Elsevier Inc. All rights reserved.)

TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.

The heterogeneity and instability of human tumors hamper straightforward identification of cancer-causing mutations through genomic approaches alone. Herein we describe a mouse model of liver cancer initiated from progenitor cells harboring defined cancer-predisposing lesions. Genome-wide analyses of tumors in this mouse model and in human hepatocellular carcinomas revealed a recurrent amplification at mouse chromosome 9qA1, the syntenic region of human chromosome 11q22. Gene-expression analyses delineated cIAP1, a known inhibitor of apoptosis, and Yap, a transcription factor, as candidate oncogenes in the amplicon. In the genetic context of their amplification, both cIAP1 and Yap accelerated tumorigenesis and were required to sustain rapid growth of amplicon-containing tumors. Furthermore, cIAP1 and Yap cooperated to promote tumorigenesis. Our results establish a tractable model of liver cancer, identify two oncogenes that cooperate by virtue of their coamplification in the same genomic locus, and suggest an efficient strategy for the annotation of human cancer genes.

It is becoming clear that "apoptotic" caspases can effect cellular processes other than cell death. A recent paper in Cell points to a novel role of the Drosophila caspase inhibitor DIAP1 as a determinant of cell migration.

Mammalian mitochondrial HtrA2/Omi was originally described as an apoptosis inducer, but rather than having extra cells, mice with mutant HtrA2/Omi suffer from a neurodegenerative disease due to progressive mitochondrial damage. This suggests that instead of promoting cell death by antagonizing inhibitor of apoptosis (IAP) proteins, the primary function of HtrA2/Omi is to handle misfolded proteins in the mitochondria.

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.

Bcl-2, one of a family of key regulators of apoptosis, was the first cell-death machinery component to be identified, but how the family functions is still not clear. Mammalian Bax, a pro-apoptotic family member, can cause yeast cells to die, and two recent yeast genetic screens shed light on how Bax might function.