Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response

Cells constantly adjust the sizes and shapes of their organelles according to need. In this study, we examine endoplasmic reticulum (ER) membrane expansion during the unfolded protein response (UPR) in the yeast Saccharomyces cerevisiae. We find that membrane expansion occurs through the generation of ER sheets, requires UPR signaling, and is driven by lipid biosynthesis. Uncoupling ER size control and the UPR reveals that membrane expansion alleviates ER stress independently of an increase in ER chaperone levels. Converting the sheets of the expanded ER into tubules by reticulon overexpression does not affect the ability of cells to cope with ER stress, showing that ER size rather than shape is the key factor. Thus, increasing ER size through membrane synthesis is an integral yet distinct part of the cellular program to overcome ER stress.     

Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.     

Impaired Cutinase Secretion in Saccharomyces cerevisiae Induces Irregular Endoplasmic Reticulum (ER) Membrane Proliferation, Oxidative Stress, and ER-Associated Degradation

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.     

The potential contribution of stromal cell-derived factor 2 (SDF2) in endoplasmic reticulum stress response in severe preeclampsia and labor-onset

Endoplasmic reticulum (ER) stress occurs when the protein folding machinery in the cell is unable to cope with newly synthesized proteins, which results in an accumulation of misfolded proteins in the ER lumen. In response, the cell activates a cellular signaling pathway known as the Unfolded Protein Response (UPR), aiming to restore cellular homeostasis. Activation and exacerbation of the UPR have been described in several human pathologies, including cancer and neurological disorders, and in some gestational diseases such as preeclampsia and gestational diabetes. This review explores the participation of stromal cell-derived factor 2 (SDF2) in UPR pathways, shows new information and discusses its exacerbation regarding protein expression in severe preeclampsia and labor, both of which are associated with ER stress.     

Unfolded protein response in chronic obstructive pulmonary disease: smoking, aging and disease: a SAD trifecta

Cigarette smoke (CS) is a risk factor for the development of chronic obstructive pulmonary disease (COPD). Oxidative stress is an immediate result of CS exposure and has the ability to modify cellular proteins. The endoplasmic reticulum (ER) is a compartment where early steps of synthesis and folding of membrane and secretory proteins takes place. Oxidative stress has been shown to interfere with protein folding in the ER and elicits the unfolded protein response (UPR). The UPR is a massive endoplasmic reticulum to the nucleus and the cellular kinase cascades signaling pathway. The UPR triggers a series of intracellular events that aim to help cells overcome the consequences of the stress or eliminate rogue cells by altering expression of genes involved in anti-oxidant defense, cell cycle progression, inflammation, and apoptosis. Recent data demonstrate that CS induces the UPR in vitro and in vivo. The timing of UPR induction in smokers and the mechanism of CS-induced UPR are areas of active investigation. The role of UPR in the protection of smoker's lungs from CS-induced oxidative stress, and its contribution to CS-induced apoptosis and inflammation, is beginning to emerge. This review discusses recent data about UPR in COPD and summarizes findings on UPR that have potential relevance to COPD.     

The Unfolded Protein Response is Triggered in Rat Neurons of the Dorsal Raphe Nucleus After Single-Prolonged Stress

The dorsal raphe nucleus (DRN) has been suggested playing an important role in the pathophysiology of post-traumatic stress disorder (PTSD), however the underlying cellular mechanisms are not fully understood. The endoplasmic reticulum (ER) is a critical organelle for synthesis of membrane and secretory proteins, and perturbations in ER lead to the unfolded protein response (UPR). In the present experiment, we hypothesized UPR may be associated with the PTSD, and there is an induction of UPR in the DRN neurons of the PTSD-like rats. We first observed the morphological changes of ER in the DRN neurons of the rats exposed to single-prolonged stress (SPS), a model of PTSD, and then we also detected the expression of ER chaperones glucose regulated protein 78 (GRP78) and glucose regulated protein (GRP94) which are two key sensors and mediators of the UPR and are considered an ER stress-specific inducible proteins using methods of western blot and immunohistochemical analysis. Our results demonstrated there were abnormal expansion of ER and up-regulation expression of GRP78 and GRP94 after SPS, which indicated that the UPR was triggered in the DRN neurons of the PTSD-like rats. These results are consistent with our speculation that UPR may be associated with the PTSD, and suggest us the UPR may be a new critical cellular mechanisms of PTSD.     

