Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Application of Cytochrome C-Related Genes in Prognosis and Treatment Prediction of Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common subtype of nonsmall cell lung cancer. Cytochrome c (Cyt c), which is produced from mitochondria, interacts with a protein called Apaf-1 to form the heptameric apoptosome. This heptameric apoptosome then activates the caspase cascade, which ultimately results in the execution of apoptosis. The purpose of our research was to discover a new prognostic model that is based on cytochrome c-related genes (CCRGs) for LUAD patients. Through LASSO regression analysis conducted on the LUAD datasets included in the TCGA datasets, a CCRGs signature was created. The diagnostic accuracy of the multigene signature was verified by an independent source using the GSE31210 and GSE72094 datasets. The GO and KEGG enrichment analysis were performed. In this study, there were 159 differentially expressed CCRGs in the TCGA dataset, while there were 68 differentially expressed CCRGs in the GSE31210 dataset. Additionally, there were 57 genes that overlapped across the two datasets. Using LASSO and Cox regression analysis, a signature consisting of 12 differentially expressed CCRGs was developed from the total of 57 such genes. On the basis of their risk ratings, patients were categorized into high-risk and low-risk categories, with low-risk patients having lower risk scores and a greater likelihood of surviving the disease. Univariate and multivariate analyses both concluded that this signature is an independent risk factor for LUAD. ROC curves demonstrated that this risk signature is capable of accurately predicting the 1-year, 2-year, 3-year, and 5-year survival rates of patients who have LUAD. The infiltration of antigen-presenting cells was higher in the low-risk group, such as aDCs, DCs, pDCs, and iDCs. The expression of multiple immune checkpoints was significantly higher in the low-risk group, such as BTLA, CD28, and CD86. Finally, we showed that the signature can be used to predict the drug sensitivity of already available or under investigational drugs. Overall, patient classification and individualized therapy options may benefit from this study's development of a powerful gene signature with high value for prognostic prediction in LUAD.

Transcriptomic responses to cytotoxic drug cisplatin in water flea Daphnia magna.

Cytotoxic drugs have been recognized by the European Union as the potential threat in the aquatic environment. As a typical cytotoxic drug, effects of long-term exposure to cisplatin at the environmentally relevant concentrations on the crustacean health and its molecular mechanism remain undetermined. In this study, the growth and reproduction of Daphnia magna resulting from cisplatin exposure were initially assessed. While the phenotypes were not altered in 2 µg L-1, 20 µg L-1, and 200 µg L-1 treatment groups, cisplatin at 500 µg L-1 significantly reduced the offspring number to 8-13 neonates in each brood, which was lower than 13-27 neonates in the control group. In addition to the delay in the time of first pregnancy, the body length was decreased by approximate 12.13% at day 7. Meanwhile, all daphnids died after exposure to 500 µg L-1 cisplatin for 17 days. Transcriptome profiling bioassays were performed for 10 days to explore the alternation at the molecular level. Briefly, 980 (257 up- and 723 down-regulated), 429 (182 up- and 247 down-regulated) and 1984 (616 up-regulated and 1368 down-regulated) genes were differentially expressed (adj p < 0.05) in low (2 µg L-1), medium (200 µg L-1) and high (500 µg L-1) cisplatin treatment groups, respectively. Differentially expressed genes were primarily enriched in the digestion and absorption, nerve conduction, endocrine interference, and circulatory related pathways. Specifically, the down-regulated digestive secretion and nutrient absorption and neuronal conduction pathways may lead to insufficient energy supply involved in growth and reproduction, and hinder ovarian development and cell growth. Down-regulation of ovarian steroids and relaxin signaling pathways may be related to the reduction of offspring number and delayed pregnancy, and reduced body length of D. magna may attribute to the enrichment of insulin secretion pathway. In addition, the death of D. magna may result from the reduced expression of genes in cardiomyocyte contraction and apoptosome processes. Taken together, this study revealed the potential toxic mechanism of cisplatin in a model water flea.

APAF1-Binding Long Noncoding RNA Promotes Tumor Growth and Multidrug Resistance in Gastric Cancer by Blocking Apoptosome Assembly.

