Direct interaction of Smac with NADE promotes TRAIL-induced apoptosis

Second mitochondria-derived activator of caspase (Smac) has been implicated in the activation of apoptosis in response to cell stress. We screened for Smac/DIABLO-binding protein for further understanding of Smac-mediated apoptosis. We identified NADE, previously known as p75NTR-associated cell death executor, as a Smac-binding protein. Smac-NADE interaction was mapped to the N-terminal region of Samc and the C-terminal region of NADE. Co-expression of NADE and Smac promotes TRAIL-induced apoptosis in MCF-7 cells. Interestingly, the co-presence of Smac and NADE inhibits XIAP-mediated Smac ubiquitination. In conclusion, our results provide the first evidence that the interaction between Smac and NADE regulates apoptosis through the inhibition of Smac ubiquitination.     

Sustained elevation of serum interleukin-18 and its association with hemophagocytic lymphohistiocytosis in XIAP deficiency

X-linked lymphoproliferative syndrome (XLP) is a rare primary immunodeficiency characterized by increased vulnerability to Epstein–Barr virus infection. XLP type 1 is caused by mutations in SH2D1A, whereas X-linked inhibitor of apoptosis (XIAP) encoded by XIAP/BIRC4 is mutated in XLP type 2. In XIAP deficiency, hemophagocytic lymphohistiocytosis (HLH) occurs more frequently and recurrence is common. However, the underlying mechanisms remain mostly unknown. We describe the characteristics of the cytokine profiles of serum samples from 10 XIAP-deficient patients. The concentration of interleukin (IL)-18 was strikingly elevated in the patients presented with HLH, and remained high after the recovery from HLH although levels of other pro-inflammatory cytokines approached the normal range. Longitudinal examination of two patients demonstrated marked exacerbation of IL-18 levels during every occasion of HLH. These findings may suggest the association between HLH susceptibility and high serum IL-18 levels in XIAP deficiency.     

XIAP associates with GSK3 and inhibits the promotion of intrinsic apoptotic signaling by GSK3

This study examined if there are interactions between two key proteins that oppositely regulate intrinsic apoptosis, X-linked inhibitor of apoptosis protein (XIAP), a key suppressor of apoptosis that binds to inhibit active caspases, and glycogen synthase kinase-3 (GSK3), which promotes intrinsic apoptosis. Immunoprecipitation of GSK3β revealed that XIAP associates with GSK3β, as do two other members of the IAP family, cIAP-1, and cIAP-2. Cell fractionation revealed that XIAP is predominantly cytosolic, cIAP-1 is predominantly nuclear and nearly all of the nuclear cIAP-1 and cIAP-2 are associated with GSK3. Expression of individual domains of XIAP demonstrated that the RING domain of XIAP associates with GSK3. Inhibition of GSK3 did not alter the binding of XIAP to active caspase-9 or caspase-3 after stimulation of apoptosis with staurosporine. However, inhibition of GSK3 reduced apoptosis and apoptosome formation, including the recruitments of caspase-9 and XIAP to Apaf-1, in response to staurosporine treatment. Cell free measurements of apoptosome-induced caspase-3 activation demonstrated that GSK3 acts upstream of the apoptosome to facilitate intrinsic apoptotic signaling. This facilitation was blocked by overexpression of XIAP. These findings indicate that the RING domain of XIAP (and probably cIAP-1 and cIAP-2) associates with GSK3, GSK3 acts upstream of the apoptosome to promote intrinsic apoptosis, and the association between XIAP and GSK3 may block the pro-apoptotic function of GSK3.     

大鼠脑缺血再灌注后Smac、XIAP和caspase-9表达及活血通络方的干预作用

目的：探讨大鼠脑缺血再灌注后线粒体通路第二种天冬氨酸特异性半胱氨酸蛋白酶激活物（Smac）、凋亡抑制蛋白（XIAP）和凋亡蛋白酶（caspase）-9的表达变化及活血通络方的干预作用机理。方法：将大鼠随机分成模型组、活血通络组,大脑中动脉栓塞再通法建立脑缺血再灌注模型。大鼠脑缺血再灌注6、12、24和48 h不同时间点进行神经功能评分,用免疫组化法检测Smac、XIAP和caspase-9阳性细胞数。结果：缺血再灌注后6 h模型组神经功能症状积分升高,缺血半暗带皮质内神经元凋亡增多,Smac、XIAP和caspase-9蛋白的表达亦有明显上升,再灌注12 h达高峰（P〈0.05,P〈0.01）,随后出现下降。活血通络方能显著降低神经功能症状积分,减少神经元凋亡,促进XIAP表达,下调Smac和caspase-9表达（P〈0.01,P〈0.05）。结论：脑缺血再灌注后脑组织Smac、XIAP和caspase-9蛋白的表达均明显增加,提示它们可能在脑缺血再灌注损伤中发挥重要作用,活血通络方可能通过促进XIAP并抑制Smac、caspase-9表达保护神经元及神经功能。     

