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ACS Omega Sep 2021The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1...
The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of the inactive "closed" form of Apaf-1. Here, a split luciferase complementation assay was used to monitor the effect of disrupting this ionic bond on apoptosome activation and caspase-3 activity in cells. The K192E mutation, predicted to disrupt the ionic interaction with D616, increased apoptosome formation and caspase activity, suggesting that this mutation favors the "open"/active form of Apaf-1. However, mutation of D616 to alanine or lysine had different effects. While both mutants favored apoptosome formation such as K192E, D616K cannot activate caspases and D616A activates caspases poorly, and not as well as wild-type Apaf-1. Thus, our data show that the ionic bond between K192 and D616 is critical for maintaining the closed form of Apaf-1 and that disrupting the interaction enhances apoptosome formation. However, our data also reveal that after apoptosome formation, D616 and K192 play a previously unsuspected role in caspase activation. The molecular explanation for this observation is yet to be elucidated.
PubMed: 34514227
DOI: 10.1021/acsomega.1c02274 -
PLoS Pathogens Dec 2023Staphylococcus aureus is a dangerous pathogen that evolved refined immuno-evasive strategies to antagonize host immune responses. This involves the biogenesis of...
Staphylococcus aureus is a dangerous pathogen that evolved refined immuno-evasive strategies to antagonize host immune responses. This involves the biogenesis of death-effector deoxyribonucleosides, which kill infectious foci-penetrating macrophages. However, the exact mechanisms whereby staphylococcal death-effector deoxyribonucleosides and coupled imbalances of intracellular deoxyribonucleotide species provoke immune cell death remain elusive. Here, we report that S. aureus systematically promotes an overload of deoxyribonucleotides to trigger mitochondrial rupture in macrophages, a fatal event that induces assembly of the caspase-9-processing apoptosome and subsequent activation of the intrinsic pathway of apoptosis. Remarkably, genetic disruption of this cascade not only helps macrophages coping with death-effector deoxyribonucleoside-mediated cytotoxicity but also enhances their infiltration into abscesses thereby ameliorating pathogen control and infectious disease outcomes in laboratory animals. Combined with the discovery of protective alleles in human CASP9, these data highlight the role of mitochondria-centered apoptosis during S. aureus infection and suggest that gene polymorphisms may shape human susceptibility toward a predominant pathogen.
Topics: Animals; Humans; Staphylococcus aureus; Nucleotides; Phagocytes; Cell Death; Apoptosis; Mitochondria; Deoxyribonucleosides
PubMed: 38157331
DOI: 10.1371/journal.ppat.1011892 -
Expert Review of Precision Medicine and... 2018Despite decades of focused research efforts, cancer remains a significant cause of morbidity and mortality. Tumor necrosis factor(TNF)-related apoptosis-inducing ligand...
INTRODUCTION
Despite decades of focused research efforts, cancer remains a significant cause of morbidity and mortality. Tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) is capable of inducing cell death selectively in cancer cells while sparing normal cells.
AREAS COVERED
In this review, the authors cover TRA therapy and strategies that have been undertaken to improve their efficacy, as well as unconventional approaches to TRAIL pathway activation including TRAIL-inducing small molecules. They also discuss mechanisms of resistance to TRAIL and the use of combination strategies to overcome it.
EXPERT COMMENTARY
Targeting the TRAIL pathway has been of interest in oncology, and although initial clinical trials of TRAIL receptor agonists (TRAs) showed limitations, novel approaches represent the future of TRAIL-based therapy.
PubMed: 30740527
DOI: 10.1080/23808993.2018.1476062 -
Cancer Research Apr 2019Although African-American (AA) patients with prostate cancer tend to develop greater therapeutic resistance and faster prostate cancer recurrence compared with...
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 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 promoter mediated cytochrome 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 promoter, cytochrome expression, caspase activation, and cell death. The lack of p-Drp1 in AA prostate cancer cells contributed to defective cytochrome release and increased resistance to apoptosis, indicating that restoration of cytochrome alone may be insufficient to induce effective apoptosis. Cytochrome deficiency promoted the acquisition of glycolytic phenotypes and mitochondrial dysfunction, whereas cytochrome 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 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.
Topics: Black or African American; Animals; Apoptosomes; Cell Line, Tumor; Cytochromes c; Humans; Male; Mice; Mice, SCID; Mitochondria; Mitochondrial Membranes; NF-kappa B; Nuclear Respiratory Factor 1; Oxidative Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc
PubMed: 30765600
DOI: 10.1158/0008-5472.CAN-18-2383 -
Medicinal Research Reviews Jul 2011Programmed cell death, apoptosis, is a highly regulated cellular pathway, responsible for the elimination of cells in the organism that are no longer needed or... (Review)
Review
Programmed cell death, apoptosis, is a highly regulated cellular pathway, responsible for the elimination of cells in the organism that are no longer needed or extensively damaged. Defects in the regulation of apoptosis could be at the molecular basis of different diseases, either when it is insufficient or excessive. The formation of the macromolecular complex, apoptosome, is a key event in this pathway, which has also been defined as the intrinsic apoptosis pathway. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated apoptotic protease-activating factor (Apaf-1), dATP, and procaspase-9. Recent studies have produced a wealth of information about the regulation and functions of Apaf-1, but additional studies aimed at elucidating its role as a signaling device at the crosstalk between different signaling pathways are needed to take advantage for the development of modulators of apoptosis pathways and possible therapeutic applications.
Topics: Apoptosomes; Apoptotic Protease-Activating Factor 1; Humans; Models, Biological
PubMed: 20099266
DOI: 10.1002/med.20198 -
Molecular Biology of the Cell May 2023The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death is not well understood. Here...
