Similar Datasets
Project description:Compared protein synthesis in Gcn2 WT vs Gcn2 KO murine PDAC cells (KRPC) in amino acid-replete or leucine-free media.
2022-10-13 | PXD028143 | Pride
Project description:GCN2 adapts the proteome to scavenging-dependent growth
| PRJNA634769 | ENA
Project description:Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer.
| S-EPMC8961722 | biostudies-literature
Project description:La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine tract (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5’TOP motif, resulting in the displacement of the eIF4E complex from TOP mRNAs. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. GCN2 inhibits TOP mRNA translation via ATF4-dependent transcriptional induction of LARP1 mRNAs and GCN1-mediated recruitment of LARP1 to stalled ribosomes. We performed ATF4 ChIP-seq experiments in both WT and GCN2 KO MEFs with or without leucine deprivation.
2022-08-31 | GSE166590 | GEO
Project description:We previously demonstrated the proof of concept for the functionality of the NUTRIREG system in T cells. This technology allows for the induction of transgene expression following the consumption of a diet deficient in an essential amino acid, by activating the GCN2-ATF4 signaling pathway. To utilize the NUTRIREG technology for therapeutic purposes in cell therapy with short-term EAA deprivation, we explored the general impact of 6-hour leucine deprivation on primary activated human T cells using RNA-seq technology. We identified 3,431 differentially expressed genes (adjusted p-value < 0.05) between T cells cultured in control media and those cultured in leucine-deprived media for 6 hours. Gene Set Enrichment Analysis revealed that "TNFα signaling via NFκB", "interferon-γ response", and "unfolded protein response" gene sets were positively enriched, while "mTORC1 signaling", "Myc targets", and "oxidative phosphorylation" gene sets were negatively enriched. T cells were cultured with or without a GCN2 inhibitor, which allowed us to assess the involvement of GCN2 kinase in differential gene expression during the 6-hour leucine deprivation. We found that 59% of the differentially expressed genes (DEGs) in our dataset were dependent on GCN2 kinase (n=2028), with 1,140 up-regulated and 888 down-regulated GCN2-dependent genes.
2025-01-20 | GSE279263 | GEO
Project description:GCN2 (General Control Nonderepressible 2) is a serine/threonine-protein kinase that controls mRNA translation in response to amino acid availability. Here we show that production and clearance of erythrocytes are controlled by GCN2. Our data highlight the importance of tissue-resident macrophages as the primary cell type mediating this effect. During different stress conditions, such as hemolysis, amino acid deficiency or hypoxia, GCN2 knockout (GCN2-/-) mice displayed resistance to anemia as compared to wild-type (GCN2+/+) mice. GCN2-/- liver macrophages display defective erythrophagocytosis and lysosome maturation. Molecular analysis of GCN2-/- cells indicates that the ATF4-NRF2 pathway is a critical downstream mediator of GCN2 in regulating RBC clearance and iron recycling. We performed NRF2 (Nfe2l2) ChIP-seq experiments in both WT and GCN2 KO MEFs with or without leucine deprivation.
2022-12-01 | GSE188460 | GEO
Project description:Background: Arginine deprivation was shown previously to inhibit retinoblastoma cell proliferation and induce cell death in vitro. However, the mechanisms by which retinoblastoma cells respond to arginine deprivation remain to be elucidated. Methods: Human derived retinoblastoma cell lines Y79 and WERI-Rb-1 were exposed to arginine depletion, and the effects on inhibiting cell growth and survival were evaluated. The study investigated potential mechanisms, including autophagy, cell cycle arrest, and apoptosis. Moreover, the roles of GCN2 and mTOR signaling pathways in these processes were examined. Results: We demonstrated that arginine deprivation effectively inhibited the growth of retinoblastoma cells in vitro. This treatment caused an increase in autophagic response. Additionally, we found that prolonged arginine shortage induced G2 cell cycle arrest and was accompanied by an increase in early apoptotic cells. Importantly, arginine depletion also caused an activation of GCN2 and an inhibition of mTOR signaling. We also discovered that the activation of SLC7A11 was regulated by GCN2 upon arginine deprivation. Knockdown of SLC7A11 rendered retinoblastoma cells partially resistant to arginine deprivation. Furthermore, we found that knockdown of GCN2 led to a decrease in autophagic response in WERI-Rb-1 cells and arrested more cells in S phase, which was accompanied by fewer apoptotic cells. Moreover, knockdown of GCN2 caused constant expression of ATF4, phosphorylation of 70S6K, and 4E-BP1 regardless of arginine deprivation. Conclusions: Collectively, our findings suggest that the GCN2-SLC7A11 axis regulates cell growth and survival upon arginine deprivation through coordinating autophagy, cell cycle arrest, and apoptosis in retinoblastoma cells. This work paves the way for the development of a novel treatment for retinoblastoma.
2024-10-28 | GSE278276 | GEO
Project description:Amino acid availability regulates translation through the action of the GCN2 and mTORC1 pathways. Low amino acids activate the eIF2α kinase GCN2 through binding of uncharged tRNAs to a histidyl-tRNA synthetase−related regulatory domain. Once activated GCN2 phosphorylates eIF2α, inhibiting ternary complex formation and translation initiation. Recent studies show that mTORC1 is particularly sensitive to arginine and leucine status, with a deprivation of these amino acids leading to a strong inhibition of mTORC1 that prevents the phosphorylation and inactivation of the translational repressor 4EBP1. Though amino acids are known regulators of translation, the effects that deficiencies of specific amino acids have on translation have yet to be determined. We demonstrate that deprivation of leucine or methionine results in large inhibitory effects on translation initiation and on polysome formation that are not replicated by overexpressing non-phosphorylatable 4EBP1 or a phosphomimetic eIF2α. Our results demonstrate that a lack of either leucine or methionine has a major impact on mRNA translation, though they act by quite different mechanisms. Leucine deprivation appears to primarily inhibit ribosome loading, whereas methionine deprivation appears to primarily impair start site recognition. These data point to a unique regulatory effect that methionine status has on translation initiation.
2018-04-04 | GSE112643 | GEO
Project description:We analyzed the transcriptomes of Wild-type and GCN2-deficient mouse embryonic stem cells after 3h of arginine or leucine starvation.
2024-01-01 | GSE195470 | GEO
Project description:Diverse environmental insults induce the integrated stress response (ISR), which features eIF2 phosphorylation and translational control that serves to restore protein homeostasis. The eIF2 kinase GCN2 is a first responder in the ISR that is activated by amino acid depletion and other unrelated stresses. Two processes are suggested to trigger an ordered process of GCN2 activation during stress: GCN2 monitoring stress via accumulating uncharged tRNAs or by stalled and colliding ribosomes. Our results suggest that while ribosomal collisions are indeed essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 via its direct association with affected tRNAs. Both process require the GCN2 regulatory domain related to histidyl tRNA synthetases. GCN2 activation by UV irradiation features lowered amino acids and increased uncharged tRNAs and ribosome collisions are dispensable. We conclude that there are multiple mechanisms that activate GCN2 during diverse stresses.
2024-03-06 | GSE239867 | GEO