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Environmental Toxicology and... Oct 2020Multidrug-resistance protein-1 facilitates the efflux of arsenic conjugated with reduced glutathione nonetheless; the relation between Mrp-1 ATPase activity and cellular...
Multidrug-resistance protein-1 facilitates the efflux of arsenic conjugated with reduced glutathione nonetheless; the relation between Mrp-1 ATPase activity and cellular GSH levels is contentious. To study this, Mrp-1-ATPase activity was measured in 5 μM arsenic trioxide exposed zebrafish hepatocytes (ZFH) and correlated with intracellular GSH levels. Alongside, mrp-1 gene expression as well as Mrp-1 protein level was also monitored. Diverse mode of Mrp-1 inhibition was reflected from differential level of Km and Vmax of Mrp-1 at different time points. 3 h post-arsenic treatment demonstrated non-competitive inhibition. At 6 h, there was significant increase in Km and ZFH death, suggesting reduced binding affinity of Mrp-1 for ATP. Increased caspase-9-cytochromeC-ATP levels (putative apoptosome), reinforced ZFH apoptosis. The increase in Vmax coupled with reduced substrate affinity of Mrp-1 suggests malfunctioning in arsenic- tolerance mechanisms. We posit the triggering glutathione level regulate arsenic tolerance in ZFH. Irreversible impairment of ATP binding to Mrp-1 culminates in arsenic-induced ZFH apoptosis.
Topics: Adenosine Triphosphate; Animals; Apoptosis; Arsenic; Cells, Cultured; Glutathione; Hepatocytes; Multidrug Resistance-Associated Proteins; Zebrafish; Zebrafish Proteins
PubMed: 32470611
DOI: 10.1016/j.etap.2020.103427 -
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 -
Autophagy Jun 2021Recently, we reported that increased expression of CASP9 pro-domain, at the endosomal membrane in response to HSP90 inhibition, mediates a cell-protective effect that...
Recently, we reported that increased expression of CASP9 pro-domain, at the endosomal membrane in response to HSP90 inhibition, mediates a cell-protective effect that does not involve CASP9 apoptotic activity. We report here that a non-apoptotic activity of endosomal membrane CASP9 facilitates the retrograde transport of IGF2R/CI-MPR from the endosomes to the trans-Golgi network, indicating the involvement of CASP9 in endosomal sorting and lysosomal biogenesis. CASP9-deficient cells demonstrate the missorting of CTSD (cathepsin D) and other acid hydrolases, accumulation of late endosomes, and reduced degradation of bafilomycin A-sensitive proteins. In the absence of CASP9, IGF2R undergoes significant degradation, and its rescue is achieved by the re-expression of a non-catalytic mutant. This endosomal activity of CASP9 is potentially mediated by herein newly identified interactions of CASP9 with the components of the endosomal membrane transport complexes. These endosomal complexes include the retromer VPS35 and the SNX dimers, SNX1-SNX5 and SNX2-SNX6, which are involved in the IGF2R retrieval mechanism. Additionally, CASP9 interacts with HGS/HRS/ESCRT-0 and the CLTC (clathrin heavy chain) that participate in the initiation of the endosomal ESCRT degradation pathway. We propose that endosomal CASP9 inhibits the endosomal membrane degradative subdomain(s) from initiating the ESCRT-mediated degradation of IGF2R, allowing its retrieval to transport-designated endosomal membrane subdomain(s). These findings are the first to identify a cell survival, non-apoptotic function for CASP9 at the endosomal membrane, a site distinctly removed from the cytoplasmic apoptosome. Via its non-apoptotic endosomal function, CASP9 impacts the retrograde transport of IGF2R and, consequently, lysosomal biogenesis.: ACTB: actin beta; ATG7: autophagy related 7; BafA1: bafilomycin A; CASP: caspase; CLTC/CHC: clathrin, heavy chain; CTSD: cathepsin D; ESCRT: endosomal sorting complexes required for transport; HEXB: hexosaminidase subunit beta; HGS/HRS/ESCRT-0: hepatocyte growth factor-regulated tyrosine kinase substrate; IGF2R/CI-MPR: insulin like growth factor 2 receptor; ILV: intraluminal vesicles; KD: knockdown; KO: knockout; M6PR/CD-MPR: mannose-6-phosphate receptor, cation dependent; MEF: murine embryonic fibroblasts; MWU: Mann-Whitney U test; PepA: pepstatin A; RAB7A: RAB7, member RAS oncogene family; SNX-BAR: sorting nexin dimers with a Bin/Amphiphysin/Rvs (BAR) domain each; TGN: trans-Golgi network; TUBB: tubulin beta; VPS26: VPS26 retromer complex component; VPS29: VPS29 retromer complex component; VPS35: VPS35 retromer complex component.
