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Physical Chemistry Chemical Physics :... Jan 2018The formation of a heptameric apoptosome is a crucial event in the intrinsic cell death pathway. Considerable progress has been made towards unraveling the constituents...
The formation of a heptameric apoptosome is a crucial event in the intrinsic cell death pathway. Considerable progress has been made towards unraveling the constituents and the structure of the apoptosome as well as the mechanism of apoptosome-mediated caspase-9 activation. However, a significant gap remains in the understanding of this process, i.e., how seven Apaf-1·cytochrome c complexes stepwisely assemble into an apoptosome. Here, we construct a biophysical model that incorporates current biochemical knowledge about the formation of apoptosome. We propose 11 elementary routes and enumerate all 2047 possible assembly pathways from the Apaf-1·cytochrome c complex to the heptameric apoptosome. By combining mathematical analysis and numerical simulation, we find that two elementary routes are the most favorable biochemical reaction routes and there are 52 optimal assembly pathways which are economical and relatively fast. Our study yields the first comprehensive analysis of apoptosome assembly and provides insights into complex assembly pathways.
Topics: Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 9; Cytochromes c; Humans; Kinetics; Models, Molecular
PubMed: 29299551
DOI: 10.1039/c7cp06726g -
Nature Cell Biology Aug 2000Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome. Here we show that the...
Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome. Here we show that the documented anti-apoptotic effect of the principal heat-shock protein, Hsp70, is mediated through its direct association with the caspase-recruitment domain (CARD) of Apaf-1 and through inhibition of apoptosome formation. The interaction between Hsp70 and Apaf-1 prevents oligomerization of Apaf-1 and association of Apaf-1 with procaspase-9. On the basis of these results, we propose that resistance to apoptosis exhibited by stressed cells and some tumours, which constitutively express high levels of Hsp70, may be due in part to modulation of Apaf-1 function by Hsp70.
Topics: Adenosine Triphosphate; Apoptosis; Apoptotic Protease-Activating Factor 1; Blotting, Western; Caspase 9; Caspases; Cell Extracts; Cell Line; Cell-Free System; Cytochrome c Group; Deoxyadenine Nucleotides; Enzyme Activation; Enzyme Precursors; Gene Expression; HSP70 Heat-Shock Proteins; Hot Temperature; Humans; Ligands; Macromolecular Substances; Precipitin Tests; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proteins
PubMed: 10934467
DOI: 10.1038/35019510 -
PloS One 2011Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by...
Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.
Topics: Amino Acid Sequence; Apoptosis; Apoptosomes; Base Sequence; Blotting, Western; Breast Neoplasms; Caspase 3; Cell Line, Tumor; DNA Primers; Enzyme Activation; Humans; Isoenzymes; Models, Molecular; Molecular Sequence Data; Real-Time Polymerase Chain Reaction; Sequence Homology, Amino Acid
PubMed: 22216167
DOI: 10.1371/journal.pone.0029058 -
European Journal of Medical Research Jun 2016Histone deacetylation, a common hallmark in malignant tumors, strongly alters the transcription of genes involved in the control of proliferation, cell survival,...
BACKGROUND
Histone deacetylation, a common hallmark in malignant tumors, strongly alters the transcription of genes involved in the control of proliferation, cell survival, differentiation and genetic stability. We have previously shown that HDAC1, HDAC2, and HDAC3 (HDAC1-3) genes encoding histone deacetylases 1-3 are upregulated in primary human hepatocellular carcinoma (HCC). The aim of this study was to characterize the functional effects of HDAC1-3 downregulation and to identify functionally important target genes of histone deacetylation in HCC.
METHODS
Therefore, HCC cell lines were treated with the histone deacetylase inhibitor (HDACi) trichostatin A and by siRNA-knockdown of HDAC1-3. Differentially expressed mRNAs were identified after siRNA-knockdown of HDAC1-3 using mRNA expression profiling. Findings were validated after siRNA-mediated silencing of HDAC1-3 using qRTPCR and Western blotting assays.
