RNASeq of Autophagy Mutant BMDMs
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE206124 | GEO | 2023/06/01
REPOSITORIES: GEO
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Project description:Xu J, Kong L, Oliver B, Li B, Creasey EA, Guzman G, Schenone M, Carey KL, Carr SA, Graham DB, Deguine J, Xavier RJ. 2023. Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine autophagy in the context of inflammatory conditions are lacking. Here, using mouse models with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production in models of inflammation and adherent-invasive Escherichia coli (AIEC) infection. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify novel mechanistic targets downstream of autophagy. Our study reveals glutamine/glutathione metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.
2023-06-06 | MSV000092110 | MassIVE
Project description:Autophagy is a highly conserved catabolic process which degrades and recycles intracellular components. To accomplish this task the surplus material (cargo) is sequestered in a double membranous vesicle termed autophagosome. To initiate the formation of autophagosome autophagy machinery is recruited to cargo in a controlled manner. One of the first factors recruited to damaged mitochondria during mitophagy (selective removal of damaged mitochondria by autophagy) are two protein kinases, ULK1/2 complex and TBK1. They both promote a subsequent recruitment of the class III phosphatidylinositol 3-kinase complex I (PI3Kc1 complex) which function is to generate the lipid phosphatidylinositol 3-phosphate (PI(3)P) on phagophore membranes. Both ULK1 and TBK1 were reported to phosphorylate several autophagy factors. Here, we probed phosphorylation of PI3Kc1 by ULK1 and TBK1 to gain a full picture of the phosphosites map of PI3Kc phosphorylated by both protein kinases. Recombinant PI3Kc1 was incubated with recombinant ULK1 complex or TBK1 in the presence of ATP and MgCl2. As a negative control, the same protein mixtures were incubated in a kinase buffer lacking MgCl2 and ATP. Samples were separated by SDS-PAGE and stained with Coomassie. The bands for each subunit of the PI3KC3-C1 complex (VPS15, VPS34, ATG14, and Beclin1) were extracted from the gel and submitted for mass spectrometry analysis.
2023-05-08 | PXD039663 | Pride
Project description:Xenophagy, also known as antibacterial autophagy, is a process of capturing and eliminating cytosolic pathogens, like Salmonella. Salmonella is the best-studied model organism for xenophagy. We present a Petri net model of Salmonella xenophagy in epithelial cells. The model is based on functional information derived from literature data and contains all known processes of Salmonella xenophagy in epithelial. The model comprises the molecular mechanism of galectin-8-dependent and ubiquitin-dependent autophagy, including regulatory processes, like nutrient-dependent regulation of autophagy and TBK1-dependent activation of the autophagy receptor, OPTN. To model the activation of TBK1, we proposed a mechanism of TBK1 activation, suggesting a spatial and temporal regulation of this process. The Petri net is connected, covered by T-invariants, and each T-invariant has a meaningful biological interpretation. We checked the model structure for consistencies and correctness. We found 16 basic functional modules, which describe different pathways of the autophagic capturing of Salmonella and reflect the basic dynamics of the system. The PN model of Salmonella xenophagy comprises 61 places, including nine logical places, and 69 transitions connected by 184 arcs.
2020-04-09 | MODEL1904150001 | BioModels
Project description:Our aim was to identify candidate transcripts that distinguish AIEC from non-invasive E. coli (NIEC) strains and might be useful for rapid and accurate identification of AIEC by culture-independent technology. We performed comparative RNA-Sequence (RNASeq) analysis using AIEC strain LF82 and NIEC strain HS during exponential and stationary growth.
2017-02-09 | GSE69020 | GEO
Project description:Autophagosome formation requires multiple autophagy-related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome-wide CRISPR screens identifying genes required for NBR1 flux in ATG7KO cells. We find that ATG7-independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1's ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin-independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor-mediated clustering of upstream autophagy factors drives continued autophagosome formation.
