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Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance., (© 2024. The Author(s).)

Both basal and glucose-stimulated insulin release occur primarily by insulin secretory granule exocytosis from pancreatic β cells, and both are needed to maintain normoglycemia. Loss of insulin-secreting β cells, accompanied by abnormal glucose tolerance, may involve simple exhaustion of insulin reserves (which, by immunostaining, appears as a loss of β cell identity), or β cell dedifferentiation, or β cell death. While various sensing and signaling defects can result in diminished insulin secretion, somewhat less attention has been paid to diabetes risk caused by insufficiency in the biosynthetic generation and maintenance of the total insulin granule storage pool. This Review offers an overview of insulin biosynthesis, beginning with the preproinsulin mRNA (translation and translocation into the ER), proinsulin folding and export from the ER, and delivery via the Golgi complex to secretory granules for conversion to insulin and ultimate hormone storage. All of these steps are needed for generation and maintenance of the total insulin granule pool, and defects in any of these steps may, weakly or strongly, perturb glycemic control. The foregoing considerations have obvious potential relevance to the pathogenesis of type 2 diabetes and some forms of monogenic diabetes; conceivably, several of these concepts might also have implications for β cell failure in type 1 diabetes.

Hemophilia A, an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII), is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low-level secretion from mammalian host cells. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress, and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here, we identified intrinsic and extrinsic factors that cause FVIII to form aggregates. We show that FVIII forms amyloid-like fibrils within the ER lumen upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperone families and their cochaperones, immunoglobulin binding protein (BiP) and calnexin/calreticulin, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed β-sheet polymerization, and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein aggregation in general and have implication for ongoing hemophilia A gene-therapy clinical trials., (© 2020 by The American Society of Hematology.)

Activating transcription factor-6 α (ATF6) is one of the three main sensors and effectors of the endoplasmic reticulum (ER) stress response and, as such, it is critical for protecting the heart and other tissues from a variety of environmental insults and disease states. In the heart, ATF6 has been shown to protect cardiac myocytes. However, its roles in other cell types in the heart are unknown. Here we show that ATF6 decreases the activation of cardiac fibroblasts in response to the cytokine, transforming growth factor β (TGFβ), which can induce fibroblast trans-differentiation into a myofibroblast phenotype through signaling via the TGFβ-Smad pathway. ATF6 activation suppressed fibroblast contraction and the induction of α smooth muscle actin (αSMA). Conversely, fibroblasts were hyperactivated when ATF6 was silenced or deleted. ATF6 thus represents a novel inhibitor of the TGFβ-Smad axis of cardiac fibroblast activation., Competing Interests: The authors declare no conflict of interest.

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of ATF6α by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human ATF6α N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-hATF6α deletion variant was cleaved to liberate active transcription activator encompassing GV-hATF6α fragment which could translocate into the nucleus. The translocated GV-hATF6α fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-hATF6α(333) represents an innovative tool to investigate regulated intramembrane proteolysis of ATF6α. It can substitute active pATF6(N) binding motif-based reporter cell lines.

The endoplasmic reticulum ( ER ) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca 2+ -antagonized mechanism, or CaATiER ( Ca 2+ - A ntagonized T ransport i nto ER ). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca 2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca 2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation ( OxPhos ) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an ' anti-Warburg ' effect., Competing Interests: JY, HB, SB, MS, AM, RM, RK No competing interests declared, (© 2019, Yong et al.)

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics.

The IRE1α/XBP1 branch of unfolded protein response (UPR) pathway has a critical function in endoplasmic reticulum (ER) expansion in plasma cells via unknown mechanisms; interestingly, another UPR branch, PERK, is suppressed during plasma cell development. Here we show that Ufbp1, a target and cofactor of the ufmylation pathway, promotes plasma cell development by suppressing the activation of PERK. By contrast, the IRE1α/XBP1 axis upregulates the expression of Ufbp1 and ufmylation pathway genes in plasma cells, while Ufbp1 deficiency impairs ER expansion in plasma cells and retards immunoglobulin production. Structure and function analysis suggests that lysine 267 of Ufbp1, the main lysine in Ufbp1 that undergoes ufmylation, is dispensable for the development of plasmablasts, but is required for immunoglobulin production and stimulation of ER expansion in IRE1α-deficient plasmablasts. Thus, Ufbp1 distinctly regulates different branches of UPR pathway to promote plasma cell development and function.

