Recycling of autophagosomal components from autolysosomes by the recycler complex
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Yueguang Rong
PROVIDER: PXD031183 | iProX | Mon Jan 24 00:00:00 GMT 2022
REPOSITORIES: iProX
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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:Here we show that Legionella SidE effectors mediate phosphoribosyl-linked serine ubiquitination (PR-Ub) of the autophagic SNARE proteins STX17 and SNAP29. On the one hand, PR-Ub modification of STX17 enhances its interaction with ATG14L, which drives the recruitment of ER membranes to the bacterial vacuole in a PI3K-dependent manner and promotes the formation of bacterial replicative vacuoles. On the other hand, PR-Ub of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) by steric hindrance and thus prevents the fusion of STX17 + bacterial vacuoles with lysosomes.
2025-05-14 | PXD058184 | Pride
Project description:Background Transforming growth factor β1 (TGF-β1) has a neuroprotective function in traumatic brain injury (TBI) through its anti-inflammatory and immunomodulatory properties. In our previous study, we found that TGF-β1 played a critical role in inhibiting apoptosis and increasing neuronal activity in murine cortical neurons following trauma. However, the precise mechanisms underlying the neuroprotective actions of TGF-β1 on the cortex require further investigation. Methods Thus, in this study, we were aimed to investigate the regulatory function of TGF-β1 on neuronal autophagy and apoptosis using an in vitro primary cortical neuron trauma-injury model. Results To establish the landscape of differentially expressed genes (DEGs) with or without TGF-β1 (10ng/ml) treatment for 24 hours, we performed RNA-sequencing. We observed significant enrichment of DEGs related to autophagy, apoptosis, and the lysosome pathway in trauma-injured cortical neurons. Additionally, transmission electron microscopy (TEM) confirmed the presence of autophagosomes as well as autophagolysosomes. Western blot analysis revealed upregulation of autophagy-related protein light chain 3 (LC3)-Ⅱ/LC3-Ⅰ, sequestosome 1 (SQSTM1)/p62, along with apoptosis-related protein Cleaved-caspase3 in trauma-injured primary cortical neurons. Furthermore, mechanically injured neurons showed an upregulation of lysosomal marker protein lysosomal marker protein (LAMP1) and lysosomal enzyme mature cathepsin D (mCTSD), but a decrease in the activity of CTSD enzyme. These results indicated that apoptosis was up-regulated in mechanically injured neurons at 24 hours, accompanied by lysosomal dysfunction and impaired autophagic flux. Notably, TGF-β1 significantly reversed these changes. Conclusions Therefore, our findings suggested that TGF-β1 exerted neuroprotective effects on mechanically injured neurons by reducing lysosomal dysfunction, decreasing the accumulation of autophagosomes and autophagolysosomes, and enhancing autophagic flux.
2024-05-10 | GSE249554 | GEO
Project description:The biogenesis of mammalian autophagosomes remains to be fully defined. Here we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally-derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. HyPAS biogenesis depends on a previously unrecognized machinery centered upon STX17 interactors, the calcium pump SERCA2, extended synaptotagmin E-SYT2, and SIGMAR1. This apparatus controls HyPAS biogenesis and defines the mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon SARS-CoV-2 infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes that emerge via convergence of secretory and endosomal pathways, and that this process is targeted by microbial factors such as coronaviral membrane modulating proteins.
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Project description:Autophagy plays an important role in preserving cellular homeostasis in pancreatic beta cells. However, the extent of autophagic flux induced in various physiological settings in vivo is unclear. In this study, we generated transgenic mice expressing pHluorin-LC3-mCherry reporter for monitoring systemic autophagic flux. Our findings revealed that autophagic flux in pancreatic islets enhanced after starvation, although suppression of the flux after short-term refeeding needs more prolonged restarvation in islets than in liver and skeletal muscle. Furthermore, heterogeneity of autophagic flux in beta cells manifested after increasing insulin resistance and intracellular calcium influx by glucose stimulation increased more in high- than low-flux beta cells, with differential gene expression based on the flux. Thus, our monitor mouse enables us to reveal physiological response and biological insight of heterogeneity in autophagic flux in pancreatic beta cells.
2023-03-15 | PXD036390 | JPOST Repository
Project description:Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitates autophagosome-lysosome fusion. Disease causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.
2023-06-18 | PXD043082 |
Project description:Autophagy is a vital cellular process that maintains organismal health by recycling damaged or superfluous cellular components within autophagosomes. This process is mediated by conserved ATG proteins, which coordinate autophagosome biogenesis and selective cargo degradation. Among these, the ubiquitin-like ATG8 protein plays a central role by linking cargo to the growing autophagosomes through interactions with selective autophagy receptors. Unlike most ATG proteins, which are represented by single or few isoforms, the ATG8 gene family is expanded in vascular plants, yet the extent of functional specialization within ATG8 isoforms remains largely unknown. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes within the same cell. To explore ATG8 specialization, we generated the nonuple Δatg8mutant lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for the specialization of ATG8 isoforms in plants and lay a foundation for investigating their roles in diverse cell types and stress conditions.
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Project description:Autophagy is a highly conserved pathway responsible for the bulk degradation of cytoplasmic material through the formation of a double-membrane structure known as the autophagosome. However, the precise mechanisms governing the transport of autophagosomes to the vacuole for degradation in plants remain largely elusive. There exists an ongoing debate about whether RAB7, a key regulatory protein, is involved in the plant autophagy pathway. In this study, we demonstrate that upon autophagy induction by BTH treatment, RABG3e, a member of the RAB7 family, exhibits a partial localization with late-stage autophagosomes in Arabidopsis root cells, and its dysfunction leads to the accumulation of enlarged multilayered autophagosomes and a significant reduction in autophagic flux. We also showed that RABG3e is recruited to autophagosomes by its GEF (guanine nucleotide exchange factor) complex, MON1-CCZ1, which is targeted through the interaction between CCZ1 and SH3P2 (SH3 domain-containing protein 2), a plant-specific autophagy regulator. Subsequently, RABG3e recruits the HOPS (homotypic fusion and vacuole protein sorting) complex, which facilitates the recruitment of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins, including SYP21, VTI12, SYP51, and VAMP711, that are essential for the fusion process. Arabidopsis mutants with dysfunction in the autophagosome-vacuole fusion process exhibit accelerated senescence and increased sensitivity to nitrogen starvation. Collectively, our findings provide new insights into the regulation of autophagosome-vacuole fusion in Arabidopsis, highlighting the essential roles of SH3P2-dependent targeting of the CCZ1-MON1-RABG3e module to late-stage autophagosomes as well as the RAB7 effector HOPS and the unique SNARE complex.
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