Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori

We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis.     

Anti-apoptotic potential of insect cellular and viral IAPs in mammalian cells

IAPs were identified as baculoviral proteins that could inhibit the apoptotic response of insect cells to infection. Of the viral IAPs, OpIAP and CpIAP can inhibit apoptosis, whereas AcIAP cannot. OpIAP and some mammalian homologues can inhibit mammalian cell death. Two mammalian IAPs bind to TNFRII associated factors (TRAFs), but the significance of this is unclear. Here we show that Drosophila cellular IAPs and two baculoviral IAPs (OpIAP and CpIAP) can inhibit mammalian cell death induced by overexpression of Caspases 1 and 2. IAPs must act on conserved components of the apoptotic mechanism, but as none of these IAPs could bind TRAF proteins, TRAFs are not likely to be important for IAP mediated apoptosis inhibition. As OpIAP protected against death induced by ligation of TNF receptor family members, but not by factor nor serum withdrawal from dependent cells, it can inhibit certain apoptotic pathways without affecting others.     

The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis

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.     

ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas

BACKGROUND: Inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All IAPs have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. IAPs physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. This allows them to function as sensors and inhibitors of death signals that emanate from a variety of pathways. RESULTS: Here we report the characterization of ML-IAP, a novel human IAP that contains a single BIR and RING finger motif. ML-IAP is a powerful inhibitor of apoptosis induced by death receptors and chemotherapeutic agents, probably functioning as a direct inhibitor of downstream effector caspases. Modeling studies of the structure of the BIR domain revealed it to closely resemble the fold determined for the BIR2 domain of X-IAP. Deletion and mutational analysis demonstrated that integrity of the BIR domain was required for anti-apoptotic function. Tissue survey analysis showed expression in a number of embryonic tissues and tumor cell lines. In particular, the majority of melanoma cell lines expressed high levels of ML-IAP in contrast to primary melanocytes, which expressed undetectable levels. These melanoma cells were significantly more resistant to drug-induced apoptosis. CONCLUSIONS: ML-IAP, a novel human IAP, inhibits apoptosis induced by death receptors and chemotherapeutic agents. The BIR of ML-IAP possesses an evolutionarily conserved fold that is necessary for anti-apoptotic activity. Elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the pathogenesis of this malignancy.     

IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding

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.     

Functional analysis of the inhibitor of apoptosis genes in <Emphasis Type="Italic">Antheraea pernyi</Emphasis> nucleopolyhedrovirus

The inhibitor of apoptosis proteins (IAP) plays an important role in cell apoptosis. We cloned two novel IAP family members, Ap-iap1 and Ap-iap2, from Antheraea pernyi nucleopolyhedrovirus (ApNPV) genome. Ap-IAP1 contains two baculoviral IAP repeat (BIR) domains followed by a RING domain, but Ap-IAP2 has only one BIR domain and RING. The result of transient expression in Spodoptera frugiperda (Sf21) showed that Ap-iap1 blocked cell apoptosis induced by actinomycin D treatment and also rescued the p35 deficient Autographa californica nucleopolyhedrovirus (AcNPV) to replicate in Sf9 cells, while Ap-iap2 does not have this function. Several Ap-IAP1 truncations were constructed to test the activity of BIRs or RING motif to inhibit cell apoptosis. The results indicated that BIRs or RING of Ap-IAP1 had equally function to inhibit cell apoptosis. Therefore deletion of above both of the above domains could not block apoptosis induced by actinomycin D or rescue the replication of AcMNPVΔp35. We also screened two phage-display peptides that might interact with Ap-IAP1.     

Baculovirus p33 Binds Human p53 and Enhances p53-Mediated Apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.     

The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1

While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus, pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection.     

The IAP family：endogenous caspase inhibitors with multiple biological activities

IAPs (inhibitors of apoptosis) are a family of proteins containing one or more characteristic BIR domains.These proteins have multiple biological activities that include binding and inhibiting caspases,regulating cell cycle progression,and modulating receptor-mediated signal transduction.Our recent studies found the IAP family members XIAP and c-IAP1 are ubiquitinated and degraded in proteasomes in response to apoptotic stimuli in T cells,and their degradation appears to be important for T cells to commit to death.In addition to three BIR domains,each of these IAPs also contains a RING finger domain. We found this region confers ubiquitin protease ligase(E3) activity to IAPs,and is responsible for the auto-ubiquitination and degradation of IAPs after an apoptotic stimulus.Given the fact that IAPs can bind a variety of proteins,such as caspases and TRAFs,it will be of interest to characterize potential substrates of the E3 activity of IAPs and the effects of ubiquitination by IAPs on signal transduction,cell cycle,and apoptosis.     