The endoplasmic reticulum (ER) is a cellular organelle responsible for folding of secretory and membrane proteins. Perturbance in ER homeostasis caused by various intrinsic/extrinsic stimuli challenges the protein-folding capacity of the ER, leading to an ER dysfunction, called ER stress. Cells have developed a defensive response to adapt and/or survive in the face of ER stress that may be detrimental to cell function and survival. When exposed to ER stress, the cell activates a complex and elaborate signaling network that includes translational modulation and transcriptional induction of genes. In addition to these autonomous responses, recent studies suggest that the stressed tissue secretes peptides or unknown factors that transfer the signal to other cells in the same or different organs, leading the organism as a whole to cope with challenges in a non-autonomous manner. In this review, we discuss the mechanisms by which cells adapt to ER stress challenges autonomously and transfer the stress signal to non-stressed cells in different organs.

The endoplasmic reticulum (ER) is a cellular organelle important for regulating calcium homeostasis, lipid metabolism, protein synthesis, and posttranslational modification and trafficking. Numerous environmental, physiological, and pathological insults disturb ER homeostasis, referred to as ER stress, in which a collection of conserved intracellular signaling pathways, termed the unfolded protein response (UPR), are activated to maintain ER function for cell survival. However, excessive and/or prolonged UPR activation leads to initiation of self-destruction through apoptosis. Excessive accumulation of lipids and their intermediate products causes metabolic abnormalities and cell death, called lipotoxicity, in peripheral organs, including the pancreatic islets, liver, muscle, and heart. Because accumulating evidence links chronic ER stress and defects in UPR signaling to lipotoxicity in peripheral tissues, understanding the role of ER stress in cell physiology is a topic under intense investigation. In this review, we highlight recent findings that link ER stress and UPR signaling to the pathogenesis of peripheral organs due to lipotoxicity., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

In eukaryotic cells, the endoplasmic reticulum is essential for the folding and trafficking of proteins that enter the secretory pathway. Environmental insults or increased protein synthesis often lead to protein misfolding in the organelle, the accumulation of misfolded or unfolded proteins - known as endoplasmic reticulum stress - and the activation of the adaptive unfolded protein response to restore homeostasis. If protein misfolding is not resolved, cells die. Endoplasmic reticulum stress and activation of the unfolded protein response help to determine cell fate and function. Furthermore, endoplasmic reticulum stress contributes to the aetiology of many human diseases.

Pancreatic β cells synthesize and secrete insulin to increase anabolic metabolism in an organism, and insulin synthesis has long been suspected to inhibit β cell replication. Recently in Cell Metabolism, Szabat et al. (2015) present evidence that deletion of Insulin genes alleviates ER stress and promotes mature β cell replication., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate NLRP1 gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to NLRP1 promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for NLRP1 induction. This study provides further evidence that links ER stress with innate.

Calcium homeostasis is central to all cellular functions and has been studied for decades. Calcium acts as a critical second messenger for both extracellular and intracellular signaling and is fundamental in cell life and death decisions (Berridge et al., 2000) [1]. The calcium gradient in the cell is coupled with an inherent ability of the divalent cation to reversibly bind multiple target biological molecules to generate an extremely versatile signaling system [2]. Calcium signals are used by the cell to control diverse processes such as development, neurotransmitter release, muscle contraction, metabolism, autophagy and cell death. "Cellular calcium overload" is detrimental to cellular health, resulting in massive activation of proteases and phospholipases leading to cell death (Pinton et al., 2008) [3]. Historically, cell death associated with calcium ion perturbations has been primarily recognized as necrosis. Recent evidence clearly associates changes in calcium ion concentrations with more sophisticated forms of cellular demise, including apoptosis (Kruman et al., 1998; Tombal et al., 1999; Lynch et al., 2000; Orrenius et al., 2003) [4-7]. Although the endoplasmic reticulum (ER) serves as the primary calcium store in the metazoan cell, dynamic calcium release to the cytosol, mitochondria, nuclei and other organelles orchestrate diverse coordinated responses. Most evidence supports that calcium transport from the ER to mitochondria plays a significant role in regulating cellular bioenergetics, production of reactive oxygen species, induction of autophagy and apoptosis. Recently, molecular identities that mediate calcium traffic between the ER and mitochondria have been discovered (Mallilankaraman et al., 2012a; Mallilankaraman et al., 2012b; Sancak et al., 2013)[8-10]. The next questions are how they are regulated for exquisite tight control of ER-mitochondrial calcium dynamics. This review attempts to summarize recent advances in the role of calcium in regulation of ER and mitochondrial function. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau., (Copyright © 2014. Published by Elsevier B.V.)

