Sample records for accompanying endoplasmic reticulum

  1. Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy

    PubMed Central

    Montague, Karli; Malik, Bilal; Gray, Anna L.; La Spada, Albert R.; Hanna, Michael G.; Szabadkai, Gyorgy

    2014-01-01

    Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington’s disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases. PMID:24898351

  2. CaMKII-dependent endoplasmic reticulum fission by whisker stimulation and during cortical spreading depolarization.

    PubMed

    Kucharz, Krzysztof; Lauritzen, Martin

    2018-04-01

    present in dendrites and spines, and was preceded by dramatic rise in intracellular Ca2+. The endoplasmic reticulum fission during spreading depolarization was more persistent, as 1 h after the depolarization cortical neurons still exhibited loss of endoplasmic reticulum continuity. Notably, endoplasmic reticulum fission was accompanied with loss of electrocorticographic activity, whereas subsequent regain of synaptic function paralleled the organelle fusion. Furthermore, blocking CaMKII activity partly rescued endoplasmic reticulum fission and markedly shortened the recovery time of brain spontaneous activity. Thus, prevention of endoplasmic reticulum fission with CaMKII inhibitors may be a novel strategy to rescue brain function in patients with migraine and a promising therapeutic avenue in the acutely injured brain.

  3. Endoplasmic reticulum stress implicated in chronic traumatic encephalopathy.

    PubMed

    Lucke-Wold, Brandon P; Turner, Ryan C; Logsdon, Aric F; Nguyen, Linda; Bailes, Julian E; Lee, John M; Robson, Matthew J; Omalu, Bennet I; Huber, Jason D; Rosen, Charles L

    2016-03-01

    Chronic traumatic encephalopathy is a progressive neurodegenerative disease characterized by neurofibrillary tau tangles following repetitive neurotrauma. The underlying mechanism linking traumatic brain injury to chronic traumatic encephalopathy has not been elucidated. The authors investigate the role of endoplasmic reticulum stress as a link between acute neurotrauma and chronic neurodegeneration. The authors used pharmacological, biochemical, and behavioral tools to assess the role of endoplasmic reticulum stress in linking acute repetitive traumatic brain injury to the development of chronic neurodegeneration. Data from the authors' clinically relevant and validated rodent blast model were compared with those obtained from postmortem human chronic traumatic encephalopathy specimens from a National Football League player and World Wrestling Entertainment wrestler. The results demonstrated strong correlation of endoplasmic reticulum stress activation with subsequent tau hyperphosphorylation. Various endoplasmic reticulum stress markers were increased in human chronic traumatic encephalopathy specimens, and the endoplasmic reticulum stress response was associated with an increase in the tau kinase, glycogen synthase kinase-3β. Docosahexaenoic acid, an endoplasmic reticulum stress inhibitor, improved cognitive performance in the rat model 3 weeks after repetitive blast exposure. The data showed that docosahexaenoic acid administration substantially reduced tau hyperphosphorylation (t = 4.111, p < 0.05), improved cognition (t = 6.532, p < 0.001), and inhibited C/EBP homology protein activation (t = 5.631, p < 0.01). Additionally the data showed, for the first time, that endoplasmic reticulum stress is involved in the pathophysiology of chronic traumatic encephalopathy. Docosahexaenoic acid therefore warrants further investigation as a potential therapeutic agent for the prevention of chronic traumatic encephalopathy.

  4. The role of the endoplasmic reticulum stress response following cerebral ischemia.

    PubMed

    Hadley, Gina; Neuhaus, Ain A; Couch, Yvonne; Beard, Daniel J; Adriaanse, Bryan A; Vekrellis, Kostas; DeLuca, Gabriele C; Papadakis, Michalis; Sutherland, Brad A; Buchan, Alastair M

    2018-06-01

    Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

  5. Activation of autophagy by unfolded proteins during endoplasmic reticulum stress.

    PubMed

    Yang, Xiaochen; Srivastava, Renu; Howell, Stephen H; Bassham, Diane C

    2016-01-01

    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  6. Cell death induced by endoplasmic reticulum stress.

    PubMed

    Iurlaro, Raffaella; Muñoz-Pinedo, Cristina

    2016-07-01

    The endoplasmic reticulum is an organelle with multiple functions. The synthesis of transmembrane proteins and proteins that are to be secreted occurs in this organelle. Many conditions that impose stress on cells, including hypoxia, starvation, infections and changes in secretory needs, challenge the folding capacity of the cell and promote endoplasmic reticulum stress. The cellular response involves the activation of sensors that transduce signaling cascades with the aim of restoring homeostasis. This is known as the unfolded protein response, which also intersects with the integrated stress response that reduces protein synthesis through inactivation of the initiation factor eIF2α. Central to the unfolded protein response are the sensors PERK, IRE1 and ATF6, as well as other signaling nodes such as c-Jun N-terminal kinase 1 (JNK) and the downstream transcription factors XBP1, ATF4 and CHOP. These proteins aim to restore homeostasis, but they can also induce cell death, which has been shown to occur by necroptosis and, more commonly, through the regulation of Bcl-2 family proteins (Bim, Noxa and Puma) that leads to mitochondrial apoptosis. In addition, endoplasmic reticulum stress and proteotoxic stress have been shown to induce TRAIL receptors and activation of caspase-8. Endoplasmic reticulum stress is a common feature in the pathology of numerous diseases because it plays a role in neurodegeneration, stroke, cancer, metabolic diseases and inflammation. Understanding how cells react to endoplasmic reticulum stress can accelerate discovery of drugs against these diseases. © 2015 FEBS.

  7. Gating Behavior of Endoplasmic Reticulum Potassium Channels of Rat Hepatocytes in Diabetes

    PubMed Central

    Ghasemi, Maedeh; Khodaei, Naser; Salari, Sajjad; Eliassi, Afsaneh; Saghiri, Reza

    2014-01-01

    Background: Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. Method: Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. Results: Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. Conclusion: We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans. PMID:24842143

  8. Gating behavior of endoplasmic reticulum potassium channels of rat hepatocytes in diabetes.

    PubMed

    Ghasemi, Maedeh; Khodaei, Naser; Salari, Sajjad; Eliassi, Afsaneh; Saghiri, Reza

    2014-07-01

    Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans.

  9. Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis.

    PubMed

    Hughes, Alexandria; Oxford, Alexandra E; Tawara, Ken; Jorcyk, Cheryl L; Oxford, Julia Thom

    2017-03-20

    Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.

  10. Endoplasmic Reticulum Stress in Ischemic and Nephrotoxic Acute Kidney Injury.

    PubMed

    Yan, Mingjuan; Shu, Shaoqun; Guo, Chunyuan; Tang, Chengyuan; Dong, Zheng

    2018-06-12

    Acute kidney injury is a medical condition characterized by kidney damage with a rapid decline of renal function, which is associated with high mortality and morbidity. Recent research has further established an intimate relationship between acute kidney injury and chronic kidney disease. Perturbations of kidney cells in acute kidney injury result in the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum, leading to unfolded protein response or endoplasmic reticulum stress. In this review, we analyze the role and regulation of endoplasmic reticulum stress in acute kidney injury triggered by renal ischemia-reperfusion and cisplatin nephrotoxicity. The balance between the two major components of unfolded protein response, the adaptive pathway and the apoptotic pathway, plays a critical role in determining the cell fate in endoplasmic reticulum stress. The adaptive pathway is evoked to attenuate translation, induce chaperones, maintain protein homeostasis, and promote cell survival. Prolonged endoplasmic reticulum stress activates the apoptotic pathway, resulting in the elimination of dysfunctional cells. Therefore, regulating ER stress in kidney cells may provide a therapeutic target in acute kidney injury.

  11. Prediction of endoplasmic reticulum resident proteins using fragmented amino acid composition and support vector machine.

    PubMed

    Kumar, Ravindra; Kumari, Bandana; Kumar, Manish

    2017-01-01

    The endoplasmic reticulum plays an important role in many cellular processes, which includes protein synthesis, folding and post-translational processing of newly synthesized proteins. It is also the site for quality control of misfolded proteins and entry point of extracellular proteins to the secretory pathway. Hence at any given point of time, endoplasmic reticulum contains two different cohorts of proteins, (i) proteins involved in endoplasmic reticulum-specific function, which reside in the lumen of the endoplasmic reticulum, called as endoplasmic reticulum resident proteins and (ii) proteins which are in process of moving to the extracellular space. Thus, endoplasmic reticulum resident proteins must somehow be distinguished from newly synthesized secretory proteins, which pass through the endoplasmic reticulum on their way out of the cell. Approximately only 50% of the proteins used in this study as training data had endoplasmic reticulum retention signal, which shows that these signals are not essentially present in all endoplasmic reticulum resident proteins. This also strongly indicates the role of additional factors in retention of endoplasmic reticulum-specific proteins inside the endoplasmic reticulum. This is a support vector machine based method, where we had used different forms of protein features as inputs for support vector machine to develop the prediction models. During training leave-one-out approach of cross-validation was used. Maximum performance was obtained with a combination of amino acid compositions of different part of proteins. In this study, we have reported a novel support vector machine based method for predicting endoplasmic reticulum resident proteins, named as ERPred. During training we achieved a maximum accuracy of 81.42% with leave-one-out approach of cross-validation. When evaluated on independent dataset, ERPred did prediction with sensitivity of 72.31% and specificity of 83.69%. We have also annotated six different

  12. Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

    PubMed Central

    Cheng, Yu-Chi; Chang, Jer-Ming; Chen, Chien-An

    2015-01-01

    Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage. PMID:25322957

  13. Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

    PubMed

    Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

    2014-10-01

    Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

  14. Endoplasmic reticulum stress and proteasomal system in amyotrophic lateral sclerosis.

    PubMed

    Karademir, Betul; Corek, Ceyda; Ozer, Nesrin Kartal

    2015-11-01

    Protein processing including folding, unfolding and degradation is involved in the mechanisms of many diseases. Unfolded protein response and/or endoplasmic reticulum stress are accepted to be the first steps which should be completed via protein degradation. In this direction, proteasomal system and autophagy play important role as the degradation pathways and controlled via complex mechanisms. Amyotrophic lateral sclerosis is a multifactorial neurodegenerative disease which is also known as the most catastrophic one. Mutation of many different genes are involved in the pathogenesis such as superoxide dismutase 1, chromosome 9 open reading frame 72 and ubiquilin 2. These genes are mainly related to the antioxidant defense systems, endoplasmic reticulum stress related proteins and also protein aggregation, degradation pathways and therefore mutation of these genes cause related disorders.This review focused on the role of protein processing via endoplasmic reticulum and proteasomal system in amyotrophic lateral sclerosis which are the main players in the pathology. In this direction, dysfunction of endoplasmic reticulum associated degradation and related cell death mechanisms that are autophagy/apoptosis have been detailed. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

    PubMed

    Chanat, Eric; Le Parc, Annabelle; Lahouassa, Hichem; Badaoui, Bouabid

    2016-06-01

    In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

  16. Penfluridol induces endoplasmic reticulum stress leading to autophagy in pancreatic cancer.

    PubMed

    Ranjan, Alok; German, Nadezhda; Mikelis, Constantinos; Srivenugopal, Kalkunte; Srivastava, Sanjay K

    2017-06-01

    Pancreatic cancer is one of the most aggressive and difficult to treat cancers. Experimental and clinical evidence suggests that high basal state autophagy in pancreatic tumors could induce resistance to chemotherapy. Recently, we have demonstrated that penfluridol suppresses pancreatic tumor growth by autophagy-mediated apoptosis both in vitro and in vivo; however, the mechanism of autophagy induction by penfluridol was not clear. Several studies have established that endoplasmic reticulum stress could lead to autophagy and inhibit tumor progression. In this study, we demonstrated that penfluridol induced endoplasmic reticulum stress in BxPC-3, AsPC-1, and Panc-1 pancreatic cancer cell lines as indicated by upregulation of endoplasmic reticulum stress markers such as binding protein (BIP), C/EBP homologous protein (CHOP) and inositol requiring 1α (IRE1α) after treatment with penfluridol in a concentration-dependent manner. Inhibiting endoplasmic reticulum stress by pretreatment with pharmacological inhibitors such as sodium phenylbutyrate and mithramycin or by silencing CHOP using CHOP small interfering RNA, blocked penfluridol-induced autophagy. These results clearly indicate that penfluridol-induced endoplasmic reticulum stress lead to autophagy in our model. Western blot analysis of subcutaneously implanted AsPC-1 and BxPC-3 tumors as well as orthotopically implanted Panc-1 tumors demonstrated upregulation of BIP, CHOP, and IRE1α expression in the tumor lysates from penfluridol-treated mice as compared to tumors from control mice. Altogether, our study establishes that penfluridol-induced endoplasmic reticulum stress leads to autophagy resulting in reduced pancreatic tumor growth. Our study opens a new therapeutic target for advanced chemotherapies against pancreatic cancer.

  17. Organization of transport from endoplasmic reticulum to Golgi in higher plants.

    PubMed

    Andreeva, A V; Zheng, H; Saint-Jore, C M; Kutuzov, M A; Evans, D E; Hawes, C R

    2000-01-01

    In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endomembrane trafficking in plant cells.

  18. Observation of endoplasmic reticulum tubules via TOF-SIMS tandem mass spectrometry imaging of transfected cells.

    PubMed

    Chini, Corryn E; Fisher, Gregory L; Johnson, Ben; Tamkun, Michael M; Kraft, Mary L

    2018-02-26

    Advances in three-dimensional secondary ion mass spectrometry (SIMS) imaging have enabled visualizing the subcellular distributions of various lipid species within individual cells. However, the difficulty of locating organelles using SIMS limits efforts to study their lipid compositions. Here, the authors have assessed whether endoplasmic reticulum (ER)-Tracker Blue White DPX ® , which is a commercially available stain for visualizing the endoplasmic reticulum using fluorescence microscopy, produces distinctive ions that can be used to locate the endoplasmic reticulum using SIMS. Time-of-flight-SIMS tandem mass spectrometry (MS 2 ) imaging was used to identify positively and negatively charged ions produced by the ER-Tracker stain. Then, these ions were used to localize the stain and thus the endoplasmic reticulum, within individual human embryonic kidney cells that contained higher numbers of endoplasmic reticulum-plasma membrane junctions on their surfaces. By performing MS 2 imaging of selected ions in parallel with the precursor ion (MS 1 ) imaging, the authors detected a chemical interference native to the cell at the same nominal mass as the pentafluorophenyl fragment from the ER-Tracker stain. Nonetheless, the fluorine secondary ions produced by the ER-Tracker stain provided a distinctive signal that enabled locating the endoplasmic reticulum using SIMS. This simple strategy for visualizing the endoplasmic reticulum in individual cells using SIMS could be combined with existing SIMS methodologies for imaging intracellular lipid distribution and to study the lipid composition within the endoplasmic reticulum.

  19. Endoplasmic Reticulum Stress and Associated ROS

    PubMed Central

    Zeeshan, Hafiz Maher Ali; Lee, Geum Hwa; Kim, Hyung-Ryong; Chae, Han-Jung

    2016-01-01

    The endoplasmic reticulum (ER) is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS). Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI)-endoplasmic reticulum oxidoreductin (ERO)-1, glutathione (GSH)/glutathione disuphide (GSSG), NADPH oxidase 4 (Nox4), NADPH-P450 reductase (NPR), and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases. PMID:26950115

  20. The shape of things to come: regulation of shape changes in endoplasmic reticulum.

    PubMed

    Paiement, J; Bergeron, J

    2001-01-01

    Shape changes in the endoplasmic reticulum control fundamental cell processes including nuclear envelope assembly in mitotic cells, calcium homeostasis in cytoplasmic domains of secreting and motile cells, and membrane traffic in the early secretion apparatus between the endoplasmic reticulum and Golgi. Opposing forces of assembly (membrane fusion) and disassembly (membrane fragmentation) ultimately determine the size and shape of this organelle. This review examines some of the regulatory mechanisms involved in these processes and how they occur at specific sites or subcompartments of the endoplasmic reticulum.

  1. Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology

    PubMed Central

    Chong, Wai Chin; Shastri, Madhur D.; Eri, Rajaraman

    2017-01-01

    The endoplasmic reticulum (ER) is a complex protein folding and trafficking organelle. Alteration and discrepancy in the endoplasmic reticulum environment can affect the protein folding process and hence, can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits endoplasmic reticulum stress. Under such stress conditions, cells exhibit reduced functional synthesis, and will undergo apoptosis if the stress is prolonged. To resolve the ER stress, cells trigger an intrinsic mechanism called an unfolded protein response (UPR). UPR is an adaptive signaling process that triggers multiple pathways through the endoplasmic reticulum transmembrane transducers, to reduce and remove misfolded proteins and improve the protein folding mechanism, in order to improve and maintain endoplasmic reticulum homeostasis. An increasing number of studies support the view that oxidative stress has a strong connection with ER stress. During the protein folding process, reactive oxygen species are produced as by-products, leading to impaired reduction-oxidation (redox) balance conferring oxidative stress. As the protein folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and enhance the production of misfolded proteins, causing further ER stress. It is proposed that endoplasmic reticulum stress and oxidative stress together play significant roles in the pathophysiology of bowel diseases. PMID:28379196

  2. Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology.

    PubMed

    Chong, Wai Chin; Shastri, Madhur D; Eri, Rajaraman

    2017-04-05

    The endoplasmic reticulum (ER) is a complex protein folding and trafficking organelle. Alteration and discrepancy in the endoplasmic reticulum environment can affect the protein folding process and hence, can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits endoplasmic reticulum stress. Under such stress conditions, cells exhibit reduced functional synthesis, and will undergo apoptosis if the stress is prolonged. To resolve the ER stress, cells trigger an intrinsic mechanism called an unfolded protein response (UPR). UPR is an adaptive signaling process that triggers multiple pathways through the endoplasmic reticulum transmembrane transducers, to reduce and remove misfolded proteins and improve the protein folding mechanism, in order to improve and maintain endoplasmic reticulum homeostasis. An increasing number of studies support the view that oxidative stress has a strong connection with ER stress. During the protein folding process, reactive oxygen species are produced as by-products, leading to impaired reduction-oxidation (redox) balance conferring oxidative stress. As the protein folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and enhance the production of misfolded proteins, causing further ER stress. It is proposed that endoplasmic reticulum stress and oxidative stress together play significant roles in the pathophysiology of bowel diseases.

  3. The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease

    DTIC Science & Technology

    2016-07-01

    AWARD NUMBER: W81XWH-14-1-0203 TITLE: The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease PRINCIPAL...1 July 2015- 30 June 2016 4. TITLE AND SUBTITLE The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease 5a... kidney targeted microbubble/ultrasound-mediated plasmid delivery. We will also examine non-targeted CRT knockdown in these mice. Aim 2.b: We will

  4. Endoplasmic reticulum stress in chondrodysplasias caused by mutations in collagen types II and X.

    PubMed

    Gawron, Katarzyna

    2016-11-01

    The endoplasmic reticulum is primarily recognized as the site of synthesis and folding of secreted, membrane-bound, and some organelle-targeted proteins. An imbalance between the load of unfolded proteins and the processing capacity in endoplasmic reticulum leads to the accumulation of unfolded or misfolded proteins and endoplasmic reticulum stress, which is a hallmark of a number of storage diseases, including neurodegenerative diseases, a number of metabolic diseases, and cancer. Moreover, its contribution as a novel mechanistic paradigm in genetic skeletal diseases associated with abnormalities of the growth plates and dwarfism is considered. In this review, I discuss the mechanistic significance of endoplasmic reticulum stress, abnormal folding, and intracellular retention of mutant collagen types II and X in certain variants of skeletal chondrodysplasia.

  5. Ibutilide protects against cardiomyocytes injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways.

    PubMed

    Wang, Yu; Wang, Yi-Li; Huang, Xia; Yang, Yang; Zhao, Ya-Jun; Wei, Cheng-Xi; Zhao, Ming

    2017-02-01

    Atrial fibrillation (AF) is a complex disease with multiple inter-relating causes culminating in rapid atrial activation and atrial structural remodeling. The contribution of endoplasmic reticulum and mitochondria stress to AF has been highlighted. As the class III antiarrhythmic agent, ibutilide are widely used to AF. This study was designed to explore whether ibutilide could treat AF by inhibiting endoplasmic reticulum stress pathways and mitochondria stress. The neonatal rat cardiomyocytes were isolated and exposed to H 2 O 2 , ibutilide was add to the culture medium 12 h. Then the cell viability, oxidative stress levels and apoptotic rate were analyzed. In addition, endoplasmic reticulum stress related protein (GRP78, GRP94, CHOP), mitochondria-dependent protein (Bax, Bcl-2) and caspase-3/9/12 were identified by real-time PCR and western blot analysis. In our results, remarkable decreased cell viability and oxidative stress levels were detected in cardiomyocytes after treating with H 2 O 2 . The apoptotic rate and the expression of proteins involved in mitochondrial stress and endoplasmic reticulum stress pathways increased. While ibutilide significantly inhibited these changes. These data suggested that ibutilide serves a protective role against H 2 O 2 -induced apoptosis of neonatal rat cardiomyocytes, and the mechanism is related to suppression of mitochondrial stress and endoplasmic reticulum stress.

  6. Endoplasmic Reticulum-Plasma Membrane Contacts Regulate Cellular Excitability.

    PubMed

    Dickson, Eamonn J

    2017-01-01

    Cells that have intrinsic electrical excitability utilize changes in membrane potential to communicate with neighboring cells and initiate cellular cascades. Excitable cells like neurons and myocytes have evolved highly specialized subcellular architectures to translate these electrical signals into cellular events. One such structural specialization is sarco-/endoplasmic reticulum-plasma membrane contact sites. These membrane contact sites are positioned by specific membrane-membrane tethering proteins and contain an ever-expanding list of additional proteins that organize information transfer across the junctional space (~ 15-25 nm distance) to shape membrane identity and control cellular excitability. In this chapter we discuss how contacts between the sarco-/endoplasmic reticulum and plasma membrane are essential for regulated excitation-contraction coupling in striated muscle and control of lipid-dependent ion channels.

  7. Endoplasmic Reticulum Stress and Obesity.

    PubMed

    Yilmaz, Erkan

    2017-01-01

    In recent years, the world has seen an alarming increase in obesity and closely associated with insulin resistance which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) play in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably other causes for obesity-related insulin resistance and inflammation. One of these appears to be endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.

  8. Endoplasmic reticulum stress in wake-active neurons progresses with aging.

    PubMed

    Naidoo, Nirinjini; Zhu, Jingxu; Zhu, Yan; Fenik, Polina; Lian, Jie; Galante, Ray; Veasey, Sigrid

    2011-08-01

    Fragmentation of wakefulness and sleep are expected outcomes of advanced aging. We hypothesize that wake neurons develop endoplasmic reticulum dyshomeostasis with aging, in parallel with impaired wakefulness. In this series of experiments, we sought to more fully characterize age-related changes in wakefulness and then, in relevant wake neuronal populations, explore functionality and endoplasmic reticulum homeostasis. We report that old mice show greater sleep/wake transitions in the active period with markedly shortened wake periods, shortened latencies to sleep, and less wake time in the subjective day in response to a novel social encounter. Consistent with sleep/wake instability and reduced social encounter wakefulness, orexinergic and noradrenergic wake neurons in aged mice show reduced c-fos response to wakefulness and endoplasmic reticulum dyshomeostasis with increased nuclear translocation of CHOP and GADD34. We have identified an age-related unfolded protein response injury to and dysfunction of wake neurons. It is anticipated that these changes contribute to sleep/wake fragmentation and cognitive impairment in aging. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

  9. Endoplasmic reticulum stress inhibition reduces hypertension through the preservation of resistance blood vessel structure and function.

    PubMed

    Carlisle, Rachel E; Werner, Kaitlyn E; Yum, Victoria; Lu, Chao; Tat, Victor; Memon, Muzammil; No, Yejin; Ask, Kjetil; Dickhout, Jeffrey G

    2016-08-01

    Our purpose was to determine if endoplasmic reticulum stress inhibition lowers blood pressure (BP) in hypertension by correcting vascular dysfunction. The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension with its normotensive control, the Wistar Kyoto rat. Animals were subjected to endoplasmic reticulum stress inhibition with 4-phenylbutyric acid (4-PBA; 1 g/kg per day, orally) for 5 weeks from 12 weeks of age. BP was measured weekly noninvasively and at endpoint with carotid arterial cannulation. Small mesenteric arteries were removed for vascular studies. Function was assessed with a Mulvany-Halpern style myograph, and structure was assessed by measurement of medial-to-lumen ratio in perfusion fixed vessels as well as three-dimensional confocal reconstruction of vessel wall components. Endoplasmic reticulum stress was assessed by quantitative real time-PCR and western blotting; oxidative stress was assessed by 3-nitrotyrosine and dihydroethidium staining. 4-PBA significantly lowered BP in SHR (vehicle 206.1 ± 4.3 vs. 4-PBA 178.9 ± 3.1, systolic) but not Wistar Kyoto. 4-PBA diminished contractility and augmented endothelial-dependent vasodilation in SHR small mesenteric arteries, as well as reducing media-to-lumen ratio. 4-PBA significantly reduced endoplasmic reticulum stress in SHR resistance vessels. Normotensive resistance vessels, treated with the endoplasmic reticulum stress-inducing agent, tunicamycin, show decreased endothelial-dependent vasodilation; this was improved with 4-PBA treatment. 3-Nitrotyrosine and dihydroethidium staining indicated that endoplasmic reticulum stress leads to reactive oxygen species generation resolvable by 4-PBA treatment. Endoplasmic reticulum stress caused endothelial-mediated vascular dysfunction contributing to elevated BP in the SHR model of human essential hypertension.

  10. Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

    PubMed

    Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

    2017-06-26

    Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

  11. The endoplasmic reticulum stress response: A link with tuberculosis?

    PubMed

    Cui, Yongyong; Zhao, Deming; Barrow, Paul Andrew; Zhou, Xiangmei

    2016-03-01

    Tuberculosis (TB) remains a major cause of mortality and morbidity in the worldwide. The endoplasmic-reticulum stress (ERS) response constitutes a cellular process that is triggered by mycobacterial infection that disturbs the folding of proteins in the endoplasmic reticulum (ER). The unfolded protein response (UPR) is induced to suspend the synthesis of early proteins and reduce the accumulation of unfolded- or misfolded proteins in the ER restoring normal physiological cell function. Prolonged or uncontrolled ERS leads to the activation of three signaling pathways (IRE1, PERK and ATF6) which directs the cell towards apoptosis. The absence of this process facilitates spread of the mycobacteria within the body. We summarize here recent advances in understanding the signaling pathway diversity governing ERS in relation to TB. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Determinant for Endoplasmic Reticulum Retention in the Luminal Domain of the Human Cytomegalovirus US3 Glycoprotein

    PubMed Central

    Lee, Sungwook; Park, Boyoun; Ahn, Kwangseog

    2003-01-01

    US3 of human cytomegalovirus is an endoplasmic reticulum resident transmembrane glycoprotein that binds to major histocompatibility complex class I molecules and prevents their departure. The endoplasmic reticulum retention signal of the US3 protein is contained in the luminal domain of the protein. To define the endoplasmic reticulum retention sequence in more detail, we have generated a series of deletion and point mutants of the US3 protein. By analyzing the rate of intracellular transport and immunolocalization of the mutants, we have identified Ser58, Glu63, and Lys64 as crucial for retention, suggesting that the retention signal of the US3 protein has a complex spatial arrangement and does not comprise a contiguous sequence of amino acids. We also show that a modified US3 protein with a mutation in any of these amino acids maintains its ability to bind class I molecules; however, such mutated proteins are no longer retained in the endoplasmic reticulum and are not able to block the cell surface expression of class I molecules. These findings indicate that the properties that allow the US3 glycoprotein to be localized in the endoplasmic reticulum and bind major histocompatibility complex class I molecules are located in different parts of the molecule and that the ability of US3 to block antigen presentation is due solely to its ability to retain class I molecules in the endoplasmic reticulum. PMID:12525649

  13. Endoplasmic reticulum stress: a novel mechanism and therapeutic target for cardiovascular diseases

    PubMed Central

    Liu, Mei-qing; Chen, Zhe; Chen, Lin-xi

    2016-01-01

    Endoplasmic reticulum is a principal organelle responsible for folding, post-translational modifications and transport of secretory, luminal and membrane proteins, thus palys an important rale in maintaining cellular homeostasis. Endoplasmic reticulum stress (ERS) is a condition that is accelerated by accumulation of unfolded/misfolded proteins after endoplasmic reticulum environment disturbance, triggered by a variety of physiological and pathological factors, such as nutrient deprivation, altered glycosylation, calcium depletion, oxidative stress, DNA damage and energy disturbance, etc. ERS may initiate the unfolded protein response (UPR) to restore cellular homeostasis or lead to apoptosis. Numerous studies have clarified the link between ERS and cardiovascular diseases. This review focuses on ERS-associated molecular mechanisms that participate in physiological and pathophysiological processes of heart and blood vessels. In addition, a number of drugs that regulate ERS was introduced, which may be used to treat cardiovascular diseases. This review may open new avenues for studying the pathogenesis of cardiovascular diseases and discovering novel drugs targeting ERS. PMID:26838072

  14. 4-Phenylbutyric Acid Reveals Good Beneficial Effects on Vital Organ Function via Anti-Endoplasmic Reticulum Stress in Septic Rats.

    PubMed

    Liu, Liangming; Wu, Huiling; Zang, JiaTao; Yang, Guangming; Zhu, Yu; Wu, Yue; Chen, Xiangyun; Lan, Dan; Li, Tao

    2016-08-01

    Sepsis and septic shock are the common complications in ICUs. Vital organ function disorder contributes a critical role in high mortality after severe sepsis or septic shock, in which endoplasmic reticulum stress plays an important role. Whether anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to sepsis and the underlying mechanisms are not known. Laboratory investigation. State Key Laboratory of Trauma, Burns and Combined Injury. Sprague-Dawley rats. Using cecal ligation and puncture-induced septic shock rats, lipopolysaccharide-treated vascular smooth muscle cells, and cardiomyocytes, effects of 4-phenylbutyric acid on vital organ function and the relationship with endoplasmic reticulum stress and endoplasmic reticulum stress-mediated inflammation, apoptosis, and oxidative stress were observed. Conventional treatment, including fluid resuscitation, vasopressin, and antibiotic, only slightly improved the hemodynamic variable, such as mean arterial blood pressure and cardiac output, and slightly improved the vital organ function and the animal survival of septic shock rats. Supplementation of 4-phenylbutyric acid (5 mg/kg; anti-endoplasmic reticulum stress), especially administered at early stage, significantly improved the hemodynamic variables, vital organ function, such as liver, renal, and intestinal barrier function, and animal survival in septic shock rats. 4-Phenylbutyric acid application inhibited the endoplasmic reticulum stress and endoplasmic reticulum stress-related proteins, such as CCAAT/enhancer-binding protein homologous protein in vital organs, such as heart and superior mesenteric artery after severe sepsis. Further studies showed that 4-phenylbutyric acid inhibited endoplasmic reticulum stress-mediated cytokine release, apoptosis, and oxidative stress via inhibition of nuclear factor-κB, caspase-3 and caspase-9, and increasing glutathione peroxidase and superoxide dismutase expression, respectively. Anti-endoplasmic

  15. [Effect of endoplasmic reticulum stress in trophocytes on the pathogenesis of intrahepatic cholestasis of pregnancy].

    PubMed

    Yu, Y; Zhou, C L; Yu, T T; Han, X J; Shi, H Y; Wang, H Z; Shen, J J; He, J

    2017-06-25

    Objective: To evaluate the effect of endoplasmic reticulum stress in trophocytes, in patients with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty-one pregnant women who were hospitalized in Women's Hospital, School of Medicine, Zhejiang University from January to December 2015 were recruited. Thirty-one women who were diagnosed as ICP were defined as the ICP group and 30 healthy pregnant women were defined as the control group. The localization and expression intensity of glucose regulated protein 78 (GRP-78) in placental tissues were detected by immunohistochemistry technique. Electronic microscope was used to observe ultra-microstructure change of the endoplasmic reticulum in trophocytes and cell line Swan71. Reverse transcription (RT)-PCR and western blot were used to investigate the expression of GRP-78 mRNA and protein in Swan 71 cell. Results: (1) GRP-78 protein was mainly expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. The protein expression of GRP-78 in placentas of the ICP group (13.2±2.4) was significantly higher than that in the control group (7.8±1.3, P< 0.01). (2) The volume of endoplasmie reticulum did not increase and the microvilli developed well, with no swelling and no expansion of endoplasmic reticulum in the control group.In the ICP group, microvilli injury, endoplasmic reticulum edema were found; the volume of endoplasmic reticulum increased, with dilation, vacuolation and significant degranulation. After treated with 100 μmol/L cholyglycine for 24 hours, universal dilatation of the endoplasmic reticulum were seen in the Swan71 cells. (3) In Swan71 cells, cholylglycine displayed a concentration-dependent up-regulation on the expression of GRP-78. The expressions of GRP-78 mRNA in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.17, 2.17±0.16, 5.47±0.36, 5.65±0.82, respectively. The expression of GRP-78 protein in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0

  16. Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.

    PubMed

    Le, Phuong U; Guay, Ginette; Altschuler, Yoram; Nabi, Ivan R

    2002-02-01

    Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.

  17. Endoplasmic reticulum stress and eIF2α phosphorylation: The Achilles heel of pancreatic β cells.

    PubMed

    Cnop, Miriam; Toivonen, Sanna; Igoillo-Esteve, Mariana; Salpea, Paraskevi

    2017-09-01

    Pancreatic β cell dysfunction and death are central in the pathogenesis of most if not all forms of diabetes. Understanding the molecular mechanisms underlying β cell failure is important to develop β cell protective approaches. Here we review the role of endoplasmic reticulum stress and dysregulated endoplasmic reticulum stress signaling in β cell failure in monogenic and polygenic forms of diabetes. There is substantial evidence for the presence of endoplasmic reticulum stress in β cells in type 1 and type 2 diabetes. Direct evidence for the importance of this stress response is provided by an increasing number of monogenic forms of diabetes. In particular, mutations in the PERK branch of the unfolded protein response provide insight into its importance for human β cell function and survival. The knowledge gained from different rodent models is reviewed. More disease- and patient-relevant models, using human induced pluripotent stem cells differentiated into β cells, will further advance our understanding of pathogenic mechanisms. Finally, we review the therapeutic modulation of endoplasmic reticulum stress and signaling in β cells. Pancreatic β cells are sensitive to excessive endoplasmic reticulum stress and dysregulated eIF2α phosphorylation, as indicated by transcriptome data, monogenic forms of diabetes and pharmacological studies. This should be taken into consideration when devising new therapeutic approaches for diabetes.

  18. [Endoplasmic reticulum stress in INS-1-3 cell associated with the expression changes of MODY gene pathway].

