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Proceedings of the National Academy of... Feb 2024The onset of apoptosis is characterized by a cascade of caspase activation, where initiator caspases are activated by a multimeric adaptor complex known as the...
The onset of apoptosis is characterized by a cascade of caspase activation, where initiator caspases are activated by a multimeric adaptor complex known as the apoptosome. In , the initiator caspase Dronc undergoes autocatalytic activation in the presence of the Dark apoptosome. Despite rigorous investigations, the activation mechanism for Dronc remains elusive. Here, we report the cryo-EM structures of an auto-inhibited Dark monomer and a single-layered, multimeric Dark/Dronc complex. Our biochemical analysis suggests that the auto-inhibited Dark oligomerizes upon binding to Dronc, which is sufficient for the activation of both Dark and Dronc. In contrast, the previously observed double-ring Dark apoptosome may represent a non-functional or "off-pathway" conformation. These findings expand our understanding on the molecular mechanism of apoptosis in .
Topics: Animals; Apoptosomes; Caspases; Drosophila; Drosophila melanogaster; Drosophila Proteins
PubMed: 38381783
DOI: 10.1073/pnas.2312784121 -
Proceedings of the National Academy of... Nov 2014Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known...
Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known as the Apaf-1 apoptosome. The molecular mechanism by which caspase-9 is activated by the Apaf-1 apoptosome remains largely unknown. Here we demonstrate that the previously reported 1:1 interaction between Apaf-1 caspase recruitment domain (CARD) and caspase-9 CARD is insufficient for the activation of caspase-9. Rather, formation of a multimeric CARD:CARD assembly between Apaf-1 and caspase-9, which requires three types of distinct interfaces, underlies caspase-9 activation. Importantly, an additional surface area on the multimeric CARD assembly is essential for caspase-9 activation. Together, these findings reveal mechanistic insights into the activation of caspase-9 by the Apaf-1 apoptosome and support the induced conformation model for initiator caspase activation by adaptor complexes.
Topics: Apoptosis Regulatory Proteins; Apoptosomes; Caspase 9; Catalysis; Enzyme Activation; Humans; Hydrogen Bonding; In Vitro Techniques; Lysine; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Protein Interaction Mapping; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Fusion Proteins
PubMed: 25313070
DOI: 10.1073/pnas.1418000111 -
Journal of Immunology (Baltimore, Md. :... Jul 2004Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to...
Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.
Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Caspases; Cytochromes c; Humans; Hydrogen Peroxide; Jurkat Cells; Mitochondria; Permeability; Proteins; Reactive Oxygen Species; fas Receptor
PubMed: 15210786
DOI: 10.4049/jimmunol.173.1.285 -
Cell Cycle (Georgetown, Tex.) Apr 2004Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1,...
Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1, recruitment and auto-processing of the apical/signaling pro-caspase-9, and coupled activation of downstream/executioner caspases like caspase 3. Evidence now indicates that certain apoptotic cascades can bypass the apoptosome and activate caspase-9 independent of the mitochondria. Recently, we have demonstrated that caspase-9 can be activated in Apaf1-mutant primary myoblasts, but not fibroblasts, in response to stimuli that are known to act via the mitochondria. Thus, apoptosomal activation of caspase-9 seems to represent only one of the routes for its activation; other pathways, some of which are yet to be discovered, can bypass the requirement for Apaf1 and activate caspase-9 in a tissue and context specific manner.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caenorhabditis elegans; Caspase 3; Caspase 9; Caspases; Endoplasmic Reticulum; Enzyme Activation; Fibroblasts; Golgi Apparatus; Humans; Lysosomes; Mice; Mitochondria; Models, Biological; Mutation; Protein Structure, Tertiary; Proteins
PubMed: 15020840
DOI: No ID Found -
European Journal of Pharmacology Aug 2015In this study, we used spilt luciferase complementation assay strategy in order to further elucidate the main role of WD-40 repeats of Apaf-1 molecules in apoptosome...
