Baculovirus IAP

Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV and IAP (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV). Using a genetic complementation assay to identify additional genes that inhibit apoptosis during infection with a p35 mutant, a gene has been isolated from Orgyia pseudotsugata NPV (OpMNPV) that is able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicts a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs are found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appears to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism (Birnbaum, 2003).

The defining structural motif of the inhibitor of apoptosis protein family is the BIR (baculovirus iap repeat), a highly conserved zinc coordination domain of approximately 70 residues. Although the BIR is required for inhibitor-of-apoptosis function, including caspase inhibition, its molecular role in antiapoptotic activity in vivo is unknown. To define the function of the BIRs, the activity was investigated of these structural motifs within Op-IAP, an efficient, virus-derived IAP. Op-IAP(1-216), a loss-of-function truncation which contains two BIRs but lacks the C-terminal RING motif, potently interfers with Op-IAP's capacity to block apoptosis induced by diverse stimuli. In contrast, Op-IAP(1-216) has no effect on apoptotic suppression by caspase inhibitor P35. Consistent with a mechanism of dominant inhibition that involves direct interaction between Op-IAP(1-216) and full-length Op-IAP, both proteins form an immunoprecipitable complex in vivo. Op-IAP also self-associates. In contrast, the RING motif-containing truncation Op-IAP(183-268) fails to interact with or interfere with Op-IAP function. Substitution of conserved residues within BIR 2 causes loss of dominant inhibition by Op-IAP(1-216) and coincides with loss of interaction with Op-IAP. Thus, residues encompassing the BIRs mediate dominant inhibition and oligomerization of Op-IAP. Consistent with dominant interference by interaction with an endogenous cellular IAP, Op-IAP(1-216) also lowers the survival threshold of cultured insect cells. Taken together, these data suggest a new model wherein the antiapoptotic function of IAP requires homo-oligomerization, which in turn mediates specific interactions with cellular apoptotic effectors (Hozak, 2000).

Baculovirus P35 is a universal suppressor of apoptosis that stoichiometrically inhibits cellular caspases in a novel cleavage-dependent mechanism. Upon caspase cleavage at Asp-87, the 10- and 25-kDa cleavage products of P35 remain tightly associated with the inhibited caspase. Mutations in the alpha-helix of the reactive site loop preceding the cleavage site abrogate caspase inhibition and antiapoptotic activity. Substitution of Pro for Val-71, which is located in the middle of this alpha-helix, produces a protein that is cleaved at the requisite Asp-87 but does not remain bound to the caspase. This loss-of-function mutation provides the opportunity to structurally analyze the conformational changes of the P35 reactive site loop after caspase cleavage. The 2.7 Å resolution crystal structure is reported of V71P-mutated P35 after cleavage by human caspase-3. The structure reveals a large movement in the carboxyl-terminal side of the reactive site loop that swings down and forms a new beta-strand that augments an existing beta-sheet. Additionally, the hydrophobic amino terminus releases and extends away from the protein core. Similar movements occur when P35 forms an inhibitory complex with human caspase-8. These findings suggest that the alpha-helix mutation may alter the sequential steps or kinetics of the conformational changes required for inhibition, thereby causing P35 loss of function (dela Cruz, 2001).

It has been suggested that the Drosophila Hid protein interacts with the baculovirus Op-IAP protein in a manner similar to that of human Smac binding to XIAP, based largely on amino acid sequence homology. The interaction between Hid and Op-IAP has been precisely mapped; the biochemical interactions between the amino terminus of Hid and BIR2 of Op-IAP are highly similar to those found between the processed amino terminus of Smac and BIR3 of XIAP. Also similar to Smac, the amino terminus of Hid must be processed to bind Op-IAP. In addition, the data also suggest that a second interaction between Hid and Op-IAP exists that does not involve the amino terminus of Hid. The evolutionary conservation of this mechanism of binding underscores its importance in apoptotic regulation. Nevertheless, interaction with Hid is not sufficient for Op-IAP to inhibit apoptosis induced by Hid overexpression or by treatment with actinomycin D, indicating that additional sequence elements are required for the anti-apoptotic function of Op-IAP (Wright, 2002).

IAP DIAP2 functions in innate immunity, in a caspase independent role, and is essential to resist gram-negative bacterial infection

The founding member of the Inhibitor of Apoptosis (IAPs) protein family was originally identified as a cell death inhibitor. However, recent evidence suggest that IAPs are multifunctional signalling devices that influence diverse biological processes. In order to investigate the in vivo function of Drosophila IAP2 diap2 null alleles were generated. diap2 deficient animals develop normally and are fully viable suggesting that diap2 is dispensable for proper development. However, these animals are 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) signalling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the one of mammalian tumour necrosis factor receptor 1 (TNFR1). diap2 mutant flies fail to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumb to bacterial infection. Genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1 and Relish. Therefore, DIAP2 functions in host-immune response to Gram-negative bacteria. In contrast, it was found that the Drosophila Traf-family member Traf2 is dispensable to resist Gram-negative bacterial infection. Taken together, these genetic data identify DIAP2 as an essential component of the Imd signalling cascade protecting the organism from infiltrating microbes (Leulier, 2006).

In vivo, DIAP2 is indispensable for Imd-mediated expression of antibacterial peptide genes. Like known mutants of the Imd pathway, flies with a mutation in the diap2 gene fail to induce expression of Attacin-A, Cecropin-A1, Defensin, Diptericin, Drosocin and Metchikowin, and mount an efficient immune reaction in response to infection by Gram-negative bacteria. Consequently, diap2 mutant flies succumb to Gram-negative bacterial infection. In contrast, such flies mount a normal Toll-dependent immune response and are resistant to infection by fungi and Gram-positive bacteria. The diap2 null mutant phenotype, therefore, demonstrates that DIAP2 is an essential component of the Imd pathway. Thus, these data are consistent with recent RNAi studies that have implicated diap2 in the Imd pathway. DIAP2 is a member of the evolutionarily conserved IAP protein family. IAPs are classified by the presence of the Baculovirus IAP Repeat (BIR) domain through which they interact with various 'client' proteins. Genetic analysis of the Drosophila IAP DIAP1 has provided some of the most compelling insights into the in vivo function of this protein family. DIAP1, the first and most extensively studied Drosophila IAP, is essential for cell survival and acts as a potent caspase inhibitor. Mutations that abrogate physical association of DIAP1 with caspases cause widespread and unrestrained caspase activation leading to cell and organismal death. In contrast to diap1, diap2 null-mutants do not show an apparent cell death phenotype and develop normally. This is unexpected because both these IAPs interact with caspases and IAP-antagonists with similar affinities. Moreover, when overexpressed, DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 can functionally substitute for DIAP1 in its ability to regulate caspases. Nevertheless, diap2 mutant animals do not show any apparent apoptosis related phenotypes during development. However, these animals appear to be sensitised to Reaper-mediated killing in the eye. The lack of any apparent gross developmental phenotype may be due to sufficiently high levels of DIAP1 protein that may thwart unscheduled caspase activation in response to loss of DIAP2. In this respect it is noteworthy that during embryonic development the levels of diap1 mRNA dramatically exceed those of diap2 (17 fold difference). Moreover, similarly to cIAP2 knock-out mice, where a cell death phenotype is revealed only after LPS challenge, phenotypic manifestation may only become apparent under certain conditions or in selective tissues. In agreement with this notion, RNAi-mediated depletion of DIAP2 has no effect on cell viability in unchallenged tissue culture cells, but significantly sensitizes S2 cells to stress-induced apoptosis (Leulier, 2006).