    PubMed

    Liu, Y T; Li, S R; Wang, Z; Xiao, J Z

    2016-09-13

    Objective: To profile the gene expression changes associated with endoplasmic reticulum stress in INS-1-3 cells induced by thapsigargin (TG) and tunicamycin (TM). Methods: Normal cultured INS-1-3 cells were used as a control. TG and TM were used to induce endoplasmic reticulum stress in INS-1-3 cells. Digital gene expression profiling technique was used to detect differentially expressed gene. The changes of gene expression were detected by expression pattern clustering analysis, gene ontology (GO) function and pathway enrichment analysis. Real time polymerase chain reaction (RT-PCR) was used to verify the key changes of gene expression. Results: Compared with the control group, there were 57 (45 up-regulated, 12 down-regulated) and 135 (99 up-regulated, 36 down-regulated) differentially expressed genes in TG and TM group, respectively. GO function enrichment analyses indicated that the main enrichment was in the endoplasmic reticulum. In signaling pathway analysis, the identified pathways were related with endoplasmic reticulum stress, antigen processing and presentation, protein export, and most of all, the maturity onset diabetes of the young (MODY) pathway. Conclusion: Under the condition of endoplasmic reticulum stress, the related expression changes of transcriptional factors in MODY signaling pathway may be related with the impaired function in islet beta cells.

  19. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

    PubMed

    Zhang, Jintao; Yi, Man; Zha, Longying; Chen, Siqiang; Li, Zhijia; Li, Cheng; Gong, Mingxing; Deng, Hong; Chu, Xinwei; Chen, Jiehua; Zhang, Zheqing; Mao, Limei; Sun, Suxia

    2016-01-01

    Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (si

  20. Dysfunction in endoplasmic reticulum-mitochondria crosstalk underlies SIGMAR1 loss of function mediated motor neuron degeneration.

    PubMed

    Bernard-Marissal, Nathalie; Médard, Jean-Jacques; Azzedine, Hamid; Chrast, Roman

    2015-04-01

    Mutations in Sigma 1 receptor (SIGMAR1) have been previously identified in patients with amyotrophic lateral sclerosis and disruption of Sigmar1 in mouse leads to locomotor deficits. However, cellular mechanisms underlying motor phenotypes in human and mouse with disturbed SIGMAR1 function have not been described so far. Here we used a combination of in vivo and in vitro approaches to investigate the role of SIGMAR1 in motor neuron biology. Characterization of Sigmar1(-/-) mice revealed that affected animals display locomotor deficits associated with muscle weakness, axonal degeneration and motor neuron loss. Using primary motor neuron cultures, we observed that pharmacological or genetic inactivation of SIGMAR1 led to motor neuron axonal degeneration followed by cell death. Disruption of SIGMAR1 function in motor neurons disturbed endoplasmic reticulum-mitochondria contacts, affected intracellular calcium signalling and was accompanied by activation of endoplasmic reticulum stress and defects in mitochondrial dynamics and transport. These defects were not observed in cultured sensory neurons, highlighting the exacerbated sensitivity of motor neurons to SIGMAR1 function. Interestingly, the inhibition of mitochondrial fission was sufficient to induce mitochondria axonal transport defects as well as axonal degeneration similar to the changes observed after SIGMAR1 inactivation or loss. Intracellular calcium scavenging and endoplasmic reticulum stress inhibition were able to restore mitochondrial function and consequently prevent motor neuron degeneration. These results uncover the cellular mechanisms underlying motor neuron degeneration mediated by loss of SIGMAR1 function and provide therapeutically relevant insight into motor neuronal diseases. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grondin, Melanie; Marion, Michel; Denizeau, Francine

    2007-07-01

    Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad frommore » the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl{sup -}/HCO{sub 3} {sup -} exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes.« less

  2. Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood–Brain Barrier Damage

    PubMed Central

    Qie, Xiaojuan; Wen, Di; Guo, Hongyan; Xu, Guanjie; Liu, Shuai; Shen, Qianchao; Liu, Yi; Zhang, Wenfang; Cong, Bin; Ma, Chunling

    2017-01-01

    Methamphetamine (METH) abuse causes serious health problems worldwide, and long-term use of METH disrupts the blood–brain barrier (BBB). Herein, we explored the potential mechanism of endoplasmic reticulum (ER) stress in METH-induced BBB endothelial cell damage in vitro and the therapeutic potential of endoplasmic reticulum stress inhibitors for METH-induced BBB disruption in C57BL/6J mice. Exposure of immortalized BMVEC (bEnd.3) cells to METH significantly decreased cell viability, induced apoptosis, and diminished the tightness of cell monolayers. METH activated ER stress sensor proteins, including PERK, ATF6, and IRE1, and upregulated the pro-apoptotic protein CHOP. The ER stress inhibitors significantly blocked the upregulation of CHOP. Knockdown of CHOP protected bEnd.3 cells from METH-induced cytotoxicity. Furthermore, METH elevated the production of reactive oxygen species (ROS) and induced the dysfunction of mitochondrial characterized by a Bcl2/Bax ratio decrease, mitochondrial membrane potential collapse, and cytochrome c. ER stress release was partially reversed by ROS inhibition, and cytochrome c release was partially blocked by knockdown of CHOP. Finally, PBA significantly attenuated METH-induced sodium fluorescein (NaFluo) and Evans Blue leakage, as well as tight junction protein loss, in C57BL/6J mice. These data suggest that BBB endothelial cell damage was caused by METH-induced endoplasmic reticulum stress, which further induced mitochondrial dysfunction, and that PBA was an effective treatment for METH-induced BBB disruption. PMID:28959203

  3. Ghrelin Ameliorates Asthma by Inhibiting Endoplasmic Reticulum Stress.

    PubMed

    Fu, Tian; Wang, Lei; Zeng, Qingdi; Zhang, Yan; Sheng, Baowei; Han, Liping

    2017-12-01

    This study aimed to confirm the ameliorative effect of ghrelin on asthma and investigate its mechanism. The murine model of asthma was induced by ovalbumin (OVA) treatment and assessed by histological pathology and airway responsiveness to methacholine. The total and differential leukocytes were counted. Tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 levels in bronchoalveolar lavage fluid were quantified by commercial kits. The protein levels in pulmonary tissues were measured by Western blot analysis. Ghrelin ameliorated the histological pathology and airway hyperresponsiveness in the OVA-induced asthmatic mouse model. Consistently, OVA-increased total and differential leukocytes and levels of tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 in bronchoalveolar lavage fluid were significantly attenuated by ghrelin. Ghrelin prevented the increased protein levels of the endoplasmic reticulum stress markers glucose regulated protein 78 and CCAAT/enhancer binding protein homologous protein and reversed the reduced levels of p-Akt in asthmatic mice. Ghrelin might prevent endoplasmic reticulum stress activation by stimulating the Akt signaling pathway, which attenuated inflammation and ameliorated asthma in mice. Ghrelin might be a new target for asthma therapy. Copyright © 2017. Published by Elsevier Inc.

  4. Lon in maintaining mitochondrial and endoplasmic reticulum homeostasis.

    PubMed

    Yang, Jieyeqi; Chen, Wenying; Zhang, Boyang; Tian, Fengli; Zhou, Zheng; Liao, Xin; Li, Chen; Zhang, Yi; Han, Yanyan; Wang, Yan; Li, Yuzhe; Wang, Guo-Qing; Shen, Xiao Li

    2018-06-01

    As a vital member of AAA+ (ATPase associated with diverse cellular activities) protein superfamily, Lon, a homo-hexameric ring-shaped protein complex with a serine-lysine catalytic dyad, is highly conserved throughout almost all prokaryotic and eukaryotic organisms. Lon protease (LONP) plays an important role in maintaining mitoproteostasis through selectively recognizing and degrading oxidatively modified mitoproteins within mitochondrial matrix, such as oxidized aconitase, phosphorylated mitochondrial transcription factor A, etc. Furthermore, the up-regulated LONP increased mitochondrial ROS generation to promote cell survival, cell proliferation, epithelial-mesenchymal transition, and cell migration, which was attributed to the up-regulation of NADH:ubiquinone oxidoreductase core subunit S8 via interaction with chaperone Lon under hypoxic or oxidative stress in tumorigenesis. In addition, Lon also participated in protein kinase RNA (PKR)-like endoplasmic reticulum kinase signaling pathway under endoplasmic reticulum (ER) stress. In short, Lon, as a pivotal stress-responsive protein that involved in the crosstalks among mitochondria, ER and nucleus, participated in multifarious important cellular processes crucial for cell survival, such as the mitochondrial protein quality control system, the mitochondrial unfolded protein response, the mtDNA maintenance, and the ER unfolded protein response.

  5. Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

    PubMed

    Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

    2017-09-02

    The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. From Endoplasmic Reticulum to Mitochondria: Absence of the Arabidopsis ATP Antiporter Endoplasmic Reticulum Adenylate Transporter1 Perturbs Photorespiration[W

    PubMed Central

    Hoffmann, Christiane; Plocharski, Bartolome; Haferkamp, Ilka; Leroch, Michaela; Ewald, Ralph; Bauwe, Hermann; Riemer, Jan; Herrmann, Johannes M.; Neuhaus, H. Ekkehard

    2013-01-01

    The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO2 concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants. PMID:23860249

  7. Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Faria, Gisele; Cardoso, Cristina R.B.; Department of Biological Sciences, Federal University of Triangulo Mineiro, Uberaba, Minas Gerais

    Chlorhexidine (CHX), widely used as antiseptic and therapeutic agent in medicine and dentistry, has a toxic effect both in vivo and in vitro. The intrinsic mechanism underlying CHX-induced cytotoxicity in eukaryotic cells is, however, still unknown. A recent study from our laboratory has suggested that CHX may induce death in cultured L929 fibroblasts via endoplasmic reticulum (ER) stress. This hypothesis was further tested by means of light and electron microscopy, quantification of apoptosis and necrosis by flow cytometry, fluorescence visualization of the cytoskeleton and endoplasmic reticulum, and evaluation of the expression of 78-kDa glucose-regulated protein 78 (Grp78), a marker ofmore » activation of the unfolded protein response (UPR) in cultured L929 fibroblasts. Our finding showing increased Grp 78 expression in CHX-treated cells and the results of flow cytometry, cytoskeleton and endoplasmic reticulum fluorescence visualization, and scanning and transmission electron microscopy allowed us to suggest that CHX elicits accumulation of proteins in the endoplasmic reticulum, which causes ER overload, resulting in ER stress and cell death either by necrosis or apoptosis. It must be pointed out, however, that this does not necessarily mean that ER stress is the only way that CHX kills L929 fibroblasts, but rather that ER stress is an important target or indicator of cell death induced by this drug.« less

  8. The endoplasmic reticulum is a hub to sort proteins toward unconventional traffic pathways and endosymbiotic organelles.

    PubMed

    Bellucci, Michele; De Marchis, Francesca; Pompa, Andrea

    2017-12-18

    The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  9. Toll-like receptor 4-induced endoplasmic reticulum stress contributes to endothelial dysfunction

    USDA-ARS?s Scientific Manuscript database

    Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Endoplasmic reticulum (ER) stress is implicated as one of the mechanisms for pathophysiology of various cardiometabolic syndromes, including insulin resistance and endothelial dysfunction. ...

  10. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

    PubMed

    Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

    2016-04-01

    It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

  11. Obesity-induced endoplasmic reticulum stress causes chronic inflammation in adipose tissue.

    PubMed

    Kawasaki, Noritaka; Asada, Rie; Saito, Atsushi; Kanemoto, Soshi; Imaizumi, Kazunori

    2012-01-01

    Adipose tissue plays a central role in maintaining metabolic homeostasis under normal conditions. Metabolic diseases such as obesity and type 2 diabetes are often accompanied by chronic inflammation and adipose tissue dysfunction. In this study, we observed that endoplasmic reticulum (ER) stress and the inflammatory response occurred in adipose tissue of mice fed a high-fat diet for a period of 16 weeks. After 16 weeks of feeding, ER stress markers increased and chronic inflammation occurred in adipose tissue. We found that ER stress is induced by free fatty acid (FFA)-mediated reactive oxygen species (ROS) generation and up-regulated gene expression of inflammatory cytokines in 3T3-L1 adipocytes. Oral administration to obese mice of chemical chaperons, which alleviate ER stress, improved chronic inflammation in adipose tissue, followed by the suppression of increased body weight and improved insulin signaling. These results indicate that ER stress plays important pathophysiological roles in obesity-induced adipose tissue dysfunction.

  12. Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions.

    PubMed Central

    Cartwright, I J; Higgins, J A

    1992-01-01

    We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound. Images Fig. 1. Fig. 3. Fig. 4. PMID:1637294

  13. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu Qiong; Department of Biochemistry and Molecular Biology, Ningbo University Medical School, Ningbo 315211; Zhan Jinbiao

    Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purifiedmore » by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.« less

  14. Mitochondrial enzymes and endoplasmic reticulum calcium stores as targets of oxidative stress in neurodegenerative diseases.

    PubMed

    Gibson, Gary E; Huang, Hsueh-Meei

    2004-08-01

    Considerable evidence indicates that oxidative stress accompanies age-related neurodegenerative diseases. Specific mechanisms by which oxidative stress leads to neurodegeneration are unknown. Two targets of oxidative stress that are known to change in neurodegenerative diseases are the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) and endoplasmic reticulum calcium stores. KGDHC activities are diminished in all common neurodegenerative diseases and the changes are particularly well documented in Alzheimer's disease (AD). A second change that occurs in cells from AD patients is an exaggerated endoplasmic reticulum calcium store [i.e., bombesin-releasable calcium stores (BRCS)]. H(2)O(2), a general oxidant, changes both variables in the same direction as occurs in disease. Other oxidants selectively alter these variables. Various antioxidants were used to help define the critical oxidant species that modifies these responses. All of the antioxidants diminish the oxidant-induced carboxy-dichlorofluorescein (cDCF) detectable reactive oxygen species (ROS), but have diverse actions on these cellular processes. For example, alpha-keto-beta-methyl-n-valeric acid (KMV) diminishes the H(2)O(2) effects on BRCS, while trolox and DMSO exaggerate the response. Acute trolox treatment does not alter H(2)O(2)-induced changes in KGDHC, whereas chronic treatment with trolox increases KGDHC almost threefold. The results suggest that KGDHC and BRCS provide targets by which oxidative stress may induce neurodegeneration and a useful tool for selecting antioxidants for reversing age-related neurodegeneration.

  15. Influence of resveratrol on endoplasmic reticulum stress and expression of adipokines in adipose tissues/adipocytes induced by high-calorie diet or palmitic acid.

    PubMed

    Chen, Li; Wang, Ting; Chen, Guanjun; Wang, Nuojin; Gui, Li; Dai, Fang; Fang, Zhaohui; Zhang, Qiu; Lu, Yunxia

    2017-03-01

    This study aimed to determine whether resveratrol treatment alleviates endoplasmic reticulum stress and changes the expression of adipokines in adipose tissues and cells. 8-week-old male C57BL/6 mice were fed a high-calorie diet (HCD group) or high-calorie diet supplemented with resveratrol (high-calorie diet  + resveratrol group) for 3 months. Insulin resistance, serum lipids and proinflammatory indices, the size and inflammatory cell infiltration in subcutaneous and visceral adipose tissues were analyzed. The gene expressions of endoplasmic reticulum stress, adipokines, and inflammatory cytokines were determined. The induced mature 3T3-L1 cells were pretreated with resveratrol and then palmitic acid, and the gene expressions of endoplasmic reticulum stress, adipokines, and inflammatory cytokines were determined. Subcutaneous and visceral adipose tissues in the high-calorie diet-fed mice exhibited adipocyte hypertrophy, inflammatory activation, and endoplasmic reticulum stress. Resveratrol alleviated high-calorie diet-induced insulin resistance and endoplasmic reticulum stress, increased expression of SIRT1, and reversed expression of adipokines in varying degrees in both subcutaneous and visceral adipose tissues. The effects of resveratrol on palmitic acid-treated adipocytes were similar to those shown in the tissues. Resveratrol treatment obviously reversed adipocyte hypertrophy and insulin resistance by attenuating endoplasmic reticulum stress and inflammation, thus increasing the expression of SIRT1 and inverting the expression of adipokines in vivo and in vitro.

  16. The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

    PubMed

    Chao de la Barca, Juan Manuel; Simard, Gilles; Amati-Bonneau, Patrizia; Safiedeen, Zainab; Prunier-Mirebeau, Delphine; Chupin, Stéphanie; Gadras, Cédric; Tessier, Lydie; Gueguen, Naïg; Chevrollier, Arnaud; Desquiret-Dumas, Valérie; Ferré, Marc; Bris, Céline; Kouassi Nzoughet, Judith; Bocca, Cinzia; Leruez, Stéphanie; Verny, Christophe; Miléa, Dan; Bonneau, Dominique; Lenaers, Guy; Martinez, M Carmen; Procaccio, Vincent; Reynier, Pascal

    2016-11-01

    Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q 2cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as

  17. Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates.

    PubMed

    Parsons, J Travis; Sun, David A; DeLorenzo, Robert J; Churn, Severn B

    2004-07-01

    Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.

  18. Endoplasmic reticulum stress in the pathogenesis of hypertension.

    PubMed

    Young, Colin N

    2017-08-01

    What is the topic of this review? This review highlights the emerging role of disruptions in endoplasmic reticulum (ER) function, namely ER stress, as a contributor to hypertension. What advances does it highlight? This review presents an integrative view of ER stress in cardiovascular control systems, including systems within the brain, kidney and peripheral vasculature, as related to development of hypertension. The endoplasmic reticulum (ER) is a cellular organelle specialized in the synthesis, folding, assembly and modification of proteins. In situations of increased protein demand, complex signalling pathways, termed the unfolded protein response, influence a series of cellular feedback loops to control ER function strictly. Although this is initially a compensatory attempt to maintain cellular homeostasis, chronic activation of the unfolded protein response, known as ER stress, leads to sustained changes in cellular function. A growing body of literature points to ER stress in diverse cardioregulatory systems, including the brain, kidney and vasculature, as central to the development of hypertension. Here, these recent findings from essential and obesity-related forms of hypertension are highlighted in an integrative manner, with discussion of the potential upstream causes and downstream consequences of ER stress. Given that hypertension is a leading medical and socio-economic global challenge, emerging findings suggest that targeting ER stress might represent a viable strategy for the treatment of hypertensive disease. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

  19. Inhibin beta E is upregulated by drug-induced endoplasmic reticulum stress as a transcriptional target gene of ATF4

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brüning, Ansgar, E-mail: ansgar.bruening@med.uni-muenchen.de; Matsingou, Christina; Brem, German Johannes

    2012-10-15

    Inhibins and activins are gonadal peptide hormones of the transforming growth factor-β super family with important functions in the reproductive system. By contrast, the recently identified inhibin βE subunit, primarily expressed in liver cells, appears to exert functions unrelated to the reproductive system. Previously shown downregulation of inhibin βE in hepatoma cells and anti-proliferative effects of ectopic inhibin βE overexpression indicated growth-regulatory effects of inhibin βE. We observed a selective re-expression of the inhibin βE subunit in HepG2 hepatoblastoma cells, MCF7 breast cancer cells, and HeLa cervical cancer cells under endoplasmic reticulum stress conditions induced by tunicamycin, thapsigargin, and nelfinavir.more » Analysis of XPB1 splicing and ATF4 activation revealed that inhibin βE re-expression was associated with induction of the endoplasmic reticulum stress reaction by these drugs. Transfection of an ATF4 expression plasmid specifically induced inhibin βE expression in HeLa cells and indicates inhibin βE as a hitherto unidentified target gene of ATF4, a key transcription factor of the endoplasmic reticulum stress response. Therefore, the inhibin βE subunit defines not only a new player but also a possible new marker for drug-induced endoplasmic reticulum stress. -- Highlights: ► Endoplasmic reticulum stress induces inhibin beta E expression. ► Inhibin beta E is regulated by the transcription factor ATF4. ► Inhibin beta E expression can be used as a marker for drug-induced ER stress.« less

  20. Endoplasmic-Reticulum Calcium Depletion and Disease

    PubMed Central

    Mekahli, Djalila; Bultynck, Geert; Parys, Jan B.; De Smedt, Humbert; Missiaen, Ludwig

    2011-01-01

    The endoplasmic reticulum (ER) as an intracellular Ca2+ store not only sets up cytosolic Ca2+ signals, but, among other functions, also assembles and folds newly synthesized proteins. Alterations in ER homeostasis, including severe Ca2+ depletion, are an upstream event in the pathophysiology of many diseases. On the one hand, insufficient release of activator Ca2+ may no longer sustain essential cell functions. On the other hand, loss of luminal Ca2+ causes ER stress and activates an unfolded protein response, which, depending on the duration and severity of the stress, can reestablish normal ER function or lead to cell death. We will review these various diseases by mainly focusing on the mechanisms that cause ER Ca2+ depletion. PMID:21441595

  1. Multivesicular body formation enhancement and exosome release during endoplasmic reticulum stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kanemoto, Soshi; Nitani, Ryota; Murakami, Tatsuhiko

    The endoplasmic reticulum (ER) plays a pivotal role in maintaining cellular homeostasis. However, numerous environmental and genetic factors give rise to ER stress by inducing an accumulation of unfolded proteins. Under ER stress conditions, cells initiate the unfolded protein response (UPR). Here, we demonstrate a novel aspect of the UPR by electron microscopy and immunostaining analyses, whereby multivesicular body (MVB) formation was enhanced after ER stress. This MVB formation was influenced by inhibition of ER stress transducers inositol required enzyme 1 (IRE1) and PKR-like ER kinase (PERK). Furthermore, exosome release was also increased during ER stress. However, in IRE1 ormore » PERK deficient cells, exosome release was not upregulated, indicating that IRE1- and PERK-mediated pathways are involved in ER stress-dependent exosome release. - Highlights: • Endoplasmic reticulum (ER) stress induces multivesicular body (MVB) formation. • ER stress transducers IRE1 and PERK mediate MVB formation. • Exosome release is enhanced after ER stress. • IRE1 or PERK deficiency blocks upregulation of ER stress-dependent exosome release.« less

  2. Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis*

    PubMed Central

    Stocki, Pawel; Chapman, Daniel C.; Beach, Lori A.; Williams, David B.

    2014-01-01

    Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins. PMID:24990953

  3. Picosecond pulsed electric fields induce apoptosis in HeLa cells via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

    PubMed

    Chen, Wen-Juan; Xiong, Zheng-Ai; Zhang, Min; Yao, Chen-Guo; Zhao, Zhong-Yong; Hua, Yuan-Yuan; Zhou, Wei

    2013-03-01

    The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

  4. HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Luo, Ying; Li, Shu-Jun; Yang, Jian

    Highlights: •Mechanisms of inflammatory response induced by HMGB1 are incompletely understood. •We found that endoplasmic reticulum stress mediate the inflammatory response induced by HMGB1. •RAGE-mediated ERS pathways are involved in those processes. •We reported a new mechanism for HMGB1 induced inflammatory response. -- Abstract: The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulummore » stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1 μg/ml) for 24 h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.« less

  5. Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kaakinen, Mika; Papponen, Hinni; Metsikkoe, Kalervo

    2008-01-15

    The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca{sup 2+}-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped virusesmore » indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.« less

  6. Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    PubMed

    Saheki, Yasunori; De Camilli, Pietro

    2017-06-20

    The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

  7. Lack of an Efficient Endoplasmic Reticulum-localized Recycling System Protects Peroxiredoxin IV from Hyperoxidation*

    PubMed Central

    Cao, Zhenbo; Subramaniam, Suraj; Bulleid, Neil J.

    2014-01-01

    Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation. PMID:24403061

  8. Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    PubMed

    Wang, Pengwei; Hawes, Chris; Hussey, Patrick J

    2017-04-01

    The endoplasmic reticulum (ER) acts as a superhighway with multiple sideroads that connects the different membrane compartments including the ER to the plasma membrane (PM). ER-PM contact sites (EPCSs) are a common feature in eukaryotic organisms, but have not been studied well in plants owing to the lack of molecular markers and to the difficulty in resolving the EPCS structure using conventional microscopy. Recently, however, plant protein complexes required for linking the ER and PM have been identified. This is a further step towards understanding the structure and function of plant EPCSs. We highlight some recent studies in this field and suggest several hypotheses that relate to the possible function of EPCSs in plants. Copyright © 2016. Published by Elsevier Ltd.

  9. Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

    PubMed Central

    Bravo, Roberto; Gutierrez, Tomás; Paredes, Felipe; Gatica, Damián; Rodriguez, Andrea E.; Pedrozo, Zully; Chiong, Mario; Parra, Valentina; Quest, Andrew F.G.; Rothermel, Beverly A.; Lavandero, Sergio

    2014-01-01

    Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders. PMID:22064245

  10. Requirement of Zinc Transporter SLC39A7/ZIP7 for Dermal Development to Fine-Tune Endoplasmic Reticulum Function by Regulating Protein Disulfide Isomerase.

    PubMed

    Bin, Bum-Ho; Bhin, Jinhyuk; Seo, Juyeon; Kim, Se-Young; Lee, Eunyoung; Park, Kyuhee; Choi, Dong-Hwa; Takagishi, Teruhisa; Hara, Takafumi; Hwang, Daehee; Koseki, Haruhiko; Asada, Yoshinobu; Shimoda, Shinji; Mishima, Kenji; Fukada, Toshiyuki

    2017-08-01

    Skin is the first area that manifests zinc deficiency. However, the molecular mechanisms by which zinc homeostasis affects skin development remain largely unknown. Here, we show that zinc-regulation transporter-/iron-regulation transporter-like protein 7 (ZIP7) localized to the endoplasmic reticulum plays critical roles in connective tissue development. Mice lacking the Slc39a7/Zip7 gene in collagen 1-expressing tissue exhibited dermal dysplasia. Ablation of ZIP7 in mesenchymal stem cells inhibited cell proliferation thereby preventing proper dermis formation, indicating that ZIP7 is required for dermal development. We also found that mesenchymal stem cells lacking ZIP7 accumulated zinc in the endoplasmic reticulum, which triggered zinc-dependent aggregation and inhibition of protein disulfide isomerase, leading to endoplasmic reticulum dysfunction. These results suggest that ZIP7 is necessary for endoplasmic reticulum function in mesenchymal stem cells and, as such, is essential for dermal development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Type 2 diabetes mellitus induces congenital heart defects in murine embryos by increasing oxidative stress, endoplasmic reticulum stress, and apoptosis.

    PubMed

    Wu, Yanqing; Reece, E Albert; Zhong, Jianxiang; Dong, Daoyin; Shen, Wei-Bin; Harman, Christopher R; Yang, Peixin

    2016-09-01

    Maternal type 1 and 2 diabetes mellitus are strongly associated with high rates of severe structural birth defects, including congenital heart defects. Studies in type 1 diabetic embryopathy animal models have demonstrated that cellular stress-induced apoptosis mediates the teratogenicity of maternal diabetes leading to congenital heart defect formation. However, the mechanisms underlying maternal type 2 diabetes mellitus-induced congenital heart defects remain largely unknown. We aim to determine whether oxidative stress, endoplasmic reticulum stress, and excessive apoptosis are the intracellular molecular mechanisms underlying maternal type 2 diabetes mellitus-induced congenital heart defects. A mouse model of maternal type 2 diabetes mellitus was established by feeding female mice a high-fat diet (60% fat). After 15 weeks on the high-fat diet, the mice showed characteristics of maternal type 2 diabetes mellitus. Control dams were either fed a normal diet (10% fat) or the high-fat diet during pregnancy only. Female mice from the high-fat diet group and the 2 control groups were mated with male mice that were fed a normal diet. At E12.5, embryonic hearts were harvested to determine the levels of lipid peroxides and superoxide, endoplasmic reticulum stress markers, cleaved caspase 3 and 8, and apoptosis. E17.5 embryonic hearts were harvested for the detection of congenital heart defect formation using India ink vessel patterning and histological examination. Maternal type 2 diabetes mellitus significantly induced ventricular septal defects and persistent truncus arteriosus in the developing heart, along with increasing oxidative stress markers, including superoxide and lipid peroxidation; endoplasmic reticulum stress markers, including protein levels of phosphorylated-protein kinase RNA-like endoplasmic reticulum kinase, phosphorylated-IRE1α, phosphorylated-eIF2α, C/EBP homologous protein, and binding immunoglobulin protein; endoplasmic reticulum chaperone gene

  12. Non-genomic STAT5-dependent effects at the endoplasmic reticulum and Golgi apparatus and STAT6-GFP in mitochondria

    PubMed Central

    Sehgal, Pravin B

    2013-01-01

    STAT protein species are well-known as transcription factors that regulate nuclear gene expression. Recent novel lines of research suggest new non-genomic functions of STAT5A/B and STAT6. It was discovered in human pulmonary arterial endothelial cells that STAT5A, including STAT5A-GFP, constitutively associated with the Golgi apparatus, and both STAT5A and B with the endoplasmic reticulum. Acute siRNA-mediated knockdown of STAT5A/B led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition of the ER structural protein reticulon-4 (RTN4; also called Nogo-B) and the ER-resident GTPase atlastin-3 (ATL3) along cyst membranes and cyst-zone boundaries, accompanied by Golgi fragmentation. Functional consequences included reduced anterograde trafficking, an ER stress response (increased GRP78/BiP) and eventual mitochondrial fragmentation. This phenotype was “non-genomic” in that it was elicited in enucleated cytoplasts. In cross-immunopanning assays STAT5A and B species associated with ATL3, and the ER-lumen spacer CLIMP63 (also called cytoskeleton-associated protein 4, CKAP4) but not RTN4. From a disease significance perspective we posit that STAT5, which is known to be affected by estradiol-17β and prolactin, represents the gender-sensitive determinant in the pathogenesis of idiopathic pulmonary hypertension (IPAH), a disease which includes ER/Golgi dysfunctions but with a 2- to 4-fold higher prevalence in postpubertal women. A separate line of recent research produced evidence for the association of STAT6-GFP, but not STAT3-GFP, STAT3-DsRed, or STAT3-Flag, with mitochondria in live-cell, immunofluorescence, and immunoelectron microscopy. An N-terminal truncation of STAT6-GFP (1–459), which lacked the SH2 domain and Tyr-phosphorylation site, constitutively associated with mitochondria. Thus, the emergent new of biology STAT proteins includes non-genomic roles—structurally and functionally—in the three

  13. Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

    PubMed

    Rosenfeld, M G; Marcantonio, E E; Hakimi, J; Ort, V M; Atkinson, P H; Sabatini, D; Kreibich, G

    1984-09-01

    Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

  14. A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

    NASA Astrophysics Data System (ADS)

    Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

    2017-06-01

    As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

  15. Arresting a Torsin ATPase Reshapes the Endoplasmic Reticulum*

    PubMed Central

    Rose, April E.; Zhao, Chenguang; Turner, Elizabeth M.; Steyer, Anna M.; Schlieker, Christian

    2014-01-01

    Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures. PMID:24275647

  16. circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica.

    PubMed

    Cao, Zhouli; Xiao, Qingling; Dai, Xiaoniu; Zhou, Zewei; Jiang, Rong; Cheng, Yusi; Yang, Xiyue; Guo, Huifang; Wang, Jing; Xi, Zhaoqing; Yao, Honghong; Chao, Jie

    2017-12-13

    Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO 2 -induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO 2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO 2 . SiO 2 induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO 2 , inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO 2 . circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO 2 . Our study elucidated a link between SiO 2 -induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

  17. Lysosome and endoplasmic reticulum quality control pathways in Niemann-Pick type C disease

    PubMed Central

    Schultz, Mark L.; Krus, Kelsey L.; Lieberman, Andrew P.

    2017-01-01

    Lysosomal storage diseases result from inherited deficiencies of lysosomal hydrolytic activities or lipid transport. Collectively, these disorders are a common cause of morbidity in the pediatric population and are often associated with severe neurodegeneration. Among this group of diseases is Niemann-Pick type C, an autosomal recessive disorder of lipid trafficking that causes cognitive impairment, ataxia and death, most often in childhood. Here, we review the current knowledge of disease pathogenesis, with particular focus on insights gleaned from genetics and the study of model systems. Critical advances in understanding mechanisms that regulate intracellular cholesterol trafficking have emerged from this work and are highlighted. We review effects of disease-causing mutations on quality control pathways involving the lysosome and endoplasmic reticulum, and discuss how they function to clear the most common mutant protein found in Niemann-Pick type C patients, NPC1-I1061T. Finally, we summarize insights into the mechanisms that degrade misfolded transmembrane proteins in the endoplasmic reticulum and how manipulating these quality control pathways may lead to the identification of novel targets for disease-modifying therapies. PMID:27026653

  18. Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum

    PubMed Central

    1985-01-01

    SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event. PMID:3905826

  19. CDK5-mediated tau accumulation triggers methamphetamine-induced neuronal apoptosis via endoplasmic reticulum-associated degradation pathway.

    PubMed

    Xiao, Ning; Zhang, Fu; Zhu, Bofeng; Liu, Chao; Lin, Zhoumeng; Wang, Huijun; Xie, Wei-Bing

    2018-08-01

    Overexposure to methamphetamine (METH) causes apoptosis in a number of cell types, particularly neuronal cells. However, the underlying mechanisms of METH-induced neuronal apoptosis remain to be elucidated. Accumulation of microtubule-associated protein Tau can lead to activation of multiple neurotoxic pathways, which is closely correlated with neuronal apoptosis. The aim of this study was to determine the role of Tau in METH-induced neuronal apoptosis. We determined the expression of two phosphorylated Tau proteins (serine 396 and threonine 231) in the human neuroblastoma SH-SY5Y cells and in the hippocampus of Sprague-Dawley rats treated with vehicle or METH using western blotting, immunohistochemical staining and immunofluorescence staining. We also measured the expression levels of the phosphorylated Tau protein, ubiquitination proteins, the intermediate products of proteasome degradation pathway, CD3-δ (a substrate of proteasome degradation pathway), endoplasmic reticulum stress signal molecule phosphorylated PERK (pPERK), and endoplasmic reticulum stress-specific apoptotic signal molecule caspase-12 in SH-SY5Y cells and in rats after inhibiting the expression of an upstream regulatory factor of phosphorylated Tau protein (CDK5) using siRNA or virus transfection. The results showed that exposure to METH significantly up-regulated the expression of phosphorylated Tau protein in vivo and in vitro and silencing the expression of CDK5 inhibited the up-regulation of phosphorylated Tau induced by METH exposure. METH exposure also significantly increased the expression of ubiquitination protein and CD3-δ and these effects were blocked by CDK5 silencing. In addition, METH exposure significantly elevated the levels of phosphorylated PERK and caspase-12 and these effects were suppressed after CDK5 silencing, which indicates that blockade of CDK5 expression can mitigate METH-induced neuronal apoptosis. These results suggest that METH can impair the endoplasmic

  20. Agonist-activated Ca2+ influx occurs at stable plasma membrane and endoplasmic reticulum junctions

    PubMed Central

    Treves, Susan; Vukcevic, Mirko; Griesser, Johanna; Armstrong, Clara-Franzini; Zhu, Michael X.; Zorzato, Fancesco

    2010-01-01

    Junctate is a 33 kDa integral protein of sarco(endo)plasmic reticulum membranes that forms a macromolecular complex with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and TRPC3 channels. TIRF microscopy shows that junctate enhances the number of fluorescent puncta on the plasma membrane. The size and distribution of these puncta are not affected by the addition of agonists that mobilize Ca2+ from Ins(1,4,5)P3-sensitive stores. Puncta are associated with a significantly larger number of peripheral junctions between endoplasmic reticulum and plasma membrane, which are further enhanced upon stable co-expression of junctate and TRPC3. The gap between the membranes of peripheral junctions is bridged by regularly spaced electron-dense structures of 10 nm. Ins(1,4,5)P3 inhibits the interaction of the cytoplasmic N-terminus of junctate with the ligand-binding domain of the Ins(1,4,5)P3 receptor. Furthermore, Ca2+ influx evoked by activation of Ins(1,4,5)P3 receptors is increased where puncta are located. We conclude that stable peripheral junctions between the plasma membrane and endoplasmic reticulum are the anatomical sites of agonist-activated Ca2+ entry. PMID:21062895

  1. Role of Endoplasmic Reticulum Stress in Metabolic Disease and Other Disorders

    PubMed Central

    Ozcan, Lale; Tabas, Ira

    2012-01-01

    Perturbations in the normal functions of the endoplasmic reticulum (ER) trigger a signaling network that coordinates adaptive and apoptotic responses. There is accumulating evidence implicating prolonged ER stress in the development and progression of many diseases, including neurodegeneration, atherosclerosis, type 2 diabetes, liver disease, and cancer. With the improved understanding of the underlying molecular mechanisms, therapeutic interventions that target the ER stress response would be potential strategies to treat various diseases driven by prolonged ER stress. PMID:22248326

  2. Met receptor inhibitor SU11274 localizes in the endoplasmic reticulum.

    PubMed

    Wiest, Edwin J; Smith, Heather Jensen; Hollingsworth, Michael A

    2018-07-02

    We discovered that SU11274, a class I c-Met inhibitor, fluoresces when excited by 488 nm laser light and showed rapid specific accumulation in distinct subcellular compartments. Given that SU11274 reduces cancer cell viability, we exploited these newly identified spectral properties to determine SU11274 intracellular distribution and accumulation in human pancreatic cancer cells. The aim of the studies reported here was to identify organelle(s) to which SU11274 is trafficked. We conclude that SU11274 rapidly and predominantly accumulates in the endoplasmic reticulum. Copyright © 2018. Published by Elsevier Inc.

