Eukaryotic translation initiation factor 5A regulates autophagy via translation of ATG3
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Michal Lubas
LAB HEAD: Anders Lund
PROVIDER: PXD008874 | Pride | 2018-04-27
REPOSITORIES: Pride
Similar Datasets
Project description:Translation of specific mRNAs is tightly regulated in primary T cells to ensure correct timing and precise delivery of protein syntheses in response to environmental cues. Translation factor eIF5a is known to be important for protein synthesis and is expected to regulate translation of certain difficult mRNA motifs such as consecutive prolines. Functional eIF5a is dynamically regulated upon T cell activation largely through addition of hypusine, a unique post-translational modification, to the mature protein and remarkably the eIF5a family are the only proteins to carry this modification. Here we show that knockout of eIF5a and its hypusination pathway affected both global and gene-specific protein synthesis, especially regulators of cytokine production and cell cycle progression. Furthermore, mRNA targets regulated by eIF5a in activated mouse primary CD8 T cells were systematically identified. AHA-labelled nascent proteins in GC7 (Dhps inhibitor)-treated and eIF5a knockout OT-1 cells (generated using CRSPR-Cas9) were measured using LC-MS. Authors: Thomas CJ Tan, Van Kelly, Xiaoyan Zhou, Tony Ly and Rose Zamoyska
2022-11-11 | PXD021063 | Pride
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.
| MSV000086106 | MassIVE
Project description:eIF5A is an essential translation elongation factor present in all eukaryotes, and the only known protein to follow a post-translational modification called hypusination. Here, we performed a wide analysis of ribosome dynamics in S. cerevisiae eIF5A depleted cells using 5Pseq (Pelechano et al. 2015 PMID 26046441). This method allows the study of ribosome dynamics, by sequencing 5’ phosphorylated mRNA co-translational degradation intermediates. Since eIF5A is an essential protein in yeast, we used two eIF5A temperature-sensitive strains containing a single Pro83 to Ser mutation (tif51A-1) and double Cys39 to Tyr and Gly118 to Asp mutations (tif51A-3) in the highly expressed gene TIF51A (HYP2) that encodes eIF5A protein (Li et al. 2011 PMID: 24923804).
2017-05-16 | GSE91064 | GEO
Project description:Innate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5A<sup>Hyp</sup>) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter rodentium, resulting in enhanced hypusination of EIF5A. EIF5A<sup>Hyp</sup> was also increased in gastric macrophages from patients with H. pylori gastritis. Furthermore, we identify the bacteria-induced immune effectors regulated by hypusination. This set of proteins includes essential constituents of antimicrobial response and autophagy. Mice with myeloid cell-specific deletion of Dhps exhibit reduced EIF5A<sup>Hyp</sup> in macrophages and increased bacterial burden and inflammation. Thus, regulation of translation through hypusination is a critical hallmark of the defense of eukaryotic hosts against pathogenic bacteria.
2021-04-06 | MTBLS607 | MetaboLights
Project description:The PI3K-PKB/c-akt-FOXO signalling network provides a major intracellular hub for regulation of cell proliferation, survival and stress resistance1. Here we report a novel function for FOXO transcription factors in regulating autophagy through modulation of intracellular glutamine levels. To identify novel transcriptional targets of this module we performed an unbiased microarray analysis after conditional activation of the key components PI3K, PKB, FOXO3 and FOXO4. Utilising this global pathway approach we identified glutamine synthetase (GS) as being transcriptionally regulated by PI3K-PKB-FOXO signalling. FOXO-mediated increase in GS expression specifically induced glutamine production independently of cell type, and this was evolutionary conserved. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes, which was dependent on GS activity. Increased GS activity resulted in increased autophagosome turnover as measured by LC3 lipidation, p62 degradation, and confocal imaging of LC3, p62, WIPI-1, ULK2 and Atg12. Inhibition of FOXO3-mediated autophagy resulted in increased apoptosis, suggesting that the induction of autophagy by FOXO3-mediated upregulation of GS is important for cellular survival. These findings reveal a novel signalling network that can directly modulate autophagy through regulation of glutamine metabolism. conditional activation of pkb and pi3k were followed in a timeseries. Each timepoint consists of 4 independent replicates, labeled with either cy3 or cy5 and put on array against time0.