Although IAPs have originally been identified as apoptosis inhibitors, recent evidence suggests that IAPs are multifunctional signalling devices that, depending on the protein they interact with, influence diverse biological processes. In this respect, it is noteworthy that IAPs also carry C-terminal RING finger domains providing them with E3 ubiquitin-protein ligase, and hence, signalling activity. Thus, in addition to inhibiting apoptosis, IAPs also fulfil functions that operate independently of their ability to control caspases and cell death. Therefore, BIR-containing proteins are more precisely referred to as BIRCs rather than IAPs. Consistent with the notion that BIRCs are multifunctional proteins, the mammalian c-IAP1 and c-IAP2 bind to caspases as well as RIP1 and TRAF320 2, two components of the TNF receptor signalling complex. c-IAP1 or c-IAP2, or both, can promote ubiquitylation and degradation of TRAF2, RIP1 and NF-kappaB kinase (IKKgamma)/NF-kappaB essential modulator (NEMO). Hence, these BIRC proteins are thought to modulate the response to TNF. More recently, another BIRC protein was identified as a important regulator of innate immune surveillance in mammals. BIRC1e (NAIP5) was found to control the intracellular pathogen Legionella pneumophila, a Gram-negative microbe that causes severe bacterial pneumonia known as Legionnaires' disease. BIRC1e protects infected host macrophages by restricting intracellular replication of this pathogen (Leulier, 2006).

The Drosophila BIRC protein DIAP2 is similarly required for innate immune responses and the resistance to Gram-negative bacterial infection. diap2 null mutants become highly susceptible to Gram-negative bacteria and fail to induce antibacterial peptide gene expression. Intriguingly, the Imd pathway, which is required for antibacterial peptide gene expression in response to Gram-negative microbes, shares significant similarities with the TNFR1 signalling cascade. The notion that the BIRC proteins c-IAP1, c-IAP2 and DIAP2 are core components of the TNFR1- and Imd-pathway, respectively, further reinforces the parallels between the mammalian TNFR1 pathway and the one of Imd of Drosophila, pointing to an evolutionary conservation of these pathways in NF-kappaB activation. Moreover, both pathways seem to rely on ubiquitin-mediated protein modifications. As in human cells, where activation of TAK1 and IKK requires the E2 ubiquitin-conjugating enzyme complex Ubc13/UEV1A, Drosophila Ubc13(Bendless)/UEV1A are similarly required for activating Tak1 and the Drosophila IKK complex. Moreover, recent RNAi data from cultured cells suggest that Drosophila Tab contributes to Imd signalling, although this still awaits in vivo validation. Therefore, similar to the TNFR1 pathway, ubiquitin-mediated protein modification is likely to activate the Tak1/Tab complex via Tab's ability to bind to Lys63-linked polyubiquitin chains, thereby recruiting Tak1 to activator platforms. In contrast to the E2 ubiquitin-conjugating enzyme complex, little is known about the nature of the E3 ubiquitin-ligase of the Imd pathway. While TRAF2 is crucial for Ubc13/UEV1A-mediated ubiquitylation in the mammalian TNFR1 pathway, it seems that for Imd signalling Traf2, the TRAF2/6 orthologue in flies, is not a critical component. Traf2 null mutation did not completely block NF-kappaB activation in Drosophila. Moreover, the data clearly indicate that Traf2 mutant flies are fully competent to mount an immune response that resists Gram-negative bacterial infection. Hence, Traf2 appears not to be essential for an effective Imd-mediated immune response. Since the Drosophila genome encodes at least three TRAF family members, it is possible that the loss of Traf2 function is complemented by other TRAF family members. Alternatively, other signalling pathways that bypass Traf2 to transmit the infection signal to NF-kappaB may exist in Drosophila. In agreement with this notion, RNAi-mediated knock-down of all three Drosophila TRAFs also did not abrogate Imd signalling in S2 cells. Thus, an E3 ubiquitin ligase different to, or in addition of Traf2 may be responsible for Imd signalling in Drosophila. Since DIAP2 carries a RING finger domain, it represents a likely candidate (Leulier, 2006).

The genetic epistatic analysis places diap2 downstream or in parallel of imd, Dredd, Tak1 and Relish. Over-expression of imd, Dredd, Tak1 and Relish failed to induce Diptericin expression in diap2 mutant animals; in wild-type animals, enforced expression of these genes (in the absence of any infection) resulted in reproducible Diptericin induction. Intriguingly, Diptericin induction following enforced expression of imd and Dredd is also blocked in Tak1 mutant animals, indicating that both DIAP2 and Tak1 are required downstream of Dredd. In contrast, kenny and ird5 seem not to be required for Diptericin induction when Dredd is over-expressed. A recent report indicates that Relish cleavage and nuclear translocation on its own is not sufficient for Diptericin expression, and that, at least in vivo, a further cooperative input from the JNK signalling pathway is required. According to this scenario the Imd signalling pathway bifurcates at the level of Tak1, with Tak1 activating the NF-kappaB as well as JNK signalling branch, both of which are required for expression of antibacterial peptide genes in the fat body. In light of this model, the observation that diap2 acts genetically downstream of imd, Dredd, Tak1 and Relish may indicate that DIAP2 functions at the level of Tak1. This view is in agreement with recent reports from Drosophila tissue culture cells, which suggest that DIAP2 is required for Tak1-mediated JNK activation. In this respect, DIAP2 functions at the same epistatic position as the putative E3 ubiquitin ligase of the Imd pathway. Future biochemical experiments will be required to test whether DIAP2 is indeed the E3 ubiquitin ligase that functions together with Ubc13/UEV1A to stimulate Tak1 (Leulier, 2006).