Lipase maturation factor 1 (Lmf1) is a critical determinant of plasma lipid metabolism, as demonstrated by severe hypertriglyceridemia associated with its mutations in mice and human subjects. Lmf1 is a chaperone localized to the endoplasmic reticulum (ER) and required for the post-translational maturation and activation of several vascular lipases. Despite its importance in plasma lipid homeostasis, the regulation of Lmf1 remains unexplored. We report here that Lmf1 expression is induced by ER stress in various cell lines and in tunicamycin (TM)-injected mice. Using genetic deficiencies in mouse embryonic fibroblasts and mouse liver, we identified the Atf6α arm of the unfolded protein response as being responsible for the up-regulation of Lmf1 in ER stress. Experiments with luciferase reporter constructs indicated that ER stress activates the Lmf1 promoter through a GC-rich DNA sequence 264 bp upstream of the transcriptional start site. We demonstrated that Atf6α is sufficient to induce the Lmf1 promoter in the absence of ER stress, and this effect is mediated by the TM-responsive cis-regulatory element. Conversely, Atf6α deficiency induced by genetic ablation or a dominant-negative form of Atf6α abolished TM stimulation of the Lmf1 promoter. In conclusion, our results indicate that Lmf1 is an unfolded protein response target gene, and Atf6α signaling is sufficient and necessary for activation of the Lmf1 promoter. Importantly, the induction of Lmf1 by ER stress appears to be a general phenomenon not restricted to lipase-expressing cells, which suggests a lipase-independent cellular role for this protein in ER homeostasis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The disruption of the energy or nutrient balance triggers endoplasmic reticulum (ER) stress, a process that mobilizes various strategies, collectively called the unfolded protein response (UPR), which reestablish homeostasis of the ER and cell. Activation of the UPR stress sensor IRE1α (inositol-requiring enzyme 1α) stimulates its endoribonuclease activity, leading to the generation of the mRNA encoding the transcription factor XBP1 (X-box binding protein 1), which regulates the transcription of genes encoding factors involved in controlling the quality and folding of proteins. We found that the activity of IRE1α was regulated by the ER oxidoreductase PDIA6 (protein disulfide isomerase A6) and the microRNA miR-322 in response to disruption of ER Ca2+ homeostasis. PDIA6 interacted with IRE1α and enhanced IRE1α activity as monitored by phosphorylation of IRE1α and XBP1 mRNA splicing, but PDIA6 did not substantially affect the activity of other pathways that mediate responses to ER stress. ER Ca2+ depletion and activation of store-operated Ca2+ entry reduced the abundance of the microRNA miR-322, which increased PDIA6 mRNA stability and, consequently, IRE1α activity during the ER stress response. In vivo experiments with mice and worms showed that the induction of ER stress correlated with decreased miR-322 abundance, increased PDIA6 mRNA abundance, or both. Together, these findings demonstrated that ER Ca2+, PDIA6, IRE1α, and miR-322 function in a dynamic feedback loop modulating the UPR under conditions of disrupted ER Ca2+ homeostasis., (Copyright © 2014, American Association for the Advancement of Science.)

Background: Recent studies link endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) to inflammatory bowel disease. Altered eIF2α phosphorylation (eIF2α-P), a regulatory hub of the UPR, was observed in mucosal tissue of patients with inflammatory bowel disease. In this study, we examined the mechanistic role of eIF2α-P in intestinal epithelial cell (IEC) function and intestinal homeostasis in mice., Methods: We generated mice with villin-Cre-mediated conditional expression of nonphosphorylatable Ser51Ala mutant eIF2α in IECs (AA mice). We analyzed AA mice under normal conditions and on challenge with oral infection of Salmonella Typhimurium or dextran sulfate sodium-induced colitis., Results: Loss of eIF2α-P did not affect the normal proliferation or differentiation of IECs. However, AA mice expressed decreased secretory proteins including lysozyme, suggesting eIF2α-P is required for Paneth cell function. The ultrastructure of AA Paneth cells exhibited a reduced number of secretory granules, a fragmented ER, and distended mitochondria under normal conditions. UPR gene expression was defective in AA IECs. Translation of Paneth cell specific messenger RNAs encoding lysozyme and cryptidins was significantly defective leading to the observed granule-deficient phenotype, which was associated with reduced ribosomal recruitment of these messenger RNAs to the ER membrane. Consequently, AA mice were more susceptible to oral Salmonella infection and dextran sulfate sodium-induced colitis., Conclusions: We conclude eIF2α phosphorylation is required for the normal function of intestinal Paneth cells and mucosal homeostasis by activating UPR signaling and promoting messenger RNA recruitment to the ER membrane for translation.

