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1.  PKR Protects Colonic Epithelium Against Colitis Through the Unfolded Protein Response and Prosurvival Signaling 
Inflammatory bowel diseases  2012;18(9):1735-1742.
Background
The dsRNA-activated protein kinase (PKR) phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α), a global regulator of protein synthesis in mammals. In addition, PKR activates several signal transduction pathways including STAT3 and AKT. PKR is activated by a number of inflammatory stimuli that are induced in the inflamed intestine. In this study we intended to determine the role of PKR in colonic epithelial cells during experimental colitis in mice.
Methods
Age- and sex-matched PKR+/+,+/− and PKR−/− littermate mice were reconstituted with wildtype bone marrow cells and subjected to dextran sodium sulfate (DSS)-induced colitis.
Results
PKR−/− mice displayed more severe clinical and histological manifestations upon DSS colitis compared with their PKR+/+,+/− litter-mates. In response to DSS colitis, the colonic epithelial cells of PKR−/− mice exhibited impaired activation of the unfolded protein response (UPR) signaling, including eIF2α phosphorylation, endoplasmic reticulum (ER) chaperone response, and ER-associated degradation (ERAD) components, as well as antioxidative stress response. In addition, the phosphorylation of STAT3 and AKT, which are protective against epithelial cell death and colonic inflammation, was also impaired in the colonic epithelial cells of PKR−/− mice upon DSS colitis.
Conclusions
These data demonstrate that PKR is a physiologically relevant transducer of inflammatory response signaling in colonic epithelial cells. PKR may promote the homeostasis and survival of intestinal epithelial cells (IECs) through eIF2α-mediated UPR activation, as well as the activation of STAT3 and AKT pathways. In the absence of PKR, the survival and proliferation of IECs was impaired, thus exacerbating intestinal inflammation.
doi:10.1002/ibd.22878
PMCID: PMC3751177  PMID: 22275310
PKR; DSS colitis; UPR; prosurvival signaling
2.  The Unfolded Protein Response and Chemical Chaperones Reduce Protein Misfolding and Colitis in Mice 
Gastroenterology  2013;144(5):989-1000.e6.
BACKGROUND & AIMS
Endoplasmic reticulum (ER) stress has been associated with development of inflammatory bowel disease. We examined the effects of ER stress–induced chaperone response and the orally active chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (PBA), which facilitate protein folding and reduce ER stress, in mice with colitis.
METHODS
We used dextran sulfate sodium (DSS) to induce colitis in mice that do not express the transcription factor ATF6α or the protein chaperone P58IPK. We examined the effects of TUDCA and PBA in cultured intestinal epithelial cells (IECs); in wild-type, P58IPK−/−, and Atf6α−/− mice with colitis; and in Il10−/− mice.
RESULTS
P58IPK−/− and Atf6α−/− mice developed more severe colitis following administration of DSS than wild-type mice. IECs from P58IPK−/− mice had excessive ER stress, and apoptotic signaling was activated in IECs from Atf6α−/− mice. Inflammatory stimuli induced ER stress signals in cultured IECs, which were reduced by incubation with TUDCA or PBA. Oral administration of either PBA or TUDCA reduced features of DSS-induced acute and chronic colitis in wild-type mice, the colitis that develops in Il10−/− mice, and DSS-induced colitis in P58IPK−/− and Atf6α−/− mice. Reduced signs of colonic inflammation in these mice were associated with significantly decreased ER stress in colonic epithelial cells.
CONCLUSIONS
The unfolded protein response induces expression of genes that encode chaperones involved in ER protein folding; these factors prevent induction of colitis in mice. Chemical chaperones such as TUDCA and PBA alleviate different forms of colitis in mice and might be developed for treatment of inflammatory bowel diseases.
doi:10.1053/j.gastro.2013.01.023
PMCID: PMC3751190  PMID: 23336977
IBD; Mouse Model; Ulcerative Colitis; Therapeutic Agent
3.  Interaction between quality control systems for ER protein folding and RNA biogenesis 
Worm  2013;2(2):e23005.