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Project description:The gastrointestinal tract is colonized by trillions of microorganisms collectively known as the gut microbiota. These microbes provide essential signals to support healthy gut function. The microbiota is separated from internal tissue by a single layer of intestinal epithelial cells that not only provides a physical barrier but also relays luminal signals to underlying gut immune cells. Altered microbiota composition including loss of anti-inflammatory microbes or outgrowth of mucosa-associated bacteria such as adherent-invasive E. coli (AIEC) are hallmarks of inflammatory disease including inflammatory bowel disease (IBD). In contrast to their hypothesized role in pathology, we recently identified select AIEC isolates that improve outcomes in mouse colitis models. These AIEC induce macrophage production of the anti-inflammatory cytokine IL-10 which limits gut inflammation and supports barrier repair. These benefits were lost if the AIEC was unable to attach to epithelial cells. However, the epithelial signaling underlying this protection remained unclear. To understand if intestinal epithelial cells signaled to immune cells after microbial attachment, we utilized human colonic organoid monolayers and found co-culture with a subset of AIEC isolates upregulated immune regulatory genes including CCL2, a macrophage recruiting chemokine. This effect was only observed in undifferenced epithelial cells, indicating epithelial stem cell recognition of microbes leads to macrophage recruitment. In vivo, antibody blockade of CCR2 abrogated the protective effect of AIEC colonization. Using bacterial transcriptome analysis, we identified high flagellin expression in AIEC isolates that activated epithelial signaling, with lost signaling in organoids deficient for TLR5, the receptor for flagellin. Together our findings suggest intestinal epithelial cells recognize microbial signals to coordinate macrophage recruitment that support intestinal repair, protecting from colitis.
2024-03-11 | GSE261040 | GEO
Project description:The gastrointestinal tract is colonized by trillions of microorganisms collectively known as the gut microbiota. These microbes provide essential signals to support healthy gut function. The microbiota is separated from internal tissue by a single layer of intestinal epithelial cells that not only provides a physical barrier but also relays luminal signals to underlying gut immune cells. Altered microbiota composition including loss of anti-inflammatory microbes or outgrowth of mucosa-associated bacteria such as adherent-invasive E. coli (AIEC) are hallmarks of inflammatory disease including inflammatory bowel disease (IBD). In contrast to their hypothesized role in pathology, we recently identified select AIEC isolates that improve outcomes in mouse colitis models. These AIEC induce macrophage production of the anti-inflammatory cytokine IL-10 which limits gut inflammation and supports barrier repair. These benefits were lost if the AIEC was unable to attach to epithelial cells. However, the epithelial signaling underlying this protection remained unclear. To understand if intestinal epithelial cells signaled to immune cells after microbial attachment, we utilized human colonic organoid monolayers and found co-culture with a subset of AIEC isolates upregulated immune regulatory genes including CCL2, a macrophage recruiting chemokine. This effect was only observed in undifferenced epithelial cells, indicating epithelial stem cell recognition of microbes leads to macrophage recruitment. In vivo, antibody blockade of CCR2 abrogated the protective effect of AIEC colonization. Using bacterial transcriptome analysis, we identified high flagellin expression in AIEC isolates that activated epithelial signaling, with lost signaling in organoids deficient for TLR5, the receptor for flagellin. Together our findings suggest intestinal epithelial cells recognize microbial signals to coordinate macrophage recruitment that support intestinal repair, protecting from colitis.
2024-03-12 | GSE261061 | GEO
Project description:TBK1 is a multifunctional kinase phosphorylating a variety of proteins triggering innate immune signaling and autophagy. TBK1 has recently attracted much attention of neurologists for its critical role for the pathogenesis of various neurologic diseases including ALS. Loss of function of TBK1 has been identified as a major cause of ALS and also as a sufficient cause for the disease. Existing data suggests that TBK1 or its interacting partners may be promising targets for therapeutic intervention.
2019-11-19 | GSE140583 | GEO
Project description:Germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B-cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh differentiation, although some malaria-infected B-cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through non-canonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC leading to BCL6 suppression, therefore failed to form GC. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon re-infection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.
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Project description:The purpose of the dataset is to analyze expression of genes induced by KRAS and regulated by TBK1; The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. An alternative strategy for targeting KRAS is to identify gene products that, when suppressed or inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference (RNAi) to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkB kinase, TBK1, was selectively essential in cells that harbor mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF- B anti-apoptotic signals involving cREL and BCL-XL that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations identify TBK1 as a potential therapeutic target in KRAS mutant tumors and establish a general approach for the rational identification of co-dependent pathways in cancer. Experiment Overall Design: Knock out of TBK1 in the contect of KRAS activation (mutant) and control (WT)
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