IAPs: what's in a name?

Originally described in insect viruses, cellular proteins with Baculoviral IAP repeat (BIR) motifs have been thought to function primarily as inhibitors of apoptosis. The subsequent finding that a subset of IAPs that contain a RING domain have ubiquitin protein ligase (E3) activity implied the presence of other functions. It is now known that IAPs are involved in mitotic chromosome segregation, cellular morphogenesis, copper homeostasis, and intracellular signaling. Here, we review the current understanding of the roles of IAPs in apoptotic and nonapoptotic processes and explore the notion that the latter represents the primary physiologic activities of IAPs.     

The Roles of IAPs with Ubiquitin Ligase Activity in the Occurrence and Development of Malignant Tumors

The inhibitors of apoptosis proteins (IAPs) are a family of highly conserved proteins involved in apoptosis. Recent studies indicate that IAPs with RING domains act as ubiquitin E3 ligases and play an important role in the occurrence and development of malignant tumors through inhibiting the caspases and regulating MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor kappa-B) signaling. The mechanisms of IAPs in malignant tumors are complex and diverse, including resistance to cell death, inflammatory response, invasion and metastasis. IAPs inhibit apoptosis through both intrinsic and extrinsic pathways. They promote inflammatory response and regulate immune response. Besides, they both promote and inhibit tumor cell migration. Recent studies indicated that IAPs are positively correlated with poor prognosis in most malignant tumors, and negatively correlated with poor prognosis in some other few malignant tumors. The conclusions above show that it will be particularly necessary to further explore the relationship among IAPs, the occurrence and development of malignant tumors and the prognosis of patients. This review summarizes the latest research of IAPs that serve as E3s, in particular XIAP (X-chromosome linked IAP), c-IAP1 (cellular IAP1), c-IAP2 (cellular IAP2) and ML-IAP (melanoma IAP), covering the structures, functions in the malignant tumors, the signaling pathways and their correlation with the development and prognosis of malignant tumors, as well as the progress of anti-tumor drugs and therapies for IAPs. Furthermore, this review explores the problems and challenges in the current studies, which may provide new directions and strategies for future research.     

The processed isoform of the translation termination factor eRF3 localizes to the nucleus to interact with the ARF tumor suppressor

The eukaryotic releasing factor eRF3 is a multifunctional protein that plays pivotal roles in translation termination as well as the initiation of mRNA decay. eRF3 also functions in the regulation of apoptosis; eRF3 is cleaved at Ala73 by an as yet unidentified protease into processed isoform of eRF3 (p-eRF3), which interacts with the inhibitors of apoptosis proteins (IAPs). The binding of p-eRF3 with IAPs leads to the release of active caspases from IAPs, which promotes apoptosis. Although full-length eRF3 is localized exclusively in the cytoplasm, p-eRF3 localizes in the nucleus as well as the cytoplasm. We here focused on the role of p-eRF3 in the nucleus. We identified leptomycin-sensitive nuclear export signal (NES) at amino acid residues 61–71 immediately upstream of the cleavage site Ala73. Thus, the proteolytic cleavage of eRF3 into p-eRF3 leads to release an amino-terminal fragment containing NES to allow the relocalization of eRF3 into the nucleus. Consistent with this, p-eRF3 more strongly interacted with the nuclear ARF tumor suppressor than full-length eRF3. These results suggest that while p-eRF3 interacts with IAPs to promote apoptosis in the cytoplasm, p-eRF3 also has some roles in regulating cell death in the nucleus.     

Doom, a product of the Drosophila mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins.

A family of baculovirus inhibitor-of-apoptosis (IAP) genes is present in mammals, insects, and baculoviruses, but the mechanism by which they block apoptosis is unknown. We have identified a protein encoded by the Drosophila mod(mdg4) gene which bound to the baculovirus IAPs. This protein induced rapid apoptosis in insect cells, and consequently we have named it Doom. Baculovirus IAPs and P35, an inhibitor of aspartate-specific cysteine proteases, blocked Doom-induced apoptosis. The carboxyl terminus encoded by the 3' exon of the doom cDNA, which distinguishes it from other mod(mdg4) cDNAs, was responsible for induction of apoptosis and engagement of the IAPs. Doom localized to the nucleus, while the IAPs localized to the cytoplasm, but when expressed together, Doom and the IAPs both localized in the nucleus. Thus, IAPs might block apoptosis by interacting with and modifying the behavior of Doom-like proteins that reside in cellular apoptotic pathways.     