Although the underlying mechanism for the impaired induction of antibacterial peptide gene expression by loss of DIAP2 remains to be defined, the genetic observations made in this study are likely to have relevance not only for innate immune responses in Drosophila but also for TNFR1 signalling in mammals. While in flies DIAP2 is indispensable for Imd signalling, genetic studies in mice have, so far, failed to uncover a physiological role for c-IAP1 and c-IAP2 in TNFR1 signalling. Since c-iap1 knock out mice carry significantly elevated levels of c-IAP2 protein, it is feasible that the increased c-IAP2 levels functionally compensate for the loss of c-IAP1. Consistently, mammalian IAPs have been reported to be under strict homeostatic control by regulating each other's protein levels, which provides a mechanistic explanation for the crosstalk among IAPs. Thus, in mammals, double knock out mice lacking both c-iap1 and c-iap2 genes will be required to study the role of c-IAP1 and c-IAP2 in TNFR1 signalling. Therefore, Drosophila, where redundancies and compensatory mechanisms are less problematic, provides an ideal model system to study caspase-independent functions of IAPs in an in vivo setting (Leulier, 2006).

IAPs in other insect species and other invertebrates

Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes, in addition to apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear. Two Caenorhabditis elegans BIRPs, BIR-1 and BIR-2, have been identified and characterized; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 is unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression does not increase cell death. Instead, embryos lacking bir-1 are unable to complete cytokinesis and they become multinucleate. This cytokinesis defect can be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. It is concluded that BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. It is suggested that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis (Fraser, 1999).

Baculoviral IAP repeat proteins (BIRPs) may affect cell death, cell division, and tumorigenesis. The C. elegans BIRP, BIR-1, localizes to chromosomes and to the spindle midzone. Embryos and fertilized oocytes lacking BIR-1 have defects in chromosome behavior, spindle midzone formation, and cytokinesis. Indistinguishable defects are observed in fertilized oocytes and embryos lacking the Aurora-like kinase AIR-2. AIR-2 is not present on chromosomes in the absence of BIR-1. Histone H3 phosphorylation and HCP-1 staining, which marks kinetochores, are reduced in the absence of either BIR-1 or AIR-2. It is proposed that BIR-1 localizes AIR-2 to chromosomes and perhaps to the spindle midzone, where AIR-2 phosphorylates proteins that affect chromosome behavior and spindle midzone organization. The human BIRP survivin, which is upregulated in tumors, can partially substitute for BIR-1 in C. elegans. Deregulation of bir-1 promotes changes in ploidy, suggesting that similar deregulation of mammalian BIRPs may contribute to tumorigenesis (Speliotes, 2000).

A homolog of the Drosophila inhibitor of apoptosis protein 1 (designated AtIAP1) has been identified in Aedes triseriatus mosquitoes. The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA is detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein is detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognizes proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 are expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures (Blitvich, 2002).

Drosophila activators of apoptosis mapping to the Reaper region function, in part, by antagonizing IAP proteins through a shared RHG motif. Reaper isolated from the Blowfly L. cuprina, triggers extensive apoptosis in Drosophila cells. Conserved regions of Reaper were tested in the context of GFP fusions and a second killing activity, distinct from the RHG, was identified. A 20 amino-acid peptide, designated R3, conferred targeting to a focal compartment and promoted membrane blebbing. Killing by the R3 fragment did not correlate with translational suppression or with reduced DIAP1 levels. Likewise, R3-induced cell deaths were only modestly suppressed by silencing of Dronc and involved no detectable association with DIAP1. Instead, a second IAP-binding domain, distinct from the R3, was identified at the C terminus of Reaper that binds to DIAP1 but fails to trigger apoptosis. Collectively, these findings are inconsistent with single effector models for cell killing by Reaper and suggest, instead, that Reaper encodes conserved bifunctional death activities that propagate through distinct effector pathways (Chen, 2004).

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, the apoptosis of the prothoracic gland was analyzed of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, prothoracic gland degeneration was shown to be effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species (Mané-Padrós, 2010).

In Drosophila, a similar ecdysteroid-dependent cascade directs the stage-specific destruction of the salivary glands. In this insect, however, the cascade converges in the induction of rpr and hid, and the consequent massive caspase activation and cell death. Interestingly, RPR and HID homologous proteins have not been reported in any insect outside Drosophila and closely related species, suggesting that although the role of 20E as inducer of cell death is conserved, the mechanisms by which the hormone controls such process would be different in hemimetabolous insects (Mané-Padrós, 2010).

This study has demonstrated that BgFTZ-F1 is a critical factor to specifically induce the degeneration of the prothoracic gland of B. germanica after the imaginal molt. Several observations support this: (1) the prothoracic gland of BgFTZ-F1 knockdown adults failed to degenerate; (2) ectopic expression of BgFTZ-F1 in mid-last nymphal instar, mediated by the reduction of BgE75, prematurely induced the degeneration of the prothoracic gland; (3) the prothoracic gland of last instar nymphs that had been simultaneously knockdown for BgE75 and BgFTZ-F1, thus preventing the ectopic expression of BgFTZ-F1 during mid-nymphal instar, also failed to degenerate; (4) the expression of BgFTZ-F1 in the prothoracic gland during the imaginal molt is significantly higher (12-fold) than during nymphal transitions, and its expression is maintained during the first days of the adult stage until the onset of prothoracic gland degeneration (Mané-Padrós, 2010).

The nuclear receptor FTZ-F1 has also been implicated in the apoptotic response in Drosophila. In this insect, βFTZ-F1 acts as a transcriptional competence factor that provides the stage-specificity for salivary gland degeneration at the onset of the pupal development. Moreover, its activity is required for the destruction of the cytoplasm, nuclear DNA fragmentation and controlling caspase levels in salivary gland cells (Mané-Padrós, 2010).

In addition, it was also demonstrated that the pro-apoptotic effect of BgFTZ-F1 is restricted to the last nymphal instar due to the anti-apoptotic effect exerted by JH before the last nymphal instar. Although JH regulates a number of developmental events in insects, its molecular mechanism of action still remains a matter of debate. The death inhibitor effect of JH has been also described in relation to the midgut remodelling that occurs during metamorphosis of the mosquito A. aegypti and the moth H. virescens. Recently, it has been also shown that the larval fat body of JH-deficient Drosophila undergoes premature degeneration and that this response can be prevented by the application of methoprene. Interestingly, however, the anti-apoptotic effect of methoprene in Drosophila was based on the inhibition of two bHLH-PAS transcription factors involved in JH action, Methoprene-tolerant and Germ-cell expressed, but was not related, in contrast to B. germanica, to the suppression of the ecdysteroid-dependent transcriptional cascade (Mané-Padrós, 2010).

