scholarly journals Balancing life with glycoconjugates: Monitoring unfolded protein response-mediated anti-angiogenic action of tunicamycin by Raman spectroscopy

2012 ◽  
Vol 84 (9) ◽  
pp. 1907-1918 ◽  
Author(s):  
Maria O. Longas ◽  
Ashok Kotapati ◽  
Kilari PVRK Prasad ◽  
Aditi Banerjee ◽  
Jesus Santiago ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Competitive Inhibitor ◽  
Protein Glycosylation ◽  
Endothelial Cell Proliferation ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
And Function ◽  
Protein Response

Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucos-aminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin inhibits capillary endothelial cell proliferation in a dose-dependent manner; (ii) treated cells are incapable of forming colonies upon its withdrawal; and (iii) tunicamycin treatment causes nuclear fragmentation. Tunicamycin-induced ER stress-mediated UPR event in these cells was studied with the aid of Raman spectroscopy, in particular, the interpretation of bands at 1672, 1684, and 1694 cm–1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells, these bands decreased in area as follows: at 1672 cm–1 by 41.85 % at 3 h and 55.39 % at 12 h; at 1684 cm–1 by 20.63 % at 3 h and 40.08 % at 12 h; and also at 1994 cm–1 by 33.33 % at 3 h and 32.92 % at 12 h, respectively. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation.

scholarly journals Intracellular XBP1-IL-24 axis dismantles cytotoxic unfolded protein response in the liver

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jianye Wang ◽  
Bian Hu ◽  
Zhicong Zhao ◽  
Haiyan Zhang ◽  
He Zhang ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Homologous Protein ◽  
Stress Signal ◽  
And Function ◽  
Protein Response ◽  
Er Homeostasis

AbstractEndoplasmic reticulum (ER) stress-associated cell death is prevalent in various liver diseases. However, the determinant mechanism how hepatocytes survive unresolved stress was still unclear. Interleukin-24 (IL-24) was previously found to promote ER stress-mediated cell death, and yet its expression and function in the liver remained elusive. Here we identified an antiapoptotic role of IL-24, which transiently accumulated within ER-stressed hepatocytes in a X-box binding protein 1 (XBP1)-dependent manner. Disruption of IL-24 increased cell death in the CCL4- or APAP-challenged mouse liver or Tm-treated hepatocytes. In contrast, pharmaceutical blockade of eukaryotic initiation factor 2α (eIF2α) or genetical ablation of C/EBP homologous protein (CHOP) restored hepatocyte function in the absence of IL-24. In a clinical setting, patients with acute liver failure manifested a profound decrease of hepatic IL-24 expression, which was associated with disease progression. In conclusion, intrinsic hepatocyte IL-24 maintains ER homeostasis by restricting the eIF2α-CHOP pathway-mediated stress signal, which might be exploited as a bio-index for prognosis or therapeutic intervention in patients with liver injury.

scholarly journals Regulation of the Unfolded Protein Response by BiP AMPylation protects photoreceptors from light-dependent degeneration

2018 ◽  
Author(s):  
Andrew T. Moehlman ◽  
Amanda K. Casey ◽  
Kelly Servage ◽  
Kim Orth ◽  
Helmut Krämer
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Synaptic Function ◽  
Transient Stress ◽  
Unfolded Protein ◽  
Response To Stress ◽  
The Unfolded Protein Response ◽  
And Function ◽  
Protein Response ◽  
Light:Dark Cycle

AbstractIn response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. Among these pathways, the Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or de-AMPylation of BiP, the major ER chaperone and regulator of the Unfolded Protein Response. This work elucidates the importance of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to stress. After 72 hours of constant light, photoreceptors of fic-null and AMPylation-resistant BiPT366A mutants, but not wild-type flies, display loss of synaptic function, disintegration of rhabdomeres, and excessive activation of ER stress reporters. Strikingly, this phenotype is reversible: photoreceptors regain their structure and function within 72 hours once returned to a standard light:dark cycle. These findings show that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands.

scholarly journals Heat Shock Protein 90 Modulates the Unfolded Protein Response by Stabilizing IRE1α

