Mammalian Unfolded Protein Response Research Articles

Oligomerization of the transmembrane domain of IRE1α in SDS micelles

IRE1α (Inositol-requiring enzyme 1 α), an endoplasmic reticulum (ER)-resident sensor for mammalian unfolded protein response, is a type I transmembrane protein which has a bifunctional enzyme containing kinase and RNase domains. Although the luminal domain and cytosolic domain of IRE1α are thought to play crucial roles in regulating the protein activity, no functional and structural studies of the transmembrane domain exist thus far. Herein, using CD spectroscopy, we report that the transmembrane domain of the IRE1α is alpha-helical in a membrane-like environment. In addition, SDS–PAGE and FRET analyses support that the transmembrane domain forms oligomers in SDS micelles. Thus, the study would provide insights into how the transmembrane domain plays a role in regulating the IRE1α protein activity.

Hyperactivity of the Ero1α Oxidase Elicits Endoplasmic Reticulum Stress but No Broad Antioxidant Response

Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.

RETRACTED ARTICLE: XBP1S protects cells from ER stress-induced apoptosis through Erk1/2 signaling pathway involving CHOP

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER), and the transcription factor X-box binding protein 1 spliced (XBP1S), a regulator of the UPR, is known to be important for ER stress (ERS)-mediated apoptosis and cell growth, but the molecular mechanism underlying these processes remains unexplored. Here, we report that knockdown of XBP1S by an siRNA silencing approach increased the expression of ERS-associated molecules. The overexpression of XBP1S stimulated, whereas its knockdown inhibited, cell proliferation in chondrocytes and chondrosarcoma cells; in addition, overexpression of XBP1S inhibited, while its repression enhanced, ERS-mediated apoptosis in chondrocytes and chondrosarcoma cells. Furthermore, XBP1S-mediated inhibition of apoptosis in response to ERS is through the Erk1/2 signaling pathway and down-regulation CHOP transcription factor. CHOP is one of the key downstream molecules known to be involved in ERS-mediated apoptosis. Collectively, these findings reveal a novel critical role of XBP1S in ERS-mediated apoptosis and the molecular mechanisms involved.

STT3B-Dependent Posttranslational N-Glycosylation as a Surveillance System for Secretory Protein

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Study on the effect of IRE1α on cell growth and apoptosis via modulation PLK1 in ER stress response

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1a by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1a induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1a can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1a-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1a in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1a may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.

Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

BackgroundLong QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins.MethodspcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined.ConclusionOur results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin was stronger after proteasome inhibition, compared to WT-hERG. These results suggest that trafficking-deficient G572R-hERG and E637K-hERG mutant proteins can activate ER stress pathways and are targeted to the proteasome for degradation. Calnexin and Calreticulin play important roles in these processes.

A Conserved Structural Determinant Located at the Interdomain Region of Mammalian Inositol-requiring Enzyme 1α

Inositol-requiring enzyme 1α (IRE1α), an endoplasmic reticulum-resident sensor for mammalian unfolded protein response, is a bifunctional enzyme containing kinase and RNase domains critical for trans-autophosphorylation and Xbp1 mRNA splicing, respectively, in response to endoplasmic reticulum stress. However, the amino acid residues important for its function and activation remain largely unexplored. Here, through analysis of IRE1α mutants associated with human somatic cancers, we have identified a highly conserved proline residue at position 830 (Pro(830)) that is critical for its structural integrity and hence, the activation of both kinase and RNase domains. Structural analysis revealed that Pro(830) may form a highly conserved structural linker with adjacent tryptophan and tyrosine residues at positions 833 and 945 (Trp(833) and Tyr(945)), thereby bridging the kinase and RNase domains. Indeed, mutation of Pro(830) to leucine (P830L) completely abolished the kinase and RNase activities, significantly decreased protein stability, and prevented oligomerization of IRE1α upon ER stress; similar observations were made for mutations of Trp(833) to alanine (W833A) and to a lesser extent for Y945A. Our finding may facilitate the identification of small molecules to compromise IRE1α function specifically.

RtcB, a Novel RNA Ligase, Can Catalyze tRNA Splicing and HAC1 mRNA Splicing in Vivo

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.

