Dataset Information

Proteomics analysis of LN308 cell line under treatment with tunicamycin and thapsigargin

ABSTRACT: Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR), an adaptive signal transduction pathway aimed at reinstating cellular homeostasis, or, if that fails, at triggering of apoptosis. The UPR plays a key role in a variety of disorders (including diabetes, neurodegenerative disorders, and inflammatory processes) and has been implicated in cancer progression and resistance to chemotherapy. However, the mechanisms and pathways by which the UPR contributes to chemoresistance are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of the UPR with two different compounds, probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes (the UPR regulon), many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control via phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2a) and subsequent induction of activating transcription factor 4 (ATF4) causes an up-regulation of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. This metabolic rewiring of the cells results in resistance to treatment with the widely-used folate anti-metabolites Methotrexate and Permetrexed.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Brain Cancer

SUBMITTER: Chi Nguyen

LAB HEAD: Robert Ahrends

PROVIDER: PXD013541 | Pride | 2020-05-06

REPOSITORIES: Pride

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Publications

A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.

Reich Stefan S Nguyen Chi D L CDL Has Canan C Steltgens Sascha S Soni Himanshu H Coman Cristina C Freyberg Moritz M Bichler Anna A Seifert Nicole N Conrad Dominik D Knobbe-Thomsen Christiane B CB Tews Björn B Toedt Grischa G Ahrends Robert R Medenbach Jan J

Nature communications 20200610 1

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transc ...[more]

PMID: 32522993

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Project description:Erguler2013 - Unfolded protein stress response The model investigates the mechanism by which UPR (unfolded protein response) outcome switches between survival and death. This model is described in the article: A mathematical model of the unfolded protein stress response reveals the decision mechanism for recovery, adaptation and apoptosis. Erguler K, Pieri M, Deltas C. BMC Syst Biol. 2013 Feb 21;7(1):16. Abstract: BACKGROUND: The unfolded protein response (UPR) is a major signalling cascade acting in the quality control ofprotein folding in the endoplasmic reticulum (ER). The cascade is known to play an accessory rolein a range of genetic and environmental disorders including neurodegenerative and cardiovasculardiseases, diabetes and kidney diseases. The three major receptors of the ER stress involved withthe UPR, i.e. IRE1a, PERK and ATF6, signal through a complex web of pathways to convey anappropriate response. The emerging behaviour ranges from adaptive to maladaptive depending on theseverity of unfolded protein accumulation in the ER; however, the decision mechanism for the switchand its timing have so far been poorly understood. RESULTS: Here, we propose a mechanism by which the UPR outcome switches between survival and death.We compose a mathematical model integrating the three signalling branches, and perform a comprehensivebifurcation analysis to investigate possible responses to stimuli. The analysis reveals threedistinct states of behaviour, low, high and intermediate activity, associated with stress adaptation, tolerance,and the initiation of apoptosis. The decision to adapt or destruct can, therefore, be understoodas a dynamic process where the balance between the stress and the folding capacity of the ER playsa pivotal role in managing the delivery of the most appropriate response. The model demonstratesfor the first time that the UPR is capable of generating oscillations in translation attenuation and theapoptotic signals, and this is supplemented with a Bayesian sensitivity analysis identifying a set ofparameters controlling this behaviour. CONCLUSIONS: This work contributes largely to the understanding of one of the most ubiquitous signalling pathwaysinvolved in protein folding quality control in the metazoan ER. The insights gained have direct consequenceson the management of many UPR-related diseases, revealing, in addition, an extended listof candidate disease modifiers. Demonstration of stress adaptation sheds light to how preconditioningmight be beneficial in manifesting the UPR outcome to prevent untimely apoptosis, and paves the wayto novel approaches for the treatment of many UPR-related conditions. In the paper, PERKA refers to the amount of phosphorylated PERK monomer. However, it refers to the active complex in the model. The complex with the model parameterization is formed of 4 monomers (n=4). So, the value of PERKA should be multiplied by 4, in order to generate the figures in the paper (eg. Figure 12). An additional parameter (tmr=10)) is used in the model. This parameter is not mentioned in the paper. The model values of kf(=10) and kr(=1) are not consistent with that of the paper (kf=100, kr=10, in the paper). However, this is corrected by the introduction of "tmr" in the model, which is multiplied with kf and kr to get the resulting values. The term "tmr" was missing in the kinetic laws of the reactions reu7 and reu8, in the original model. This has been corrected as per the author's request. This model is hosted on BioModels Database and identified by: MODEL1302180000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the UPR. Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest a component of UPR induction in arv1? strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, C/EBP homologous protein (CHOP) and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis resulting in a disruption of ER integrity, one consequence of which is induction of the UPR. Yeast strains were grown to mid-logarithmic stage (A600=0.5-0.6) for RNA extraction and hybridization on Ye6100 or S98 Affymetrix gene chips. The control array (sample name C) was carried out in duplicate. The ARV1 mutant array (sample name A) was carried out in triplicate. Both ARV1 mutants Ye6100 arrays were compared to the same control Ye6100 array. Strains represent different isolates of the same genotype, mating type and genetic background (w303). Sturley lab collection (SCY) strains include SCY328 and SCY2004 for controls and SCY820 and SCY840 for ARV1 mutants.

Dataset Information

Proteomics analysis of LN308 cell line under treatment with tunicamycin and thapsigargin

Publications

A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.

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