RESULTS
mRNA profiling identified apoptotic protease-activating factor 1 (Apaf1) to be significantly upregulated after HDAC inhibition (HLE siRNA#1/siRNA#2 p < 0.05, HLF siRNA#1/siRNA#2 p < 0.05). As a component of the apoptosome, a caspase-activating complex, Apaf1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. Using annexin V, a significant increase in apoptosis could also be shown in HLE (siRNA #1 p = 0.0034) and HLF after siRNA against HDAC1-3 (Fig. 3a, b). In parallel, caspase-9 activity was increased after siRNA-knockdown of HDAC1-3 leading to enhanced apoptosis after HDAC inhibition (Fig. 3c, d).
CONCLUSIONS
The present data show that siRNA-knockdown of HDAC1-3 plays a major role in mediating apoptotic response to HDAC inhibitors through regulation of Apaf1.
Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Blotting, Western; Carcinoma, Hepatocellular; Caspases; Cell Proliferation; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylases; Humans; Liver Neoplasms; Mitochondria; Prognosis; RNA, Messenger; RNA, Small Interfering; Real-Time Polymerase Chain Reaction; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured
PubMed: 27342975
DOI: 10.1186/s40001-016-0217-x -
The Journal of Experimental Medicine Mar 2003Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the...
Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the "apoptosome" as a result of the cell stress-associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell-free extracts readily supported Apaf-1-dependent caspase activation, suggesting that these cells may assemble cytochrome c-independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1-dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1-dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspases; Cell Fractionation; Cell-Free System; Cells, Cultured; Cytochrome c Group; Deoxyadenine Nucleotides; Enzyme Activation; Fluorescent Dyes; Humans; Mitochondria; Mitochondrial Proteins; Neutrophils; Proteins
PubMed: 12615903
DOI: 10.1084/jem.20021862 -
International Journal of Molecular... 2012Apoptosis, also called programmed cell death, is an orderly cellular suicide program that is critical for the development, immune regulation and homeostasis of a... (Review)
Review
Apoptosis, also called programmed cell death, is an orderly cellular suicide program that is critical for the development, immune regulation and homeostasis of a multi-cellular organism. Failure to control this process can lead to serious human diseases, including many types of cancer, neurodegenerative diseases, and autoimmununity. The process of apoptosis is mediated by the sequential activation of caspases, which are cysteine proteases. Initiator caspases, such as caspase-2, -8, -9, and -10, are activated by formation of caspase-activating complexes, which function as a platform to recruit caspases, providing proximity for self-activation. Well-known initiator caspase-activating complexes include (1) DISC (Death Inducing Signaling Complex), which activates caspases-8 and 10; (2) Apoptosome, which activates caspase-9; and (3) PIDDosome, which activates caspase-2. Because of the fundamental biological importance of capases, many structural and biochemical studies to understand the molecular basis of assembly mechanism of caspase-activating complexes have been performed. In this review, we summarize previous studies that have examined the structural and biochemical features of caspase-activating complexes. By analyzing the structural basis for the assembly mechanism of the caspase-activating complex, we hope to provide a comprehensive understanding of caspase activation by these important oligomeric complexes.
Topics: Apoptosis; Apoptosomes; Caspases; Death Domain Receptor Signaling Adaptor Proteins; Enzyme Activation; Humans; Multienzyme Complexes; Protein Structure, Tertiary; Signal Transduction
PubMed: 22606010
DOI: 10.3390/ijms13044807 -
The Journal of Biological Chemistry May 2013Maturation of the single-chain caspase-9 zymogen through autoproteolytic processing is mediated by the Apaf-1 apoptosome at the onset of apoptosis. Processed caspase-9...
Maturation of the single-chain caspase-9 zymogen through autoproteolytic processing is mediated by the Apaf-1 apoptosome at the onset of apoptosis. Processed caspase-9 and the apoptosome form a holoenzyme with robust proteolytic activity that is 2-3 orders of magnitude higher than that of free processed caspase-9. An unresolved important question is the role of caspase-9 processing, with some experimental data suggesting its dispensability. In this study, we demonstrate that, in contrast to wild-type caspase-9, the unprocessed single-chain caspase-9 triple mutant E306A/D315A/D330A (Casp9-TM) could no longer be adequately activated by the apoptosome. Compared with the protease activity of wild-type caspase-9, that of Casp9-TM in the presence of the apoptosome was drastically reduced. The crippled protease activity of Casp9-TM in the presence of the apoptosome is likely attributable to a markedly reduced ability of Casp9-TM to form homodimers. These data identify an essential role for the autoproteolytic processing of caspase-9 in its activation.
