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.     

Smac Mimetic SM-164 Potentiates APO2L/TRAIL- and Doxorubicin-Mediated Anticancer Activity in Human Hepatocellular Carcinoma Cells

Background

The members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics.

Methods

Four HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms.

Results

Although SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation.

Conclusions

Smac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.     

Inhibitors of apoptosis proteins in experimental benign prostatic hyperplasia: effects of serenoa repens,selenium and lycopene

Background

The apoptosis machinery is a promising target against benign prostatic hyperplasia (BPH). Inhibitors of apoptosis proteins (IAPs) modulate apoptosis by direct inhibition of caspases. Serenoa Repens (SeR) may be combined with other natural compounds such as Lycopene (Ly) and Selenium (Se) to maximize its therapeutic activity in BPH. We investigated the effects of SeR, Se and Ly, alone or in association, on the expression of four IAPs, cIAP-1, cIAP-2, NAIP and survivin in rats with experimental testosterone-dependent BPH. Moreover, caspase-3, interleukin-6 (IL-6) and prostate specific membrane antigen (PSMA) have been evaluated.Rats were administered, daily, with testosterone propionate (3 mg/kg/sc) or its vehicle for 14 days. Testosterone injected animals (BPH) were randomized to receive vehicle, SeR (25 mg/kg/sc), Se (3 mg/kg/sc), Ly (1 mg/kg/sc) or the SeR-Se-Ly association for 14 days. Animals were sacrificed and prostate removed for analysis.

Results

BPH animals treated with vehicle showed unchanged expression of cIAP-1 and cIAP-2 and increased expression of NAIP, survivin, caspase-3, IL-6 and PSMA levels when compared with sham animals. Immunofluorescence studies confirmed the enhanced expression of NAIP and survivin with a characteristic pattern of cellular localization. SeR-Se-Ly association showed the highest efficacy in reawakening apoptosis; additionally, this therapeutic cocktail significantly reduced IL-6 and PSMA levels. The administration of SeR, Se and Ly significantly blunted prostate overweight and growth; moreover, the SeR-Se-Ly association was most effective in reducing prostate enlargement and growth by 43.3% in treated animals.

Conclusions

The results indicate that IAPs may represent interesting targets for drug therapy of BPH.     

Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells

Background  

During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.     

Apoptosis of resident and inflammatory macrophages before and during the inflammatory response of the virgin bovine mammary gland

Background  

Macrophages may play a prominent role in defense of the bovine mammary gland, and their functionality is necessary for successful eradication of bacterial pathogens. In contrast to necrosis, however, apoptosis has not yet been studied in macrophages from bovine mammary glands. Therefore, the aim of this study was to confirm the occurrence of apoptosis in macrophages from resting heifer mammary glands and during the inflammatory response.     

Developmental expression of survivin during embryonic submandibular salivary gland development

Background  

The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival.     

Host Insect Inhibitor-of-Apoptosis SfIAP Functionally Replaces Baculovirus IAP but Is Differentially Regulated by Its N-Terminal Leader

