Endoplasmic Reticulum Enrollment in Alzheimer’s Disease

Alzheimer??s disease (AD) poses a huge challenge for society and health care worldwide as molecular pathogenesis of the disease is poorly understood and curative treatment does not exist. The mechanisms leading to accelerated neuronal cell death in AD are still largely unknown, but accumulation of misfolded disease-specific proteins has been identified as potentially involved. In the present review, we describe the essential role of endoplasmic reticulum (ER) in AD. Despite the function that mitochondria may play as the central major player in the apoptotic process, accumulating evidence highlights ER as a critical organelle in AD. Stress that impairs ER physiology leads to accumulation of unfolded or misfolded proteins, such as amyloid ?? (A??) peptide, the major component of amyloid plaques. In an attempt to ameliorate the accumulation of unfolded proteins, ER stress triggers a protective cellular mechanism, which includes the unfolded protein response (UPR). However, when activation of the UPR is severe or prolonged enough, the final cellular outcome is pathologic apoptotic cell death. Distinct pathways can be activated in this process, involving stress sensors such as the JNK pathway or ER chaperones such as Bip/GRP94, stress modulators such as Bcl-2 family proteins, or even stress effectors such as caspase-12. Here, we detail the involvement of the ER and associated stress pathways in AD and discuss potential therapeutic strategies targeting ER stress.     

The Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle–mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERES.     

Endoplasmic Reticulum Oxidoreductin-1α (Ero1α) Improves Folding and Secretion of Mutant Proinsulin and Limits Mutant Proinsulin-induced Endoplasmic Reticulum Stress

Upon chronic up-regulation of proinsulin synthesis, misfolded proinsulin can accumulate in the endoplasmic reticulum (ER) of pancreatic β-cells, promoting ER stress and type 2 diabetes mellitus. In Mutant Ins-gene-induced Diabetes of Youth (MIDY), misfolded mutant proinsulin impairs ER exit of co-expressed wild-type proinsulin, limiting insulin production and leading to eventual β-cell death. In this study we have investigated the hypothesis that increased expression of ER oxidoreductin-1α (Ero1α), despite its established role in the generation of H₂O₂, might nevertheless be beneficial in limiting proinsulin misfolding and its adverse downstream consequences. Increased Ero1α expression is effective in promoting wild-type proinsulin export from cells co-expressing misfolded mutant proinsulin. In addition, we find that upon increased Ero1α expression, some of the MIDY mutants themselves are directly rescued from ER retention. Secretory rescue of proinsulin-G(B23)V is correlated with improved oxidative folding of mutant proinsulin. Indeed, using three different variants of Ero1α, we find that expression of either wild-type or an Ero1α variant lacking regulatory disulfides can rescue mutant proinsulin-G(B23)V, in parallel with its ability to provide an oxidizing environment in the ER lumen, whereas beneficial effects were less apparent for a redox-inactive form of Ero1. Increased expression of protein disulfide isomerase antagonizes the rescue provided by oxidatively active Ero1. Importantly, ER stress induced by misfolded proinsulin was limited by increased expression of Ero1α, suggesting that enhancing the oxidative folding of proinsulin may be a viable therapeutic strategy in the treatment of type 2 diabetes.     

Switch Between Apoptosis and Autophagy: Radiation-Induced Endoplasmic Reticulum Stress?

The induction of cell death by radiation has largely been attributed to pro-apoptotic mechanisms. Autophagy, an alternative form of programmed cell death, has recently been shown to contribute significantly to anti-neoplastic effects of radiation therapy. In light of this, ER stress has been shown to trigger both apoptosis and autophagy, and act as an important mediator linking the two programmed cell death pathways. Recent data reveal that ER stress leads to activation of autophagosome formation with LC3 conversion via either PERK-eIF2α pathway or IRE1-JNK pathway. In this focused review, we summarize the main molecular mediators that control cellular “switches” between apoptosis and autophagy pathways by utilizing radiation therapy as a model.     

Endoplasmic Reticulum Stress Stimulates p53 Expression through NF-κB Activation

Background

Induction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.

Principal Findings

In this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.

Significance

Taken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.     