Stress signaling from the endoplasmic reticulum: A central player in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the misfolding and aggregation of distinct proteins in affected tissues, however, the pathogenic cause of disease remains unknown. Recent evidence indicates that endoplasmic reticulum (ER) stress plays a central role in ALS pathogenesis. ER stress activates the unfolded protein response (UPR), a homeostatic response to misfolded proteins. The UPR is initially protective by up-regulation of specific ER stress-regulated genes and inhibition of general protein translation. However, long-term ER stress leads to cell death via apoptotic signaling, thus providing a link to neurodegeneration. Activation of the UPR is one of the earliest events in affected motor neurons of transgenic rodent models expressing ALS-linked mutant superoxide dismutase 1 (SOD1). Recently, genetic manipulation of ER stress in several different SOD1 mouse models was shown to alter disease onset and progression, implicating an active role for the UPR in disease mechanisms. Furthermore, mutations to vesicle-associated membrane protein-associated protein B (VAPB), an ER transmembrane protein involved in ER stress regulation, also cause some cases of familial ALS. ER stress also occurs in spinal cord tissues of human sporadic ALS patients, and recent evidence suggests that perturbation of the ER could occur in ALS cases associated with TAR DNA binding protein 43 (TDP-43), fused in sarcoma (FUS) and valosin containing protein (VCP). Together these findings implicate ER stress as a potential upstream mechanism involved in both familial and sporadic forms of ALS.     

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.     

Proteostasis regulation at the endoplasmic reticulum: a new perturbation site for targeted cancer therapy

To deal with the constant challenge of protein misfolding in the endoplasmic reticulum (ER), eukaryotic cells have evolved an ER protein quality control (ERQC) mechanism that is integrated with an adaptive stress response. The ERQC pathway is comprised of factors residing in the ER lumen that function in the identification and retention of aberrantly folded proteins, factors in the ER membrane for retrotranslocation of misfolded polypeptides, and enzymes in the cytosol that degrade retrotranslocated proteins. The integrated stress response (termed ER stress or unfolded protein response, UPR) contains several signaling branches elicited from the ER membrane, which fine-tune the rate of protein synthesis and entry into the ER to match the ER folding capacity. The fitness of the cell, particularly those bearing a high secretory burden, is critically dependent on functional integrity of the ER, which in turn relies on these stress-attenuating mechanisms to maintain protein homeostasis, or proteostasis. Aberrant proteostasis can trigger cellular apoptosis, making these adaptive stress response systems attractive targets for perturbation in treatment of cell malignancies. Here, we review our current understanding of how the cell preserves ER proteostasis and discuss how we may harness the mechanistic information on this process to develop new cancer therapeutics.     

Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.     

ER stress and the unfolded protein response

Conformational diseases are caused by mutations altering the folding pathway or final conformation of a protein. Many conformational diseases are caused by mutations in secretory proteins and reach from metabolic diseases, e.g. diabetes, to developmental and neurological diseases, e.g. Alzheimer's disease. Expression of mutant proteins disrupts protein folding in the endoplasmic reticulum (ER), causes ER stress, and activates a signaling network called the unfolded protein response (UPR). The UPR increases the biosynthetic capacity of the secretory pathway through upregulation of ER chaperone and foldase expression. In addition, the UPR decreases the biosynthetic burden of the secretory pathway by downregulating expression of genes encoding secreted proteins. Here we review our current understanding of how an unfolded protein signal is generated, sensed, transmitted across the ER membrane, and how downstream events in this stress response are regulated. We propose a model in which the activity of UPR signaling pathways reflects the biosynthetic activity of the ER. We summarize data that shows that this information is integrated into control of cellular events, which were previously not considered to be under control of ER signaling pathways, e.g. execution of differentiation and starvation programs.     