Topics: Autophagy; Caspase 9; Endosomes; HeLa Cells; Humans; Microtubule-Associated Proteins; Protein Transport; Receptor, IGF Type 2; Vesicular Transport Proteins; trans-Golgi Network
PubMed: 32397873
DOI: 10.1080/15548627.2020.1761742 -
Cell Death & Disease May 2020Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic...
Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic myoblasts inducing differentiation in neighboring cells. Here we show for the first time that caspase-3 is activated in the myoblasts undergoing differentiation. Using RNAi, we also demonstrate that differentiation requires both cytochrome c and Apaf-1, and by using a new pharmacological approach, we show that apoptosome formation is required. We also show that Bid, whose cleavage links caspase-2 to the mitochondrial death pathway, was required for differentiation, and that the caspase cleavage product, tBid, was generated during differentiation. Taken together, these data suggest that myoblast differentiation requires caspase-2 activation of the mitochondrial death pathway, and that this occurs in the cells that differentiate. Our data also reveal a hierarchy of caspases in differentiation with caspase-2 upstream of apoptosome activation, and exerting a more profound control of differentiation, while caspases downstream of the apoptosome primarily control cell fusion.
Topics: Animals; Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; BH3 Interacting Domain Death Agonist Protein; Caspase 2; Caspase 3; Caspase Inhibitors; Cell Differentiation; Cell Fusion; Cell Line; Cyclohexanones; Cytochromes c; Enzyme Activation; Gene Knockdown Techniques; Humans; Mice; Muscle Fibers, Skeletal; Myoblasts; RNA, Small Interfering
PubMed: 32366831
DOI: 10.1038/s41419-020-2502-4 -
Cell Death and Differentiation Oct 2020The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is...
The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is the apoptosome, a platform for the activation of pro-caspase 9 (PC9). Recent findings have shown that autocleavage of PC9 to Caspase 9 (C9) p35/p12 not only permits XIAP-mediated C9 inhibition but also temporally shuts down apoptosome activity, forming a molecular timer. In order to delineate the combined contributions of XIAP and the apoptosome molecular timer to apoptosis execution we utilised a systems modelling approach. We demonstrate that cooperative recruitment of PC9 to the apoptosome, based on existing PC9-apoptosome interaction data, is important for efficient formation of PC9 homodimers, autocatalytic cleavage and dual regulation by XIAP and the molecular timer across biologically relevant PC9 and APAF1 concentrations. Screening physiologically relevant concentration ranges of apoptotic proteins, we discovered that the molecular timer can prevent apoptosis execution in specific scenarios after complete or partial mitochondrial outer membrane permeabilisation (MOMP). Furthermore, its ability to prevent apoptosis is intricately tied to a synergistic combination with XIAP. Finally, we demonstrate that simulations of these processes are prognostic of survival in stage III colorectal cancer and that the molecular timer may promote apoptosis resistance in a subset of patients. Based on our findings, we postulate that the physiological function of the molecular timer is to aid XIAP in the shutdown of caspase-mediated apoptosis execution. This shutdown potentially facilitates switching to pro-inflammatory caspase-independent responses subsequent to Bax/Bak pore formation.
Topics: Apoptosis; Caspase 9; Colorectal Neoplasms; Humans; Mitochondria; Mitochondrial Membranes; X-Linked Inhibitor of Apoptosis Protein
PubMed: 32341447
DOI: 10.1038/s41418-020-0545-9 -
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 -
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 -
Biochemistry. Biokhimiia Feb 2020Cytotoxic T lymphocytes and natural killer cells eliminate infected cells from the organism by triggering programmed cell death (apoptosis). The contents of the lytic... (Review)
Review
Cytotoxic T lymphocytes and natural killer cells eliminate infected cells from the organism by triggering programmed cell death (apoptosis). The contents of the lytic granules of killer cells, including pore-forming proteins perforins and proteolytic enzymes granzymes, are released with the following penetration of the released proteins into the target cells. Granzyme B initiates mitochondria-dependent apoptosis via (i) proapoptotic Bid protein, (ii) Mcl-1 and Bim proteins, or (iii) p53 protein. As a result, cytochrome c is released from the mitochondria into the cytoplasm, causing formation of apoptosomes that initiate the proteolytic cascade of caspase activation. Granzymes M, H, and F cause cell death accompanied by the cytochrome c release from the mitochondria. Granzyme A induces generation of reactive oxygen species (ROS), which promotes translocation of the endoplasmic reticulum-associated SET complex to the nucleus where it is cleaved by granzyme A, leading to the activation of nucleases that catalyze single-strand DNA breaks. Granzymes A and B penetrate into the mitochondria and cleave subunits of the respiratory chain complex I. One of the complex I subunits is also a target for caspase-3. Granzyme-dependent damage to complex I leads to the ROS generation and cell death.
