Most metastatic cancers remain incurable due to the emergence of apoptosis-resistant clones, fuelled by intratumour heterogeneity and tumour evolution. To improve treatment, therapies should not only kill cancer cells but also activate the immune system against the tumour to eliminate any residual cancer cells that survive treatment. While current cancer therapies rely heavily on apoptosis - a largely immunologically silent form of cell death - there is growing interest in harnessing immunogenic forms of cell death such as necroptosis. Unlike apoptosis, necroptosis generates second messengers that act on immune cells in the tumour microenvironment, alerting them of danger. This lytic form of cell death optimizes the provision of antigens and adjuvanticity for immune cells, potentially boosting anticancer treatment approaches by combining cellular suicide and immune response approaches. In this Review, we discuss the mechanisms of necroptosis and how it activates antigen-presenting cells, drives cross-priming of CD8+ T cells and induces antitumour immune responses. We also examine the opportunities and potential drawbacks of such strategies for exposing cancer cells to immunological attacks..

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies..

Cell death and inflammation are closely linked arms of the innate immune response to combat infection and tissue malfunction. Recent advancements in our understanding of the intricate signals originating from dying cells have revealed that cell death serves as more than just an end point. It facilitates the exchange of information between the dying cell and cells of the tissue microenvironment, particularly immune cells, alerting and recruiting them to the site of disturbance. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is emerging as a critical stress sentinel that functions as a molecular switch, governing cellular survival, inflammatory responses and immunogenic cell death signalling. Its tight regulation involves multiple layers of post-translational modifications. In this Review, we discuss the molecular mechanisms that regulate RIPK1 to maintain homeostasis and cellular survival in healthy cells, yet drive cell death in a context-dependent manner. We address how RIPK1 mutations or aberrant regulation is associated with inflammatory and autoimmune disorders and cancer. Moreover, we tease apart what is known about catalytic and non-catalytic roles of RIPK1 and discuss the successes and pitfalls of current strategies that aim to target RIPK1 in the clinic..

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs..

Precise vascular patterning is crucial for normal growth and development. The ERG transcription factor drives Delta-like ligand 4 (DLL4)/Notch signalling and is thought to act as a pivotal regulator of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC-specific focal adhesion kinase (FAK)-knockout (KO) and point-mutant FAK-knock-in mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid immunoprecipitation mass spectrometry of endogenous proteins identified that endothelial nuclear-FAK interacts with the deubiquitinase USP9x and the ubiquitin ligase TRIM25. Further in silico analysis confirms that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel-sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x..

Protein-protein interactions (PPIs) represent the main mode of the proteome organization in the cell. In the last decade, several large-scale representations of PPI networks have captured generic aspects of the functional organization of network components but mostly lack the context of cellular states. However, the generation of context-dependent PPI networks is essential for structural and systems-level modeling of biological processes-a goal that remains an unsolved challenge. Here we describe an experimental/computational strategy to achieve a modeling of PPIs that considers contextual information. This strategy defines the composition, stoichiometry, temporal organization, and cellular requirements for the formation of target assemblies. We used this approach to generate an integrated model of the formation principles and architecture of a large signalosome, the TNF-receptor signaling complex (TNF-RSC). Overall, we show that the integration of systems- and structure-level information provides a generic, largely unexplored link between the modular proteome and cellular function..

The changes that drive differentiation facilitate the emergence of abnormal cells that need to be removed before they contribute to further development or the germline. Consequently, in mice in the lead-up to gastrulation, ∼35% of embryonic cells are eliminated. This elimination is caused by hypersensitivity to apoptosis, but how it is regulated is poorly understood. Here, we show that upon exit of naive pluripotency, mouse embryonic stem cells lower their mitochondrial apoptotic threshold, and this increases their sensitivity to cell death. We demonstrate that this enhanced apoptotic response is induced by a decrease in mitochondrial fission due to a reduction in the activity of dynamin-related protein 1 (DRP1). Furthermore, we show that in naive pluripotent cells, DRP1 prevents apoptosis by promoting mitophagy. In contrast, during differentiation, reduced mitophagy levels facilitate apoptosis. Together, these results indicate that during early mammalian development, DRP1 regulation of mitophagy determines the apoptotic response..

Selective autophagy is a catabolic route that turns over specific cellular material for degradation by lysosomes, and whose role in the regulation of innate immunity is largely unexplored. Here, we show that the apical kinase of the Drosophila immune deficiency (IMD) pathway Tak1, as well as its co-activator Tab2, are both selective autophagy substrates that interact with the autophagy protein Atg8a. We also present a role for the Atg8a-interacting protein Sh3px1 in the downregulation of the IMD pathway, by facilitating targeting of the Tak1/Tab2 complex to the autophagy platform through its interaction with Tab2. Our findings show the Tak1/Tab2/Sh3px1 interactions with Atg8a mediate the removal of the Tak1/Tab2 signaling complex by selective autophagy. This in turn prevents constitutive activation of the IMD pathway in Drosophila. This study provides mechanistic insight on the regulation of innate immune responses by selective autophagy..

The receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a key mediator of regulated cell death and inflammation. Recent studies suggest that RIPK1 inhibition would fundamentally improve the therapy of RIPK1-dependent organ damage in stroke, myocardial infarction, kidney failure, and systemic inflammatory response syndrome. Additionally, it could ameliorate or prevent multi-organ failure induced by cytokine release in the context of hyperinflammation, as seen in COVID-19 patients. Therefore, we searched for a RIPK1 inhibitor and present the aromatic antiepileptic and FDA-approved drug primidone (Liskantin®) as a potent inhibitor of RIPK1 activation in vitro and in a murine model of TNFα-induced shock, which mimics the hyperinflammatory state of cytokine release syndrome. Furthermore, we detected for the first time RIPK1 activation in the respiratory tract epithelium of hospitalized patients who tested positive for SARS-CoV-2 infection. Our data provide a strong rationale for evaluating the drug primidone in conditions of hyperinflammation in humans..

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system..

The overexpression of the protein tyrosine kinase, Focal adhesion kinase (FAK), in endothelial cells has implicated its requirement in angiogenesis and tumour growth, but how pericyte FAK regulates tumour angiogenesis is unknown. We show that pericyte FAK regulates tumour growth and angiogenesis in multiple mouse models of melanoma, lung carcinoma and pancreatic B-cell insulinoma and provide evidence that loss of pericyte FAK enhances Gas6-stimulated phosphorylation of the receptor tyrosine kinase, Axl with an upregulation of Cyr61, driving enhanced tumour growth. We further show that pericyte derived Cyr61 instructs tumour cells to elevate expression of the proangiogenic/protumourigenic transmembrane receptor Tissue Factor. Finally, in human melanoma we show that when 50% or more tumour blood vessels are pericyte-FAK negative, melanoma patients are stratified into those with increased tumour size, enhanced blood vessel density and metastasis. Overall our data uncover a previously unknown mechanism of tumour growth by pericytes that is controlled by pericyte FAK..

Cell competition is an emerging principle that eliminates suboptimal or potentially dangerous cells. For 'unfit' cells to be detected, their competitive status needs to be compared to the collective fitness of cells within a tissue. Here we report that the NMDA receptor controls cell competition of epithelial cells and Myc supercompetitors in the Drosophila wing disc. While clonal depletion of the NMDA receptor subunit NR2 results in their rapid elimination via the TNF/Eiger>JNK signalling pathway, local over-expression of NR2 causes NR2 cells to acquire supercompetitor-like behaviour that enables them to overtake the tissue through clonal expansion that causes, but also relies on, the killing of surrounding cells. Consistently, NR2 is utilised by Myc clones to provide them with supercompetitor status. Mechanistically, we find that the JNK>PDK signalling axis in 'loser' cells reprograms their metabolism, driving them to produce and transfer lactate to winners. Preventing lactate transfer from losers to winners abrogates NMDAR-mediated cell competition. Our findings demonstrate a functional repurposing of NMDAR in the surveillance of tissue fitness..

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti-tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre-clinical application of an in vivo treatment protocol for soft-tissue sarcoma that directly engages RIPK1-mediated immunogenic cell death. We find that RIPK1-mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1-induced cell death in patients with advanced disease at limb extremities..

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-β3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-β3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell β3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by β3-integrin, providing a previously unrecognized mechanism of cancer growth control..

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..

Different forms of regulated cell death-like apoptosis and necroptosis contribute to the pathophysiology of clinical conditions including ischemia-reperfusion injury, myocardial infarction, sepsis, and multiple sclerosis. In particular, the kinase activity of the receptor-interacting serine/threonine protein kinase 1 (RIPK1) is crucial for cell fate in inflammation and cell death. However, despite its involvement in pathological conditions, no pharmacologic inhibitor of RIPK1-mediated cell death is currently in clinical use. Herein, we screened a collection of clinical compounds to assess their ability to modulate RIPK1-mediated cell death. Our small-scale screen identified the anti-epilepsy drug Phenhydan® as a potent inhibitor of death receptor-induced necroptosis and apoptosis. Accordingly, Phenhydan® blocked activation of necrosome formation/activation as well as death receptor-induced NF-κB signaling by influencing the membrane function of cells, such as lipid raft formation, thus exerting an inhibitory effect on pathophysiologic cell death processes. By targeting death receptor signaling, the already FDA-approved Phenhydan® may provide new therapeutic strategies for inflammation-driven diseases caused by aberrant cell death..