Purpose of Review: The endoplasmic reticulum (ER) maintains cellular metabolic homeostasis by coordinating protein synthesis, secretion activities, lipid biosynthesis and calcium (Ca²⁺) storage. In this review, we will discuss how altered ER homeostasis contributes to dysregulation of hepatic lipid metabolism and contributes to liver-associated metabolic diseases., Recent Findings: Perturbed ER functions or accumulation of unfolded protein in the ER leads to the activation of the unfolded protein response (UPR) to protect the cell from ER stress. Recent findings pinpoint the key regulatory role of the UPR in hepatic lipid metabolism and demonstrate the potential causal mechanism of ER stress in metabolic dysregulation including diabetes and obesity., Summary: A wide range of factors can alter the protein-folding environment in the ER of hepatocytes and contribute to dysregulation of hepatic lipid metabolism and liver disease. The UPR constitutes a series of adaptive programs that preserve ER protein-folding environment and maintain hepatic lipid homeostasis. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human liver-associated metabolic diseases.

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.

Disulfide bond formation in the endoplasmic reticulum (ER) requires the sequential transfer of electrons from thiol residues to protein disulfide isomerase and ER oxidase 1, with the final reduction of molecular oxygen to form hydrogen peroxide. Conditions that perturb correct protein folding lead to accumulation of misfolded proteins in the ER lumen, which induce ER stress and oxidative stress. Oxidative damage of cellular macromolecules is a common marker of aging and various pathological conditions including diabetes, cancer, and neurodegenerative disease. As accumulating evidence suggests a tight connection between the ER stress and oxidative stress, analysis of appropriate markers becomes particularly important. Here, we describe methods to analyze markers of oxidative damage associated with ER stress., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The unfolded protein response (UPR) is an intracellular stress-signaling pathway that counteracts the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Because defects in ER protein folding are associated with many pathological states, including metabolic, neurologic, genetic, and inflammatory diseases, it is important to understand how the UPR maintains ER protein-folding homeostasis. All metazoans have conserved the fundamental UPR transducers IRE1, ATF6, and PERK. In Caenorhabditis elegans, the UPR is required to prevent larval lethality and intestinal degeneration. Although ire-1-null worms are viable, they are particularly sensitive to ER stress. To identify genes that are required for development of ire-1-null worms, we performed a comprehensive RNA interference screen to find 10 genes that exhibit synthetic growth and intestinal defects with the ire-1(v33) mutant but not with atf-6(tm1153) or pek-1(ok275) mutants. The expression of two of these genes, exos-3 and F48E8.6, was induced by ER stress, and their knockdown in a wild-type strain caused ER stress. Because these genes encode subunits of the exosome complex that functions in mRNA surveillance, we analyzed other gene products required for nonsense-mediated mRNA decay (NMD). Our results demonstrate that defects in smg-1, smg-4, and smg-6 in C. elegans and SMG6 in mammalian cells cause ER stress and sensitize to the lethal effects of ER stress. Although ER stress did not activate mRNA surveillance complex assembly, ER stress did induce SMG6 expression, and NMD regulators were constitutively localized to the ER. Importantly, the findings demonstrate a unique and fundamental interaction where NMD-mediated mRNA quality control is required to prevent ER stress.

