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Hypoxia increases neutrophil-driven matrix destruction after exposure to Mycobacterium tuberculosis.Scientific Reports Jul 2018The importance of neutrophils in the pathology of tuberculosis (TB) has been recently established. We demonstrated that TB lesions in man are hypoxic, but how...
The importance of neutrophils in the pathology of tuberculosis (TB) has been recently established. We demonstrated that TB lesions in man are hypoxic, but how neutrophils in hypoxia influence lung tissue damage is unknown. We investigated the effect of hypoxia on neutrophil-derived enzymes and tissue destruction in TB. Human neutrophils were stimulated with M. tuberculosis (M.tb) or conditioned media from M.tb-infected monocytes (CoMTB). Neutrophil matrix metalloproteinase-8/-9 and elastase secretion were analysed by luminex array and gelatin zymography, gene expression by qPCR and cell viability by flow cytometry. Matrix destruction was investigated by confocal microscopy and functional assays and neutrophil extracellular traps (NETs) by fluorescence assay. In hypoxia, neutrophil MMP-8 secretion and gene expression were up-regulated by CoMTB. MMP-9 activity and neutrophil elastase (NE) secretion were also increased in hypoxia. Hypoxia inhibited NET formation and both neutrophil apoptosis and necrosis after direct stimulation by M.tb. Hypoxia increased TB-dependent neutrophil-mediated matrix destruction of Type I collagen, gelatin and elastin, the main structural proteins of the human lung. Dimethyloxalylglycin (DMOG), which stabilizes hypoxia-inducible factor-1α, increased neutrophil MMP-8 and -9 secretion. Hypoxia in our cellular model of TB up-regulated pathways that increase neutrophil secretion of MMPs that are implicated in matrix destruction.
Topics: Apoptosomes; Cell Line; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lung; Matrix Metalloproteinase 8; Matrix Metalloproteinase 9; Monocytes; Mycobacterium tuberculosis; Neutrophils; Pancreatic Elastase; Signal Transduction; Tuberculosis; Up-Regulation
PubMed: 30065292
DOI: 10.1038/s41598-018-29659-1 -
Biochemical and Biophysical Research... Mar 2020Neuroglobin is a heme protein present in the nervous system cells of mammals and other organisms. Although cytoprotective effects of neuroglobin on neuronal damage have...
Neuroglobin is a heme protein present in the nervous system cells of mammals and other organisms. Although cytoprotective effects of neuroglobin on neuronal damage have been reported, the physiological mechanisms of neuroglobin function remain unknown. In recent years, a role for neuroglobin as a reductant for extramitochondrial cytochrome c has been proposed. According to this hypothesis, cytoplasmic neuroglobin can interact with cytochrome c released from the mitochondria and reduce its heme group to the ferrous state, thus preventing cytochrome c-dependent assembly of the apoptosome. The interaction of neuroglobin and cytochrome c has been studied by surface plasmon resonance techniques and molecular dynamics, however the empirical evidence on the specific residues of neuroglobin and cytochrome c involved in the interaction is scarce and indirect. This study analyzes the role of five negatively charged residues in the neuroglobin surface putatively involved in the interaction with cytochrome c - Glu60, Asp63, Asp73, Glu 87 and Glu151 - by site-directed mutagenesis. Characterization of the electron transfer between neuroglobin mutants and cytochrome c indicates that Asp73 is critical for the interaction, and Glu60, Asp63 and Glu87 also contribute to the neuroglobin-cytochrome c interaction. Based on the results, structures and binding surfaces for the neuroglobin-cytochrome c complex compatible with the experimental observations are proposed. These data can guide further studies on neuroglobin function and its involvement in cytochrome c signaling cascades.
Topics: Animals; Apoptosis; Apoptosomes; Binding Sites; Cytochromes c; Electron Transport; Horses; Molecular Docking Simulation; Neuroglobin; Protein Binding; Protein Interaction Maps; Recombinant Proteins; Static Electricity
PubMed: 31937411
DOI: 10.1016/j.bbrc.2019.12.089 -
Hepatobiliary & Pancreatic Diseases... Jun 2017Liver inflammation or hepatitis is a result of pluripotent interactions of cell death molecules, cytokines, chemokines and the resident immune cells collectively called... (Review)
Review
BACKGROUND
Liver inflammation or hepatitis is a result of pluripotent interactions of cell death molecules, cytokines, chemokines and the resident immune cells collectively called as microenvironment. The interplay of these inflammatory mediators and switching of immune responses during hepatotoxic, viral, drug-induced and immune cell-mediated hepatitis decide the fate of liver pathology. The present review aimed to describe the mechanisms of liver injury, its relevance to human liver pathology and insights for the future therapeutic interventions.
DATA SOURCES
The data of mouse hepatic models and relevant human liver diseases presented in this review are systematically collected from PubMed, ScienceDirect and the Web of Science databases published in English.