The polypeptide chain-releasing factor GSPT1/eRF3 is proteolytically processed into an IAP-binding protein

Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation.     

Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis

Despite the potential of the inhibitor of apoptosis proteins (IAPs) to block cytochrome c-dependent caspase activation, the critical function of IAPs in regulating mammalian apoptosis remains unclear. We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell. Despite being expressed at equivalent levels, endogenous IAPs afforded no protection against cytochrome c-induced apoptosis in naive pheochromocytoma (PC12) cells, but were remarkably effective in doing so in neuronally differentiated cells. Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity. Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells. These data illustrate specifically how the apoptotic pathway acquires increased regulation with cellular differentiation, and are the first to show that IAP function and apoptosome activity are coupled in cells.     

Inhibitor of apoptosis proteins (IAPs) as regulatory factors of hepatic apoptosis

IAPs are a group of regulatory proteins that are structurally related. Their conserved homologues have been identified in various organisms. In human, eight IAP members have been recognized based on baculoviral IAP repeat (BIR) domains. IAPs are key regulators of apoptosis, cytokinesis and signal transduction. The antiapoptotic property of IAPs depends on their professional role for caspases. IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. IAPs impede apoptotic process via membrane receptor-dependent (extrinsic) cascade and mitochondrial dependent (intrinsic) pathway. IAP-mediated apoptosis affects the progression of liver diseases. Therapeutic options of liver diseases may depend on the understanding toward mechanisms of the IAP-mediated apoptosis.     

The 3D's of apoptosis: death,degradation and DIAPs

Inhibitor of apoptosis proteins (IAPs) are critical regulators of apoptosis. Recent evidence suggests that in some situations, induction of apoptosis initiates general repression of translation, as well as the targeted ubiquitination and degradation of IAPs.     

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.     

Impact of protein kinase CK2 on inhibitor of apoptosis proteins in prostate cancer cells

We have previously demonstrated that protein kinase CK2 is a potent suppressor of apoptosis in cells subjected to diverse mediators of apoptosis. The process of apoptosis involves a complex series of molecules localized in various cellular compartments. Among the various proteins that modulate apoptotic activity are inhibitors of apoptosis proteins (IAPs) which are elevated in cancers and have been proposed to block caspase activity. We have examined the impact of CK2 signal on these proteins in prostate cancer cells. Cellular IAPs demonstrate distinct localization and responsiveness to altered CK2 expression or activity in the cytoplasmic and nuclear matrix fractions. Modulation of cellular CK2 by various approaches impacts on cellular IAPs such that inhibition or downregulation of CK2 results in reduction in these proteins. Further, IAPs are also reduced when cells are treated with sub-optimal concentrations of chemical inhibitors of CK2 combined with low or sub-optimal levels of apoptosis-inducing agents (such as etoposide) suggesting that downregulation of CK2 sensitizes cells to induction of apoptosis which may be related to attenuation of IAPs. Decreased IAP protein levels in response to apoptotic agents such as TNFalpha or TRAIL were potently blocked upon forced overexpression of CK2 in cells. Together, our results suggest that one of the modes of CK2-mediated modulation of apoptotic activity is via its impact on cellular IAPs.     

Apoptotic effect of fludarabine is independent of expression of IAPs in B-cell chronic lymphocytic leukemia

Despite the efficiency of fludarabine in the induction of clinical responses in B-cell chronic lymphocytic leukemia (B-CLL) patients, resistance to this drug has been documented. The present study tested whether resistance to fludarabine is related to the expression of inhibitor of apoptosis proteins (IAPs) family members. We analyzed the expression of c-IAP1, c-IAP2 and XIAP, by immunocytochemistry, in 30 blood samples from B-CLL patients and correlated protein expression to fludarabine-induced apoptosis estimated by an annexin-V assay. Expression of c-IAP1, c-IAP2 and XIAP were found predominantly in the cytoplasm, and a wide range of staining intensities was observed among distinct samples. No correlation was found between the levels of IAPs expression and prognostic factors such as age, gender, lymphocyte doubling time, white blood cell count or previous treatment. The expression of IAPs also failed to predict the sensitivity to fludarabine-induced apoptosis. Alternative pathways of cell death may explain the independence of fludarabine-induced apoptosis from the high expression of IAPs.     

Cellular inhibitor of apoptosis 1 and 2 are ubiquitin ligases for the apoptosis inducer Smac/DIABLO

Inhibitors of apoptosis (IAPs) are crucial regulators of programmed cell death. The mechanism by which IAPs prevent apoptosis has previously 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. Here we show that 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.     