��-Helical Domains Promote Translocation of Intrinsically Disordered Polypeptides into the Endoplasmic Reticulum

Co-translational import into the endoplasmic reticulum (ER) is primarily controlled by N-terminal signal sequences that mediate targeting of the ribosome-nascent chain complex to the Sec61/translocon and initiate the translocation process. Here we show that after targeting to the translocon the secondary structure of the nascent polypeptide chain can significantly modulate translocation efficiency. ER-targeted polypeptides dominated by unstructured domains failed to efficiently translocate into the ER lumen and were subjected to proteasomal degradation via a co-translocational/preemptive pathway. Productive ER import could be reinstated by increasing the amount of α-helical domains, whereas more effective ER signal sequences had only a minor effect on ER import efficiency of unstructured polypeptides. ER stress and overexpression of p58^IPK promoted the co-translocational degradation pathway. Moreover polypeptides with unstructured domains at their N terminus were specifically targeted to proteasomal degradation under these conditions. Our study indicates that extended unstructured domains are signals to dispose ER-targeted proteins via a co-translocational, preemptive quality control pathway.To ensure cellular homeostasis and to preclude toxic effects of aberrant protein conformers quality control mechanisms have evolved to recognize and degrade non-functional and misfolded proteins. In the cytosol the ubiquitin-proteasome system is the main pathway for regulated protein turnover (for reviews, see Refs. 1–3). Moreover the proteasome is part of a quality control system designated endoplasmic reticulum (ER)⁵-associated degradation (ERAD), which mediates post-translational degradation of non-native proteins generated in the ER. ERAD is the primary response to eliminate non-native ER proteins. It involves recognition of non-native polypeptides by ER-resident proteins and retrograde transport to the cytosol where proteasomal degradation occurs (for reviews, see Refs. 4–6). In case ERAD substrates accumulate in the ER lumen intracellular signaling pathways are induced that are collectively called the unfolded protein response (for reviews, see Refs. 7 and 8). Recently a preemptive, co-translocational quality control pathway was described that operates before translocation into the ER is completed (9, 10). Regulated translocation could act as an early quality control step to prevent an overload of the ER with non-native proteins. This regulation relies on an interplay between intrinsic features of the polypeptides and accessory factors able to modulate the translocation efficiency (for a review, see Ref. 11). Although numerous factors are known to be involved in ERAD less is known about mediators of the preemptive, co-translocational quality control pathway. In one study p58^IPK was identified as a key regulator (9), whereas in another it was shown that ER translocation during conditions of acute ER stress is controlled by the signal peptide (10). Indeed the signal peptide is an important intrinsic determinant. Although an exceptionally diverse set of sequences can target proteins for ER import it has been demonstrated that the translocation efficiency is modulated in a signal peptide sequence-specific manner (for reviews, see Refs. 12–14). Another attractive candidate for an intrinsic factor to regulate translocation is the folding state of the nascent polypeptide chain. Formation of secondary structure occurs already in the ribosome exit tunnel (15–18). Moreover it was shown that the polypeptide structure within the ribosomal exit tunnel can modulate translocation of distal parts of the nascent chain (19).The mammalian prion protein (PrP) is a suitable model protein to study whether formation of secondary structure could modulate translocation efficiency because it has an intriguing modular composition: the N-terminal domain spanning 120 amino acids is flexibly disordered followed by a highly structured C-terminal domain of ∼110 amino acids. This autonomously folding domain contains three α-helical regions and a short, two-stranded β-sheet (20–22). Notably folding of the C-terminal domain is one of the most rapid folding reactions measured in vitro (23). Interestingly previous studies indicated that the C-terminal folded domain of PrP is necessary and sufficient to promote ER import. In cultured cells and neurons of transgenic animals PrP-S230X (also known as PrPΔGPI or GPI⁻PrP), a mutant devoid of the C-terminal glycosylphosphatidylinositol (GPI) anchor signal peptide, is efficiently imported into the ER and secreted (24–27). However, by deleting parts of the α-helical domains located in the C-terminal domain a fraction is directed to proteasomal degradation in the cytosol (28).We have now analyzed the underlying mechanisms of this impaired ER import and show that ER-targeted proteins require a certain amount of structured domains to be imported into the ER. In addition, our study indicates that extended unstructured domains are signals for a preemptive/co-translocational degradation pathway.     

ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway

Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.     

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.     

Selenoprotein S/SEPS1 Modifies Endoplasmic Reticulum Stress in Z Variant ��1-Antitrypsin Deficiency

Z α₁-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFκB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFκB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFκB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.SEPS1 (selenoprotein S, VIMP, Tanis, or SelS) is a selenoprotein found in the endoplasmic reticulum (ER)³ membrane. SEPS1 participates in the processing and removal of misfolded proteins from the ER to the cytosol, where they are polyubiquitinated and degraded through the proteasome (1). SEPS1 can be induced by ER stress (2) and has been shown in macrophages to be protective from pharmacological ER stress agent-induced apoptosis (3).The endoplasmic reticulum is one of the largest cell organelles. It serves many essential functions, including production of all components of cellular membranes, proteins, lipids, and sterols (4). Only correctly folded proteins are transported out of the ER, whereas incompletely folded proteins are retained in the organelle to complete the folding process or be targeted for destruction. ER stress is defined as an imbalance between the cellular demand for ER function and capacity of the organelle. It is characterized by a number of intracellular responses. These responses include the ER overload response (EOR), the unfolded protein response (UPR), and apoptosis.α₁-Antitrypsin (AAT) deficiency is a disease characterized by early onset emphysema and liver disease (5). The mutant Z form of this autosomal co-dominant disease occurs in >95% of all individuals with AAT deficiency (6). Liver disease occurs in ∼10% of all homozygous neonates who develop hepatitis and cholestasis. A proportion of these children progress to liver failure, requiring liver transplantation (7, 8). Cirrhosis can also occur in adults without a preceding history of childhood liver disease. The mutant Z AAT polymerizes and accumulates in the ER, leaving only 15% of ZAAT secreted (9, 10). This accumulation of abnormal protein in the ER gives rise to ER stress, which is believed to contribute to the liver disease that results from AAT deficiency. The cells respond to this perturbation by inducing the expression of novel genes whose products aim to restore normal ER function (11). SEPS1 is an example of a molecular chaperone that serves to augment the capacity of the ER for protein folding and degradation.In this paper, we investigate the role of SEPS1 in regulating the cellular response to ER stress in HepG2 cells transfected with the ZAAT transgene. We investigate the effect of SEPS1 on the UPR component of this response by measuring grp78 promoter activity, a UPR-up-regulated gene that functions as a molecular chaperone, and by detecting activated ATF6, which occurs downstream to the activation of grp78. The EOR component of ER stress is investigated by measuring NFκB activation.We study the effect of selenium supplementation on the action of SEPS1 to see if the function of this selenoprotein can be enhanced and, if so, through which pathways, looking specifically at grp78 promoter activity, ATF6 activation, and NFκB activation and also at glutathione peroxidase (GPx) activity and at the anti-inflammatory 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) pathway. GPx is a selenoprotein whose activity can be readily assayed. This was used as a measure of selenoprotein activity.The role of SEPS1 in conformational diseases has not been evaluated. These diseases are caused by inherited or acquired modifications in protein structure, where specific proteins undergo a conformational rearrangement, causing deposition within cellular compartments, such as the ER. This can lead to devastating results. AAT deficiency is one such disease, but the group includes Alzheimer, Parkinson, and Huntington diseases and cystic fibrosis.     

Multiple Mechanism–Mediated Retention of a Defective Brassinosteroid Receptor in the Endoplasmic Reticulum of Arabidopsis

Endoplasmic reticulum–mediated quality control (ERQC) is a well-studied process in yeast and mammals that retains and disposes misfolded/unassembled polypeptides. By contrast, how plants exert quality control over their secretory proteins is less clear. Here, we report that a mutated brassinosteroid receptor, bri1-5, that carries a Cys69Tyr mutation, is retained in the ER by an overvigilant ERQC system involving three different retention mechanisms. We demonstrate that bri1-5 interacts with two ER chaperones, calnexin and binding protein (BiP), and is degraded by a proteasome-independent endoplasmic reticulum–associated degradation (ERAD). Mutations in components of the calnexin/calreticulin cycle had little effect on the fidelity of the Arabidopsis thaliana ERQC for bri1-5 retention. By contrast, overexpression of bri1-5, treatment with an ERAD inhibitor, RNA interference–mediated BiP silencing, or simultaneous mutations of Cys-69 and its partner Cys-62 can mitigate this quality control, resulting in significant suppression of the bri1-5 phenotype. Thus, bri1-5 is an excellent model protein to investigate plant ERQC/ERAD in a model organism.     