In summary, these results led a model for developmentally regulated PCD in the hemimetabolous B. germanica. In this model, JH is responsible to hold back the degeneration of the prothoracic gland during nymphal-nymphal transitions. The low levels of BgFTZ-F1 observed at the end of the penultimate nymphal instar would be below a critical threshold necessary to overcome the BgIAP1-mediated inhibition of prothoracic gland degeneration. During the last nymphal instar, however, the specific and dramatic up-regulation of BgFTZ-F1 in the prothoracic gland at the end of the instar (12 fold higher than in the penultimate instar) would overcome, in the absence of JH, the inhibitory effect of BgIAP1 making the prothoracic gland competent to execute the cell death program (Mané-Padrós, 2010).

This study shows that prothoracic gland degeneration in the nymphal-adult transition of B. germanica show conserved but also divergent features with respect to the highly derived and thoroughly studied species Drosophila melanogaster. This underlines the importance of investigating basal, less modified species, in order to understand the evolutionary trends and mechanisms that led to the highly sophisticated holometabolan mode of metamorphosis (Mané-Padrós, 2010).

Interaction of IAP with caspases inhibits cell death

During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome has been examined. A multi-step process is proposed, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex (Bratton, 2001).

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. XIAP associates with the active caspase-9-Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevent caspase-9 association with XIAP and its concomitant inhibition. The N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. These studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis (Srinivasula, 2001).

The IAPs potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 A resolution. The structure reveals that the BIR3 domain forms a heterodimer with a caspase-9 monomer. Strikingly, the surface of caspase-9 that interacts with BIR3 also mediates its homodimerization. Monomeric caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases (Shiozaki, 2003).

The inhibitor of apoptosis proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 Å resolution. The structure reveals that the BIR3 domain forms a heterodimer with a caspase-9 monomer. Strikingly, the surface of caspase-9 that interacts with BIR3 also mediates its homodimerization. Monomeric caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases (Shiozaki, 2003).

The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site [DQLD(330)], which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome has been assessed. The effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation has been assessed, as well as the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. Cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms results in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposes a novel p10 NH(2)-terminal peptide motif (AISS) that retains the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP (Zou, 2003).

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocks proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocks apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibits human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors (Zoog, 2002).

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. Site-directed mutagenesis of BIR2 and its linker were used to determine the mechanism of executioner caspase inhibition by XIAP. The BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, it is predicted that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP (Scott, 2005).

The fundamental mechanism of specific protein interactions is usually conserved during protein evolution. According to conservation of mechanism, the two units of XIAP that inhibit caspases should preserve a fundamental interaction strategy. On the basis of structural studies, this concept could be questioned. The key elements of caspase-9 inhibition by BIR3 are the IBM interacting groove and the C-terminal helix. In contrast, the key element of caspase-3 and -7 inhibition by BIR2 seems to be the completely nonconserved N-terminal linker region. The most conserved surface structure of BIR domains is the IBM interacting groove. It is found on many BIR domains including the BIR2 and BIR3 of XIAP, and the BIR1 and BIR2 of an ortholog in Drosophila melanogaster, DIAP1. The surface groove of DIAP1 BIR2 is involved in binding and ubiquitination of Dronc, the initiator caspase in flies. In addition, the IBM interacting groove of DIAP1 BIR1 is absolutely required for inhibition of the executioner caspase DrICE by an unknown mechanism. It is suggested that the IBM interacting groove is a conserved interaction element of BIR domains and that for XIAP BIR2 it confers tight inhibition of caspase-3 and -7 by providing a second binding site (Scott, 2005).

Both BIR2 and BIR3 inhibit their target caspases by a two-site interaction mechanism. They have conserved a functional IBM interacting groove that participates in inhibition by binding neoepitopes revealed following activation of their target enzymes. This interaction, primarily a docking contact, represents the conserved mechanism and also provides a platform for regulation by antagonists Smac/DIABLO and HtrA2. The primary inhibition site, however, is mechanistically different for each domain: blocking the active site in caspase-3 and -7, or dissociating the dimer of caspase-9. It is far from clear how the inhibitory mechanism diverged. It is much clearer that these distinct mechanisms direct the exquisite specificity that allows XIAP BIR domains to target selectively individual caspases in a way that other inhibitory strategies, both natural and artificial have yet to achieve (Scott, 2005).

Peptides that bind to IAPs

Peptides targeting the X-inhibitor of apoptosis protein (XIAP) have been analyzed. XIAP functions as a caspase inhibitor and is a member of the IAP family of proteins. IAPs are often overexpressed in cancers and leukemias and are associated with an unfavorable clinical prognosis. Peptides from a phage library have been selected by using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from two independent screenings by using different libraries. Phage-displaying variations of the consensus sequence bind specifically to the BIR2 domain of XIAP but not to other IAPs. The interaction is specific, because it can be blocked by the cognate synthetic peptides in a dose-dependent manner. Phage displaying the XIAP-binding motif CEFESC bind to the BIR2 domain of XIAP with an estimated dissociation constant of 1.8 nm as determined by surface plasmon resonance. Protein-protein interaction assays reveal that caspase-3 and caspase-7 (but not caspase-8) block the binding of the CEFESC phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. In fact, the sequence EFES is homologous to a loop unique to the executioner caspase-3 and caspase-7 that are targeted by XIAP. An internalizing version of the XIAP-binding peptide identified in these screenings (PFKQ) can induce programmed cell death in leukemia cells. Peptides interacting with XIAP can serve as prototypes for the design of low molecular weight modulators of apoptosis (Tamm, 2003).

Smac/Diablo releases IAPs from caspases initiating the apoptotic signaling process

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE were used, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. Since transfection of cells with DIABLO is able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases (Verhagen, 2000).

MIHA is an inhibitor of apoptosis protein that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. After UV radiation, MIHA prevents apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functions after release of cytochrome c and DIABLO from the mitochondria and is able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO binds to MIHA and disrupts MIHA's association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis (Ekert, 2001).

Inhibitor of apoptosis proteins (IAPs) interact with and inhibit caspases-3, -7, and -9. This interaction can be inhibited by Smac/DIABLO, a polypeptide released from mitochondria upon initiation of the apoptotic signaling process. The first 4-8 N-terminal amino acids of Smac/DIABLO fused to the Drosophila Antennapaedia penetratin sequence (a carrier peptide) enhance the induction of apoptosis and long term antiproliferative effects of diverse antineoplastic agents including paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin (SN-38), and doxorubicin in MCF-7 breast cancer cells. Similar effects were observed in additional breast cancer and immortalized cholangiocyte cell lines. The Smac-penetratin fusion peptide crosses the cellular membrane, binds XIAP and cIAP1, displaces caspase-3 from cytoplasmic aggregates, and enhances drug-induced caspase action in situ. These studies demonstrate that inhibition of IAP proteins can modulate the efficacy of antineoplastic agents (Arnt, 2002).

