Moerke, C., Jaco, I., Dewitz, C., Müller, T., Jacobsen, A.V., Gautheron, J., Fritsch, J., Schmitz, J., Bräsen, J.H., Günther, C., et al.
(2019). The anticonvulsive Phenhydan® suppresses extrinsic cell death. Cell death & differentiation,
Legrand, A.J., Konstantinou, M., Goode, E.F. & Meier, P.
(2019). The Diversification of Cell Death and Immunity: Memento Mori. Molecular cell,
Annibaldi, A. & Meier, P.
(2018). Checkpoints in TNF-Induced Cell Death: Implications in Inflammation and Cancer. Trends in molecular medicine,
Barry, R., John, S.W., Liccardi, G., Tenev, T., Jaco, I., Chen, C.-., Choi, J., Kasperkiewicz, P., Fernandes-Alnemri, T., Alnemri, E., et al.
(2018). SUMO-mediated regulation of NLRP3 modulates inflammasome activity. Nat commun,
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..
Galluzzi, L., Vitale, I., Aaronson, S.A., Abrams, J.M., Adam, D., Agostinis, P., Alnemri, E.S., Altucci, L., Amelio, I., Andrews, D.W., et al.
(2018). Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell death & differentiation,
Salla, M., Aguayo-Ortiz, R., Danmaliki, G.I., Zare, A., Said, A., Moore, J., Pandya, V., Manaloor, R., Fong, S., Blankstein, A.R., et al.
(2018). Identification and Characterization of Novel Receptor-Interacting Serine/Threonine‐Protein Kinase 2 Inhibitors Using Structural Similarity Analysis. Journal of pharmacology and experimental therapeutics,
Annibaldi, A. & Meier, P.
(2018). Ripk1 and haematopoiesis: a case for LUBAC and Ripk3. Cell death differ,
Jaco, I., Annibaldi, A., Lalaoui, N., Wilson, R., Tenev, T., Laurien, L., Kim, C., Jamal, K., Wicky John, S., Liccardi, G., et al.
(2017). MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death. Mol cell,
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..
Horn, S., Hughes, M.A., Schilling, R., Sticht, C., Tenev, T., Ploesser, M., Meier, P., Sprick, M.R., MacFarlane, M. & Leverkus, M., et al.
(2017). Caspase-10 Negatively Regulates Caspase-8-Mediated Cell Death, Switching the Response to CD95L in Favor of NF-κB Activation and Cell Survival. Cell rep,
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..
Wallberg, F., Tenev, T. & Meier, P.
(2016). Time-Lapse Imaging of Cell Death. Cold spring harbor protocols,
Meier, P. & Banreti, A.
(2016). Tissue Repair: How to Inflame Your Neighbours. Current biology,
Orme, M.H., Liccardi, G., Moderau, N., Feltham, R., Wicky-John, S., Tenev, T., Aram, L., Wilson, R., Bianchi, K., Morris, O., et al.
(2016). The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles. Nature communications,
Plaza-Menacho, I., Barnouin, K., Barry, R., Borg, A., Orme, M., Chauhan, R., Mouilleron, S., Martínez-Torres, R.J., Meier, P. & McDonald, N.Q., et al.
(2016). RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements. Cell reports,
Morris, O., Liu, X., Domingues, C., Runchel, C., Chai, A., Basith, S., Tenev, T., Chen, H., Choi, S., Pennetta, G., et al.
(2016). Signal Integration by the IκB Protein Pickle Shapes Drosophila Innate Host Defense. Cell host microbe,
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. .
Neyen, C., Runchel, C., Schüpfer, F., Meier, P. & Lemaitre, B.
(2016). The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors. Nature immunology,
Meier, P., Morris, O. & Broemer, M.
(2015). Ubiquitin-Mediated Regulation of Cell Death, Inflammation, and Defense of Homeostasis. ,
Silke, J. & Meier, P.
(2013). Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation. Cold spring harbor perspectives in biology,
Borst, G.R., McLaughlin, M., Kyula, J.N., Neijenhuis, S., Khan, A., Good, J., Zaidi, S., Powell, N.G., Meier, P., Collins, I., et al.
(2013). Targeted Radiosensitization by the Chk1 Inhibitor SAR-020106. International journal of radiation oncology biology physics,
(2012). Ubiquitin-mediated regulation of RhoGTPase signalling: IAPs and HACE1 enter the fray. Embo journal,
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..