Unlabelled: cAMP responsive element-binding protein, hepatocyte specific (CREBH), is a liver-specific transcription factor localized in the endoplasmic reticulum (ER) membrane. Our previous work demonstrated that CREBH is activated by ER stress or inflammatory stimuli to induce an acute-phase hepatic inflammation. Here, we demonstrate that CREBH is a key metabolic regulator of hepatic lipogenesis, fatty acid (FA) oxidation, and lipolysis under metabolic stress. Saturated FA, insulin signals, or an atherogenic high-fat diet can induce CREBH activation in the liver. Under the normal chow diet, CrebH knockout mice display a modest decrease in hepatic lipid contents, but an increase in plasma triglycerides (TGs). After having been fed an atherogenic high-fat (AHF) diet, massive accumulation of hepatic lipid metabolites and significant increase in plasma TG levels were observed in the CrebH knockout mice. Along with the hypertriglyceridemia phenotype, the CrebH null mice displayed significantly reduced body-weight gain, diminished abdominal fat, and increased nonalcoholic steatohepatitis activities under the AHF diet. Gene-expression analysis and chromatin-immunoprecipitation assay indicated that CREBH is required to activate the expression of the genes encoding functions involved in de novo lipogenesis, TG and cholesterol biosynthesis, FA elongation and oxidation, lipolysis, and lipid transport. Supporting the role of CREBH in lipogenesis and lipolysis, forced expression of an activated form of CREBH protein in the liver significantly increases accumulation of hepatic lipids, but reduces plasma TG levels in mice., Conclusion: All together, our study shows that CREBH plays a key role in maintaining lipid homeostasis by regulating the expression of the genes involved in hepatic lipogenesis, FA oxidation, and lipolysis under metabolic stress. The identification of CREBH as a stress-inducible metabolic regulator has important implications in the understanding and treatment of metabolic disease., (Copyright © 2011 American Association for the Study of Liver Diseases.)

The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol-requiring enzyme 1 α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1α-mediated signalling in the liver, we generated hepatocyte-specific Ire1α-null mice by utilizing an albumin promoter-controlled Cre recombinase-mediated deletion. Deletion of Ire1α caused defective induction of genes encoding functions in ER-to-Golgi protein transport, oxidative protein folding, and ER-associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro-apoptotic UPR trans-activators. We show that IRE1α is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte-specific Ire1α deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1α represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer-binding protein (C/EBP) β, C/EBPδ, peroxisome proliferator-activated receptor γ (PPARγ), and enzymes involved in triglyceride biosynthesis. IRE1α was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1α in preventing intracellular lipid accumulation, mice with hepatocyte-specific deletion of Ire1α developed severe hepatic steatosis after treatment with an ER stress-inducing anti-cancer drug Bortezomib, upon expression of a misfolding-prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1α as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

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 splice 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. Herein we discuss each branch of the UPR and how they may impact hepatocyte function in different pathologic states., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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 eIF2alpha (eukaryotic initiation factor 2alpha) 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, eIF2alpha phosphorylation is required for accumulation of XBP1s. We propose that the signalling via phosphorylated eIF2alpha is required for maximum induction of Cat-1 transcription during the UPR by inducing the accumulation of both ATF4 and XBP1s.

Inositol-requiring 1 (IRE1)/X-box-binding protein 1 (XBP1)-mediated signalling 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.

Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin overproduction causes anemia of inflammation, whereas its deficiency leads to hemochromatosis. Inflammation and iron are known extracellular stimuli for hepcidin expression. We found that endoplasmic reticulum (ER) stress also induces hepcidin expression and causes hypoferremia and spleen iron sequestration in mice. CREBH (cyclic AMP response element-binding protein H), an ER stress-activated transcription factor, binds to and transactivates the hepcidin promoter. Hepcidin induction in response to exogenously administered toxins or accumulation of unfolded protein in the ER is defective in CREBH knockout mice, indicating a role for CREBH in ER stress-regulated hepcidin expression. The regulation of hepcidin by ER stress links the intracellular response involved in protein quality control to innate immunity and iron homeostasis.

The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2alpha plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2alpha phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting approximately 90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2alpha, suggesting that this pathway may be broadly utilized by stresses that activate eIF2alpha kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.

The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies is characterized by the accumulation of ordered polymers of mutant neuroserpin within the endoplasmic reticulum of neurones. We show here that intracellular neuroserpin polymers activate NF-kappaB by a pathway that is independent of the IRE1, ATF6, and PERK limbs of the canonical unfolded protein response but is dependent on intracellular calcium. This pathway provides a mechanism for cells to sense and react to the accumulation of folded structures of mutant serpins within the endoplasmic reticulum. Our results provide strong support for the endoplasmic reticulum overload response being independent of the unfolded protein response.