  3. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders

    PubMed Central

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-01-01

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article. PMID:28208663

  4. Endoplasmic Reticulum Stress and Type 2 Diabetes

    PubMed Central

    Back, Sung Hoon; Kaufman, Randal J.

    2013-01-01

    Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause β-cell dysfunction and death. PMID:22443930

  5. Endoplasmic reticulum-dependent redox reactions control endoplasmic reticulum-associated degradation and pathogen entry.

    PubMed

    Walczak, Christopher P; Bernardi, Kaleena M; Tsai, Billy

    2012-04-15

    Protein misfolding within the endoplasmic reticulum (ER) is managed by an ER quality control system that retro-translocates aberrant proteins into the cytosol for proteasomal destruction. This process, known as ER-associated degradation, utilizes the action of ER redox enzymes to accommodate the disulfide-bonded nature of misfolded proteins. Strikingly, various pathogenic viruses and toxins co-opt these redox components to reach the cytosol during entry. These redox factors thus regulate critical cellular homeostasis and host-pathogen interactions. Recent studies identify specific members of the protein disulfide isomerase (PDI) family, which use their chaperone and catalytic activities, in engaging both misfolded ER proteins and pathogens. The precise molecular mechanism by which a dedicated PDI family member disrupts the disulfide bonds in the misfolded ER proteins and pathogens, as well as how they act to unfold these substrates to promote their ER-to-cytosol membrane transport, remain poorly characterized. How PDI family members distinguish folded versus misfolded ER substrates remains enigmatic. What physical characteristics surrounding a substrate's disulfide bond instruct PDI that it is mispaired or native? For the pathogens, as their disulfide bonds normally serve a critical role in providing physical support, what conformational changes experienced in the host enable their disulfide bonds to be disrupted? A combination of more rigorous biochemical and high-resolution structural studies should begin to address these questions.

  6. Selective modulation of endoplasmic reticulum stress markers in prostate cancer cells by a standardized mangosteen fruit extract.

    PubMed

    Li, Gongbo; Petiwala, Sakina M; Pierce, Dana R; Nonn, Larisa; Johnson, Jeremy J

    2013-01-01

    The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER) machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE) was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs) procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation.

  7. Selective Modulation of Endoplasmic Reticulum Stress Markers in Prostate Cancer Cells by a Standardized Mangosteen Fruit Extract

    PubMed Central

    Li, Gongbo; Petiwala, Sakina M.; Pierce, Dana R.; Nonn, Larisa; Johnson, Jeremy J.

    2013-01-01

    The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER) machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE) was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs) procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation. PMID:24367485

  8. Sirtuin 1 protects the aging heart from contractile dysfunction mediated through the inhibition of endoplasmic reticulum stress-mediated apoptosis in cardiac-specific Sirtuin 1 knockout mouse model.

    PubMed

    Hsu, Yu-Juei; Hsu, Shih-Che; Hsu, Chiao-Po; Chen, Yen-Hui; Chang, Yung-Lung; Sadoshima, Junichi; Huang, Shih-Ming; Tsai, Chien-Sung; Lin, Chih-Yuan

    2017-02-01

    The longevity regulator Sirtuin 1 is an NAD + -dependent histone deacetylase that regulates endoplasmic reticulum stress and influences cardiomyocyte apoptosis during cardiac contractile dysfunction induced by aging. The mechanism underlying Sirtuin 1 function in cardiac contractile dysfunction related to aging has not been completely elucidated. We evaluated cardiac contractile function, endoplasmic reticulum stress, apoptosis, and oxidative stress in 6- and 12month-old cardiac-specific Sirtuin 1 knockout (Sirt1 -/- ) and control (Sirt1 f/f ) mice using western blotting and immunohistochemistry. Mice were injected with a protein disulphide isomerase inhibitor. For in vitro analysis, cultured H9c2 cardiomyocytes were exposed to either a Sirtuin 1 inhibitor or activator, with or without a mitochondrial inhibitor, to evaluate the effects of Sirtuin 1 on endoplasmic reticulum stress, nitric oxide synthase expression, and apoptosis. The effects of protein disulphide isomerase inhibition on oxidative stress and ER stress-related apoptosis were also investigated. Compared with 6-month-old Sirt1 f/f mice, marked impaired contractility was observed in 12-month-old Sirt1 -/- mice. These findings were consistent with increased endoplasmic reticulum stress and apoptosis in the myocardium. Measures of oxidative stress and nitric oxide synthase expression were significantly higher in Sirt1 -/- mice compared with those in Sirt1 f/f mice at 6months. In vitro experiments revealed increased endoplasmic reticulum stress-mediated apoptosis in H9c2 cardiomyocytes treated with a Sirtuin 1 inhibitor; the effects were ameliorated by a Sirtuin 1 activator. Moreover, consistent with the in vitro findings, impaired cardiac contractility was demonstrated in Sirt1 -/- mice injected with a protein disulphide isomerase inhibitor. The present study demonstrates that the aging heart is characterized by contractile dysfunction associated with increased oxidative stress and endoplasmic reticulum

  9. Identification and Functional Characterization of Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) in Pericytes.

    PubMed

    Bischoff, Florian C; Werner, Astrid; John, David; Boeckel, Jes-Niels; Melissari, Maria-Theodora; Grote, Phillip; Glaser, Simone F; Demolli, Shemsi; Uchida, Shizuka; Michalik, Katharina M; Meder, Benjamin; Katus, Hugo A; Haas, Jan; Chen, Wei; Pullamsetti, Soni S; Seeger, Werner; Zeiher, Andreas M; Dimmeler, Stefanie; Zehendner, Christoph M

    2017-08-04

    Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays ( P <0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1α ( P <0.001), ATF6 ( P <0.01), and soluble BiP ( P <0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure ( P <0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). Here, we show that HypERlnc regulates human pericyte function and the

  10. Relevance of Endoplasmic Reticulum Stress Cell Signaling in Liver Cold Ischemia Reperfusion Injury

    PubMed Central

    Folch-Puy, Emma; Panisello, Arnau; Oliva, Joan; Lopez, Alexandre; Castro Benítez, Carlos; Adam, René; Roselló-Catafau, Joan

    2016-01-01

    The endoplasmic reticulum (ER) is involved in calcium homeostasis, protein folding and lipid biosynthesis. Perturbations in its normal functions lead to a condition called endoplasmic reticulum stress (ERS). This can be triggered by many physiopathological conditions such as alcoholic steatohepatitis, insulin resistance or ischemia-reperfusion injury. The cell reacts to ERS by initiating a defensive process known as the unfolded protein response (UPR), which comprises cellular mechanisms for adaptation and the safeguarding of cell survival or, in cases of excessively severe stress, for the initiation of the cell death program. Recent experimental data suggest the involvement of ERS in ischemia/reperfusion injury (IRI) of the liver graft, which has been considered as one of major problems influencing outcome after liver transplantation. The purpose of this review is to summarize updated data on the molecular mechanisms of ERS/UPR and the consequences of this pathology, focusing specifically on solid organ preservation and liver transplantation models. We will also discuss the potential role of ERS, beyond the simple adaptive response and the regulation of cell death, in the modification of cell functional properties and phenotypic changes. PMID:27231901

  11. Endoplasmic reticulum proteostasis impairment in aging.

    PubMed

    Martínez, Gabriela; Duran-Aniotz, Claudia; Cabral-Miranda, Felipe; Vivar, Juan P; Hetz, Claudio

    2017-08-01

    Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one-third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  12. Terasaki Spiral Ramps in the Rough Endoplasmic Reticulum

    NASA Astrophysics Data System (ADS)

    Guven, Jemal; Huber, Greg; Valencia, Dulce María

    2014-10-01

    We present a model describing the morphology as well as the assembly of "Terasaki ramps," the recently discovered helicoidal connections linking adjacent sheets of the rough endoplasmic reticulum (ER). The fundamental unit is a localized symmetric double-ramped "parking garage" formed by two separated gently pitched, approximately helicoidal, ramps of opposite chiralities. This geometry is stabilized by a short-range repulsive interaction between ramps associated with bending energy which opposes the long-range attraction associated with tension. The ramp inner boundaries are themselves stabilized by the condensation of membrane-shaping proteins along their length. A mechanism for parking garage self-assembly is proposed involving the nucleation of dipoles at the center of tubular three-way junctions within the smooth ER. Our predictions are compared with the experimental data.

  13. Snapshot: implications for melatonin in endoplasmic reticulum homeostasis

    PubMed Central

    Hu, Wei; Ma, Zhiqiang; Di, Shouyin; Jiang, Shuai; Li, Yue; Fan, Chongxi

    2016-01-01

    The endoplasmic reticulum (ER) is an important intracellular membranous organelle. Previous studies have demonstrated that the ER is responsible for protein folding and trafficking, lipid synthesis and the maintenance of calcium homeostasis. Interestingly, the morphology and structure of the ER were recently found to be important. Melatonin is a hormone that anticipates the daily onset of darkness in mammals, and it is well known that melatonin acts as an antioxidant by scavenging free radicals and increasing the activity of antioxidant enzymes in the body. Notably, the existing evidence demonstrates that melatonin is involved in ER homeostasis, particularly in the morphology of the ER, indicating a potential protective role of melatonin. This review discusses the existing knowledge regarding the implications for the involvement of melatonin in ER homeostasis. PMID:27759160

  14. Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

    PubMed

    Hong, Seong-Ho; Chang, Seung-Hee; Cho, Kyung-Cho; Kim, Sanghwa; Park, Sungjin; Lee, Ah Young; Jiang, Hu-Lin; Kim, Hyeon-Jeong; Lee, Somin; Yu, Kyeong-Nam; Seo, Hwi Won; Chae, Chanhee; Kim, Kwang Pyo; Park, Jongsun; Cho, Myung-Haing

    2016-10-04

    Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies.

  15. Dipeptidyl peptidase-4 inhibitor, vildagliptin, inhibits pancreatic beta cell apoptosis in association with its effects suppressing endoplasmic reticulum stress in db/db mice.

    PubMed

    Wu, Yan-ju; Guo, Xin; Li, Chun-jun; Li, Dai-qing; Zhang, Jie; Yang, Yiping; Kong, Yan; Guo, Hang; Liu, De-min; Chen, Li-ming

    2015-02-01

    Vildagliptin promotes beta cell survival by inhibiting cell apoptosis. It has been suggested that chronic ER (endoplasmic reticulum) stress triggers beta cell apoptosis. The objective of the study is to explore whether the pro-survival effect of vildagliptin is associated with attenuation of endoplasmic reticulum stress in islets of db/db mice. Vildagliptin was orally administered to db/db mice for 6 weeks, followed by evaluation of beta cell apoptosis by caspase3 activity and TUNEL staining method. Endoplasmic reticulum stress markers were determined with quantitative RT-PCR, immunohistochemistry and immunoblot analysis. After 6 weeks of treatment, vildagliptin treatment increased plasma active GLP-1 levels (22.63±1.19 vs. 11.69±0.44, P<0.001), inhibited beta cell apoptosis as demonstrated by lower amounts of TUNEL staining nuclei (0.37±0.03 vs. 0.55±0.03, P<0.01) as well as decreased caspase3 activity (1.48±0.11 vs. 2.67±0.13, P<0.01) in islets of diabetic mice compared with untreated diabetic group. Further, vildagliptin treatment down-regulated several genes related to endoplasmic reticulum stress including TRIB3 (tribbles homolog 3) (15.9±0.4 vs. 33.3±1.7, ×10⁻³, P<0.001), ATF-4(activating transcription factor 4) (0.83±0.06 vs. 1.42±0.02, P<0.001) and CHOP(C/EBP homologous protein) (0.07±0.01 vs. 0.16±0.01, P<0.001). Vildagliptin promoted beta cell survival in db/db mice in association with down-regulating markers of endoplasmic reticulum stress including TRIB3, ATF-4 as well as CHOP. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

    PubMed Central

    Behnke, O.; Moe, H.

    1964-01-01

    In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells. PMID:14206428

  17. Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival

    PubMed Central

    Sun, Shengyi; Shi, Guojun; Han, Xuemei; Francisco, Adam B.; Ji, Yewei; Mendonça, Nuno; Liu, Xiaojing; Locasale, Jason W.; Simpson, Kenneth W.; Duhamel, Gerald E.; Kersten, Sander; Yates, John R.; Long, Qiaoming; Qi, Ling

    2014-01-01

    Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involved in endoplasmic reticulum-associated degradation (ERAD). Sel1L’s physiological importance in mammalian ERAD, however, remains to be established. Here, using the inducible Sel1L knockout mouse and cell models, we show that Sel1L is indispensable for Hrd1 stability, ER homeostasis, and survival. Acute loss of Sel1L leads to premature death in adult mice within 3 wk with profound pancreatic atrophy. Contrary to current belief, our data show that mammalian Sel1L is required for Hrd1 stability and ERAD function both in vitro and in vivo. Sel1L deficiency disturbs ER homeostasis, activates ER stress, attenuates translation, and promotes cell death. Serendipitously, using a biochemical approach coupled with mass spectrometry, we found that Sel1L deficiency causes the aggregation of both small and large ribosomal subunits. Thus, Sel1L is an indispensable component of the mammalian Hrd1 ERAD complex and ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival. PMID:24453213

  18. The Endoplasmic Reticulum Stress Response in Neuroprogressive Diseases: Emerging Pathophysiological Role and Translational Implications.

    PubMed

    Morris, Gerwyn; Puri, Basant K; Walder, Ken; Berk, Michael; Stubbs, Brendon; Maes, Michael; Carvalho, André F

    2018-03-29

    The endoplasmic reticulum (ER) is the main cellular organelle involved in protein synthesis, assembly and secretion. Accumulating evidence shows that across several neurodegenerative and neuroprogressive diseases, ER stress ensues, which is accompanied by over-activation of the unfolded protein response (UPR). Although the UPR could initially serve adaptive purposes in conditions associated with higher cellular demands and after exposure to a range of pathophysiological insults, over time the UPR may become detrimental, thus contributing to neuroprogression. Herein, we propose that immune-inflammatory, neuro-oxidative, neuro-nitrosative, as well as mitochondrial pathways may reciprocally interact with aberrations in UPR pathways. Furthermore, ER stress may contribute to a deregulation in calcium homoeostasis. The common denominator of these pathways is a decrease in neuronal resilience, synaptic dysfunction and even cell death. This review also discusses how mechanisms related to ER stress could be explored as a source for novel therapeutic targets for neurodegenerative and neuroprogressive diseases. The design of randomised controlled trials testing compounds that target aberrant UPR-related pathways within the emerging framework of precision psychiatry is warranted.

  19. Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration.

    PubMed

    Feng, Juan; Lü, Silin; Ding, Yanhong; Zheng, Ming; Wang, Xian

    2016-06-01

    Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

  20. Silver nanoparticles induce SH-SY5Y cell apoptosis via endoplasmic reticulum- and mitochondrial pathways that lengthen endoplasmic reticulum-mitochondria contact sites and alter inositol-3-phosphate receptor function.

    PubMed

    Li, Lin; Cui, Jiahui; Liu, Zi; Zhou, Xuejiao; Li, Zengqiang; Yu, Yang; Jia, Yuanyuan; Zuo, Daiying; Wu, Yingliang

    2018-03-15

    Silver nanoparticles (AgNPs) have many medical and commercial applications, but their effects on human health are poorly understood. The aim of this study was to assess the effect of AgNPs on the human neuroblastoma cell line SH-SY5Y and to explore their potential mechanisms of action. We found that AgNPs decreased SH-SY5Y cell viability in a dose- and time-dependent manner. Exposure to AgNPs activated endoplasmic reticulum (ER) stress, as reflected by upregulated expression of glucose-regulated protein 78 (GRP78), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α), C/EBP homology protein (CHOP), spliced X-box binding protein-1 (XBP1), and phosphorylated inositol-requiring enzyme (p-IRE), all of which are involved in the cellular unfolded protein response. Prolonged exposure of cells to AgNPs damaged calcium (Ca 2+ ) homeostasis, increased the length of contact sites between the ER and mitochondria, altered IP 3 R function by the increased levels of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in the ER and enhanced mitochondrial Ca 2+ uptake. Finally, Ca 2+ overload and disrupted homeostasis in the mitochondria triggered apoptotic cell death. Our results suggest that caution should be exercised in the use of AgNPs in humans. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Targeting the Endoplasmic Reticulum Unfolded Protein Response to Counteract the Oxidative Stress-Induced Endothelial Dysfunction

    PubMed Central

    Moltedo, Ornella; Faraonio, Raffaella

    2018-01-01

    In endothelial cells, the tight control of the redox environment is essential for the maintenance of vascular homeostasis. The imbalance between ROS production and antioxidant response can induce endothelial dysfunction, the initial event of many cardiovascular diseases. Recent studies have revealed that the endoplasmic reticulum could be a new player in the promotion of the pro- or antioxidative pathways and that in such a modulation, the unfolded protein response (UPR) pathways play an essential role. The UPR consists of a set of conserved signalling pathways evolved to restore the proteostasis during protein misfolding within the endoplasmic reticulum. Although the first outcome of the UPR pathways is the promotion of an adaptive response, the persistent activation of UPR leads to increased oxidative stress and cell death. This molecular switch has been correlated to the onset or to the exacerbation of the endothelial dysfunction in cardiovascular diseases. In this review, we highlight the multiple chances of the UPR to induce or ameliorate oxidative disturbances and propose the UPR pathways as a new therapeutic target for the clinical management of endothelial dysfunction. PMID:29725497

  2. Lipolysis Response to Endoplasmic Reticulum Stress in Adipose Cells*

    PubMed Central

    Deng, Jingna; Liu, Shangxin; Zou, Liangqiang; Xu, Chong; Geng, Bin; Xu, Guoheng

    2012-01-01

    In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues. PMID:22223650

  3. Endoplasmic Reticulum Stress Induces Myostatin High Molecular Weight Aggregates and Impairs Mature Myostatin Secretion.

    PubMed

    Sachdev, Rishibha; Kappes-Horn, Karin; Paulsen, Lydia; Duernberger, Yvonne; Pleschka, Catharina; Denner, Philip; Kundu, Bishwajit; Reimann, Jens; Vorberg, Ina

    2018-03-15

    Sporadic inclusion body myositis (sIBM) is the most prevalent acquired muscle disorder in the elderly with no defined etiology or effective therapy. Endoplasmic reticulum stress and deposition of myostatin, a secreted negative regulator of muscle growth, have been implicated in disease pathology. The myostatin signaling pathway has emerged as a major target for symptomatic treatment of muscle atrophy. Here, we systematically analyzed the maturation and secretion of myostatin precursor MstnPP and its metabolites in a human muscle cell line. We find that increased MsntPP protein levels induce ER stress. MstnPP metabolites were predominantly retained within the endoplasmic reticulum (ER), also evident in sIBM histology. MstnPP cleavage products formed insoluble high molecular weight aggregates, a process that was aggravated by experimental ER stress. Importantly, ER stress also impaired secretion of mature myostatin. Reduced secretion and aggregation of MstnPP metabolites were not simply caused by overexpression, as both events were also observed in wildtype cells under ER stress. It is tempting to speculate that reduced circulating myostatin growth factor could be one explanation for the poor clinical efficacy of drugs targeting the myostatin pathway in sIBM.

  4. The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Naranmandura, Hua, E-mail: narenman@zju.edu.cn; Xu, Shi; Koike, Shota

    The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulummore » (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase

  5. ERdj5 sensitizes neuroblastoma cells to endoplasmic reticulum stress-induced apoptosis.

    PubMed

    Thomas, Christophoros G; Spyrou, Giannis

    2009-03-06

    Down-regulation of the unfolded protein response (UPR) can be therapeutically valuable in cancer treatment, and endoplasmic reticulum (ER)-resident chaperone proteins may thus be targets for developing novel chemotherapeutic strategies. ERdj5 is a novel ER chaperone that regulates the ER-associated degradation of misfolded proteins through its associations with EDEM and the ER stress sensor BiP. To investigate whether ERdj5 can regulate ER stress signaling pathways, we exposed neuroblastoma cells overexpressing ERdj5 to ER stress inducers. ERdj5 promoted apoptosis in tunicamycin, thapsigargin, and bortezomib-treated cells. To provide further evidence that ERdj5 induces ER stress-regulated apoptosis, we targeted Bcl-2 to ER of ERdj5-overexpressing cells. Targeting the Bcl-2 to ER prevented the apoptosis induced by ER stress inducers but not by non-ER stress apoptotic stimuli, suggesting induction of ER stress-regulated apoptosis by ERdj5. ERdj5 enhanced apoptosis by abolishing the ER stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) and the subsequent translational repression. ERdj5 was found to inhibit the eIF2alpha phosphorylation under ER stress through inactivating the pancreatic endoplasmic reticulum kinase. The compromised integrated stress response observed in ERdj5-overexpressing ER-stressed cells due to repressed eIF2alpha phosphorylation correlated with impaired neuroblastoma cell resistance under ER stress. These results demonstrate that ERdj5 decreases neuroblastoma cell survival by down-regulating the UPR, raising the possibility that this protein could be a target for anti-tumor approaches.

  6. Structural reorganization of the fungal endoplasmic reticulum upon induction of mycotoxin biosynthesis

    DOE PAGES

    Boenisch, Marike Johanne; Broz, Karen Lisa; Purvine, Samuel Owen; ...

    2017-03-13

    Eukaryotic cells routinely compartmentalize metabolic pathways to particular organelles for biosynthetic purposes. Relatively few studies have addressed the cellular localization of pathways for secondary metabolites synthesis. In this study, the phytopathogenic fungus Fusarium graminearum reorganized its endoplasmic reticulum (ER) when triggered to produce mycotoxins, both in vitro and in planta. Fluorescence tagged biosynthetic proteins were found to co-localize with the modified ER as confirmed by co-fluorescence and co-purification with known ER proteins. Microscopy, cell sorting, and proteomics were applied in this FICUS collaborative effort.

  7. The VCP/p97 and YOD1 Proteins Have Different Substrate-dependent Activities in Endoplasmic Reticulum-associated Degradation (ERAD).

    PubMed

    Sasset, Linda; Petris, Gianluca; Cesaratto, Francesca; Burrone, Oscar R

    2015-11-20

    Endoplasmic reticulum-associated degradation (ERAD) is an essential quality control mechanism of the folding state of proteins in the secretory pathway that targets unfolded/misfolded polypeptides for proteasomal degradation. The cytosolic p97/valosin-containing protein is an essential ATPase for degradation of ERAD substrates. It has been considered necessary during retro-translocation to extract proteins from the endoplasmic reticulum that are otherwise supposed to accumulate in the endoplasmic reticulum lumen. The activity of the p97-associated deubiquitinylase YOD1 is also required for substrate disposal. We used the in vivo biotinylation retro-translocation assay in mammalian cells under conditions of impaired p97 or YOD1 activity to directly discriminate their requirements and diverse functions in ERAD. Using different ERAD substrates, we found that both proteins participate in two distinct retro-translocation steps. For CD4 and MHC-Iα, which are induced to degradation by the HIV-1 protein Vpu and by the CMV immunoevasins US2 and US11, respectively, p97 and YOD1 have a retro-translocation-triggering role. In contrast, for three other spontaneous ERAD model substrates (NS1, NHK-α1AT, and BST-2/Tetherin), p97 and YOD1 are required in the downstream events of substrate deglycosylation and proteasomal degradation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. [Effect and mechanism of endoplasmic reticulum stress on cisplatin resistance in ovarian carcinoma].

    PubMed

    Tian, Jing; Hu, Xiaoming; Qu, Quanxin

    2014-05-01

    The study intended to investigate the effect and mechanism of endoplasmic reticulum stress on cisplatin resistance in ovarian carcinoma. RT-PCR and Western blot were used to test the expression of mTOR and Beclin1 mRNA and protein in ovarian cancer SKOV3 cells after saquinavir induction. MTT assay was used to analyze the influence of saquinavir on cisplatin sensitivity in SKOV3 cells. The IC50 of SKOV3 cells was (5.490 ± 1.148) µg/ml. After induced by Saquinavair 10 µmol/L and 20 µmol/L, the IC50 of SKOV3 cells was increased to (11.199 ± 0.984) µg/ml and (14.906 ± 2.015) µg/ml, respectively. It suggested that the sensitivity of ovarian cancer cells to cisplatin was decreased significantly (P = 0.001). The expression of mTOR and Beclin1 mRNA and protein was significantly different among the five groups: the (Saquinavair+DDP) group of, Saquinavair group, LY294002 group, DDP group and control group (P < 0.001) . The expressions of mTOR and Beclin1 mRNA were highest in the (Saquinavair+DDP) group, 0.684 ± 0.072 and 0.647 ± 0.047, respectively; Secondly, the Saquinavair group, 0.577 ± 0.016 and 0.565 ± 0.037, respectively. The expressions of mTOR and Beclin1 proteins were also highest in the (Saquinavair+DDP) group, 0.624 ± 0.058 and 0.924 ± 0.033, respectively, followed by the Saquinavair group, 0.544 ± 0.019 and 0.712 ± 0.024. 3-MA inhibited the autophagy and restored cisplatin sensitivity in the SKOV3 cells after Saquinavir induced ER stress (P < 0.001). Saquinavir can effectively induce endoplasmic reticulum stress in SKOV3 cells. Endoplasmic reticulum stress can decrease the sensitivity to cisplatin in SKOV3 cells. The mechanism of the decrease of sensitivity to cisplatin in SKOV3 cells may be that ERS regulates cell autophagy through the mTOR and Beclin1 pathways. ERS of tumor cells and autophagy may become a new target to improve the therapeutic effect of chemotherapy and to reverse the drug resistance in tumor treatment.

  9. Rheumatoid arthritis as a hyper-endoplasmic-reticulum-associated degradation disease.

    PubMed

    Yamasaki, Satoshi; Yagishita, Naoko; Tsuchimochi, Kaneyuki; Nishioka, Kusuki; Nakajima, Toshihiro

    2005-01-01

    We introduce Synoviolin as a novel pathogenic factor in rheumatoid arthritis (RA). Experimental studies indicate that this endoplasmic reticulum (ER)-resident E3 ubiquitin ligase has important functions in the ER-associated degradation (ERAD) system, an essential system for ER homeostasis. Overexpression of Synoviolin in mice causes arthropathy with synovial hyperplasia, whereas heterozygous knockdown results in increased apoptosis of synovial cells and resistance to collagen-induced arthritis in mice. On the basis of these experimental data, we propose that excess elimination of unfolded proteins (that is, 'hyper-ERAD') by overexpression of Synoviolin triggers synovial cell overgrowth and hence a worsening of RA. Further analysis of the hyper-ERAD system may permit the complex pathomechanisms of RA to be uncovered.

  10. Inositol trisphosphate modification of ion transport in rough endoplasmic reticulum.

    PubMed

    Muallem, S; Schoeffield, M; Pandol, S; Sachs, G

    1985-07-01

    The ion transport properties of the rough endoplasmic reticulum (RER) from liver have been defined by using measurements of active and potential gradient-driven transport. The Ca2+ pump is shown to be electrogenic, and both ATP and potential difference is able to drive vanadate-inhibitable Ca2+ uptake into the RER. ATP-dependent Ca2+ transport into the RER depends on the presence of tetraethylammonium-sensitive cation conductance and a furosemide-inhibited cation/chloride cotransport pathway. Inositol trisphosphate does not affect either of the monovalent ion translocation systems but activates a Ca2+ conductance in the RER, allowing efflux of RER Ca2+ stores into the cytosol in exchange for K+ uptake.

  11. Inositol trisphosphate modification of ion transport in rough endoplasmic reticulum.

    PubMed Central

    Muallem, S; Schoeffield, M; Pandol, S; Sachs, G

    1985-01-01

    The ion transport properties of the rough endoplasmic reticulum (RER) from liver have been defined by using measurements of active and potential gradient-driven transport. The Ca2+ pump is shown to be electrogenic, and both ATP and potential difference is able to drive vanadate-inhibitable Ca2+ uptake into the RER. ATP-dependent Ca2+ transport into the RER depends on the presence of tetraethylammonium-sensitive cation conductance and a furosemide-inhibited cation/chloride cotransport pathway. Inositol trisphosphate does not affect either of the monovalent ion translocation systems but activates a Ca2+ conductance in the RER, allowing efflux of RER Ca2+ stores into the cytosol in exchange for K+ uptake. PMID:3874400

  12. The endoplasmic reticulum: structure, function and response to cellular signaling.

    PubMed

    Schwarz, Dianne S; Blower, Michael D

    2016-01-01

    The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, sheets and the nuclear envelope. Several proteins that contribute to the overall architecture and dynamics of the ER have been identified, but many questions remain as to how the ER changes shape in response to cellular cues, cell type, cell cycle state and during development of the organism. Here we discuss what is known about the dynamics of the ER, what questions remain, and how coordinated responses add to the layers of regulation in this dynamic organelle.

  13. Murine Intracisternal A Type Particles Fail to Separate from the Membrane of the Endoplasmic Reticulum

    PubMed Central

    Perk, Kalman; Dahlberg, John E.

    1974-01-01

    Analysis of serial sections of murine cells containing intracisternal A particles revealed that over 99% of all A particles remain in a budding configuration. This indicates that these particles fail to detach from the membrane of the endoplasmic reticulum. This observation explains how, despite their intracellular abundance in certain murine tumors, no extracellular A-type particles can be found. Images PMID:4431082

  14. The Endoplasmic Reticulum-Associated Degradation Pathways of Budding Yeast

    PubMed Central

    Thibault, Guillaume; Ng, Davis T.W.

    2012-01-01

    Protein misfolding is a common cellular event that can produce intrinsically harmful products. To reduce the risk, quality control mechanisms are deployed to detect and eliminate misfolded, aggregated, and unassembled proteins. In the secretory pathway, it is mainly the endoplasmic reticulum-associated degradation (ERAD) pathways that perform this role. Here, specialized factors are organized to monitor and process the folded states of nascent polypeptides. Despite the complex structures, topologies, and posttranslational modifications of client molecules, the ER mechanisms are the best understood among all protein quality-control systems. This is the result of convergent and sometimes serendipitous discoveries by researchers from diverse fields. Although major advances in ER quality control and ERAD came from all model organisms, this review will focus on the discoveries culminating from the simple budding yeast. PMID:23209158

  15. Ticagrelor protects against AngII-induced endothelial dysfunction by alleviating endoplasmic reticulum stress.

    PubMed

    Wang, Xiaoyu; Han, Xuejie; Li, Minghui; Han, Yu; Zhang, Yun; Zhao, Shiqi; Li, Yue

    2018-05-16

    Ticagrelor has been reported to decrease cardiovascular mortality compared with clopidogrel. This benefit cannot be fully explained by the more efficient platelet inhibition. Many studies demonstrated that ticagrelor improved endothelial function, leaving the mechanism elusive though. The present study aims to investigate whether ticagrelor protects against endothelial dysfunction induced by angiotensinII (AngII) through alleviating endoplasmic reticulum (ER) stress. Male Sprague Dawley rats were infused with AngII or vehicle and administrated with ticagrelor or vehicle for 14 days. Reactive oxygen species (ROS) was detected. Aortas from normal mice were incubated with endoplasmic reticulum stress inducer tunicamycin with or without ticagrelor. Vasorecactivity was measured on wire myography. Rat aortic endothelial cells (RAECs) were pretreated with ticagrelor followed by AngII or tunicamycin. Endothelial nitric oxide synthase (eNOS) phosphorylation and ER stress markers were determined by western blotting. Impaired endothelial function, induction of ER stress, reduced eNOS phosphorylation and elevated ROS generation was restored by ticagrelor treatment in vivo. In addition, tunicamycin induced endothelial dysfunction was improved by ticagrelor. In vitro, the induction of ER stress and inhibited eNOS phosphorylation in REACs exposed to AngII as well as tunicamycin was reversed by co-culturing with ticagrelor. In conclusion, ticagrelor protects against AngII-induced endothelial dysfunction via alleviating ER stress. Copyright © 2017. Published by Elsevier Inc.

  16. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

    PubMed Central

    Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

  17. Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes.

    PubMed

    Suzuki, Akiko; Endo, Takeshi

    2002-02-06

    We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.

  18. Regulation of Protein Secretion Through Controlled Aggregation in the Endoplasmic Reticulum

    NASA Astrophysics Data System (ADS)

    Rivera, Victor M.; Wang, Xiurong; Wardwell, Scott; Courage, Nancy L.; Volchuk, Allen; Keenan, Terence; Holt, Dennis A.; Gilman, Michael; Orci, Lelio; Cerasoli, Frank; Rothman, James E.; Clackson, Tim

    2000-02-01

    A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

  19. Hydrogen Gas Attenuates Myocardial Ischemia Reperfusion Injury Independent of Postconditioning in Rats by Attenuating Endoplasmic Reticulum Stress-Induced Autophagy.

