Dataset Information

Pharmacological induction of selective endoplasmic reticulum retention as a novel strategy for cancer therapy

ABSTRACT: We analysed the difference in the surface proteome of human cells that were either subjected to ER stress (induced by thapsigargin (Tg)) only or were additionally treated with a pharmacological inhibitor against the eIF2α kinase PERK, which is localized in the endoplasmic reticulum. Compared to single ER stress, several important plasma-membrane associated kinases, showed a significant downregulation under PERK inhibition.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Liver, Permanent Cell Line Cell

DISEASE(S): Hepatocellular Carcinoma

SUBMITTER: Christian Preisinger

LAB HEAD: Boaz Tirosh

PROVIDER: PXD014709 | Pride | 2020-03-02

REPOSITORIES: Pride

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Pharmacological induction of selective endoplasmic reticulum retention as a strategy for cancer therapy.

Mahameed Mohamed M Boukeileh Shatha S Obiedat Akram A Darawshi Odai O Dipta Priya P Rimon Amit A McLennan Gordon G Fassler Rosi R Reichmann Dana D Karni Rotem R Preisinger Christian C Wilhelm Thomas T Huber Michael M Tirosh Boaz B

Nature communications 20200311 1

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or a ...[more]

PMID: 32161259

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Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

Project description:Disruption of protein folding in the endoplasmic reticulum triggers the Unfolded Protein Response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PERK phosphorylation of the alpha subunit of eIF2 (eIF2~P), which represses global translation coincident with preferential translation of mRNAs, such as ATF4 and CHOP, that serve to implement the UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKa as being subject to both translation and transcriptional induction during eIF2~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKalpha mRNA involves the stress-induced relief of two inhibitory uORFs in the 5'-leader of the transcript. Depletion of IBTKalpha by shRNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKalpha is a key regulator in determining cell fate during the UPR. We used a genome-wide microarray approach to determine how individual mRNAs were differentially translated during endoplasmic reticulum stress. A microarray analysis from our laboratory identified gene transcripts suggested to be under translation control in mouse embryonic fibroblast (MEF) cells following a 6 hour treatment with thapsigargin, a potent inducer of ER stress, or no stress. The mRNAs were separated by sucrose gradient analyses to yield three fractions, those transcripts associated with large polysomes (?4 ribosomes per mRNA), those associated with monosome, disomes, or trisomes, and those fractionated at the top of the gradient with free ribosomes. RNA was extracted from sucrose gradients corresponding to these fractions and hybridized on Affymetrix microarrays. In parallel, we also measured total levels for each gene transcript in the presence or absence of thapsigargin treatment to address transcription regulation coincident with translational control. Please note that the treatment plus fractionation based on association with different numbers of ribosomes did yield different populations of mRNAs, which resulted in considerable variation in normalized data across the samples.

Project description:INS1-EGFP-L10a stable cell lines were induced with Doxycycline (Dox) for 24 hours, then either untreated or treated for 30 minutes with 1uM Thapsigargin to induce ER stress. Total RNA was purified with Trizol, and RIP RNA samples were extracted by immunoaffinity purification using an EGFP antibody We aimed to determine which genes are enriched under ER stress at the polyribosomal level. To do this we compared expression profiles of Total RNA with and without ER stress, and Immunoaffinity purified RNA (RIP) with and without ER stress. Microarray results of INS-1 EGFP-L10a cells either untreated or treated with 1uM Thapsigargin (Tg) in triplicate. Both Total RNA and RNA isolated from ribosomal Immunoprecipitation (RIP) When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this M-bM-^@M-^\Terminal UPRM-bM-^@M-^]. TXNIP becomes rapidly induced by hyperactivated IRE1a, an ER bifunctional kinase/endoribonuclease (RNase). IRE1a controls TXNIP mRNA stability by reducing levels of a TXNIP destabilizing micro-RNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing Caspase-1 cleavage and interleukin 1b (IL-1b) secretion. Txnip gene deletion reduces pancreatic b-cell death during ER stress, and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1a RNase inhibitors suppress TXNIP production to block IL-1b secretion. In summary, the IRE1a-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death, and may be targeted to develop new treatments for degenerative diseases driven by ER stress. There are 12 samples total all in INS-1 EGFP L10a cells- 3 untreated total RNA (reference sample), 3 treated with 1uM Tg 30 min total RNA, 3 untreated RIP RNA (reference sample), 3 treated with 1uM for 30 min RIP RNA