Analysis of inhibitor of apoptosis protein family expression during mammary gland development

Background  

Inhibitors-of-Apoptosis-Proteins (IAPs) are an evolutionarily conserved family of proteins capable of regulating several facets of apoptosis. IAPs are frequently dysregulated in cancer, but their role in the regulation of apoptosis during developmental processes is not fully understood. Here we examined the expression of IAPs during the post-natal development of the mouse mammary gland, which is a tissue that exhibits a profound induction of apoptosis during involution.     

The inhibitor of apoptosis protein-binding domain of Smac is not essential for its proapoptotic activity

Smac/DIABLO, a recently identified inhibitor of apoptosis protein (IAP)-binding protein, is released from the mitochondria during apoptosis and reportedly potentiates apoptosis by relieving the inhibition of IAPs on caspases. We now describe the molecular characterization of Smac beta, an alternatively spliced form of Smac, which lacks the mitochondrial-targeting sequence found in Smac and has a cortical distribution in both human embryonic kidney 293 and breast epithelial tumor MCF-7 cells. Smac beta, which binds IAPs in vitro, does not bind IAPs in intact cells due to cellular processing and removal of its NH(2)-terminal IAP-binding domain. Despite its inability to interact with IAPs in cells, processed Smac beta is proapoptotic, as demonstrated by its ability to potentiate apoptosis induced by both death receptor and chemical stimuli. Furthermore, expression of a NH(2)-terminally truncated Smac mutant (Delta75), which lacks the entire IAP-interacting domain, potentiates apoptosis to the same extent as Smac and Smac beta. Our data support the hypothesis that the main proapoptotic function of Smac and Smac beta is due to a mechanism other than IAP binding.     

The ARTS Connection: Role of ARTS in Apoptosis and Cancer

ARTS is an unusual septin that is localized to mitochondria in living cells promotes apoptosis by antagonizing IAPs. ARTS functions as a tumor suppressor in Acute Lymphoblatic Leukemia (ALL) and is lost in more than of leukemic patients. The loss of ARTS is specific as levels of H5, a closely related non-apoptotic septin protein derived from the same gene, were unaffected. Thus, ARTS, a new member in the mitochondrial pro-apoptoticarsenal, provides a link between mitochondria, apoptosis and cancer.     

Neutralization of Smac/Diablo by inhibitors of apoptosis (IAPs). A caspase-independent mechanism for apoptotic inhibition

Numerous members of the IAP family can suppress apoptotic cell death in physiological settings. Whereas certain IAPs directly inhibit caspases, the chief proteolytic effectors of apoptosis, the protective effects of other IAPs do not correlate well with their caspase inhibitory activities, suggesting the involvement of alternative cytoprotective abilities. To examine this issue, we have characterized the protective effects of an ancestral, baculoviral IAP (Op-IAP) in mammalian cells. We show that although Op-IAP potently inhibited Bax-mediated apoptosis in human cells, Op-IAP failed to directly inhibit mammalian caspases. However, Op-IAP efficiently bound the IAP antagonist Smac/Diablo, thereby preventing Smac/Diablo-mediated inhibition of cellular IAPs. Whereas reduction of Smac/Diablo protein levels in the absence of Op-IAP prevented Bax-mediated apoptosis, overexpression of Smac/Diablo neutralized Op-IAP-mediated protection, and an Op-IAP variant unable to bind Smac/Diablo failed to prevent apoptosis. Finally, Op-IAP catalyzed the ubiquitination of Smac/Diablo, an activity that contributed to Op-IAP-mediated inhibition of apoptosis. These data show that cytoprotective IAPs can inhibit apoptosis through the neutralization of IAP antagonists, rather than by directly inhibiting caspases.     

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.     

IAP家族分子与肿瘤靶向治疗

凋亡抑制因子(inhibitor of apoptosis proteins,IAPs)是一类高度保守的内源性抗细胞凋亡因子家族,主要通过抑制Caspase活性和参与调节核因子NF-κB的作用而抑制细胞凋亡。细胞抗凋亡机制在肿瘤发生、发展以及肿瘤耐药性形成中发挥重要作用。肿瘤细胞高表达IAPs是导致肿瘤细胞抵抗凋亡的关键。细胞凋亡调控异常与肿瘤细胞耐药密切相关,增强肿瘤细胞对化疗药物的敏感性成为近年来肿瘤治疗的重要策略之一。该文综述了IAP家族蛋白的结构、生物学特性及其作为肿瘤治疗靶点的研究进展。