Smac/DIABLO is a mitochondrial protein that is proteolytically processed and released during apoptosis along with cytochrome c and other proapoptotic factors. Once in the cytosol, Smac protein binds to inhibitors of apoptosis proteins and disrupts the ability of the IAPs to inhibit caspases 3, 7, and 9. The requirement for mitochondrial processing and release has complicated efforts to delineate the effect of Smac overexpression and IAP inhibition on cell death processes. In this report, a novel expression system is documented that uses ubiquitin fusions to express mature, biologically active Smac in the cytosol of transfected cells. Processing of the ubiquitin-Smac fusions is rapid and complete and generates mature Smac protein initiating correctly with the Ala-Val-Pro-Ile tetrapeptide sequence that is required for proper function. The biological activity of this exogenous protein was demonstrated by its interaction with X-linked IAP, one of the most potent of the IAPs. The presence of mature Smac is not sufficient to trigger apoptosis of healthy cells. However, cells with excess Smac protein were greatly sensitized to apoptotic triggers such as etoposide exposure. Cancer cells typically display deregulated apoptotic pathways, including Bcl2 overexpression, thereby suppressing the release of cytochrome c and Smac. The ability to circumvent the requirement for mitochondrial processing and release is critical to developing Smac as a possible gene therapy payload in cancer chemosensitization (Hunter, 2003).

Survivin is a member of the inhibitor of apoptosis protein family that has been implicated in both apoptosis inhibition and cell cycle control. However, understandings of its inhibitory mechanism and subcellular localization remain controversial. Evidence is provided that Survivin physically interacts with Smac/DIABLO both in vitro and in vivo. A point mutation (D71R) in the baculovirus IAP repeat motif and a C-terminal deletion mutant (Surv-BIR) of Survivin result in failure to bind to Smac/DIABLO and these modifications abrogate the ability of Survivin to inhibit apoptosis. The N-terminal of mature Smac/DIABLO is absolutely required for Survivin.Smac complex formation. Subcellular distributions of Survivin and Smac/DIABLO show that they co-localize within the cytosol during interphase. In addition, Survivin is incapable of binding to caspase. Co-presence of Smac/DIABLO and XIAP is required for Survivin to inhibit caspase cleavage in a cell-free system. In conclusion, these results provide the first evidence that the interaction between Smac/DIABLO and Survivin is an essential step underlying the inhibition of apoptosis induced by Taxol (Song, 2003).

IAPs are ubiquitin ligases that function to promote protein degradation

To determine why proteasome inhibitors prevent thymocyte death, an examination was carried out to see whether proteasomes degrade anti-apoptotic molecules in cells induced to undergo apoptosis. The c-IAP1 and XIAP inhibitors of apoptosis are selectively lost in glucocorticoid- or etoposide-treated thymocytes in a proteasome-dependent manner before death. IAPs catalyzes their own ubiquitination in vitro, an activity requiring the RING domain. Overexpressed wild-type c-IAP1, but not a RING domain mutant, is spontaneously ubiquitinated and degraded, and stably expressed XIAP lacking the RING domain is relatively resistant to apoptosis-induced degradation and, correspondingly, more effective at preventing apoptosis than wild-type XIAP. Autoubiquitination and degradation of IAPs may be a key event in the apoptotic program (Yang, 2000).

The mechanism by which IAPs prevent apoptosis has been attributed to the direct inhibition of caspases. The function of mammalian IAPs is counteracted by cell death inducer second mitochondria-derived activator of caspases (Smac)/DIABLO during apoptosis. cIAP1 and cIAP2 are E3 ubiquitin-protein isopeptide ligases (ubiquitin ligases) for Smac. cIAPs stimulate Smac ubiquitination both in vivo and in vitro, leading to Smac degradation. cIAP1 and cIAP2 associate with overlapping but distinct subsets of E2 (ubiquitin carrier protein) ubiquitin-conjugating enzymes. The substrate-dependent E3 activity of cIAPs is mediated by their RING domains and is dependent on the specific interactions between cIAPs and Smac. Similarly, Drosophila IAP1 also possesses ubiquitin ligase activity that mediates the degradation of the Drosophila apoptosis inducers Grim and HID. These results suggest a novel and conserved mechanism by which IAPs block apoptosis through the degradation of death inducers (Hu, 2003).

Mutation of IAPs

X-linked IAP (XIAP), cloned due to its sequence homology with other family members, prevents apoptosis by binding to active caspases 3, 7, and 9 in vitro. XIAP transcripts can be found in a variety of tissues, and the protein levels are regulated both transcriptionally and posttranscriptionally. To better understand the function of XIAP in normal cells, mice deficient in XIAP were generated through homologous gene targeting. The resulting mice were viable, and histopathological analysis did not reveal any differences between XIAP-deficient and wild-type mice. No defects were detected in induction of caspase-dependent or -independent apoptosis in cells from the gene-targeted mice. One change was observed in cells derived from XIAP-deficient mice: the levels of c-IAP1 and c-IAP2 protein were increased. This suggests that there exists a compensatory mechanism that leads to upregulation of other family members when XIAP expression is lost. The changes in c-IAP1 and c-IAP2 expression may provide functional compensation for loss of XIAP during development or in the induction of apoptosis (Harlin, 2001).

Transcriptional regulation of IAPs

The c-Jun NH2-terminal kinase (JNK) can cause cell death by activating the mitochondrial apoptosis pathway. However, JNK is also capable of signaling cell survival. The mechanism that accounts for the dual role of JNK in apoptosis and survival signaling has not been established. JNK-stimulated survival signaling can be mediated by JunD. The JNK/JunD pathway can collaborate with NF-kappaB to increase antiapoptotic gene expression, including cIAP-2. This observation accounts for the ability of JNK to cause either survival or apoptosis in different cellular contexts. Furthermore, these data illustrate the general principal that signal transduction pathway integration is critical for the ability of cells to mount an appropriate biological response to a specific challenge (Lamb, 2003).