RESULTS
The hepatotoxic liver injury in mice induced by the metabolites of CCl, acetaminophen or alcohol represent necrotic cell death with activation of cytochrome pathway, formation of reactive oxygen species (ROS) and mitochondrial damage. The Fas or TNF-alpha induced apoptotic liver injury was dependent on activation of caspases, release of cytochrome c and apoptosome formation. The ConA-hepatitis demonstrated the involvement of TRAIL-dependent necrotic/necroptotic cell death with activation of RIPK1/3. The alpha-GalCer-induced liver injury was mediated by TNF-alpha. The LPS-induced hepatitis involved TNF-alpha, Fas/FasL, and perforin/granzyme cell death pathways. The MHV3 or Poly(I:C) induced liver injury was mediated by natural killer cells and TNF-alpha signaling. The necrotic ischemia-reperfusion liver injury was mediated by hypoxia, ROS, and pro-inflammatory cytokines; however, necroptotic cell death was found in partial hepatectomy. The crucial role of immune cells and cell death mediators in viral hepatitis (HBV, HCV), drug-induced liver injury, non-alcoholic fatty liver disease and alcoholic liver disease in human were discussed.
CONCLUSIONS
The mouse animal models of hepatitis provide a parallel approach for the study of human liver pathology. Blocking or stimulating the pathways associated with liver cell death could unveil the novel therapeutic strategies in the management of liver diseases.
Topics: Animals; Apoptosis; Cell Communication; Chemical and Drug Induced Liver Injury; Cytokines; Disease Models, Animal; Fas Ligand Protein; Hepatitis, Viral, Animal; Host-Pathogen Interactions; Humans; Inflammation Mediators; Liver; Mice; Necrosis; Oxidative Stress; Reactive Oxygen Species; Signal Transduction; Species Specificity
PubMed: 28603092
DOI: 10.1016/s1499-3872(17)60014-6 -
Communications Biology Aug 2020Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans...
Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans apoptosis has several unique features compared with human apoptosis. To date, there has been no detailed analysis of apoptosis regulators in nematodes other than C. elegans. Here, we discovered BCL-2 orthologues in 89 free-living and parasitic nematode taxa representing four evolutionary clades (I, III, IV and V). Unlike in C. elegans, 15 species possess multiple (two to five) BCL-2-like proteins, and some do not have any recognisable BCL-2 sequences. Functional studies provided no evidence that BAX/BAK proteins have evolved in nematodes, and structural studies of a BCL-2 protein from the basal clade I revealed it lacks a functionally important feature of the C. elegans orthologue. Clade I CED-4/APAF-1 proteins also possess WD40-repeat sequences associated with apoptosome assembly, not present in C. elegans, or other nematode taxa studied.
Topics: Animals; Apoptosis; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Genes, Helminth; Mice; Phylogeny; Protein Domains; Protein Structure, Secondary; Proto-Oncogene Proteins c-bcl-2; Signal Transduction
PubMed: 32859965
DOI: 10.1038/s42003-020-01208-5 -
Scientific Reports Jan 2018Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian...
Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian effector caspase containing a CARD that is similar to the amino terminal CARD of mammalian capsase-9, and we named it procaspase-3/9. Recombinant procaspase-3/9 expressed at 15 °C was cleaved to form active caspase-3/9 which has DEVDase activity. Microinjection of the active caspase-3/9 into starfish oocytes/eggs induced apoptosis. An antibody against the recombinant protein recognized endogenous procaspase-3/9 in starfish oocytes, which was cleaved upon apoptosis in aged unfertilized eggs. These results indicate that caspase-3/9 is an effector caspase in starfish. To verify the mechanism of caspase-3/9 activation, we cloned starfish Apaf-1 containing a CARD, a NOD, and 11 WD40 repeat regions, and we named it sfApaf-1. Recombinant sfApaf-1 CARD interacts with recombinant caspase-3/9 CARD and with endogenous procaspase-3/9 in cell-free preparations made from starfish oocytes, causing the formation of active caspase-3/9. When the cell-free preparation without mitochondria was incubated with inactive recombinant procaspase-3/9 expressed at 37 °C, DEVDase activity increased and apoptosome-like complexes were formed in the high molecular weight fractions containing both sfApaf-1 and cleaved caspase-3/9. These results suggest that sfApaf-1 activation is not dependent on cytochrome c.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Asterina; Caspase 3; Caspase 9; Caspases, Effector; Cloning, Molecular; Gene Expression Regulation, Enzymologic; Peptide Hydrolases; Recombinant Proteins; Zygote
PubMed: 29371610
DOI: 10.1038/s41598-018-19845-6 -
Journal of Molecular Biology Feb 2022Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines...
Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines and immune effectors upon cell bursting. This form of cell death has been increasingly well-characterized in mammals and can occur using alternative routes however, across phyla, there has been little evidence for the existence of pyroptosis. Here we provide evidence for an ancient origin of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to wounds and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory-based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects and relies on ancient apoptotic machinery to induce proto-pyroptosis. Further, we unveil key players upstream in the activation of cell death in these cells including the apoptosome which may play an alternative role akin to the inflammasome in proto-pyroptosis. Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system.
Topics: Animals; Apoptosomes; Caspases; Drosophila melanogaster; Inflammasomes; Mammals; Pyroptosis
PubMed: 34756921
DOI: 10.1016/j.jmb.2021.167333 -
Proceedings of the National Academy of... Nov 2014Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known...
Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known as the Apaf-1 apoptosome. The molecular mechanism by which caspase-9 is activated by the Apaf-1 apoptosome remains largely unknown. Here we demonstrate that the previously reported 1:1 interaction between Apaf-1 caspase recruitment domain (CARD) and caspase-9 CARD is insufficient for the activation of caspase-9. Rather, formation of a multimeric CARD:CARD assembly between Apaf-1 and caspase-9, which requires three types of distinct interfaces, underlies caspase-9 activation. Importantly, an additional surface area on the multimeric CARD assembly is essential for caspase-9 activation. Together, these findings reveal mechanistic insights into the activation of caspase-9 by the Apaf-1 apoptosome and support the induced conformation model for initiator caspase activation by adaptor complexes.
Topics: Apoptosis Regulatory Proteins; Apoptosomes; Caspase 9; Catalysis; Enzyme Activation; Humans; Hydrogen Bonding; In Vitro Techniques; Lysine; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Protein Interaction Mapping; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Fusion Proteins
PubMed: 25313070
DOI: 10.1073/pnas.1418000111 -
Cell Reports Nov 2022Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we...
Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.
Topics: Humans; Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Melanoma; Microphthalmia-Associated Transcription Factor; Protein Kinase Inhibitors; Salvage Therapy
PubMed: 36351409
DOI: 10.1016/j.celrep.2022.111601 -
Molecular Nutrition & Food Research May 2016Prolonged endoplasmic reticulum (ER) stress has lost the function of protein folding capacity and the ER accumulation of unfolded proteins that eventually triggers...
SCOPE
Prolonged endoplasmic reticulum (ER) stress has lost the function of protein folding capacity and the ER accumulation of unfolded proteins that eventually triggers apoptosis. Oxysterols are emerging as contributing factors in atherogenesis known to involve macrophage apoptosis. This study determined the inhibitory effect of α-asarone present in purple perilla, on 7β-hydroxycholesterol-induced macrophage apoptosis, targeting against ER stress signaling pathway.
METHODS AND RESULTS
J774A1 murine macrophages were exposed to 28 μM 7β-hydroxycholesterol and treated with 1-10 μM α-asarone. Macrophage apoptosis and ER stress were examined by and α-Asarone blocked 7β-hydroxycholesterol-induced DNA fragmentation and apoptosome formation. Immunoblotting showed that the oxysterol activated the ER transmembrane resident kinases of IRE1α, PERK and ATF4 and triggered caspase-12 signaling cascades, which was reversed by α-asarone. Additionally, 7β-hydroxycholesterol activated TRAF2-ASK1-JNK1/2 complex following the IRE1α activation, and α-asarone blunted such IRE1α-mediated pathway. Real-time PCR and dual-luciferase reporter analyses revealed that α-asarone reduced transcriptional activation of ER stress-responsive genes including XBP1 and CHOP by 7β-hydroxycholesterol. Finally, α-asarone disturbed oxysterol-elicited signaling of PERK and ATF4 responsible for CHOP induction.
CONCLUSION
α-Asarone blocked 7β-hydroxycholesterol-induced macrophage apoptosis through allaying ER stress-specific signaling involving caspase activation and CHOP induction. α-Asarone was an anti-atherosclerotic agent antagonizing ER stress-mediated macrophage apoptosis by 7β-hydroxycholesterol.
Topics: Activating Transcription Factor 4; Allylbenzene Derivatives; Animals; Anisoles; Apoptosis; Apoptosomes; Caspase 12; Cell Line; DNA Fragmentation; Endoplasmic Reticulum Stress; Endoribonucleases; Hydroxycholesterols; Macrophages; Mice; Protein Serine-Threonine Kinases; Signal Transduction; X-Box Binding Protein 1; eIF-2 Kinase
PubMed: 26893256
DOI: 10.1002/mnfr.201500750 -
Proceedings of the National Academy of... Nov 2014
Topics: Animals; Apoptosomes; Biophysics; Caenorhabditis elegans Proteins; Calcium-Binding Proteins; Caspases; China; Cryoelectron Microscopy; Crystallography, X-Ray; History, 20th Century; History, 21st Century; Humans; Models, Molecular; National Academy of Sciences, U.S.; Smad Proteins; United States; Universities
PubMed: 25331881
DOI: 10.1073/pnas.1418405111