scholarly journals IRE-1α regulates expression of ubiquitin specific peptidases during hypoxic response in U87 glioma cells

2016 ◽  
Vol 3 (1) ◽  
Author(s):  
Oleksandr H. Minchenko ◽  
Dariia O. Tsymbal ◽  
Dmytro O. Minchenko ◽  
Olena O. Riabovol ◽  
Oleh V. Halkin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Tumor Growth ◽  
Glioma Cells ◽  
Stress Signaling ◽  
Hypoxic Stress ◽  
Wild Type ◽  
Ubiquitin System ◽  
Genes Expression ◽  
Evolutionarily Conserved ◽  
Hypoxic Treatment

AbstractIRE-1α (inositol requiring enzyme-1α), the most evolutionarily conserved of the endoplasmic reticulum stress signaling pathways, is highly implicated in sustaining the proliferation of glioma cells and subsequent tumor growth, which is decreased by the inhibition of IRE-1α. To explore the IRE-1α mediated regulation of ubiquitin system in glioma cells, the expression of a subset of ubiquitin specific peptidases (USP) and of ubiquitin activating enzyme E1-like protein/autophagy related 7 (GSA7/ATG7) genes was studied, during hypoxic stress in wild type and U87 glioma cells with inhibited IRE-1α. Hypoxic treatment of wild type glioma cells leads to the up-regulation of USP25 and the concomitant downregulation of USP1, USP10, USP14, and GSA7 genes. USP4 and USP22 genes expression did not significantly change with hypoxic treatment. Inhibition of IRE-1α activity led to up-regulation of USP1, USP4, USP10, USP22, and USP25, while USP14 and GSA7 genes were down-regulated. Therefore, IRE-1α activity modifies substrate-targeting specificity to proteasome during hypoxic stress, which in turn can affect cell survival. Inhibition of IRE-1α correlates directly with deregulation of ubiquitin specific peptidases and GSA7 in a fashion that ultimately slows tumor growth.

scholarly journals Regulation of Protein Synthesis by Hypoxia via Activation of the Endoplasmic Reticulum Kinase PERK and Phosphorylation of the Translation Initiation Factor eIF2α

2002 ◽  
Vol 22 (21) ◽  
pp. 7405-7416 ◽  
Author(s):  
Constantinos Koumenis ◽  
Christine Naczki ◽  
Marianne Koritzinsky ◽  
Sally Rastani ◽  
Alan Diehl ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Prolonged Exposure ◽  
Response To Therapy ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Hypoxic Stress ◽  
Wild Type

ABSTRACT Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2α on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2α, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2α attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2α kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2α. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2α and reduced inhibition of protein synthesis in response to hypoxia. PERK−/− mouse embryo fibroblasts failed to phosphorylate eIF2α and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2α and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.

scholarly journals Sec61p Serves Multiple Roles in Secretory Precursor Binding and Translocation into the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (12) ◽  
pp. 3455-3473 ◽  
Author(s):  
Marinus Pilon ◽  
Karin Römisch ◽  
Dong Quach ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Receptor Site ◽  
Multiple Roles ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
Evolutionarily Conserved ◽  
Cold Sensitive ◽  
A Subunit

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles ofsec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression ofSEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

scholarly journals Inhibition of ERN1 modifies the hypoxic regulation of the expression of TP53-related genes in U87 glioma cells

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Dmytro O. Minchenko ◽  
Serhii V. Danilovskyi ◽  
Iryna V. Kryvdiuk ◽  
Taia V. Bakalets ◽  
Nadia M. Lypova ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Tumor Growth ◽  
Topoisomerase I ◽  
Glioma Cells ◽  
Fine Tuning ◽  
Endoplasmic Reticulum Stress Response ◽  
Expression Levels ◽  
Hypoxic Conditions ◽  
P53 Signaling

AbstractInhibition of ERN1 (endoplasmic reticulum to nuclei 1), the major signalling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of tumor protein 53 (TP53)- related genes such as TOPORS (topoisomerase I binding, arginine/serine-rich, E3 ubiquitin protein ligase), TP53BP1 (TP53 binding protein 1), TP53BP2, SESN1 (sestrin 1), NME6 (non-metastatic cells 6), and ZMAT3 (zinc finger, Matrin-type 3) in glioma cells expressing dominantnegative ERN1 under baseline and hypoxic conditions. We demonstrated that inhibition of ERN1 function in U87 glioma cells resulted in increased expression of RYBP, TP53BP2, and SESN1 genes, but decreased expression of TP53BP1, TOPORS, NME6, and ZMAT3 genes. Moreover, inhibition of ERN1 affected hypoxia-mediated changes in expression of TP53-related genes and their magnitude. Indeed, hypoxia has no effect on expression of TP53BP1 and SESN1 in control cells, while resulted in increased expression of these genes in cells with inhibited ERN1 function. Magnitude of hypoxia-mediated changes in expression levels of RYBP and TP53BP2 was gene specific and more robust in the case of TP53BP2. Hypoxiamediated decrease in expression levels of TOPORS was more prominent if ERN1 was inhibited. Present study demonstrates that fine-tuning of the expression of TP53- associated genes depends upon endoplasmic reticulum stress signaling under normal and hypoxic conditions. Inhibition of ERN1 branch of endoplasmic reticulum stress response correlates with deregulation of p53 signaling and slower tumor growth.