The survival signaling role of JNK in the response to TNF contrasts with the effects of JNK to mediate apoptosis in response to the exposure of cells to environmental stress. How can one signal transduction pathway mediate two very different responses? There are two general mechanisms that could account for these different roles of JNK in apoptosis signaling. These mechanisms are not mutually exclusive. One mechanism is represented by the time course of JNK activation. Studies of MAP kinases indicate that the time course of activation can determine the cellular response. This may also apply to the response of cells to JNK activation. Sustained JNK activation is required for apoptotic signaling and is sufficient for apoptosis. In contrast, TNF causes transient JNK activation. These considerations indicate that transient JNK activation may be important for mediating a survival response in TNF-treated cells and that chronic JNK activation may contribute to apoptotic responses. A second mechanism that may account for the different roles of JNK in apoptosis signaling is that the biological consequence of JNK function may depend upon the activation state of other signal transduction pathways. For example, increased AKT activation can suppress the apoptotic effects of activated JNK. A plausible hypothesis is that the JNK signaling pathway may cooperate with other signaling pathways to mediate cell survival (e.g., NF-kappaB and AKT). For example, target genes that are induced by the antiapoptotic NF-kappaB pathway may contain JNK-responsive elements in their promoters (e.g., AP-1 sites). The cIAP-2 gene represents an example of this class of gene. JNK increases the expression of such genes in cells with activated NF-kappaB and thus increases cell survival. In contrast, in the absence of a survival pathway that can cooperate with JNK, sustained JNK activation may lead to apoptosis (Lamb, 2003 and references therein).

This analysis demonstrates that JNK mediates a survival response in cells treated with TNF. The role of JNK is mediated by the transcription factor JunD, which can collaborate with NF-kappaB to increase the expression of prosurvival genes, including cIAP-2. In the absence of activated NF-kappaB, the JNK pathway mediates an apoptotic response. Together, these data provide a mechanism that can account for the dual ability of JNK to cause either survival or apoptosis in different cellular contexts (Lamb, 2003).

Regulation of translation of IAPs

The X-chromosome-linked inhibitor of apoptosis, XIAP, is the most powerful and ubiquitous intrinsic inhibitor of apoptosis. Translation of XIAP is controlled by a potent internal ribosome entry site (IRES) element. IRES-mediated translation of XIAP is increased in response to cellular stress, suggesting the critical role for IRES translation during cellular stress. Heterogeneous nuclear ribonucleoproteins C1 and C2 (hnRNPC1 and hnRNPC2) are part of the RNP complex that forms on XIAP IRES. Furthermore, the cellular levels of hnRNPC1 and hnRNPC2 parallel the activity of XIAP IRES and the overexpression of hnRNPC1 and hnRNPC2 specifically enhance translation of XIAP IRES, suggesting that hnRNPC1 and hnRNPC2 may modulate XIAP expression. Given the central role of XIAP in the regulation of apoptosis these results are important for an understanding of the control of apoptosis (Holcik, 2003).

IAPs and development

Apoptosis is an essential physiologic process used in almost all tissues to remove damaged or superfluous cells. However, the early embryos are unique because no cell death is found up to the blastocyst stage during normal development. Survivin, a member of the IAP family, is capable of binding to caspases to modulate their functions. The expression of survivin has been examined along with its role in preventing apoptosis in mouse preimplantation embryos. Transcripts for survivin and a splice variant lacking exon 2 are detected from unfertilized oocytes up to hatched blastocyst stage. At the protein level, survivin is also detected at all stages of early embryos. The antisense approach was used to demonstrate the role of survivin on embryo development. Development of early embryos treated with antisense survivin oligonucleotides is arrested at the morula or early blastocyst stage with disruption of tubulin formation and abnormal nuclei, associated with apoptosis. The effect of the antisense is enhanced by cotreatment with an apoptosis-inducing reagent, staurosporine. In contrast, apoptosis induced by the antisense treatment is inhibited by caspase-3 and -9 inhibitors. These results indicate that survivin is an essential antiapoptotic gene expressed in preimplantation embryos and could protect the embryos from apoptosis by inhibiting an apoptotic pathway involving caspases (Kawamura, 2003).

IAPs and viral infection

Many viruses belonging to diverse viral families with differing structure and replication strategies induce apoptosis both in cultured cells in vitro and in tissues in vivo. Despite this fact, little is known about the specific cellular apoptotic pathways induced during viral infection. Reovirus-induced apoptosis of HEK cells is initiated by death receptor activation but requires augmentation by mitochondrial apoptotic pathways for its maximal expression. Reovirus infection of HEK cells is associated with selective cytosolic release of the mitochondrial proapoptotic factors cytochrome c and Smac/DIABLO, but not the release of apoptosis-inducing factor. Release of these factors is not associated with loss of mitochondrial transmembrane potential and is blocked by overexpression of Bcl-2. Stable expression of caspase-9b, a dominant-negative form of caspase-9, blocks reovirus-induced caspase-9 activation but fails to significantly reduce activation of the key effector caspase, caspase-3. Smac/DIABLO enhances apoptosis through its action on cellular inhibitor of apoptosis proteins (IAPs). Reovirus infection is associated with selective down-regulation of cellular IAPs, including c-IAP1, XIAP, and survivin, effects that are blocked by Bcl-2 expression, establishing the dependence of IAP down-regulation on mitochondrial events. Taken together, these results are consistent with a model in which Smac/DIABLO-mediated inhibition of IAPs, rather than cytochrome c-mediated activation of caspase-9, is the key event responsible for mitochondrial augmentation of reovirus-induced apoptosis. These studies provide the first evidence for the association of Smac/DIABLO with virus-induced apoptosis (Kominsky, 2002).

Binding of mammalian IAPs to reaper-like motifs

The inhibitor-of-apoptosis proteins (IAPs) play a critical role in the regulation of apoptosis by binding and inhibiting caspases. Reaper family proteins and Smac/DIABLO use a conserved amino-terminal sequence to bind to IAPs in flies and mammals, respectively, blocking their ability to inhibit caspases and thus promoting apoptosis. The serine protease Omi/HtrA2 has been identified as a second mammalian XIAP-binding protein with a Reaper-like motif. This protease autoprocesses to form a protein with amino-terminal homology to Smac/DIABLO and Reaper family proteins. Full-length Omi/HtrA2 is localized to mitochondria but fails to interact with XIAP. Mitochondria also contain processed Omi/HtrA2, which, following apoptotic insult, translocates to the cytosol, where it interacts with XIAP. Overexpression of Omi/HtrA2 sensitizes cells to apoptosis, and its removal by RNA interference reduces cell death. Omi/HtrA2 thus extends the set of mammalian proteins with Reaper-like function that are released from the mitochondria during apoptosis (Martins, 2002).

Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis (Yang, 2003).

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated (Verhagen, 2002).