Endoplasmic reticulum stress and lipids in health and diseases

The endoplasmic reticulum (ER) is a complex and dynamic organelle that regulates many cellular pathways, including protein synthesis, protein quality control, and lipid synthesis. When one or multiple ER roles are dysregulated and saturated, the ER enters a stress state, which, in turn, activates the highly conserved unfolded protein response (UPR). By sensing the accumulation of unfolded proteins or lipid bilayer stress (LBS) at the ER, the UPR triggers pathways to restore ER homeostasis and eventually induces apoptosis if the stress remains unresolved. In recent years, it has emerged that the UPR works intimately with other cellular pathways to maintain lipid homeostasis at the ER, and so does at cellular levels. Lipid distribution, along with lipid anabolism and catabolism, are tightly regulated, in part, by the ER. Dysfunctional and overwhelmed lipid-related pathways, independently or in combination with ER stress, can have reciprocal effects on other cellular functions, contributing to the development of diseases. In this review, we summarize the current understanding of the UPR in response to proteotoxic stress and LBS and the breadth of the functions mitigated by the UPR in different tissues and in the context of diseases.     

Effect of the unfolded protein response on ER protein export: a potential new mechanism to relieve ER stress

The unfolded protein response (UPR) is an adaptive cellular response that aims to relieve endoplasmic reticulum (ER) stress via several mechanisms, including inhibition of protein synthesis and enhancement of protein folding and degradation. There is a controversy over the effect of the UPR on ER protein export. While some investigators suggested that ER export is inhibited during ER stress, others suggested the opposite. In this article, their conflicting studies are analyzed and compared in attempt to solve this controversy. The UPR appears indeed to enhance ER export, possibly via multiple mechanisms. However, another factor, which is the integrity of the folding machinery/environment inside ER, determines whether ER export will appear increased or decreased during experimentation. Also, different methods of stress induction appear to have different effects on ER export. Thus, improvement of ER export may represent a new mechanism by which the UPR alleviates ER stress. This may help researchers to understand how the UPR works inside cells and how to manipulate it to alter cell fate during stress, either to promote cell survival or death. This may open up new approaches for the treatment of ER stress-related diseases.     

Cellular responses to endoplasmic reticulum stress and apoptosis

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly—misfolded proteins—are retained in the ER and their accumulation may constitute a form of stress to the cell—ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.     

Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression

The unfolded protein response (UPR) is a cellular recovery mechanism activated by endoplasmic reticulum (ER) stress. The UPR is coordinated with the ER-associated degradation (ERAD) to regulate the protein load at the ER. In the present study, we tested how membrane protein biogenesis is regulated through the UPR in epithelia, using the cystic fibrosis transmembrane conductance regulator (CFTR) as a model. Pharmacological methods such as proteasome inhibition and treatment with brefeldin A and tunicamycin were used to induce ER stress and activate the UPR as monitored by increased levels of spliced XBP1 and BiP mRNA. The results indicate that activation of the UPR is followed by a significant decrease in genomic CFTR mRNA levels without significant changes in the mRNA levels of another membrane protein, the transferrin receptor. We also tested whether overexpression of a wild-type CFTR transgene in epithelia expressing endogenous wild-type CFTR activated the UPR. Although CFTR maturation is inefficient in this setting, the UPR was not activated. However, pharmacological induction of ER stress in these cells also led to decreased endogenous CFTR mRNA levels without affecting recombinant CFTR message levels. These results demonstrate that under ER stress conditions, endogenous CFTR biogenesis is regulated by the UPR through alterations in mRNA levels and posttranslationally by ERAD, whereas recombinant CFTR expression is regulated only by ERAD. endoplasmic reticulum-associated degradation; messenger ribonucleic acid     

HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response

Background

The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance.

Methods and Findings

The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2–5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells.

Conclusions

Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.     

Mediators of endoplasmic reticulum stress-induced apoptosis

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.