The endoplasmic reticulum (ER) is the intracellular organelle responsible for the synthesis, folding and assembly of proteins destined for secretion and the endomembrane system of the cell. ER quality control (ERQC) is an intensively studied surveillance mechanism that selectively degrades misfolded proteins to ensure that only properly folded proteins exit the ER en route to the Golgi compartment. Proper protein folding is indispensable for the differentiation and function of cells that secrete high levels of protein and defects in protein folding are implicated in many pathologies, including metabolic, genetic, neurodegenerative and inflammatory diseases. Accumulation of misfolded proteins in the ER activates an adaptive set of signaling pathways, collectively known as the unfolded protein response (UPR), to resolve protein misfolding and restore ER homeostasis. Nonsense-mediated RNA decay (NMD) is an RNA surveillance system that selectively degrades nascent mRNAs containing premature termination codons (PTCs). Recently, we used a genetic screen to identify genes that interact with UPR signaling in C. elegans. These studies identified NMD-associated genes that are required for ER protein folding homeostasis. These findings link the quality control systems required for ER protein folding and RNA biogenesis, provide new insights into mechanisms of ERQC and have implications on diseases of ER dysfunction and therapeutic approaches based on NMD inhibition. Here, we discuss the biological significance of these findings and future directions for study.
doi:10.4161/worm.23005
PMCID: PMC3704444  PMID: 24058870
endoplasmic reticulum stress; unfolded protein response; nonsense-mediated RNA decay; quality control; protein folding; premature termination codons
4.  The impact of the unfolded protein response on human disease 
The Journal of Cell Biology  2012;197(7):857-867.
A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease.
doi:10.1083/jcb.201110131
PMCID: PMC3384412  PMID: 22733998
5.  Glycoprotein folding and quality-control mechanisms in protein-folding diseases 
Disease Models & Mechanisms  2014;7(3):331-341.
Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC) system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER). A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.
doi:10.1242/dmm.014589
PMCID: PMC3944493  PMID: 24609034
N-glycosylation; Glycoprotein folding; ER quality control; ER-associated degradation; ER export
6.  iRhoms: ERADicating the messenger in growth control signaling 
Developmental cell  2011;20(4):414-416.
iRhoms are inactive rhomboid-like pseudoproteases that lack essential catalytic residues. Although iRhoms are highly conserved in metazoan species, little is known about their function. In a recent issue of Cell, Zettl et al. (2011) show that iRhoms regulate growth factor signaling through endoplasmic reticulum-associated protein degradation (ERAD).
doi:10.1016/j.devcel.2011.04.003
PMCID: PMC3408039  PMID: 21497754
7.  Endoplasmic Reticulum Stress in Liver Disease 
Journal of Hepatology  2010;54(4):795-809.
The unfolded protein response (UPR) is activated upon the accumulation of misfolded proteins in the endoplasmic reticulum (ER), that are sensed by the binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78). The accumulation of unfolded proteins sequesters BiP so it dissociates from three ER-transmembrane transducers leading to their activation. These transducers are inositol requiring (IRE) 1α, PKR-like ER kinase (PERK) and activating transcription factor (ATF) 6α. PERK phosphorylates eukaryotic initiation factor 2 alpha (eIF2α) resulting in global mRNA translation attenuation, and concurrently selectively increases the translation of several mRNAs, including the transcription factor ATF4, and its downstream target CHOP. IRE1α has kinase and endoribonuclease (RNase) activities. IRE1α autophosphorylation activates the RNase activity to cleave XBP1 mRNA, to produce the active transcription factor sXBP1. IRE1α activation also recruits and activates the stress kinase JNK. ATF6α transits to the Golgi compartment where it is cleaved by intramembrane proteolysis to generate a soluble active transcription factor. These UPR pathways act in concert to increase ER content, expand the ER protein folding capacity, degrade misfolded proteins, and reduce the load of new proteins entering the ER. All of these are geared toward adaptation to resolve the protein folding defect. Faced with persistent ER stress, adaptation starts to fail and apoptosis occurs, possibly mediated through calcium perturbations, reactive oxygen species, and the proapoptotic transcription factor CHOP. The UPR is activated in several liver diseases; including obesity associated fatty liver disease, viral hepatitis and alcohol-induced liver injury, all of which are associated with steatosis, raising the possibility that ER stress-dependent alteration in lipid homeostasis is the mechanism that underlies the steatosis. Hepatocyte apoptosis is a pathogenic event in several liver diseases, and may be linked to unresolved ER stress. If this is true, restoration of ER homeostasis prior to ER stress-induced cell death may provide a therapeutic rationale in these diseases. Here we discuss each branch of the UPR and how they may impact hepatocyte function in different pathologic states.
doi:10.1016/j.jhep.2010.11.005
PMCID: PMC3375108  PMID: 21145844
8.  eIF2α phosphorylation bypasses premature senescence caused by oxidative stress and pro-oxidant antitumor therapies 
Aging (Albany NY)  2013;5(12):884-901.