2002 ◽  
Vol 22 (24) ◽  
pp. 8506-8513 ◽  
Author(s):  
Monica G. Marcu ◽  
Melissa Doyle ◽  
Anne Bertolotti ◽  
David Ron ◽  
Linda Hendershot ◽  
...  
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Specific Inhibitor ◽  
Transcriptional Response ◽  
Heat Shock Protein 90 ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Stress Dependent ◽  
And Function ◽  
Protein Response

ABSTRACT The molecular chaperone HSP90 regulates stability and function of multiple protein kinases. The HSP90-binding drug geldanamycin interferes with this activity and promotes proteasome-dependent degradation of most HSP90 client proteins. Geldanamycin also binds to GRP94, the HSP90 paralog located in the endoplasmic reticulum (ER). Because two of three ER stress sensors are transmembrane kinases, namely IRE1α and PERK, we investigated whether HSP90 is necessary for the stability and function of these proteins. We found that HSP90 associates with the cytoplasmic domains of both kinases. Both geldanamycin and the HSP90-specific inhibitor, 514, led to the dissociation of HSP90 from the kinases and a concomitant turnover of newly synthesized and existing pools of these proteins, demonstrating that the continued association of HSP90 with the kinases was required to maintain their stability. Further, the previously reported ability of geldanamycin to stimulate ER stress-dependent transcription apparently depends on its interaction with GRP94, not HSP90, since geldanamycin but not 514 led to up-regulation of BiP. However, this effect is eventually superseded by HSP90-dependent destabilization of unfolded protein response signaling. These data establish a role for HSP90 in the cellular transcriptional response to ER stress and demonstrate that chaperone systems on both sides of the ER membrane serve to integrate this signal transduction cascade.

scholarly journals KSHV Activates Unfolded Protein Response Sensors but Suppresses Downstream Transcriptional Responses to Support Lytic Replication

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 116
Author(s):  
Benjamin P. Johnston ◽  
Eric S. Pringle ◽  
Craig McCormick
Keyword(s):  
Transcription Factor ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Lytic Replication ◽  
Cell Protein ◽  
Dependent Manner ◽  
Transcriptional Responses ◽  
Unfolded Protein ◽  
Virion Production ◽  
Protein Response

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure the proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading the ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins, ATF6, PERK and IRE1, initiate a stress-mitigating transcription program that accelerates the catabolism of misfolded proteins, while increasing the ER folding capacity. Kaposi’s sarcoma-associated herpesvirus (KSHV) can be reactivated from latency through the chemical induction of ER stress, which causes an accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and were required for efficient KSHV lytic replication. The genetic or pharmacologic inhibitions of each UPR sensor reduced virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted: (1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; (2) PERK phosphorylated eIF2, but ATF4 did not accumulate; (3) IRE1 caused XBP1–mRNA splicing, but the XBP1s protein did not accumulate and the XBP1s-responsive genes were not transcribed. The complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in epithelial cells. Taken together, these findings indicate that, while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.

scholarly journals Pan-Sigma Receptor Modulator RC-106 Induces Terminal Unfolded Protein Response In In Vitro Pancreatic Cancer Model

2020 ◽  
Vol 21 (23) ◽  
pp. 9012
Author(s):  
Michela Cortesi ◽  
Alice Zamagni ◽  
Sara Pignatta ◽  
Michele Zanoni ◽  
Chiara Arienti ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Cell Lines ◽  
Transcriptional Gene Silencing ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Protein Response

Pancreatic cancer (PC) remains one of the most lethal cancers worldwide. Sigma receptors (SRs) have been proposed as cancer therapeutic targets. Their main localization suggests they play a potential role in ER stress and in the triggering of the unfolded protein response (UPR). Here, we investigated the mechanisms of action of RC-106, a novel pan-SR modulator, to characterize therapeutically exploitable role of SRs in tumors. Two PC cell lines were used in all the experiments. Terminal UPR activation was evaluated by quantifying BiP, ATF4 and CHOP by Real-Time qRT-PCR, Western Blot, immunofluorescence and confocal microscopy. Cell death was studied by flow cytometry. Post-transcriptional gene silencing was performed to study the interactions between SRs and UPR key proteins. RC-106 activated ER stress sensors in a dose- and time-dependent manner. It also induced ROS production accordingly with ATF4 upregulation at the same time reducing cell viability of both cell lines tested. Moreover, RC-106 exerted its effect through the induction of the terminal UPR, as shown by the activation of some of the main transducers of this pathway. Post-transcriptional silencing studies confirmed the connection between SRs and these key proteins. Overall, our data highlighted a key role of SRs in the activation of the terminal UPR pathway, thus indicating pan-SR ligands as candidates for targeting the UPR in pancreatic cancer.