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease-activating factor 1 (APAF1)-binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA-binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3-mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase-9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC.

The concept of intrinsic versus extrinsic apoptosis.

Regulated cell death is a vital and dynamic process in multicellular organisms that maintains tissue homeostasis and eliminates potentially dangerous cells. Apoptosis, one of the better-known forms of regulated cell death, is activated when cell-surface death receptors like Fas are engaged by their ligands (the extrinsic pathway) or when BCL-2-family pro-apoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both the intrinsic and extrinsic pathways of apoptosis lead to the activation of a family of proteases, the caspases, which are responsible for the final cell demise in the so-called execution phase of apoptosis. In this review, I will first discuss the most common types of regulated cell death on a morphological basis. I will then consider in detail the molecular pathways of intrinsic and extrinsic apoptosis, discussing how they are activated in response to specific stimuli and are sometimes overlapping. In-depth knowledge of the cellular mechanisms of apoptosis is becoming more and more important not only in the field of cellular and molecular biology but also for its translational potential in several pathologies, including neurodegeneration and cancer.

Contribution of Apaf-1 to the pathogenesis of cancer and neurodegenerative diseases.

Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders at one end of the spectrum and cancer at the other end. Generally speaking, differentiated cells like cardiomyocytes, skeletal myocytes and neurons exhibit low levels of Apaf-1 (Apoptotic protease activating factor 1) protein suggesting that down-regulation of Apaf-1 is an important event contributing to the resistance of these cells to apoptosis. Nonetheless, upregulation of Apaf-1 has not emerged as a common phenomenon in pathologies associated with enhanced neuronal cell death, i.e., neurodegenerative diseases. In cancer, on the other hand, Apaf-1 downregulation is a common phenomenon, which occurs through various mechanisms including mRNA hyper-methylation, gene methylation, Apaf-1 localization in lipid rafts, inhibition by microRNAs, phosphorylation, and interaction with specific inhibitors. Due to the diversity of these mechanisms and involvement of other factors, defining the exact contribution of Apaf-1 to the development of cancer in general and neurodegenerative disorders, in particular, is complicated. The current review is an attempt to provide a comprehensive image of Apaf-1's contribution to the pathologies observed in cancer and neurodegenerative diseases with the emphasis on the therapeutic aspects of Apaf-1 as an important target in these pathologies.

Molecular insights on cytochrome c and nucleotide regulation of apoptosome function and its implication in cancer.

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction. Mutation of these lysine residues abrogates the apoptosome formation causing inhibition of caspase activation. Using in-silico molecular docking, we have identified Cyt c-binding interface on Apaf-1. Although mutant Cyt c shows higher affinity for Apaf-1, the presence of Cyt c-WT restores the apoptosome activity. ATP addition modulates only mutant Cyt c binding to Apaf-1 but not WT Cyt c binding to Apaf-1. Using TCGA and cBioPortal, we identified multiple mutations in both Apaf-1 and Cyt c that are predicted to interfere with apoptosome assembly. We also demonstrate that transcript levels of various enzymes involved with dATP or ATP synthesis are increased in various cancers. Silencing of nucleotide metabolizing enzymes such as ribonucleotide reductase subunit M1 (RRM1) and ATP-producing glycolytic enzymes PKM2 attenuated ATP production and enhanced caspase activation. These findings suggest important role for lysine residues of Cyt c and nucleotides in the regulation of apoptosome-dependent apoptotic cell death as well as demonstrate how these mutations and nucleotides may have a pivotal role in human diseases such as cancer.

ECRG4 acts as a tumor suppressor gene frequently hypermethylated in human breast cancer.

