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PROVIDER: PRJNA977212 | ENA |
REPOSITORIES: ENA
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Project description:A lysosomal delivery mechanism required for autophagosome degradation
| PRJEB62651 | ENA
Project description:Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.
2024-08-02 | PXD042079 | Pride
Project description:Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. We identified a highly conserved transmembrane protein named RNAseK as a novel regulator of autophagosome degradation. Analyses of RNAseK knockout cells revealed that, while autophagosome maturation was intact, cargo degradation was severely disrupted. Importantly, lysosomal protease activity and acidification remained intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions showed reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 led to a defect in autophagosome clearance. In addition, the lysosomal fraction of RNAseK-depleted cells exhibited an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, our findings identified a lysosomal hydrolase delivery pathway required to mediate efficient autolysosome degradation.
2024-07-05 | PXD042727 | Pride
Project description:Autophagy-lysosomal degradation is an evolutionarily conserved process key to cellular homeostasis, differentiation, and stress survival, which is particularly important to the pathophysiology of the cardiovascular system. What is more, both experimental and clinical observations indicate that autophagy-lysosomal degradation affects correct cardiac morphogenesis, and in particular valve development. However, it is still unclear which cells upregulate autophagy-lysosomal degradation and for which specific cellular processes it is required. Here, we introduce novel zebrafish transgenic models to visualize autophagosomes and lysosomes in vivo and to follow their temporal and cellular localization in the larval heart. This allowed us to determine the kinetics of autophagosome and lysosome vesicle formation and to observe significant accumulation of lysosomal vesicles during the development of the atrioventricular and bulboventricular valves. We then addressed the functional role of lysosomal degradation in cardiovascular development using a spns1 mutant as a zebrafish model of lysosomal impairment. We found that spns1 mutants displayed morphologically and functionally abnormal heart development, including abnormal endocardial organization, impaired cardiac valve formation and high incidence of retrograde blood flow. Single-nuclear transcriptome analysis revealed endocardial-specific differences in the expression of lysosome-related genes and alterations of notch1 signaling in the mutant larval heart. Further, endocardial-specific overexpression of spns1 and notch1 rescued features of valve formation and function as well as overall cardiac morphogenesis. Altogether, our study provides an improved description of the autophagy and lysosomal events that take place during zebrafish heart development and reveals a cell-autonomous role of lysosomal processing during cardiac valve formation upstream of notch1 signaling.
2024-10-25 | GSE246850 | GEO
Project description:The activation of cellular quality control pathways to maintain metabolic homeostasis and mitigate diverse cellular stresses is emerging as a critical growth and survival mechanism in many cancers. Autophagy, a highly conserved cellular self-degradative process, is a key player in the initiation and maintenance of pancreatic ductal adenocarcinoma (PDA). However, the regulatory circuits that activate autophagy, and how they enable reprogramming of PDA cell metabolism are unknown. We now show that autophagy regulation in PDA occurs as part of a broader program that coordinates activation of lysosome biogenesis, function and nutrient scavenging, through constitutive activation of the MiT/TFE family of bHLH transcription factors. In PDA cells, the MiT/TFE proteins - MITF, TFE3 and TFEB - override a regulatory mechanism that controls their nuclear translocation, resulting in their constitutive activation. By orchestrating the expression of a coherent network of genes that induce high levels of lysosomal catabolic function, the MiT/TFE factors are required for proliferation and tumorigenicity of PDA cells. Importantly, unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosomal activation is specifically required to maintain intracellular AA pools in PDA. This AA flux is part of a program that is essential for metabolic homeostasis and bioenergetics of PDA but not for their non-transformed counterparts. These results identify the MiT/TFE transcription factors as master regulators of the autophagy-lysosomal system in PDA and demonstrate a central role of the autophagosome-lysosome compartment in maintaining tumor cell metabolism through alternative amino acid acquisition and utilization. Examination of mRNA levels in pancreatic ductal adenocarcinoma (PDA) cell line 8988T after treatment with siRNA for control or TFE3
2015-04-03 | E-GEOD-62077 | biostudies-arrayexpress
Project description:Autophagy is a highly conserved lysosomal degrative pathway important for maintaining cellular homeostasis. Much of our current knowledge of autophagy is focused on the initiation steps with the later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling become evident. Autophagy can function in both cell survival and cell death, however the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remains to be established. Here we use a proteomic analysis of larval midguts during degradation identifying a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. We show that Cathepsin L deficient Drosophila larval midgut cells, accumulate autophagic vesicles due to a block in autophagic flux, yet midgut degradation in not compromised. The accumulation of large aberrant autolysosomes in the absence of Cathepsin function appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material rather and not due to a defect in autophagosome-lysosome fusion. Finally, we find that other Cathepsins are also required for proper autolysosomal degradation in Drosophila larval midgut cells and that this is conserved in mammalian cells. Our findings suggest that Cathepsins play an important degrative role in the autolysosome to maintain autophagy flux, by balancing autophagosome production and turnover (to prevent autophagic stress).