A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6alpha (ATF6alpha), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6alpha drives ER expansion and can do so in the absence of XBP1(S). Overexpression of active ATF6alpha induces PtdCho biosynthesis and modulates the CDP-choline pathway differently than does enforced expression of XBP1(S). These data indicate that ATF6alpha and XBP1(S) have the ability to regulate lipid biosynthesis and ER expansion by mechanisms that are at least partially distinct. These studies reveal further complexity in the potential relationships between UPR pathways, lipid production and ER biogenesis.

The unfolded protein response (UPR) is linked to metabolic dysfunction, yet it is not known how endoplasmic reticulum (ER) disruption might influence metabolic pathways. Using a multilayered genetic approach, we find that mice with genetic ablations of either ER stress-sensing pathways (ATF6alpha, eIF2alpha, IRE1alpha) or of ER quality control (p58(IPK)) share a common dysregulated response to ER stress that includes the development of hepatic microvesicular steatosis. Rescue of ER protein processing capacity by the combined action of UPR pathways during stress prevents the suppression of a subset of metabolic transcription factors that regulate lipid homeostasis. This suppression occurs in part by unresolved ER stress perpetuating expression of the transcriptional repressor CHOP. As a consequence, metabolic gene expression networks are directly responsive to ER homeostasis. These results reveal an unanticipated direct link between ER homeostasis and the transcriptional regulation of metabolism, and suggest mechanisms by which ER stress might underlie fatty liver disease.

Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.

Prolonged accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in ER stress-mediated apoptosis. Cyclophilins are protein chaperones that accelerate the rate of protein folding through their peptidyl-prolyl cis-trans isomerase (PPIase) activity. In this study, we demonstrated that ER stress activates the expression of the ER-localized cyclophilin B (CypB) gene through a novel ER stress response element. Overexpression of wild-type CypB attenuated ER stress-induced cell death, whereas overexpression of an isomerase activity-defective mutant, CypB/R62A, not only increased Ca(2+) leakage from the ER and ROS generation, but also decreased mitochondrial membrane potential, resulting in cell death following exposure to ER stress-inducing agents. siRNA-mediated inhibition of CypB expression rendered cells more vulnerable to ER stress. Finally, CypB interacted with the ER stress-related chaperones, Bip and Grp94. Taken together, we concluded that CypB performs a crucial function in protecting cells against ER stress via its PPIase activity.

Unlabelled: Loss of the nuclear hormone receptor hepatocyte nuclear factor 4alpha (HNF4alpha) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a severe disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4alpha, with expression of 567 genes reduced by > or =2.5-fold (P < or = 0.05) in Hnf4alpha(-/-) fetal livers. Although many of these genes are direct targets, HNF4alpha has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4alpha for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4alpha might reveal roles for HNF4alpha in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4alpha. Analyses of genomic DNA revealed an HNF4alpha binding site upstream of the CrebH coding sequence that was occupied by HNF4alpha in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH(-/-) mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)-stress response, CrebH(-/-) mice displayed reduced expression of acute phase response proteins., Conclusion: These data implicate HNF4alpha in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH.

The biogenesis of endoplasmic reticulum (ER) exit sites (ERES) involves the formation of phosphatidylinositol-4 phosphate (PI4) and Sec16, but it is entirely unknown how ERES adapt to variations in cargo load. Here, we studied acute and chronic adaptive responses of ERES to an increase in cargo load for ER export. The acute response (within minutes) to increased cargo load stimulated ERES fusion events, leading to larger but less ERES. Silencing either PI4-kinase IIIalpha (PI4K-IIIalpha) or Sec16 inhibited the acute response. Overexpression of secretory cargo for 24 h induced the unfolded protein response (UPR), upregulated COPII, and the cells formed more ERES. This chronic response was insensitive to silencing PI4K-IIIalpha, but was abrogated by silencing Sec16. The UPR was required as the chronic response was absent in cells lacking inositol-requiring protein 1. Mathematical model simulations further support the notion that increasing ERES number together with COPII levels is an efficient way to enhance the secretory flux. These results indicate that chronic and acute increases in cargo load are handled differentially by ERES and are regulated by different factors.

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 (PKCtheta) is required for ER stress-induced autophagy. We show that ER stress, in a Ca(2+)-dependent manner, induces PKCtheta 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 PKCtheta block the ER stress-induced autophagic response. PKCtheta activation is not required for autophagy induced by amino acid starvation, and PKCtheta 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.