Why do cells have so many ways to die? Why does "cellular suicide" exist at all? In the war against pathogens and rogue cells, organisms developed cellular suicide as a last resort. Fighting an evolutionary arms race, cell death pathways have adapted and multiplied to cover the complexity of the foes the immune system faces. In this review, we discuss the different types of cell death, the underlying signaling events, and their unequal ability to trigger an immune response. We also comment on how to use our knowledge of cell death signaling to improve the efficacy of cancer treatment. We argue that cell death is integral to the immune response and acts as a beacon, a second messenger, that guides both immune system and tissue micro-environment to ensure tissue repair and homeostasis. Memento mori-"remember you must die"-as failure to do so opens the way to chronic infection and cancer..

Tumor necrosis factor (TNF) is a proinflammatory cytokine that coordinates tissue homeostasis by regulating cytokine production, cell survival, and cell death. However, how life and death decisions are made in response to TNF is poorly understood. Many inflammatory pathologies are now recognized to be driven by aberrant TNF-induced cell death, which, in most circumstances, depends on the kinase Receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Recent advances have identified ubiquitin (Ub)-mediated phosphorylation of RIPK1 as belonging to crucial checkpoints for cell fate in inflammation and infection. A better understanding of these checkpoints might lead to new approaches for the treatment of chronic inflammatory diseases fueled by aberrant RIPK1-induced cell death, and/or reveal novel strategies for anticancer immunotherapies, harnessing the ability of RIPK1 to trigger immunogenic cell death..

The NLRP3 inflammasome responds to infection and tissue damage, and rapidly escalates the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. How the NLRP3 inflammasome is negatively regulated is poorly understood. Here we show that NLRP3 inflammasome activation is suppressed by sumoylation. NLRP3 is sumoylated by the SUMO E3-ligase MAPL, and stimulation-dependent NLRP3 desumoylation by the SUMO-specific proteases SENP6 and SENP7 promotes NLRP3 activation. Defective NLRP3 sumoylation, either by NLRP3 mutation of SUMO acceptor lysines or depletion of MAPL, results in enhanced caspase-1 activation and IL-1β release. Conversely, depletion of SENP7 suppresses NLRP3-dependent ASC oligomerisation, caspase-1 activation and IL-1β release. These data indicate that sumoylation of NLRP3 restrains inflammasome activation, and identify SUMO proteases as potential drug targets for the treatment of inflammatory diseases..

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field..

Receptor-interacting protein kinase 2 (RIP2 or RICK, herein referred to as RIPK2) is linked to the pathogen pathway that activates nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) and autophagic activation. Using molecular modeling (docking) and chemoinformatics analyses, we used the RIPK2/ponatinib crystal structure and searched in chemical databases for small molecules exerting binding interactions similar to those exerted by ponatinib. The identified RIPK2 inhibitors potently inhibited the proliferation of cancer cells by > 70% and also inhibited NFκB activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggests effectiveness to resolve inflammation with low toxicity to the animals. Thus, our identified RIPK2 inhibitor may offer possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cholangitis, and pancreatitis..

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Tumor necrosis factor (TNF) is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2) regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2's E3 activity or RIPK1's ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF..

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..

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..

Apoptosis is an evolutionary conserved cell death mechanism, which requires activation of initiator and effector caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian Caspase-2 and Caspase-9, has an N-terminal CARD domain that recruits Dronc into the apoptosome for activation. In addition to its role in apoptosis, Dronc also has non-apoptotic functions such as compensatory proliferation. One mechanism to control the activation of Dronc is ubiquitylation. However, the mechanistic details of ubiquitylation of Dronc are less clear. For example, monomeric inactive Dronc is subject to non-degradative ubiquitylation in living cells, while ubiquitylation of active apoptosome-bound Dronc triggers its proteolytic degradation in apoptotic cells. Here, we examined the role of non-degradative ubiquitylation of Dronc in living cells in vivo, i.e. in the context of a multi-cellular organism. Our in vivo data suggest that in living cells Dronc is mono-ubiquitylated on Lys78 (K78) in its CARD domain. This ubiquitylation prevents activation of Dronc in the apoptosome and protects cells from apoptosis. Furthermore, K78 ubiquitylation plays an inhibitory role for non-apoptotic functions of Dronc. We provide evidence that not all of the non-apoptotic functions of Dronc require its catalytic activity. In conclusion, we demonstrate a mechanism whereby Dronc's apoptotic and non-apoptotic activities can be kept silenced in a non-degradative manner through a single ubiquitylation event in living cells..

Formation of the death-inducing signaling complex (DISC) initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival..

