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Genes & Development Aug 2013It is now well appreciated that the apoptosome, which governs caspase-dependent cell death, also drives nonapoptotic caspase activation to remodel cells. However, the...
It is now well appreciated that the apoptosome, which governs caspase-dependent cell death, also drives nonapoptotic caspase activation to remodel cells. However, the determinants that specify whether the apoptosome acts to kill or remodel have yet to be identified. Here we report that Tango7 collaborates with the Drosophila apoptosome to drive a caspase-dependent remodeling process needed to resolve individual sperm from a syncytium. In these cells, Tango7 is required for caspase activity and localizes to the active apoptosome compartment via its C terminus. Furthermore, Tango7 directly stimulates the activity of this complex in vitro. We propose that Tango7 specifies the Drosophila apoptosome as an effector of cellular remodeling.
Topics: Animals; Apoptosomes; Aryl Hydrocarbon Receptor Nuclear Translocator; Caspases; Drosophila Proteins; Drosophila melanogaster; Fertility; Genetic Variation; Male; Mutation; Spermatogenesis; Spermatozoa
PubMed: 23913920
DOI: 10.1101/gad.219287.113 -
Cell Apr 2010The CED-4 homo-oligomer or apoptosome is required for initiation of programmed cell death in Caenorhabditis elegans by facilitating autocatalytic activation of the CED-3...
The CED-4 homo-oligomer or apoptosome is required for initiation of programmed cell death in Caenorhabditis elegans by facilitating autocatalytic activation of the CED-3 caspase zymogen. How the CED-4 apoptosome assembles and activates CED-3 remains enigmatic. Here we report the crystal structure of the complete CED-4 apoptosome and show that it consists of eight CED-4 molecules, organized as a tetramer of an asymmetric dimer via a previously unreported interface among AAA(+) ATPases. These eight CED-4 molecules form a funnel-shaped structure. The mature CED-3 protease is monomeric in solution and forms an active holoenzyme with the CED-4 apoptosome, within which the protease activity of CED-3 is markedly stimulated. Unexpectedly, the octameric CED-4 apoptosome appears to bind only two, not eight, molecules of mature CED-3. The structure of the CED-4 apoptosome reveals shared principles for the NB-ARC family of AAA(+) ATPases and suggests a mechanism for the activation of CED-3.
Topics: Amino Acid Sequence; Animals; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium-Binding Proteins; Caspases; Crystallography, X-Ray; Models, Molecular; Sequence Alignment; X-Ray Diffraction
PubMed: 20434985
DOI: 10.1016/j.cell.2010.03.017 -
FEBS Open Bio Jul 2019The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new...
The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase-based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease-Activating Factor-1 (Apaf-1) molecules and induces cell death by activating caspase-9. Apaf-1-dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf-1 fused to an N-terminal fragment of luciferase binds to Apaf-1 fused to a C-terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill-defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Cell Death; Cytochromes c; HEK293 Cells; Humans; Luciferases; Pentachlorophenol; Signal Transduction; Small Molecule Libraries; Toxicity Tests
PubMed: 31033240
DOI: 10.1002/2211-5463.12646 -
Nature Communications Nov 2016According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form...
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.
Topics: Animals; Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Dimerization; Mice; Phosphotransferases; Protein Kinases; Sf9 Cells; Spodoptera; Tumor Suppressor Proteins
PubMed: 27882936
DOI: 10.1038/ncomms13565 -
Proceedings of the National Academy of... Feb 2017Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The...
Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structure-guided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub. The interface between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the CARD of free caspase-9 strongly inhibits its proteolytic activity. These structural and biochemical findings demonstrate that the apoptosome activates caspase-9 at least in part through sequestration of the inhibitory CARD domain.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 9; Caspase Activation and Recruitment Domain; Cryoelectron Microscopy; Enzyme Activation; Holoenzymes; Humans; Models, Molecular; Mutation; Protein Binding; Protein Domains; Protein Multimerization
PubMed: 28143931
DOI: 10.1073/pnas.1620626114 -
Structure (London, England : 1993) Jan 2011The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator...
The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ∼6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.
Topics: Animals; Apoptosomes; Caspases; Cryoelectron Microscopy; Drosophila Proteins; Drosophila melanogaster; Protein Binding; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Structural Homology, Protein
PubMed: 21220123
DOI: 10.1016/j.str.2010.10.009 -
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 -
Oncotarget Apr 2017As the most intensively studied initiator caspase, caspase-9 is a key player in the intrinsic or mitochondrial pathway which is involved in various stimuli, including... (Review)
Review
As the most intensively studied initiator caspase, caspase-9 is a key player in the intrinsic or mitochondrial pathway which is involved in various stimuli, including chemotherapies, stress agents and radiation. Caspase-9 is activated on the apoptosome complex to remain catalytic status and is thought of involving homo-dimerization monomeric zymogens. Failing to activate caspase-9 has profound physiological and pathophysiological outcomes, leading to degenerative and developmental disorders even cancer. To govern the apoptotic commitment process appropriately, plenty of proteins and small molecules involved in regulating caspase-9. Therefore, this review is to summarize recent pertinent literature on the comprehensive description of the molecular events implicated in caspase-9 activation and inhibition, as well as the clinical trials in progress to give deep insight into caspase-9 for suppressing cancer. We hope that our concerns will be helpful for further clinical studies addressing the roles of caspase-9 and its regulators demanded to identify more effective solutions to overcome intrinsic apoptosis-related diseases especially cancer.
Topics: Animals; Apoptosis; Caspase 9; Humans; Phosphorylation; Signal Transduction
PubMed: 28177918
DOI: 10.18632/oncotarget.15098 -
Biochimie Apr 2017Apoptosis, a form of programmed cell death, is responsible for eliminating damaged or unnecessary cells in multicellular organisms. Various types of intracellular stress... (Review)
Review
Apoptosis, a form of programmed cell death, is responsible for eliminating damaged or unnecessary cells in multicellular organisms. Various types of intracellular stress trigger apoptosis by induction of cytochrome c release from mitochondria into the cytosol. Apoptotic protease activating factor-1 (Apaf-1) is a key molecule in the intrinsic or mitochondrial pathway of apoptosis, which oligomerizes in response to cytochrome c release and forms a large complex known as apoptosome. Procaspase-9, an initiator caspase in the mitochondrial pathway, is recruited and activated by the apoptosome leading to downstream caspase-3 processing. Various cellular proteins and small molecules can modulate apoptosome formation and function directly or indirectly. Despite recent progress in understanding the mitochondrial pathway of apoptosis, numerous questions such as the molecular mechanism of Apaf-1 oligomerization and caspase-9 activation remain poorly understood. In addition, reports have emerged showing non-apoptotic functions for Apaf-1. The current review summarizes the latest findings regarding structure-function relationship of Apaf-1 as well as its modifiers.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9
PubMed: 28192157
DOI: 10.1016/j.biochi.2017.02.001 -
The Journal of Biological Chemistry Mar 2003The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to...
The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD(330)), which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposed a novel p10 NH(2)-terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.
Topics: Amino Acid Sequence; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cytochrome c Group; Enzyme Activation; Humans; Hydrolysis; Kinetics; Molecular Sequence Data; Proteins; Recombinant Proteins; Sequence Homology, Amino Acid; X-Linked Inhibitor of Apoptosis Protein
PubMed: 12506111
DOI: 10.1074/jbc.M204783200