    PubMed

    Gao, Yunan; Yang, Hongxiao; Chi, Jing; Xu, Qiannan; Zhao, Luqi; Yang, Weijia; Liu, Weifan; Yang, Wei

    2017-01-01

    To study the effect of inhaling hydrogen gas on myocardial ischemic/reperfusion(I/R) injury in rats. Seventy male Wistar albino rats were divided into five groups at random as the sham group (Sham). The I/R group (I/R), The ischemic postconditioning group (IPo), The I/R plus hydrogen group (IH2) and the ischemic postconditioning plus hydrogen group (IPoH2). The Sham group was without coronary occlusion. In I/R group, Ischemic/reperfusion injury was induced by coronary occlusion for 1 hour. Followed by 2 hours of reperfusion. In the IPo and IPoH2 group, four cycles of 1 min reperfusion/1 min ischemia was given at the end of 1 hour coronary occlusion. While 2% hydrogen was administered by inhalation 5 min before reperfusion till 2 hours after reperfusion in both the IPoH2 and IH2 group. The heart and blood samples were harvested at the end of the surgical protocol. Then the myocardium cell endoplasmic reticulum(ER) stress and autophagy was observed by electron microscope. In addition, the cardiac ER stress and autophagy related proteins expression were detected by Western blotting analysis. Both inhaling 2% hydrogen and ischemic postconditioning treatment reduced the ischemic size and serum troponin I level in rats with I/R injury, and inhaling hydrogen showed a more curative effect compared with ischemic postconditioning treatment. Meanwhile inhaling hydrogen showed a better protective effect in attenuating tissue reactive oxygen species. Malondialdehyde levels and immunoreactivities against 8-hydroxy-2'-deoxyguanosine and inhibiting cardiac endoplasmic reticulum stress and down-regulating autophagy as compared with ischemic postconditioning treatment. These results revealed a better protective effect of hydrogen on myocardial ischemic/reperfusion injury in rats by attenuating endoplasmic reticulum stress and down-regulating autophagy compared with ischemic postconditioning treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

  20. Bacteria, the endoplasmic reticulum and the unfolded protein response: friends or foes?

    PubMed

    Celli, Jean; Tsolis, Renée M

    2015-02-01

    The unfolded protein response (UPR) is a cytoprotective response that is aimed at restoring cellular homeostasis following physiological stress exerted on the endoplasmic reticulum (ER), which also invokes innate immune signalling in response to invading microorganisms. Although it has been known for some time that the UPR is modulated by various viruses, recent evidence indicates that it also has multiple roles during bacterial infections. In this Review, we describe how bacteria interact with the ER, including how bacteria induce the UPR, how subversion of the UPR promotes bacterial proliferation and how the UPR contributes to innate immune responses against invading bacteria.

  1. Protein-accumulating cells and dilated cisternae of the endoplasmic reticulum in three glucosinolate-containing genera: Armoracia, Capparis, Drypetes.

    PubMed

    Jørgensen, L B; Behnke, H D; Mabry, T J

    1977-01-01

    Three glucosinolate-containing species, Armoracia rusticana Gaertner, Meyer et Scherbius (Brassicaceae), Capparis cynophallophora L. (Capparaceae) and Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae), are shown by both light and electron microscopy to contain protein-accumulating cells (PAC). The PAC of Armoracia and Copparis (former "myrosin cells") occur as idioblasts. The PAC of Drypetes are usual members among axial phloem parenchyma cells rather than idioblasts. In Drypetes the vacuoles of the PAC are shown ultrastructurally to contain finely fibrillar material and to originate from local dilatations of the endoplasmic reticulum. The vacuoles in PAC of Armoracia and Capparis seem to originate in the same way; but ultrastructurally, their content is finely granular. In addition, Armoracia and Capparis are shown by both light and electron microscopy to contain dilated cisternae (DC) of the endoplasmic reticulum in normal parenchyma cells, in accord with previous findings for several species within Brassicaceae. The relationship of PAC and DC to glucosinolates and the enzyme myrosinase is discussed.

  2. Interaction of Mitochondria with the Endoplasmic Reticulum and Plasma Membrane in Calcium Homeostasis, Lipid Trafficking and Mitochondrial Structure.

    PubMed

    Szymański, Jędrzej; Janikiewicz, Justyna; Michalska, Bernadeta; Patalas-Krawczyk, Paulina; Perrone, Mariasole; Ziółkowski, Wiesław; Duszyński, Jerzy; Pinton, Paolo; Dobrzyń, Agnieszka; Więckowski, Mariusz R

    2017-07-20

    Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca 2 + handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism.

  3. Cytochrome P450 Organization and Function Are Modulated by Endoplasmic Reticulum Phospholipid Heterogeneity.

    PubMed

    Brignac-Huber, Lauren M; Park, Ji Won; Reed, James R; Backes, Wayne L

    2016-12-01

    Cytochrome P450s (P450s) comprise a superfamily of proteins that catalyze numerous monooxygenase reactions in animals, plants, and bacteria. In eukaryotic organisms, these proteins not only carry out reactions necessary for the metabolism of endogenous compounds, but they are also important in the oxidation of exogenous drugs and other foreign compounds. Eukaryotic P450 system proteins generally reside in membranes, primarily the endoplasmic reticulum or the mitochondrial membrane. These membranes provide a scaffold for the P450 system proteins that facilitate interactions with their redox partners as well as other P450s. This review focuses on the ability of specific lipid components to influence P450 activities, as well as the role of the membrane in P450 function. These studies have shown that P450s and NADPH-cytochrome P450 reductase appear to selectively associate with specific phospholipids and that these lipid-protein interactions influence P450 activities. Finally, because of the heterogeneous nature of the endoplasmic reticulum as well as other biologic membranes, the phospholipids are not arranged randomly but associate to generate lipid microdomains. Together, these characteristics can affect P450 function by 1) altering the conformation of the proteins, 2) influencing the P450 interactions with their redox partners, and 3) affecting the localization of the proteins into specific membrane microdomains. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Anomalous Diffusion Reports on the Interaction of Misfolded Proteins with the Quality Control Machinery in the Endoplasmic Reticulum

    PubMed Central

    Malchus, Nina; Weiss, Matthias

    2010-01-01

    A multitude of transmembrane proteins enters the endoplasmic reticulum (ER) as unfolded polypeptide chains. During their folding process, they interact repetitively with the ER's quality control machinery. Here, we have used fluorescence correlation spectroscopy to probe these interactions for a prototypical transmembrane protein, VSVG ts045, in vivo. While both folded and unfolded VSVG ts045 showed anomalous diffusion, the unfolded protein had a significantly stronger anomaly. This difference subsided when unfolded VSVG ts045 was in a complex with its chaperone calnexin, or when a mutant form of VSVG ts045 with only one glycan was used. Our experimental data and accompanying simulations suggest that the folding sensor of the quality control (UGT1) oligomerizes unfolded VSVG ts045, leading to a more anomalous/obstructed diffusion. In contrast, calnexin dissolves the oligomers, rendering unfolded VSVG ts045 more mobile, and hence prevents poisoning of the ER. PMID:20713018

  5. Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly.

    PubMed

    Romero-Brey, Inés; Bartenschlager, Ralf

    2016-06-07

    The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles.

  6. Endoplasmic reticulum stress is induced in the human placenta during labour

    PubMed Central

    Veerbeek, J.H.W.; Tissot Van Patot, M.C.; Burton, G.J.; Yung, H.W.

    2015-01-01

    Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in vitro. Here, we explored whether exposure to labour represents an in vivo model for the study of acute placental ER stress. ER stress markers, GRP78, P-eIF2α and XBP-1, were significantly higher in laboured placentas than in Caesarean-delivered controls localised mainly in the syncytiotrophoblast. The similarities to changes observed in PE/IUGR placentas suggest exposure to labour can be used to investigate induction of ER stress in pathological placentas. PMID:25434970

  7. Endoplasmic Reticulum and the Unfolded Protein Response: Dynamics and Metabolic Integration

    PubMed Central

    Bravo, Roberto; Parra, Valentina; Gatica, Damián; Rodriguez, Andrea E.; Torrealba, Natalia; Paredes, Felipe; Wang, Zhao V.; Zorzano, Antonio; Hill, Joseph A.; Jaimovich, Enrique; Quest, Andrew F.G.; Lavandero, Sergio

    2013-01-01

    The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of reestablishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies. PMID:23317820

  8. A few positively charged residues slow movement of a polypeptide chain across the endoplasmic reticulum membrane.

    PubMed

    Yamagishi, Marifu; Onishi, Yukiko; Yoshimura, Shotaro; Fujita, Hidenobu; Imai, Kenta; Kida, Yuichiro; Sakaguchi, Masao

    2014-08-26

    Many polypeptide chains are translocated across and integrated into the endoplasmic reticulum membrane through protein-conducting channels. During the process, amino acid sequences of translocating polypeptide chains are scanned by the channels and classified to be retained in the membrane or translocated into the lumen. We established an experimental system with which the kinetic effect of each amino acid residue on the polypeptide chain movement can be analyzed with a time resolution of tens of seconds. Positive charges greatly slow movement; only two lysine residues caused a remarkable slow down, and their effects were additive. The lysine residue was more effective than arginine. In contrast, clusters comprising three residues of each of the other 18 amino acids had little effect on chain movement. We also demonstrated that a four lysine cluster can exert the effect after being fully exposed from the ribosome. We concluded that as few as two to three residues of positively charged amino acids can slow the movement of the nascent polypeptide chain across the endoplasmic reticulum membrane. This effect provides a fundamental basis of the topogenic function of positively charged amino acids.

  9. Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum

    PubMed Central

    1995-01-01

    It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin- like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP- Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides. PMID:7642696

  10. Early Neurodegeneration in the Brain of a Child Without Functional PKR-like Endoplasmic Reticulum Kinase.

    PubMed

    Bruch, Julius; Kurz, Carolin; Vasiljevic, Alexandre; Nicolino, Marc; Arzberger, Thomas; Höglinger, Günter U

    2015-08-01

    We report the first detailed examination of the brain of a patient with Wolcott-Rallison syndrome. Wolcott-Rallison syndrome is an extremely rare clinical manifestation of a lack of protein kinase R-like endoplasmic reticulum kinase (PERK) function caused by mutations in the PERK gene EIF2AK3. Protein kinase R-like endoplasmic reticulum kinase is thought to play a significant pathogenetic role in several neurodegenerative diseases, including Alzheimer disease, other tauopathies, and Parkinson disease. The brain of a male patient aged 4 years 7 months showed pathologic and immunohistochemical evidence that the absence of PERK for several years is sufficient to induce early changes reminiscent of various neurodegenerative conditions. These include neurofibrillary tangles (as in progressive supranuclear palsy), FUS-immunopositive and p62-immunopositive neurons, and reactive glial changes. We also detected an increased amount of p62-positive puncta coimmunostaining for LC3 and ubiquitin, suggesting changes in autophagic flux. Studying a human brain with absent PERK function presents the opportunity to assess the long-term consequences of nonfunctioning of PERK in the presence of all of the compensatory mechanisms that are normally active in a living human, thereby confirming the importance of PERK for autophagy in the brain and for neurodegeneration.

  11. Form follows function: the importance of endoplasmic reticulum shape.

    PubMed

    Westrate, L M; Lee, J E; Prinz, W A; Voeltz, G K

    2015-01-01

    The endoplasmic reticulum (ER) has a remarkably complex structure, composed of a single bilayer that forms the nuclear envelope, along with a network of sheets and dynamic tubules. Our understanding of the biological significance of the complex architecture of the ER has improved dramatically in the last few years. The identification of proteins and forces required for maintaining ER shape, as well as more advanced imaging techniques, has allowed the relationship between ER shape and function to come into focus. These studies have also revealed unexpected new functions of the ER and novel ER domains regulating alterations in ER dynamics. The importance of ER structure has become evident as recent research has identified diseases linked to mutations in ER-shaping proteins. In this review, we discuss what is known about the maintenance of ER architecture, the relationship between ER structure and function, and diseases associated with defects in ER structure.

  12. 4-Phenylbutyrate Benefits Traumatic Hemorrhagic Shock in Rats by Attenuating Oxidative Stress, Not by Attenuating Endoplasmic Reticulum Stress.

    PubMed

    Yang, Guangming; Peng, Xiaoyong; Hu, Yi; Lan, Dan; Wu, Yue; Li, Tao; Liu, Liangming

    2016-07-01

    Vascular dysfunction such as vascular hyporeactivity following severe trauma and shock is a major cause of death in injured patients. Oxidative stress and endoplasmic reticulum stress play an important role in vascular dysfunction. The objective of the present study was to determine whether or not 4-phenylbutyrate can improve vascular dysfunction and elicit antishock effects by inhibiting oxidative and endoplasmic reticulum stress. Prospective, randomized, controlled laboratory experiment. State key laboratory of trauma, burns, and combined injury. Five hundred and fifty-two Sprague-Dawley rats. Rats were anesthetized, and a model of traumatic hemorrhagic shock was established by left femur fracture and hemorrhage. The effects of 4-phenylbutyrate (5, 20, 50, 100, 200, and 300 mg/kg) on vascular reactivity, animal survival, hemodynamics, and vital organ function in traumatic hemorrhagic shock rats and cultured vascular smooth muscle cells, and the relationship to oxidative stress and endoplasmic reticulum stress was observed. Lower doses of 4-phenylbutyrate significantly improved the vascular function, stabilized the hemodynamics, and increased the tissue blood flow and vital organ function in traumatic hemorrhagic shock rats, and markedly improved the survival outcomes. Among all dosages observed in the present study, 20 mg/kg of 4-phenylbutyrate had the best effect. Further results indicated that 4-phenylbutyrate significantly inhibited the oxidative stress, decreased shock-induced oxidative stress index such as the production of reactive oxygen species, increased the antioxidant enzyme levels such as superoxide dismutase, catalase, and glutathione, and improved the mitochondrial function by inhibiting the opening of the mitochondrial permeability transition pore in rat artery and vascular smooth muscle cells. In contrast, 4-phenylbutyrate did not affect the changes of endoplasmic reticulum stress markers following traumatic hemorrhagic shock. Furthermore, 4

  13. Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

    PubMed Central

    Ji, Wei-Ke; Fan, Xintao; Strack, Stefan

    2017-01-01

    Drp1 is a dynamin guanosine triphosphatase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51, and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is associated with ER in mammalian cells and is distinct from mitochondrial or peroxisomal Drp1 populations. Subpopulations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, and ER bound) and mitochondrial division, whereas Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division. PMID:29158231

  14. Lactobacillus acidophilus suppresses intestinal inflammation by inhibiting endoplasmic reticulum stress.

    PubMed

    Kim, Da Hye; Kim, Soochan; Lee, Jin Ha; Kim, Jae Hyeon; Che, Xiumei; Ma, Hyun Woo; Seo, Dong Hyuk; Kim, Tae Il; Kim, Won Ho; Kim, Seung Won; Cheon, Jae Hee

    2018-06-22

    Nuclear factor kappa B (NF-κB) activation and endoplasmic reticulum (ER) stress signaling play significant roles in the pathogenesis of inflammatory bowel disease (IBD). Thus, we evaluated whether new therapeutic probiotics have anti-colitic effects and we investigated their mechanisms related to NF-κB and ER-stress pathways. Luciferase, nitric oxide (NO), and cytokine assays using HT-29 or RAW264.7 cells were conducted. Mouse colitis was induced using dextran sulfate sodium (DSS) and confirmed by disease activity index and histology. Macrophages and T-cell subsets in isolated peritoneal cavity cells (PCCs) and splenocytes were analyzed by flow cytometry. Gene and cytokine expression profiles were determined using RT-PCR. Lactobacillus acidophilus (LA1) and Pediococcus pentosaceus inhibited NO production in RAW264.7 cells, but only LA1 inhibited Tnfa and induced Il10 expression. LA1 increased the life span of DSS-treated mice and attenuated the severity of colitis by inducing M2 macrophages in PCCs and Th2 and Treg cells in splenocytes. The restoration of goblet cells in the colon was accompanied by the induction of Il10 expression and the suppression of proinflammatory cytokines. Additionally, we found that LA1 exerts an anti-colitic effect by improving ER stress in HT-29 cells as well as in vivo. We showed that LA1 significantly interferes with ER stress and suppresses NF-κB activation. Our findings suggest that LA1 can be used as a potent immunomodulator in IBD treatment and the regulation of ER stress may have significant implications in treating IBD. This article is protected by copyright. All rights reserved.

  15. Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt

    PubMed Central

    Marini, Cecilia; Ravera, Silvia; Buschiazzo, Ambra; Bianchi, Giovanna; Orengo, Anna Maria; Bruno, Silvia; Bottoni, Gianluca; Emionite, Laura; Pastorino, Fabio; Monteverde, Elena; Garaboldi, Lucia; Martella, Roberto; Salani, Barbara; Maggi, Davide; Ponzoni, Mirco; Fais, Franco; Raffaghello, Lizzia; Sambuceti, Gianmario

    2016-01-01

    Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This “Warburg effect” represents a standard to diagnose and monitor tumor aggressiveness with 18F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that 18F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy. PMID:27121192

  16. Ovalbumin-derived precursor peptides are transferred sequentially from gp96 and calreticulin to MHC I in the endoplasmic reticulum

    PubMed Central

    Kropp, Laura E.; Garg, Manish; Binder, Robert J.

    2010-01-01

    Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC I are associated with several chaperones. Hsp70, hsp90 and the TCP1-ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC I and hence presentation of the MHC I-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC I heavy chains and β2-microglobulin as a trimolecular complex. PMID:20410492

  17. Oxidative Processing of Latent Fas in the Endoplasmic Reticulum Controls the Strength of Apoptosis

    PubMed Central

    Anathy, Vikas; Roberson, Elle; Cunniff, Brian; Nolin, James D.; Hoffman, Sidra; Spiess, Page; Guala, Amy S.; Lahue, Karolyn G.; Goldman, Dylan; Flemer, Stevenson; van der Vliet, Albert; Heintz, Nicholas H.; Budd, Ralph C.; Tew, Kenneth D.

    2012-01-01

    We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241–252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis. PMID:22751926

  18. A plasma membrane-type Ca[sup 2+]-ATPase of 120 kilodaltons on the endoplasmic reticulum from carrot (Daucus carota) cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, F.H.; Ratterman, D.M.; Sze, H.

    1993-06-01

    Cytosolic Ca[sup 2+] levels are regulated in part by Ca[sup 2+]-pumping ATPases that export Ca[sup 2+] from the cytoplasm; The types and properties of Ca[sup 2+] pumps in plants are not well understood. The kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca[sup 2+]-ATPase from suspension-cultured carrot (Daucus carota) cells are characterized. Only one Ca[sup 2+]-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [[gamma]-[sup 32]P]ATP. Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La[sup 3+], and decreased by hydroxylamine, confirming its identification as an intermediate of amore » phosphorylated-type Ca[sup 2+]-translocating ATPase. The 120-kD Ca[sup 2+]-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca[sup 2+]-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca[sup 2+]-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a K[sub m] for Ca[sup 2+] of 1 to 2 [mu]m and a K[sub m] for ATP of 67 nm. Relative affinities of substrates, determined by competition experiments, were 0.075 [mu]m for ATP, 1 [mu]m for ADP, 100 [mu]m for ITP, and 250 [mu]m for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca[sup 2+]-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 [mu]m. Calmodulin (1 [mu]m) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca[sup 2+]-ATPase is similar but not identical to animal plasma membrane-type Ca[sup 2+]-ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca[sup 2+] pump may reside on the cortical endoplasmic reticulum, thought to play a major role in anchoring the cytoskeleton and in facilitating secretion. 34 refs., 9 figs., 3 tabs.« less

  19. Retrograde traffic from the Golgi to the endoplasmic reticulum.

    PubMed

    Spang, Anne

    2013-06-01

    Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels--SNARE proteins--to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years.

  20. Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

    PubMed Central

    Spang, Anne

    2013-01-01

    Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels—SNARE proteins—to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years. PMID:23732476

  1. The Role of Endoplasmic Reticulum Stress in Human Pathology

    PubMed Central

    Oakes, Scott A.; Papa, Feroz R.

    2017-01-01

    Numerous genetic and environmental insults impede the ability of cells to properly fold and posttranslationally modify secretory and transmembrane proteins in the endoplasmic reticulum (ER), leading to a buildup of misfolded proteins in this organelle—a condition called ER stress. ER-stressed cells must rapidly restore protein-folding capacity to match protein-folding demand if they are to survive. In the presence of high levels of misfolded proteins in the ER, an intracellular signaling pathway called the unfolded protein response (UPR) induces a set of transcriptional and translational events that restore ER homeostasis. However, if ER stress persists chronically at high levels, a terminal UPR program ensures that cells commit to self-destruction. Chronic ER stress and defects in UPR signaling are emerging as key contributors to a growing list of human diseases, including diabetes, neurodegeneration, and cancer. Hence, there is much interest in targeting components of the UPR as a therapeutic strategy to combat these ER stress–associated pathologies. PMID:25387057

  2. STIM proteins and the endoplasmic reticulum-plasma membrane junctions.

    PubMed

    Carrasco, Silvia; Meyer, Tobias

    2011-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

  3. Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides.

    PubMed

    Ito, Yukishige; Takeda, Yoichi

    2012-01-01

    Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules.

  4. Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

    PubMed Central

    Romero-Brey, Inés; Bartenschlager, Ralf

    2016-01-01

    The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles. PMID:27338443

  5. Endoplasmic reticulum stress is induced in the human placenta during labour.

    PubMed

    Veerbeek, J H W; Tissot Van Patot, M C; Burton, G J; Yung, H W

    2015-01-01

    Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in vitro. Here, we explored whether exposure to labour represents an in vivo model for the study of acute placental ER stress. ER stress markers, GRP78, P-eIF2α and XBP-1, were significantly higher in laboured placentas than in Caesarean-delivered controls localised mainly in the syncytiotrophoblast. The similarities to changes observed in PE/IUGR placentas suggest exposure to labour can be used to investigate induction of ER stress in pathological placentas. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Hydrogen-rich saline attenuates hippocampus endoplasmic reticulum stress after cardiac arrest in rats.

    PubMed

    Gao, Yu; Gui, Qinfang; Jin, Li; Yu, Pan; Wu, Lin; Cao, Liangbin; Wang, Qiang; Duan, Manlin

    2017-02-15

    Hydrogen-rich saline can selectively scavenge reactive oxygen species (ROS) and protect brain against ischemia reperfusion (I/R) injury. Endoplasmic reticulum stress (ERS) has been implicated in the pathological process of cerebral ischemia. However, very little is known about the role of hydrogen-rich saline in mediating pathophysiological reactions to ERS after I/R injury caused by cardiac arrest. The rats were randomly divided into three groups, sham group (n=30), ischemia/reperfusion group (n=40) and hydrogen-rich saline group (n=40). The rats in experimental groups were subjected to 4min of cardiac arrest and followed by resuscitation. Then they were randomized to receive 5ml/kg of either hydrogen-rich saline or normal saline. Hydrogen-rich saline significantly improves survival rate and neurological function. The beneficial effects of hydrogen-rich saline were associated with decreased levels of oxidative products, as well as the increased levels of antioxidant enzymes. Furthermore, the protective effects of hydrogen-rich saline were accompanied by the increased activity of glucose-regulated protein 78 (GRP78), the decreased activity of cysteinyl aspartate specific proteinase-12 (caspase-12) and C/EBP homologous protein (CHOP). Hydrogen-rich saline attenuates brain I/R injury may through inhibiting hippocampus ERS after cardiac arrest in rats. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Suppression of the endoplasmic reticulum calcium pump during zebrafish gastrulation affects left-right asymmetry of the heart and brain.

    PubMed

    Kreiling, Jill A; Balantac, Zaneta L; Crawford, Andrew R; Ren, Yuexin; Toure, Jamal; Zchut, Sigalit; Kochilas, Lazaros; Creton, Robbert

    2008-01-01

    Vertebrate embryos generate striking Ca(2+) patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca(2+) during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca(2+) along the dorsal-ventral axis, with higher Ca(2+) concentrations in the ventral margin and lower Ca(2+) concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca(2+) pump with 0.5 microM thapsigargin elevates cytosolic Ca(2+) in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left-right asymmetry. Brain defects include a left-right reversal of pitx2 expression in the dorsal diencephalon and a left-right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca(2+) pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca(2+) pump during gastrulation inhibits expression of no tail (ntl) and left-right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer's vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca(2+) plays a role in Kupffer's vesicle function, influencing ciliary motility and translating the vesicle's counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca(2+) plays an additional role in the formation of Kupffer's vesicle.

  8. Decursin enhances TRAIL-induced apoptosis through oxidative stress mediated- endoplasmic reticulum stress signalling in non-small cell lung cancers.

    PubMed

    Kim, Jaekwang; Yun, Miyong; Kim, Eun-Ok; Jung, Deok-Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon; Kim, Sung-Hoon

    2016-03-01

    The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL-induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL-resistant non-small-cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer-binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down-regulated expression of survivin and Bcl-xL in TRAIL-resistant NSCLC cells. ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL-resistant NSCLC cell lines, partly via up-regulation of DR5. © 2015 The British Pharmacological Society.

  9. Decursin enhances TRAIL‐induced apoptosis through oxidative stress mediated‐ endoplasmic reticulum stress signalling in non‐small cell lung cancers

    PubMed Central

    Kim, Jaekwang; Yun, Miyong; Kim, Eun‐Ok; Jung, Deok‐Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon

    2016-01-01

    Background and Purpose The TNF‐related apoptosis‐inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL‐induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Experimental Approach Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Key Results Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL‐resistant non‐small‐cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer‐binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down‐regulated expression of survivin and Bcl‐xL in TRAIL‐resistant NSCLC cells. Conclusions and Implications ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL‐resistant NSCLC cell lines, partly via up‐regulation of DR5. PMID:26661339

  10. Cyclosporine A-Sensitive, Cyclophilin B-Dependent Endoplasmic Reticulum-Associated Degradation

    PubMed Central

    Luban, Jeremy; Molinari, Maurizio

    2010-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIs) catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER)-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA) selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB) as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-LS substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells. PMID:20927389

  11. Endoplasmic Reticulum Stress in Sepsis

    PubMed Central

    Khan, Mohammad Moshahid; Yang, Weng-Lang; Wang, Ping

    2015-01-01

    Sepsis is an enormous public health issue and the leading cause of death in critically ill patients in intensive care units (ICU). Overwhelming inflammation, characterized by cytokine storm, oxidative threats, and neutrophil sequestration is an underlying component of sepsis-associated organ failure. Despite recent advances in sepsis research, there is still no effective treatment available beyond the standard of care and supportive therapy. To reduce sepsis-related mortality, a better understanding of the biological mechanism associated with the sepsis is essential. Endoplasmic reticulum (ER), a subcellular organelle is responsible for the facilitation of protein folding and assembly and involved in several other physiological activities. Under the stress and inflammation condition, ER loses the homeostasis in its function, which is termed as ER stress. During ER stress, unfolded protein response (UPR) is activated to restore ER function to its normal balance. However, once the stress is beyond the compensatory capacity of UPR or protracted, the apoptosis would be initiated by triggering cell injuries, even to cell death. As such, ER stress and UPR are reported to be implicated in several pathological and inflammatory conditions. Although the detrimental role of ER stress during infections has been demonstrated, there is growing evidences that ER stress participate in the pathogenesis of sepsis. In this review, we summarize the current research in the context of ER stress and UPR signaling associated with sepsis and its related clinical conditions, such as trauma- hemorrhage, and ischemia/reperfusion (I/R) injury. We also discuss the potential implication of ER stress as a novel therapeutic target and prognostic marker in patients with sepsis. PMID:26125088

  12. Endoplasmic reticulum mediated signaling in cellular microdomains

    PubMed Central

    Biwer, Lauren; Isakson, Brant E

    2016-01-01

    The endoplasmic reticulum (ER) is a prime mediator of cellular signaling due to its functions as an internal cellular store for calcium, as well as a site for synthesis of proteins and lipids. Its peripheral network of sheets and tubules facilitate calcium and lipid signaling, especially in areas of the cell that are more distant to the main cytoplasmic network. Specific membrane proteins shape the peripheral ER architecture and influence the network stability in order to project into restricted spaces. The signaling microdomains are anatomically separate from the cytoplasm as a whole and exhibit localized protein, ion channel and cytoskeletal element expression. Signaling can also occur between the ER and other organelles, such as the Golgi or mitochondria. Lipids made in the ER membrane can be sent to the Golgi via specialized transfer proteins and specific phospholipid synthases are enriched at ER-mitochondria junctions to more efficiently expedite phospholipid transfer. As a hub for protein and lipid synthesis, a store for intracellular calcium [Ca2+]i, and a mediator of cellular stress, the ER is an important cellular organelle. Its ability to organize into tubules and project into restricted spaces allows for discrete and temporal signaling, which is important for cellular physiology and organism homeostasis. PMID:26973141

  13. Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei.

    PubMed

    Ooi, Cher-Pheng; Smith, Terry K; Gluenz, Eva; Wand, Nadina Vasileva; Vaughan, Sue; Rudenko, Gloria

    2018-06-01

    The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi. © 2018 The Authors. Traffic published by John Wiley & Sons Ltd.

  14. p53 and Ca(2+) signaling from the endoplasmic reticulum: partners in anti-cancer therapies.

    PubMed

    Bittremieux, Mart; Bultynck, Geert

    2015-01-01

    Ca(2+) transfer from the endoplasmic reticulum (ER) to the mitochondria critically controls cell survival and cell death decisions. Different oncogenes and deregulation of tumor suppressors exploit this mechanism to favor the survival of altered, malignant cells. Two recent studies of the Pinton team revealed a novel, non-transcriptional function of cytosolic p53 in cell death. During cell stress, p53 is recruited to the ER and the ER-mitochondrial contact sites. This results in augmented ER Ca(2+) levels by enhancing sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity, ultimately promoting mitochondrial Ca(2+) overload. The boosting of "toxic" Ca(2+) signaling by p53 appears to be a critical component of the cell death-inducing properties of chemotherapeutic agents and anti-cancer treatments, like photodynamic stress. Strikingly, the resistance of p53-deficient cancer cells to these treatments could be overcome by facilitating Ca(2+) transfer between the ER and the mitochondria via overexpression of SERCA or of the mitochondrial Ca(2+) uniporter (MCU). Importantly, these concepts have also been supported by in vivo Ca(2+) measurements in tumor masses in mice. Collectively, these studies link for the first time the major tumor suppressor, p53, to Ca(2+) signaling in dictating cell-death outcomes and by the success of anti-cancer treatments.

  15. Thermal Shock Induces Host Proteostasis Disruption and Endoplasmic Reticulum Stress in the Model Symbiotic Cnidarian Aiptasia.

    PubMed

    Oakley, Clinton A; Durand, Elysanne; Wilkinson, Shaun P; Peng, Lifeng; Weis, Virginia M; Grossman, Arthur R; Davy, Simon K

    2017-06-02

    Coral bleaching has devastating effects on coral survival and reef ecosystem function, but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography-tandem mass spectrometry to compare the effects of rapidly (33.5 °C, 24 h) and gradually (30 and 33.5 °C, 12 days) elevated temperatures on the proteome of the model symbiotic anemone Aiptasia. We identified 2133 proteins in Aiptasia, 136 of which were differentially abundant between treatments. Thermal shock, but not acclimation, resulted in significant abundance changes in 104 proteins, including those involved in protein folding and synthesis, redox homeostasis, and central metabolism. Nineteen abundant structural proteins showed particularly reduced abundance, demonstrating proteostasis disruption and potential protein synthesis inhibition. Heat shock induced antioxidant mechanisms and proteins involved in stabilizing nascent proteins, preventing protein aggregation and degrading damaged proteins, which is indicative of endoplasmic reticulum stress. Host proteostasis disruption occurred before either bleaching or symbiont photoinhibition was detected, suggesting host-derived reactive oxygen species production as the proximate cause of thermal damage. The pronounced abundance changes in endoplasmic reticulum proteins associated with proteostasis and protein turnover indicate that these processes are essential in the cellular response of symbiotic cnidarians to severe thermal stress.

  16. Tributyltin-induced endoplasmic reticulum stress and its Ca{sup 2+}-mediated mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Isomura, Midori; Kotake, Yaichiro, E-mail: yaichiro@hiroshima-u.ac.jp; Masuda, Kyoichi

    2013-10-01

    Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca{sup 2+} signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca{sup 2+} homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers suchmore » as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca{sup 2+} depletion, and to test this idea, we examined the effect of TBT on intracellular Ca{sup 2+} concentration using fura-2 AM, a Ca{sup 2+} fluorescent probe. TBT increased intracellular Ca{sup 2+} concentration in a TBT-concentration-dependent manner, and Ca{sup 2+} increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca{sup 2+} concentration by releasing Ca{sup 2+} from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca{sup 2+} release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca{sup 2+}.« less

  17. Melatonin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress.

    PubMed

    Lin, Yu Wen; Chen, Tsung Ying; Hung, Chia Yang; Tai, Shih Huang; Huang, Sheng Yang; Chang, Che Chao; Hung, Hsin Yi; Lee, E Jian

    2018-07-01

    Endoplasmic reticulum (ER) stress plays a vital role in mediating ischemic reperfusion damage in brain. In this study, we evaluated whether melatonin inhibits ER stress in cultured neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to transient focal cerebral ischemia. Sprague-Dawley rats were treated with melatonin (5 mg/kg) or control at reperfusion onset after transient occlusion of the right middle cerebral artery (MCA) for 90 min. Brain infarction and hemorrhage within infarcts were measured. The expression of ER stress proteins of phosphorylation of PRKR‑like endoplasmic reticulum kinase (p-PERK), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by western blotting and immunohistochemistry analysis. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method, cleaved caspase-3 and cytochrome c were used to investigate cell apoptosis in OGD-induced cultured neurons. Our results demonstrated that animals treated with melatonin had significantly reduced infarction volumes and individual cortical lesion sizes as well as increased numbers of surviving neurons. Melatonin can significantly modulate protein levels by decreasing both p-PERK and p-eIF2α in the ischemic core and penumbra. Moreover, the expressions of ATF4 and CHOP were restrained in the ischemic core and penumbra, respectively. Furthermore, pretreatment with melatonin at 10-100 µM effectively reduced the levels of p-PERK and p-eIF2α in cultured neurons after OGD injury. Melatonin treatment also effectively decreased neuron apoptosis resulting from OGD-induced neuron injury. These results indicate that melatonin effectively attenuated post-ischemic ER stress after ischemic stroke.

  18. The Endoplasmic Reticulum: A Central Player in Cell Signalling and Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Llewelyn Roderick, H.; Berridge, Michael J.; Bootman, Martin D.