A review of the mammalian unfolded protein response.

Proteins requiring post-translational modifications such as N-linked glycosylation are processed in the endoplasmic reticulum (ER). A diverse array of cellular stresses can lead to dysfunction of the ER and ultimately to an imbalance between protein-folding capacity and protein-folding load. Cells monitor protein folding by an inbuilt quality control system involving both the ER and the Golgi apparatus. Unfolded or misfolded proteins are tagged for degradation via ER-associated degradation (ERAD) or sent back through the folding cycle. Continued accumulation of incorrectly folded proteins can also trigger the unfolded protein response (UPR). In mammalian cells, UPR is a complex signaling program mediated by three ER transmembrane receptors: activating transcription factor 6 (ATF6), inositol requiring kinase 1 (IRE1) and double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK). UPR performs three functions, adaptation, alarm, and apoptosis. During adaptation, the UPR tries to reestablish folding homeostasis by inducing the expression of chaperones that enhance protein folding. Simultaneously, global translation is attenuated to reduce the ER folding load while the degradation rate of unfolded proteins is increased. If these steps fail, the UPR induces a cellular alarm and mitochondrial mediated apoptosis program. UPR malfunctions have been associated with a wide range of disease states including tumor progression, diabetes, as well as immune and inflammatory disorders. This review describes recent advances in understanding the molecular structure of UPR in mammalian cells, its functional role in cellular stress, and its pathophysiology.

Hypomorphic Mutation in the Site-1 Protease Mbtps1 Endows Resistance to Persistent Viral Infection in a Cell-Specific Manner

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

The endoplasmic reticulum stress response and diabetic kidney disease

The endoplasmic reticulum (ER) folds and modifies proteins; however, during conditions of cellular stress, unfolded proteins accumulate in the ER and activate the unfolded protein response (UPR). The UPR, also referred to as the ER stress response, activates three distinct signaling cascades that are designed to globally reduce transcription and translation. The three major arms of the mammalian UPR include 1) protein kinase RNA (PKR)-like ER kinase (PERK), 2) inositol-requiring protein-1 (IRE1α), and 3) activating transcription factor-6 (ATF6) pathways. The PERK pathway rapidly attenuates protein translation, whereas the ATF6 and IRE1α cascades transcriptionally upregulate ER chaperone genes that promote proper folding and ER-associated degradation (ERAD) of proteins. This integrated response in turn allows the folding machinery of the ER to catch up with the backlog of unfolded proteins. The ER stress response plays a role in a number of pathophysiological processes, including pancreatic β-cell failure and apoptosis. The goals of the current review are to familiarize investigators with cellular and tissue activation of this response in the rodent and human diabetic kidney. Additionally, we will review therapeutic modulators of the ER stress response and discuss their efficacy in models of diabetic kidney disease. The ER stress response has both protective and deleterious features. A better understanding of the molecular pathways regulated during this process in a cell- and disease-specific manner could reveal novel therapeutic strategies in chronic renal diseases, including diabetic kidney disease.

Vertebrate Unfolded Protein Response: Mammalian Signaling Pathways Are Conserved in Medaka Fish

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ER stress signal is sensed and transmitted by a transmembrane protein(s) in the ER. The number of these transducers has increased with evolution, one in yeast, three in worm and fly, and five in mammals. Here, we examined medaka fish, Oryzias latipes, as a vertebrate model organism, and found that the medaka genome encodes five UPR transducers. Analysis of a medaka embryonic cell line revealed that the mammalian UPR signaling mechanisms are very well conserved. Thus, XBP1 mRNA, which encodes the transcription factor XBP1 downstream of the IRE1 pathway, was spliced in response to ER stress, resulting in production of the active form of XBP1. Translation was generally attenuated in response to ER stress, which paradoxically induced the translation of ATF4, the transcription factor downstream of the PERK pathway. ATF6 was constitutively synthesized as a transmembrane protein and activated by ER stress-induced proteolysis. Results obtained with the overexpression of active ATF6α, ATF6β, and XBP1 strongly suggested that ATF6α plays a major role in upregulating the major ER chaperone BiP, contrary to the case in non-vertebrates, in which the IRE1 pathway is essential to the induction of BiP. Physiological ER stress occurring during embryonic development was visualized using transgenic medaka carrying the enhanced green fluorescent protein gene under the control of the BiP promoter. Thus, analysis of the vertebrate UPR using medaka will help provide a more comprehensive understanding of the biology and physiology of the UPR.