Human breast cancer is a malignant form of tumor with a relatively high mortality rate. Although esophageal cancer-related gene 4 (ECRG4) is thought to be a possible potent tumor suppressor gene that acts to suppress breast cancer, its precise role in this disease is not understood. Herein, we assess the correlation between ECRG4 expression and DNA methylation, probing the potential epigenetic regulation of ECRG4 in breast cancer. We analyzed ECRG4 promoter methylation via methylation-specific PCR (MSPCR), bisulfite sequencing, and a promoter reporter assay in human breast cancer cell lines and samples. Gene expression was assessed by quantitative real-time PCR (qPCR), while protein levels were assessed by Western blotting. CCK8 assays were used to quantify cell growth; Esophageal cancer-related gene 4 wound healing assays were used to assess cellular migration, while flow cytometry was used to assess apoptosis and cell cycle progression. Apoptosome formation was validated via CO-IP and Western blotting. We found that human breast cancer samples exhibited increased methylation of the ECRG4 promoter and decreased ECRG4 expression. Remarkably, the down-regulation of ECRG4 was highly associated with promoter methylation, and its expression could be re-activated via 5-aza-2'-deoxycytidine treatment to induce demethylation. ECRG4 overexpression impaired breast cancer cell proliferation and migration, and led to G0/G1 cell cycle phase arrest. Moreover, ECRG4 induced the formation of the Cytc/Apaf-1/caspase-9 apoptosome and promoted breast cancer cell apoptosis. ECRG4 is silenced in human breast cancer cells and cell lines, likely owing to promoter hypermethylation. ECRG4 may act as a tumor suppressor, inhibiting proliferation and migration, inducing G0/G1 phase arrest and apoptosis via the mitochondrial apoptotic pathway.

Cytochrome c Deficiency Confers Apoptosome and Mitochondrial Dysfunction in African-American Men with Prostate Cancer.

Although African-American (AA) patients with prostate cancer tend to develop greater therapeutic resistance and faster prostate cancer recurrence compared with Caucasian-American (CA) men, the molecular mechanisms of this racial prostate cancer disparity remain undefined. In this study, we provide the first comprehensive evidence that cytochrome c deficiency in AA primary tumors and cancer cells abrogates apoptosome-mediated caspase activation and contributes to mitochondrial dysfunction, thereby promoting therapeutic resistance and prostate cancer aggressiveness in AA men. In AA prostate cancer cells, decreased nuclear accumulation of nuclear respiration factor 1 (Nrf1) and its subsequent loss of binding to the cytochrome c promoter mediated cytochrome c deficiency. The activation of cellular Myc (c-Myc) and NF-κB or inhibition of AKT prevented nuclear translocation of Nrf1. Genetic and pharmacologic inhibition of c-Myc and NF-κB or activation of AKT promoted Nrf1 binding to cytochrome c promoter, cytochrome c expression, caspase activation, and cell death. The lack of p-Drp1S616 in AA prostate cancer cells contributed to defective cytochrome c release and increased resistance to apoptosis, indicating that restoration of cytochrome c alone may be insufficient to induce effective apoptosis. Cytochrome c deficiency promoted the acquisition of glycolytic phenotypes and mitochondrial dysfunction, whereas cytochrome c restoration via inhibition of c-Myc and NF-κB or activation of AKT attenuated glycolysis in AA prostate cancer cells. Inhibition of c-Myc and NF-κB enhanced the efficacy of docetaxel in tumor xenografts. Therefore, restoring cytochrome c may overcome therapeutic resistance and prostate cancer aggressiveness in AA men. Overall, this study provides the first comprehensive experimental, mechanistic, and clinical evidence for apoptosome and mitochondrial dysfunction in prostate cancer racial disparity. SIGNIFICANCE: Mechanistic insights on prostate cancer health disparity among American men provide novel approaches to restore mitochondrial function, which can address therapeutic resistance and aggressiveness in African-American men with prostate cancer.

Menadione-induced apoptosis in U937 cells involves Bid cleavage and stefin B degradation.

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.

Modulation of Mcl-1 transcription by serum deprivation sensitizes cancer cells to cisplatin.