    In addition to being the principle intracellular Ca 2+ store, the endoplasmic reticulum (ER) is the initial site of synthesis and folding of membrane and secretory proteins. These two roles of the ER are intimately linked. First, the function of many proteins involved in Ca 2+ handling are modulated by Ca 2+ and second, ER lumenal Ca 2+ modulates protein synthesis and folding. Within the ER, Ca 2+ is stored by low affinity high capacity Ca 2+ binding proteins and is maintained at a free concentration between 0.1 and 1 μM relative to 0.1 μM cytosolic Ca 2+. This concentration gradient is maintained by the action of the Sarco-Endoplasmic Reticulum ATPases (SERCa) which hydrolyse ATP to pump Ca 2+ into the ER. Following stimulation Ca 2+ is released from the ER through several classes of ligand gated channels. The most well characterized of these being the inositol 1,4,5-trisphosphate receptor ( IP 3 R) and the Ryanodine receptor (RyR) families of proteins. This release of Ca 2+ results in a drop of ER free Ca 2+ to levels as low as 10 μM. This decrease in lumenal Ca 2+ inhibits further release through the channels and increases the rate of re-sequestration of Ca 2+ into the ER by the SERCa pumps. Under these conditions, in addition to effects on Ca 2+ handling proteins, protein synthesis is inhibited, chaperones dissociate from their substrates, secondary modifications of proteins are inhibited and the retention of many proteins within the ER is lost. Furthermore, a signalling cascade resulting in the up-regulation of many proteins involved in protein folding and Ca 2+ homeostasis is initiated. This review will focus on the proteins involved in the regulation ER lumenal Ca 2+ and the role of ER lumenal Ca 2+ in cell signalling and protein synthesis.

  19. A physical/psychological and biological stress combine to enhance endoplasmic reticulum stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mondal, Tapan Kumar; Emeny, Rebecca T.; Gao, Donghong

    The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2 h, host defenses against bacterial growth are suppressed 2–3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidationmore » (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes. - Highlights: • Cold-restraint (physical/psychological stress) induces early oxidative stress. • The oxidative stress relates to catecholamine signaling beta-adrenoceptors. • Physical/psychological stress combines infection enhancing inflammation. • Endoplasmic

  20. Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress.

    PubMed

    Takayanagi, Sayuri; Fukuda, Riga; Takeuchi, Yuuki; Tsukada, Sakiko; Yoshida, Kenichi

    2013-01-01

    In the endoplasmic reticulum (ER), secretory and membrane proteins are properly folded and modified, and the failure of these processes leads to ER stress. At the same time, unfolded protein response (UPR) genes are activated to maintain homeostasis. Despite the thorough characterization of the individual gene regulation of UPR genes to date, further investigation of the mutual regulation among UPR genes is required to understand the complex mechanism underlying the ER stress response. In this study, we aimed to reveal a gene regulatory network formed by UPR genes, including immunoglobulin heavy chain-binding protein (BiP), X-box binding protein 1 (XBP1), C/EBP [CCAAT/enhancer-binding protein]-homologous protein (CHOP), PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring 1 (IRE1), activating transcription factor 6 (ATF6), and ATF4. For this purpose, we focused on promoter-luciferase reporters for BiP, XBP1, and CHOP genes, which bear an ER stress response element (ERSE), and p5 × ATF6-GL3, which bears an unfolded protein response element (UPRE). We demonstrated that the luciferase activities of the BiP and CHOP promoters were upregulated by all the UPR genes, whereas those of the XBP1 promoter and p5 × ATF6-GL3 were upregulated by all the UPR genes except for BiP, CHOP, and ATF4 in HeLa cells. Therefore, an ERSE- and UPRE-centered gene regulatory network of UPR genes could be responsible for the robustness of the ER stress response. Finally, we revealed that BiP protein was degraded when cells were treated with DNA-damaging reagents, such as etoposide and doxorubicin; this finding suggests that the expression level of BiP is tightly regulated at the post-translational level, rather than at the transcriptional level, in the presence of DNA damage.

  1. Temperature-sensitive, Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

    USDA-ARS?s Scientific Manuscript database

    Changes in ambient temperature represent a major physiological challenge to poikilothermic organisms that requires rapid adjustments in the composition of cellular membranes in order to preserve overall membrane dynamics and integrity. In plants, the endoplasmic reticulum-localized omega-3 fatty ac...

  2. The role of endoplasmic reticulum stress and insulin resistance in the occurrence of goose fatty liver.

    PubMed

    Geng, Tuoyu; Xia, Lili; Li, Fuyuan; Xia, Jing; Zhang, Yihui; Wang, Qianqian; Yang, Biao; Montgomery, Sean; Cui, Hengmi; Gong, Daoqing

    2015-09-11

    In mammals, insulin resistance (IR) is required for the occurrence of non-alcoholic fatty liver disease, and endoplasmic reticulum stress (ERS) contributes to IR. As geese have physiological and metabolic characteristics different from mammals, it is unclear whether these mechanisms also underlie the occurrence of goose fatty liver. To address this, 70-day-old geese were treated with an ERS inducer or overfed, and variables associated with ERS or IR were subsequently determined. The data indicated that the group of geese treated with the ERS inducer for 20d appeared to be more intolerant to blood glucose than the control group, and their livers showed features of hepatic steatosis, suggesting ERS can induce IR and hepatic steatosis in geese. In contrast, overfeeding did not induce ERS, probably due to the upregulated expression of fatty acid desaturases, but induced higher fasting/postprandial blood glucose as well as glucose intolerance in geese, which was accompanied by a dramatic increase of liver weight. Taken together, these findings delineated the role of ERS and IR in the occurrence of goose fatty liver. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum.

    PubMed

    Kitamura, Takuya; Seki, Naoya; Kihara, Akio

    2017-03-28

    Although normal fatty acids (FAs) are degraded via β-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2 -deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

  4. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

    PubMed

    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-07-25

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

  5. Endoplasmic Reticulum Stress and Lipid Metabolism: Mechanisms and Therapeutic Potential

    PubMed Central

    Basseri, Sana; Austin, Richard C.

    2012-01-01

    The endoplasmic reticulum (ER) plays a crucial role in protein folding, assembly, and secretion. Disruption of ER homeostasis may lead to accumulation of misfolded or unfolded proteins in the ER lumen, a condition referred to as ER stress. In response to ER stress, a signal transduction pathway known as the unfolded protein response (UPR) is activated. UPR activation allows the cell to cope with an increased protein-folding demand on the ER. Recent studies have shown that ER stress/UPR activation plays a critical role in lipid metabolism and homeostasis. ER-stress-dependent dysregulation of lipid metabolism may lead to dyslipidemia, insulin resistance, cardiovascular disease, type 2 diabetes, and obesity. In this paper, we examine recent findings illustrating the important role ER stress/UPR signalling pathways play in regulation of lipid metabolism, and how they may lead to dysregulation of lipid homeostasis. PMID:22195283

  6. Regulation of endoplasmic reticulum turnover by selective autophagy.

    PubMed

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-06-18

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

  7. The Endoplasmic Reticulum and the Unfolded Protein Response

    PubMed Central

    Malhotra, Jyoti D.; Kaufman, Randal J.

    2009-01-01

    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. PMID:18023214

  8. Permethrin potentiates adipogenesis via intracellular calcium and endoplasmic reticulum stress-mediated mechanisms in 3T3-L1 adipocytes.

    PubMed

    Xiao, Xiao; Qi, Weipeng; Clark, John M; Park, Yeonhwa

    2017-11-01

    Permethrin, a pyrethroid insecticide, was previously reported to promote adipogenesis in vitro and weight gain in vivo. The mechanism by which permethrin promotes adipogenesis/obesity, however, has not been fully explored. Intracellular calcium and endoplasmic reticulum (ER) stress have been reported to be linked with adipogenesis and obesity. Because pyrethroid insecticides have been determined to influence intracellular calcium and ER stress in vitro, the purpose of this current study was to investigate whether permethrin potentiates adipogenesis via a change in intracellular calcium, leading to endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. 3T3-L1 cells were exposed to four different concentrations of permethrin (0.01, 0.1, 1 & 10 μM) for 6 days during differentiation. Treatment of permethrin increased intracellular calcium level in a concentration-dependent manner. Similarly, permethrin treatment increased protein levels of ER stress markers in a concentration-dependent manner. These data suggest that intracellular calcium and ER stress may be involved in permethrin-induced adipogenesis of 3T3-L1 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Tomato Ribonuclease LX with the Functional Endoplasmic Reticulum Retention Motif HDEF Is Expressed during Programmed Cell Death Processes, Including Xylem Differentiation, Germination, and Senescence1

    PubMed Central

    Lehmann, Karin; Hause, Bettina; Altmann, Dorit; Köck, Margret

    2001-01-01

    We have studied the subcellular localization of the acid S-like ribonuclease (RNase) LX in tomato (Lycopersicon esculentum Mill.) cells using a combination of biochemical and immunological methods. It was found that the enzyme, unexpectedly excluded from highly purified vacuoles, accumulates in the endoplasmic reticulum. The evidence that RNase LX is a resident of the endoplasmic reticulum (ER) is supported by an independent approach showing that the C-terminal peptide HDEF of RNase LX acts as an alternative ER retention signal in plants. For functional testing, the cellular distribution of chimeric protein constructs based on a marker protein, Brazil nut (Bertholletia excelsa) 2S albumin, was analyzed immunochemically in transgenic tobacco (Nicotiana tabacum) plants. Here, we report that the peptide motif is necessary and sufficient to accumulate 2S albumin constructs of both vacuolar and extracellular final destinations in the ER. We have shown immunochemically that RNase LX is specifically expressed during endosperm mobilization and leaf and flower senescence. Using immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. These results support a physiological function of RNase LX in selective cell death processes that are also thought to involve programmed cell death. It is assumed that RNase LX accumulates in an ER-derived compartment and is released by membrane disruption into the cytoplasma of those cells that are intended to undergo autolysis. These processes are accompanied by degradation of cellular components supporting a metabolic recycling function of the intracellular RNase LX. PMID:11598219

  10. Carrier-mediated translocation of uridine diphosphate glucose into the lumen of endoplasmic reticulum-derived vesicles from rat liver.

    PubMed Central

    Vanstapel, F; Blanckaert, N

    1988-01-01

    Radiolabeled UDPGlc incubated with rough endoplasmic reticulum (RER)-derived microsomes from rat liver became associated with the vesicles. This microsomal uptake of nucleotide sugar was time and temperature dependent. Analysis of the molecular species containing radiolabel revealed that initial uptake represented entry of predominantly intact UDPGlc in the microsomes. Conclusive evidence for proper translocation of UDPGlc across the microsomal membrane into the intravesicular space was obtained by demonstrating that UDPGlc was transported into an osmotically sensitive compartment. Microsomal uptake of UDPGlc exhibited features characteristic of carrier-mediated transport including saturation, specificity, and countertransport. Inhibition and trans-stimulation studies showed that other uridine-containing nucleotide sugars and 5'-UMP were substrates of the postulated microsomal carrier system for UDPGlc, while cytosine- or guanosine-containing nucleotides and non-5'-uridine monophosphates were, at best, very poor substrates. UDPGlc translocation activities were lower in smooth microsomal fractions than in the RER-derived vesicles, indicating that contamination with Golgi membranes could not be responsible for microsomal transport of UDPGlc. Our findings suggest that rat liver endoplasmic reticulum possesses a carrier system mediating proper translocation of UDPGlc and 5'-uridine-substituted structural analogues across the membrane. PMID:3417868

  11. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    PubMed

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  13. The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

    PubMed Central

    Le Gall, Sylvie; Neuhof, Andrea; Rapoport, Tom

    2004-01-01

    The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of ∼5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER. PMID:14617815

  14. Aflatoxin B1 affects apoptosis and expression of death receptor and endoplasmic reticulum molecules in chicken spleen.

    PubMed

    Zhu, Panpan; Zuo, Zhicai; Zheng, Zhixiang; Wang, Fengyuan; Peng, Xi; Fang, Jing; Cui, Hengmin; Gao, Caixia; Song, Hetao; Zhou, Yi; Liu, Xici

    2017-11-21

    Aflatoxin B 1 (AFB 1 ) is a natural product of the Aspergillus genus of molds, which grow on several foodstuffs stored in hot moist conditions, and is among the most potent hepatocarcinogens and immunosuppression presently known. The latter was related to the up-regulated apoptosis of immune organs. However, the effect of expression of death receptor and endoplasmic reticulum molecules in AFB 1 -induced apoptosis of chicken splenocytes was largely unknown. The objective of this study was to investigate this unknown field. One hundred and forty four one-day-old chickens were randomly divided into control group (0 mg/kg AFB 1 ) and AFB 1 group (0.6 mg/kg AFB 1 ), respectively and fed with AFB 1 for 21 days. Histological observation demonstrated that AFB 1 caused slight congestion and lymphocytic depletion in the spleen. TUNEL and flow cytometry assays showed the excessive apoptosis of splenocytes provoked by AFB 1 . Moreover, quantitative real-time PCR analysis revealed that AFB 1 induced the elevated mRNA expression of Fas, FasL, TNF-α, TNF-R 1 , Caspase-3, Caspase-8, Caspase-10, Grp78 and Grp94 in the spleen. These findings suggested that AFB 1 could lead the excessive apoptosis and alter the expression of death receptor and endoplasmic reticulum molecules in chicken spleen.

  15. A natural compound jaceosidin ameliorates endoplasmic reticulum stress and insulin resistance via upregulation of SERCA2b.

    PubMed

    Ouyang, Zijun; Li, Wanshuai; Meng, Qianqian; Zhang, Qi; Wang, Xingqi; Elgehama, Ahmed; Wu, Xudong; Shen, Yan; Sun, Yang; Wu, Xuefeng; Xu, Qiang

    2017-05-01

    Increased endoplasmic reticulum (ER) stress has emerged as a vital contributor to dysregulated glucose homeostasis, and impaired function of sarco-endoplasmic reticulum Ca 2+ -ATPase 2b (SERCA2b) is one of the central mechanisms underlying ER stress. In this study, we reported that SERCA2b upregulation contributed to the amelioration of ER stress and insulin resistance by a small natural compound jaceosidin. In a model of differentiated C2C12 myotubes, jaceosidin-triggered SERCA2b upregulation enhanced insulin sensitivity and decreased ER stress. Moreover, the activity of Ca 2+ -ATPase in thapsigargin-treated myotubes was also augmented by jaceosidin. Furthermore, jaceosidin significantly suppressed blood glucose levels, improved glucose tolerance and lowered body weight, but did not alter food intake in insulin-resistant obese mice. In addition, this compound markedly reduced lipid accumulation, suppressed the expression of lipogenic genes in liver and ameliorated liver injury. The ameliorative effects of jaceosidin were due to its ability to reduce ER stress via increasing the expression of SERCA2b in the muscles of obese mice. Taken together, jaceosidin could improve ER stress and attenuate insulin resistance via SERCA2b upregulation in mice skeletal muscles. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles

    PubMed Central

    Harris, James; Schwinn, Nicole; Mahoney, James A; Lin, Hsi-Hsien; Shaw, Michael; Howard, Chris J; da Silva, Rosangela P; Gordon, Siamon

    2006-01-01

    Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease. CPVL is up-regulated during the maturation of monocytes (MO) to macrophages, although the protein can be seen in both. In primary macrophages, CPVL is glycosylated with high mannose residues and colocalizes with markers for endoplasmic reticulum, while in MO it is more disperse and less clearly associated with endoplasmic reticulum. CPVL is highly expressed in lamellipodia and membrane ruffles, which also concentrate markers of the secretory pathway (MIP-1α and tumour necrosis factor-α) and major histocompatibility complex (MHC) class I and II molecules. CPVL can be seen on early latex bead and Candida albicans phagosomes, but it is not retained in the maturing phagosome, unlike MHC class I/II. CPVL has a mixed cytosolic and membrane-associated localization but is not detectable on the outer plasma membrane. We propose that CPVL may be involved in antigen processing, the secretory pathway and/or in actin remodelling and lamellipodium formation. PMID:16436111

  17. Friend or foe: Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer

    PubMed Central

    Chen, Shaohua; Zhang, Daohai

    2015-01-01

    The endoplasmic reticulum (ER) protein 29 (ERp29) is a molecular chaperone that plays a critical role in protein secretion from the ER in eukaryotic cells. Recent studies have also shown that ERp29 plays a role in cancer. It has been demonstrated that ERp29 is inversely associated with primary tumor development and functions as a tumor suppressor by inducing cell growth arrest in breast cancer. However, ERp29 has also been reported to promote epithelial cell morphogenesis, cell survival against genotoxic stress and distant metastasis. In this review, we summarize the current understanding on the biological and pathological functions of ERp29 in cancer and discuss the pivotal aspects of ERp29 as “friend or foe” in epithelial cancer. PMID:25709888

  18. Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs

    PubMed Central

    Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W.; Schalek, Richard; Hayworth, Kenneth J.; Hand, Arthur R.; Yankova, Maya; Huber, Greg; Lichtman, Jeff W.; Rapoport, Tom A.; Kozlov, Michael M.

    2013-01-01

    The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used novel staining and automated ultra-thin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell. PMID:23870120

  19. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins

    PubMed Central

    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-01-01

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function. DOI: http://dx.doi.org/10.7554/eLife.23882.001 PMID:28742022

  20. Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs.

    PubMed

    Terasaki, Mark; Shemesh, Tom; Kasthuri, Narayanan; Klemm, Robin W; Schalek, Richard; Hayworth, Kenneth J; Hand, Arthur R; Yankova, Maya; Huber, Greg; Lichtman, Jeff W; Rapoport, Tom A; Kozlov, Michael M

    2013-07-18

    The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Terasaki Ramps in the Endoplasmic Reticulum: Structure, Function and Formation

    NASA Astrophysics Data System (ADS)

    Huber, Greg; Guven, Jemal; Valencia, Dulce-Maria

    2015-03-01

    The endoplasmic reticulum (ER) has long been considered an exceedingly important and complex cellular organelle in eukaryotes (like you). It is a membrane structure, part folded lamellae, part tubular network, that both envelopes the nucleus and threads its way outward, all the way to the cell's periphery. Despite the elegant mechanics of bilayer membranes offered by the work of Helfrich and Canham, as far as the ER is concerned, theory has mostly sat on the sidelines. However, refined imaging of the ER has recently revealed beautiful and subtle geometrical forms - simple geometries, from the mathematical point of view - which some have called a ``parking garage for ribosomes.'' I'll review the discovery and physics of Terasaki ramps and discuss their relation to cell-biological questions, such as ER and nuclear-membrane re-organization during mitosis. Rather than being a footnote in a textbook on differential geometry, these structures suggest answers to a number of the ER's structure-function problems.

  2. Potential Role of Endoplasmic Reticulum Stress in Pathogenesis of Diabetic Retinopathy.

    PubMed

    Sánchez-Chávez, Gustavo; Hernández-Ramírez, Ernesto; Osorio-Paz, Ixchel; Hernández-Espinosa, Claudia; Salceda, Rocío

    2016-05-01

    Diabetes mellitus is a metabolic disease that leads to several complications which include retinopathy. Multiple biochemical abnormalities have been proposed to explain the development of retinopathy, including oxidative stress. Although the existence of oxidative stress has been established in the retina from long standing diabetic animals, pathogenesis and progression of retinopathy remain unclear. In order to gain insight into the pathogenesis of diabetic retinopathy, we analyzed the levels of different oxidative stress biomarkers in the retina at early stages during the progress of streptozotocin-induced diabetes. No significant changes in glutathione content, expression of NADPH-oxidase, levels of lipid peroxidation, nor production of free radicals were observed in the retina up to 45 days of diabetes induction. Likewise, a transient decrease in aconitase activity, parallel to an increase in the superoxide dismutase activity was observed at 20 days of hyperglycemia, suggesting a high capacity of retina to maintain its redox homeostasis, at least at early stages of diabetes. Nonetheless, we found an early and time-dependent increase in the levels of oxidized proteins, which was not affected by the administration of the antioxidant quercetin. Also, positive immunoreactivity to the reticulum stress protein CHOP was found in glial Müller cells of diabetic rat retinas. These findings suggest the occurrence of endoplasmic reticulum stress as a primary event in retina pathogenesis in diabetes.

  3. Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCA) Contribute to GPCR-Mediated Taste Perception

    PubMed Central

    Iguchi, Naoko; Ohkuri, Tadahiro; Slack, Jay P.; Zhong, Ping; Huang, Liquan

    2011-01-01

    The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory), bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca2+ concentration ([Ca2+]c) for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca2+]c from the cytosol of taste receptor cells (TRCs) and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca2+ clearance in TRCs, we sought the molecules involved in [Ca2+]c regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family that sequesters cytosolic Ca2+ into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca2+ release for signaling. Serca3-knockout (KO) mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca2+ clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception. PMID:21829714

  4. Ebselen alters cellular oxidative status and induces endoplasmic reticulum stress in rat hippocampal astrocytes.

    PubMed

    Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; de la Casa-Resino, Irene; Martinez-Ruiz, Antonio; Perez-Lopez, Marcos; Portilla, Juan C; Salido, Gines M; Gonzalez, Antonio

    2016-05-16

    Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. Because of its properties, it may be protective against injury to the nervous tissue. However, evidence suggests that its glutathione peroxidase activity could underlie certain deleterious actions on cell physiology. In this study we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular oxidative status, cytosolic free-Ca(2+) concentration ([Ca(2+)]c), setting of endoplasmic reticulum stress and phosphorylation of glial fibrillary acidic protein and major mitogen-activated protein kinases were analyzed. Our results show that ebselen induced a concentration-dependent increase in the generation of reactive oxygen species in the mitochondria. We observed a concentration-dependent increase in global cysteine oxidation and in the level of malondialdehyde in the presence of ebselen. We also detected increases in catalase, glutathione S-transferase and glutathione reductase activity. Ebselen also evoked a concentration-dependent increase in [Ca(2+)]c. Moreover, we observed a concentration-dependent increase in the phosphorylation of the unfolded protein response markers, eukaryotic translation initiation factor 2α and X-box binding protein 1. Finally, ebselen also induced an increase in the phosphorylation of glial fibrillary acidic protein, SAPK/JNK, p38 MAPK and p44/42 MAPK. Our results provide strong evidence that implicate endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in an oxidative damage of cells in the presence of ebselen. The compound thus might exert deleterious actions on astrocyte physiology that could compromise their function. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Hyperhomocysteinemia, endoplasmic reticulum stress, and alcoholic liver injury

    PubMed Central

    Ji, Cheng; Kaplowitz, Neil

    2004-01-01

    Deficiencies in vitamins or other factors (B6, B12, folic acid, betaine) and genetic disorders for the metabolism of the non-protein amino acid-homocysteine (Hcy) lead to hyperhomocysteinemia (HHcy). HHcy is an integral component of several disorders including cardiovascular disease, neurodegeneration, diabetes and alcoholic liver disease. HHcy unleashes mediators of inflammation such as NFκB, IL-1β, IL-6, and IL-8, increases production of intracellular superoxide anion causing oxidative stress and reducing intracellular level of nitric oxide (NO), and induces endoplasmic reticulum (ER) stress which can explain many processes of Hcy-promoted cell injury such as apoptosis, fat accumulation, and inflammation. Animal models have played an important role in determining the biological effects of HHcy. ER stress may also be involved in other liver diseases such as α1-antitrypsin (α1-AT) deficiency and hepatitis C and/or B virus infection. Future research should evaluate the possible potentiative effects of alcohol and hepatic virus infection on ER stress-induced liver injury, study potentially beneficial effects of lowering Hcy and preventing ER stress in alcoholic humans, and examine polymorphism of Hcy metabolizing enzymes as potential risk-factors for the development of HHcy and liver disease. PMID:15188490

  6. Endoplasmic reticulum stress in kidney function and disease.

    PubMed

    Taniguchi, Mai; Yoshida, Hiderou

    2015-07-01

    Recently, a number of papers have reported that endoplasmic reticulum (ER) stress is involved in the onset of various kidney diseases, but the pathological mechanisms responsible have not been clarified. In this review, we summarize recent findings on this issue and try to clarify the pathology of ER stress-induced kidney diseases. ER stress is evoked in various kidney diseases, including diabetic nephropathy, renal fibrosis, inflammation or osmolar contrast-induced renal injury, ischemia-reperfusion, genetic mutations of renal proteins, proteinuria and cyclosporine A treatment. In some cases, chemical chaperones, such as 4-phenylbutyrate and taurodeoxycholic acid, relieve the symptoms, indicating that ER stress-induced apoptosis of renal cells is one of the major causes of certain kidney diseases. Actually, the ER stress response provides protection against some kidney diseases, although the PERK-ATF4-CHOP pathway of the ER stress response is proapoptotic in some kidney diseases. The disposal of unfolded proteins by autophagy is also protective for some ER stress-induced kidney diseases. Because ER stress is a major cause of some kidney diseases, the ER stress response and autophagy, which deal with unfolded proteins that accumulate in the ER, are promising therapeutic targets in acute and chronic kidney diseases.

  7. Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Wang, Guoliang; Liu, Shenghua; Wang, Li; Meng, Liukun; Cui, Chuanjue; Zhang, Hao; Hu, Shengshou; Ma, Ning; Wei, Yingjie

    2017-01-01

    Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH. PMID:28255266

  8. The Unfolded Protein Response: At the Intersection between Endoplasmic Reticulum Function and Mitochondrial Bioenergetics.

    PubMed

    Carreras-Sureda, Amado; Pihán, Philippe; Hetz, Claudio

    2017-01-01

    Endoplasmic reticulum (ER) to mitochondria communication has emerged in recent years as a signaling hub regulating cellular physiology with a relevant contribution to diseases including cancer and neurodegeneration. This functional integration is exerted through discrete interorganelle structures known as mitochondria-associated membranes (MAMs). At these domains, ER/mitochondria physically associate to dynamically adjust metabolic demands and the response to stress stimuli. Here, we provide a focused overview of how the ER shapes the function of the mitochondria, giving a special emphasis to the significance of local signaling of the unfolded protein response at MAMs. The implications to cell fate control and the progression of cancer are also discussed.

  9. The Endoplasmic Reticulum Coat Protein II Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis*

    PubMed Central

    Fryer, Lee G. D.; Jones, Bethan; Duncan, Emma J.; Hutchison, Claire E.; Ozkan, Tozen; Williams, Paul A.; Alder, Olivia; Nieuwdorp, Max; Townley, Anna K.; Mensenkamp, Arjen R.; Stephens, David J.; Dallinga-Thie, Geesje M.; Shoulders, Carol C.

    2014-01-01

    Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions. PMID:24338480

  10. C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C - structural features and membrane interactions.

    PubMed

    Casals, Cristina; Johansson, Hanna; Saenz, Alejandra; Gustafsson, Magnus; Alfonso, Carlos; Nordling, Kerstin; Johansson, Jan

    2008-02-01

    Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is alpha-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (beta-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC-membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.

  11. The glutathione mimic ebselen inhibits oxidative stress but not endoplasmic reticulum stress in endothelial cells.

    PubMed

    Ahwach, Salma Makhoul; Thomas, Melanie; Onstead-Haas, Luisa; Mooradian, Arshag D; Haas, Michael J

    2015-08-01

    Reactive oxygen species are associated with cardiovascular disease, diabetes, and atherosclerosis, yet the use of antioxidants in clinical trials has been ineffective at improving outcomes. In endothelial cells, high-dextrose-induced oxidative stress and endoplasmic reticulum stress promote endothelial dysfunction leading to the recruitment and activation of peripheral blood lymphocytes and the breakdown of barrier function. Ebselen, a glutathione peroxidase 1 (GPX1) mimic, has been shown to improve β-cell function in diabetes and prevent atherosclerosis. To determine if ebselen inhibits both oxidative stress and endoplasmic reticulum (ER) stress in endothelial cells, we examined its effects in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) with and without high-dextrose. Oxidative stress and ER stress were measured by 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence and ER stress alkaline phosphatase assays, respectively. GPX1 over-expression and knockdown were performed by transfecting cells with a GPX1 expression construct or a GPX1-specific siRNA, respectively. Ebselen inhibited dextrose-induced oxidative stress but not ER stress in both HUVEC and HCAEC. Ebselen also had no effect on tunicamycin-induced ER stress in HCAEC. Furthermore, augmentation of GPX1 activity directly by sodium selenite supplementation or transfection of a GPX1 expression plasmid decreased dextrose-induced oxidative stress but not ER stress, while GPX1 knockout enhanced oxidative stress but had no effect on ER stress. These results suggest that ebselen targets only oxidative stress but not ER stress. Copyright © 2015. Published by Elsevier Inc.

  12. Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

    PubMed

    Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

    2016-04-01

    Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  13. Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting Endoplasmic Reticulum (ER) Stress Induced by Disturbed Flow.

    PubMed

    Chung, Jihwa; Kim, Kyoung Hwa; Lee, Seok Cheol; An, Shung Hyun; Kwon, Kihwan

    2015-10-01

    Disturbed blood flow with low-oscillatory shear stress (OSS) is a predominant atherogenic factor leading to dysfunctional endothelial cells (ECs). Recently, it was found that disturbed flow can directly induce endoplasmic reticulum (ER) stress in ECs, thereby playing a critical role in the development and progression of atherosclerosis. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid, has long been used to treat chronic cholestatic liver disease and is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, its role in atherosclerosis remains unexplored. In this study, we demonstrated the anti-atherogenic activity of UDCA via inhibition of disturbed flow-induced ER stress in atherosclerosis. UDCA effectively reduced ER stress, resulting in a reduction in expression of X-box binding protein-1 (XBP-1) and CEBP-homologous protein (CHOP) in ECs. UDCA also inhibits the disturbed flow-induced inflammatory responses such as increases in adhesion molecules, monocyte adhesion to ECs, and apoptosis of ECs. In a mouse model of disturbed flow-induced atherosclerosis, UDCA inhibits atheromatous plaque formation through the alleviation of ER stress and a decrease in adhesion molecules. Taken together, our results revealed that UDCA exerts anti-atherogenic activity in disturbed flow-induced atherosclerosis by inhibiting ER stress and the inflammatory response. This study suggests that UDCA may be a therapeutic agent for prevention or treatment of atherosclerosis.

  14. Pancreatic and Duodenal Homeobox Protein 1 (Pdx-1) Maintains Endoplasmic Reticulum Calcium Levels through Transcriptional Regulation of Sarco-endoplasmic Reticulum Calcium ATPase 2b (SERCA2b) in the Islet β Cell*

    PubMed Central

    Johnson, Justin S.; Kono, Tatsuyoshi; Tong, Xin; Yamamoto, Wataru R.; Zarain-Herzberg, Angel; Merrins, Matthew J.; Satin, Leslie S.; Gilon, Patrick; Evans-Molina, Carmella

    2014-01-01

    Although the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in β cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of β cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/− mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca2+ regulation in Pdx-1-deficient states. PMID:25271154

  15. Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells

    NASA Technical Reports Server (NTRS)

    Zheng, H. Q.; Staehelin, L. A.

    2001-01-01

    The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.

  16. Monoamine oxidase-dependent endoplasmic reticulum-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes.

    PubMed

    Deshwal, Soni; Forkink, Marleen; Hu, Chou-Hui; Buonincontri, Guido; Antonucci, Salvatore; Di Sante, Moises; Murphy, Michael P; Paolocci, Nazareno; Mochly-Rosen, Daria; Krieg, Thomas; Di Lisa, Fabio; Kaludercic, Nina

    2018-02-19

    Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon.

  17. The endoplasmic reticulum in plant immunity and cell death

    PubMed Central

    Eichmann, Ruth; Schäfer, Patrick

    2012-01-01

    The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants. PMID:22936941

  18. The endoplasmic reticulum in plant immunity and cell death.

    PubMed

    Eichmann, Ruth; Schäfer, Patrick

    2012-01-01

    The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants.

  19. Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress

    PubMed Central

    Mecha, M; Torrao, A S; Mestre, L; Carrillo-Salinas, F J; Mechoulam, R; Guaza, C

    2012-01-01

    Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 μM CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFNγ through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPARγ receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2α, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2α induced by LPS/IFNγ. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the ‘oligoprotective' effects of CBD during inflammation. PMID:22739983

  20. Maturation of hepatic lipase. Formation of functional enzyme in the endoplasmic reticulum is the rate-limiting step in its secretion.

    PubMed

    Ben-Zeev, Osnat; Doolittle, Mark H

    2004-02-13

    Among three lipases in the lipase gene family, hepatic lipase (HL), lipoprotein lipase, and pancreatic lipase, HL exhibits the lowest intracellular specific activity (i.e. minimal amounts of catalytic activity accompanied by massive amounts of inactive lipase mass in the endoplasmic reticulum (ER)). In addition, HL has a distinctive sedimentation profile, where the inactive mass overlaps the region containing active dimeric HL and trails into progressively larger molecular forms. Eventually, at least half of the HL inactive mass in the ER reaches an active, dimeric conformation (t(1/2) = 2 h) and is rapidly secreted. The remaining inactive mass is degraded. HL maturation occurs in the ER and is strongly dependent on binding to calnexin in the early co-/post-translational stages. Later stages of HL maturation occur without calnexin assistance, although inactive HL at all stages appears to be associated in distinct complexes with other ER proteins. Thus, unlike other lipases in the gene family, HL maturation is the rate-limiting step in its secretion as a functional enzyme.

  1. Identification of Two Novel Endoplasmic Reticulum Body-Specific Integral Membrane Proteins1[W][OA

    PubMed Central

    Yamada, Kenji; Nagano, Atsushi J.; Nishina, Momoko; Hara-Nishimura, Ikuko; Nishimura, Mikio

    2013-01-01

    The endoplasmic reticulum (ER) body, a large compartment specific to the Brassicales, accumulates β-glucosidase and possibly plays a role in the defense against pathogens and herbivores. Although the ER body is a subdomain of the ER, it is unclear whether any ER body-specific membrane protein exists. In this study, we identified two integral membrane proteins of the ER body in Arabidopsis (Arabidopsis thaliana) and termed them MEMBRANE PROTEIN OF ENDOPLASMIC RETICULUM BODY1 (MEB1) and MEB2. In Arabidopsis, a basic helix-loop-helix transcription factor, NAI1, and an ER body component, NAI2, regulate ER body formation. The expression profiles of MEB1 and MEB2 are similar to those of NAI1, NAI2, and ER body β-glucosidase PYK10 in Arabidopsis. The expression of MEB1 and MEB2 was reduced in the nai1 mutant, indicating that NAI1 regulates the expression of MEB1 and MEB2 genes. MEB1 and MEB2 proteins localize to the ER body membrane but not to the ER network, suggesting that these proteins are specifically recruited to the ER body membrane. MEB1 and MEB2 physically interacted with ER body component NAI2, and they were diffused throughout the ER network in the nai2 mutant, which has no ER body. Heterologous expression of MEB1 and MEB2 in yeast (Saccharomyces cerevisiae) suppresses iron and manganese toxicity, suggesting that MEB1 and MEB2 are metal transporters. These results indicate that the membrane of ER bodies has specific membrane proteins and suggest that the ER body is involved in defense against metal stress as well as pathogens and herbivores. PMID:23166355

  2. Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation.