scholarly journals Downregulation of miR-17-92 cluster by PERK fine-tunes unfolded protein response mediated apoptosis

2020 ◽  
Author(s):  
Danielle E. Read ◽  
Ananya Gupta ◽  
Karen Cawley ◽  
Laura Fontana ◽  
Patrizia Agostinis ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Proximal Region ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Promoter Deletion Analysis ◽  
Protein Response

AbstractAn important event in the unfolded protein response (UPR) is the activation of the endoplasmic reticulum kinase PERK (EIF2AK3). The PERK signalling branch first mediates a prosurvival response, which switches into a proapoptotic response upon prolonged ER stress. However, the molecular mechanisms of PERK-mediated cell death are not well understood. Here we show that expression of the primary miR-17-92 transcript and mature miRNAs belonging to miR-17-92 cluster is decreased during UPR. We found that activity of miR-17-92 promoter reporter was reduced during UPR in a PERK-dependent manner. We show that activity of miR-17-92 promoter is repressed by ectopic expression of ATF4 and NRF2. The promoter deletion analysis and ChIP assays mapped the region responding to UPR-mediated repression to site in the proximal region of the miR-17-92 promoter. Hypericin-mediated photo-oxidative ER damage reduced the expression of miRNAs belonging to miR-17-92 cluster in wild-type but not in PERK-deficient cells. Importantly, ER stress-induced apoptosis was inhibited upon miR-17-92 overexpression in SH-SY5Y and H9c2 cells. Our results reveal a novel function for NRF2, where repression of miR-17-92 cluster by NRF2 plays an important role in ER stress-mediated apoptosis. The data presented here provides mechanistic details how sustained PERK signalling via NRF2 mediated repression of miR-17-92 cluster can potentiate cell death.

scholarly journals The unfolded protein response in Pichia pastoris without external stressing stimuli

2020 ◽  
Vol 20 (7) ◽  
Author(s):  
Yasmin Nabilah Binti Mohd Fauzee ◽  
Naoki Taniguchi ◽  
Yuki Ishiwata-Kimata ◽  
Hiroshi Takagi ◽  
Yukio Kimata
Keyword(s):  
Pichia Pastoris ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Gene Knockout ◽  
Transmembrane Protein ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Stress Dependent ◽  
Protein Response

ABSTRACT Dysfunction or capacity shortage of the endoplasmic reticulum (ER) is cumulatively called ER stress and provokes the unfolded protein response (UPR). In various yeast species, the ER-located transmembrane protein Ire1 is activated upon ER stress and performs the splicing reaction of HAC1 mRNA, the mature form of which is translated into a transcription factor protein that is responsible for the transcriptome change on the UPR. Here we carefully assessed the splicing of HAC1 mRNA in Pichia pastoris (Komagataella phaffii) cells. We found that, inconsistent with previous reports by others, the HAC1 mRNA was substantially, but partially, spliced even without ER-stressing stimuli. Unlike Saccharomyces cerevisiae, growth of P. pastoris was significantly retarded by the IRE1-gene knockout mutation. Moreover, P. pastoris cells seemed to push more abundant proteins into the secretory pathway than S. cerevisiae cells. We also suggest that P. pastoris Ire1 has the ability to control its activity stringently in an ER stress-dependent manner. We thus propose that P. pastoris cells are highly ER-stressed possibly because of the high load of endogenous proteins into the ER.

scholarly journals Intracellular XBP1-IL-24 axis dismantles cytotoxic unfolded protein response in the liver

2019 ◽  
Author(s):  
Jianye Wang ◽  
Bian Hu ◽  
Zhicong Zhao ◽  
Haiyan Zhang ◽  
He Zhang ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Homologous Protein ◽  
Stress Signal ◽  
And Function ◽  
Protein Response ◽  
Er Homeostasis

AbstractEndoplasmic reticulum (ER) stress-associated cell death is prevalent in various liver diseases. However, the determinant mechanism how hepatocytes survive unresolved stress was still unclear. Interleukin-24 (IL-24) was previously found to promote ER stress-mediated cell death, and yet its expression and function in the liver remained elusive. Here we identified an anti-apoptotic role of IL-24, which transiently accumulated within ER-stressed hepatocytes in a X-box binding protein 1 (XBP1)-dependent manner. Disruption of IL-24 increased cell death in the CCL4- or APAP-challenged mouse liver or Tm-treated hepatocytes. In contrast, pharmaceutical blockade of eukaryotic initiation factor 2α (eIF2α) or genetical ablation of C/EBP homologous protein (CHOP) restored hepatocyte function in the absence of IL-24. In a clinical setting, patients with acute liver failure manifested a profound decrease of hepatic IL-24 expression, which was associated with disease progression. In conclusion, intrinsic hepatocyte IL-24 maintains ER homeostasis by restricting the eIF2α-CHOP pathway-mediated stress signal, which might be exploited as a bio-index for prognosis or therapeutic intervention in patients with liver injury.

scholarly journals Downregulation of miR-17-92 Cluster by PERK Fine-Tunes Unfolded Protein Response Mediated Apoptosis

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 30
Author(s):  
Danielle E. Read ◽  
Ananya Gupta ◽  
Karen Cawley ◽  
Laura Fontana ◽  
Patrizia Agostinis ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Proximal Region ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Promoter Deletion Analysis ◽  
Protein Response

An important event in the unfolded protein response (UPR) is activation of the endoplasmic reticulum (ER) kinase PERK. The PERK signalling branch initially mediates a prosurvival response, which progresses to a proapoptotic response upon prolonged ER stress. However, the molecular mechanisms of PERK-mediated cell death are not well understood. Here we show that expression of the primary miR-17-92 transcript and mature miRNAs belonging to the miR-17-92 cluster are decreased during UPR. We found that miR-17-92 promoter reporter activity was reduced during UPR in a PERK-dependent manner. Furthermore, we show that activity of the miR-17-92 promoter is repressed by ectopic expression of ATF4 and NRF2. Promoter deletion analysis mapped the region responding to UPR-mediated repression to a site in the proximal region of the miR-17-92 promoter. Hypericin-mediated photo-oxidative ER damage reduced the expression of miRNAs belonging to the miR-17-92 cluster in wild-type but not in PERK-deficient cells. Importantly, ER stress-induced apoptosis was inhibited upon miR-17-92 overexpression in SH-SY5Y and H9c2 cells. Our results reveal a novel function for ATF4 and NRF2, where repression of the miR-17-92 cluster plays an important role in ER stress-mediated apoptosis. Mechanistic details are provided for the potentiation of cell death via sustained PERK signalling mediated repression of the miR-17-92 cluster.

scholarly journals Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Andrew T Moehlman ◽  
Amanda K Casey ◽  
Kelly Servage ◽  
Kim Orth ◽  
Helmut Krämer
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Synaptic Function ◽  
Constant Light ◽  
Unfolded Protein ◽  
Response To Stress ◽  
The Unfolded Protein Response ◽  
And Function ◽  
Protein Response ◽  
Light:Dark Cycle

In response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. Among these pathways, the Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or de-AMPylation of BiP, the major ER chaperone and regulator of the Unfolded Protein Response. This work elucidates the importance of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to stress. After 72 hr of constant light, photoreceptors of fic-null and AMPylation-resistant BiPT366A mutants, but not wild-type flies, display loss of synaptic function, disintegration of rhabdomeres, and excessive activation of ER stress reporters. Strikingly, this phenotype is reversible: photoreceptors regain their structure and function within 72 hr once returned to a standard light:dark cycle. These findings show that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands.

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