BACKGROUND: The development of approaches that increase therapeutic effects of anti-cancer drugs is one of the most important tasks of oncology. Caloric restriction in vivo or serum deprivation (SD) in vitro has been shown to be an effective tool for sensitizing cancer cells to chemotherapeutic drugs. However, the detailed mechanisms underlying the enhancement of apoptosis in cancer cells by SD remain to be elucidated. METHODS: Flow cytometry, caspase activity assay and western blotting were used for cell death rate evaluation. Western blotting, gel-filtration, siRNA approach and qRT-PCR were used to elucidate the mechanism underlying cell death potentiation upon SD. RESULTS: We demonstrated that SD sensitizes cancer cells to treatment with chemotherapeutic agent cisplatin. This effect is independent on activation of caspases-2 and -8, apical caspases triggering apoptosis in response to genotoxic stress. SD potentiates cell death via downregulation of the anti-apoptotic protein Mcl-1. In fact, SD reduces the Mcl-1 mRNA level, which consequently decreases the Mcl-1 protein level and renders cells more susceptible to apoptosis induction via the formation of apoptosome. CONCLUSIONS: Mcl-1 protein is an important regulator of sensitivity of cancer cells to apoptotic stimuli upon SD. GENERAL SIGNIFICANCE: This study identifies Mcl-1 as a new target for the sensitization of human cancer cells to cell death by SD, which is of great significance for the development of efficient anti-cancer therapies.

Effect and mechanism of hyaluronic acid on the neurotoxic injury of lidocaine.

Hyaluronic acid (HA) is used to aid tissue repair and is a characterized inhibitor of TRPV1 channels. In this study, we investigated the effects of HA on lidocaine induced neurotoxicity and its mechanism of action. U87-MG cells with low (U87-MG-shTRPV1) or high (U87-MG-TRPV1) TRPV1 expression were studied. The control group was treated with lidocaine. The experimental group was treated with lidocaine and HA. Flow cytometry was used to assess the intracellular calcium concentration ([Ca2+] i) and cell apoptosis. Cell viability was detected by MTT assays. Compared to the control group, [Ca2+]i of U87-MG-TRPV1 and U87-MG cells were lower at T3, T4 and T5 (p < 0.05), apoptosis rates of U87-MG and U87-MG-TRPV1 cells were lower (p<0.05), and the cell viability of U87-MG and U87-MG-TRPV1 cells were higher in the experimental group (p<0.05). HA reduces the toxic damage of lidocaine through blocking Ca2+ influx through TRPV1 channels, preventing Ca2+ overload, leading to nerve cell protection.

The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities.

According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.

Alpha fetoprotein antagonizes apoptosis induced by paclitaxel in hepatoma cells in vitro.

Hepatocellular carcinoma (HCC) cell resistance to the effects of paclitaxel has not been adequately addressed. In this study, we found that paclitaxel significantly inhibited the viability of HLE, Bel 7402 and L-02 cells in a dose- and time-dependent manner. HLE cells and L-02 cells resisted the cytotoxicity of paclitaxel when transfected with pcDNA3.1-afp vectors. However, Bel 7402 cell sensitivity to paclitaxel was increased when transfected with alpha fetoprotein (AFP)-siRNA. Bel 7402 cell resistance to paclitaxel was associated with the expression of the "stemness" markers CD44 and CD133. Paclitaxel significantly inhibited growth and promoted apoptosis in HLE cells and L-02 cells by inducing fragmentation of caspase-3 and inhibiting the expression of Ras and Survivin, but pcDNA3.1-afp vectors prevented these effects. However, paclitaxel could not significantly promote the cleavage of caspase-3 or suppress the expression of Ras and Survivin in Bel 7402 cells. Silenced expression of AFP may be synergistic with paclitaxel to restrain proliferation and induce apoptosis, enhance cleavage of caspase-3, and suppress the expression of Ras and Survivin. Taken together, AFP may be an important molecule acting against paclitaxel-inhibited proliferation and induced apoptosis in HCC cells via repressing the activity of caspase-3 and stimulating the expression of Ras and Survivin. Targeted inhibition of AFP expression after treatment with paclitaxel is an available strategy for the therapy of patients with HCC.

Dietary compound α-asarone alleviates ER stress-mediated apoptosis in 7ß-hydroxycholesterol-challenged macrophages.