The best approach to distinguish between necrosis and apoptosis is time-lapse video microscopy. This technique enables a biological process to be photographed at regular intervals over a period, which may last from a few hours to several days, and can be applied to cells in culture or in vivo. We have established two time-lapse microscopy methods based on different ways of calculating cell death: semiautomated and automated. In the semiautomated approach, cell death can be visualized by staining with combinations of Alexa Fluor 647-conjugated Annexin V and Sytox Green (SG), or Annexin V(FITC) and Propidium iodide (PI). The automated method is similar except that all cells are labeled with dyes. This allows faster quantification of data. To this end Cell Tracker Green is used to label all cells at time zero in combination with PI and Alexa Fluor 647-conjugated Annexin V. Necrotic cell death is accompanied by either simultaneous labeling with Annexin V and PI or SG (double-positive), or direct PI or SG staining. Additionally, necrotic cells display characteristic morphology, such as cytoplasmic swelling. In contrast to necrosis where membrane permeabilization is an early event, cells that die by apoptosis lose their membrane permeability relatively late. Therefore, the time between Annexin V staining and PI or SG uptake (double-positive) can be used to distinguish necrosis from apoptosis. This protocol describes the analysis of cell death by time-lapse imaging of HT1080 and L929 cells stained with these dyes, but it can be readily adapted to other cell types of interest..

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New work in Drosophila has identified a link between dying cells and compensatory proliferation of neighbouring survivor cells. Activation of initiator caspases in the dying cells stimulates the production of hydrogen peroxide, which orchestrates tissue repair via macrophages and TNF signalling..

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events..

Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. We describe Pickle, a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-κB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships..

Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM) segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery..

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Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death..

Cell death and inflammation are ancient processes of fundamental biological importance in both normal physiology and human disease pathologies. The recent observation that apoptosis regulatory components have dual roles in cell death and inflammation suggests that these proteins function, not primarily to kill, but to coordinate tissue repair and remodeling. This perspective unifies cell death components as positive regulators of tissue repair that replaces malfunctioning or damaged tissues and enhances the resilience of epithelia to insult. It is now recognized that cells that die by apoptosis do not do so silently, but release a variety of paracrine signals to communicate with their cellular environment to ensure tissue regeneration, and wound healing. Moreover, inflammatory signaling pathways, such as those emanating from the TNF receptor or Toll-related receptors, take part in cell competition to eliminate developmentally aberrant clones. Ubiquitylation has emerged as crucial mediator of signal transduction in cell death and inflammation. Here, we focus on recent advances on ubiquitin-mediated regulation of cell death and inflammation, and how this is used to regulate the defense of homeostasis..

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The EMBO Journal, 14–28 (2012); published online November 25 2011 Activation of members of the Rho-like family of guanosine triphosphatases GTPases (RhoGTPases) controls diverse physiological processes and is frequently found in cancer, contributing to tumour malignancy, cancer cell migration, invasion and metastasis. While the regulation of nucleotide binding to RhoGTPases is well understood, little is currently known regarding the molecular mechanisms through which RhoGTPase signalling is regulated by ubiquitylation. Two reports in this issue of The EMBO Journal and Developmental Cell now identify inhibitor of apoptosis (IAP) proteins and HACE1 as E3 ubiquitin (Ub)-protein ligases for Rac1 regulating Rac1 levels and activity..

Caspases have been extensively studied as critical initiators and executioners of cell death pathways. However, caspases also take part in non-apoptotic signalling events such as the regulation of innate immunity and activation of nuclear factor-κB (NF-κB). How caspases are activated under these conditions and process a selective set of substrates to allow NF-κB signalling without killing the cell remains largely unknown. Here, we show that stimulation of the Drosophila pattern recognition protein PGRP-LCx induces DIAP2-dependent polyubiquitylation of the initiator caspase DREDD. Signal-dependent ubiquitylation of DREDD is required for full processing of IMD, NF-κB/Relish and expression of antimicrobial peptide genes in response to infection with Gram-negative bacteria. Our results identify a mechanism that positively controls NF-κB signalling via ubiquitin-mediated activation of DREDD. The direct involvement of ubiquitylation in caspase activation represents a novel mechanism for non-apoptotic caspase-mediated signalling..

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E3 ligases mediate the covalent attachment of ubiquitin to target proteins thereby enabling ubiquitin-dependent signaling. Unraveling how E3 ligases are regulated is important because miscontrolled ubiquitylation can lead to disease. Cellular inhibitor of apoptosis (cIAP) proteins are E3 ligases that modulate diverse biological processes such as cell survival, proliferation, and migration. Here, we have solved the structure of the caspase recruitment domain (CARD) of cIAP1 and identified that it is required for cIAP1 autoregulation. We demonstrate that the CARD inhibits activation of cIAP1's E3 activity by preventing RING dimerization, E2 binding, and E2 activation. Moreover, we show that the CARD is required to suppress cell proliferation and migration. Further, CARD-mediated autoregulation is also necessary to maximally suppress caspase-8-dependent apoptosis and vascular tree degeneration in vivo. Taken together, our data reveal mechanisms by which the E3 ligase activity of cIAP1 is controlled, and how its deregulation impacts on cell proliferation, migration and cell survival..