Topics: Animals; Cell Death; Granzymes; Humans; Mitochondria; Reactive Oxygen Species
PubMed: 32093590
DOI: 10.1134/S0006297920020017 -
Molecular and Cellular Biochemistry Apr 2020Circadian rhythms help organisms adapt to changes of external environment by regulating energy metabolism and remaining the balance of homeostasis. Numerous researches...
Circadian gene Clock participates in mitochondrial apoptosis pathways by regulating mitochondrial membrane potential, mitochondria out membrane permeablization and apoptosis factors in AML12 hepatocytes.
Circadian rhythms help organisms adapt to changes of external environment by regulating energy metabolism and remaining the balance of homeostasis. Numerous researches have proved that the physiological function of liver was precisely controlled by circadian rhythms. Clock, one of core circadian genes, has been demonstrated to regulate the oxidative phosphorylation process of mitochondrial, which provides energy for living cells and acts as one of the hub for apoptosis. However, whether Clock gene regulates mitochondrial apoptosis pathways in liver cells remains less explored. In the present study, we used lentiviral vector to establish a stable AML12 cell lines which were capable of expressing specific shRNA to interfere the expression of Clock gene and investigated the effect of Clock on mitochondrial apoptosis pathways. Herein, we found that the interference of Clock gene could significantly suppress mitochondrial apoptosis pathways by stabilizing mitochondrial membrane potential and inhibiting mitochondria out membrane permeablization, which might be a result of lower expression of BAD and BIM proteins. Moreover, the interference of Clock gene could downregulate the expression of mitochondrial apoptosis factors, i.e. AIF, CYCS, APAF-1 and SMAC, which will suppress the formation of apoptosome and the process of DNA degradation to further inhibit apoptosis process. This work provides an insight on the important role of Clock gene participating in mitochondrial apoptosis pathways of hepatocytes and unveils a probable pathogenesis of how circadian rhythm regulates liver diseases.
Topics: Animals; Apoptosis; CLOCK Proteins; Cell Line; Cell Membrane Permeability; Gene Knockdown Techniques; Gene Regulatory Networks; Hepatocytes; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondrial Membranes
PubMed: 32067140
DOI: 10.1007/s11010-020-03701-1 -
Microbial Cell (Graz, Austria) Dec 2019Viruses and other genetic parasites are present in virtually all forms of life. This chronic condition has led to diverse host cell adaptations such as CRISPR and RNAi,...
Viruses and other genetic parasites are present in virtually all forms of life. This chronic condition has led to diverse host cell adaptations such as CRISPR and RNAi, whose functions attenuate these parasites. It is hypothesized that programmed cell death (PCD) is an additional adaptation whose origins reside in viral defense. A core event of apoptotic PCD is the regulated release of mitochondrial inter-membrane space proteins into the cytosol, following which these apoptogenic proteins bring about the demise of the cell. The most well studied example of this is found in animals, where the release of mitochondrial cytochrome C nucleates the formation of the apoptosome, which then activates caspase mediated cell death. The release of mitochondrial proteins contributes to PCD in diverse organisms lacking the apoptosome, indicating that regulated mitochondrial release predates the evolution of canonical apoptosis. Using the budding yeast , we recently confirmed an early study showing that Nuc1, a homolog of the mitochondrial apoptotic driver protein Endonuclease G, attenuates cytosolic double stranded RNA (dsRNA) viruses, which are endemic to yeast and many other organisms. Viral attenuation by Nuc1 occurs most prominently during meiosis and in association with its developmentally programmed relocation from the mitochondria to the cytosol. Intriguingly, meiotic viral attenuation by Nuc1 occurs within the context of meiotic PCD of the superfluous mother cell that we have also discovered. These findings are discussed here.
PubMed: 32025511
DOI: 10.15698/mic2020.02.705