The endoplasmic reticulum is responsible for much of a cell's protein synthesis and folding, but it also has an important role in sensing cellular stress. Recently, it has been shown that the endoplasmic reticulum mediates a specific set of intracellular signalling pathways in response to the accumulation of unfolded or misfolded proteins, and these pathways are collectively known as the unfolded-protein response. New observations suggest that the unfolded-protein response can initiate inflammation, and the coupling of these responses in specialized cells and tissues is now thought to be fundamental in the pathogenesis of inflammatory diseases. The knowledge gained from this emerging field will aid in the development of therapies for modulating cellular stress and inflammation.

Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.

The endoplasmic reticulum (ER) is the entry site into the secretory pathway for newly synthesized proteins destined for the cell surface or released into the extracellular milieu. The study of protein folding and trafficking within the ER is an extremely active area of research that has provided novel insights into many disease processes. Cells have evolved mechanisms to modulate the capacity and quality of the ER protein-folding machinery to prevent the accumulation of unfolded or misfolded proteins. These signaling pathways are collectively termed the unfolded protein response (UPR). The UPR sensors signal a transcriptional response to expand the ER folding capacity, increase degradation of malfolded proteins, and limit the rate of mRNA translation to reduce the client protein load. Recent genetic and biochemical evidence in both humans and mice supports a requirement for the UPR to preserve ER homeostasis and prevent the beta-cell failure that may be fundamental in the etiology of diabetes. Chronic or overwhelming ER stress stimuli associated with metabolic syndrome can disrupt protein folding in the ER, reduce insulin secretion, invoke oxidative stress, and activate cell death pathways. Therapeutic interventions to prevent polypeptide-misfolding, oxidative damage, and/or UPR-induced cell death have the potential to improve beta-cell function and/or survival in the treatment of diabetes.

The endoplasmic reticulum (ER) is recognized primarily as the site of synthesis and folding of secreted and membrane-bound proteins. The ER provides stringent quality control systems to ensure that only correctly folded, functional proteins are released from the ER and that misfolded proteins are degraded. The efficient functioning of the ER is essential for most cellular activities and for survival. 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. ER transmembrane proteins detect the onset of ER stress and initiate highly specific signaling pathways collectively called the "unfolded protein response" (UPR) to restore normal ER functions. However, if ER homeostasis cannot be reestablished in response to intense or prolonged ER stress, the UPR induces ER stress-associated apoptosis to protect the organism by removing the stressed cells that produce misfolded or malfunctioning proteins. This chapter summarizes current understanding of ER stress-induced apoptosis and reliable methods to examine ER stress and apoptosis in mammalian cells. Since the liver is the major organ dealing with metabolic or pathological stress and is responsible for the detoxification of chemical compounds, the experimental protocols described here focus on identification and characterization of ER stress-induced apoptosis in mouse liver.

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.

The endoplasmic reticulum (ER) is a well-orchestrated protein-folding machine composed of protein chaperones, proteins that catalyze protein folding, and sensors that detect the presence of misfolded or unfolded proteins. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed toward a degradative pathway. The unfolded protein response (UPR) is an intracellular signaling pathway that coordinates ER protein-folding demand with protein-folding capacity and is essential to adapt to homeostatic alterations that cause protein misfolding. These include changes in intraluminal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. The ER provides a unique oxidizing folding-environment that favors the formation of the disulfide bonds. Accumulating evidence suggests that protein folding and generation of reactive oxygen species (ROS) as a byproduct of protein oxidation in the ER are closely linked events. It has also become apparent that activation of the UPR on exposure to oxidative stress is an adaptive mechanism to preserve cell function and survival. Persistent oxidative stress and protein misfolding initiate apoptotic cascades and are now known to play predominant roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis, and neurodegenerative diseases.

Cells respond to the accumulation of unfolded proteins by activating signal transduction cascades that improve protein folding. One example of such a cascade is the unfolded protein response (UPR), which senses protein folding stress in the endoplasmic reticulum (ER) and leads to improvement in the protein folding and processing capacity of the organelle. A central paradox of the UPR, and indeed of all such stress pathways, is that the response is designed to facilitate both adaptation to stress and apoptosis, depending upon the nature and severity of the stressor. Understanding how the UPR can allow for adaptation, instead of apoptosis, is of tremendous physiological importance. Recent advances have improved our understanding of ER stress and the vertebrate UPR, which suggest possible mechanisms by which cells adapt to chronic stress.