A better understanding of the mechanisms through which anticancer drugs exert their effects is essential to improve combination therapies. While studying how genotoxic stress kills cancer cells, we discovered a large ∼2MDa cell death-inducing platform, referred to as "Ripoptosome." It contains the core components RIP1, FADD, and caspase-8, and assembles in response to genotoxic stress-induced depletion of XIAP, cIAP1 and cIAP2. Importantly, it forms independently of TNF, CD95L/FASL, TRAIL, death-receptors, and mitochondrial pathways. It also forms upon Smac-mimetic (SM) treatment without involvement of autocrine TNF. Ripoptosome assembly requires RIP1's kinase activity and can stimulate caspase-8-mediated apoptosis as well as caspase-independent necrosis. It is negatively regulated by FLIP, cIAP1, cIAP2, and XIAP. Mechanistically, IAPs target components of this complex for ubiquitylation and inactivation. Moreover, we find that etoposide-stimulated Ripoptosome formation converts proinflammatory cytokines into prodeath signals. Together, our observations shed new light on fundamental mechanisms by which chemotherapeutics may kill cancer cells..

The inhibitors of apoptosis (IAP) proteins cIAP1 and cIAP2 have recently emerged as key ubiquitin-E3 ligases regulating innate immunity and cell survival. Much of our knowledge of these IAPs stems from studies using pharmacological inhibitors of IAPs, dubbed Smac mimetics (SMs). Although SMs stimulate auto-ubiquitylation and degradation of cIAPs, little is known about the molecular determinants through which SMs activate the E3 activities of cIAPs. In this study, we find that SM-induced rapid degradation of cIAPs requires binding to tumour necrosis factor (TNF) receptor-associated factor 2 (TRAF2). Moreover, our data reveal an unexpected difference between cIAP1 and cIAP2. Although SM-induced degradation of cIAP1 does not require cIAP2, degradation of cIAP2 critically depends on the presence of cIAP1. In addition, degradation of cIAP2 also requires the ability of the cIAP2 RING finger to dimerise and to bind to E2s. This has important implications because SM-mediated degradation of cIAP1 causes non-canonical activation of NF-κB, which results in the induction of cIAP2 gene expression. In the absence of cIAP1, de novo synthesised cIAP2 is resistant to the SM and suppresses TNFα killing. Furthermore, the cIAP2-MALT1 oncogene, which lacks cIAP2's RING, is resistant to SM treatment. The identification of mechanisms through which cancer cells resist SM treatment will help to improve combination therapies aimed at enhancing treatment response.Cell Death and Differentiation advance online publication, 18 February 2011; doi:10.1038/cdd.2011.10..

Deregulation of innate immune signalling and cell death form the basis of most human disease pathogenesis. Inhibitor of APoptosis (IAP) protein-family members are frequently overexpressed in cancer and contribute to tumour cell survival, chemo-resistance, disease progression and poor prognosis. Although best known for their ability to regulate caspases, IAPs also influence ubiquitin-dependent pathways that modulate innate immune signalling by activation of NF-κB. Recent advances in our understanding of the molecular mechanisms through which IAPs influence cell death and innate immune responses have provided new insights into novel strategies for treatment of cancer. In this review we discuss our current understanding of IAP-mediated NF-κB signalling, as well as elaborate on unexpected insights into the involvement of IAPs in regulating the 'Ripoptosome', a novel intrinsic cell death-inducing platform. We propose an evolutionarily conserved concept whereby IAPs function as guardians of killer platforms such as the apoptosome in Drosophila and the Ripoptosome in mammals.Cell Death and Differentiation advance online publication, 18 November 2011; doi:10.1038/cdd.2011.163..

The realization that alterations in inhibitor of apoptosis (IAP) proteins are found in many types of human cancer and are associated with chemoresistance, disease progression and poor prognosis, has sparked a worldwide frenzy in the development of small pharmacological inhibitors of IAPs. The development of such inhibitors has radically changed our knowledge of the signalling processes that are regulated by IAPs. Recent studies indicate that IAPs not only regulate caspases and apoptosis, but also modulate inflammatory signalling and immunity, mitogenic kinase signalling, proliferation and mitosis, as well as cell invasion and metastasis..

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..

The processes of dying are as tightly regulated as those of growth and proliferation, and together they establish a finely tuned balance that ensures proper organ size and function. Failure in the regulation of these responses lies at the heart of many human diseases. Certain members of the inhibitor of apoptosis (IAP) protein family function as important gatekeepers of cell death and survival. While IAPs can regulate cell death by controlling caspases, they also modulate other signalling processes that impact on cell viability. Probably the most important contribution of IAPs to cell survival and tumorigenesis resides in the ability of a number of IAPs to act as ubiquitin-E3 ligases regulating NF-κB signalling. Here, we discuss the latest insights into the ubiquitin-related roles of IAPs and how this contributes to the survival of cells and the organism..

Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides through two NF-kappa B signaling pathways-IMD and Toll. The IMD pathway is triggered by DAP-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan receptors is thought to involve K63 ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. We now show that PGN stimulation causes caspase-mediated cleavage of the imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N terminus. A functional IBM is required for the association of cleaved IMID with the ubiquitin E3-ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63 ubiquitination in immune-induced NF-kappa B signaling..

Ubiquitin is a protein modifier that is conjugated to target proteins either as a single moiety or as polyubiquitin chains. Over the past several years, an increasing number of ubiquitin ligases and ubiquitin-deconjugating enzymes have been identified; these modulate cell survival by degradative and non-degradative means. Mutations that affect ubiquitin-mediated signalling are tightly linked to various human pathologies including tumorigenesis. Unravelling how the ubiquitin-signal is conjugated, edited and 'read' is crucial to understanding cellular processes such as endocytic trafficking, NF-kappaB signalling, gene expression, DNA repair and apoptosis. In this review, we summarize recent advances that start to elucidate how the ubiquitin message is used as a versatile tool to regulate apoptosis, for example in the conjugation of ubiquitin to caspases. This results in steric interference with substrate entry and allosteric conformational impairment of the catalytic pocket of the caspase..

Regulation of apoptosis is crucial to ensure cellular viability, and failure to do so is linked to several human pathologies. The apoptotic cell death programme culminates in the activation of caspases, a family of highly specific cysteine proteases essential for the destruction of the cell. Although best known for their role in executing apoptosis, caspases also play important signalling roles in non-apoptotic processes, such as regulation of actin dynamics, innate immunity, cell proliferation, differentiation and survival. Under such conditions, caspases are activated without killing the cell. Caspase activation and activity is subject to complex regulation, and various cellular and viral inhibitors have been identified that control the activity of caspases in their apoptotic and non-apoptotic roles. Members of the Inhibitor of APoptosis (IAP) protein family ensure cell viability in Drosophila by directly binding to caspases and regulating their activities in a ubiquitin-dependent manner. The observation that IAPs are essential for cell survival in Drosophila, and are frequently deregulated in human cancer, contributing to tumourigenesis, chemoresistance, disease progression and poor patient survival, highlights the importance of this family of caspase regulators in health and disease. Here we summarise recent advances from Drosophila that start to elucidate how the cellular response to caspase activation is modulated by IAPs and their regulators..

A flurry of recent revelations is challenging the current dogma on how ubiquitin-dependent processes culminate in the activation of NF-kappaB by TNF. Here, we integrate these findings into a model for TNF-R1 signaling-and underscore the importance of individual components, including linear ubiquitin chains-which allows for the remarkable versatility of the ubiquitin system..

The Drosophila NF-kappa B transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after Gram-negative bacterial infection. Relish is a bipartite NF-kappa B precursor protein, with an N-terminal Rel homology domain and a C-terminal I kappa B-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappa B module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila I kappa B kinase (IKK) and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. In this report, we show that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish..

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis..

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of lmd signaling (PIMS), is required to suppress the lmd innate immune signaling pathway in response to commensal bacteria. pims expression is lmd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of lmd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced lmd response following exposure to bacterial infections..

There is a growing interest in the mechanisms that control the apoptosis cascade during development and adult life. To investigate the regulatory events that trigger apoptosis in whole tissues, we have devised a genetically encoded caspase sensor that can be detected in live and fixed tissue by standard confocal microscopy. The sensor comprises two fluorophores, mRFP, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP), that are linked by an efficient and specific: caspase-sensitive site. Upon caspase activation, the sensor is cleaved and eGFP translocates to the nucleus, leaving mRFP at membranes. This is detected before other markers of apoptosis, including anti-cleaved caspase 3 immunoreactivity. Moreover, the sensor does not perturb normal developmental apoptosis and is specific, as cleavage does not occur in Drosophila embryos that are unable to activate the apoptotic cascade. Importantly, dying cells can be recognized in live embryos, thus opening the way for in vivo imaging. As expected from the high conservation of caspases, it is also cleaved in dying cells of chick embryos. it is therefore likely to be generally useful to track the spatiotemporal pattern of caspase activity in a variety of species..

Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 "activation" and nondegradative polyubiquitylation..

