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Nature Catalysis Mar 2020Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising...
Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximity-induced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein-protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity, and additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami-based protein assembly platforms have the potential to inform the function of other multi-enzyme complexes involved in inflammation, innate immunity and cell death.
PubMed: 32190819
DOI: 10.1038/s41929-019-0403-7 -
Structure (London, England : 1993) May 2010Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the...
Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the holoapoptosome by site-directed thrombinolysis. A structure of the resulting apoptosome-pc-9 CARD complex was then determined at approximately 9.5 A resolution. In our model, the central hub is constructed like other AAA+ protein rings but also contains novel features. At higher radius, the regulatory region of each Apaf-1 is comprised of tandem seven and eight blade beta-propellers with cytochrome c docked between them. Remarkably, Apaf-1 CARDs are disordered in the ground state. During activation, each Apaf-1 CARD interacts with a pc-9 CARD and these heterodimers form a flexibly tethered "disk" that sits above the central hub. When taken together, the data reveal conformational changes during Apaf-1 assembly that allow pc-9 activation. The model also provides a plausible explanation for the effects of NOD mutations that have been mapped onto the central hub.
Topics: Apoptosomes; Caspase 9; Cytochrome c Group; Cytochromes c; Humans; Proteins
PubMed: 20462491
DOI: 10.1016/j.str.2010.04.001 -
Genes & Development Feb 2015Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog,...
Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome.
Topics: Animals; Apoptosomes; Caspases; Drosophila; Drosophila Proteins; Enzyme Activation; Models, Molecular; Protein Binding; Protein Structure, Tertiary
PubMed: 25644603
DOI: 10.1101/gad.255877.114 -
Molecular Aspects of Medicine Dec 2022Regulated cell death is defined as genetically encoded pathways that lead towards the demise of cells. In mammals, cell demise can be either inflammatory or... (Review)
Review
Regulated cell death is defined as genetically encoded pathways that lead towards the demise of cells. In mammals, cell demise can be either inflammatory or non-inflammatory, depending on whether the mechanism of death results in cell rupture or not. Inflammatory cell death can lead towards acute and chronic disease. Therefore, it becomes important to distinguish the mechanisms that result in these different inflammatory cell death outcomes. Apoptosis is a non-inflammatory form of cell death where cells resist rupture. In contrast, pyroptosis and necroptosis are inflammatory forms of cell death principally because of release of pro-inflammatory mediators from cells undergoing lysis. This review focusses on the mechanisms of these different cell death outcomes with specific emphasis on the caspase family of proteolytic enzymes.
Topics: Animals; Humans; Caspases; Inflammasomes; Pyroptosis; Necroptosis; Inflammation; Apoptosis; Mammals
PubMed: 35248371
DOI: 10.1016/j.mam.2022.101085 -
Cell Death and Differentiation Mar 2007Although sympathetic neurons are a well-studied model for neuronal apoptosis, the role of the apoptosome in activating caspases in these neurons remains debated. We find...
Although sympathetic neurons are a well-studied model for neuronal apoptosis, the role of the apoptosome in activating caspases in these neurons remains debated. We find that the ability of sympathetic neurons to undergo apoptosis in response to nerve growth factor (NGF) deprivation is completely dependent on having an intact apoptosome pathway. Genetic deletion of Apaf-1, caspase-9, or caspase-3 prevents apoptosis after NGF deprivation, and importantly, allows these neurons to recover and survive long-term following readdition of NGF. The inability of caspase-3 deficient sympathetic neurons to undergo apoptosis is particularly striking, as apoptosis in dermal fibroblasts and cortical neurons proceeds even in the absence of caspase-3. Our results show that in contrast to dermal fibroblasts and cortical neurons, sympathetic neurons express no detectable levels of caspase-7. The strict requirement for an intact apoptosome, coupled with a lack of effector caspase redundancy, provides sympathetic neurons with a markedly increased control over their apoptotic pathway.
