Inhibitor of Apoptosis (IAP) Proteins–Modulators of Cell Death and Inflammation

Misregulated innate immune signaling and cell death form the basis of much human disease pathogenesis. Inhibitor of apoptosis (IAP) protein family members are frequently overexpressed in cancer and contribute to tumor cell survival, chemo-resistance, disease progression, and poor prognosis. Although best known for their ability to regulate caspases, IAPs also influence ubiquitin (Ub)-dependent pathways that modulate innate immune signaling via activation of nuclear factor κB (NF-κB). Recent research into IAP biology has unearthed unexpected roles for this group of proteins. In addition, the advances in our understanding of the molecular mechanisms that IAPs use to regulate cell death and innate immune responses have provided new insights into disease states and suggested novel intervention strategies. Here we review the functions assigned to those IAP proteins that act at the intersection of cell death regulation and inflammatory signaling.Apoptosis represents a fundamental biological process that relies on the activation of caspases. Inhibitor of apoptosis (IAP) proteins represent a group of negative regulators of both caspases and cell death. Although best known for their ability to regulate caspases and cell death, it is now clear that they function as arbiters of diverse biological processes (Gyrd-Hansen and Meier 2010). Most prominently, IAPs control ubiquitin (Ub)-dependent signaling events that regulate activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways that in turn drive expression of genes important for inflammation, immunity, cell migration, and cell survival. IAPs thereby function as E3 Ub ligases, mediating the transfer of Ub from E2s to target substrates. This in turn modulates the signaling process through regulating protein stability as well as via nondegradative means (see below for details). Many of the cellular processes controlled by IAPs are frequently deregulated in cancer and, directly or indirectly, contribute to disease initiation, tumor maintenance, and/or progression, making IAPs obvious targets for anticancer therapy (LaCasse et al. 2008). Accordingly, small pharmacological inhibitors of IAPs, frequently referred to as Smac-mimetics (SM), were developed and are currently undergoing clinical trials for the treatment of cancer (LaCasse et al. 2008). The use of SMs in preclinical tumor models and clinical trials has provided compelling evidence for the therapeutic benefit of IAP inhibition.     

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 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.     

Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition

Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis of apoptotic substrates. IAPs occlude the active sites of caspases to which they are bound and can function as ubiquitin ligases. IAPs are also reported to ubiquitinate themselves and caspases. Several proteins induce apoptosis, at least in part, by binding and inhibiting IAPs. Among these are the Drosophila melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal IAP-binding motif. We report here that Rpr not only inhibits IAP function, but also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation required both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibited protein translation. As the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis.     

Reaper is regulated by IAP-mediated ubiquitination

In most cases, apoptotic cell death culminates in the activation of the caspase family of cysteine proteases, leading to the orderly dismantling and elimination of the cell. The IAPs (inhibitors of apoptosis) comprise a family of proteins that oppose caspases and thus act to raise the apoptotic threshold. Disruption of IAP-mediated caspase inhibition has been shown to be an important activity for pro-apoptotic proteins in Drosophila (Reaper, HID, and Grim) and in mammalian cells (Smac/DIABLO and Omi/HtrA2). In addition, in the case of the fly, these proteins are able to stimulate the ubiquitination and degradation of IAPs by a mechanism involving the ubiquitin ligase activity of the IAP itself. In this report, we show that the Drosophila RHG proteins (Reaper, HID, and Grim) are themselves substrates for IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, we also show that regulation of Reaper by ubiquitination is a significant factor in determining its biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance.     