SCOPE: Prolonged endoplasmic reticulum (ER) stress has lost the function of protein folding capacity and the ER accumulation of unfolded proteins that eventually triggers apoptosis. Oxysterols are emerging as contributing factors in atherogenesis known to involve macrophage apoptosis. This study determined the inhibitory effect of α-asarone present in purple perilla, on 7ß-hydroxycholesterol-induced macrophage apoptosis, targeting against ER stress signaling pathway. METHODS AND RESULTS: J774A1 murine macrophages were exposed to 28 µM 7ß-hydroxycholesterol and treated with 1-10 µM α-asarone. Macrophage apoptosis and ER stress were examined by and α-Asarone blocked 7ß-hydroxycholesterol-induced DNA fragmentation and apoptosome formation. Immunoblotting showed that the oxysterol activated the ER transmembrane resident kinases of IRE1α, PERK and ATF4 and triggered caspase-12 signaling cascades, which was reversed by α-asarone. Additionally, 7ß-hydroxycholesterol activated TRAF2-ASK1-JNK1/2 complex following the IRE1α activation, and α-asarone blunted such IRE1α-mediated pathway. Real-time PCR and dual-luciferase reporter analyses revealed that α-asarone reduced transcriptional activation of ER stress-responsive genes including XBP1 and CHOP by 7ß-hydroxycholesterol. Finally, α-asarone disturbed oxysterol-elicited signaling of PERK and ATF4 responsible for CHOP induction. CONCLUSION: α-Asarone blocked 7ß-hydroxycholesterol-induced macrophage apoptosis through allaying ER stress-specific signaling involving caspase activation and CHOP induction. α-Asarone was an anti-atherosclerotic agent antagonizing ER stress-mediated macrophage apoptosis by 7ß-hydroxycholesterol.

The Cellular Apoptosis Susceptibility Protein (CAS) Promotes Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis and Cell Proliferation.

A signature event during the cell intrinsic apoptotic pathway is mitochondrial outer membrane permeabilization, leading to formation of the apoptosome, a caspase activation complex. The cellular apoptosis susceptibility protein (CAS) can facilitate apoptosome assembly by stimulating nucleotide exchange on Apaf-1 following binding of cytochrome c. We report here that CAS expression itself is up-regulated during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and knockdown of CAS renders cells resistant to TRAIL. We find that TRAIL induces up-regulation of CAS in a posttranscriptional, caspase-8-dependent manner through degradation of cIAP1, an E3 ligase that targets CAS for ubiquitin-dependent proteasomal degradation. We identified a novel signaling pathway whereby caspase-8 engages a feedforward cascade that leads to CAS up-regulation and amplifies the apoptotic signal. Furthermore, in silico analysis revealed that expression of CAS is up-regulated at both the mRNA and DNA levels in human breast tumors, consistent with its role in promoting cell proliferation. Overexpression of various oncogenes led to CAS up-regulation in non-transformed cells. Intriguingly, oncogene-induced CAS up-regulation also resulted in greater susceptibility to TRAIL-induced cell death, consistent with its proapoptotic function. These findings suggest that CAS plays contrasting roles in proliferation and apoptosis and that overexpression of CAS in tumors could serve as a potential biomarker to guide therapeutic choices.

Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death.

Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer.

Role of the salt bridge between glutamate 546 and arginine 907 in preservation of autoinhibited form of Apaf-1.

Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited form inside the cytosol. WRD-domain of Apaf-1 has a critical role in the preservation of auto-inhibited form; however the underlying mechanism is unclear. It seems the salt bridges between WRD and NOD domains are involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investigated the effect of E546-R907 salt bridge on the maintenance of auto-inhibited form of human Apaf-1. E546 is mutated to glutamine (Q) and arginine (R). Over-expression of wild type Apaf-1 and its E546Q and E546R variants in HEK293T cells does not induce apoptosis unlike - HL-60 cancer cell line. In vitro apoptosome formation assay showed that all variants are cytochrome c and dATP dependent to form apoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results suggest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behavior of Apaf-1 variants for in vitro apoptosome formation in HEK293T cell is similar to exogenous wild type Apaf-1. Wild type and its variants can form apoptosome in HEK293T cell with different procaspase-3 processing pattern in the presence and absence of exogenous cytochrome c and dATP.

BARF1 gene silencing triggers caspase-dependent mitochondrial apoptosis in Epstein-Barr virus-positive malignant cells.

Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and downregulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.