Role of XIAP in inhibiting cisplatin-induced caspase activation in non-small cell lung cancer cells: a small molecule Smac mimic sensitizes for chemotherapy-induced apoptosis by enhancing caspase-3 activation

X-linked IAP (XIAP) suppresses apoptosis by binding to initiator caspase-9 and effector caspases-3 and -7. Smac/DIABLO that is released from mitochondria during apoptosis can relieve its inhibitory activity. Here we investigated the role of XIAP in the previously found obstruction of chemotherapy-induced caspase-9 activation in non-small cell lung cancer (NSCLC) cells. Endogenously expressed XIAP bound active forms of both caspase-9 and caspase-3. However, downregulation of XIAP using shRNA or disruption of XIAP/caspase-9 interaction using a small molecule Smac mimic were unable to significantly induce caspase-9 activity, indicating that despite a strong binding potential of XIAP to caspase-9 it is not a major determinant in blocking caspase-9 in NSCLC cells. Although unable to revert caspase-9 blockage, the Smac mimic was able to enhance cisplatin-induced apoptosis, which was accompanied by increased caspase-3 activity. Additionally, a more detailed analysis of caspase activation in response to cisplatin indicated a reverse order of activation, whereby caspase-3 cleaved caspase-9 yielding an inactive form. Our findings indicate that the use of small molecule Smac mimic, when combined with an apoptotic trigger, may have therapeutic potential for the treatment of NSCLC.     

Integrity of ATP binding site is essential for effective inhibition of the intrinsic apoptosis pathway by NAIP

The importance of the ATP binding site of human Neuronal Apoptosis Inhibitory Protein (NAIP) on its ability in prevention of intrinsic apoptotic pathway was investigated. Thus, ATP binding lysine 476 of NAIP, which is located at the Nucleotide Binding Oligomerization Domain (NOD) was mutated to threonine and the effect of this mutation on autoproteolysis of procaspase-9 and the cleavage of procaspase-3 by apoptosome was investigated. Formation of apoptosome was induced by the addition of cytochrome c and dATP to lysates of HeLa cells transfected with pcDNA-NAIP or pcDNA-NAIP (K476T). Full length wild type NAIP prevented the cleavage of both procaspase-9 to caspase-9 and procaspase-3 to caspase-3. However, K476T variant of NAIP did not block autocleavage of procaspase-9 efficiently. Furthermore, cleavage pattern of procaspase-9 was altered in the presence of mutant NAIP. Interestingly no effect on the procaspase-3 cleavage by apoptosome was observed. The presence of NOD domain by itself had no effect on autocleavage of procaspase-9 yet slightly reduced the cleavage of procaspase-3 by apoptosome. Pull down experiment showed direct interaction of the NOD domain of NAIP with the CARD-NOD domain of Apoptotic Protease Activating Factor 1 (APAF-1). The physical association of these domains was confirmed by pull-down assays. These observations taken with previous findings indicate that the integrity of the NOD domain is essential for effective inhibition of procaspase-9 and procaspase-3 cleavage by the NAIP protein.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.     

Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3

Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.     

Bax/Bak-dependent,Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.     

Inhibitory Phosphorylation of GSK-3 by CaMKII Couples Depolarization to Neuronal Survival

Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3α and GSK-3β, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3α/β. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3α/β mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3β protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90^RSK. CaMKII associated with and phosphorylated GSK-3α/β. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca²⁺/calmodulin/CaMKII/GSK-3 pathway that couples depolarization to neuronal survival.     

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.     

cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

Although early studies of inhibitor of apoptosis proteins (IAPs) suggested that cIAP1 directly binds and inhibits caspases similarly to X-linked IAP (XIAP), a recent one found that micromolar concentrations of cIAP1 only weakly inhibit caspase-3, -7, or -9. Here, we show that cIAP1 specifically and cooperatively blocks the cytochrome c-dependent apoptosome in vitro. Hence, cIAP1 prevented the activation of procaspase-3 but had no effect on the processing of procaspase-9 or the activity of prior activated caspase-3. Like cIAP1, XIAP had no effect on procaspase-9 processing and was a more potent inhibitor of procaspase-3 activation than of already activated caspase-3 activity. Inhibition of procaspase-3 activation depended on BIR2 and BIR3 of cIAP1 and was independent of BIR1, RING, CARD, and UBA domains. Smac prevented cIAP1 from inhibiting procaspase-3 activation and reversed the inhibition by prior addition of cIAP1. A procaspase-9 mutant (D315A) that cannot produce the p12 subunit was resistant to inhibition by cIAP1. Therefore, the N-terminal Ala-Thr-Pro-Phe motif of the p12 subunit of the caspase-9 apoptosome facilitates apoptosome blockade. Consequently, cIAP1 cooperatively interacts with oligomerized processed caspase-9 in the apoptosome and blocks procaspase-3 activation.     

