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.     

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.     

Smac/Diablo antagonizes ubiquitin ligase activity of inhibitor of apoptosis proteins

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through binding to active caspases and antagonizing their function. IAP function can be neutralized by Smac/Diablo, an IAP-binding protein that is released from mitochondria during apoptosis. In addition to their ability to interact with caspases, certain IAPs also display ubiquitin-protein isopeptide ligase activity because of the presence of a RING domain. However, it is not known whether the ubiquitin-protein isopeptide ligase activities of human IAPs contribute to their apoptosis inhibitory activity or whether this IAP property can be modulated through association with Smac/Diablo. Here we demonstrate that the ubiquitin ligase activities of XIAP, and to a lesser extent c-IAP-1 and c-IAP2, are potently repressed through binding to Smac/Diablo. We also show that mutation of the XIAP RING domain rendered this IAP a less effective inhibitor of apoptosis, suggesting that the ubiquitin ligase activity of XIAP contributes to its anti-apoptotic function. These data suggest that Smac/Diablo potentiates apoptosis by simultaneously antagonizing caspase-IAP interactions and repressing IAP ubiquitin ligase activities.     

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.     

IAPS and ubiquitylation

The Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death, whose amplification has been correlated with tumor progression. Due to the presence of a RING domain, IAP proteins are classed as ubiquitin ligases and regulate cell survival by orchestrating a variety of ubiquitin modifications. Ubiquitin protein modification is fundamental in cell signaling and different ubiquitin modifications may label proteins for destruction, relocalization or provide a recruitment platform for ubiquitin binding proteins. Ubiquitin performs a myriad of different functions because it can be conjugated to a large range of target proteins through numerous different types of ubiquitin linkages. Despite the fact that ubiquitin is extremely versatile, the E3s such as the IAPs provide an important level of control due to their specificity for certain substrates. Several recent reviews have discussed the role of IAPs in regulating immune signaling so we have therefore focused our review on the interplay between IAPs and ubiquitin and discussed the importance of this relationship for the regulation of themselves, specific substrates and various cell death and survival signaling 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.     

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.     

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.     

Mathematical modeling of the regulation of caspase-3 activation and degradation

Caspases are thought to be important players in the execution process of apoptosis. Inhibitors of apoptosis (IAPs) are able to block caspases and therefore apoptosis. The fact that a subgroup of the IAP family inhibits active caspases implies that not each caspase activation necessarily leads to apoptosis. In such a scenario, however, processed and enzymically active caspases should somehow be removed. Indeed, IAP-caspase complexes covalently bind ubiquitin, resulting in degradation by the 26S proteasome. Following release from mitochondria, IAP antagonists (e.g. second mitochondrial activator of caspases (Smac)) inactivate IAPs. Moreover, although pro-apoptotic factors such as irradiation or anti-cancer drugs may release Smac from mitochondria in tumor cells, high cytoplasmic survivin and ML-IAP levels might be able to neutralize it and, consequently, IAPs would further be able to bind activated caspases. Here, we propose a simple mathematical model, describing the molecular interactions between Smac deactivators, Smac, IAPs, and caspase-3, including the requirements for both induction and prevention of apoptosis, respectively. In addition, we predict a novel mechanism of caspase-3 degradation that might be particularly relevant in long-living cells.     

The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination

Human cIAP1 protein is a member of the inhibitor of apoptosis proteins (IAPs) that are involved in apoptosis regulation and an increasing number of other functions, including cell cycle and intracellular signal transduction. In order to identify novel proteins involved in cIAP1 regulation, we performed a yeast two-hybrid screen and identified an F-box protein Fbxo7 as a cIAP1 interacting protein. Co-immunoprecipitation assay showed that cIAP1 can interact with Fbxo7 in human cells. When co-expressed in cells, cIAP1 and Fbxo7 co-localized remarkably both in the cytoplasm and nucleus, and considerable amounts of these often co-localized at one or few distinct Golgi-like structures close to the nucleus. Furthermore, we showed that overexpression of Fbxo7 promotes the ubiquitination of cIAP1. Since F-box proteins are specificity determining subunits of SCF ubiquitin protein ligases, our results suggest that Fbxo7 can mediate the ubiquitination of cIAP1 by SCF ubiquitin protein ligase and thus have important implication in the regulation of cIAP1 function.     

Structural analysis of the UBA domain of X-linked inhibitor of apoptosis protein reveals different surfaces for ubiquitin-binding and self-association

Background

Inhibitor of apoptosis proteins (IAPs) belong to a pivotal antiapoptotic protein family that plays a crucial role in tumorigenesis, cancer progression, chemoresistance and poor patient-survival. X-linked inhibitor of apoptosis protein (XIAP) is a prominent member of IAPs attracting intense research because it has been demonstrated to be a physiological inhibitor of caspases and apoptosis. Recently, an evolutionarily conserved ubiquitin-associated (UBA) domain was identified in XIAP and a number of RING domain-bearing IAPs. This has placed the IAPs in the group of ubiquitin binding proteins. Here, we explore the three-dimensional structure of the XIAP UBA domain (XIAP-UBA) and how it interacts with mono-ubiquitin and diubiquitin conjugates.

Principal Findings

The solution structure of the XIAP-UBA domain was determined by NMR spectroscopy. XIAP-UBA adopts a typical UBA domain fold of three tightly packed α-helices but with an additional N-terminal 3₁₀ helix. The XIAP-UBA binds mono-ubiquitin as well as Lys48-linked and linear-linked diubiquitins at low-micromolar affinities. NMR analysis of the XIAP-UBA–ubiquitin interaction reveals that it involves the classical hydrophobic patches surrounding Ile44 of ubiquitin and the conserved MGF/LV motif surfaces on XIAP-UBA. Furthermore, dimerization of XIAP-UBA was observed. Mapping of the self-association surface of XIAP-UBA reveals that the dimerization interface is formed by residues in the N-terminal 3₁₀ helix, helix α1 and helix α2, separate from the ubiquitin-binding surface.

Conclusion

Our results provide the first structural information of XIAP-UBA and map its interaction with mono-ubiquitin, Lys48-linked and linear-linked diubiquitins. The notion that XIAP-UBA uses different surfaces for ubiquitin-binding and self-association provides a plausible model to explain the reported selectivity of XIAP in binding polyubiquitin chains with different linkages.     

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates.     

The USP19 deubiquitinase regulates the stability of c-IAP1 and c-IAP2

The inhibitors of apoptosis (IAPs) are critical regulators of apoptosis and other fundamental cellular processes. Many IAPs are RING domain-containing ubiquitin E3 ligases that control the stability of their interacting proteins. However, how IAP stability is regulated remains unclear. Here we report that USP19, a deubiquitinating enzyme, interacts with cellular IAP 1 (c-IAP1) and c-IAP2. Knockdown of USP19 decreases levels of both c-IAPs, whereas overexpression of USP19 results in a marked increase in c-IAP levels. USP19 effectively removes ubiquitin from c-IAPs in vitro, but it stabilizes c-IAPs in vivo mainly through deubiquitinase-independent mechanisms. The deubiquitinase activity is involved in the stabilization of USP19 itself, which is facilitated by USP19 self-association. Functionally, knockdown of USP19 enhances TNFα-induced caspase activation and apoptosis in a c-IAP1 and 2-dependent manner. These results suggest that the self-ubiquitin ligase activity of c-IAPs is inhibited by USP19 and implicate deubiquitinating enzymes in the regulation of IAP stability.     

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.     

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

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

A novel ubiquitin fusion system bypasses the mitochondria and generates biologically active Smac/DIABLO

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 (IAP) 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, we document a novel expression system using 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 was 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.     

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.     

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.