X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO

Members of the IAP (inhibitor of apoptosis) family function as anti-apoptotic proteins by binding directly to caspase-3, -7, and -9 to inhibit their activities. During apoptosis, the activities of IAPs are relieved by a second mitochondria-derived caspase activator, named Smac/DIABLO. Some IAPs have a C-terminal RING finger domain that has been identified as the essential motif for the activity of ubiquitin ligase (E3). Here we show that X-linked IAP (XIAP) mediates the polyubiquitination of caspase-9 and Smac. The large subunit of mature caspase-9 was polyubiquitinated by XIAP in vitro, while procaspase-9 was not. Furthermore, the polyubiquitinated form of caspase-9 accumulated in an XIAP-dependent manner in intact cells. The ubiquitination of caspase-9 was significantly inhibited in the presence of mature Smac, whereas XIAP was also found to promote the polyubiquitination of cytosolic Smac both in vitro and in intact cells. These ubiquitination reactions require the RING finger domain of XIAP. These findings suggest that XIAP functions as ubiquitin ligase toward mature caspase-9 and Smac to inhibit apoptosis.     

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.     

Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.     

Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides

The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. Our study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins.     

Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases.

Several human inhibitor of apoptosis (IAP) family proteins function by directly inhibiting specific caspases in a mechanism that does not require IAP cleavage. In this study, however, we demonstrate that endogenous XIAP is cleaved into two fragments during apoptosis induced by the tumor necrosis factor family member Fas (CD95). The two fragments produced comprise the baculoviral inhibitory repeat (BIR) 1 and 2 domains (BIR1-2) and the BIR3 and RING (BIR3-Ring) domains of XIAP. Overexpression of the BIR1-2 fragment inhibits Fas-induced apoptosis, albeit at significantly reduced efficiency compared with full-length XIAP. In contrast, overexpression of the BIR3-Ring fragment results in a slight enhancement of Fas-directed apoptosis. Thus, cleavage of XIAP may be one mechanism by which cell death programs circumvent the anti-apoptotic barrier posed by XIAP. Interestingly, ectopic expression of the BIR3-Ring fragment resulted in nearly complete protection from Bax-induced apoptosis. Use of purified recombinant proteins revealed that BIR3-Ring is a specific inhibitor of caspase-9 whereas BIR1-2 is specific for caspases 3 and 7. Therefore XIAP possesses two different caspase inhibitory activities which can be attributed to distinct domains within XIAP. These data may provide an explanation for why IAPs have evolved with multiple BIR domains.     

Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.     

SMAC negatively regulates the anti-apoptotic activity of melanoma inhibitor of apoptosis (ML-IAP).

Inhibitors of apoptosis (IAPs) physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. X-linked IAP (X-IAP) is a prototype IAP family member that inhibits several caspases, the effector proteases of apoptosis. The inhibitory activity of X-IAP is regulated by SMAC, a protein that is processed to its active form upon receipt of a death stimulus. Cleaved SMAC binds X-IAP and antagonizes its anti-apoptotic activity. Here we show that melanoma IAP (ML-IAP), a potent anti-cell death protein and caspase inhibitor, physically interacts with SMAC through its BIR (baculovirus IAP repeat) domain. In addition to binding full-length SMAC, ML-IAP BIR associates with SMAC peptides that are derived from the amino terminus of active, processed SMAC. This high affinity interaction is very specific and can be completely abolished by single amino acid mutations either in the amino terminus of active SMAC or in the BIR domain of ML-IAP. In cells expressing ML-IAP and X-IAP, SMAC coexpression or addition of SMAC peptides abrogates the ability of the IAPs to inhibit cell death. These results demonstrate the feasibility of using SMAC peptides as a way to sensitize IAP-expressing cells to pro-apoptotic stimuli such as chemotherapeutic agents.     

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.     

Livin, a novel inhibitor of apoptosis protein family member

A novel human inhibitor of apoptosis protein (IAP) family member termed Livin was identified, containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. The mRNA for livin was not detectable by Northern blot in most normal adult tissues with the exception of the placenta, but was present in developmental tissues and in several cancer cell lines. Highest levels were observed in two melanoma-derived cell lines, G361 and SK-Mel29. Transfection of livin in HeLa cells resulted in protection from apoptosis induced by expression of FADD, Bax, RIP, RIP3, and DR6. Similar to other IAP family members, the anti-apoptotic activity of Livin was dependent on the BIR domain. Livin was also capable of inhibiting DEVD-like caspase activity triggered by tumor necrosis factor-alpha. In vitro binding studies demonstrated a direct interaction between Livin and the active form of the downstream caspases, caspase-3 and -7, that was dependent on the BIR domain of Livin. In addition, the unprocessed and cleaved forms of caspase-9 co-immunoprecipitated with Livin in vivo, and recombinant Livin could inhibit the activation of caspase-9 induced by Apaf-1, cytochrome c, and dATP. The subcellular distribution of the transfected Livin was analyzed by immunofluorescence. Both Livin and Survivin were expressed in the nucleus and in a filamentous pattern throughout the cytoplasm. In contrast to the apoptotic activity, the COOH-terminal RING domain mediated its subcellular localization patterning. Further studies found that transfection of an antisense construct against livin could trigger apoptosis specifically in cell lines expressing livin mRNA. This was associated with an increase in DNA fragmentation and in DEVD-like caspase activity. Thus, disruption of Livin may provide a strategy to induce apoptosis in certain cancer cells.