TEX264 drives selective autophagy of DNA lesions to promote DNA repair and cell survival
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PROVIDER: PRJNA1013058 | ENA |
REPOSITORIES: ENA
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Project description:DNA repair and autophagy are distinct biological processes vital for cell survival. Although autophagy helpsmaintain genome stability, there is no evidence of its direct role in the repair of DNA lesions. We discoveredthat lysosomes process topoisomerase 1 cleavage complexes (TOP1cc) DNA lesions in vertebrates. Selectivedegradation of TOP1cc by autophagy directs DNA damage repair and cell survival at clinically relevantdoses of topoisomerase 1 inhibitors. TOP1cc are exported from the nucleus to lysosomes through a transientalteration of the nuclear envelope and independent of the proteasome. Mechanistically, the autophagy receptorTEX264 acts as a TOP1cc sensor at DNA replication forks, triggering TOP1cc processing by theQ8 p97 ATPase and mediating the delivery of TOP1cc to lysosomes in an MRE11-nuclease- and ATR-kinasedependentmanner. We found an evolutionarily conserved role for selective autophagy in DNA repair that enQ2ables cell survival, protects genome stability, and is clinically relevant for colorectal cancer patients.
2024-09-03 | PXD046037 | Pride
Project description:DNA repair and autophagy are distinct biological processes vital for cell survival. Although autophagy helps maintain genome stability, there is no evidence of its direct role in the repair of DNA lesions. We discovered that in human cells, lysosomes process Topoisomerase 1-cleavage complexes (TOP1cc) DNA lesion. Selective degradation of TOP1cc by autophagy directs DNA damage repair and cell survival at clinically relevant doses of Topoisomerase 1 inhibitors. TOP1cc are exported from the nucleus to lysosomes through transient alteration of the nuclear envelope, and independent of the proteasome. Mechanistically, the autophagy receptor TEX264 acts as a TOP1cc sensor at DNA replication forks, triggering TOP1cc processing by the p97 ATPase and mediating TOP1cc delivery to lysosomes dependent on MRE11 nuclease and ATR kinase. We found an evolutionary conserved role for selective autophagy in DNA damage repair that enables cell survival, protects genome stability, and is clinically relevant for colorectal cancer patients.
2024-09-01 | GSE242298 | GEO
Project description:Overexpression of the selective autophagy receptor AtNBR1
| PRJNA506042 | ENA
Project description:Autophagy is a conserved metabolic pathway that is central to many diseases. Recently, there has been a lot of interest in targeting autophagy with small molecule inhibitors as a possible therapeutic strategy. However, many of the compounds used for autophagy are non-selective. Here, we explored the inhibition of autophagy in pancreatic cancer cells using established selective small molecule inhibitors and discovered an unexpected link between the autophagy pathway and progression through the cell cycle. RNA-Seq analysis revealed that treatments with inhibitors that have different autophagy pathway targets block cell replication and activate other metabolic pathways to compensate for the blockade in autophagy. An unbiased screen looking for known drugs that might synergize with autophagy inhibition revealed new combination treatments that might provide a blueprint for therapeutic approaches to pancreatic cancer. The drugs quizartinib and THZ1 showed a strong synergistic effect in pancreatic cells with autophagy inhibition.
2022-12-05 | GSE206261 | GEO
Project description:Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargoes to the membrane bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we discover a selective autophagy pathway for protein condensates formed by endocytic proteins. In this pathway, the endocytic yeast protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis, does not involve other known selective autophagy receptors, but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates – at the plasma membrane and in autophagic bodies – shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy pathway. Our results imply an important role of autophagy in surveillance of membraneless compartments
2020-11-23 | PXD019083 | Pride
Project description:Cargo receptors are well established elements of the selective autophagy in all eukaryotes. The NBR1-like family of such receptors is evolutionarily conserved, however plants have only one member of the family, which is a functional and structural hybrid of its two animal counterparts, p62 (SQSTM1) and NBR1. Examination of plants overexpressing NBR1 indicates that they are oversensitive to TOR inhibitors and have more autophagosomes than the parental lines. The observed changes in shoots suggested induction of ABA signalling pathway, what was further corroborated by the germination tests and the assay of ABA level. The transcriptome changes in roots, such as lower level of snoRNA and some tRNAs, suggested inhibition of ribosome biogenesis, what is supported by identification of RPS6, an important component of TOR-dependent translational control, as a novel partner of NBR1. Grant number: 2014/15/B/NZ3/04854 Grant funding source: National Science Centre (Poland) Grantee Name: Agnieszka Sirko Grant title: Upstream regulators and cellular targets of the selective autophagy cargo receptor AtNBR1 in different phases of sulfur deficiency in plants.
