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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 -
PloS One 2019Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles...
Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington's disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.
Topics: Apoptotic Protease-Activating Factor 1; Caspase 9; Cullin Proteins; Enzyme Activation; Etoposide; HEK293 Cells; Humans; Leupeptins; Protein Binding; Ubiquitination; bcl-X Protein
PubMed: 31329620
DOI: 10.1371/journal.pone.0219782 -
Journal of Biological Inorganic... Mar 2024Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human...
Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.
Topics: Cytochromes c; Humans; Animals; Mice; Mutation; Protein Conformation; Enzyme Activation; Species Specificity; Molecular Dynamics Simulation; Caspases
PubMed: 38472487
DOI: 10.1007/s00775-024-02044-2 -
Cells May 2021Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer...
Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method.
Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500-3000 nm) reminiscent of apoptosomes, (ii) EVs (100-500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80-300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20-200 nm) containing unidentified vesicles/particles, and (v) EVs (10-70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producinga GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.
Topics: Cell Differentiation; Exosomes; Extracellular Vesicles; Humans; Macrophages; Mouth Neoplasms; Tumor Microenvironment; Tumor-Associated Macrophages
PubMed: 34071980
DOI: 10.3390/cells10061328 -
Cell Death Discovery Apr 2022Oxidative stress is a state in which the accumulation of reactive oxygen species exceeds the capacity of cellular antioxidant systems. Both apoptosis and necrosis are...
Oxidative stress is a state in which the accumulation of reactive oxygen species exceeds the capacity of cellular antioxidant systems. Both apoptosis and necrosis are observed under oxidative stress, and we have reported that these two forms of cell death are induced in HO-stimulated HeLa cells depending on the concentration of HO. Weak HO stimulation induces apoptosis, while strong HO stimulation induces necrosis. However, the detailed mechanisms controlling the switching between these forms of cell death depending on the level of oxidative stress remain elusive. Here, we found that NAD metabolism is a key factor in determining the form of cell death in HO-stimulated HeLa cells. Under both weak and strong HO stimulation, intracellular nicotinamide adenine dinucleotide (NAD) was depleted to a similar extent by poly (ADP-ribose) (PAR) polymerase 1 (PARP1)-dependent consumption. However, the intracellular NAD concentration recovered under weak HO stimulation but not under strong HO stimulation. NAD recovery was mediated by nicotinamide (NAM) phosphoribosyltransferase (NAMPT)-dependent synthesis via the NAD salvage pathway, which was suggested to be impaired only under strong HO stimulation. Furthermore, downstream of NAD, the dynamics of the intracellular ATP concentration paralleled those of NAD, and ATP-dependent caspase-9 activation via apoptosome formation was thus impaired under strong HO stimulation. Collectively, these findings suggest that NAD dynamics balanced by PARP1-dependent consumption and NAMPT-dependent production are important to determine the form of cell death activated under oxidative stress.
PubMed: 35410407
DOI: 10.1038/s41420-022-01007-3 -
Cancers Oct 2019Colorectal cancer (CRC) is a leading killer cancer worldwide and one of the most common malignancies with increasing incidences of mortality. Guggulsterone (GS) is a...
Colorectal cancer (CRC) is a leading killer cancer worldwide and one of the most common malignancies with increasing incidences of mortality. Guggulsterone (GS) is a plant sterol used for treatment of various ailments such as obesity, hyperlipidemia, diabetes, and arthritis. In the current study, anti-cancer effects of GS in human colorectal cancer cell line HCT 116 was tested, potential targets identified using mass spectrometry-based label-free shotgun proteomics approach and key pathways validated by proteome profiler antibody arrays. Comprehensive proteomic profiling identified 14 proteins as significantly dysregulated. Proteins involved in cell proliferation/migration, tumorigenesis, cell growth, metabolism, and DNA replication were downregulated while the protein with functional role in exocytosis/tumor suppression was found to be upregulated. Our study evidenced that GS treatment altered expression of Bcl-2 mediated the mitochondrial release of cytochrome c which triggered the formation of apoptosome as well as activation of caspase-3/7 leading to death of HCT 116 cells via intrinsic apoptosis pathway. GS treatment also induced expression of p53 protein while p21 expression was unaltered with no cell cycle arrest. In addition, GS was found to inhibit NF-kB signaling in colon cancer cells by quelling the expression of its regulated gene products Bcl-2, cIAP-1, and survivin.
PubMed: 31581454
DOI: 10.3390/cancers11101478 -
Frontiers in Neurology 2019Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that leads to the death of neurons and oligodendrocytes, which cannot be measured...
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that leads to the death of neurons and oligodendrocytes, which cannot be measured in living subjects. Physiological cellular death, otherwise known as apoptosis, progresses through a series of stages which culminates in the discharge of cellular contents into vesicles known as apoptotic bodies (ABs) or apoptosomes. These ABs can be detected in bodily fluids as Annexin-V-positive vesicles of 0.5-4.0 μm in size. In addition, the origin of these ABs might be detected by staining for cell-specific surface markers. Thus, we investigated whether quantifications of the total and CNS cell-specific ABs in the cerebrospinal fluid (CSF) of patients provided any clinical value in MS. Extracellular vesicles, from CSF of 64 prospectively-acquired subjects, were collected in a blinded fashion using ultra-centrifugation. ABs were detected by flow cytometry using bead-enabled size-gating and Annexin-V-staining. The origin of these ABs was further classified by staining the vesicles for cell-specific surface markers. Upon unblinding, we evaluated the differences between diagnostic categories and correlations with clinical measures. There were no statistically significant differences in the numbers of total or any cell-specific ABs across different disease diagnostic subgroups and no significant correlations with any of the tested clinical measures of CNS tissue destruction, disability, MS activity, and severity (i.e., rates of disability accumulation). Overlap of cell surface markers suggests inability to reliably determine origin of ABs using antibody-based flow cytometry. These negative data suggest that CNS cells in MS either die by non-apoptotic mechanisms or die in frequencies indistinguishable by current assays from apoptosis of other cells, such as immune cells performing immunosurveillance in healthy conditions.
PubMed: 31849814
DOI: 10.3389/fneur.2019.01241 -
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