Inhibitor of apoptosis proteins (IAPs) prevent apoptosis through direct inhibition of caspases. The serine protease HtrA2/Omi has an amino-terminal IAP interaction motif like that found in Reaper, which displaces IAPs from caspases, leading to enhanced caspase activity. The cell death-promoting properties of HtrA2/Omi are not only exerted through its capacity to oppose IAP inhibition of caspases but also through its integral serine protease activity. Peptide libraries were used to determine the optimal substrate sequence for cleavage by HtrA2 and also the preferred binding sequence for its PDZ domain. Using these peptides, it has been show that the PDZ domain of HtrA2/Omi suppresses the proteolytic activity unless it is engaged by a binding partner. Subjecting HtrA2/Omi to heat shock treatment also increases its protease activity. Unexpectedly, binding of X-linked inhibitor of apoptosis protein (XIAP) to the Reaper motif of HtrA2/Omi results in a marked increase in proteolytic activity, suggesting a new role for IAPs. When HtrA2/Omi is released from mitochondria following an apoptotic stimulus, binding to IAPs may switch their function from caspase inhibition to serine protease activation. Thus although IAP overexpression can suppress caspase activation, it could have the opposite effect on HtrA2/Omi-dependent cell death. This, together with the ability of HtrA2/Omi to degrade IAPs, may limit the overall cellular protection that can be provided by these proteins (Martins, 2003).

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays have demonstrated that Omi/HtrA2 degrades inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, these data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis (Srinivasula, 2003).

XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. This study reports an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. DIAP1 was identified as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. The data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing β-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program (Hanson, 2012).

Conversion of the Wnt transcription factor TCF/Lef from a transcriptional repressor to an activator is a critical event in Wnt signal transduction, yet understanding of how this switch occurs in cells is limited. The current model, based primarily on reconstitution studies using purified proteins, proposes direct displacement of the transcriptional corepressor Gro/TLE by the coactivator β-catenin through competition for overlapping binding sites on TCF/Lef (Hanson, 2012).

The data suggest a model in which XIAP constitutively binds and ubiquitylates non-TCF-bound Gro/TLE in the nucleus, thereby limiting the amount of Gro/TLE available to form corepressor complexes with TCF/Lef. In the presence of a Wnt signal, XIAP is recruited to TCF/Lef transcriptional complexes where it promotes dissociation of Gro/TLE. The experiments were not able to distinguish whether XIAP ubiquitylates Gro/TLE bound to TCF/Lef to promote its dissociation or ubiquitylates dissociated Gro/TLE, thereby blocking its reassociation. Regardless, ubiquitylation of Gro/TLE by TCF/Lef-bound XIAP further decreases the affinity of Gro/TLE for TCF/Lef, thereby allowing efficient recruitment and binding of the transcriptional coactivator β-catenin to TCF/Lef that is required to initiate a Wnt-specific transcriptional program. The mechanism by which XIAP is recruited to TCF/Lef transcriptional complexes is unknown, although the results demonstrating that lithium can also induce recruitment of XIAP to TCF/Lef suggest that GSK3 activity plays an important role in regulating this process (Hanson, 2012).

This proposed model for Wnt-mediated transcriptional activation parallels the findings of Sierra (2006) who proposed that inactivation of Wnt target gene transcription similarly occurs as a multistep process. That data suggest that APC and β-TRCP (an E3 ligase) mediate removal of β-catenin from Lef1 to allow for subsequent TLE1 binding. Together, these experiments and the current study have revealed that transcriptional activation and inactivation in the Wnt pathway are highly regulated processes (Hanson, 2012).

β-catenin protein levels are tightly regulated in the cell via continual synthesis and degradation by the β-catenin destruction complex. Why, then, would a cell evolve an additional layer of regulation for Wnt transcriptional activation, as is proposed in this study, as opposed to a simpler mechanism based solely on bimolecular association between β-catenin and TCF/Lef? It is proposed that this Wnt signaling circuitry provides a mechanism to dampen transcriptional noise without a corresponding loss in sensitivity. Binding of Gro/TLE to TCF/Lef allows the system to be resistant to stochastic fluxes in β-catenin levels in the absence of Wnt pathway activation. In the presence of a Wnt signal, a coincident circuit involving nuclear accumulation of β-catenin and recruitment of XIAP to TCF/Lef is established. Such circuitry ensures that transcriptional activation only occurs upon Wnt ligand binding and provides an additional mechanism for reducing spontaneous activity. Sensitivity to a Wnt signal is maintained by the facilitated removal of Gro/TLE from TCF/Lef, which ensures that even low levels of β-catenin would be sufficient to bind TCF/Lef and activate transcription (Hanson, 2012).

Support for this model comes from a study showing that β-catenin levels change only modestly (∼2- to 6-fold) upon Wnt signaling in human cells and Xenopus embryos. It is unlikely that this degree of nuclear β-catenin accumulation is sufficient to effectively displace Gro/TLE from TCF/Lef. This suggests that a facilitated mechanism for Gro/TLE removal is required prior to β-catenin-TCF/Lef complex formation (Hanson, 2012).

The data indicate that XIAP may also influence the nuclear pool of Gro/TLE that is available to form corepressor complexes with TCF/Lef. This study found that XIAP is associated with Gro/TLE in the presence and absence of Wnt signaling. Additionally, whereas ubiquitylated Gro/TLE is readily observed in total cellular lysates, only the nonubiquitylated form of Gro/TLE binds to TCF/Lef. This suggests a model in which XIAP functions to constitutively ubiquitylate free Gro/TLE to control the pool of Gro/TLE that can bind TCF/Lef. The data also suggest the presence of an as yet unidentified deubiquitylase (DUB) that facilitates removal of ubiquitin from Gro/TLE, which would allow TCF/Lef binding. Cycles of monoubiquitylation and deubiquitylation have been shown to regulate activity of the transcriptional activators Smad4, p53, and FoxO. This study provides evidence for a similar mode of regulation of a transcriptional repressor (Hanson, 2012).