2020-04-20 | GSE122705 | GEO
Project description:The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis (Survivin, HIF1α, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1, BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers, in response to DNA damage caused by ionizing radiation,
2012-10-17 | GSE41627 | GEO
Project description:RNA viruses co-opt host intracellular structures to create specialized replication sites and advance infection. The response of the host to this membrane disruption is poorly understood. In this study, we explore Arabidopsis thaliana's reaction to three distinct viruses that commandeer varying cellular compartments. Our findings reveal autophagy is significantly induced within systemically infected tissues, with autophagy disruption rendering plants highly sensitive to infection. Contrary to being an antiviral defense, quantitative analyses of viral RNA and proteomes establish autophagy as a critical component of the host's tolerance mechanism. Further analysis of mitochondrial hijacking by the Turnip Crinkle Virus has shown that despite perturbing mitochondrial integrity, the virus does not trigger a typical mitophagy response. Instead, viral infection activates a distinct selective autophagy mechanism, where oligomeric metabolic enzymes moonlight as selective autophagy receptors and degrade key executors of defense and cell death. Altogether, our study reveals an autophagy-regulated metabolic rheostats that gauges cellular integrity during viral infection.
2024-07-23 | GSE264734 | GEO
Project description:The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis (Survivin, HIF1α, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1, BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers, in response to DNA damage caused by ionizing radiation, Polysome isolation was performed by separation of ribosome-bound mRNAs in sucrose gradients. Beckman Ultra-Clear centrifuge tubes were loaded with 5.5 ml of 50% sucrose in low salt buffer (LSB: 200 mM Tris pH7.4 in DEPC H2O, 100 mM NaCl, 30 mM MgCl2) with 1:1000 RNasin (Fermentas) and 100 μg/mL cycloheximide (CHX) in ethanol and incubated at 4°C horizontally overnight. Media was removed from cell cultures, replaced with media containing 100 μg/mL CHX, cells incubated at 37°C for 10 min to halt protein synthesis, and trypsinized in trypsin/EDTA containing 100 μg/mL CHX. Cells were washed twice in PBS containing CHX, RNasin and Roche complete EDTA-free protease inhibitor tablet, lysed in LSB with CHX and RNasin. Lysates were added to a Dounce homogenizer and incubated on ice for 3 min before addition of Triton detergent buffer (1.2% Triton N-100, 0.2M sucrose in LSB) and homogenization. Samples were transferred to cold sterile Eppendorf centrifuge tubes and centrifuged at 13,000 x RPM for 10 min at 4°C. Post-nuclear supernatants were transferred to centrifuge tubes containing 100 μL Heparin solution (10 mg/mL heparin, 1.5M NaCl in LSB) with RNasin and CHX, applied to a sucrose gradient. Gradients were centrifuged at 36K RPM at 4°C for 2 h, and supernatants recovered with an ISCO-UV fractionator. Samples were recovered into Eppendorf centrifuge tubes containing 40 μL RNase-free 0.5M EDTA and kept on ice. One volume acidic phenol-chloroform was added to each sample, which were then mixed and centrifuged at 10,000 x g for 15 min. The acidic phenol-chloroform extraction was repeated with the aqueous phase, which was then extracted twice via chloroform extraction. RNA was precipitated at -20°C overnight by addition of isopropanol with 0.1 vol NaOAc, pH5.2. The following day, RNA pellets were recovered by centrifugation, washed with 70% ethanol, air-dried and resuspended in sterile H2O. RNA from fractions was then pooled based on their relative rate of translation, with fractions containing 4 or more ribosomes considered heavily translated and used for gene chip analysis. The RNA quality was examined via the Agilent Technologies kit.
2012-10-17 | E-GEOD-41627 | biostudies-arrayexpress
Project description:This method, termed Repair Assisted Damage Detection sequencing (RADD-seq), uses repair enzymes to excise the DNA lesions leaving a single-strand gap. The damage sites are then repaired in-vitro with biotinylated nucleotides, followed by DNA fragmentation, immunoprecipitation and sequencing. Reads are mapped back to the reference genome, indicating the locations of damage sites.
2021-02-17 | GSE166843 | GEO