2012-07-21 | E-GEOD-35701 | biostudies-arrayexpress
Project description:The PI3K-PKB/c-akt-FOXO signalling network provides a major intracellular hub for regulation of cell proliferation, survival and stress resistance1. Here we report a novel function for FOXO transcription factors in regulating autophagy through modulation of intracellular glutamine levels. To identify novel transcriptional targets of this module we performed an unbiased microarray analysis after conditional activation of the key components PI3K, PKB, FOXO3 and FOXO4. Utilising this global pathway approach we identified glutamine synthetase (GS) as being transcriptionally regulated by PI3K-PKB-FOXO signalling. FOXO-mediated increase in GS expression specifically induced glutamine production independently of cell type, and this was evolutionary conserved. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes, which was dependent on GS activity. Increased GS activity resulted in increased autophagosome turnover as measured by LC3 lipidation, p62 degradation, and confocal imaging of LC3, p62, WIPI-1, ULK2 and Atg12. Inhibition of FOXO3-mediated autophagy resulted in increased apoptosis, suggesting that the induction of autophagy by FOXO3-mediated upregulation of GS is important for cellular survival. These findings reveal a novel signalling network that can directly modulate autophagy through regulation of glutamine metabolism. conditional activation of foxo3 and foxo4 were followed in a timeseries. Each timepoint consists of 4 independent replicates, labeled with either cy3 or cy5 and put on array against time0 as reference.
2012-07-21 | E-GEOD-35703 | biostudies-arrayexpress
Project description:The eukaryotic translation factor eIF5A plays an essential role in translation elongation, especially across stretches of prolines and charged amino acids, and in translation termination. Although eIF5A is subjected to hypusination, a post-translational modification unique to this protein, how hypusination contributes to the eIF5A function remains elusive. Here we investigated the cellular defects induced by hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane). Through proteome, translatome and transcriptome analysis, we found that GC7 decreased a subset of mitochondrial proteins and DNA (mtDNA). Furthermore, chemical genomic screening using barcoded shRNA libraries identified genes for particular proteins involved in polyamine metabolism and a mitochondrial protein MPV17L2 as those altering the cytotoxicity induced by GC7. Depletion of MPV17L2 led to hypersensitivity to GC7 as well as the decreases in mitochondrial proteins and mtDNA. Moreover, metabolome analysis revealed that MPV17L2 depletion and GC7 treatment additively decreased the amount of proline and asparagine. Our results indicate that MPV17L2 displays a synthetic lethal interaction with the eIF5A hypusination targeted by GC7. The eukaryotic translation factor eIF5A plays an essential role in translation elongation, especially across stretches of prolines and charged amino acids, and in translation termination. Although eIF5A is subjected to hypusination, a post-translational modification unique to this protein, how hypusination contributes to the eIF5A function remains elusive. Here we investigated the cellular defects induced by hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane). Through proteome, translatome and transcriptome analysis, we found that GC7 decreased a subset of mitochondrial proteins and DNA (mtDNA). Furthermore, chemical genomic screening using barcoded shRNA libraries identified genes for particular proteins involved in polyamine metabolism and a mitochondrial protein MPV17L2 as those altering the cytotoxicity induced by GC7. Depletion of MPV17L2 led to hypersensitivity to GC7 as well as the decreases in mitochondrial proteins and mtDNA. Moreover, metabolome analysis revealed that MPV17L2 depletion and GC7 treatment additively decreased the amount of proline and asparagine. Our results indicate that MPV17L2 displays a synthetic lethal interaction with the eIF5A hypusination targeted by GC7. numerous molecules serving as valuable therapeutics. The marine natural product girolline has been described as an inhibitor of protein synthesis. Here, we demonstrate that it is not a general translation inhibitor but represents a sequence-specific modulator of translation factor eIF5A. Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling, primarily on AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in ribosome-associated quality control (RQC) and is important in maintaining the efficiency of translational progress. Girolline, therefore, provides a potent tool compound for understanding the interplay between protein production and quality control in a physiological setting and offers a new and selective means of modulating gene expression. numerous molecules serving as valuable therapeutics. The marine natural product girolline has27 been described as an inhibitor of protein synthesis. Here, we demonstrate that it is not a general28 translation inhibitor but represents a sequence-specific modulator of translation factor eIF5A.29 Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling, primarily on30 AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in31 ribosome-associated quality control (RQC) and is important in maintaining the efficiency of32 translational progress. Girolline, therefore, provides a potent tool compound for understanding33 the interplay between protein production and quality control in a physiological setting and offers34 a new and selective means of modulating gene expression.