Glucolipotoxicity Impairs Ceramide Flow from the Endoplasmic Reticulum to the Golgi Apparatus in INS-1 β-Cells

Accumulating evidence suggests that glucolipotoxicity, arising from the combined actions of elevated glucose and free fatty acid levels, acts as a key pathogenic component in type II diabetes, contributing to β-cell dysfunction and death. Endoplasmic reticulum (ER) stress is among the molecular pathways and regulators involved in these negative effects, and ceramide accumulation due to glucolipotoxicity can be associated with the induction of ER stress. Increased levels of ceramide in ER may be due to enhanced ceramide biosynthesis and/or decreased ceramide utilization. Here, we studied the effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells in order to gain insights into the molecular mechanism(s) of glucolipotoxicity. We showed that glucolipotoxicity inhibited ceramide utilization for complex sphingolipid biosynthesis, thereby reducing the flow of ceramide from the ER to Golgi. Glucolipotoxicity impaired both vesicular- and CERT-mediated ceramide transport through (1) the decreasing of phospho-Akt levels which in turn possibly inhibits vesicular traffic, and (2) the reducing of the amount of active CERT mainly due to a lower protein levels and increased protein phosphorylation to prevent its localization to the Golgi. In conclusion, our findings provide evidence that glucolipotoxicity-induced ceramide overload in the ER, arising from a defect in ceramide trafficking may be a mechanism that contributes to dysfunction and/or death of β-cells exposed to glucolipotoxicity.     

C/EBP-β Regulates Endoplasmic Reticulum Stress–Triggered Cell Death in Mouse and Human Models

Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-® variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-® regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death.     

Extracellular α-Synuclein Modulates Iron Metabolism Related Proteins via Endoplasmic Reticulum Stress in MES23.5 Dopaminergic Cells

Alpha-synuclein plays a vital role in the pathology of Parkinson’s disease (PD). Spreading of α-synuclein in neighboring cells was believed to contribute to progression in PD. How α-synuclein transmission affects adjacent cells is not full elucidated. Here, we used recombinant α-synuclein to mimic intercellular transmitted α-synuclein in MES23.5 dopaminergic cells, to investigate whether and how it could modulate iron metabolism. The results showed that α-synuclein treatment up-regulated divalent metal transporter 1 (DMT1) and down-regulated iron transporter (FPN), also up-regulated iron regulatory protein 1 (IRP1) protein levels and hepcidin mRNA levels. Endocytosis inhibitor dynasore pretreatment completely abolished and even reversed the upregulation of DMT1 and IRP1 induced by α-synuclein, however, FPN down-regulation was partially blocked by dynasore. Autophagy-inducing agent rapamycin reversed DMT1 up-regulation and FPN down-regulation, and fully blocked the upregulation of IRP1. Elevated hepcidin levels induced by α-synuclein was fully blocked by dynasore pretreatment, however, even higher with rapamycin pretreatment. Alpha-synuclein treatment triggered endoplasmic reticulum (ER) stress. ER stress inducer thapsigargin induced similar responses elicited by α-synuclein. ER stress inhibitor salubrinal blocked the up-regulation of IRP1 and hepcidin, as well as DMT1 up-regulation and FPN down-regulation, also dramatically abolished cAMP-response elements binding protein phosphorylation induced by α-synuclein. Taken together, these finding indicated that extracellular α-synuclein could regulate cellular iron metabolism, probably mediated by ER stress. It provides novel evidence to elucidate the relationships between transmitted α-synuclein and iron metabolism disturbance in PD.