Understanding IAP function and regulation: a view from Drosophila

Apoptosis is an active form of cell suicide that results in the orderly death and phagocytosis of cells during normal development and in the adult. Many death signals lead to the activation of members of a family of cysteine proteases known as caspases. These proteins act to transduce death signals from different cellular compartments and they cleave a number of cellular proteins, leading ultimately to many of the biochemical and morphological events associated with death. Many mechanisms act to inhibit cell death upstream of caspase activation. However, only one family of cellular proteins, the inhibitors of apoptosis (IAPs), has been identified that inhibit caspase activation and/or activity. The observations that IAP function is essential for cell survival in Drosophila, and that IAP expression is deregulated in many forms of cancer in humans, argue that IAPs are important cell death inhibitors and that deregulation of their function is likely to be important in human disease. Here we review IAP function, with particular reference to insights that study of the Drosophila IAPs has provided. We also discuss some directions for future study.     

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 RING domain of cIAP1 mediates the degradation of RING-bearing inhibitor of apoptosis proteins by distinct pathways

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR-mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.     

Regulators of IAP function: coming to grips with the grim reaper

Inhibitor of apoptosis proteins (IAPs) are a conserved class of proteins that control apoptosis in both vertebrates and invertebrates. They exert their anti-apoptotic function through inhibition of caspases, the principal executioners of apoptotic cell death. Recent advances in vertebrates and Drosophila have demonstrated that IAPs use ubiquitin conjugation to control the stability, and thus the activity, of select target proteins. The Drosophila IAP1 gene is an instructive example: it employs at least two distinct ubiquitin-dependent mechanisms of protein destruction. The apoptosis-inducing genes grim, reaper and hid modulate these mechanisms, and determine the outcome.     

(Un)expected roles of c-IAPs in apoptotic and NFκB signaling pathways

A family of anti-apoptotic regulators known as inhibitor of apoptosis (IAP) proteins block cell death in response to diverse stimuli. In spite of the fact that cellular IAP1and 2 (c IAP1 and 2) were discovered more than 12 years ago, their physiological roles have remained obscure. Several molecular mechanisms were proposed to explain their anti-apoptotic activity, ranging from direct inhibition and ubiquitination of pro-apoptotic molecules, to the activation of pro-survival signaling. New findings present a surprising and complex twists. On the one hand, c IAP1 and c IAP2 suppress Tumor Necrosis Factor α (TNFα) stimulated cell death by preventing formation of the TNF Receptor 1 (TNFR1) pro-apoptotic signaling complex. On the other hand, they regulate pro-survival NFκB signaling pathways: in the non-canonical pathway, by ubiquitination of NFκB-inducing kinase (NIK), and in the canonical pathway, by a yet-to-be-defined mechanism. In addition, c IAPs self-regulate their protein levels through RING domain mediated auto-ubiquitination. Here, we discuss the most recent progress in our understanding of the biological roles of c-IAPs, as well as the implications of targeting c IAPs for therapeutic intervention.     

泛素链的形成机理

泛素化是真核细胞中重要的蛋白质翻译后修饰过程,通过靶向蛋白质降解或其他信号途径参与多种细胞功能.底物蛋白的多聚泛素化修饰是一个持续的过程,其中不仅涉及复杂泛素系统相关酶的参与,而且存在更为复杂的结构上相互作用与泛素链组装机理.不同的泛素链修饰决定了底物蛋白下游的不同命运,泛素结合酶E2在泛素链形成中的重要作用受到越来越多的关注.对泛素链形成机理的深入研究与认识有利于发现与泛素系统相关的疾病靶点和利用泛素化调控方法进行治疗.本综述总结了E2和E3如何决定不同泛素链形成的机制和相关的结构信息,以及两种不同的泛素链组装机制.     

Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.     

IAPs, RINGs and ubiquitylation

The inhibitor of apoptosis (IAP) proteins all contain one or more baculoviral IAP repeat motifs, through which they interact with various other proteins. Many IAPs also have another zinc-binding motif, the RING domain, which can recruit E2 ubiquitin-conjugating enzymes and catalyse the transfer of ubiquitin onto target proteins. The number of targets of IAP-mediated ubiquitylation is increasing and recent results indicate that outcomes following ubiquitylation are tantalizingly complex. As well as regulating other proteins, the IAPs themselves are controlled by ubiquitin-mediated degradation.     