    PubMed

    Spiro, R G

    2004-05-01

    Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc(3)Man(9)GlcNAc(2) glycans after trimming with endoplasmic reticulum (ER) alpha-glucosidases and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man(8)GlcNAc(2) isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc(2) along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man(5)GlcNAc(1) by the action of cytosolic endo-beta- N-acetylglucosaminidase and alpha-mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc(3)Man(9)GlcNAc(2)-P-P-Dol, and the unfolded protein

  3. Endoplasmic Reticulum Stress in Beta Cells and Development of Diabetes

    PubMed Central

    Fonseca, Sonya G.; Burcin, Mark; Gromada, Jesper; Urano, Fumihiko

    2009-01-01

    The endoplasmic reticulum (ER) is a cellular compartment responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. A myriad of pathological and physiological factors perturb ER function and cause dysregulation of ER homeostasis, leading to ER stress. ER stress elicits a signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can relieve stress, cells can produce the proper amount of proteins and maintain ER homeostasis. If the UPR, however, fails to maintain ER homeostasis, cells will undergo apoptosis. Activation of the UPR is critical to the survival of insulin-producing pancreatic β-cells with high secretory protein production. Any disruption of ER homeostasis in β-cells can lead to cell death and contribute to the pathogenesis of diabetes. There are several models of ER stress-mediated diabetes. In this review, we outline the underlying molecular mechanisms of ER stress-mediated β-cell dysfunction and death during the progression of diabetes. PMID:19665428

  4. Role of Flippases in Protein Glycosylation in the Endoplasmic Reticulum

    PubMed Central

    Rush, Jeffrey S.

    2015-01-01

    Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane. The glycans are first assembled as precursor glycolipids, on the cytosolic surface of the ER, which are tethered to the membrane by attachment to a long-chain polyisoprenyl phosphate (dolichol) containing a reduced α-isoprene. The lipid-anchored building blocks then migrate transversely (flip) across the ER membrane to the lumenal surface, where final assembly of the glycan is completed. This strategy allows the cell to export high-energy biosynthetic intermediates as lipid-bound glycans, while constraining the glycosyl donors to the site of assembly on the membrane surface. This review focuses on the flippases that participate in protein glycosylation in the ER. PMID:26917968

  5. CCPG1, a cargo receptor required for reticulophagy and endoplasmic reticulum proteostasis.

    PubMed

    Smith, Matthew D; Wilkinson, Simon

    2018-06-19

    The importance of selective macroautophagy/autophagy in cellular health is increasingly evident. The selective degradation of portions of the endoplasmic reticulum (ER), or reticulophagy, is an emerging example but requires further mechanistic detail and broad evidence of physiological relevance. In a recent study, we identified CCPG1, an ER-resident transmembrane protein that can bind to Atg8-family proteins and, independently and discretely, to RB1CC1/FIP200. Both of these interactions are required to facilitate CCPG1's function as a reticulophagy cargo receptor. CCPG1 transcripts are inducible by ER stress, providing a direct link between ER stress and reticulophagy. In vivo, CCPG1 prevents the hyper-accumulation of insoluble protein within the ER lumen of pancreatic acinar cells and alleviates ER stress. Accordingly, CCPG1 loss sensitizes the exocrine pancreas to tissue injury.

  6. Flurbiprofen ameliorated obesity by attenuating leptin resistance induced by endoplasmic reticulum stress.

    PubMed

    Hosoi, Toru; Yamaguchi, Rie; Noji, Kikuko; Matsuo, Suguru; Baba, Sachiko; Toyoda, Keisuke; Suezawa, Takahiro; Kayano, Takaaki; Tanaka, Shinpei; Ozawa, Koichiro

    2014-03-01

    Endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins, is involved in the development of obesity. We demonstrated that flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAID), exhibited chaperone activity, which reduced protein aggregation and alleviated ER stress-induced leptin resistance, characterized by insensitivity to the actions of the anti-obesity hormone leptin. This result was further supported by flurbiprofen attenuating high-fat diet-induced obesity in mice. The other NSAIDs tested did not exhibit such effects, which suggested that this anti-obesity action is mediated independent of NSAIDs. Using ferriteglycidyl methacrylate beads, we identified aldehyde dehydrogenase as the target of flurbiprofen, but not of the other NSAIDs. These results suggest that flurbiprofen may have unique pharmacological properties that reduce the accumulation of unfolded proteins and may represent a new class of drug for the fundamental treatment of obesity.

  7. Dysfunctional tubular endoplasmic reticulum constitutes a pathological feature of Alzheimer's disease.

    PubMed

    Sharoar, M G; Shi, Q; Ge, Y; He, W; Hu, X; Perry, G; Zhu, X; Yan, R

    2016-09-01

    Pathological features in Alzheimer's brains include mitochondrial dysfunction and dystrophic neurites (DNs) in areas surrounding amyloid plaques. Using a mouse model that overexpresses reticulon 3 (RTN3) and spontaneously develops age-dependent hippocampal DNs, here we report that DNs contain both RTN3 and REEPs, topologically similar proteins that can shape tubular endoplasmic reticulum (ER). Importantly, ultrastructural examinations of such DNs revealed gradual accumulation of tubular ER in axonal termini, and such abnormal tubular ER inclusion is found in areas surrounding amyloid plaques in biopsy samples from Alzheimer's disease (AD) brains. Functionally, abnormally clustered tubular ER induces enhanced mitochondrial fission in the early stages of DN formation and eventual mitochondrial degeneration at later stages. Furthermore, such DNs are abrogated when RTN3 is ablated in aging and AD mouse models. Hence, abnormally clustered tubular ER can be pathogenic in brain regions: disrupting mitochondrial integrity, inducing DNs formation and impairing cognitive function in AD and aging brains.

  8. Endoplasmic Reticulum Stress in Arterial Smooth Muscle Cells: A Novel Regulator of Vascular Disease

    PubMed Central

    Furmanik, Malgorzata; Shanahan, Catherine M.

    2017-01-01

    Cardiovascular disease continues to be the leading cause of death in industrialised societies. The idea that the arterial smooth muscle cell (ASMC) plays a key role in regulating many vascular pathologies has been gaining importance, as has the realisation that not enough is known about the pathological cellular mechanisms regulating ASMC function in vascular remodelling. In the past decade endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been recognised as a stress response underlying many physiological and pathological processes in various vascular cell types. Here we summarise what is known about how ER stress signalling regulates phenotypic switching, trans/dedifferentiation and apoptosis of ASMCs and contributes to atherosclerosis, hypertension, aneurysms and vascular calcification.

  9. Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

    PubMed

    Lazar, Catalin; Uta, Mihaela; Petrescu, Stefana Maria; Branza-Nichita, Norica

    2017-02-01

    Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation. © 2016 John Wiley & Sons Ltd.

  10. Dietary toxins, endoplasmic reticulum (ER) stress and diabetes.

    PubMed

    Hettiarachchi, Kalindi D; Zimmet, Paul Z; Myers, Mark A

    2008-05-01

    The incidence of Type 1 diabetes has been increasing at a rate too rapid to be due to changes in genetic risk. Instead changes in environmental factors are the likely culprit. The endoplasmic reticulum (ER) plays an important role in the production of newly synthesized proteins and interference with these processes leads to ER stress. The insulin-producing beta cells are particularly prone to ER stress as a result of their heavy engagement in insulin production. Increasing evidence suggests ER stress is central to initiation and progression of Type 1 diabetes. An early environmental exposure, such as toxins and viral infections, can impart a significant physiological load on beta cells to initiate abnormal processing of proinsulin, ER stress and insulin secretory defects. Release of altered proinsulin from the beta cells early in life may trigger autoimmunity in those with genetic susceptibility leading to cytokine-induced nitric oxide production and so exacerbating ER stress in beta cells, ultimately leading to apoptosis of beta cells and diabetes. Here we suggest that ER stress is an inherent cause of beta cell dysfunction and environmental factors, in particular dietary toxins derived from Streptomyces in infected root vegetables, can impart additional stress that aggravates beta cell death and progression to diabetes. Furthermore, we propose that the increasing incidence of Type 1 diabetes may be accounted for by increased dietary exposure to ER-stress-inducing Streptomyces toxins.

  11. Selective export of autotaxin from the endoplasmic reticulum.

    PubMed

    Lyu, Lin; Wang, Baolu; Xiong, Chaoyang; Zhang, Xiaotian; Zhang, Xiaoyan; Zhang, Junjie

    2017-04-28

    Autotaxin (ATX) or ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) is a secretory glycoprotein and functions as the key enzyme for lysophosphatidic acid generation. The mechanism of ATX protein trafficking is largely unknown. Here, we demonstrated that p23, a member of the p24 protein family, was the protein-sorting receptor required for endoplasmic reticulum (ER) export of ATX. A di-phenylalanine (Phe-838/Phe-839) motif in the human ATX C-terminal region was identified as a transport signal essential for the ATX-p23 interaction. Knockdown of individual Sec24 isoforms by siRNA revealed that ER export of ATX was impaired only if Sec24C was down-regulated. These results suggest that ATX is selectively exported from the ER through a p23, Sec24C-dependent pathway. In addition, it was found that AKT signaling played a role in ATX secretion regulation to facilitate ATX ER export by enhancing the nuclear factor of activated T cell-mediated p23 expression. Furthermore, the di-hydrophobic amino acid motifs (FY) also existed in the C-terminal regions of human ENPP1 and ENPP3. Such a p23, Sec24C-dependent selective ER export mechanism is conserved among these ENPP family members. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The Herbal Medicine Cordyceps sinensis Protects Pancreatic Beta Cells from Streptozotocin-Induced Endoplasmic Reticulum Stress.

    PubMed

    Liu, Hong; Cao, Diyong; Liu, Hua; Liu, Xinghai; Mai, Wenli; Lan, Haitao; Huo, Wen; Zheng, Qian

    2016-08-01

    Our previous work found that Cordyceps sinensis (CS) improves the activity and secretory function of pancreatic islet beta cells. The objective was to observe a further possible role of CS in the protection of insulin-secreting cells. A rat model of type 2 diabetes mellitus was developed with streptozotocin (STZ) and a high-energy fat diet (HFD). CS was administered in the successful model of rats with type 2 diabetes. After 4 weeks, the biochemistry index of blood samples was measured, and pathologic observation was performed by immunohistochemistry. In the rats with type 2 diabetes induced by a HFD and STZ, the levels of fasting blood glucose and fasting insulin were elevated, and the insulin sensitivity index was decreased. Pathologic examination found an increased number of apoptotic cells, an elevated protein expression of pro-apoptotic C/EBP homologous protein (CHOP) and an increased c-Jun level by means of JNK phosphorylation, responsive to the endoplasmic reticulum stress of islet beta cells. With treatment by CS for 4 weeks, the elevated levels of both fasting blood glucose and fasting insulin in the rats with type 2 diabetes were significantly lower, and the decreased insulin sensitivity index was reversed. Compared to the control rats with type 2 diabetes, CS application significantly reduced the number of apoptotic cells and decreased protein expression of both CHOP and c-Jun. The herbal compound CS could protect pancreatic beta cells from the pro-apoptotic endoplasmic reticulum stress induced by HFD-STZ. This suggests an alternative approach to treating type 2 diabetes. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  13. Endoplasmic reticulum membrane potassium channel dysfunction in high fat diet induced stress in rat hepatocytes

    PubMed Central

    Khodaee, Naser; Ghasemi, Maedeh; Saghiri, Reza; Eliassi, Afsaneh

    2014-01-01

    In a previous study we reported the presence of a large conductance K+ channel in the membrane of endoplasmic reticulum (ER) from rat hepatocytes. The channel open probability (Po) appeared voltage dependent and reached to a minimum 0.2 at +50 mV. Channel activity in this case was found to be totally inhibited at ATP concentration 2.5 mM, glibenclamide 100 µM and tolbutamide 400 µM. Existing evidence indicates an impairment of endoplasmic reticulum functions in ER stress condition. Because ER potassium channels have been involved in several ER functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the ER potassium channel function is altered in a high fat diet model of ER stress. Male Wistar rats were made ER stress for 2 weeks with a high fat diet. Ion channel incorporation of ER stress model into the bilayer lipid membrane allowed the characterization of K+ channel. Our results indicate that the channel Po was significantly increased at voltages above +30 mV. Interestingly, addition of ATP 7.5 mM, glibenclamide 400 µM and tolbutamide 2400 µM totally inhibited the channel activities, 3-fold, 4-fold and 6-fold higher than that in the control groups, respectively. Our results thus demonstrate a modification in the ER K+ channel gating properties and decreased sensitivity to drugs in membrane preparations coming from ER high fat model of ER stress, an effect potentially linked to a change in ER K+ channel subunits in ER stress condition. Our results may provide new insights into the cellular mechanisms underlying ER dysfunctions in ER stress. PMID:26417322

  14. N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes.

    PubMed

    Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

    2013-03-01

    Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg(-1)). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.

  15. N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes

    PubMed Central

    Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

    2013-01-01

    Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg−1). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes. PMID:23353715

  16. Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro.

    PubMed

    Xue, Hongxia; Gan, Fang; Qian, Gang; Hu, Junfa; Hao, Shu; Xu, Jing; Chen, Xingxiang; Huang, Kehe

    2017-01-10

    This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress.

  17. Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

    PubMed Central

    Xue, Hongxia; Gan, Fang; Qian, Gang; Hu, Junfa; Hao, Shu; Xu, Jing; Chen, Xingxiang; Huang, Kehe

    2017-01-01

    This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress. PMID:28071725

  18. Unraveling the function of Arabidopsis thaliana OS9 in the endoplasmic reticulum-associated degradation of glycoproteins.

    PubMed

    Hüttner, Silvia; Veit, Christiane; Schoberer, Jennifer; Grass, Josephine; Strasser, Richard

    2012-05-01

    In the endoplasmic reticulum, immature polypeptides coincide with terminally misfolded proteins. Consequently, cells need a well-balanced quality control system, which decides about the fate of individual proteins and maintains protein homeostasis. Misfolded and unassembled proteins are sent for destruction via the endoplasmic reticulum-associated degradation (ERAD) machinery to prevent the accumulation of potentially toxic protein aggregates. Here, we report the identification of Arabidopsis thaliana OS9 as a component of the plant ERAD pathway. OS9 is an ER-resident glycoprotein containing a mannose-6-phosphate receptor homology domain, which is also found in yeast and mammalian lectins involved in ERAD. OS9 fused to the C-terminal domain of YOS9 can complement the ERAD defect of the corresponding yeast Δyos9 mutant. An A. thaliana OS9 loss-of-function line suppresses the severe growth phenotype of the bri1-5 and bri1-9 mutant plants, which harbour mutated forms of the brassinosteroid receptor BRI1. Co-immunoprecipitation studies demonstrated that OS9 associates with Arabidopsis SEL1L/HRD3, which is part of the plant ERAD complex and with the ERAD substrates BRI1-5 and BRI1-9, but only the binding to BRI1-5 occurs in a glycan-dependent way. OS9-deficiency results in activation of the unfolded protein response and reduces salt tolerance, highlighting the role of OS9 during ER stress. We propose that OS9 is a component of the plant ERAD machinery and may act specifically in the glycoprotein degradation pathway.

  19. Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum-plasma membrane contact sites.

    PubMed

    Siao, Wei; Wang, Pengwei; Voigt, Boris; Hussey, Patrick J; Baluska, Frantisek

    2016-11-01

    Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum-plasma membrane (ER-PM) contact sites in leaf and root cells. The ER-PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER-PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER-PM contact sites. The VAP27-1-enriched ER-PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER-PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER-PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. Endoplasmic Reticulum Stress and Unfolded Protein Response Pathways: Potential for Treating Age-related Retinal Degeneration

    PubMed Central

    Haeri, Mohammad; Knox, Barry E

    2012-01-01

    Accumulation of misfolded proteins in the endoplasmic reticulum (ER) and their aggregation impair normal cellular function and can be toxic, leading to cell death. Prolonged expression of misfolded proteins triggers ER stress, which initiates a cascade of reactions called the unfolded protein response (UPR). Protein misfolding is the basis for a variety of disorders known as ER storage or conformational diseases. There are an increasing number of eye disorders associated with misfolded proteins and pathologic ER responses, including retinitis pigmentosa (RP). Herein we review the basic cellular and molecular biology of UPR with focus on pathways that could be potential targets for treating retinal degenerative diseases. PMID:22737387

  1. Progranulin causes adipose insulin resistance via increased autophagy resulting from activated oxidative stress and endoplasmic reticulum stress.

    PubMed

    Guo, Qinyue; Xu, Lin; Li, Huixia; Sun, Hongzhi; Liu, Jiali; Wu, Shufang; Zhou, Bo

    2017-01-31

    Progranulin (PGRN) has recently emerged as an important regulator for insulin resistance. However, the direct effect of progranulin in adipose insulin resistance associated with the autophagy mechanism is not fully understood. In the present study, progranulin was administered to 3T3-L1 adipocytes and C57BL/6 J mice with/without specific inhibitors of oxidative stress and endoplasmic reticulum stress, and metabolic parameters, oxidative stress, endoplasmic reticulum stress and autophagy markers were assessed. Progranulin treatment increased iNOS expression, NO synthesis and ROS generation, and elevated protein expressions of CHOP, GRP78 and the phosphorylation of PERK, and caused a significant increase in Atg7 and LC3-II protein expression and a decreased p62 expression, and decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and glucose uptake, demonstrating that progranulin activated oxidative stress and ER stress, elevated autophagy and induced insulin insensitivity in adipocytes and adipose tissue of mice. Interestingly, inhibition of iNOS and ER stress both reversed progranulin-induced stress response and increased autophagy, protecting against insulin resistance in adipocytes. Furthermore, the administration of the ER stress inhibitor 4-phenyl butyric acid reversed the negative effect of progranulin in vivo. Our findings showed the clinical potential of the novel adipokine progranulin in the regulation of insulin resistance, suggesting that progranulin might mediate adipose insulin resistance, at least in part, by inducing autophagy via activated oxidative stress and ER stress.

  2. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

    PubMed Central

    Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

    2017-01-01

    Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

  3. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

    PubMed

    Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

    2017-01-01

    Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

  4. Flurbiprofen ameliorated obesity by attenuating leptin resistance induced by endoplasmic reticulum stress

    PubMed Central

    Hosoi, Toru; Yamaguchi, Rie; Noji, Kikuko; Matsuo, Suguru; Baba, Sachiko; Toyoda, Keisuke; Suezawa, Takahiro; Kayano, Takaaki; Tanaka, Shinpei; Ozawa, Koichiro

    2014-01-01

    Endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins, is involved in the development of obesity. We demonstrated that flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAID), exhibited chaperone activity, which reduced protein aggregation and alleviated ER stress-induced leptin resistance, characterized by insensitivity to the actions of the anti-obesity hormone leptin. This result was further supported by flurbiprofen attenuating high-fat diet-induced obesity in mice. The other NSAIDs tested did not exhibit such effects, which suggested that this anti-obesity action is mediated independent of NSAIDs. Using ferriteglycidyl methacrylate beads, we identified aldehyde dehydrogenase as the target of flurbiprofen, but not of the other NSAIDs. These results suggest that flurbiprofen may have unique pharmacological properties that reduce the accumulation of unfolded proteins and may represent a new class of drug for the fundamental treatment of obesity. Subject Categories Metabolism; Pharmacology & Drug Discovery PMID:24421337

  5. Dietary Cocoa Powder Improves Hyperlipidemia and Reduces Atherosclerosis in apoE Deficient Mice through the Inhibition of Hepatic Endoplasmic Reticulum Stress.

    PubMed

    Guan, Hua; Lin, Yan; Bai, Liang; An, Yingfeng; Shang, Jianan; Wang, Zhao; Zhao, Sihai; Fan, Jianglin; Liu, Enqi

    2016-01-01

    Cocoa powder is rich in flavonoids, which have many beneficial effects on human health, including antioxidative and anti-inflammatory effects. The aim of our study was to investigate whether the intake of cocoa powder has any influence on hyperlipidemia and atherosclerosis and examine the underlying molecular mechanisms. We fed apoE knockout mice a Western diet supplemented with either 0.2% (low group) or 2% (high group) cocoa powder for 12 weeks. The groups fed dietary cocoa powder showed a significant reduction in both plasma cholesterol levels and aortic atherosclerosis compared to the control group. Analysis of mRNA profiling of aortic atherosclerotic lesions revealed that the expression of several genes related to apoptosis, lipid metabolism, and inflammation was significantly reduced, while the antiapoptotic gene Bcl2 was significantly increased in the cocoa powder group compared to the control. RT-PCR analysis along with Western blotting revealed that a diet containing cocoa powder inhibited the expression of hepatic endoplasmic reticulum stress. These data suggest that cocoa powder intake improves hyperlipidemia and atherosclerosis, and such beneficial effects are possibly mediated through the suppression of hepatic endoplasmic reticulum stress.

  6. TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Fu-Tao; Ding, Yi; Shah, Zahir

    Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels ofmore » the related signaling molecules (TNFα, TNFR{sub 1}, TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR{sub 1} was suppressed with its siRNA. The protein levels of TNFα, TNFR{sub 1} and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR{sub 1} and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR{sub 1}, Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR{sub 1}–siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR{sub 1} pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a

  7. Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

    NASA Technical Reports Server (NTRS)

    Hong, B.; Ichida, A.; Wang, Y.; Gens, J. S.; Pickard, B. G.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    1999-01-01

    A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

  8. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

    PubMed

    Kim, Jong Hun; Lee, Eunjung; Friedline, Randall H; Suk, Sujin; Jung, Dae Young; Dagdeviren, Sezin; Hu, Xiaodi; Inashima, Kunikazu; Noh, Hye Lim; Kwon, Jung Yeon; Nambu, Aya; Huh, Jun R; Han, Myoung Sook; Davis, Roger J; Lee, Amy S; Lee, Ki Won; Kim, Jason K

    2018-04-01

    Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78 -/- ) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78 -/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78 -/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78 -/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

  9. Endoplasmic reticulum stress and IRE-1 signaling cause apoptosis in colon cancer cells in response to andrographolide treatment.

    PubMed

    Banerjee, Aditi; Ahmed, Hafiz; Yang, Peixin; Czinn, Steven J; Blanchard, Thomas G

    2016-07-05

    The plant metabolite andrographolide induces cell cycle arrest and apoptosis in cancer cells. The mechanism(s) by which andrographolide induces apoptosis however, have not been elucidated. The present study was performed to determine the molecular events that promote apoptosis in andrographolide treated cells using T84, HCT116 and COLO 205 colon cancer cell lines. Andrographolide was determined to limit colony formation and Ki67 expression, alter nuclear morphology, increase cytoplasmic histone-associated-DNA-fragments, and increase cleaved caspase-3 levels. Andrographolide also induced significantly higher expression of endoplasmic reticulum (ER) stress proteins GRP-78 and IRE-1 by 48 h but not PERK or ATF6. Apoptosis signaling molecules BAX, spliced XBP-1 and CHOP were also significantly increased. Moreover, chemical inhibition of ER stress or IRE-1 depletion with siRNA in andrographolide treated cells significantly limited expression of IRE-1 and CHOP as determined by immunofluorescence staining, real time PCR, or immunobloting. This was accompanied by a decreased BAX/Bcl-2 ratio. Andrographolide significantly promotes cancer cell death compared to normal cells. These data demonstrate that andrographolide associated ER stress contributes to apoptosis through the activation of a pro-apoptotic GRP-78/IRE-1/XBP-1/CHOP signaling pathway.

  10. Lipid droplet formation on opposing sides of the endoplasmic reticulum

    PubMed Central

    Sturley, Stephen L.; Hussain, M. Mahmood

    2012-01-01

    In animal cells, the primary repositories of esterified fatty acids and alcohols (neutral lipids) are lipid droplets that form on the lumenal and/or cytoplasmic side of the endoplasmic reticulum (ER) membrane. A monolayer of amphipathic lipids, intermeshed with key proteins, serves to solubilize neutral lipids as they are synthesized and desorbed. In specialized cells, mobilization of the lipid cargo for delivery to other tissues occurs by secretion of lipoproteins into the plasma compartment. Serum lipoprotein assembly requires an obligate structural protein anchor (apolipoprotein B) and a dedicated chaperone, microsomal triglyceride transfer protein. By contrast, lipid droplets that form on the cytoplasmic face of the ER lack an obligate protein scaffold or any required chaperone/lipid transfer protein. Mobilization of neutral lipids from the cytosol requires regulated hydrolysis followed by transfer of the products to different organelles or export from cells. Several proteins play a key role in controlling droplet number, stability, and catabolism; however, it is our premise that their formation initiates spontaneously, solely as a consequence of neutral lipid synthesis. This default pathway directs droplets into the cytoplasm where they accumulate in many lipid disorders. PMID:22701043

  11. Proteomic analysis of endoplasmic reticulum stress responses in rice seeds.

    PubMed

    Qian, Dandan; Tian, Lihong; Qu, Leqing

    2015-09-23

    The defects in storage proteins secretion in the endosperm of transgenic rice seeds often leads to endoplasmic reticulum (ER) stress, which produces floury and shrunken seeds, but the mechanism of this response remains unclear. We used an iTRAQ-based proteomics analysis of ER-stressed rice seeds due to the endosperm-specific suppression of OsSar1 to identify changes in the protein levels in response to ER stress. ER stress changed the expression of 405 proteins in rice seed by >2.0- fold compared with the wild-type control. Of these proteins, 140 were upregulated and 265 were downregulated. The upregulated proteins were mainly involved in protein modification, transport and degradation, and the downregulated proteins were mainly involved in metabolism and stress/defense responses. A KOBAS analysis revealed that protein-processing in the ER and degradation-related proteasome were the predominant upregulated pathways in the rice endosperm in response to ER stress. Trans-Golgi protein transport was also involved in the ER stress response. Combined with bioinformatic and molecular biology analyses, our proteomic data will facilitate our understanding of the systemic responses to ER stress in rice seeds.

  12. Membrane glycerolipid equilibrium under endoplasmic reticulum stress in Arabidopsis thaliana.

    PubMed

    Yu, Chao-Yuan; Nguyen, Van Cam; Chuang, Ling; Kanehara, Kazue

    2018-06-02

    Endoplasmic reticulum (ER) is an indispensable organelle for secretory protein synthesis as well as metabolism of phospholipids and their derivatives in eukaryotic cells. Various external and internal factors may cause an accumulation of aberrant proteins in the ER, which causes ER stress and activates cellular ER stress responses to cope with the stress. In animal research, molecular mechanisms for protein quality control upon ER stress are well documented; however, how cells maintain lipid homeostasis under ER stress is an emerging issue. The ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE), two major phospholipid classes, is important under ER stress in animal cells. However, in seed plants, no study has reported on the changes in membrane lipid content under ER stress, although a number of physiologically important environmental stresses, such as heat and salinity, induce ER stress. Here, we investigated membrane glycerolipid metabolism under ER stress in Arabidopsis. ER stress transcriptionally affected PC and PE biosynthesis pathways differentially, with no significant changes in membrane glycerolipid content. Our results suggest that higher plants maintain membrane lipid equilibrium during active transcription of phospholipid biosynthetic genes under ER stress. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Proteostasis: bad news and good news from the endoplasmic reticulum.

    PubMed

    Noack, Julia; Brambilla Pisoni, Giorgia; Molinari, Maurizio

    2014-01-01

    The endoplasmic reticulum (ER) is an intracellular compartment dedicated to the synthesis and maturation of secretory and membrane proteins, totalling about 30% of the total eukaryotic cells proteome. The capacity to produce correctly folded polypeptides and to transport them to their correct intra- or extracellular destinations relies on proteostasis networks that regulate and balance the activity of protein folding, quality control, transport and degradation machineries. Nutrient and environmental changes, pathogen infection aging and, more relevant for the topics discussed in this review, mutations that impair attainment of the correct 3D structure of nascent polypeptide chains may compromise the activity of the proteostasis networks with devastating consequences on cells, organs and organisms' homeostasis. Here we present a review of mechanisms regulating folding and quality control of proteins expressed in the ER, and we describe the protein degradation and the ER stress pathways activated by the expression of misfolded proteins in the ER lumen. Finally, we highlight select examples of proteopathies (also known as conformational disorders or protein misfolding diseases) caused by protein misfolding in the ER and/or affecting cellular proteostasis and therapeutic interventions that might alleviate or cure the disease symptoms.

  14. Chlamydiae interaction with the endoplasmic reticulum: contact, function and consequences.

    PubMed

    Derré, Isabelle

    2015-07-01

    Chlamydiae and chlamydiae-related organisms are obligate intracellular bacterial pathogens. They reside in a membrane-bound compartment termed the inclusion and have evolved sophisticated mechanisms to interact with cellular organelles. This review focuses on the nature, the function(s) and the consequences of chlamydiae-inclusion interaction with the endoplasmic reticulum (ER). The inclusion membrane establishes very close contact with the ER at specific sites termed ER-inclusion membrane contact sites (MCSs). These MCSs are constituted of a specific set of factors, including the C. trachomatis effector protein IncD and the host cell proteins CERT and VAPA/B. Because CERT and VAPA/B have a demonstrated role in the non-vesicular trafficking of lipids between the ER and the Golgi, it was proposed that Chlamydia establish MCSs with the ER to acquire host lipids. However, the recruitment of additional factors to ER-inclusion MCSs, such as the ER calcium sensor STIM1, may suggest additional functions unrelated to lipid acquisition. Finally, chlamydiae interaction with the ER appears to induce the ER stress response, but this response is quickly dampened by chlamydiae to promote host cell survival. © 2015 John Wiley & Sons Ltd.

  15. Endoplasmic Reticulum - Plasma Membrane Crosstalk Mediated by the Extended Synaptotagmins.

    PubMed

    Saheki, Yasunori

    2017-01-01

    The endoplasmic reticulum (ER) possesses multiplicity of functions including protein synthesis, membrane lipid biogenesis, and Ca 2+ storage and has broad localization throughout the cell. While the ER and most other membranous organelles are highly interconnected via vesicular traffic that relies on membrane budding and fusion reactions, the ER forms direct contacts with virtually all other membranous organelles, including the plasma membrane (PM), without membrane fusion. Growing evidence suggests that these contacts play major roles in cellular physiology, including the regulation of Ca 2+ homeostasis and signaling and control of cellular lipid homeostasis. Extended synaptotagmins (E-Syts) are evolutionarily conserved family of ER-anchored proteins that tether the ER to the PM in PM PI(4,5)P 2 -dependent and cytosolic Ca 2+ -regulated manner. In addition, E-Syts possess a cytosolically exposed lipid-harboring module that confers the ability to transfer/exchange glycerolipids between the ER and the PM at E-Syts-mediated ER-PM contacts. In this chapter, the functions of ER-PM contacts and their role in non-vesicular lipid transport with special emphasis on the crosstalk between the two bilayers mediated by E-Syts will be discussed.

  16. Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response

    PubMed Central

    McKibbin, Craig; Mares, Alina; Piacenti, Michela; Williams, Helen; Roboti, Peristera; Puumalainen, Marjo; Callan, Anna C.; Lesiak-Mieczkowska, Karolina; Linder, Stig; Harant, Hanna; High, Stephen; Flitsch, Sabine L.; Whitehead, Roger C.; Swanton, Eileithyia

    2011-01-01

    Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ESI (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ESI causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation. PMID:22145777

  17. The failure to express a protein disulphide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice.

    PubMed

    Han, Xiaohua; Wang, Yihua; Liu, Xi; Jiang, Ling; Ren, Yulong; Liu, Feng; Peng, Cheng; Li, Jingjing; Jin, Ximing; Wu, Fuqing; Wang, Jiulin; Guo, Xiuping; Zhang, Xin; Cheng, Zhijun; Wan, Jianmin

    2012-01-01

    The rice somaclonal mutant T3612 produces small grains with a floury endosperm, caused by the loose packing of starch granules. The positional cloning of the mutation revealed a deletion in a gene encoding a protein disulphide isomerase-like enzyme (PDIL1-1). In the wild type, PDIL1-1 was expressed throughout the plant, but most intensely in the developing grain. In T3612, its expression was abolished, resulting in a decrease in the activity of plastidial phosphorylase and pullulanase, and an increase in that of soluble starch synthase I and ADP-glucose pyrophosphorylase. The amylopectin in the T3612 endosperm showed an increase in chains with a degree of polymerization 8-13 compared with the wild type. The expression in the mutant's endosperm of certain endoplasmic reticulum stress-responsive genes was noticeably elevated. PDIL1-1 appears to play an important role in starch synthesis. Its absence is associated with endoplasmic reticulum stress in the endosperm, which is likely to underlie the formation of the floury endosperm in the T3612 mutant.

  18. A Molecular Fluorescent Probe for Targeted Visualization of Temperature at the Endoplasmic Reticulum

    PubMed Central

    Arai, Satoshi; Lee, Sung-Chan; Zhai, Duanting; Suzuki, Madoka; Chang, Young Tae

    2014-01-01

    The dynamics of cellular heat production and propagation remains elusive at a subcellular level. Here we report the first small molecule fluorescent thermometer selectively targeting the endoplasmic reticulum (ER thermo yellow), with the highest sensitivity reported so far (3.9%/°C). Unlike nanoparticle thermometers, ER thermo yellow stains the target organelle evenly without the commonly encountered problem of aggregation, and successfully demonstrates the ability to monitor intracellular temperature gradients generated by external heat sources in various cell types. We further confirm the ability of ER thermo yellow to monitor heat production by intracellular Ca2+ changes in HeLa cells. Our thermometer anchored at nearly-zero distance from the ER, i.e. the heat source, allowed the detection of the heat as it readily dissipated, and revealed the dynamics of heat production in real time at a subcellular level. PMID:25330751

  19. Melatonin reduces endoplasmic reticulum stress and corneal dystrophy-associated TGFBIp through activation of endoplasmic reticulum-associated protein degradation.

    PubMed

    Choi, Seung-Il; Lee, Eunhee; Akuzum, Begum; Jeong, Jang Bin; Maeng, Yong-Sun; Kim, Tae-Im; Kim, Eung Kweon

    2017-10-01

    Endoplasmic reticulum (ER) stress is emerging as a factor for the pathogenesis of granular corneal dystrophy type 2 (GCD2). This study was designed to investigate the molecular mechanisms underlying the protective effects of melatonin on ER stress in GCD2. Our results showed that GCD2 corneal fibroblasts were more susceptible to ER stress-induced death than were wild-type cells. Melatonin significantly inhibited GCD2 corneal cell death, caspase-3 activation, and poly (ADP-ribose) polymerase 1 cleavage caused by the ER stress inducer, tunicamycin. Under ER stress, melatonin significantly suppressed the induction of immunoglobulin heavy-chain-binding protein (BiP) and activation of inositol-requiring enzyme 1α (IRE1α), and their downstream target, alternative splicing of X-box binding protein 1(XBP1). Notably, the reduction in BiP and IRE1α by melatonin was suppressed by the ubiquitin-proteasome inhibitor, MG132, but not by the autophagy inhibitor, bafilomycin A1, indicating involvement of the ER-associated protein degradation (ERAD) system. Melatonin treatment reduced the levels of transforming growth factor-β-induced protein (TGFBIp) significantly, and this reduction was suppressed by MG132. We also found reduced mRNA expression of the ERAD system components HRD1 and SEL1L, and a reduced level of SEL1L protein in GCD2 cells. Interestingly, melatonin treatments enhanced SEL1L levels and suppressed the inhibition of SEL1L N-glycosylation caused by tunicamycin. In conclusion, this study provides new insights into the mechanisms by which melatonin confers its protective actions during ER stress. The results also indicate that melatonin might have potential as a therapeutic agent for ER stress-related diseases including GCD2. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Denamur, Sophie; Boland, Lidvine

    Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasomemore » by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.« less

  1. Estrogen receptor α protects pancreatic β-cells from apoptosis by preserving mitochondrial function and suppressing endoplasmic reticulum stress.