The inhibitor-of-apoptosis (IAP) proteins encoded by baculoviruses bear a striking resemblance to the cellular IAP homologs of their invertebrate hosts. By virtue of the acquired selective advantage of blocking virus-induced apoptosis, baculoviruses may have captured cellular IAP genes that subsequently evolved for virus-specific objectives. To compare viral and host IAPs, we defined antiapoptotic properties of SfIAP, the principal cellular IAP of the lepidopteran host Spodoptera frugiperda. We report here that SfIAP prevented virus-induced apoptosis as well as viral Op-IAP3 (which is encoded by the Orgyia pseudotsugata nucleopolyhedrovirus) when overexpressed from the baculovirus genome. Like Op-IAP3, SfIAP blocked apoptosis at a step prior to caspase activation. Both of the baculovirus IAP repeats (BIRs) were required for SfIAP function. Moreover, deletion of the C-terminal RING motif generated a loss-of-function SfIAP that interacted and dominantly interfered with wild-type SfIAP. Like Op-IAP3, wild-type SfIAP formed intracellular homodimers, suggesting that oligomerization is a functional requirement for both cellular and viral IAPs. SfIAP possesses a ∼100-residue N-terminal leader domain, which is absent among all viral IAPs. Remarkably, deletion of the leader yielded a fully functional SfIAP with dramatically increased protein stability. Thus, the SfIAP leader contains an instability motif that may confer regulatory options for cellular IAPs that baculovirus IAPs have evolved to bypass for maximal stability and antiapoptotic potency. Our findings that SfIAP and viral IAPs have common motifs, share multiple biochemical properties including oligomerization, and act at the same step to block apoptosis support the hypothesis that baculoviral IAPs were derived by acquisition of host insect IAPs.Apoptosis is a prevalent host cell response to virus infection. Representing an important antivirus defense, apoptotic cell death can limit multiplication and virus dissemination in the host. Thus, the mechanisms by which a host organism detects a viral intruder and initiates the apoptotic response are critical to the outcome of the infection for both the host and virus. The cellular inhibitor-of-apoptosis (IAP) proteins are important candidates for sensing virus infection and determining cell fate by virtue of their central position in the apoptosis pathway (reviewed in references 35, 36, and 44). Affirming their importance in regulation of apoptosis, IAPs are encoded by multiple DNA viruses, including baculoviruses, entomopoxviruses, iridoviruses, and African swine fever virus (reviewed in 3). Nonetheless, the molecular mechanisms by which viral IAPs regulate virus-induced apoptosis and how they biochemically differ from cellular IAPs are poorly understood.The IAPs were first discovered in baculoviruses because of their capacity to prevent virus-induced apoptosis and thereby facilitate virus multiplication (4, 8). The baculovirus IAPs bear a striking resemblance to the cellular IAPs carried by the host insects that they infect. Cellular IAPs are a highly conserved family of survival factors that regulate developmental and stress-induced apoptosis, as well as inflammation, the cell cycle, and other signaling processes (35, 38, 44). Importantly, misregulation or overexpression of IAPs is associated with neoplasia and tumor chemoresistance (24, 49). The IAPs are defined by the presence of one or more ∼80-residue baculovirus IAP repeat (BIR) domains. The BIRs consist of a conserved Zn²⁺-coordinating arrangement of Cys and His residues (CCHC) that interact with diverse proteins, including the cysteinyl aspartate-specific proteases called caspases that execute apoptosis (reviewed in 16 and 37). The antiapoptotic activity of some, but not all, IAPs is derived from their ability to bind and neutralize caspases (reviewed in 35 and 44). The BIRs also interact with proapoptotic factors that contain IAP binding motifs (IBMs). IBM-containing factors have the capacity to bind and dissociate the IAP-caspase complex, thereby liberating active caspases to execute apoptosis (16, 35, 36, 48). Many IAPs, including viral IAPs, also possess a C-terminal RING domain, which is a Zn²⁺-coordinating motif with E3-ubiquitin ligase activity, which can contribute to antiapoptotic activity (48).The best-studied baculovirus IAP is Op-IAP3, which is encoded by Orgyia pseudotsugata nucleopolyhedrovirus. This small IAP (268 residues) contains two BIRs and a C-terminal RING (Fig. ​(Fig.1A).1A). Both BIRs are required for Op-IAP3 antiapoptotic activity (19, 50, 53). Truncation of the Op-IAP3 RING creates a loss-of-function dominant inhibitor (19). Op-IAP3''s capacity to form a complex with this RING-lacking (RINGless) dominant inhibitor and with itself suggests that oligomerization is necessary for IAP function. Upon overexpression, Op-IAP3 blocks apoptosis triggered by diverse signals in cells from certain insects and mammals, suggesting that it acts through a conserved mechanism (7, 11, 15, 33, 51, 54, 56). In the baculovirus host moth Spodoptera frugiperda (Lepidoptera: Noctuidae), Op-IAP3 prevents apoptosis by blocking the activation of effector caspases (25, 32, 40). However, in contrast to host insect IAPs, Op-IAP3 fails to inhibit active caspases (45, 51, 54). Thus, the host cell target(s) and the mechanism by which they are neutralized by this viral IAP remain unclear.Open in a separate windowFIG. 1.SfIAP structure and mutagenesis. (A) Viral and cellular IAPs. Viral Op-IAP3 (268 residues) and SfIAP (377 residues) each contain two BIR motifs (black boxes) and an E3 ligase RING domain (cross-hatched box). Each representing a potential start site, four methionines (M1 to M4) exist in the N-terminal leader of SfIAP. (B) SfIAP^M4 mutations. SfIAP^M4 (281 residues) begins with the M4 methionine. SfIAP^M4ΔR (227 residues) lacks the C-terminal RING. Amino acid substitutions of Zn-coordinating residues are indicated. An epitope tag (HA) was inserted at the N terminus. (C) Marker rescue assay. The antiapoptotic activity of wild-type or mutated forms of SfIAP^M4 was assayed by virus marker rescue in which replication of p35-deficient vΔp35/lacZ was restored in proportion to the antiapoptotic activity of the mutated Sfiap gene acquired by integration of the SfIAP-encoding plasmid (2). Virus yields were determined by plaque assay using apoptosis-sensitive SF21 cells. Antiapoptotic activity is reported as the ratio of nonapoptotic, lacZ-expressing plaques produced by transfection of the indicated Sfiap to those produced by wild-type Sfiap. Values shown are the averages ± standard deviations obtained from triplicate transfections.Among the cellular IAPs, SfIAP from Spodoptera frugiperda is most closely related to viral Op-IAP3. SfIAP (Fig. ​(Fig.1A)1A) is 42% identical to Op-IAP3, with a higher degree of amino acid identity localized to its two BIRs and C-terminal RING (20). As the principal IAP in Spodoptera, SfIAP suppresses a constitutive push toward apoptosis (34); ablation of SfIAP leads to immediate apoptosis of cultured Spodoptera cells. Upon overexpression, SfIAP also rescues the multiplication of apoptosis-inducing baculoviruses and can prevent apoptosis in certain mammalian cell lines (20, 26). In contrast to viral Op-IAP3, SfIAP can bind and inhibit caspases, including Spodoptera frugiperda caspase-1 (Sf-caspase-1) and human caspase-9 (20, 45). Thus, despite their structural similarities, there exist fundamental differences in the biochemical activities of these two IAPs. Importantly, SfIAP fails to prevent baculovirus-induced apoptosis when produced at endogenous levels in permissive Spodoptera cells. Thus, it is expected that SfIAP also possesses regulatory motifs that respond to cellular signals triggered upon virus infection.SfIAP provides an unprecedented opportunity to investigate the functional and evolutionary relationships between host and viral IAPs and to test the intriguing hypothesis that viral IAPs were acquired by host gene capture (21). We have investigated the biochemical properties of SfIAP as a means to define its molecular mechanisms and to test its relatedness to viral IAPs. We report here that SfIAP shares many biochemical and functional features with viral IAPs. Like Op-IAP3, overexpressed SfIAP prevented virus-induced apoptosis at a step upstream of caspase activation by a mechanism that required BIR1, BIR2, and the RING. SfIAP formed a complex with itself and with a RINGless dominant inhibitor, suggesting that oligomerization is also required for function of cellular IAPs. Unlike viral IAPs, SfIAP possesses an N-terminal leader, which modulates intracellular SfIAP levels and may respond to apoptotic signals to regulate cell survival. Our data are consistent with a model in which baculoviruses acquired a host cell IAP and modified it for virus-specific needs, thereby increasing virus fitness by preventing virus-induced apoptosis.     

Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-κB-dependent pathway

Introduction  

Methotrexate (MTX) induces macrophage apoptosis in vitro, but there is not much evidence for increased synovial macrophage apoptosis in MTX-treated patients. Macrophage apoptosis is reported, however, during clinical response to anti-tumor necrosis factor-alpha (TNF-α) treatments. This implies that TNF-α promotes macrophage survival and suggests that TNF-α may protect against MTX-induced apoptosis. We, therefore, investigated this proposal and the macrophage signaling pathways underlying it.     

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye

Background  

Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, ras ^KP , which causes excessive apoptosis in the Drosophila eye.     

Defining the antigen receptor-dependent regulatory network that induces arrest of cycling immature B-lymphocytes

Background  

Engagement of the antigen receptor on immature B-lymphocytes leads to cell cycle arrest, and subsequent apoptosis. This is an essential process for eliminating self reactive B cells during its different stages of development. However, the mechanism by which it is achieved is not completely understood.     

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.     

Valproic acid inhibits neural progenitor cell death by activation of NF-κB signaling pathway and up-regulation of Bcl-XL

Background  

At the beginning of neurogenesis, massive brain cell death occurs and more than 50% of cells are eliminated by apoptosis along with neuronal differentiation. However, few studies were conducted so far regarding the regulation of neural progenitor cells (NPCs) death during development. Because of the physiological role of cell death during development, aberration of normal apoptotic cell death is detrimental to normal organogenesis.     

Effect of the degree of ischaemic injury and reoxygenation time on the type of myocardial cell death in man: role of caspases

Background  

The importance of apoptosis in the injury sustained by the human myocardium during ischaemia and reoxygenation and the underlying mechanisms remain unclear. To quantify apoptosis and necrosis induced by simulated ischaemia/reoxygenation in the human atrial myocardium, free-hand sections of right atrial appendage (n = 8/group) were subjected to 90 minutes simulated ischaemia followed by 2, 8 and 24 hours reoxygenation.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).     

MiR-204 regulates cardiomyocyte autophagy induced by ischemia-reperfusion through LC3-II

Background  

Autophagy plays a significant role in myocardial ischemia-reperfusion (IR) injury. So it is important to inhibit autophagy to protect cardiomyocytes besides anti-apoptosis. MiRNA has been demonstrated to protect cardiomyocytes against apoptosis during IR, while whether it has anti-autophagy effect has not been known. The aim of this study was to investigate whether miR-204 regulated autophagy by regulating LC3-II protein, which is the marker of autophagosome during myocardial IR injury.     

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.     

Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

Background  

During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-β isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.