Systematic in vivo RNAi analysis of putative components of the Drosophila cell death machinery

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.     

Diverse domains of THREAD/DIAP1 are required to inhibit apoptosis induced by REAPER and HID in Drosophila

Significant amounts of apoptosis take place during Drosophila development. The proapoptotic genes reaper (rpr), grim, and head involution defective (hid) are required for virtually all embryonic apoptosis. The proteins encoded by these genes share a short region of homology at their amino termini. The Drosophila IAP homolog THREAD/DIAP1 (TH/DIAP1), encoded by the thread (th) gene, negatively regulates apoptosis during development. It has been proposed that RPR, GRIM, and HID induce apoptosis by binding and inactivating TH/DIAP1. The region of homology between the three proapoptotic proteins has been proposed to bind to the conserved BIR2 domain of TH/DIAP1. Here, we present an analysis of loss-of-function and gain-of-function alleles of th, which indicates that additional domains of TH/DIAP1 are necessary for its ability to inhibit death induced by RPR, GRIM, and HID. In addition, that analysis of loss-of-function mutations demonstrates that th is necessary to block apoptosis very early in embryonic development. This may reflect a requirement to block maternally provided RPR and HID, or it may indicate another function of the TH/DIAP1 protein.     

DIAP2 functions as a mechanism-based regulator of drICE that contributes to the caspase activity threshold in living cells

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.     

Dissection of DIAP1 functional domains via a mutant replacement strategy

Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of active caspases. Drosophila IAP1 (DIAP1) activity is required to keep cells from undergoing apoptosis. The central cell death regulators Reaper and Hid induce apoptosis very rapidly by inhibiting DIAP1 function. We have developed a system for replacing endogenous DIAP1 with mutant forms of the protein, allowing us to examine the roles of various domains of the protein in living and dying cells. We found that DIAP1 is cleaved by a caspase early after the initiation of apoptosis. This cleavage is required for DIAP1 degradation, but Rpr and Hid can still initiate apoptosis in the absence of cleavage. The cleavage of DIAP1 promotes DIAP1 degradation in a manner dependent on the function of the ubiquitin ligase function of the DIAP1 ring domain. This ring domain function is required for Hid-induced apoptosis. We propose a model that synthesizes our data with those of other laboratories and provide a consistent model for DIAP1 function in living and dying cells.     

Reaper-mediated inhibition of DIAP1-induced DTRAF1 degradation results in activation of JNK in Drosophila

Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death is largely unknown. Here we show through a genetic screen that a mutant of Drosophila melanogaster tumour-necrosis factor receptor-associated factor 1 (DTRAF1) is a dominant suppressor of Reaper-induced cell death. We show that Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates DTRAF1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of DTRAF1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of DTRAF1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of DTRAF1-mediated JNK activation in Drosophila cells. Taken together, our results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin#150;proteasome pathway.     

The inflammasome: first line of the immune response to cell stress

The NALP3-inflammasome is a protein complex that stimulates caspase-1 activation to promote the processing and secretion of proinflammatory cytokines. Recent work indicates that the NALP3-inflammasome can be activated by endogenous "danger signals" as well as compounds associated with pathogens (Kanneganti et al., 2006; Mariathasan et al., 2006, Martinon et al., 2006; Sutterwala et al., 2006). Here, we discuss new insights into the regulation of caspase-1 activity in the inflammatory response.     

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. Here we report the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc.     

Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1

Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Here, we report that Drosophila melanogaster IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. We show that Rpr, but not Hid (head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, we found ubcD1, which suppresses rpr-induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro. Our results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting its degradation.     

RalB signaling: a bridge between inflammation and cancer

A connection between the genetic events that lead to tumor formation and the signaling pathways of the innate immune response has been established. In this issue, Chien et al. (2006) show that the RalB GTPase regulates the IKK family member TBK1, providing an unexpected link between the signaling pathways that promote inflammation and cancer. In tumor cells the RalB/TBK1 pathway inhibits apoptosis and in nontumorigenic cells it stimulates an innate immune response.     

Hid,Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Here we show that Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis.     

Molecular mechanisms of DrICE inhibition by DIAP1 and removal of inhibition by Reaper, Hid and Grim

The Drosophila melanogaster inhibitor of apoptosis protein DIAP1 suppresses apoptosis in part through inhibition of the effector caspase DrICE. The pro-death proteins Reaper, Hid and Grim (RHG) induce apoptosis by antagonizing DIAP1 function. However, the underlying molecular mechanisms remain unknown. Here we demonstrate that DIAP1 directly inhibits the catalytic activity of DrICE through its BIR1 domain and this inhibition is countered effectively by the RHG proteins. Inhibition of DrICE by DIAP1 occurs only after the cleavage of its N-terminal 20 amino acids and involves a conserved surface groove on BIR1. Crystal structures of BIR1 bound to the RHG peptides show that the RHG proteins use their N-terminal IAP-binding motifs to bind to the same surface groove, hence relieving DIAP1-mediated inhibition of DrICE. These studies define novel molecular mechanisms for the inhibition and activation of a representative D. melanogaster effector caspase.     

How Do I Fatten Thee? Let Me Count the Ways…

Glycerol-3-phosphate acyltransferase (GPAT) initiates the synthesis of triacylglycerol and phospholipids and, in the process, regulates the formation of several lipid metabolites known to be intracellular signaling molecules. The recent identification of a new GPAT isoform (Cao et al., 2006) suggests a role for GPAT isoforms in nutrient-mediated signaling.     

Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis

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.     

Drosophila Morgue is an F box/ubiquitin conjugase domain protein important for grim-reaper mediated apoptosis

In Drosophila melanogaster, apoptosis is controlled by the integrated actions of the Grim-Reaper (Grim-Rpr) and Drosophila Inhibitor of Apoptosis (DIAP) proteins (reviewed in refs 1 4). The anti-apoptotic DIAPs bind to caspases and inhibit their proteolytic activities. DIAPs also bind to Grim-Rpr proteins, an interaction that promotes caspase activity and the initiation of apoptosis. Using a genetic modifier screen, we identified four enhancers of grim-reaper-induced apoptosis that all regulate ubiquitination processes: uba-1, skpA, fat facets (faf), and morgue. Strikingly, morgue encodes a unique protein that contains both an F box and a ubiquitin E2 conjugase domain that lacks the active site Cys required for ubiquitin linkage. A reduction of morgue activity suppressed grim-reaper-induced cell death in Drosophila. In cultured cells, Morgue induced apoptosis that was suppressed by DIAP1. Targeted morgue expression downregulated DIAP1 levels in Drosophila tissue, and Morgue and Rpr together downregulated DIAP1 levels in cultured cells. Consistent with potential substrate binding functions in an SCF ubiquitin E3 ligase complex, Morgue exhibited F box-dependent association with SkpA and F box-independent association with DIAP1. Morgue may thus have a key function in apoptosis by targeting DIAP1 for ubiquitination and turnover.     

If the prophet does not come to the mountain: dynamics of signaling complexes in NF-kappaB activation

Recruitment of the NF-kappaB-activating IKK signaling complex to the TNF receptor is shown to be driven by induced binding of NEMO, a regulatory component of this complex, to K63-linked polyubiquitin chains attached to RIP1, a receptor-associated adaptor protein (Ea et al., 2006 [in a recent issue of Molecular Cell]; Li et al., 2006; Wu et al., 2006a).     

The Drosophila inhibitor of apoptosis (IAP) DIAP2 is dispensable for cell survival, required for the innate immune response to gram-negative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of IAP-binding apoptosis inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.     

Acetylation of Werner protein at K1127 and K1117 is important for nuclear trafficking and DNA repair

Werner syndrome is a rare autosomal recessive disorder where Werner (WRN) gene is mutated. Being a nucleolar protein, during DNA damage, WRN translocates at the damage site where its catalytic function is required in DNA repair. Several studies have indicated that WRN acetylation may modulate WRN trafficking and catalytic function (Blander et al., 2002; Lozada et al., 2014). Among the six acetylation sites in WRN protein identified by mass-spectrometry analysis (Li et al., 2010) we here explore the role of acetylation sites in C-terminal of WRN (K1127, K1117, K1389, K1413) because the C- terminal domain is the hub for protein- protein interaction and DNA binding activity (Brosh et al. [4]; Muftuoglu et al., 2008; Huang et al., 2006). To explore their functional activity, we created mutations in these sites by changing the acetylation residue lysine (K) to a non-acetylation residue arginine (R) and expressed them in WRN mutant cell lines. We observed that K1127R and K1117R mutants are sensitive to the DNA damaging agents etoposide and mitomycin C and display deficient DNA repair. Importantly, deacetylation of WRN by SIRT1 (Mammalian Sir2) is necessary for restoration of WRN localization at nucleoli after completion of DNA repair. Among all putative acetylation sites, K1127R, K1117R and the double mutant K1127R/K1117R showed significantly delayed re-entry to the nucleolus after damage recovery, even when SIRT1 is overexpressed. These mutants showed partial interaction with SIRT1 compared to WT WRN. Thus, our results suggest that K1127 and K1117 are the major sites of acetylation, necessary for DNA repair. These results elucidate the mechanism by which SIRT1 regulates WRN trafficking via these acetylation sites during DNA damage.     

Unification of the genera Nonlabens, Persicivirga, Sandarakinotalea and Stenothermobacter into a single emended genus, Nonlabens, and description of Nonlabens agnitus sp. nov

Phylogenetic analyses based on 16S rRNA gene sequences showed that a bacterial isolate, designated JC2678(T), represents a distinct phyletic line within the suprageneric monophyletic clade containing the genera Nonlabens, Persicivirga, Stenothermobacter and Sandarakinotalea. The polyphasic data presented in this study demonstrated that the members belonging to the Nonlabens-like clade overall constitute a single genus. Therefore, it is proposed to transfer the members of genera Persicivirga O'Sullivan et al. 2006, Stenothermobacter Lau et al. 2006 and Sandarakinotalea Khan et al. 2006 to the genus Nonlabens Lau et al. 2005. Thus, P. dokdonensis (Yoon et al. 2006) Nedashkovskaya et al. 2009, P. ulvanivorans Barbeyron et al. 2010, P. xylanidelens O'Sullivan et al. 2006, Sandarakinotalea sediminis Khan et al. 2006 and Stenothermobacter spongiae Lau et al. 2006 should be transferred to Nonlabens dokdonensis comb. nov., Nonlabens ulvanivorans comb. nov., Nonlabens xylanidelens comb. nov., Nonlabens sediminis comb. nov. and Nonlabens spongiae comb. nov., respectively. In addition, strain JC2678(T) (=KACC 14155(T)=JCM 17109(T)) is proposed to constitute a novel species belonging to the genus Nonlabens with the name of Nonlabens agnitus sp. nov.