scholarly journals Inhibition of IRE1 signaling affects expression of a subset genes encoding for TNF-related factors and receptors and modifies their hypoxic regulation in U87 glioma cells

2016 ◽  
Vol 3 (1) ◽  
Author(s):  
Oleksandr H. Minchenko ◽  
Iryna V. Kryvdiuk ◽  
Dmytro O. Minchenko ◽  
Olena O. Riabovol ◽  
Oleh V. Halkin
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Endoplasmic Reticulum Stress ◽  
Tumor Growth ◽  
Glioma Cells ◽  
Dominant Negative ◽  
Gene Expressions ◽  
Related Factors ◽  
Genes Encoding ◽  
Glioma Growth

AbstractInhibition of IRE1 (inositol requiring enzyme-1), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth and proliferation of glioma cells. To elucidate the role of IRE1- mediated glioma growth, we studied the expression of a subset genes encoding for TNF (tumor necrosis factor)- related factors and receptors and their hypoxic regulation in U87 glioma cells overexpressing dominant-negative IRE1 (dnIRE1). We demonstrated that the expression of TNFAIP1, TNFRSF10D, TNFRSF21, TNFRSF11B, TNFSF7, and LITAF genes is increased in glioma cells with modified IRE1; however, TNFRSF10B, TRADD, and TNFAIP3 is down-regulated in these cells as compared to their control counterparts. We did not find TNFRSF1A gene expression to change significantly under this experimental condition. In control glioma cells, hypoxia leads to the up-regulated expression of TNFAIP1, TNFAIP3, TRADD, and TNFRSF10D genes and the concomitant down-regulation of TNFRSF21, TNFRSF11B, and LITAF genes; while, TNFRSF10B and TNFRSF1A genes are resistant to hypoxic treatment. However, inhibition of IRE1 modifies the hypoxic regulation of LITAF, TNFRSF21, TNFRSF11B, and TRADD genes and introduces hypoxia-induced sensitivity to TNFRSF10B, TNFRSF1A, and TNFSF7 gene expressions. Furthermore, knockdown by siRNA of TNFRSF21 mRNA modifies the hypoxic effect on the IRE1-dependent rate of proliferation and cell death in U87 glioma cells. The present study demonstrates that fine-tuned manipulation of the expression of TNF-related factors and receptors directly relating to cell death and proliferation, is mediated by an effector of endoplasmic reticulum stress, IRE1, as well as by hypoxia in a gene-specific manner. Thus, inhibition of the kinase and endoribonuclease activities of IRE1 correlates with deregulation of TNF-related factors and receptors in a manner that is gene specific and thus slows tumor growth.

scholarly journals Inhibition of kinase and endoribonuclease activity of ERN1/IRE1α affects expression of proliferationrelated genes in U87 glioma cells

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Oleksandr H. Minchenko ◽  
Dariia O. Tsymbal ◽  
Dmytro O. Minchenko ◽  
Michel Moenner ◽  
Olena V. Kovalevska ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Endoplasmic Reticulum Stress ◽  
Tumor Growth ◽  
Mrna Level ◽  
Glioma Cells ◽  
Dominant Negative ◽  
Fine Tuning ◽  
Gene Expressions ◽  
Endoribonuclease Activity

AbstractInhibition of ERN1/IRE1α (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme-1α), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F1), and HOXC6 (homeobox C6) in U87 glioma cells overexpressing dominant-negative ERN1/IRE1α defective in endoribonuclease (dnr-ERN1) as well as defective in both kinase and endonuclease (dn-ERN1) activity of ERN1/IRE1α. We have demonstrated that the expression of all studied genes is decreased at the mRNA level in cells with modified ERN1/IRE1α; TBX3, however, is increased in these cells as compared to control glioma cells. Changes in protein levels of E2F8, HOXC6, ATF3, and TBX3 corresponded to changes in mRNAs levels. We also found that two mutated ERN1/IRE1α have differential effects on the expression of studied transcripts. The presence of kinase and endonuclease deficient ERN1/IRE1α in glioma cells had a less profound effect on the expression of E2F8, HOXC6, and TBX3 genes than the blockade of the endoribonuclease activity of ERN1/IRE1α alone. Kinase and endonuclease deficient ERN1/IRE1α suppresses ATF3 and FOXF1 gene expressions, while inhibition of only endoribonuclease of ERN1/IRE1α leads to the up-regulation of these gene transcripts. The present study demonstrates that fine-tuning of the expression of proliferation related genes is regulated by ERN1/IRE1α an effector of endoplasmic reticulum stress. Inhibition of ERN1/IRE1α, especially its endoribonuclease activity, correlates with deregulation of proliferation related genes and thus slower tumor growth.

scholarly journals Effect of glucose deprivation on the expression of genes encoding glucocorticoid receptor and some related factors in ERN1-knockdown U87 glioma cells

