Importance of Atg5, Atg16L1 and Atg7 in mouse bone marrow-derived macrophages infected for 48h with M. tuberculosis Erdman
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ABSTRACT:
PROVIDER: PRJNA940447 | ENA |
REPOSITORIES: ENA
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Project description:To identify cellular pathways altered in autophagy-deficient macrophages during M. tuberculosis infection, we performed 3' Tag-RNA-seq on RNA harvested from Atg5fl/fl LysMcre+/+, Atg16L1fl/fl LysMcre+/+ and Atg7fl/fl LysMcre+/+ mouse bone marrow derived macrophages, and their respective LysMwt/wt control cells, both after 48 hours of infection with M. tuberculosis Erdman and or in mock-infection controls.
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Project description:Mycobacterium tuberculosis impairs host lysosomal trafficking pathways. Xenophagy and LC3-associated phagocytosis (LAP) are lysosomal trafficking pathways that normally clear intracellular microbes. The xenophagy and LAP pathways depend upon ATG5 and ATG7, which result in the lipidation of LC3-I to LC3-II. How Mtb undermines these innate immune defenses of the host in not well understood. We used a transposon sequencing (Tn-seq) screen to identify bacterial factors that are required for the bacilli to resist ATG5 and ATG7-dependent processes. We found that that mutants defective in production of PDIM are attenuated in murine macrophages, and they are able to survive in macrophages lacking ATG5 or ATG7.
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2019-10-01 | GSE122652 | GEO
Project description:Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn's disease patients carrying the Crohn's disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells. Experiment Overall Design: 4 Samples: 2 replicates of Atg16-hypomorph Paneth cells and 2 replicates of Wildtype Paneth cells.
2008-11-15 | E-GEOD-13512 | biostudies-arrayexpress
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2012-01-05 | GSE34863 | GEO
Project description:Type-I (α/β) and -II (γ) interferons (IFN), through an incompletely understood combination of redundant and unique mechanisms, are essential for host resistance to viral infection. We report a requirement for the Atg5-Atg12/Atg16L1 autophagosome elongation complex in IFNγ-mediated control of murine norovirus in macrophages. We use microarrays to compare transcriptional changes induced in control and Atg5 deficient macrophages by IFNγ treatment. Bone marrow derived macrophages were generated from Atg5flox/flox and Atg5flox/flox+LysM cre mice and treated with media alone or 100U/ml of IFNγ for 14 hrs. RNA was extracted using Trizol (Invitrogen) and 10 µg of RNA hybridized to Affymetrix microarrays.
2012-01-05 | E-GEOD-34863 | biostudies-arrayexpress
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2021-04-14 | GSE159793 | GEO
Project description:Glioblastoma (GBM) carries a dismal prognosis largely due to acquired resistance to the standard treatment, which incorporates the chemotherapy temozolomide (TMZ). Inhibiting the proteasomal pathway is an emerging strategy, where combination treatments are under clinical investigation. We hypothesized that pre-treatment of GBM with bortezomib (BTZ) might sensitize glioblastoma to TMZ by abolishing autophagy survival signals to augment DNA damage and apoptosis. P3 patient-derived GBM cells as well as the tumor cell lines U87, HF66, A172 and T98G were investigated for clonogenic survival after single or combined treatment with TMZ and BTZ in vitro. Change in autophagic flux was examined after experimental treatments in conjunction with inhibitors of autophagy or downregulation of autophagy-related genes -5 and -7 (ATG5 and ATG7, respectively). Autophagic flux was increased in TMZ-resistant P3 and T98G cells as indicated by diminished levels of the autophagy markers LC3A/B-II and increased STX17, higher protein degradation and no formation of p62 bodies nor induction of apoptosis. In contrast, BTZ treatment attenuated ULK1 mRNA, total and phosphorylated protein, and accumulated LC3A/B-II, p62 and autophagosomes analogously to Baf1 and chloroquine autophagy inhibitors. These autophagosomes did not fuse with lysosomes, indicated by attenuated STX17 expression and reduced degradation of long-lived proteins, which culminated in enhanced caspase-3/8 dependent apoptosis. BTZ synergistically enhanced TMZ efficacy, attenuated tumor cell proliferation, triggered ATM/Chk2 DNA damage signalling to further augment caspase-3/8 mediated apoptosis in the TMZ resistant P3 and T98G GBM cells. Genetic or chemical inhibition of autophagy (with CRISPR-CAs9 ATG5, ATG7 shRNA, MRT68921 or VPS34-IN1) abrogated BTZ efficacy and rescued BTZ+ TMZ treated GBM cells from death. We conclude that Bortezomib ameliorates temozolomide resistance through ATG5/7-dependent abrogated autophagic flux and may be amenable in combination treatment regimens for TMZ refractory GBM patients.
2023-03-10 | PXD021828 | Pride
Project description:Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn's disease patients carrying the Crohn's disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.
2008-11-08 | GSE13512 | GEO
Project description:In this project, we employed a dynamic mass spectrometry-based proteomic approach to obtain global maps of basal autophagic flux in human primary fibroblasts (HCA2-hTert). The data provide a comparison of protein degradation rates between wildtype and autophagy deficient (Atg5-/- and Atg7-/-) cells. The media utilized for isotopic labeling was Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100U/mL penicillin, 100U/mL streptomycin. Prior to labeling, cells were gradually adapted to unlabeled media over the course of multiple passages. Cells were then plated at a density of 500,000 cells per 10 cm plate. 8 days after plating, the confluent quiescent cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-Arginine:HCl (13C6, 99%) and L-Lysine:2HCl (13C6, 99%)(Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L and 15% dialyzed fetal bovine serum (Thermo scientific). Cells were collected after 0d, 2d, 4d, 6d of labeling, washed with PBS and cell pellets were frozen prior to further analysis. In order to assess the precision of our measurements, biological replicate experiments were conducted for WT+vector and Atg5-/- experiments and cells were collected after 4d for analysis
2016-06-14 | PXD003562 | Pride