Relative timing of redistribution of cytochrome c and Smac/DIABLO from mitochondria during apoptosis assessed by double immunocytochemistry on mammalian cells

Redistribution of cytochrome c and Smac/DIABLO from mitochondria occurs during apoptosis, although the relative timing of their release is not well characterized. Double immunocytochemistry was utilized here to study quantitatively the patterns of release of cytochrome c and Smac/DIABLO from mitochondria in single cells. Human osteosarcoma cells and murine embryonic cortical neurons were analyzed during apoptosis induced by staurosporine. In osteosarcoma cells treated with staurosporine for 24 h, a substantial proportion of cells (36%) released cytochrome c from the mitochondria before Smac/DIABLO. In contrast, these proteins were released mostly concordantly in neurons; only a minority of cells (< or = 15%) released cytochrome c without Smac/DIABLO (or vice versa) from mitochondria. Patterns of release in either cell type were unaltered by addition of the caspase inhibitor, zVAD-fmk. The double immunocytochemistry procedure facilitated clear definition of the temporal release of cytochrome c and Smac/DIABLO from mitochondria in intact apoptotic cells, enabling us to demonstrate for the first time that their mutual redistribution during apoptosis varies between different cell types.     

Inhibition of JNK2 and JNK3 by JNK inhibitor IX induces prometaphase arrest-dependent apoptotic cell death in human Jurkat T cells

Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G₂/M arrest, phosphorylation of Bcl-2, Mcl-1, and Bim, Δψm loss, and activation of Bak and caspase cascade. These JNKi-induced apoptotic events were abrogated by Bcl-2 overexpression, whereas G₂/M arrest, cyclin B1 up-regulation, Cdk1 activation, and phosphorylation of Bcl-2 family proteins were sustained. In the concomitant presence of the G₁/S blocking agent aphidicolin and JNKi, the cells underwent G₁/S arrest and failed to induce all apoptotic events. The JNKi-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by the Cdk1 inhibitor. Immunofluorescence microscopic analysis revealed that mitotic spindle defect and prometaphase arrest were the underlying factors for the G₂/M arrest. These results demonstrate that JNKi-induced mitochondrial apoptosis was caused by microtubule damage-mediated prometaphase arrest, prolonged Cdk1 activation, and phosphorylation of Bcl-2 family proteins in Jurkat T cells.     

Phosphorylation of glycosyl-phosphatidylinositol by phosphatidylinositol 3-kinase changes its properties as a substrate for phospholipases

Phosphatidylinositol 3-kinases (PI3K) phosphorylate the 3-position of the inositol ring of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate. It is not clear whether PI3K can phosphorylate the inositol group in other biomolecules. We sought to determine whether PI3K was able to use glycosyl-phosphatidylinositol (GPI) as a substrate. This phospholipid may exist either in free form (GPIfree) or forming a lipid anchor (GPIanchor) for the attachment of extracellular proteins to the plasma membrane. We demonstrate the specific PI3K-mediated phosphorylation of the inositol 3-hydroxyl group within both types of GPI by incubating this phospholipid with immunoprecipitated PI3K. The phosphorylated product behaves in HPLC as a derivative of a PI3K lipid product. To our knowledge, this is the first demonstration that PI3K uses lipid substrates other than phosphoinositides. Further, we show that this has potential functional consequences. When GPIfree is phosphorylated, it becomes a poorer substrate for GPI-specific phospholipase D, but a better substrate for phosphatidylinositol-specific phospholipase C. These phosphorylation events may constitute the basis of a previously undescribed signal transduction mechanism.     

小檗碱通过JNK通路调控柯萨奇病毒B3感染心肌细胞凋亡的作用

小檗碱是具有细胞保护作用的生物碱,能够在柯萨奇病毒B3(CVB3)感染引起的病毒性心肌炎小鼠中发挥心肌保护作用,但具体的机制未阐明。在内皮细胞中,小檗碱通过c-Jun氨基末端激酶(JNK)通路抑制细胞凋亡,因此本研究将分析小檗碱通过JNK通路调控CVB3感染心肌细胞凋亡的作用。H9c2心肌细胞分为对照组(不含药物的DMEM处理)、模型组(含CVB3的DMEM处理)、小檗碱组(含CVB3及小檗碱的DMEM处理)、小檗碱+JNK质粒组(含CVB3、小檗碱、JNK质粒的DMEM处理),检测细胞凋亡率、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、活性氧(ROS)、丙二醛(MDA)的含量、p-JNK、cleaved caspase-3、bax、bcl-2的表达量。结果显示,模型组的细胞凋亡率、TNF-α、IL-6、ROS、MDA的含量、p-JNK、cleaved caspase-3、bax的表达量高于对照组,bcl-2的表达量低于对照组(P<0.05);小檗碱组的细胞凋亡率、TNF-α、IL-6、ROS、MDA的含量、p-JNK、cleaved caspase-3、bax的表达量低于模型组,bcl-2的表达量高于模型组(P<0.05);小檗碱+JNK质粒组的细胞凋亡率、TNF-α、IL-6、ROS、MDA的含量、p-JNK、cleaved caspase-3、bax的表达量高于小檗碱组,bcl-2的表达量低于小檗碱组(P<0.05)。以上结果表明小檗碱对CVB3感染心肌细胞的凋亡具有抑制作用,抑制JNK通路是介导这一作用可能的分子机制。     

Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal.