70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 142
Author(s):  
K. Gutierrez ◽  
W. G. Glanzner ◽  
N. Dicks ◽  
R. C. Bohrer ◽  
L. G. Currin ◽  
...  
Keyword(s):  
Er Stress ◽  
Gene Editing ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Unfolded Protein ◽  
Guide Rnas ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Er Homeostasis

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9) + 1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9 + 1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9 + sgRNA-1 + sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1 + sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Student’s t-test or Tukey–Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a = 59.8 ± 4.9%, b = 58.8 ± 5.3%, c = 68.86 ± 2.2%, d = 66.4 ± 5.9%, and e = 70.10 ± 1.9%), but development to the blastocyst stage was substantially reduced (P < 0.05) in the groups injected with Cas9 + sgRNAs (a = 18 ± 4.5%, b = 16 ± 1.5%, and c = 5.3 ± 2.8%) compared with controls (d = 33.7 ± 6.2% and e = 31.4 ± 1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9 + sgRNA-1 + sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.

scholarly journals ER stress causes widespread protein aggregation and prion formation

2017 ◽  
Vol 216 (8) ◽  
pp. 2295-2304 ◽  
Author(s):  
Norfadilah Hamdan ◽  
Paraskevi Kritsiligkou ◽  
Chris M. Grant
Keyword(s):  
Protein Aggregation ◽  
Er Stress ◽  
Secretory Pathway ◽  
Cellular Protein ◽  
Protein Homeostasis ◽  
Er Quality Control ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Er Homeostasis

Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.

scholarly journals Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress inSaccharomyces cerevisiae

2009 ◽  
Vol 20 (5) ◽  
pp. 1493-1508 ◽  
Author(s):  
Shi-Xiong Tan ◽  
Mariati Teo ◽  
Yuen T. Lam ◽  
Ian W. Dawes ◽  
Gabriel G. Perrone
Keyword(s):  
Endoplasmic Reticulum ◽  
Superoxide Dismutase ◽  
Er Stress ◽  
Stress Sensitivity ◽  
Pentose Phosphate ◽  
Unfolded Protein ◽  
Genome Wide ◽  
The Unfolded Protein Response ◽  
Protein Response

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.

scholarly journals The Impact of Endoplasmic Reticulum-Associated Protein Modifications, Folding and Degradation on Lung Structure and Function

2021 ◽  
Vol 12 ◽  
Author(s):  
Emily M. Nakada ◽  
Rui Sun ◽  
Utako Fujii ◽  
James G. Martin
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Unfolded Protein ◽  
Lung Structure ◽  
Selective Translation ◽  
The Unfolded Protein Response ◽  
And Function ◽  
Protein Response ◽  
Er Homeostasis ◽  
The Impact

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and induces the unfolded protein response (UPR) and other mechanisms to restore ER homeostasis, including translational shutdown, increased targeting of mRNAs for degradation by the IRE1-dependent decay pathway, selective translation of proteins that contribute to the protein folding capacity of the ER, and activation of the ER-associated degradation machinery. When ER stress is excessive or prolonged and these mechanisms fail to restore proteostasis, the UPR triggers the cell to undergo apoptosis. This review also examines the overlooked role of post-translational modifications and their roles in protein processing and effects on ER stress and the UPR. Finally, these effects are examined in the context of lung structure, function, and disease.

scholarly journals Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

2021 ◽  
Author(s):  
Chao Li
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Immune Cell ◽  
Macrophage Polarization ◽  
Intestinal Epithelial ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) stress triggers a series of signaling and transcriptional events termed the unfolded protein response (UPR). Severe ER stress is associated with the development of fibrosis in different organs including lung, liver, kidney, heart, and intestine. ER stress is an essential response of epithelial and immune cells in the pathogenesis of inflammatory bowel disease (IBD) including Crohn&rsquo;s disease. Intestinal epithelial cells are susceptible to ER stress-mediated damage due to secretion of a large amount of proteins that are involved in mucosal defense. In other cells, ER stress is linked to myofibroblast activation, extracellular matrix production, macrophage polarization, and immune cell differentiation. This review focuses on the role of UPR in the pathogenesis in IBD from an immunologic perspective. The roles of macrophage and mesenchymal cells in the UPR from in vitro and in vivo animal models are discussed. The links between ER stress and other signaling pathways such as senescence and autophagy are introduced. Recent advances in the understanding of the epigenetic regulation of UPR signaling are also updated here. The future directions of development of the UPR research and therapeutic strategies to manipulate ER stress levels are also reviewed.

scholarly journals Inhibiting P2Y12 in Macrophages Induces Endoplasmic Reticulum Stress and Promotes an Anti-Tumoral Phenotype

2020 ◽  
Vol 21 (21) ◽  
pp. 8177
Author(s):  
Nataša Pavlović ◽  
Maria Kopsida ◽  
Pär Gerwins ◽  
Femke Heindryckx
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Adenosine Diphosphate ◽  
In Vitro Methods ◽  
Unfolded Protein ◽  
Key Players ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
G Protein Coupled

The P2Y12 receptor is an adenosine diphosphate responsive G protein-coupled receptor expressed on the surface of platelets and is the pharmacologic target of several anti-thrombotic agents. In this study, we use liver samples from mice with cirrhosis and hepatocellular carcinoma to show that P2Y12 is expressed by macrophages in the liver. Using in vitro methods, we show that inhibition of P2Y12 with ticagrelor enhances tumor cell phagocytosis by macrophages and induces an anti-tumoral phenotype. Treatment with ticagrelor also increases the expression of several actors of the endoplasmic reticulum (ER) stress pathways, suggesting activation of the unfolded protein response (UPR). Inhibiting the UPR with tauroursodeoxycholic acid (Tudca) diminishes the pro-phagocytotic effect of ticagrelor, thereby indicating that P2Y12 mediates macrophage function through activation of ER stress pathways. This could be relevant in the pathogenesis of chronic liver disease and cancer, as macrophages are considered key players in these inflammation-driven pathologies.

scholarly journals Endoplasmic reticulum stress and the development of endothelial dysfunction

2017 ◽  
Vol 312 (3) ◽  
pp. H355-H367 ◽  
Author(s):  
M. L. Battson ◽  
D. M. Lee ◽  
C. L. Gentile
Keyword(s):  
Endoplasmic Reticulum ◽  
Endothelial Dysfunction ◽  
Er Stress ◽  
Critical Role ◽  
Unfolded Protein ◽  
Vasoactive Substances ◽  
Important Regulator ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Er Homeostasis

The vascular endothelium plays a critical role in cardiovascular homeostasis, and thus identifying the underlying causes of endothelial dysfunction has important clinical implications. In this regard, the endoplasmic reticulum (ER) has recently emerged as an important regulator of metabolic processes. Dysfunction within the ER, broadly termed ER stress, evokes the unfolded protein response (UPR), an adaptive pathway that aims to restore ER homeostasis. Although the UPR is the first line of defense against ER stress, chronic activation of the UPR leads to cell dysfunction and death and has recently been implicated in the pathogenesis of endothelial dysfunction. Numerous risk factors for endothelial dysfunction can induce ER stress, which may in turn disrupt endothelial function via direct effects on endothelium-derived vasoactive substances or by activating other pathogenic cellular networks such as inflammation and oxidative stress. This review summarizes the available data linking ER stress to endothelial dysfunction.

3-O-Hydroxytyrosol glucuronide and 4-O-hydroxytyrosol glucuronide reduce endoplasmic reticulum stress in vitro

2015 ◽  
Vol 6 (10) ◽  
pp. 3275-3281 ◽  
Author(s):  
Elena Giordano ◽  
Olivier Dangles ◽  
Njara Rakotomanomana ◽  
Silvia Baracchini ◽  
Francesco Visioli
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Unfolded Protein ◽  
Atherosclerosis Development ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) stress is important for atherosclerosis development and is mediated by the unfolded protein response (UPR).

scholarly journals Different Forms of ER Stress in Chondrocytes Result in Short Stature Disorders and Degenerative Cartilage Diseases: New Insights by Cartilage-Specific ERp57 Knockout Mice

2018 ◽  
Vol 2018 ◽  
pp. 1-14 ◽  
Author(s):  
Yvonne Rellmann ◽  
Rita Dreier
Keyword(s):  
Er Stress ◽  
Short Stature ◽  
Knockout Mice ◽  
Skeletal Development ◽  
Unfolded Protein ◽  
Cartilage Diseases ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Er Homeostasis ◽  
Protein Disulfide

Cartilage is essential for skeletal development by endochondral ossification. The only cell type within the tissue, the chondrocyte, is responsible for the production of macromolecules for the extracellular matrix (ECM). Before proteins and proteoglycans are secreted, they undergo posttranslational modification and folding in the endoplasmic reticulum (ER). However, the ER folding capacity in the chondrocytes has to be balanced with physiological parameters like energy and oxygen levels. Specific cellular conditions, e.g., a high protein demand, or pathologic situations disrupt ER homeostasis and lead to the accumulation of poorly folded or misfolded proteins. This state is called ER stress and induces a cellular quality control system, the unfolded protein response (UPR), to restore homeostasis. Different mouse models with ER stress in chondrocytes display comparable skeletal phenotypes representing chondrodysplasias. Therefore, ER stress itself seems to be involved in the pathogenesis of these diseases. It is remarkable that chondrodysplasias with a comparable phenotype arise independent from the sources of ER stress, which are as follows: (1) mutations in ECM proteins leading to aggregation, (2) deficiencies in ER chaperones, (3) mutations in UPR signaling factors, or (4) deficiencies in the degradation of aggregated proteins. In any case, the resulting UPR substantially impairs ECM protein synthesis, chondrocyte proliferation, and/or differentiation or regulation of autophagy and apoptosis. Notably, chondrodysplasias arise no matter if single or multiple events are affected. We analyzed cartilage-specific ERp57 knockout mice and demonstrated that the deficiency of this single protein disulfide isomerase, which is responsible for formation of disulfide bridges in ECM glycoproteins, is sufficient to induce ER stress and to cause an ER stress-related bone phenotype. These mice therefore qualify as a novel model for the analysis of ER stress in chondrocytes. They give new insights in ER stress-related short stature disorders and enable the analysis of ER stress in other cartilage diseases, such as osteoarthritis.

scholarly journals Coordination of stress, Ca2+, and immunogenic signaling pathways by PERK at the endoplasmic reticulum

2016 ◽  
Vol 397 (7) ◽  
pp. 649-656 ◽  
Author(s):  
Alexander R. van Vliet ◽  
Abhishek D. Garg ◽  
Patrizia Agostinis
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Signaling Pathways ◽  
Unfolded Protein ◽  
Independent Functions ◽  
Intracellular Ca ◽  
Stress Signals ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Er Homeostasis

AbstractThe endoplasmic reticulum (ER) is the main coordinator of intracellular Ca2+signaling, protein synthesis, and folding. The ER is also implicated in the formation of contact sites with other organelles and structures, including mitochondria, plasma membrane (PM), and endosomes, thereby orchestrating through interorganelle signaling pathways, a variety of cellular responses including Ca2+homeostasis, metabolism, and cell death signaling. Upon loss of its folding capacity, incited by a number of stress signals including those elicited by various anticancer therapies, the unfolded protein response (UPR) is launched to restore ER homeostasis. The ER stress sensor protein kinase RNA-like ER kinase (PERK) is a key mediator of the UPR and its role during ER stress has been largely recognized. However, growing evidence suggests that PERK may govern signaling pathways through UPR-independent functions. Here, we discuss emerging noncanonical roles of PERK with particular relevance for the induction of danger or immunogenic signaling and interorganelle communication.

scholarly journals The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant

2018 ◽  
Vol 475 (5) ◽  
pp. 923-929 ◽  
Author(s):  
Stanley M.H. Chan ◽  
Mark P. Lowe ◽  
Ashton Bernard ◽  
Alyson A. Miller ◽  
Terence P. Herbert
Keyword(s):  
Er Stress ◽  
Cultured Cells ◽  
Specific Inhibitor ◽  
Unfolded Protein ◽  
Ic50 Values ◽  
A Cell ◽  
Target Effect ◽  
The Unfolded Protein Response ◽  
Protein Response

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic β-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes.

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