Neuroprotective effects of stanniocalcin 2 following kainic acid-induced hippocampal degeneration in ICR mice

Stanniocalcin 2 (STC2), the paralog of STC1, has been shown to act as a novel target of the mammalian unfolded protein response. We investigated the potential neuroprotective actions of STC2 against kainic acid toxicity in the hippocampus of ICR mice. STC2-treated mice experienced less neuronal cell loss in the CA3 area of the hippocampus. Also, microglial activation and heme oxygenase 1 expression were attenuated in the hippocampus of STC2-treated mice. To confirm whether STC2 regulates microglial activation directly, nitric oxide levels were measured in BV2 cells cultured with and without 10 nM STC2. STC2 decreased the level of nitric oxide induced by lipopolysaccharide (LPS) treatment significantly. Also, STC2 pretreatment significantly decreased TNF-α and IL-1β expression induced by LPS treatment. These observations demonstrated that STC2 exerts neuroprotective actions against excitotoxic insults through the inhibition of microglial activation.

Prolonged endoplasmic reticulum stress promotes mislocalization of immunoglobulins to the cytoplasm

Signal peptide-dependent insertion of newly synthesized proteins into the endoplasmic reticulum (ER) is a multi-step process, whose fidelity varies with the identity of the protein and the cell type. ER translocation of prions is sensitive to conditions of acute ER stress in a manner that pre-emptively prevents their aggregation and proteo-toxicity. While this has been documented for extreme ER stress conditions and for a special type of proteins, the impact of chronic ER stress on protein translocation in general has not been well characterized. The unfolded protein response (UPR) is a cytoprotective signaling pathway activated by ER stress. The transcription factor X-box-binding protein 1 (XBP-1) is a key element of the mammalian UPR, which is activated in response to ER stress. Deletion of XBP-1 generates constitutive chronic ER stress conditions. Chronic ER stress can also be produced pharmacologically, for example by prolonged treatment with proteasome inhibitors, which abrogates XBP-1 activation. We tested the impact of chronic ER stress on protein insertion into the ER with special emphasis on antibody secreting cells (ASCs), as these cells cope physiologically with prolonged stress conditions. We show that XBP-1 in plasmablasts and fibroblasts controls the ER translocation of US2, a viral-encoded protein with a priori poor insertion efficiency. Using monoclonal antibodies that preferentially recognize ER-mis-inserted μ Ig chains we demonstrate that prolonged treatment of plasmablasts with proteasome inhibitors, as well as deletion of XBP-1, impaired the translocation of μ chains to the ER. Our data suggest that ASCs under prolonged ER stress conditions endure cytoplasmic mislocalization of Ig proteins. This mislocalization may further explain the exquisite sensitivity of multiple myeloma to proteasome inhibitors.

Divest Yourself of a Preconceived Idea: Transcription Factor ATF6 Is Not a Soluble Protein!

The unfolded protein response (UPR), an evolutionarily conserved transcriptional induction program that is coupled with intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, is activated to cope with ER stress and to maintain the homeostasis of the ER. In 1996, we isolated a basic leucine zipper protein, which had been previously named activating transcription factor (ATF)6, as a candidate transcription factor responsible for the mammalian UPR. Subsequent analysis, however, was confounding. The problem was eventually tracked down to an unusual property of ATF6: rather than being a soluble nuclear protein, as expected for an active transcription factor, ATF6 was instead synthesized as a transmembrane protein embedded in the ER, which was activated by ER stress-induced proteolysis. ATF6 was thus unique: an ER stress sensor/transducer that is involved in all steps of the UPR, from the sensing step in the ER to the transcriptional activation step in the nucleus.