Topics: Animals; Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 7; Caspase 9; Cells, Cultured; Cerebral Cortex; Dermis; Enzyme Activation; Fibroblasts; Gene Expression Regulation, Enzymologic; Mice; Mice, Knockout; Nerve Growth Factor; Neurons; Signal Transduction; Sympathetic Nervous System
PubMed: 16932756
DOI: 10.1038/sj.cdd.4402024 -
Toxicology Letters Mar 2001Transforming growth factor-beta1 (TGF-beta1), is involved in controlling liver size, by inducing apoptotic cell death in hepatocytes. However the mechanism by which... (Review)
Review
Transforming growth factor-beta1 (TGF-beta1), is involved in controlling liver size, by inducing apoptotic cell death in hepatocytes. However the mechanism by which TGF-beta(1) induces caspase activation and cell death is unknown. Apoptosis can be initiated either by receptor-mediated (e.g. Fas/CD95) or non-receptor chemically mediated (stress-induced) processes. With Fas/CD95 receptor mediated cell death, a multi-protein complex (DISC) is assembled at the plasma membrane, which activates the downstream caspases and cell death. In stress-mediated apoptosis, a cytosolic DISC equivalent, the apoptosome is formed that activates the effector caspases. We have characterised this complex in THP.1 cells, and shown that this is a cytochrome c dependent process that induces the formation of an approximately 700 kDa apoptosome caspase processing complex. This is formed by oligomerisation of apoptotic protease-activating factor 1 (Apaf-1), and recruitment and processing of caspase-9. We have now shown that TGF-beta1-induced apoptosis also occurs via the release of cytochrome c and the subsequent oligomerisation of Apaf-1 into an approximately 700 kDa apoptosome complex. Our studies show that, even though TGF-beta1 induction of apoptosis is a receptor-mediated event, it operates through the mitochondrial/Apaf-1 caspase activation pathway that appears to act as a common execution pathway for many diverse apoptotic stimuli.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspases; Cytochrome c Group; Enzyme Activation; Hepatocytes; Humans; Molecular Weight; Proteins; Transforming Growth Factor beta; fas Receptor
PubMed: 11323189
DOI: 10.1016/s0378-4274(01)00283-1 -
Sensors (Basel, Switzerland) Mar 2020The apoptotic protease-activating factor 1 (Apaf-1) split luciferase biosensor has been used as a biological tool for the detection of early stage of apoptosis. The...
The apoptotic protease-activating factor 1 (Apaf-1) split luciferase biosensor has been used as a biological tool for the detection of early stage of apoptosis. The effect of doxorubicin in a cell-based assay and the addition of cytochrome and ATP in a cell-free system have been used to test the functionality of the reporter for the detection of apoptosome formation. Here, our data established a drug- and cytochrome /ATP-independent way of apoptosis induction relying on the expression of the biosensor itself to induce formation of apoptosome. Overexpression of Apaf-1 constructs led to increased split luciferase activity and caspase-3 activity in the absence of any drug treatment. Caspase-3 activity was significantly inhibited when caspase-9DN was co-overexpressed, while the activity of the Apaf1 biosensor was significantly increased. Our results show that the Apaf-1 biosensor does not detect etoposide-induced apoptosis.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Biosensing Techniques; Caspase 3; Cell Survival; Enzyme Activation; Etoposide; HEK293 Cells; Humans; Luciferases
PubMed: 32210205
DOI: 10.3390/s20061782 -
Molecular and Cellular Biology Nov 2006The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using...
The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 8; Caspase 9; Cells, Cultured; Cycloheximide; Cytochromes c; Enzyme Activation; Fibroblasts; Lysosomes; Mice; Mice, Knockout; Mitochondria; Protein Synthesis Inhibitors; Tumor Necrosis Factor-alpha
PubMed: 16966373
DOI: 10.1128/MCB.00716-06 -
Cell Death and Differentiation Nov 2004Deficiency of the apoptosome component Apaf1 leads to accumulation of supernumerary brain cells in mouse embryos. We observed that neural precursor cells (NPCs) in...
Deficiency of the apoptosome component Apaf1 leads to accumulation of supernumerary brain cells in mouse embryos. We observed that neural precursor cells (NPCs) in Apaf1(-/-) embryos escape programmed cell death, proliferate and retain their potential to differentiate. To evaluate the circumstances of Apaf1(-/-) NPC survival and investigate their fate under neurodegenerative conditions, we established cell lines of embryonic origin (ETNA). We found that Apaf1(-/-) NPCs resist common apoptotic stimuli and neurodegenerative inducers such as amyloid-beta peptide (typical of Alzheimer's disease) and mutant G93A superoxide dismutase 1 (typical of familial amyotrophic lateral sclerosis). Similar results were obtained in Apaf1(-/-) primary cells. When death is prevented by Apaf1 deficiency, cytochrome c is released from mitochondria and rapidly degraded by the proteasome, but mitochondria remain intact. Under these conditions, neither activation by cleavage of initiator caspases nor release of alternative apoptotic inducers from mitochondria takes place. In addition, NPCs can still differentiate, as revealed by neurite outgrowth and expression of differentiation markers. Our findings imply that the mitochondrion/apoptosome pathway is the main route of proneural and neural cells to death and that its inhibition prevents them from dismantling in neurodegenerative conditions. Indeed, the ETNA cell model is ideally suited for exploring the potential of novel cell therapies for the treatment of human neurodegenerations.
Topics: Amyloid beta-Peptides; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Blotting, Western; Bromodeoxyuridine; Caspases; Cell Death; Cell Differentiation; Cell Proliferation; Cell Survival; Immunohistochemistry; Immunoprecipitation; Membrane Potentials; Mice; Mice, Transgenic; Microscopy, Fluorescence; Mitochondria; Nerve Degeneration; Neurodegenerative Diseases; Neurons; Peptide Fragments; Plasmids; Proteins; Time Factors; Transgenes
PubMed: 15257302
DOI: 10.1038/sj.cdd.4401476 -
The Journal of Biological Chemistry Jun 2014Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in...
Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs.
Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosomes; Caspases; Cell Differentiation; Cell Line; Cytochromes c; Embryonic Stem Cells; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Time Factors
PubMed: 24755221
DOI: 10.1074/jbc.M113.536730