Apoptosis represents a fundamental biological process that relies on the activation of caspases. Inhibitor of apoptosis (IAP) proteins represent a group of negative regulators of both caspases and cell death. The current model dictates that IAPs suppress apoptosis by blocking the catalytic pocket of effector caspases thereby preventing substrate entry. Here, we provide evolutionary evidence for the functional interplay between insect IAPs and the N-end rule-associated ubiquitylation machinery in neutralising effector caspases and cell death. We find that IAPs require 'priming' in order to function as antiapoptotic molecules. Consistently, we demonstrate that the antiapoptotic activity of diverse insect IAPs is activated by effector caspases, providing the cell with a sensitive strategy to monitor and neutralise active caspases. Almost 300 million years of evolutionary selection pressure has preserved a caspase cleavage site in insect IAPs that, following processing by a caspase, exposes a binding motif for the N-end-rule-associated degradation machinery. Recruitment of this ubiquitylation machinery into the 'cleaved-IAP: caspase' complex provides a mechanism to negatively regulate effector caspases and block apoptosis. Furthermore, comparisons between cellular and several viral IAPs suggest differences in their modes of action, as OpIAP3, CpGV-IAP3 and HcNPV-IAP3 fail to associate with several effector caspases. Evolutionary conservation of the N-end-rule degradation pathway in IAP-mediated regulation of apoptosis further corroborates the physiological relevance of this ubiquitylation-associated process..

Cell death, as a foil to cell expansion, has become one of the most intensively studied research areas of modern biology. Almost every aspect of life is intimately enmeshed with the proper regulation of cell death, which either contributes to or forms the basis of most human disease pathogenesis. Now, with our ever-expanding knowledge of the various death pathways, comes the hope that we might harness cell death--by either inhibition or promotion--to translate the concepts into cure..

In addition to their well-known function in apoptosis, caspases are also important in several nonapoptotic processes. How caspase activity is restrained and shut down under such nonapoptotic conditions remains unknown. Here, we show that Drosophila melanogaster inhibitor of apoptosis protein 2 (DIAP2) controls the level of caspase activity in living cells. Animals that lack DIAP2 have higher levels of drICE activity. Although diap2-deficient cells remain viable, they are sensitized to apoptosis following treatment with sublethal doses of x-ray irradiation. We find that DIAP2 regulates the effector caspase drICE through a mechanism that resembles the one of the caspase inhibitor p35. As for p35, cleavage of DIAP2 is required for caspase inhibition. Our data suggest that DIAP2 forms a covalent adduct with the catalytic machinery of drICE. In addition, DIAP2 also requires a functional RING finger domain to block cell death and target drICE for ubiquitylation. Because DIAP2 efficiently interacts with drICE, our data suggest that DIAP2 controls drICE in its apoptotic and nonapoptotic roles..

Despite the identification of numerous key players of the cell death machinery, little is known about their physiological role. Using RNA interference (RNAi) in vivo, we have studied the requirement of all Drosophila caspases and caspase-adaptors in different paradigms of apoptosis. Of the seven caspases, Dronc, drICE, Strica and Decay are rate limiting for apoptosis. Surprisingly, Hid-mediated apoptosis requires a broader range of caspases than apoptosis initiated by loss of the caspase inhibitor DIAP1, suggesting that Hid causes apoptosis not only by antagonizing DIAP1 but also by activating DIAP1-independent caspase cascades. While Hid killing requires Strica, Decay, Dronc/Dark and drICE, apoptosis triggered by DIAP1 depletion merely relied upon Dronc/Dark and drICE. Furthermore, we found that overexpression of DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 regulates caspases directly. Consistently, we show that DIAP2 binds active drICE. Since DIAP2 associates with Hid, we propose a model whereby Hid co-ordinately targets both DIAP1 and DIAP2 to unleash drICE..

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes..

Some members of the inhibitor of apoptosis (IAP) family suppress apoptosis by neutralizing caspases. The current model suggests that all caspase-regulatory IAPs function as direct enzyme inhibitors, blocking effector caspases by binding to their catalytically active pockets. Here we show that IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Whereas XIAP binds directly to the active-site pockets of effector caspases, we find that regulation of effector caspases by Drosophila IAP1 (DIAP1) requires an evolutionarily conserved IAP-binding motif (IBM) at the neo-amino terminus of the large caspase subunit. Remarkably, unlike XIAP, DIAP1-sequestered effector caspases remain catalytically active, suggesting that DIAP1 does not function as a bona fide enzyme inhibitor. Moreover, we demonstrate that the mammalian IAP c-IAP1 interacts with caspase-7 in an exclusively IBM-dependent, but active site pocket-independent, manner that is mechanistically similar to DIAP1. The importance of IBM-mediated regulation of effector-caspases in vivo is substantiated by the enhanced apoptotic potency of IBM-mutant versions of drICE, DCP-1 and caspase-7..