Until recently, most studies have focused on transcriptional coactivator activity because it was generally believed that corepressors are abundant proteins subject to little regulation. It is becoming clear, however, that corepressor activity is highly complex and can be controlled through a variety of mechanisms. This study shows that the corepressor Gro/TLE is regulated by ubiquitylation in a manner that may be Wnt pathway specific. Gro/TLE has been shown to participate in transcriptional repression of multiple signaling pathways. The corepressor function of Gro/TLE occurs locally through its binding to DNA-bound transcription factors (primarily via its C-terminal WD40 domain) and histone deacetylase recruitment and globally via its N-terminal Q domain, which mediates oligomerization to alter chromatin structure and mediate long-range repression. The finding that XIAP ubiquitylates Gro/TLE on its N-terminal Q domain (which disrupts TCF/Lef binding) without disrupting its capacity to oligomerize suggests that XIAP modification of Gro/TLE may specifically affect its Wnt repressive function. This possibility is consistent with the observation that XIAP knockdown has no observable effect on Notch signaling. In the absence of Notch signaling, Gro/TLE normally binds to the Hairless protein to repress Notch target gene activation by the transcription factor, Suppressor of Hairless. Binding to Hairless occurs via the C-terminal WD40 domain of Gro/TLE. Thus, ubiquitin modifications of Gro/TLE on its N-terminal Q domain would not be expected to disrupt its interaction with Hairless in the Notch pathway or other pathways in which repression by Gro/TLE occurs via the WD40 domain or via Gro/TLE oligomerization (Hanson, 2012).

The identification of XIAP as a critical Wnt pathway component provides a link between apoptosis and Wnt signaling and represents a mechanism by which a cell could coordinate survival and proliferation. Wnt signaling has been shown to inhibit apoptosis and to be required for XIAP expression in cancer cells. Thus, XIAP may be part of a positive feedback loop involving Wnt pathway-induced proliferation and inhibition of apoptosis. Surprisingly, XIAP knockout mice have no obvious apoptotic or Wnt phenotypes, as would be expected given its important role in apoptotic inhibition and the findings that XIAP is required for Wnt signaling in cultured human cells and in Xenopus embryos. Only exon 1 of XIAP was deleted in the knockout mouse. Thus, it is possible that there is readthrough that permits expression of the C-terminal region of XIAP, which includes the RING domain. Alternatively, other IAP family members or E3 ligases might compensate for XIAP function when it is deleted in the mouse (Hanson, 2012).

These findings may have important clinical implications, as XIAP is upregulated in a majority of human cancers and inhibitors of XIAP are currently in clinical trials. Drug development has been largely focused on developing small molecule and peptide Smac mimetics that bind to the BIR domains of XIAP to inhibit its antiapoptotic function. This study shows that the critical role of XIAP in Wnt signaling depends on its E3 ligase RING domain and is distinct from its antiapoptotic functions. The results predict that small molecules targeting the RING domain of XIAP rather than its BIR domains would represent more selective inhibitors of Wnt signaling. Alternatively, drugs targeting both antiapoptotic and pro-Wnt functions of XIAP may be particularly effective against Wnt-driven cancers. Recent findings indicate that inducing apoptosis results in 'compensatory proliferation' of surrounding surviving cells due to release of mitogenic signals (e.g., Wnt) from dying cells, suggesting that drugs targeting both aspects of XIAP function may be particularly useful anticancer therapies even in non-Wnt-driven tumors (Hanson, 2012).

Cellular Inhibitor of apoptosis protein 1 ubiquitinates endonuclease G but does not affect endonuclease G-mediated cell death

Inhibitors of Apoptosis Proteins (IAPs) are evolutionarily well conserved and have been recognized as the key negative regulators of apoptosis. Recently, the role of IAPs as E3 ligases through the Ring domain was revealed. Using proteomic analysis to explore potential target proteins of DIAP1, Drosophila Endonuclease G (dEndoG), which is known as an effector of caspase-independent cell death, was identified. This study demonstrates that human EndoG interacts with IAPs, including human cellular Inhibitor of Apoptosis Protein 1 (cIAP1). EndoG was ubiquitinated by IAPs in vitro and in human cell lines. Interestingly, cIAP1 was capable of ubiquitinating EndoG in the presence of wild-type and mutant Ubiquitin, in which all lysines except K63 were mutated to arginine. cIAP1 expression did not change the half-life of EndoG and cIAP1 depletion did not alter its levels. Expression of dEndoG 54310, in which the mitochondrial localization sequence was deleted, led to cell death that could not be suppressed by DIAP1 in S2 cells. Moreover, EndoG-mediated cell death induced by oxidative stress in HeLa cells was not affected by cIAP1. Therefore, these results indicate that IAPs interact and ubiquitinate EndoG via K63-mediated isopeptide linkages without affecting EndoG levels and EndoG-mediated cell death, suggesting that EndoG ubiquitination by IAPs may serve as a regulatory signal independent of proteasomal degradation (Seo, 2014).

IAPs and tumors

The identification of antigens associated with tumor destruction is a major goal of cancer immunology. Vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor generates potent, specific, and long-lasting antitumor immunity through improved tumor antigen presentation by dendritic cells and macrophages. A phase I clinical trial of this immunization strategy in patients with disseminated melanoma has revealed the consistent induction in distant metastases of dense T and B cell infiltrates that effectuated substantial tumor necrosis and fibrosis. To delineate the target antigens of this vaccine-stimulated tumor destruction, a melanoma cDNA expression library was screened with postimmunization sera from a long-term responding patient (K030). High-titer IgG antibodies recognized melanoma inhibitor of apoptosis protein (ML-IAP), a caspase antagonist containing a single baculoviral IAP repeat and a COOH-terminal RING domain. Although K030 harbored antibodies to ML-IAP at the time of study entry, multiple courses of vaccination over 4 years increased antibody titers and elicited isotype switching. Moreover, lymphocyte infiltrates in necrotic metastases included CD4+ and CD8+ T cells specific for ML-IAP, as revealed by proliferation, tetramer, enzyme-linked immunospot, and cytotoxicity analysis. Whereas melanoma cells in densely infiltrated lesions show strong ML-IAP expression by immunohistochemistry, lethal disease progression i s associated with the loss of ML-IAP staining and the absence of lymphocyte infiltrates. These findings demonstrate that ML-IAP can serve as a target for immune-mediated tumor destruction, but that antigen-loss variants can accomplish immune escape (Schmollinger, 2003).

IAPs involvement in p53 dependent apoptosis

A Drosophila p53 protein has been identified that mediates apoptosis via a novel pathway involving the activation of the Reaper gene and subsequent inhibition of the inhibitors of apoptosis (IAPs). The present study found that CIAP1, a major mammalian homolog of Drosophila IAPs, is irreversibly inhibited (cleaved) during p53-dependent apoptosis and this cleavage is mediated by a serine protease. Serine protease inhibitors that block CIAP1 cleavage inhibit p53-dependent apoptosis. Furthermore, activation of the p53 protein increases the transcription of the HTRA2 gene, which encodes a serine protease that interacts with CIAP1 and potentiates apoptosis. These results demonstrate that the mammalian p53 protein may activate apoptosis through a novel pathway functionally similar to that in Drosophila, which involves HTRA2 and subsequent inhibition of CIAP1 by cleavage (Jin, 2003).

thread: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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