In this study, we used spilt luciferase complementation assay strategy in order to further elucidate the main role of WD-40 repeats of Apaf-1 molecules in apoptosome formation. In the presence of ATP and cytochrome c, Apaf-1 monomers oligomerize and provide a platform for the activation of procaspase-9 and subsequently procaspase-3/7. For a detailed biochemical and structural investigation of Apaf-1 function and apoptosome formation, several studies have been made in recent years. However, many questions related to in vivo evaluation of this phenomenon have been persisting to answer. Some of the most important of these questions are related to WD-40 repeats at the carboxy terminus of Apaf-1 and its function in apoptosome complex formation and caspase activation. When truncated Apaf-1 molecules conjugated with luciferase fragments place in close proximity, light signal emits and real time evaluation of protein-protein interactions becomes possible. Here, we observed, for the first time, the autoassembly of truncated Apaf-1 molecules disappeared after several hours without any caspase-3/7 activation. However, we observed that, truncated Apaf-1 molecules can activate caspase-3/7 upon the induction of apoptosis via doxorubicin. Moreover, oscillation in luciferase activity upon complementation was revealed which implicates the dynamism of apoptosome formation.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Biological Clocks; HEK293 Cells; Humans; Point Mutation; Protein Binding
PubMed: 25895636
DOI: 10.1016/j.ejphar.2015.04.008 -
Journal of Virology Dec 2017Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) gene....
Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) gene. is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1-procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions. Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.
Topics: Animals; Ankyrin Repeat; Apoptosis; Apoptosis Regulatory Proteins; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 9; HeLa Cells; Host-Pathogen Interactions; Humans; Jurkat Cells; Saccharomyces cerevisiae; Staurosporine; Vaccinia virus
PubMed: 28904196
DOI: 10.1128/JVI.01385-17 -
Cancer Biology & Therapy Feb 2007The apoptosome is a multiprotein complex mediating the mitochondrial pathway of cell death. Its importance during development has been clearly demonstrated by knocking...
The apoptosome is a multiprotein complex mediating the mitochondrial pathway of cell death. Its importance during development has been clearly demonstrated by knocking out key genes in mouse. APAF1 is the core protein of the apoptosome and its dosage is also critical in various cancer types, i.e., melanoma, germ line tumor, gastrointestinal cancer and B-type chronic lymphocytic leukemia. This is generally due to inactivation of the APAF1 locus by epigenetic phenomena or by activity of promoter regulators. We investigated the putative roles of the apoptosome in pancreatic ductal adenocarcinoma (PDAC). We found that both APAF1 mRNA and protein are dysregulated in human PDAC samples. Similarly, several PDAC cell lines exhibited variable levels of both APAF1 protein and mRNA. The response to cell death induction and its biochemical features were assessed by treatment of each line with commonly used chemotherapeutic agents. We found that the apoptosome pathway was not functional in most cell lines upon cytochrome c release from mitochondria. In addition, we restored APAF1 and Caspase-9 dosage in Panc-1 cells, where the apoptosome is downregulated, by overexpressing the murine cDNA of the two molecules, and we improved the death response to chemotherapeutic agents.
Topics: Animals; Antineoplastic Agents; Apoptosomes; Apoptotic Protease-Activating Factor 1; Carcinoma, Pancreatic Ductal; Caspase 9; Cell Death; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mice; Pancreatic Neoplasms
PubMed: 17224646
DOI: 10.4161/cbt.6.2.3622 -
Structure (London, England : 1993) Jan 2017In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of...
In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.
Topics: Animals; Apoptosis; Apoptosomes; Caspases; Cryoelectron Microscopy; Deoxyadenine Nucleotides; Drosophila Proteins; Drosophila melanogaster; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Protein Structure, Secondary; Serine
PubMed: 27916517
DOI: 10.1016/j.str.2016.11.002 -
Journal of Cell Science May 2020Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and...
Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 9; Cell Death; Cytochromes c; HEK293 Cells; Humans
PubMed: 32461338
DOI: 10.1242/jcs.242636 -
Biochimica Et Biophysica Acta.... Jan 2020Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis....
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.
Topics: Alanine; Amino Acid Substitution; Apoptosomes; Apoptotic Protease-Activating Factor 1; Case-Control Studies; Cell Transformation, Neoplastic; Cells, Cultured; Cytochromes c; Female; Humans; Lysine; Male; Models, Molecular; Molecular Docking Simulation; Mutant Proteins; Neoplasms; Nucleotides; PC-3 Cells; Protein Binding; Protein Interaction Mapping; Protein Multimerization; Signal Transduction
PubMed: 31678591
DOI: 10.1016/j.bbamcr.2019.118573