Meinander, A., Runchel, C., Tenev, T., Chen, L., Kim, C.-., Ribeiro, P.S., Broemer, M., Leulier, F., Zvelebil, M., Silverman, N., et al.
(2012). Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling. Embo j,
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..
Lopez, J., John, S.W., Tenev, T., Rautureau, G.J., Hinds, M.G., Francalanci, F., Wilson, R., Broemer, M., Santoro, M.M., Day, C.L., et al.
(2011). CARD-Mediated Autoinhibition of cIAP1's E3 Ligase Activity Suppresses Cell Proliferation and Migration. Mol cell,
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..
Tenev, T., Bianchi, K., Darding, M., Broemer, M., Langlais, C., Wallberg, F., Zachariou, A., Lopez, J., Macfarlane, M., Cain, K., et al.
(2011). The Ripoptosome, a Signaling Platform that Assembles in Response to Genotoxic Stress and Loss of IAPs. Mol cell,
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..
Lee, T.V., Fan, Y., Wang, S.A., Srivastava, M., Broemer, M., Meier, P. & Bergmann, A.
(2011). Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase DRONC Independent of Protein Degradation. Plos genet,
Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition ("undead'' cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in "undead'' cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs..
Darding, M., Feltham, R., Tenev, T., Bianchi, K., Benetatos, C., Silke, J. & Meier, P.
(2011). Molecular determinants of Smac mimetic induced degradation of cIAP1 and cIAP2. Cell death differ,
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..
Darding, M. & Meier, P.
(2011). IAPs: Guardians of RIPK1. Cell death differ,
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..
Tenev, T., Bianchi, K., Darding, M., Broemer, M., Langlais, C., Wallberg, F., Zachariou, A., Lopez, J., MacFarlane, M., Cain, K., et al.
(2011). The Ripoptosome, a Signaling Platform that Assembles in Response to Genotoxic Stress and Loss of IAPs (vol 43, pg 432, 2011). Mol cell,
Gyrd-Hansen, M. & Meier, P.
(2010). IAPs: from caspase inhibitors to modulators of NF-kappa B, inflammation and cancer. Nat rev cancer,
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..
Broemer, M., Tenev, T., Rigbolt, K.T., Hempel, S., Blagoev, B., Silke, J., Ditzel, M. & Meier, P.
(2010). Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases. Mol cell,
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..
Lopez, J. & Meier, P.
(2010). To fight or die - inhibitor of apoptosis proteins at the crossroad of innate immunity and death. Curr opin cell biol,
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..
Paquette, N., Broemer, M., Aggarwal, K., Chen, L., Husson, M., Erturk-Hasdemir, D., Reichhart, J.M., Meier, P. & Silverman, N.
(2010). Caspase-Mediated Cleavage, IAP Binding, and Ubiquitination: Linking Three Mechanisms Crucial for Drosophila NF-kappa B Signaling. Mol cell,
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..
Broemer, M. & Meier, P.
(2009). Ubiquitin-mediated regulation of apoptosis. Trends cell biol,
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..
Orme, M. & Meier, P.
(2009). Inhibitor of apoptosis proteins in Drosophila: gatekeepers of death. Apoptosis,
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..
Bianchi, K. & Meier, P.
(2009). A tangled web of ubiquitin chains: breaking news in TNF-R1 signaling. Mol cell,
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..
Erturk-Hasdemir, D., Broemer, M., Leulier, F., Lane, W.S., Paquette, N., Hwang, D., Kim, C.H., Stoven, S., Meier, P. & Silverman, N., et al.
(2009). Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes. P natl acad sci usa,
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..
Gyrd-Hansen, M., Darding, M., Miasari, M., Santoro, M.M., Zender, L., Xue, W., Tenev, T., da Fonseca, P.C., Zvelebil, M., Bujnicki, J.M., et al.
(2008). IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-kappaB as well as cell survival and oncogenesis. Nat cell biol,
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..
Lhocine, N., Ribeiro, P.S., Buchon, N., Wepf, A., Wilson, R., Tenev, T., Lemaitre, B., Gstaiger, M., Meier, P. & Leulier, F., et al.
(2008). PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell host microbe,
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..