Drosophila morgue and the intersection between protein ubiquitination and programmed cell death

In Drosophila, cell survival decisions are mediated by the integrated functions of the Grim-Reaper death activators and Inhibitor-of-Apoptosis-Proteins (IAPs), such as DIAP1, to regulate caspase activities. We recently identified a gene that enhances the actions of the Grim-Reaper proteins and negatively regulates the levels of DIAP1 protein. This gene, morgue, encodes a novel protein that contains both an F box and a ubiquitin conjugase domain. Interestingly, the Morgue conjugase domain lacks the active site cysteine required for covalent linkage to ubiquitin. Morgue could target IAPs and other proteins for ubiquitination and proteasome-dependent turnover by acting either in an SCF ubiquitin E3 ligase complex, or as a ubiquitin E2 conjugase enzyme variant (UEV) in conjunction with a catalytically active E2 conjugase. Morgue is evolutionarily conserved, as a Morgue ortholog was identified from the mosquito, Anopheles gambiae. Elucidation of morgue function should provide novel insights into the mechanisms of ubiquitination and programmed cell death.     

Inhibitor of apoptosis proteins and apoptosis

Apoptosis is a physiological cell death process that plays a critical role in development, homeostasis, and immune defense of multicellular animals. Inhibitor of apoptosis proteins (IAPs) constitute a family of proteins that possess between one and three baculovirus IAP repeats. Some of them also have a really interesting new gene finger domain, and can prevent cell death by binding and inhibiting active caspases, but are regulated by IAP antagonists. Some evidence also indicates that IAP can modulate the cell cycle and signal transduction. The three main factors, IAPs, IAP antagonists, and caspases, are involved in regulating the progress of apoptosis in many species. Many studies and assumptions have been focused on the anfractuous interactions between these three main factors to explore their real functional model in order to develop potential anticancer drugs. In this review, we describe the classification, molecular structures, and properties of IAPs and discuss the mechanisms of apoptosis. We also discuss the promising significance of clinical applications of IAPs in the diagnosis and treatment of malignancy.     

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.     

Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs

Apoptosis is a physiological cell death process important for development, homeostasis and the immune defence of multicellular animals. The key effectors of apoptosis are caspases, cysteine proteases that cleave after aspartate residues. The inhibitor of apoptosis (IAP) family of proteins prevent cell death by binding to and inhibiting active caspases and are negatively regulated by IAP-binding proteins, such as the mammalian protein DIABLO/Smac. IAPs are characterized by the presence of one to three domains known as baculoviral IAP repeat (BIR) domains and many also have a RING-finger domain at their carboxyl terminus. More recently, a second group of BIR-domain-containing proteins (BIRPs) have been identified that includes the mammalian proteins Bruce and Survivin as well as BIR-containing proteins in yeasts and Caenorhabditis elegans. These Survivin-like BIRPs regulate cytokinesis and mitotic spindle formation. In this review, we describe the IAPs and other BIRPs, their evolutionary relationships and their subcellular and tissue localizations.     

Morgue mediates apoptosis in the Drosophila melanogaster retina by promoting degradation of DIAP1

Inhibitor of apoptosis proteins (IAPs) provide a critical barrier to inappropriate apoptotic cell death through direct binding and inhibition of caspases. We demonstrate that degradation of IAPs is an important mechanism for the initiation of apoptosis in vivo. Drosophila Morgue, a ubiquitin conjugase-related protein, promotes DIAP1 down-regulation in the developing retina to permit selective programmed cell death. Morgue complexes with DIAP1 in vitro and mediates DIAP1 degradation in a manner dependent on the Morgue UBC domain. Reaper (Rpr) and Grim, but not Hid, also promote the degradation of DIAP1 in vivo, suggesting that these proteins promote cell death through different mechanisms.