Eukaryotic cells respond to various forms of stress by blocking mRNA translation initiation via the phosphorylation of the alpha (α) subunit of eIF2 at serine 51 (S51) (eIFαP). An important role of eIF2αP is the regulation of redox homeostasis and adaptation of cells to oxidative stress. Herein, we demonstrate that eIF2αP guards cells from intracellular reactive oxygen species (ROS) via the inhibition of senescence. Specifically, genetic inactivation of either eIF2αP or eIF2α kinase PERK in primary mouse or human fibroblasts leads to proliferative defects associated with increased DNA damage, G2/M accumulation and induction of premature senescence. Impaired proliferation of either PERK or eIF2αP-deficient primary cells is caused by increased ROS and restored by anti-oxidant treatment. Contrary to primary cells, immortalized mouse fibroblasts or human tumor cells become tolerant to elevated intracellular ROS levels caused by impaired eIF2αP. However, eIF2αP-deficient human tumor cells are highly susceptible to extrinsic ROS generated by the pro-oxidant drug doxorubicin by undergoing premature senescence. Our work demonstrates that eIF2αP determines cell destiny through its capacity to control senescence in response to oxidative stress. Also, inhibition of eIF2αP may be a suitable means to increase the anti-tumor effects of pro-oxidant drugs through the induction of senescence.
PMCID: PMC3883705  PMID: 24334569
eIF2; protein phosphorylation; mRNA translation; reactive oxygen species; DNA damage; cellular senescence; doxorubicin
9.  UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum 
Molecular Biology of the Cell  2013;24(17):2597-2608.
The N-glycans promote solubility in the ER even for mutant glycoproteins, such as mutant α1-antitrypsin. This study shows that enzymatic monoglucosylation activity of the enzyme UGGT1 and lectin chaperone abundance are required for N-glycans to provide maximum solubility to the misfolded substrate.
Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.
doi:10.1091/mbc.E13-02-0101
PMCID: PMC3756913  PMID: 23864712
10.  IRE1, a double-edged sword in pre-miRNA slicing and cell death 
Developmental cell  2012;23(5):921-923.
IRE1α, the most conserved transducer of the unfolded protein response, plays critical roles in many biological processes and cell fate decisions. Reporting in Science, Upton et al. (2012) broadened our understanding of IRE1a as a cell-death executioner, showing that upon ER stress, IRE1α degrades microRNAs to promote translation of caspase-2.
doi:10.1016/j.devcel.2012.10.025
PMCID: PMC3684431  PMID: 23153490
11.  XBP1 is a regulatory hub that links endoplasmic reticulum homeostasis with innate immunity and metabolism 
EMBO molecular medicine  2010;2(6):189-192.
WIS summary
IRE1/XBP1-mediated signaling represents the most conserved branch of the unfolded protein response. A series of recent studies reveal novel and potentially ancient roles for this pathway in the coordination of metabolic and immune responses.
doi:10.1002/emmm.201000076
PMCID: PMC3042734  PMID: 20533428
12.  IRE1α -XBP1s Induces PDI Expression to Increase MTP Activity for Hepatic VLDL Assembly and Lipid Homeostasis 
Cell metabolism  2012;16(4):473-486.