2024-03-31 | GSE244063 | GEO
Project description:IFN-g primes macrophages for enhanced inflammatory activation by TLRs and microbial killing, but little is known about the regulation of cell metabolism or mRNA translation during priming. We found that IFN-g regulates macrophage metabolism and translation in an integrated manner by targeting mTORC1 and MNK pathways that converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of the central metabolic regulator mTORC1 by IFN-g was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages revealed that IFN-g selectively modulates the macrophage translatome to promote inflammation, further reprogram metabolic pathways, and modulate protein synthesis. These results add IFN-g-mediated metabolic reprogramming and translational regulation as key components of classical inflammatory macrophage activation. RPF and RNAseq libraries were generated from mock or IFN-g-primed human macrophages. Cells were stimulated with Pam3Cys and harvested at 4 hours. Libraries were generated using protocol modified from Illumina Truseq technology.
2015-06-29 | E-GEOD-66809 | biostudies-arrayexpress
Project description:IFN-g primes macrophages for enhanced inflammatory activation by TLRs and microbial killing, but little is known about the regulation of cell metabolism or mRNA translation during priming. We found that IFN-g regulates macrophage metabolism and translation in an integrated manner by targeting mTORC1 and MNK pathways that converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of the central metabolic regulator mTORC1 by IFN-g was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages revealed that IFN-g selectively modulates the macrophage translatome to promote inflammation, further reprogram metabolic pathways, and modulate protein synthesis. These results add IFN-g-mediated metabolic reprogramming and translational regulation as key components of classical inflammatory macrophage activation. microRNA-seq libraries were generated from mock or IFN-g-primed human macrophages. Cells were stimulated with or without Pam3Cys and harvested at 4 hours Libraries were generated using Illumina Truseq small RNA technology.
2015-06-29 | E-GEOD-66808 | biostudies-arrayexpress
Project description:The PI3K-PKB/c-akt-FOXO signalling network provides a major intracellular hub for regulation of cell proliferation, survival and stress resistance1. Here we report a novel function for FOXO transcription factors in regulating autophagy through modulation of intracellular glutamine levels. To identify novel transcriptional targets of this module we performed an unbiased microarray analysis after conditional activation of the key components PI3K, PKB, FOXO3 and FOXO4. Utilising this global pathway approach we identified glutamine synthetase (GS) as being transcriptionally regulated by PI3K-PKB-FOXO signalling. FOXO-mediated increase in GS expression specifically induced glutamine production independently of cell type, and this was evolutionary conserved. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes, which was dependent on GS activity. Increased GS activity resulted in increased autophagosome turnover as measured by LC3 lipidation, p62 degradation, and confocal imaging of LC3, p62, WIPI-1, ULK2 and Atg12. Inhibition of FOXO3-mediated autophagy resulted in increased apoptosis, suggesting that the induction of autophagy by FOXO3-mediated upregulation of GS is important for cellular survival. These findings reveal a novel signalling network that can directly modulate autophagy through regulation of glutamine metabolism.
2012-07-22 | GSE35703 | GEO