    PubMed

    Zhou, Zhenqi; Ribas, Vicent; Rajbhandari, Prashant; Drew, Brian G; Moore, Timothy M; Fluitt, Amy H; Reddish, Britany R; Whitney, Kate A; Georgia, Senta; Vergnes, Laurent; Reue, Karen; Liesa, Marc; Shirihai, Orian; van der Bliek, Alexander M; Chi, Nai-Wen; Mahata, Sushil K; Tiano, Joseph P; Hewitt, Sylvia C; Tontonoz, Peter; Korach, Kenneth S; Mauvais-Jarvis, Franck; Hevener, Andrea L

    2018-03-30

    Estrogen receptor α (ERα) action plays an important role in pancreatic β-cell function and survival; thus, it is considered a potential therapeutic target for the treatment of type 2 diabetes in women. However, the mechanisms underlying the protective effects of ERα remain unclear. Because ERα regulates mitochondrial metabolism in other cell types, we hypothesized that ERα may act to preserve insulin secretion and promote β-cell survival by regulating mitochondrial-endoplasmic reticulum (EndoRetic) function. We tested this hypothesis using pancreatic islet-specific ERα knockout (PERαKO) mice and Min6 β-cells in culture with Esr1 knockdown (KD). We found that Esr1-KD promoted reactive oxygen species production that associated with reduced fission/fusion dynamics and impaired mitophagy. Electron microscopy showed mitochondrial enlargement and a pro-fusion phenotype. Mitochondrial cristae and endoplasmic reticulum were dilated in Esr1-KD compared with ERα replete Min6 β-cells. Increased expression of Oma1 and Chop was paralleled by increased oxygen consumption and apoptosis susceptibility in ERα-KD cells. In contrast, ERα overexpression and ligand activation reduced both Chop and Oma1 expression, likely by ERα binding to consensus estrogen-response element sites in the Oma1 and Chop promoters. Together, our findings suggest that ERα promotes β-cell survival and insulin secretion through maintenance of mitochondrial fission/fusion-mitophagy dynamics and EndoRetic function, in part by Oma1 and Chop repression.

  2. Structural reorganization of the fungal endoplasmic reticulum upon induction of mycotoxin biosynthesis.

    PubMed

    Boenisch, Marike Johanne; Broz, Karen Lisa; Purvine, Samuel Owen; Chrisler, William Byron; Nicora, Carrie Diana; Connolly, Lanelle Reine; Freitag, Michael; Baker, Scott Edward; Kistler, Harold Corby

    2017-03-13

    Compartmentalization of metabolic pathways to particular organelles is a hallmark of eukaryotic cells. Knowledge of the development of organelles and attendant pathways under different metabolic states has been advanced by live cell imaging and organelle specific analysis. Nevertheless, relatively few studies have addressed the cellular localization of pathways for synthesis of fungal secondary metabolites, despite their importance as bioactive compounds with significance to medicine and agriculture. When triggered to produce sesquiterpene (trichothecene) mycotoxins, the endoplasmic reticulum (ER) of the phytopathogenic fungus Fusarium graminearum is reorganized both in vitro and in planta. Trichothecene biosynthetic enzymes accumulate in organized smooth ER with pronounced expansion at perinuclear- and peripheral positions. Fluorescence tagged trichothecene biosynthetic proteins co-localize with the modified ER as confirmed by co-fluorescence and co-purification with known ER proteins. We hypothesize that changes to the fungal ER represent a conserved process in specialized eukaryotic cells such as in mammalian hepatocytes and B-cells.

  3. Unfolded Protein Response of the Endoplasmic Reticulum in Tumor Progression and Immunogenicity

    PubMed Central

    Yoo, Yoon Seon; Han, Hye Gyeong

    2017-01-01

    The endoplasmic reticulum (ER) is a pivotal regulator of folding, quality control, trafficking, and targeting of secreted and transmembrane proteins, and accordingly, eukaryotic cells have evolved specialized machinery to ensure that the ER enables these proteins to acquire adequate folding and maturation in the presence of intrinsic and extrinsic insults. This adaptive capacity of the ER to intrinsic and extrinsic perturbations is important for maintaining protein homeostasis, which is termed proteostasis. Failure in adaptation to these perturbations leads to accumulation of misfolded or unassembled proteins in the ER, which is termed ER stress, resulting in the activation of unfolded protein response (UPR) of the ER and the execution of ER-associated degradation (ERAD) to restore homeostasis. Furthermore, both of the two axes play key roles in the control of tumor progression, inflammation, immunity, and aging. Therefore, understanding UPR of the ER and subsequent ERAD will provide new insights into the pathogenesis of many human diseases and contribute to therapeutic intervention in these diseases. PMID:29430279

  4. Targeting the hallmarks of cancer with therapy-induced endoplasmic reticulum (ER) stress

    PubMed Central

    Garg, Abhishek D; Maes, Hannelore; van Vliet, Alexander R; Agostinis, Patrizia

    2015-01-01

    The endoplasmic reticulum (ER) is at the center of a number of vital cellular processes such as cell growth, death, and differentiation, crosstalk with immune or stromal cells, and maintenance of proteostasis or homeostasis, and ER functions have implications for various pathologies including cancer. Recently, a number of major hallmarks of cancer have been delineated that are expected to facilitate the development of anticancer therapies. However, therapeutic induction of ER stress as a strategy to broadly target multiple hallmarks of cancer has been seldom discussed despite the fact that several primary or secondary ER stress-inducing therapies have been found to exhibit positive clinical activity in cancer patients. In the present review we provide a brief historical overview of the major discoveries and milestones in the field of ER stress biology with important implications for anticancer therapy. Furthermore, we comprehensively discuss possible strategies enabling the targeting of multiple hallmarks of cancer with therapy-induced ER stress. PMID:27308392

  5. Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

    PubMed Central

    Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

    2012-01-01

    The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

  6. Dehydroascorbic acid-induced endoplasmic reticulum stress and leptin resistance in neuronal cells.

    PubMed

    Thon, Mina; Hosoi, Toru; Ozawa, Koichiro

    2016-09-16

    Due to its anti-obesity effects, an adipocyte-derived hormone, leptin, has become important for the treatment of obesity. However, most obese subjects are in a state of leptin resistance, and endoplasmic reticulum (ER) stress is suggested to be involved in the pathophysiology of leptin resistance. Dehydroascorbic acid (DHAA), an oxidized form of vitamin C, was found to be increased in diabetes. In the present study, we investigated the possible effects of DHAA on the activation of ER stress and leptin resistance. A human neuroblastoma cell line, stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb), was treated with DHAA. We found that DHAA upregulated ER stress-related genes such as GRP78, CHOP, and spliced XBP1. Moreover, leptin-induced STAT3 phosphorylation was hindered by DHAA. These results suggested that increases in the levels of DHAA might be harmful to neurons, contributing to defective leptin-responsive signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. The endoplasmic reticulum stress response in aging and age-related diseases

    PubMed Central

    Brown, Marishka K.; Naidoo, Nirinjini

    2012-01-01

    The endoplasmic reticulum(ER) is a multifunctional organelle within which protein folding, lipid biosynthesis, and calcium storage occurs. Perturbations such as energy or nutrient depletion, disturbances in calcium or redox status that disrupt ER homeostasis lead to the misfolding of proteins, ER stress and up-regulation of several signaling pathways coordinately called the unfolded protein response (UPR). The UPR is characterized by the induction of chaperones, degradation of misfolded proteins and attenuation of protein translation. The UPR plays a fundamental role in the maintenance of cellular homeostasis and thus is central to normal physiology. However, sustained unresolved ER stress leads to apoptosis. Aging linked declines in expression and activity of key ER molecular chaperones and folding enzymes compromise proper protein folding and the adaptive response of the UPR. One mechanism to explain age associated declines in cellular functions and age-related diseases is a progressive failure of chaperoning systems. In many of these diseases, proteins or fragments of proteins convert from their normally soluble forms to insoluble fibrils or plaques that accumulate in a variety of organs including the liver, brain or spleen. This group of diseases, which typically occur late in life includes Alzheimer's, Parkinson's, type II diabetes and a host of less well known but often equally serious conditions such as fatal familial insomnia. The UPR is implicated in many of these neurodegenerative and familial protein folding diseases as well as several cancers and a host of inflammatory diseases including diabetes, atherosclerosis, inflammatory bowel disease and arthritis. This review will discuss age-related changes in the ER stress response and the role of the UPR in age-related diseases. PMID:22934019

  8. Inhibition of endoplasmic reticulum stress improves coronary artery function in type 2 diabetic mice.

    PubMed

    Choi, Soo-Kyoung; Lim, Mihwa; Yeon, Soo-In; Lee, Young-Ho

    2016-06-01

    What is the central question of this study? Endoplasmic reticulum (ER) stress has been reported to be involved in type 2 diabetes; however, the role of exacerbated ER stress in vascular dysfunction in type 2 diabetes remains unknown. What is the main finding and its importance? The main findings of this study are that ER stress is increased in the coronary arteries in type 2 diabetes, and inhibition of ER stress using taurine-conjugated ursodeoxycholic acid improves vascular function, which is associated with normalization of the myogenic response and endothelium-dependent relaxation. Vascular dysfunction is a major complication in type 2 diabetes. Although endoplasmic reticulum (ER) stress has been suggested to be a contributory factor in cardiovascular diseases, the relationship between ER stress and vascular dysfunction in type 2 diabetes remains unclear. Thus, in the present study, we examined whether ER stress contributes to coronary artery dysfunction and whether inhibition of ER stress ameliorates vascular function in type 2 diabetes. Type 2 diabetic mice and their control counterparts were treated with an ER stress inhibitor (taurine-conjugated ursodeoxycholic acid, 150 mg kg(-1)  day(-1) , by i.p. injection) for 2 weeks or not treated. The myogenic response and endothelium-dependent relaxation were measured in pressurized coronary arteries. In type 2 diabetic mice, blood glucose and body weight were elevated compared with control mice. The myogenic response was potentiated and endothelium-dependent relaxation impaired in coronary arteries from the type 2 diabetic mice. Interestingly, treatment with the ER stress inhibitor normalized the myogenic responses and endothelium-dependent relaxation. These data were associated with an increase in ER stress marker expression or phosphorylation (IRE1-XBP-1 and PERK-eIF2α) in type 2 diabetic mice, which were reduced by treatment with the ER stress inhibitor. Inhibition of ER stress normalizes the myogenic

  9. Curcumin induces endoplasmic reticulum stress-associated apoptosis in human papillary thyroid carcinoma BCPAP cells via disruption of intracellular calcium homeostasis.

    PubMed

    Zhang, Li; Cheng, Xian; Xu, Shichen; Bao, Jiandong; Yu, Huixin

    2018-06-01

    Thyroid cancer is the most common endocrine tumor. Our previous studies have demonstrated that curcumin can induce apoptosis in human papillary thyroid carcinoma BCPAP cells. However, the underlined mechanism has not been clearly elucidated. Endoplasmic reticulum (ER) is a major organelle for synthesis, maturation, and folding proteins as well as a large store for Ca. Overcoming chronically activated ER stress by triggering pro-apoptotic pathways of the unfolded protein response (UPR) is a novel strategy for cancer therapeutics. Our study aimed to uncover the ER stress pathway involved in the apoptosis caused by curcumin. BCPAP cells were treated with different doses of curcumin (12.5-50 μM). Annexin V/PI double staining was used to determine cell apoptosis. Rhod-2/AM calcium fluorescence probe assay was performed to measure the calcium level of endoplasmic reticulum. Western blot was used to examine the expression of ER stress marker C/EBP homologous protein 10 (CHOP) and glucose-regulated protein 78 (GRP78). X-box binding protein1 (XBP-1) spliced form was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Curcumin significantly inhibited anchorage-independent cell growth and induced apoptosis in BCPAP cells. Curcumin induced ER stress and UPR responses in a dose- and time-dependent manner, and the chemical chaperone 4-phenylbutyrate (4-PBA) partially reversed the antigrowth activity of curcumin. Moreover, curcumin significantly increased inositol-requiring enzyme 1α (IRE1α) phosphorylation and XBP-1 mRNA splicing to induce a subsets of ER chaperones. Increased cleavage of activating transcription factor 6 (ATF6), which enhances expression of its downstream target CHOP was also observed. Furthermore, curcumin induced intracellular Ca influx through inhibition of the sarco-endoplasmic reticulum ATPase 2A (SERCA2) pump. The increased cytosolic Ca then bound to calmodulin to activate calcium/calmodulin-dependent protein kinase II (Ca

  10. Sarcoplasmic reticulum-mitochondria communication in cardiovascular pathophysiology.

    PubMed

    Lopez-Crisosto, Camila; Pennanen, Christian; Vasquez-Trincado, Cesar; Morales, Pablo E; Bravo-Sagua, Roberto; Quest, Andrew F G; Chiong, Mario; Lavandero, Sergio

    2017-06-01

    Repetitive, calcium-mediated contractile activity renders cardiomyocytes critically dependent on a sustained energy supply and adequate calcium buffering, both of which are provided by mitochondria. Moreover, in vascular smooth muscle cells, mitochondrial metabolism modulates cell growth and proliferation, whereas cytosolic calcium levels regulate the arterial vascular tone. Physical and functional communication between mitochondria and sarco/endoplasmic reticulum and balanced mitochondrial dynamics seem to have a critical role for optimal calcium transfer to mitochondria, which is crucial in calcium homeostasis and mitochondrial metabolism in both types of muscle cells. Moreover, mitochondrial dysfunction has been associated with myocardial damage and dysregulation of vascular smooth muscle proliferation. Therefore, sarco/endoplasmic reticulum-mitochondria coupling and mitochondrial dynamics are now viewed as relevant factors in the pathogenesis of cardiac and vascular diseases, including coronary artery disease, heart failure, and pulmonary arterial hypertension. In this Review, we summarize the evidence related to the role of sarco/endoplasmic reticulum-mitochondria communication in cardiac and vascular muscle physiology, with a focus on how perturbations contribute to the pathogenesis of cardiovascular disorders.

  11. Chronic Sleep Fragmentation During the Sleep Period Induces Hypothalamic Endoplasmic Reticulum Stress and PTP1b-Mediated Leptin Resistance in Male Mice

    PubMed Central

    Hakim, Fahed; Wang, Yang; Carreras, Alba; Hirotsu, Camila; Zhang, Jing; Peris, Eduard; Gozal, David

    2015-01-01

    Background: Sleep fragmentation (SF) is highly prevalent and may constitute an important contributing factor to excessive weight gain and the metabolic syndrome. Increased endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) leading to the attenuation of leptin receptor signaling in the hypothalamus leads to obesity and metabolic dysfunction. Methods: Mice were exposed to SF and sleep control (SC) for varying periods of time during which ingestive behaviors were monitored. UPR pathways and leptin receptor signaling were assessed in hypothalami. To further examine the mechanistic role of ER stress, changes in leptin receptor (ObR) signaling were also examined in wild-type mice treated with the ER chaperone tauroursodeoxycholic acid (TUDCA), as well as in CHOP −/+ transgenic mice. Results: Fragmented sleep in male mice induced increased food intake starting day 3 and thereafter, which was preceded by increases in ER stress and activation of all three UPR pathways in the hypothalamus. Although ObR expression was unchanged, signal transducer and activator of transcription 3 (STAT3) phosphorylation was decreased, suggesting reduced ObR signaling. Unchanged suppressor of cytokine signaling-3 (SOCS3) expression and increases in protein-tyrosine phosphatase 1B (PTP1B) expression and activity emerged with SF, along with reduced p-STAT3 responses to exogenous leptin. SF-induced effects were reversed following TUDCA treatment and were absent in CHOP −/+ mice. Conclusions: Sleep fragmentation (SF) induces hyperphagic behaviors and reduced leptin signaling in hypothalamus that are mediated by activation of endoplasmic reticulum (ER) stress, and ultimately lead to increased PTP1B activity. ER stress pathways are therefore potentially implicated in SF-induced weight gain and metabolic dysfunction, and may represent a viable therapeutic target. Citation: Hakim F, Wang Y, Carreras A, Hirotsu C, Zhang J, Peris E, Gozal D. Chronic sleep

  12. Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for β cell function.

    PubMed

    Lortz, S; Lenzen, S; Mehmeti, I

    2015-08-01

    Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing. © 2015 Society for Endocrinology.

  13. Endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo

    PubMed Central

    Colla, Emanuela; Coune, Philippe; Liu, Ying; Pletnikova, Olga; Troncoso, Juan C; Iwatsubo, Takeshi; Schneider, Bernard L.; Lee, Michael K.

    2012-01-01

    Accumulation of misfolded α-synuclein (αS) is mechanistically linked to neurodegeneration in Parkinson's disease (PD) and other α-synucleinopathies. However, how αS causes neurodegeneration is unresolved. Because cellular accumulation of misfolded proteins can lead to endoplasmic reticulum stress/unfolded protein response (ERS/UPR), chronic ERS could contribute to neurodegeneration in α-synucleinopathy. Using the A53T mutant human αS transgenic (A53TαS Tg) mouse model of α-synucleinopathy, we show that disease onset in the αS Tg model is coincident with induction of ER chaperone in neurons exhibiting αS pathology. However, the neuronal ER chaperone induction was not accompanied by the activation of phospho-eIF2α, indicating that α-synucleinopathy is associated with abnormal UPR that could promote cell death. Induction of ERS/UPR was associated with increased levels of ER/microsomal (ER/M) associated αS monomers and aggregates. Significantly, human PD cases also exhibit higher relative levels of ER/M αS than the control cases. Moreover, αS interacts with ER chaperones and overexpression of αS sensitizes neuronal cells to ERS-induced toxicity, suggesting that αS may have direct impact on ER function. This view is supported by the presence of ERS-activated caspase-12 and the accumulation of ER-associated polyubiquitin. More important, treatment with Salubrinal, an anti-ERS compound, significantly attenuates disease manifestations in both the A53TαS Tg mouse model and the Adeno-associated Virus-transduced rat model of A53T α S-dependent dopaminergic neurodegeneration. Our data indicate that the accumulation αS within ER leads to chronic ER stress conditions that contribute to neurodegeneration in α-synucleinopathies. Attenuating chronic ERS could be an effective therapy for PD and other α-synucleinopathies. PMID:22399753

  14. Fisetin induces apoptosis and endoplasmic reticulum stress in human non-small cell lung cancer through inhibition of the MAPK signaling pathway.

    PubMed

    Kang, Kyoung Ah; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Ryu, Yea Seong; Oh, Min Chang; Kwon, Taeg Kyu; Chae, Sungwook; Hyun, Jin Won

    2016-07-01

    Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid compound, is currently being investigated for its anticancer effect in various cancer models, including lung cancer. Recent studies show that fisetin induces cell growth inhibition and apoptosis in the human non-small cell lung cancer line NCI-H460. In this study, we investigated whether fisetin can induce endoplasmic reticulum (ER) stress-mediated apoptosis in NCI-H460 cells. Fisetin induced mitochondrial reactive oxygen species (ROS) and characteristic signs of ER stress: ER staining; mitochondrial Ca(2+) overload; expression of ER stress-related proteins; glucose-regulated protein (GRP)-78, phosphorylation of protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylation of eukaryotic initiation factor-2 α subunit; cleavage of activating transcription factor-6; phosphorylation of inositol-requiring kinase-1 and splicing of X-box transcription factor-1; induction of C/EBP homologous protein and cleaved caspase-12. siRNA-mediated knockdown of CHOP and ATF-6 attenuated fisetin-induced apoptotic cell death. In addition, fisetin induced phosphorylation of ERK, JNK, and p38 MAPK. Moreover, silencing of the MAPK signaling pathway prevented apoptotic cell death. In summary, our results indicate that, in NCI-H460 cells, fisetin induces apoptosis and ER stress that is mediated by induction of the MAPK signaling pathway.

  15. Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase 'Synoviolin'.

    PubMed

    Yamasaki, Satoshi; Yagishita, Naoko; Sasaki, Takeshi; Nakazawa, Minako; Kato, Yukihiro; Yamadera, Tadayuki; Bae, Eunkyung; Toriyama, Sayumi; Ikeda, Rie; Zhang, Lei; Fujitani, Kazuko; Yoo, Eunkyung; Tsuchimochi, Kaneyuki; Ohta, Tomohiko; Araya, Natsumi; Fujita, Hidetoshi; Aratani, Satoko; Eguchi, Katsumi; Komiya, Setsuro; Maruyama, Ikuro; Higashi, Nobuyo; Sato, Mitsuru; Senoo, Haruki; Ochi, Takahiro; Yokoyama, Shigeyuki; Amano, Tetsuya; Kim, Jaeseob; Gay, Steffen; Fukamizu, Akiyoshi; Nishioka, Kusuki; Tanaka, Keiji; Nakajima, Toshihiro

    2007-01-10

    Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin-null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

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

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-04

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. HLA-B35 Upregulates Endothelin-1 and Downregulates Endothelial Nitric Oxide Synthase via Endoplasmic Reticulum Stress Response in Endothelial Cells

    PubMed Central

    Lenna, Stefania; Townsend, Danyelle M.; Tan, Filemon K.; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

    2013-01-01

    The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1mRNAand protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension. PMID:20335527

  18. HLA-B35 upregulates endothelin-1 and downregulates endothelial nitric oxide synthase via endoplasmic reticulum stress response in endothelial cells.

    PubMed

    Lenna, Stefania; Townsend, Danyelle M; Tan, Filemon K; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

    2010-05-01

    The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

  19. Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

    PubMed Central

    Skehel, P. A.; Fabian-Fine, R.; Kandel, E. R.

    2000-01-01

    VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum–resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis. PMID:10655491

  20. The role of albumin and endoplasmic reticulum in pathogenesis Preeclampsia. Changes of GRP78 and placental VEGF in preeclampsia.

    PubMed

    Aditiawarman

    2014-07-01

    Preeclampsia remains the highest causes of maternal mortality in Indonesia. This disease is known as "the disease of theories" since the etiology of this disease still obscure, but it is clearly accepted that this disease is correlated with placenta. The incidence of this disease increased in dr. Soetomo General Hospital during economic crisis in Indonesia, as nutritional problem occurred, but correlation between nutrition to preeclampsia still unclear. This study was conducted to find out the influence of hypoalbuminemia caused by alteration of amino acids and changes of GRP78 and placental VEGF in preeclampsia. It was cross-sectional study among 10 patients with preeclampsia albumin ⩽ gram/dL and 9 patients with preeclampsia albumin >3g/dL. Placenta collected was homogenized and blood was separated from the cells. Placental lysat, serum and plasma were stored -87°C until laboratory analysis for GRP78 and VEGF. Data were analyzed using student t test, Manova. Pearson correlation, simple and multiple regressions. There were alterations of amino acid's profile among albumin ⩽3g/dL compare with albumin >3g/dL. GRP78 concentration on albumin ⩽3g/dL group was higher, but were not significant. Weak negative correlation between albumins to GRP78 was found. Negative correlation were found in the groups of albumin ⩽3g/dL - GRP⩽425μg/dL, but significant result only on albumin ⩽3g/dL - GRP⩽425μg/dL. Positive correlation were found in the group of albumin >3g/dL - GRP>425μg/dL. Response of Endoplasmic Reticulum Stress appeared different depends on levels of albumin (stressor) and GRP78 (stress response protein). Placental VEGF were significantly lower in albumin ⩽3g/dL (p<0.05). Correlation between GRP78 on placental VEGF was negative with coefficient correlation was 3.8%. Placental VEGF profoundly decreased as correlation between albumin and GRP78 stronger. Alteration of amino acids profile influenced Albumin concentration. Hypoalbuminemia was a stressor

  1. Limonoid Compounds Inhibit Sphingomyelin Biosynthesis by Preventing CERT Protein-dependent Extraction of Ceramides from the Endoplasmic Reticulum*

    PubMed Central

    Hullin-Matsuda, Françoise; Tomishige, Nario; Sakai, Shota; Ishitsuka, Reiko; Ishii, Kumiko; Makino, Asami; Greimel, Peter; Abe, Mitsuhiro; Laviad, Elad L.; Lagarde, Michel; Vidal, Hubert; Saito, Tamio; Osada, Hiroyuki; Hanada, Kentaro; Futerman, Anthony H.; Kobayashi, Toshihide

    2012-01-01

    To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism. PMID:22605339

  2. Endoplasmic Reticulum Chaperon Tauroursodeoxycholic Acid Attenuates Aldosterone-Infused Renal Injury

    PubMed Central

    Guo, Honglei; Li, Hongmei; Ling, Lilu

    2016-01-01

    Aldosterone (Aldo) is critically involved in the development of renal injury via the production of reactive oxygen species and inflammation. Endoplasmic reticulum (ER) stress is also evoked in Aldo-induced renal injury. In the present study, we investigated the role of ER stress in inflammation-mediated renal injury in Aldo-infused mice. C57BL/6J mice were randomized to receive treatment for 4 weeks as follows: vehicle infusion, Aldo infusion, vehicle infusion plus tauroursodeoxycholic acid (TUDCA), and Aldo infusion plus TUDCA. The effect of TUDCA on the Aldo-infused inflammatory response and renal injury was investigated using periodic acid-Schiff staining, real-time PCR, Western blot, and ELISA. We demonstrate that Aldo leads to impaired renal function and inhibition of ER stress via TUDCA attenuates renal fibrosis. This was indicated by decreased collagen I, collagen IV, fibronectin, and TGF-β expression, as well as the downregulation of the expression of Nlrp3 inflammasome markers, Nlrp3, ASC, IL-1β, and IL-18. This paper presents an important role for ER stress on the renal inflammatory response to Aldo. Additionally, the inhibition of ER stress by TUDCA negatively regulates the levels of these inflammatory molecules in the context of Aldo. PMID:27721575

  3. Coordination of Endoplasmic Reticulum (ER) Signaling During Maize Seed Development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boston, Rebecca S.

    2010-11-20

    Seed storage reserves represent one of the most important sources of renewable fixed carbon and nitrogen found in nature. Seeds are well-adapted for diverting metabolic resources to synthesize storage proteins as well as enzymes and structural proteins needed for their transport and packaging into membrane bound storage protein bodies. Our underlying hypothesis is that the endoplasmic reticulum (ER) stress response provides the critical cellular control of metabolic flux required for optimal accumulation of storage reserves in seeds. This highly conserved response is a cellular mechanism to monitor the protein folding environment of the ER and restore homeostasis in the presencemore » of unfolded or misfolded proteins. In seeds, deposition of storage proteins in protein bodies is a highly specialized process that takes place even in the presence of mutant proteins that no longer fold and package properly. The capacity of the ER to deposit these aberrant proteins in protein bodies during a period that extends several weeks provides an excellent model for deconvoluting the ER stress response of plants. We have focused in this project on the means by which the ER senses and responds to functional perturbations and the underlying intracellular communication that occurs among biosynthetic, trafficking and degradative pathways for proteins during seed development.« less

  4. [Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte].

    PubMed

    Ji, Ying-Lei; Yan, Jun; Wang, Yan-Sha; Liu, Yi-Chang; Gu, Zhen-Yong

    2014-02-01

    To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis. Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting. LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis. ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.

  5. ORP5/ORP8 localize to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function.

    PubMed

    Galmes, Romain; Houcine, Audrey; van Vliet, Alexander R; Agostinis, Patrizia; Jackson, Catherine L; Giordano, Francesca

    2016-06-01

    The oxysterol-binding protein (OSBP)-related proteins ORP5 and ORP8 have been shown recently to transport phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) at ER-PM contact sites. PS is also transferred from the ER to mitochondria where it acts as precursor for mitochondrial PE synthesis. Here, we show that, in addition to ER-PM contact sites, ORP5 and ORP8 are also localized to ER-mitochondria contacts and interact with the outer mitochondrial membrane protein PTPIP51. A functional lipid transfer (ORD) domain was required for this localization. Interestingly, ORP5 and ORP8 depletion leads to defects in mitochondria morphology and respiratory function. © 2016 The Authors.

  6. Fluorescence methods for analysis of interactions between Ca(2+) signaling, lysosomes, and endoplasmic reticulum.

    PubMed

    Prole, David L; López-Sanjurjo, Cristina I; Tovey, Stephen C; Taylor, Colin W

    2015-01-01

    The endoplasmic reticulum (ER) is both the major source of intracellular Ca(2+) for cell signaling and the organelle that forms the most extensive contacts with the plasma membrane and other organelles. Lysosomes fulfill important roles in degrading cellular materials and in cholesterol handling, but they also contribute to Ca(2+) signaling by both releasing and sequestering Ca(2+). Interactions between ER and other Ca(2+)-transporting membranes, notably mitochondria and the plasma membrane, often occur at sites where the two membranes are closely apposed, allowing local Ca(2+) signaling between them. These interactions are often facilitated by scaffold proteins. Recent evidence suggests similar local interactions between ER and lysosomes. We describe simple fluorescence-based methods that allow the interplay between Ca(2+) signals, the ER, and lysosomes to be examined. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP.

    PubMed

    Henderson, Mark J; Wires, Emily S; Trychta, Kathleen A; Yan, Xiaokang; Harvey, Brandon K

    2015-09-06

    The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

  8. Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress.

    PubMed

    Lee, Samuel; Min Kim, Soo; Dotimas, James; Li, Letitia; Feener, Edward P; Baldus, Stephan; Myers, Ronald B; Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T

    2014-06-01

    The endoplasmic reticulum (ER) is responsible for protein folding, modification, and trafficking. Accumulation of unfolded or misfolded proteins represents the condition of ER stress and triggers the unfolded protein response (UPR), a key mechanism linking supply of excess nutrients to insulin resistance and type 2 diabetes in obesity. The ER harbors proteins that participate in protein folding including protein disulfide isomerases (PDIs). Changes in PDI activity are associated with protein misfolding and ER stress. Here, we show that thioredoxin-interacting protein (Txnip), a member of the arrestin protein superfamily and one of the most strongly induced proteins in diabetic patients, regulates PDI activity and UPR signaling. We found that Txnip binds to PDIs and increases their enzymatic activity. Genetic deletion of Txnip in cells and mice led to increased protein ubiquitination and splicing of the UPR regulated transcription factor X-box-binding protein 1 (Xbp1s) at baseline as well as under ER stress. Our results reveal Txnip as a novel direct regulator of PDI activity and a feedback mechanism of UPR signaling to decrease ER stress. © 2014 Brigham and Women's Hospital. Published under the terms of the CC BY 4.0 license.

  9. Celecoxib enhances radiosensitivity of hypoxic glioblastoma cells through endoplasmic reticulum stress

    PubMed Central

    Suzuki, Kenshi; Gerelchuluun, Ariungerel; Hong, Zhengshan; Sun, Lue; Zenkoh, Junko; Moritake, Takashi; Tsuboi, Koji

    2013-01-01

    Background Refractoriness of glioblastoma multiforme (GBM) largely depends on its radioresistance. We investigated the radiosensitizing effects of celecoxib on GBM cell lines under both normoxic and hypoxic conditions. Methods Two human GBM cell lines, U87MG and U251MG, and a mouse GBM cell line, GL261, were treated with celecoxib or γ-irradiation either alone or in combination under normoxic and hypoxic conditions. Radiosensitizing effects were analyzed by clonogenic survival assays and cell growth assays and by assessing apoptosis and autophagy. Expression of apoptosis-, autophagy-, and endoplasmic reticulum (ER) stress–related genes was analyzed by immunoblotting. Results Celecoxib significantly enhanced the radiosensitivity of GBM cells under both normoxic and hypoxic conditions. In addition, combined treatment with celecoxib and γ-irradiation induced marked autophagy, particularly in hypoxic cells. The mechanism underlying the radiosensitizing effect of celecoxib was determined to be ER stress loading on GBM cells. Conclusion Celecoxib enhances the radiosensitivity of GBM cells by a mechanism that is different from cyclooxygenase-2 inhibition. Our results indicate that celecoxib may be a promising radiosensitizing drug for clinical use in patients with GBM. PMID:23658321

  10. The Endoplasmic Reticulum Chaperone GRP170: From Immunobiology to Cancer Therapeutics

    PubMed Central

    Wang, Hongxia; Pezeshki, Abdul Mohammad; Yu, Xiaofei; Guo, Chunqing; Subjeck, John R.; Wang, Xiang-Yang

    2014-01-01

    Glucose-regulated protein 170 (GRP170) is the largest member of glucose-regulated protein family that resides in the endoplasmic reticulum (ER). As a component of the ER chaperone network, GRP170 assists in protein folding, assembly, and transportation of secretory or transmembrane proteins. The well documented cytoprotective activity of intracellular GRP170 due to its intrinsic chaperoning property has been shown to provide a survival benefit in cancer cells during tumor progression or metastasis. Accumulating evidence shows that extracellular GRP170 displays a superior capacity in delivering tumor antigens to specialized antigen-presenting cells for cross-presentation, resulting in generation of an anti-tumor immune response dependent on cytotoxic CD8+ T cells. This unique feature of GRP170 provides a molecular basis for using GRP170 as an immunostimulatory adjuvant to develop a recombinant vaccine for therapeutic immunization against cancers. This review summarizes the latest findings in understanding the biological effects of GRP170 on cell functions and tumor progression. The immunomodulating activities of GRP170 during interactions with the innate and adaptive arms of the immune system as well as its therapeutic applications in cancer immunotherapy will be discussed. PMID:25629003

  11. Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

    PubMed

    Gomes-da-Silva, Lígia C; Zhao, Liwei; Bezu, Lucillia; Zhou, Heng; Sauvat, Allan; Liu, Peng; Durand, Sylvère; Leduc, Marion; Souquere, Sylvie; Loos, Friedemann; Mondragón, Laura; Sveinbjørnsson, Baldur; Rekdal, Øystein; Boncompain, Gaelle; Perez, Franck; Arnaut, Luis G; Kepp, Oliver; Kroemer, Guido

    2018-05-28

    Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function. © 2018 The Authors.

  12. Fluoride-elicited developmental testicular toxicity in rats: Roles of endoplasmic reticulum stress and inflammatory response

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Shun; Jiang, Chunyang; Department of Thoracic Surgery, Tianjin Union Medicine Centre, 190 Jieyuan Road, Hongqiao District, Tianjin 300121, Tianjin

    Long-term excessive fluoride intake is known to be toxic and can damage a variety of organs and tissues in the human body. However, the molecular mechanisms underlying fluoride-induced male reproductive toxicity are not well understood. In this study, we used a rat model to simulate the situations of human exposure and aimed to evaluate the roles of endoplasmic reticulum (ER) stress and inflammatory response in fluoride-induced testicular injury. Sprague–Dawley rats were administered with sodium fluoride (NaF) at 25, 50 and 100 mg/L via drinking water from pre-pregnancy to gestation, birth and finally to post-puberty. And then the testes of malemore » offspring were studied at 8 weeks of age. Our results demonstrated that fluoride treatment increased MDA accumulation, decreased SOD activity, and enhanced germ cell apoptosis. In addition, fluoride elevated mRNA and protein levels of glucose-regulated protein 78 (GRP78), inositol requiring ER-to-nucleus signal kinase 1 (IRE1), and C/EBP homologous protein (CHOP), indicating activation of ER stress signaling. Furthermore, fluoride also induced testicular inflammation, as manifested by gene up-regulation of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in a nuclear factor-κB (NF-κB)-dependent manner. These were associated with marked histopathological lesions including injury of spermatogonia, decrease of spermatocytes and absence of elongated spermatids, as well as severe ultrastructural abnormalities in testes. Taken together, our results provide compelling evidence that ER stress and inflammation would be novel and significant mechanisms responsible for fluoride-induced disturbance of spermatogenesis and germ cell loss in addition to oxidative stress. - Highlights: • We used a rat model to simulate the situations of human fluoride (F) exposure. • Developmental F exposure induces testicular damage related with oxidative

  13. Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

    PubMed

    Robinson, Philip J; Pringle, Marie Anne; Woolhead, Cheryl A; Bulleid, Neil J

    2017-04-28

    The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Endoplasmic Reticulum Stress Signaling in Mammalian Oocytes and Embryos: Life in the Balance

    PubMed Central

    Latham, Keith E.