2019 ◽  
Vol 53 (4) ◽  
pp. 237-249 ◽  
Author(s):  
Olena O. Riabovol ◽  
Dariia O. Tsymbal ◽  
Dmytro O. Minchenko ◽  
Kateryna M. Lebid-Biletska ◽  
Myroslava Y. Sliusar ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Glioma Cells ◽  
Glucose Deprivation ◽  
Expression Level ◽  
Enzyme Function ◽  
Deprivation Condition ◽  
Stress Signaling ◽  
Expression Of Genes ◽  
Glioma Growth ◽  
Effect Of Glucose

AbstractObjective. The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation.Methods. The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction.Results. It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition.Conclusions. Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.

scholarly journals Salicylic Acid Accumulation Controlled by LSD1 Is Essential in Triggering Cell Death in Response to Abiotic Stress

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 962
Author(s):  
Maciej Jerzy Bernacki ◽  
Anna Rusaczonek ◽  
Weronika Czarnocka ◽  
Stanisław Karpiński
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Abiotic Stress ◽  
Wild Type ◽  
Pr Genes ◽  
Genes Expression ◽  
Salicylic Acid Accumulation ◽  
Cell Death Phenotype ◽  
Insight Into

Salicylic acid (SA) is well known hormonal molecule involved in cell death regulation. In response to a broad range of environmental factors (e.g., high light, UV, pathogens attack), plants accumulate SA, which participates in cell death induction and spread in some foliar cells. LESION SIMULATING DISEASE 1 (LSD1) is one of the best-known cell death regulators in Arabidopsis thaliana. The lsd1 mutant, lacking functional LSD1 protein, accumulates SA and is conditionally susceptible to many biotic and abiotic stresses. In order to get more insight into the role of LSD1-dependent regulation of SA accumulation during cell death, we crossed the lsd1 with the sid2 mutant, caring mutation in ISOCHORISMATE SYNTHASE 1(ICS1) gene and having deregulated SA synthesis, and with plants expressing the bacterial nahG gene and thus decomposing SA to catechol. In response to UV A+B irradiation, the lsd1 mutant exhibited clear cell death phenotype, which was reversed in lsd1/sid2 and lsd1/NahG plants. The expression of PR-genes and the H2O2 content in UV-treated lsd1 were significantly higher when compared with the wild type. In contrast, lsd1/sid2 and lsd1/NahG plants demonstrated comparability with the wild-type level of PR-genes expression and H2O2. Our results demonstrate that SA accumulation is crucial for triggering cell death in lsd1, while the reduction of excessive SA accumulation may lead to a greater tolerance toward abiotic stress.

scholarly journals Knockout of Putative Tumor Suppressor Aldh1l1 in Mice Reprograms Metabolism to Accelerate Growth of Tumors in a Diethylnitrosamine (DEN) Model of Liver Carcinogenesis

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3219
Author(s):  
Natalia I. Krupenko ◽  
Jaspreet Sharma ◽  
Halle M. Fogle ◽  
Peter Pediaditakis ◽  
Kyle C. Strickland ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Chemical Carcinogenesis ◽  
Dependent Manner ◽  
Wild Type ◽  
Liver Carcinogenesis ◽  
Wild Type Littermate ◽  
Tumor Multiplicity ◽  
Accelerate Growth ◽  
Putative Tumor Suppressor ◽  
Formyltetrahydrofolate Dehydrogenase

Cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is commonly downregulated in human cancers through promoter methylation. We proposed that ALDH1L1 loss promotes malignant tumor growth. Here, we investigated the effect of the Aldh1l1 mouse knockout (Aldh1l1−/−) on hepatocellular carcinoma using a chemical carcinogenesis model. Fifteen-day-old male Aldh1l1 knockout mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with 20 μg/g body weight of DEN (diethylnitrosamine). Mice were sacrificed 10, 20, 28, and 36 weeks post-DEN injection, and livers were examined for tumor multiplicity and size. We observed that while tumor multiplicity did not differ between Aldh1l1−/− and Aldh1l1+/+ animals, larger tumors grew in Aldh1l1−/− compared to Aldh1l1+/+ mice at 28 and 36 weeks. Profound differences between Aldh1l1−/− and Aldh1l1+/+ mice in the expression of inflammation-related genes were seen at 10 and 20 weeks. Of note, large tumors from wild-type mice showed a strong decrease of ALDH1L1 protein at 36 weeks. Metabolomic analysis of liver tissues at 20 weeks showed stronger differences in Aldh1l1+/+ versus Aldh1l1−/− metabotypes than at 10 weeks, which underscores metabolic pathways that respond to DEN in an ALDH1L1-dependent manner. Our study indicates that Aldh1l1 knockout promoted liver tumor growth without affecting tumor initiation or multiplicity.

scholarly journals Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

1994 ◽  
Vol 301 (2) ◽  
pp. 577-583 ◽  
Author(s):  
K Oda ◽  
J Cheng ◽  
T Saku ◽  
N Takami ◽  
M Sohda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Aspartic Acid ◽  
Chimeric Protein ◽  
Attachment Site ◽  
In Vitro Translation ◽  
Placental Alkaline Phosphatase ◽  
Wild Type ◽  
Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

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