Bardet, P.L., Kolahgar, G., Mynett, A., Miguel-Aliaga, I., Briscoe, J., Meier, P. & Vincent, J.P.
(2008). A fluorescent reporter of caspase activity for live imaging. P natl acad sci usa,
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..
Ditzel, M., Broemer, M., Tenev, T., Bolduc, C., Lee, T.V., Rigbolt, K.T., Elliott, R., Zvelebil, M., Blagoev, B., Bergmann, A., et al.
(2008). Inactivation of effector caspases through nondegradative polyubiquitylation. Mol cell,
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..
Tenev, T., Ditzel, M., Zachariou, A. & Meier, P.
(2007). The antiapoptotic activity of insect IAPs requires activation by an evolutionarily conserved mechanism. Cell death differ,
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..
Meier, P. & Vousden, K.H.
(2007). Lucifer's labyrinth--ten years of path finding in cell death. Mol cell,
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..
Ribeiro, P.S., Kuranaga, E., Tenev, T., Leulier, F., Miura, M. & Meier, P.
(2007). DIAP2 functions as a mechanism-based regulator of drICE that contributes to the caspase activity threshold in living cells. J cell biol,
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..
Leulier, F., Ribeiro, P.S., Palmer, E., Tenev, T., Takahashi, K., Robertson, D., Zachariou, A., Pichaud, F., Ueda, R. & Meier, P., et al.
(2006). Systematic in vivo RNAi analysis of putative components of the Drosophila cell death machinery. Cell death differ,
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..
Leulier, F., Lhocine, N., Lemaitre, B. & Meier, P.
(2006). The Drosophila inhibitor of apoptosis protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection. Mol cell biol,
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..
Tenev, T., Zachariou, A., Wilson, R., Ditzel, M. & Meier, P.
(2005). IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Nat cell biol,
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..
Ditzel, M. & Meier, P.
(2005). Ubiquitylation in apoptosis: DIAP1's (N-)en(d)igma. Cell death differ,
Ditzel, M., Wilson, R., Tenev, T., Zachariou, A., Paul, A., Deas, E. & Meier, P.
(2003). Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nat cell biol,
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..
Meier, P. & Silke, J.
(2003). Programmed cell death: Superman meets Dr Death. Nat cell biol,
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..
Zachariou, A., Tenev, T., Goyal, L., Agapite, J., Steller, H. & Meier, P.
(2003). IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding. Embo j,
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..
Tenev, T., Zachariou, A., Wilson, R., Paul, A. & Meier, P.
(2002). Jafrac2 is an IAP antagonist that promotes cell death by liberating Dronc from DIAP1. Embo j,
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..
Wilson, R., Goyal, L., Ditzel, M., Zachariou, A., Baker, D.A., Agapite, J., Steller, H. & Meier, P.
(2002). The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat cell biol,
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..
Ditzel, M. & Meier, P.
(2002). IAP degradation: decisive blow or altruistic sacrifice?. Trends cell biol,
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..
Meier, P., Finch, A. & Evan, G.
(2000). Apoptosis in development. Nature,
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..
Meier, P., neyen, C., runchel, C., schupfer, F. & lemaitre, B.
The regulatory isoform rPGRP-LC resolves immune activation through receptor clearance via ESCRT-mediated trafficking. Nature immunology,
Meier, P., kata, H., ditzel, M. & bergmann, A.
An inhibitory mono-ubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways. Plos genetics,
Annibaldi, A., Wicky John, S., Vanden Berghe, T., Swatek, K., Ruan, J., Liccardi, G., Bianchi, K., Komander, D., Choi, S., Coillie, S., et al.
Ubiquitin-mediated regulation of RIPK1 kinase activity, independent of IKK and MK2. Molecular cell,
Feltham, R., Jamal, K., Tenev, T., Liccardi, G., Jaco, I., Monteiro Domingues, C., Morris, O., Wicky John, S., Annibaldi, A., Widya, M., et al.
Mind bomb regulates cell death during TNF signaling by suppressing RIPK1’s cytotoxic potential. Cell reports,
Liccardi, G., Ramos Garcia, L., Tenev, T., Annibaldi, A., Legrand, A., Robertson, D., Darding, M., Bianchi, K. & Meier, P.
RIPK1 and Caspase-8 ensure chromosome stability independently of their role in cell death and inflammation. Molecular cell,