Direct proteasome binding and subsequent degradation of unspliced XBP-1 prevent its intracellular aggregation

The non-canonical splicing of XBP-1 mRNA is a hallmark of the mammalian unfolded protein response (UPR). The proteasomal degradation of unspliced XBP-1 (XBP-1u) facilitates the termination of the UPR. Thus, understanding the mechanism of XBP-1u degradation may allow control over UPR duration and intensity. We show that XBP-1u interacts with purified 20S proteasomes through its unstructured C-terminus, which leads to its degradation in a manner that autonomously opens the proteasome gate. In living cells, the C-terminus of XBP-1u accumulates in aggresome structures in the presence of proteasome inhibitors. We propose that direct proteasomal degradation of XBP-1u prevents its intracellular aggregation. Structured summary MINT- 7302217: XBP1-u (uniprotkb: P17861-1) binds (MI: 0407) to Proteasome subunit alpha 7.2 (uniprotkb: O14818) by pull down (MI: 0096) MINT- 7302148: Vimentin (uniprotkb: P08670) and XBP1-u (uniprotkb: P17861-1) colocalize (MI: 0403) by fluorescence microscopy (MI: 0416) MINT- 7302163: XBP1-u (uniprotkb: P17861-1) binds (MI: 0407) to Proteasome subunit alpha 5 (uniprotkb: P28066) by pull down (MI: 0096) MINT- 7302186: XBP1-u (uniprotkb: P17861-1) binds (MI: 0407) to Proteasome subunit alpha 6 (uniprotkb: P60900) by pull down (MI: 0096)

Unconventional splicing ofXBP1mRNA occurs in the cytoplasm during the mammalian unfolded protein response

XBP1 is a key transcription factor that regulates the mammalian unfolded protein response. Its expression is regulated by unconventional mRNA splicing that is carried out by endonuclease IRE1 and a specific, as yet unknown, RNA ligase in response to the accumulation of unfolded proteins in the ER. Conventional mRNA splicing occurs only in the nucleus, but it has remained unclear whether unconventional splicing of XBP1 mRNA takes place in the nucleus, cytoplasm or both. Here, we show that the catalytic domain of IRE1 contains a nuclear exclusion signal to prevent IRE1 from mislocalizing to the nucleus. In addition, RNA ligase, which joins XBP1 exons cleaved by IRE1 was detected in the cytoplasm but not in the nucleus. Moreover, the cytoplasm contained large amounts of unspliced XBP1 mRNA compared with the nucleus. Most unspliced XBP1 mRNA was converted to spliced mRNA by unconventional splicing even if de novo transcription was blocked, suggesting that cytoplasmic XBP1 mRNA, not nuclear XBP1 mRNA, is a major substrate for unconventional splicing. From these observations, we concluded that unconventional splicing of XBP1 mRNA occurs predominantly in the cytoplasm.

The mammalian UPR boosts glycoprotein ERAD by suppressing the proteolytic downregulation of ER mannosidase I

The secretory pathway provides a physical route through which only correctly folded gene products are delivered to the eukaryotic cell surface. The efficiency of endoplasmic reticulum (ER)-associated degradation (ERAD), which orchestrates the clearance of structurally aberrant proteins under basal conditions, is boosted by the unfolded protein response (UPR) as one of several means to relieve ER stress. However, the underlying mechanism that links the two systems in higher eukaryotes has remained elusive. Herein, the results of transient expression, RNAi-mediated knockdown and functional studies demonstrate that the transcriptional elevation of EDEM1 boosts the efficiency of glycoprotein ERAD through the formation of a complex that suppresses the proteolytic downregulation of ER mannosidase I (ERManI). The results of site-directed mutagenesis indicate that this capacity does not require that EDEM1 possess inherent mannosidase activity. A model is proposed in which ERManI, by functioning as a downstream effector target of EDEM1, represents a checkpoint activation paradigm by which the mammalian UPR coordinates the boosting of ERAD.

Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway

The UPR (unfolded protein response) pathway comprises three signalling cascades mediated by the ER (endoplasmic reticulum) stress-sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPbeta (CCAAT/enhancer-binding protein beta) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared with triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box-binding protein 1), ATF6alpha or ATF6beta expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, ASNS NSRE-1 and NSRE-2, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian ER-stress-responsive sequences.

2. Luman/CREB3 and LRF, novel players in the mammalian unfolded protein response

2. Luman/CREB3 and LRF, novel players in the mammalian unfolded protein response