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Some members of the inhibitor of apoptosis (IAP) protein family block apoptosis by binding to and neutralizing active caspases. We recently demonstrated that a physical association between IAP and caspases alone is insufficient to regulate caspases in vivo and that an additional level of control is provided by IAP-mediated ubiquitination of both itself and the associated caspases. Here we show that Drosophila IAP 1 (DIAP1) is degraded by the 'N-end rule' pathway and that this process is indispensable for regulating apoptosis. Caspase-mediated cleavage of DIAP1 at position 20 converts the more stable pro-N-degron of DIAP1 into the highly unstable, Asn-bearing, DIAP1 N-degron of the N-end rule degradation pathway. Thus, DIAP1 represents the first known metazoan substrate of the N-end rule pathway that is targeted for degradation through its amino-terminal Asn residue. We demonstrate that the N-end rule pathway is required for regulation of apoptosis induced by Reaper and Hid expression in the Drosophila melanogaster eye. Our data suggest that DIAP1 instability, mediated through caspase activity and subsequent exposure of the N-end rule pathway, is essential for suppression of apoptosis. We suggest that DIAP1 safeguards cell viability through the coordinated mutual destruction of itself and associated active caspases..

This year's Cold Spring Harbor meeting on programmed cell death (September 17-21, 2003), organised by Craig Thompson and Junying Yuan, was proof that the 'golden age' of research in this field is far from over. There was a flurry of fascinating insights into the regulation of diverse apoptotic pathways and unexpected non-apoptotic roles for some of the key apoptotic regulators and effectors. In addition to their role in cell death, components of the apoptotic molecular machinery are now known to also function in a variety of essential cellular processes, such as regulating glucose homeostasis, lipid metabolism, cell proliferation and differentiation..

The Drosophila inhibitor of apoptosis protein DIAP1 ensures cell viability by directly inhibiting caspases. In cells destined to die this IAP-mediated inhibition of caspases is overcome by IAP-antagonists. Genetic evidence indicates that IAP-antagonists are non-equivalent and function synergistically to promote apoptosis. Here we provide biochemical evidence for the non-equivalent mode of action of Reaper, Grim, Hid and Jafrac2. We find that these IAP-antagonists display differential and selective binding to specific DIAP1 BIR domains. Consistently, we show that each DIAP1 BIR region associates with distinct caspases. The differential DIAP1 BIR interaction seen both between initiator and effector caspases and within IAP-antagonist family members suggests that different IAP-antagonists inhibit distinct caspases from interacting with DIAP1. Surprisingly, we also find that the caspase-binding residues of XIAP predicted to be strictly conserved in caspase-binding IAPs, are absent in DIAP1. In contrast to XIAP, residues C-terminal to the DIAP1 BIR1 domain are indispensable for caspase association. Our studies on DIAP1 and caspases expose significant differences between DIAP1 and XIAP suggesting that DIAP1 and XIAP inhibit caspases in different ways..

Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival in Drosophila and appear to neutralize the cell death machinery by binding to and ubiquitylating pro-apoptotic caspases. Cell death is triggered when "Reaper-like" proteins bind to IAPs and liberate caspases from IAPs. We have identified the thioredoxin peroxidase Jafrac2 as an IAP-interacting protein in Drosophila cells that harbours a conserved N-terminal IAP-binding motif. In healthy cells, Jafrac2 resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Mature Jafrac2 interacts genetically and biochemically with DIAP1 and promotes cell death in tissue culture cells and the Drosophila developing eye. In common with Rpr, Jafrac2-mediated cell death is contingent on DIAP1 binding because mutations that abolish the Jafrac2-DIAP1 interaction suppress the eye phenotype caused by Jafrac2 expression. We show that Jafrac2 displaces Dronc from DIAP1 by competing with Dronc for the binding of DIAP1, consistent with the idea that Jafrac2 triggers cell death by liberating Dronc from DIAP1-mediated inhibition..

Members of the Inhibitor of Apoptosis Protein (IAP) family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper (Rpr), Grim and Hid (head involution defective) all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. Here, we show that in vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, we show that the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. Our data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination..

Some members of the inhibitors of apoptosis protein (IAPs) family block apoptosis by binding and neutralizing pro-apoptotic caspases. A glut of recent papers, predominantly utilizing the power of Drosophila genetics, has attributed a novel mode of action as to how this IAP-mediated apoptosis 'roadblock' is overcome in cells fated to die. Ubiquitin-mediated degradation and general inhibition of protein translation are now thought to downregulate IAP levels, resulting in unrestrained death-inducing caspase activity. Although IAP degradation might well be a key event in the initiation of apoptosis, we propose an alternative interpretation of the recent results on IAP degradation. We argue that the instability of IAPs might actually shed light on their normal, anti-apoptotic mode of action. In healthy cells, IAPs could safeguard cell viability through the coordinated mutual destruction of themselves and associated IAP antagonists..

Essential to the construction, maintenance and repair of tissues is the ability to induce suicide of supernumerary, misplaced or damaged cells with high specificity and efficiency. Study of three principal organisms - the nematode, fruitfly and mouse - indicate that cell suicide is implemented through the activation of an evolutionarily conserved molecular programme intrinsic to all metazoan cells. Dysfunctions in the regulation or execution of cell suicide are implicated in a wide range of developmental abnormalities and diseases..