The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death is not well understood. Here we show that in response to DNA damage, human cells form a juxtanuclear F-actin-rich territory that coordinates the organized progression of apoptosome assembly to caspase activation. This cytoskeletal compartment is created by the actin nucleation factors JMY, WHAMM, and the Arp2/3 complex, and it excludes proteins that inhibit JMY and WHAMM activity. Within the territory, mitochondria undergo outer membrane permeabilization and JMY localization overlaps with punctate structures containing the core apoptosome components cytochrome and Apaf-1. The F-actin-rich area also encompasses initiator caspase-9 and clusters of a cleaved form of executioner caspase-3 but restricts accessibility of the caspase inhibitor XIAP. The clustering and potency of caspase-3 activation are positively regulated by the amount of actin polymerized by JMY and WHAMM. These results indicate that JMY-mediated actin reorganization functions in apoptotic signaling by coupling the biogenesis of apoptosomes to the localized processing of caspases.
Topics: Humans; Actins; Caspase 3; Apoptosomes; Apoptosis; Caspases; Actin Cytoskeleton; DNA Damage; Membrane Proteins; Microtubule-Associated Proteins
PubMed: 36920061
DOI: 10.1091/mbc.E22-04-0119 -
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 -
The FEBS Journal Nov 2009Cell death by the process of apoptosis plays important roles in development, tissue homeostasis, diseases and drug responses. The cysteine aspartyl protease caspase-9... (Review)
Review
Cell death by the process of apoptosis plays important roles in development, tissue homeostasis, diseases and drug responses. The cysteine aspartyl protease caspase-9 plays a central role in the mitochondrial or intrinsic apoptotic pathway that is engaged in response to many apoptotic stimuli. Caspase-9 is activated in a large multimeric complex, the apoptosome, which is formed with apoptotic peptidase activating factor 1 (Apaf-1) in response to the release of cytochrome c from mitochondria. Once activated, caspase-9 cleaves and activates the effector caspases 3 and 7 to bring about apoptosis. This pathway is tightly regulated at multiple steps, including apoptosome formation and caspase-9 activation. Recent work has shown that caspase-9 is the direct target for regulatory phosphorylation by multiple protein kinases activated in response to extracellular growth/survival factors, osmotic stress or during mitosis. Here, we review these advances and discuss the possible roles of caspase-9 phosphorylation in the regulation of apoptosis during development and in pathological states, including cancer.
Topics: Amino Acid Sequence; Animals; Apoptosis; Autophagy; CDC2 Protein Kinase; Caspase 9; Caspase Inhibitors; Cyclin B; Cyclin B1; DNA Damage; Extracellular Signal-Regulated MAP Kinases; Humans; Molecular Sequence Data; Phosphorylation; p38 Mitogen-Activated Protein Kinases
PubMed: 19788417
DOI: 10.1111/j.1742-4658.2009.07330.x -
Experimental Cell Research Jul 2012Cell death is critical to the normal functioning of multi-cellular organisms, playing a central role in development, immunity, inflammation, and cancer progression. Two... (Review)
Review
Cell death is critical to the normal functioning of multi-cellular organisms, playing a central role in development, immunity, inflammation, and cancer progression. Two cell death mechanisms, apoptosis and necroptosis, are dependent on the formation of distinct multi-protein complexes including the DISC, Apoptosome, Piddosome and Necrosome following the induction of cell death by specific stimuli. The role of several of these key multi-protein signalling platforms, namely the DISC, TNFR1 complex I/II, the Necrosome and Ripoptosome, in mediating these pathways will be discussed, as well as the open questions and potential therapeutic benefits of understanding their underlying mechanisms.
Topics: Animals; Cell Death; Humans; Multiprotein Complexes; Receptors, Death Domain; Signal Transduction
PubMed: 22542855
DOI: 10.1016/j.yexcr.2012.04.005 -
Oncogene Oct 2008Mitochondrial outer membrane permeabilization (MOMP) constitutes one of the major checkpoint(s) of apoptotic and necrotic cell death. Recently, the permeabilization of... (Review)
Review
Mitochondrial outer membrane permeabilization (MOMP) constitutes one of the major checkpoint(s) of apoptotic and necrotic cell death. Recently, the permeabilization of yet another organelle, the lysosome, has been shown to initiate a cell death pathway, in specific circumstances. Lysosomal membrane permeabilization (LMP) causes the release of cathepsins and other hydrolases from the lysosomal lumen to the cytosol. LMP is induced by a plethora of distinct stimuli including reactive oxygen species, lysosomotropic compounds with detergent activity, as well as some endogenous cell death effectors such as Bax. LMP is a potentially lethal event because the ectopic presence of lysosomal proteases in the cytosol causes digestion of vital proteins and the activation of additional hydrolases including caspases. This latter process is usually mediated indirectly, through a cascade in which LMP causes the proteolytic activation of Bid (which is cleaved by the two lysosomal cathepsins B and D), which then induces MOMP, resulting in cytochrome c release and apoptosome-dependent caspase activation. However, massive LMP often results in cell death without caspase activation; this cell death may adopt a subapoptotic or necrotic appearance. The regulation of LMP is perturbed in cancer cells, suggesting that specific strategies for LMP induction might lead to novel therapeutic avenues.
Topics: Animals; Cell Death; Cell Membrane Permeability; Humans; Intracellular Membranes; Lysosomes; Neoplasms; Signal Transduction
PubMed: 18955971
DOI: 10.1038/onc.2008.310