Summary
The unfolded protein response (UPR) is a signaling pathway required to maintain endoplasmic reticulum (ER) homeostasis and hepatic lipid metabolism. Here, we identify an essential role for the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α)-X-box binding protein 1 (XBP1) arm of the UPR in regulation of hepatic very low-density lipoprotein (VLDL) assembly and secretion. Hepatocyte-specific deletion of Ire1α reduces lipid partitioning into the ER lumen and impairs the assembly of triglyceride (TG)-rich VLDL, but does not affect TG synthesis, de novo lipogenesis, or the synthesis or secretion of apolipoprotein B (apoB). The defect in VLDL assembly is, at least in part, due to decreased microsomal triglyceride-transfer protein (MTP) activity resulting from reduced protein disulfide isomerase (PDI) expression. Collectively, our findings reveal a key role for the IRE1α-XBP1s-PDI axis in linking ER homeostasis with regulation of VLDL production and hepatic lipid homeostasis that may provide a therapeutic target for disorders of lipid metabolism.
doi:10.1016/j.cmet.2012.09.003
PMCID: PMC3569089  PMID: 23040069
13.  Temporal clustering of gene expression links the metabolic transcription factor HNF4α to the ER stress-dependent gene regulatory network 
Frontiers in Genetics  2013;4:188.
The unfolded protein response (UPR) responds to disruption of endoplasmic reticulum (ER) function by initiating signaling cascades that ultimately culminate in extensive transcriptional regulation. Classically, this regulation includes genes encoding ER chaperones, ER-associated degradation factors, and others involved in secretory protein folding and processing, and is carried out by the transcriptional activators that are produced as a consequence of UPR activation. However, up to half of the mRNAs regulated by ER stress are downregulated rather than upregulated, and the mechanisms linking ER stress and UPR activation to mRNA suppression are poorly understood. To begin to address this issue, we used a “bottom-up” approach to study the metabolic gene regulatory network controlled by the UPR in the liver, because ER stress in the liver leads to lipid accumulation, and fatty liver disease is the most common liver disease in the western world. qRT-PCR profiling of mouse liver mRNAs during ER stress revealed that suppression of the transcriptional regulators C/EBPα, PPARα, and PGC-1α preceded lipid accumulation, and was then followed by suppression of mRNAs encoding key enzymes involved in fatty acid oxidation and lipoprotein biogenesis and transport. Mice lacking the ER stress sensor ATF6α, which experience persistent ER stress and profound lipid accumulation during challenge, were then used as the basis for a functional genomics approach that allowed genes to be grouped into distinct expression profiles. This clustering predicted that ER stress would suppress the activity of the metabolic transcriptional regulator HNF4α—a finding subsequently confirmed by chromatin immunopreciptation at the Cebpa and Pgc1a promoters. Our results establish a framework for hepatic gene regulation during ER stress and suggest that HNF4α occupies the apex of that framework. They also provide a unique resource for the community to further explore the temporal regulation of gene expression during ER stress in vivo.
doi:10.3389/fgene.2013.00188
PMCID: PMC3781334  PMID: 24069029
ER stress; fatty liver; functional genomics; gene regulatory network; lipid metabolism
14.  Domain compatibility in Ire1 kinase is critical for the Unfolded Protein Response 
FEBS letters  2010;584(14):3203-3208.
The unfolded phrotein response is a mechanism to cope with endoplasmic reticulum stress. In Saccharomyces cerevisiae, Ire1 senses the stress and mediates a signaling cascade to upregulate responsive genes through an unusual HAC1 mRNA splicing. The splicing requires interconnected activity (kinase and endoribonuclease) of Ire1 to cleave HAC1 mRNA at the non-canonical splice sites before translation into Hac1 transcription factor. Analysis of the truncated kinase domain from Ire1 homologs revealed that this domain is highly conserved. Characterization by domain swapping indicated that a functional ATP/ADP binding domain is minimally required. However the overall domain compatibility is critical for eliciting its full endoribonuclease function.
doi:10.1016/j.febslet.2010.06.003
PMCID: PMC3762510  PMID: 20541549
Unfolded protein response; Ire1; Domain swapping; HAC1 splicing; protein kinase; endoribonuclease
15.  ER Stress and Its Functional Link to Mitochondria: Role in Cell Survival and Death 
The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca2+ transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.
If protein misfolding in the ER is not resolved by the unfolded protein response (UPR), apoptosis is triggered. The is regulated by Ca2+ transfer from the ER to the mitochondria.
doi:10.1101/cshperspect.a004424
PMCID: PMC3181038  PMID: 21813400
16.  Regulation of ER stress-induced macroautophagy by protein kinase C 
Autophagy  2008;4(6):841-843.