Topics: Amino Acid Substitution; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 9; Enzyme Activation; Enzyme Precursors; Humans; Mutation, Missense; Protein Multimerization; Proteolysis
PubMed: 23572523
DOI: 10.1074/jbc.M112.441568 -
Current Opinion in Cell Biology Dec 2010Multi-cellular animals have evolved a variety of mechanisms to respond to diverse apoptotic stimuli. In general these proceed through activation of apical caspases and... (Review)
Review
Multi-cellular animals have evolved a variety of mechanisms to respond to diverse apoptotic stimuli. In general these proceed through activation of apical caspases and culminate in executioner caspase activation and cell death. Because of the breadth of possible initiators, various molecular platforms are used to trigger different apical caspases. Although some common protein domains are used to assemble the apoptosome, the PIDDosome and death receptor complexes, an array of checks-and-balances are employed to ensure appropriate activation. Notwithstanding, these pathways share the underlying principle of proximity-dependent activation and post-translational modification. Here we will describe our current structural understanding of assembly and regulation of these signaling platforms.
Topics: Animals; Apoptosomes; Caspases; Cell Death; Enzyme Activation; Models, Molecular; Protein Structure, Tertiary; Signal Transduction; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
PubMed: 20817427
DOI: 10.1016/j.ceb.2010.08.004 -
British Journal of Cancer Dec 2003Renal cell carcinoma (RCC) responds very poorly to chemo- or radiotherapy. Renal cell carcinoma cell lines have been described to be resistant to apoptosis-inducing...
Renal cell carcinoma (RCC) responds very poorly to chemo- or radiotherapy. Renal cell carcinoma cell lines have been described to be resistant to apoptosis-inducing stimuli and to lack caspase expression. Here, we provide a structural and functional assessment of the apoptosome, the central caspase-activating signalling complex and a candidate for apoptosis-inactivating mutations. Cells from RCC cell lines and clinical samples isolated from RCC patients were included. Apoptosome function was measured as quantitative activation of caspases in protein extracts. In all five cell lines and in 19 out of 20 primary clear cell RCC samples, the expression of apoptosome components and caspase activation appeared normal. Of the four nonclear cell RCC that could be included, both oncocytomas gave no response to cytochrome c (in one case, no Apaf-1 was detected), one chromophobe RCC lacked caspase-9 and failed to activate caspase-3 in response to cytochrome c, and one papillary RCC showed good caspase activation despite the lack of caspase-7. Experiments utilising a peptide derived from Smac/DIABLO gave no indication that inhibitor of apoptosis proteins might exert an inhibiting effect in primary clear cell RCC. Thus, the apoptosome signalling complex is intact in human (clear cell) RCC, and an apoptosis defect must be located at other, probably upstream, sites.
Topics: Apoptosis; Apoptosis Inducing Factor; Carcinoma, Renal Cell; Caspases; Cytochrome c Group; Enzyme Activation; Flavoproteins; Humans; Inhibitor of Apoptosis Proteins; Kidney; Kidney Neoplasms; Membrane Proteins; Multienzyme Complexes; Proteins; Signal Transduction; Tumor Cells, Cultured; Ubiquitin
PubMed: 14647151
DOI: 10.1038/sj.bjc.6601436 -
Proceedings of the National Academy of... Dec 2005Apoptosis in metazoans is executed by a group of intracellular proteases named caspases. One of the caspase-activating pathways in mammals is initiated by the release of...
Apoptosis in metazoans is executed by a group of intracellular proteases named caspases. One of the caspase-activating pathways in mammals is initiated by the release of cytochrome c from mitochondria to cytosol, where it binds to Apaf-1 to form a procaspase-9-activating heptameric protein complex named apoptosome. We report here the reconstitution of this pathway with purified recombinant Apaf-1, procaspase-9, procaspase-3, and cytochrome c from horse heart. Apaf-1 contains a dATP as a cofactor. Cytochrome c binding to Apaf-1 induces hydrolysis of dATP to dADP, which is subsequently replaced by exogenous dATP. The dATP hydrolysis and exchange on Apaf-1 are two required steps for apoptosome formation.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspases; Cytochromes c; Deoxyadenine Nucleotides; Enzyme Activation; Horses; Hydrolysis; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Organelles; Proteins
PubMed: 16251271
DOI: 10.1073/pnas.0507900102