In vertebrates, three proteins--PERK, IRE1alpha, and ATF6alpha--sense protein-misfolding stress in the ER and initiate ER-to-nucleus signaling cascades to improve cellular function. The mechanism by which this unfolded protein response (UPR) protects ER function during stress is not clear. To address this issue, we have deleted Atf6alpha in the mouse. ATF6alpha is neither essential for basal expression of ER protein chaperones nor for embryonic or postnatal development. However, ATF6alpha is required in both cells and tissues to optimize protein folding, secretion, and degradation during ER stress and thus to facilitate recovery from acute stress and tolerance to chronic stress. Challenge of Atf6alpha null animals in vivo compromises organ function and survival despite functional overlap between UPR sensors. These results suggest that the vertebrate ATF6alpha pathway evolved to maintain ER function when cells are challenged with chronic stress and provide a rationale for the overlap among the three UPR pathways.

The preemptive quality control (pQC) pathway protects cells from acute endoplasmic reticulum (ER) stress by attenuating translocation of nascent proteins despite their targeting to translocons at the ER membrane. Here, we investigate the hypothesis that the DnaJ protein p58(IPK) plays an essential role in this process via HSP70 recruitment to the cytosolic face of translocons for extraction of translocationally attenuated nascent chains. Our analyses revealed that the heightened stress sensitivity of p58-/- cells was not due to an impairment of the pQC pathway or elevated ER substrate burden during acute stress. Instead, the lesion was in the protein processing capacity of the ER lumen, where p58(IPK) was found to normally reside in association with BiP. ER lumenal p58(IPK) could be coimmunoprecipitated with a newly synthesized secretory protein in vitro and stimulated protein maturation upon overexpression in cells. These results identify a previously unanticipated location for p58(IPK) in the ER lumen where its putative function as a cochaperone explains the stress-sensitivity phenotype of knockout cells and mice.

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) activates the unfolded protein response, also known as the ER stress response. We previously demonstrated that ER stress induces transcription of the ER Golgi intermediate compartment protein ERGIC-53. To investigate the molecular events that regulate unfolded protein response-mediated induction of the gene, we have analyzed the transcriptional regulation of ERGIC-53. We found that the ERGIC-53 promoter contains a single cis-acting element that mediates induction of the gene by thapsigargin and other ER stress-causing agents. This ER stress response element proved to retain a novel structure and to be highly conserved in mammalian ERGIC-53 genes. The ER stress response element identified contains a 5'-end CCAAT sequence that constitutively binds NFY/CBF and, 9 nucleotides away, a 3'-end region (5'-CCCTGTTGGCCATC-3') that is equally important for ER stress-mediated induction of the gene. This sequence is the binding site for endogenous YY1 at the 5'-CCCTGTTGG-3' part and for undefined factors at the CCATC 3'-end. ATF6 alpha-YY1, but not XBP1, interacted with the ERGIC-53 regulatory region and activated ERGIC-53 ER stress response element-dependent transcription. A molecular model for the transcriptional regulation of the ERGIC-53 gene is proposed.

The mammalian translocon-associated protein (TRAP) complex comprises four transmembrane protein subunits in the endoplasmic reticulum. The complex associates with the Sec61 translocon, although its function in vivo remains unknown. Here, we show the involvement of the TRAP complex in endoplasmic reticulum-associated degradation (ERAD). All four subunits are induced simultaneously by endoplasmic reticulum stresses from the X-box-binding protein 1/inositol-requiring 1alpha pathway. RNA interference knockdown of each subunit causes disruption of the native complex and significant delay in the degradation of various ERAD substrates, including the alpha1-antitrypsin null Hong Kong variant (NHK). In a pulse-chase experiment, the TRAP complex associated with NHK at a late stage, indicating its involvement in the ERAD pathway rather than in biosynthesis of nascent polypeptides in the endoplasmic reticulum. In addition, the TRAP complex bound preferentially to misfolded proteins rather than correctly folded wild-type substrates. Thus, the TRAP complex induced by the unfolded protein response pathway might discriminate ERAD substrates from correctly folded substrates, accelerating degradation.

Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc(3)Man(9)GlcNAc(2) (G(3)M(9)Gn(2)) from the lipid-linked oligosaccharide (LLO) glucose(3)mannose(9)N-acetylglucosamine(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) on Ser(51) by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G(3)M(9)Gn(2)-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2alpha Ser(51) phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser(51) kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome., Competing Interests: Competing interests. The authors have declared that no competing interests exist.