2022-02-15 | PXD016675 | Pride
Project description:Purpose: Endosomal-lysosomal system is one of the pivotal degradation system for a varieties of extracellular substances, of which dysfunction has been indicated to be associated with cardiovascular and neurodegenerative diseases. This degradation process consists of multiple steps; uptake of extracellular molecules, endosomal formation and its transportation to lysosomes, and digestion by lysosomal enzymes. Whereas TFEB, is a transcriptional factor, has been well studied as a master regulator of cellular uptake and lysosomal function, a key regulatory mechanism for endosomal maturation remains unclear. We previously found that isorhamnetin, a dietary flavonoid, enhanced the endosomal-lysosomal proteolysis in J774.1 macrophage-like cell line, which was independent on mTORC1-TFEB axis. In this study, we analyzed the molecular mechanism of activated endosomal-lysosomal degradation by isorhamnetin.
2022-02-15 | GSE167009 | GEO
Project description:Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrate the biogenesis of autophagosomes, however, the origin of the required membranes is incompletely understood. Here, we applied sensitized pooled CRISPR screens and identified the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, we found that TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β-oxidation of fatty acids. TMEM41B localizes to the endoplasmic reticulum (ER) and interacts with SIGMAR1, a chaperone which regulates calcium and lipid transfer at ER contact sites with mitochondria and lipid droplets. Taken together, we propose that TMEM41B is a novel regulator of autophagosome biogenesis and ER lipid mobilization.
2018-08-20 | PXD010207 | Pride
Project description:Lysosomal dysfunction is considered pathogenic in Alzheimer Disease (AD). Loss of Presenilin-1(PSEN1) function causing early onset AD impedes acidification via defective vATPase V0a1 subunit delivery to lysosomes. We report that isoproterenol and related β2-adrenergic agonists re-acidify lysosomes in PSEN1 KO cells and fibroblasts from PSEN1 familial AD(FAD) patients, restores lysosomal calcium homeostasis and proteolysis, and reverses impaired autophagy flux. We identify a novel rescue mechanism involving PKA-mediated facilitated delivery of ClC-7 to lysosomes, which stimulates chloride influx and reverses markedly lowered Cl- content of PSEN1 KO lysosomes. Notably, PSEN1 loss-of-function impedes ER-to-lysosome delivery of ClC-7, thus accounting for lysosomal Cl- deficits that compound pH deficits due to deficient vATPase function. Transcriptomics of PSEN1-deficient cells reveal strongly down-regulated ER-to-lysosome transport pathways and reversibility by isoproterenol. Our findings uncover a broadened PSEN1 role in lysosomal ion homeostasis and novel pH modulation of lysosomes through β-adrenergic regulation of ClC-7, which can be therapeutically modulated.
2020-02-29 | GSE136894 | GEO
Project description:The activation of cellular quality control pathways to maintain metabolic homeostasis and mitigate diverse cellular stresses is emerging as a critical growth and survival mechanism in many cancers. Autophagy, a highly conserved cellular self-degradative process, is a key player in the initiation and maintenance of pancreatic ductal adenocarcinoma (PDA). However, the regulatory circuits that activate autophagy, and how they enable reprogramming of PDA cell metabolism are unknown. We now show that autophagy regulation in PDA occurs as part of a broader program that coordinates activation of lysosome biogenesis, function and nutrient scavenging, through constitutive activation of the MiT/TFE family of bHLH transcription factors. In PDA cells, the MiT/TFE proteins - MITF, TFE3 and TFEB - override a regulatory mechanism that controls their nuclear translocation, resulting in their constitutive activation. By orchestrating the expression of a coherent network of genes that induce high levels of lysosomal catabolic function, the MiT/TFE factors are required for proliferation and tumorigenicity of PDA cells. Importantly, unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosomal activation is specifically required to maintain intracellular AA pools in PDA. This AA flux is part of a program that is essential for metabolic homeostasis and bioenergetics of PDA but not for their non-transformed counterparts. These results identify the MiT/TFE transcription factors as master regulators of the autophagy-lysosomal system in PDA and demonstrate a central role of the autophagosome-lysosome compartment in maintaining tumor cell metabolism through alternative amino acid acquisition and utilization.
2015-04-03 | GSE62077 | GEO