The endoplasmic reticulum (ER) is the primary site for folding and quality control for proteins destined to the cell surface and intracellular organelles. A variety of cellular insults alter ER homeostasis to disrupt protein folding, cause the accumulation of misfolded proteins, and activate an autophagic response. However, the molecular signaling pathways required for ER stress-induced autophagy are largely unknown. Recently, we discovered that a novel-type protein kinase C family member (PKCθ) is required for ER stress-induced autophagy. We show that ER stress, in a Ca2+-dependent manner, induces PKCθ phosphorylation within the activation loop and localization with LC3-II in punctate cytoplasmic structures. Pharmacological inhibition, siRNA-mediated knockdown, or transdominant-negative mutant expression of PKCθ block the ER stress-induced autophagic response. PKCθ activation is not required for autophagy induced by amino acid starvation, and PKCθ activation in response to ER stress does not require either the mTOR kinase or the unfolded protein response signaling pathways. Herein, we review and discuss the significance of these findings with respect to regulation of autophagy in response to ER stress.
PMCID: PMC2770887  PMID: 18670192
unfolded protein response; protein kinase Cθ; calcium; autophagy; endoplasmic reticulum; autophagosome
17.  Thioredoxin-interacting protein mediates ER stress-induced β cell death through initiation of the inflammasome 
Cell metabolism  2012;16(2):265-273.
SUMMARY
Recent clinical and experimental evidence suggests that endoplasmic reticulum (ER) stress contributes to the life-and-death decisions of β cells during the progression of type 1 and type 2 diabetes. Although crosstalk between inflammation and ER stress has been suggested to play a significant role in β cell dysfunction and death, a key molecule connecting ER stress to inflammation has not been identified. Here we report that thioredoxin-interacting protein (TXNIP) is a critical signaling node that links ER stress and inflammation. TXNIP is induced by ER stress through the PERK and IRE1 pathways, induces IL-1β mRNA transcription, activates IL-1β production by the NLRP3 inflammasome, and mediates ER stress-mediated β cell death. Collectively, our results suggest that TXNIP is a potential therapeutic target for diabetes and ER stress-related human diseases such as Wolfram syndrome.
doi:10.1016/j.cmet.2012.07.005
PMCID: PMC3418541  PMID: 22883234
18.  The Endoplasmic Reticulum and the Unfolded Protein Response 
The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.
doi:10.1016/j.semcdb.2007.09.003
PMCID: PMC2706143  PMID: 18023214
Endoplasmic reticulum; unfolded protein response; ER; secretory pathway; apoptosis
19.  ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death 
Nature cell biology  2013;15(5):481-490.
Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.
doi:10.1038/ncb2738
PMCID: PMC3692270  PMID: 23624402
20.  Beta-Cell Failure, Stress, and Type 2 Diabetes 
The New England journal of medicine  2011;365(20):1931-1933.
doi:10.1056/NEJMcibr1109442
PMCID: PMC3684425  PMID: 22087686
21.  Endoplasmic Reticulum Stress and Type 2 Diabetes 
Annual review of biochemistry  2012;81:767-793.
Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause β-cell dysfunction and death.
doi:10.1146/annurev-biochem-072909-095555
PMCID: PMC3684428  PMID: 22443930
unfolded protein response; ER stress; free fatty acid; glucose; pancreatic β-cell
22.  Regulation of Apoptosis by the Unfolded Protein Response 
Summary
In eukaryotic cells, the endoplasmic reticulum (ER) serves many specialized functions including biosynthesis and assembly of membrane and secretory proteins, calcium storage and production of lipids and sterols. As a plant for protein folding and posttranslational modification, the ER provides stringent quality control systems to ensure that only correctly folded proteins exit the ER and unfolded or misfolded proteins are retained and ultimately degraded. Biochemical, physiological, and pathological stimuli that interfere with ER function can disrupt ER homeostasis, impose stress to the ER, and subsequently cause accumulation of unfolded or misfolded proteins in the ER lumen. To deal with accumulation of unfolded or misfolded proteins, the cell has evolved highly specific signaling pathways collectively called the “unfolded protein response” (UPR) to restore normal ER functions. However, if the overload of unfolded or misfolded proteins in the ER is not resolved, the prolonged UPR will induce ER stress-associated programmed cell death, apoptosis, to protect the organism by removing the stressed cells. In this chapter, we summarize our current understanding of UPR-induced apoptosis and various methods to detect ER stress and apoptosis in mammalian cells.
doi:10.1007/978-1-60327-017-5_14
PMCID: PMC3684430  PMID: 19609758
Apoptosis; Endoplasmic Reticulum Stress; Unfolded Protein Response
23.  Temporal regulation of Cat-1 (cationic amino acid transporter-1) gene transcription during endoplasmic reticulum stress 
The Biochemical journal  2010;429(1):215-224.
Expression of the Cat-1 gene (cationic amino acid transporter-1) is induced in proliferating cells and in response to a variety of stress conditions. The expression of the gene is mediated via a TATA-less promoter. In the present study we show that an Sp1 (specificity protein 1)-binding site within a GC-rich region of the Cat-1 gene controls its basal expression and is important for induction of the gene during the UPR (unfolded protein response). We have shown previously that induction of Cat-1 gene expression during the UPR requires phosphorylation of the translation initiation factor eIF2α (eukaryotic initiation factor 2α) by PERK (protein-kinase-receptor-like endoplasmic reticulum kinase), one of the signalling pathways activated during the UPR. This leads to increased translation of the transcription factor ATF4 (activating transcription factor 4). We also show that a second signalling pathway is required for sustained transcriptional induction of the Cat-1 gene during the UPR, namely activation of IRE1 (inositol-requiring enzyme 1) leading to alternative splicing of the mRNA for the transcription factor XBP1 (X-box-binding protein 1). The resulting XBP1s (spliced XBP1) can bind to an ERSE (endoplasmic-reticulum-stress-response-element), ERSE-II-like, that was identified within the Cat-1 promoter. Surprisingly, eIF2α phosphorylation is required for accumulation of XBP1s. We propose that the signalling via phosphorylated eIF2α is required for maximum induction of Cat-1 transcription during the UPR by inducing the accumulation of both ATF4 and XBP1s.
doi:10.1042/BJ20100286
PMCID: PMC3684439  PMID: 20408811
activating transcription factor 4 (ATF4); cationic amino acid transporter-1 (Cat-1); endoplasmic reticulum stress; specificity protein 1 (Sp1); unfolded protein response; X-box-binding protein 1 (XBP1)
24.  Tumor Suppression by PTEN Requires the Activation of the PKR-eIF2α Phosphorylation Pathway 
Science signaling  2009;2(102):ra85.
Inhibition of protein synthesis by phosphorylation of the a subunit of eukaryotic translation initiation factor 2 (eIF2) at Ser51 occurs as a result of the activation of a family of kinases in response to various forms of stress. Although some consequences of eIF2α phosphorylation are cytoprotective, phosphorylation of eIF2α by RNA-dependent protein kinase (PKR) is largely proapoptotic and tumor suppressing. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is a tumor suppressor protein that is mutated or deleted in various human cancers, with functions that are mediated through phosphatase-dependent and -independent pathways. Here, we demonstrate that the eIF2α phosphorylation pathway is downstream of PTEN. Inactivation of PTEN in human melanoma cells reduced eIF2α phosphorylation, whereas reconstitution of PTEN-null human glioblastoma or prostate cancer cells with either wild-type PTEN or phosphatase-defective mutants of PTEN induced PKR activity and eIF2α phosphorylation. The antiproliferative and proapoptotic effects of PTEN were compromised in mouse embryonic fibroblasts that lacked PKR or contained a phosphorylation-defective variant of eIF2α. Induction of the pathway leading to phosphorylation of eIF2α required an intact PDZ-binding motif in PTEN. These findings establish a link between tumor suppression by PTEN and inhibition of protein synthesis that is independent of PTEN's effects on phosphoinositide 3′-kinase signaling.
doi:10.1126/scisignal.2000389
PMCID: PMC3684442  PMID: 20029030
25.  Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response 
Nucleic Acids Research  2013;41(14):6960-6974.
Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane–bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane–bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription.
doi:10.1093/nar/gkt429
PMCID: PMC3737538  PMID: 23716639

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