    2015-01-01

    Mammalian oocytes and embryos are exquisitely sensitive to a wide range of insults related to physical stress, chemical exposure, and exposures to adverse maternal nutrition or health status. Although cells manifest specific responses to various stressors, many of these stressors intersect at the endoplasmic reticulum, where disruptions in protein folding and production of reactive oxygen species initiate downstream signaling events. These signals modulate mRNA translation and gene transcription, leading to recovery, activation of autophagy, or with severe and prolonged stress, apoptosis. ER stress signaling has recently come to the fore as a major contributor to embryo demise. Accordingly, agents that modulate or inhibit ER stress signaling have yielded beneficial effects on embryo survival and long-term developmental potential. We review here the mechanisms of ER stress signaling, their connections to mammalian oocytes and embryos, and the promising indications that interventions in this pathway may provide new opportunities for improving mammalian reproduction and health. PMID:25805126

  15. Tribbles 3 Mediates Endoplasmic Reticulum Stress-Induced Insulin Resistance in Skeletal Muscle

    PubMed Central

    Koh, Ho-Jin; Toyoda, Taro; Didesch, Michelle M.; Lee, Min-Young; Sleeman, Mark W.; Kulkarni, Rohit N.; Musi, Nicolas; Hirshman, Michael F.; Goodyear, Laurie J.

    2013-01-01

    Endoplasmic Reticulum (ER) stress has been linked to insulin resistance in multiple tissues but the role of ER stress in skeletal muscle has not been explored. ER stress has also been reported to increase tribbles 3 (TRB3) expression in multiple cell lines. Here, we report that high fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases TRB3 and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signaling. Incubation of C2C12 cells and mouse skeletal muscle with ER stressors thapsigargin and tunicamycin increases TRB3 and impairs insulin signaling and glucose uptake, effects reversed in cells overexpressing RNAi for TRB3 and in muscles from TRB3 knockout mice. Furthermore, TRB3 knockout mice are protected from high fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that TRB3 mediates ER stress-induced insulin resistance in skeletal muscle. PMID:23695665

  16. TANGO1 recruits ERGIC membranes to the endoplasmic reticulum for procollagen export

    PubMed Central

    Santos, António JM; Raote, Ishier; Scarpa, Margherita; Brouwers, Nathalie; Malhotra, Vivek

    2015-01-01

    Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process. DOI: http://dx.doi.org/10.7554/eLife.10982.001 PMID:26568311

  17. Peroxisomes, lipid droplets, and endoplasmic reticulum “hitchhike” on motile early endosomes

    PubMed Central

    Guimaraes, Sofia C.; Schuster, Martin; Bielska, Ewa; Dagdas, Gulay; Kilaru, Sreedhar; Meadows, Ben R.A.; Schrader, Michael

    2015-01-01

    Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell. PMID:26620910

  18. GABAB receptor cell surface export is controlled by an endoplasmic reticulum gatekeeper

    PubMed Central

    Doly, Stéphane; Shirvani, Hamasseh; Gäta, Gabriel; Meye, Frank; Emerit, Michel-Boris; Enslen, Hervé; Achour, Lamia; Pardo-Lopez, Liliana; Kwon, Yang Seung; Armand, Vincent; Gardette, Robert; Giros, Bruno; Gassmann, Martin; Bettler, Bernhard; Mameli, Manuel; Darmon, Michèle; Marullo, Stefano

    2016-01-01

    Summary Endoplasmic reticulum (ER) release and cell surface export of many G protein-coupled receptors (GPCRs), are tightly regulated. For GABAB receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell surface receptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gate-keepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function. PMID:26033241

  19. Endoplasmic Reticulum Stress in Obesity and Obesity-Related Disorders: An Expanded View

    PubMed Central

    Pagliassotti, M.J.; Kim, P. Y.; Estrada, A.L.; Stewart, C.M.; Gentile, C.L.

    2016-01-01

    The Endoplasmic Reticulum (ER) is most notable for its central roles in calcium ion storage, lipid biosynthesis, and protein sorting and processing. By virtue of its extensive membrane contact sites that connect the ER to most other organelles and to the plasma membrane, the ER can also regulate diverse cellular processes including inflammatory and insulin signaling, nutrient metabolism, and cell proliferation and death via a signaling pathway called the unfolded protein response (UPR). Chronic UPR activation has been observed in liver and/or adipose tissue of dietary and genetic murine models of obesity, and in human obesity and non-alcoholic fatty liver disease (NAFLD). Activation of the UPR in obesity and obesity-related disorders likely has two origins. One linked to classic ER stress involving the ER lumen and one linked to alterations to the ER membrane environment. This review discusses both of these origins and also considers the role of post-translational protein modifications, such as acetylation and palmitoylation, and ER-mitochondrial interactions to obesity-mediated impairments in the ER and activation of the UPR. PMID:27506731

  20. AGR2, an Endoplasmic Reticulum Protein, Is Secreted into the Gastrointestinal Mucus

    PubMed Central

    Bergström, Joakim H.; Berg, Katarina A.; Rodríguez-Piñeiro, Ana M.; Stecher, Bärbel; Johansson, Malin E. V.; Hansson, Gunnar C.

    2014-01-01

    The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2−/− mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine. PMID:25111734

  1. Implication of STARD5 and cholesterol homeostasis disturbance in the endoplasmic reticulum stress-related response induced by pro-apoptotic aminosteroid RM-133.

    PubMed

    Perreault, Martin; Maltais, René; Kenmogne, Lucie Carolle; Létourneau, Danny; LeHoux, Jean-Guy; Gobeil, Stéphane; Poirier, Donald

    2018-02-01

    The aminosteroid derivative RM-133 is an effective anticancer molecule for which proof of concept has been achieved in several mouse xenograph models (HL-60, MCF-7, PANC-1 and OVCAR-3). To promote this new family of molecules toward a clinical phase 1 trial, the mechanism of action governing the anticancer properties of the representative candidate RM-133 needs to be characterized. In vitro experiments were first used to determine that RM-133 causes apoptosis in cancer cells. Then, using proteomic and transcriptomic experiments, RM-133 cytotoxicity was proven to be achieved via the endoplasmic reticulum (ER)-related apoptosis, which characterizes RM-133 as an endoplasmic reticulum stress aggravator (ERSA) anticancer drug. Furthermore, an shRNA-genome-wide screening has permitted to identify the steroidogenic acute regulator-related lipid transfer protein 5 (STARD5) as a major player in the RM-133 ER-related apoptosis mechanism, which was validated by an in vitro binding experiment. Altogether, the results presented herein suggest that RM-133 provokes a disturbance of cholesterol homeostasis via the implication of STARD5, which delivers an ERSA molecule to the ER. These results will be a springboard for RM-133 in its path toward clinical use. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Modeling Endoplasmic Reticulum Network Maintenance in a Plant Cell.

    PubMed

    Lin, Congping; White, Rhiannon R; Sparkes, Imogen; Ashwin, Peter

    2017-07-11

    The endoplasmic reticulum (ER) in plant cells forms a highly dynamic network of complex geometry. ER network morphology and dynamics are influenced by a number of biophysical processes, including filament/tubule tension, viscous forces, Brownian diffusion, and interactions with many other organelles and cytoskeletal elements. Previous studies have indicated that ER networks can be thought of as constrained minimal-length networks acted on by a variety of forces that perturb and/or remodel the network. Here, we study two specific biophysical processes involved in remodeling. One is the dynamic relaxation process involving a combination of tubule tension and viscous forces. The other is the rapid creation of cross-connection tubules by direct or indirect interactions with cytoskeletal elements. These processes are able to remodel the ER network: the first reduces network length and complexity whereas the second increases both. Using live cell imaging of ER network dynamics in tobacco leaf epidermal cells, we examine these processes on ER network dynamics. Away from regions of cytoplasmic streaming, we suggest that the dynamic network structure is a balance between the two processes, and we build an integrative model of the two processes for network remodeling. This model produces quantitatively similar ER networks to those observed in experiments. We use the model to explore the effect of parameter variation on statistical properties of the ER network. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghoshal, Kankana; Theilmann, Jane; Reade, Ron

    2014-11-15

    Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified includingmore » the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication.« less

  4. Vascular Nox (NADPH Oxidase) Compartmentalization, Protein Hyperoxidation, and Endoplasmic Reticulum Stress Response in Hypertension.

    PubMed

    Camargo, Livia L; Harvey, Adam P; Rios, Francisco J; Tsiropoulou, Sofia; Da Silva, Renée de Nazaré Oliveira; Cao, Zhenbo; Graham, Delyth; McMaster, Claire; Burchmore, Richard J; Hartley, Richard C; Bulleid, Neil; Montezano, Augusto C; Touyz, Rhian M

    2018-07-01

    Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O 2 - (lucigenin), H 2 O 2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1α [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1α sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1-XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1-XBP1 pathway of the ER stress

  5. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pan, Mu-Yun; Shen, Yuh-Chiang; National Research Institute of Chinese Medicine, Taipei, Taiwan

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ERmore » stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

  6. Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

    PubMed Central

    Helm, Jared R.; Bentley, Marvin; Thorsen, Kevin D.; Wang, Ting; Foltz, Lauren; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C.

    2014-01-01

    Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery. PMID:25006245

  7. The Effect of Bornyl cis-4-Hydroxycinnamate on Melanoma Cell Apoptosis Is Associated with Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

    PubMed Central

    Yang, Tzu-Yen; Wu, Yu-Jen; Chang, Chi-I; Wu, Mei-Li

    2018-01-01

    Bornyl cis-4-hydroxycinnamate, an active compound isolated from Piper betle stems, was investigated in terms of its effects on A2058 and A375 melanoma cell proliferation and protein expression in this study. We used flow cytometric analysis to examine the early stages of apoptosis induced by bornyl cis-4-hydroxycinnamate in the two melanoma cell lines and employed comparative proteomic analysis to investigate the effects of this compound on protein expression in A375 cells. Master maps generated by PDQuest software from two-dimensional electrophoresis (2-DE) analysis of A375 cells showed that the expression levels of 35 proteins were significantly altered, with 18 proteins upregulated and 17 downregulated. The proteomics study identified several proteins that are involved in mitochondrial dysfunction and endoplasmic reticulum stress (ER stress), in addition to apoptosis-associated proteins, including prohibitin, hypoxia-upregulated protein 1, stress 70 protein, 78 kDa glucose-regulated protein (GRP78), and protein deglycase DJ-1 (protein DJ-1) in melanoma cells exposed to bornyl cis-4-hydroxycinnamate. The treatment also resulted in a marked decline of the mitochondrial membrane potential, in cytochrome C release into the cytosol, in the activation of Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad), caspase-3, and caspase-9, and in the decreased expression of p-Bad, B-cell lymphoma 2 (Bcl-2), Bcl-xl, and induced myeloid leukemia cell differentiation protein-1 (Mcl-1), indicating that apoptosis induced by bornyl cis-4-hydroxycinnamate was mediated by the mitochondria through the caspase-dependent pathway. Also, salubrinal (an eukaryotic initiation factor 2α inhibitor; eIF2α inhibitor) was able to protect the cells from bornyl cis-4-hydroxycinnamate-induced apoptosis. Bornyl cis-4-hydroxycinnamate-related cell death also implied that the protein kinase R-like endoplasmic reticulum kinase (PERK)–eIF2α–ATF4–CHOP signal pathways

  8. Peroxisomal Ascorbate Peroxidase Resides within a Subdomain of Rough Endoplasmic Reticulum in Wild-Type Arabidopsis Cells1

    PubMed Central

    Lisenbee, Cayle S.; Heinze, Michael; Trelease, Richard N.

    2003-01-01

    Previously we reported (R.T. Mullen, C.S. Lisenbee, J.A. Miernyk, R.N. Trelease [1999] Plant Cell 11: 2167–2185) that overexpressed ascorbate peroxidase (APX), a peroxisomal membrane protein, sorted indirectly to Bright Yellow-2 cell peroxisomes via a subdomain of the endoplasmic reticulum (ER; peroxisomal endoplasmic reticulum [pER]). More recently, a pER-like compartment also was identified in pumpkin (Cucurbita pepo) and transformed Arabidopsis cells (K. Nito, K. Yamaguchi, M. Kondo, M. Hayashi, M. Nishimura [2001] Plant Cell Physiol 42: 20–27). Here, we characterize more extensively the localization of endogenous Arabidopsis peroxisomal APX (AtAPX) in cultured wild-type Arabidopsis cells (Arabidopsis var. Landsberg erecta). AtAPX was detected in peroxisomes, but not in an ER subcompartment, using immunofluorescence microscopy. However, AtAPX was detected readily with immunoblots in both peroxisomal and ER fractions recovered from sucrose (Suc) density gradients. Most AtAPX in microsomes (200,000g, 1 h pellet) applied to gradients exhibited a Mg2+-induced shift from a distribution throughout gradients (approximately 18%–40% [w/w] Suc) to ≥42% (w/w) Suc regions of gradients, including pellets, indicative of localization in rough ER vesicles. Immunogold electron microscopy of the latter fractions verified these findings. Further analyses of peroxisomal and rough ER vesicle fractions revealed that AtAPX in both fractions was similarly associated with and located mostly on the cytosolic face of the membranes. Thus, at the steady state, endogenous peroxisomal AtAPX resides at different levels in rough ER and peroxisomes. Collectively, these findings show that rather than being a transiently induced sorting compartment formed in response to overexpressed peroxisomal APX, portions of rough ER (pER) in wild-type cells serve as a constitutive sorting compartment likely involved in posttranslational routing of constitutively synthesized peroxisomal APX. PMID

  9. Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver

    PubMed Central

    Li, Yu; Xu, Shanqin; Giles, Amber; Nakamura, Kazuto; Lee, Jong Woo; Hou, Xiuyun; Donmez, Gizem; Li, Ji; Luo, Zhijun; Walsh, Kenneth; Guarente, Leonard; Zang, Mengwei

    2011-01-01

    Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of human type 2 diabetes (T2DM). Although SIRT1 has a therapeutic effect on metabolic deterioration in T2DM, the precise mechanisms by which SIRT1 improves insulin resistance remain unclear. Here, we demonstrate that adenovirus-mediated overexpression of SIRT1 in the liver of diet-induced insulin-resistant low-density lipoprotein receptor-deficient mice and of genetically obese ob/ob mice attenuates hepatic steatosis and ameliorates systemic insulin resistance. These beneficial effects were associated with decreased mammalian target of rapamycin complex 1 (mTORC1) activity, inhibited the unfolded protein response (UPR), and enhanced insulin receptor signaling in the liver, leading to decreased hepatic gluconeogenesis and improved glucose tolerance. The tunicamycin-induced splicing of X-box binding protein-1 and expression of GRP78 and CHOP were reduced by resveratrol in cultured cells in a SIRT1-dependent manner. Conversely, SIRT1-deficient mouse embryonic fibroblasts challenged with tunicamycin exhibited markedly increased mTORC1 activity and impaired ER homeostasi and insulin signaling. These effects were abolished by mTORC1 inhibition by rapamycin in human HepG2 cells. These studies indicate that SIRT1 serves as a negative regulator of UPR signaling in T2DM and that SIRT1 attenuates hepatic steatosis, ameliorates insulin resistance, and restores glucose homeostasis, largely through the inhibition of mTORC1 and ER stress.—Li, Y., Xu, S., Giles, A., Nakamura, K., Lee, J. W., Hou, X., Donmez, G., Li, J., Luo, Z., Walsh, K., Guarente, L., Zang, M. Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver. PMID:21321189

  10. Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chang, Yoke-Chen; Wang, James D.; Svoboda, Kathy K.

    The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing frommore » 24 h to 168 h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72 h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24 h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury. - Highlights: ► We demonstrated ER stress response in the mouse ear vesicant model. ► We described the asymmetrical nature of wound repair in the MEVM. ► We identified the distribution of various ER stress markers in the MEVM.« less

  11. Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.

    PubMed

    Woods, Alison J; Roberts, Marnie S; Choudhary, Jyoti; Barry, Simon T; Mazaki, Yuichi; Sabe, Hisataka; Morley, Simon J; Critchley, David R; Norman, Jim C

    2002-02-22

    Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.

  12. Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin'

    PubMed Central

    Yamasaki, Satoshi; Yagishita, Naoko; Sasaki, Takeshi; Nakazawa, Minako; Kato, Yukihiro; Yamadera, Tadayuki; Bae, Eunkyung; Toriyama, Sayumi; Ikeda, Rie; Zhang, Lei; Fujitani, Kazuko; Yoo, Eunkyung; Tsuchimochi, Kaneyuki; Ohta, Tomohiko; Araya, Natsumi; Fujita, Hidetoshi; Aratani, Satoko; Eguchi, Katsumi; Komiya, Setsuro; Maruyama, Ikuro; Higashi, Nobuyo; Sato, Mitsuru; Senoo, Haruki; Ochi, Takahiro; Yokoyama, Shigeyuki; Amano, Tetsuya; Kim, Jaeseob; Gay, Steffen; Fukamizu, Akiyoshi; Nishioka, Kusuki; Tanaka, Keiji; Nakajima, Toshihiro

    2007-01-01

    Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin-null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin. PMID:17170702

  13. Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD)*

    PubMed Central

    Lee, Jae-Jin; Park, Joon Kyu; Jeong, Jaeho; Jeon, Hyesung; Yoon, Jong-Bok; Kim, Eunice EunKyeong; Lee, Kong-Joo

    2013-01-01

    Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys48-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation. PMID:23293021

  14. Tauroursodeoxycholic Acid Attenuates Lipid Accumulation in Endoplasmic Reticulum-Stressed Macrophages

    PubMed Central

    Hua, Yinan; Kandadi, Machender R.; Zhu, Meijun; Ren, Jun; Sreejayan, Nair

    2011-01-01

    Background/Aim Recent evidence suggests that endoplasmic reticulum (ER) stress provoked under diabetic conditions augments the expression of scavenger receptors on macrophages, promoting the uptake of oxidized low-density lipoprotein (ox-LDL) uptake and atherogenesis. The aim of the present study was to test the hypothesis that the chemical chaperone tauroursodeoxycholic acid (TUDCA) attenuates lipid accumulation in macrophages subjected to ER stress. Methods Cultured human macrophages were subjected to ER-stress by treating them with tunicamycin. Lipid-uptake by macrophages subjected to ER-stress in the presence or absence of TUDCA was assessed by oil red O staining and by assessing the cellular uptake of Dil-ox-LDL by fluorescence measurement. Protein levels and phosphorylation status of ER stress markers, insulin-signalling molecules and scavenger receptor were assessed by Western blotting. Results Treatment of cultured human macrophages with the ER-stressor tunicamycin caused an increase in the protein levels of CD-36, and augmentation of lipid-uptake both of which were inhibited by TUDCA. TUDCA-treatment inhibited tunicamycin-induced ER-stress as evidenced by the attenuation of phosphorylation of eukaryotic translation initiation factor-2α and glucose reactive protein-78. In addition, TUDCA improved insulin signaling in macrophages by augmenting Akt-phosphorylation and blunting c-Jun N-terminal kinase activity. Conclusion Inhibition of macrophage ER-stress may represent a potential strategy in preventing atherogenesis under diabetic conditions. PMID:19834331

  15. BiP clustering facilitates protein folding in the endoplasmic reticulum.

    PubMed

    Griesemer, Marc; Young, Carissa; Robinson, Anne S; Petzold, Linda

    2014-07-01

    The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.

  16. Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum.

    PubMed

    Cianciola, Nicholas L; Chung, Stacey; Manor, Danny; Carlin, Cathleen R

    2017-03-15

    Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDα via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDα. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich "lipid rafts." Collectively, these data show that RIDα utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes. IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune

  17. Glucosylated free oligosaccharides are biomarkers of endoplasmic- reticulum alpha-glucosidase inhibition.

    PubMed

    Alonzi, Dominic S; Neville, David C A; Lachmann, Robin H; Dwek, Raymond A; Butters, Terry D

    2008-01-15

    The inhibition of ER (endoplasmic reticulum) alpha-glucosidases I and II by imino sugars, including NB-DNJ (N-butyl-deoxynojirimycin), causes the retention of glucose residues on N-linked oligosaccharides. Therefore, normal glycoprotein trafficking and processing through the glycosylation pathway is abrogated and glycoproteins are directed to undergo ERAD (ER-associated degradation), a consequence of which is the production of cytosolic FOS (free oligosaccharides). Following treatment with NB-DNJ, FOS were extracted from cells, murine tissues and human plasma and urine. Improved protocols for analysis were developed using ion-exchange chromatography followed by fluorescent labelling with 2-AA (2-aminobenzoic acid) and purification by lectin-affinity chromatography. Separation of 2-AA-labelled FOS by HPLC provided a rapid and sensitive method that enabled the detection of all FOS species resulting from the degradation of glycoproteins exported from the ER. The generation of oligosaccharides derived from glucosylated protein degradation was rapid, reversible, and time- and inhibitor concentration-dependent in cultured cells and in vivo. Long-term inhibition in cultured cells and in vivo indicated a slow rate of clearance of glucosylated FOS. In mouse and human urine, glucosylated FOS were detected as a result of transrenal excretion and provide unique and quantifiable biomarkers of ER-glucosidase inhibition.

  18. Parkin regulation of CHOP modulates susceptibility to cardiac endoplasmic reticulum stress.

    PubMed

    Han, Kim; Hassanzadeh, Shahin; Singh, Komudi; Menazza, Sara; Nguyen, Tiffany T; Stevens, Mark V; Nguyen, An; San, Hong; Anderson, Stasia A; Lin, Yongshun; Zou, Jizhong; Murphy, Elizabeth; Sack, Michael N

    2017-05-18

    The regulatory control of cardiac endoplasmic reticulum (ER) stress is incompletely characterized. As ER stress signaling upregulates the E3-ubiquitin ligase Parkin, we investigated the role of Parkin in cardiac ER stress. Parkin knockout mice exposed to aortic constriction-induced cardiac pressure-overload or in response to systemic tunicamycin (TM) developed adverse ventricular remodeling with excessive levels of the ER regulatory C/EBP homologous protein CHOP. CHOP was identified as a Parkin substrate and its turnover was Parkin-dose and proteasome-dependent. Parkin depletion in cardiac HL-1 cells increased CHOP levels and enhanced susceptibility to TM-induced cell death. Parkin reconstitution rescued this phenotype and the contribution of excess CHOP to this ER stress injury was confirmed by reduction in TM-induced cell death when CHOP was depleted in Parkin knockdown cardiomyocytes. Isogenic Parkin mutant iPSC-derived cardiomyocytes showed exaggerated ER stress induced CHOP and apoptotic signatures and myocardium from subjects with dilated cardiomyopathy showed excessive Parkin and CHOP induction. This study identifies that Parkin functions to blunt excessive CHOP to prevent maladaptive ER stress-induced cell death and adverse cardiac ventricular remodeling. Additionally, Parkin is identified as a novel post-translational regulatory moderator of CHOP stability and uncovers an additional stress-modifying function of this E3-ubiquitin ligase.

  19. The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation

    PubMed Central

    Preston, G. Michael; Brodsky, Jeffrey L.

    2017-01-01

    The endoplasmic reticulum (ER) serves as a warehouse for factors that augment and control the biogenesis of nascent proteins entering the secretory pathway. In turn, this compartment also harbors the machinery that responds to the presence of misfolded proteins by targeting them for proteolysis via a process known as ER-associated degradation (ERAD). During ERAD, substrates are selected, modified with ubiquitin, removed from the ER, and then degraded by the cytoplasmic 26S proteasome. While integral membrane proteins can directly access the ubiquitination machinery that resides in the cytoplasm or on the cytoplasmic face of the ER membrane, soluble ERAD substrates within the lumen must be retrotranslocated from this compartment. In either case, nearly all ERAD substrates are tagged with a polyubiquitin chain, a modification that represents a commitment step to degrade aberrant proteins. However, increasing evidence indicates that the polyubiquitin chain on ERAD substrates can be further modified, serves to recruit ERAD-requiring factors, and may regulate the ERAD machinery. Amino acid side chains other than lysine on ERAD substrates can also be modified with ubiquitin, and post-translational modifications that affect substrate ubiquitination have been observed. Here, we summarize these data and provide an overview of questions driving this field of research. PMID:28159894

  20. Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang, C.-H., E-mail: chtang@mail.cmu.edu.t; Graduate Institute of Basic Medical Science, China Medical University, Taichung Taiwan; Chiu, Y.-C.

    Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromalmore » cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.« less

  1. Loss of Mitofusin 2 Promotes Endoplasmic Reticulum Stress*

    PubMed Central

    Ngoh, Gladys A.; Papanicolaou, Kyriakos N.; Walsh, Kenneth

    2012-01-01

    The outer mitochondrial membrane GTPase mitofusin 2 (Mfn2) is known to regulate endoplasmic reticulum (ER) shape in addition to its mitochondrial fusion effects. However, its role in ER stress is unknown. We report here that induction of ER stress with either thapsigargin or tunicamycin in mouse embryonic fibroblasts leads to up-regulation of Mfn2 mRNA and protein levels with no change in the expression of the mitochondrial shaping factors Mfn1, Opa1, Drp1, and Fis1. Genetic deletion of Mfn2 but not Mfn1 in mouse embryonic fibroblasts or cardiac myocytes in mice led to an increase in the expression of the ER chaperone proteins. Genetic ablation of Mfn2 in mouse embryonic fibroblasts amplified ER stress and exacerbated ER stress-induced apoptosis. Deletion of Mfn2 delayed translational recovery through prolonged eIF2α phosphorylation associated with decreased GADD34 and p58IPK expression and elevated C/EBP homologous protein induction at late time points. These changes in the unfolded protein response were coupled to increased cell death reflected by augmented caspase 3/7 activity, lactate dehydrogenase release from cells, and an increase in propidium iodide-positive nuclei in response to thapsigargin or tunicamycin treatment. In contrast, genetic deletion of Mfn1 did not affect ER stress-mediated increase in ER chaperone synthesis or eIF2α phosphorylation. Additionally, ER stress-induced C/EBP homologous protein, GADD34, and p58IPK induction and cell death were not affected by loss of Mfn1. We conclude that Mfn2 but not Mfn1 is an ER stress-inducible protein that is required for the proper temporal sequence of the ER stress response. PMID:22511781

  2. [Activation of endoplasmic reticulum stress and its effect on osteogenic differentiation induced by micropit/nanotube topography].

    PubMed

    Shi, M Q; Song, W; Han, T X; Chang, B; Zhang, Y M

    2017-02-09

    Objective: To explore the activation of endoplasmic reticulum stress (ERS) in bone marrow mesenchymal stem cell (BMMSC) and its effect on osteogenic differentiation induced by micropit/nanotube topography (MNT), so as to provide guidance for the topography design of biomaterials. Methods: Four sample groups were fabricated: polishing control group (polished titanium, PT, no treatment), thapsigargin treatment (TG, 0.1 μmol/L TG treated for 9 h), MNT5 and MNT20 (anodized at 5 V and 20 V after acid etching). Scanning electron microscope (SEM) was used to observe the topography of Ti samples. The alkaline phosphatase (ALP) production, collagen secretion and extracellular matrix (ECM) mineralization of BMMSC (osteogenic induced for 7, 14 and 21 d) on Ti samples were detected to evaluate the osteogenic differentiation. After 12 h incubation, the shape and size of ER was examined using a transmission electron microscope (TEM), and ERS-related genes including immunoglobulin heavy chain binding protein (BiP), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) were detected by quantitative real-time PCR (qRT-PCR). Results: After 7, 14 and 21 d of induction, the ALP production, collagen secretion and ECM mineralization in TG and MNT20 all significantly increased compared to PT ( P< 0.05). The cells grown on TG, MNT5 and MNT20 surfaces displayed gross distortions of the ER. Compared to PT, BiP, PERK, ATF4 mRNA expression in TG was respectively 1.87±0.10, 2.24±0.35, 1.85±0.14; BiP, ATF4 mRNA expression in MNT5 were respectively 1.27±0.09, 1.25±0.04; BiP, PERK, ATF4 mRNA expression in MNT20 were respectively 1.44±0.09, 2.40±0.60, 1.48±0.05 ( P< 0.05). Conclusions: MNT triggered different degree of ERS, and the activated ERS may promote MNT-induced osteogenic differentiation.

  3. Biochemical Requirements of Virus Wrapping by the Endoplasmic Reticulum: Involvement of ATP and Endoplasmic Reticulum Calcium Store during Envelopment of African Swine Fever Virus

    PubMed Central

    Cobbold, Christian; Brookes, Sharon M.; Wileman, Thomas

    2000-01-01

    Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae. PMID:10666244

  4. Endoplasmic Reticulum-Localized Two-Photon-Absorbing Boron Dipyrromethenes as Advanced Photosensitizers for Photodynamic Therapy.

    PubMed

    Zhou, Yimin; Cheung, Ying-Kit; Ma, Chao; Zhao, Shirui; Gao, Di; Lo, Pui-Chi; Fong, Wing-Ping; Wong, Kam Sing; Ng, Dennis K P

    2018-05-10

    Two advanced boron dipyrromethene (BODIPY) based photosensitizers have been synthesized and characterized. With a glibenclamide analogous moiety, these compounds can localize in the endoplasmic reticulum (ER) of HeLa human cervical carcinoma cells and HepG2 human hepatocarcinoma cells. The BODIPY π skeleton is conjugated with two styryl or carbazolylethenyl groups, which can substantially red-shift the Q-band absorption and fluorescence emission and impart two-photon absorption (TPA) property to the chromophores. The TPA cross section of the carbazole-containing analogue reaches a value of 453 GM at 1010 nm. These compounds also behave as singlet oxygen generators with high photostability. Upon irradiation at λ > 610 nm, these photosensitizers cause photocytotoxicity to these two cell lines with IC 50 values down to 0.09 μM, for which the cell death is triggered mainly by ER stress. The two-photon photodynamic activity of the distyryl derivative upon excitation at λ = 800 nm has also been demonstrated.

  5. Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

    PubMed

    Fox, Philip D; Haberkorn, Christopher J; Weigel, Aubrey V; Higgins, Jenny L; Akin, Elizabeth J; Kennedy, Matthew J; Krapf, Diego; Tamkun, Michael M

    2013-09-01

    In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

  6. Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

    PubMed Central

    Fox, Philip D.; Haberkorn, Christopher J.; Weigel, Aubrey V.; Higgins, Jenny L.; Akin, Elizabeth J.; Kennedy, Matthew J.; Krapf, Diego; Tamkun, Michael M.

    2013-01-01

    In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking. PMID:23864710

  7. Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity.

    PubMed

    Arruda, Ana Paula; Pers, Benedicte M; Parlakgül, Güneş; Güney, Ekin; Inouye, Karen; Hotamisligil, Gökhan S

    2014-12-01

    Proper function of the endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site has been linked to pathophysiological states, including metabolic diseases. Although the ER and mitochondria play distinct cellular roles, these organelles also form physical interactions with each other at sites defined as mitochondria-associated ER membranes (MAMs), which are essential for calcium, lipid and metabolite exchange. Here we show that in the liver, obesity leads to a marked reorganization of MAMs resulting in mitochondrial calcium overload, compromised mitochondrial oxidative capacity and augmented oxidative stress. Experimental induction of ER-mitochondria interactions results in oxidative stress and impaired metabolic homeostasis, whereas downregulation of PACS-2 or IP3R1, proteins important for ER-mitochondria tethering or calcium transport, respectively, improves mitochondrial oxidative capacity and glucose metabolism in obese animals. These findings establish excessive ER-mitochondrial coupling as an essential component of organelle dysfunction in obesity that may contribute to the development of metabolic pathologies such as insulin resistance and diabetes.

  8. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

    PubMed Central

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders. PMID:26984393

  9. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    PubMed

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  10. Renal endoplasmic reticulum stress is coupled to impaired autophagy in a mouse model of GSD Ia.

    PubMed

    Farah, Benjamin L; Landau, Dustin J; Wu, Yajun; Sinha, Rohit A; Loh, Alwin; Bay, Boon-Huat; Koeberl, Dwight D; Yen, Paul M

    2017-11-01

    GSD Ia (von Gierke Disease, Glycogen Storage Disease Type Ia) is a devastating genetic disorder with long-term sequelae, such as non-alcoholic fatty liver disease and renal failure. Down-regulated autophagy is involved in the development of hepatic metabolic dysfunction in GSD Ia; however, the role of autophagy in the renal pathology is unknown. Here we show that autophagy is impaired and endoplasmic reticulum (ER) stress is increased in the kidneys of a mouse model of GSD Ia. Induction of autophagy by rapamycin also reduces this ER stress. Taken together, these results show an additional role for autophagy down-regulation in the pathogenesis of GSD Ia, and provide further justification for the use of autophagy modulators in GSD Ia. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Endoplasmic reticulum stress in obesity and obesity-related disorders: An expanded view.

    PubMed

    Pagliassotti, Michael J; Kim, Paul Y; Estrada, Andrea L; Stewart, Claire M; Gentile, Christopher L

    2016-09-01

    The endoplasmic reticulum (ER) is most notable for its central roles in calcium ion storage, lipid biosynthesis, and protein sorting and processing. By virtue of its extensive membrane contact sites that connect the ER to most other organelles and to the plasma membrane, the ER can also regulate diverse cellular processes including inflammatory and insulin signaling, nutrient metabolism, and cell proliferation and death via a signaling pathway called the unfolded protein response (UPR). Chronic UPR activation has been observed in liver and/or adipose tissue of dietary and genetic murine models of obesity, and in human obesity and non-alcoholic fatty liver disease (NAFLD). Activation of the UPR in obesity and obesity-related disorders likely has two origins. One linked to classic ER stress involving the ER lumen and one linked to alterations to the ER membrane environment. This review discusses both of these origins and also considers the role of post-translational protein modifications, such as acetylation and palmitoylation, and ER-mitochondrial interactions to obesity-mediated impairments in the ER and activation of the UPR. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells.

    PubMed

    Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

    2015-11-09

    This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway.

  13. EGFR inhibition attenuates diabetic nephropathy through decreasing ROS and endoplasmic reticulum stress

    PubMed Central

    Zhong, Peng; Wang, Jingying; Weng, Qiaoyou; Qian, Yuanyuan; Han, Jibo; Zou, Chunpeng; Liang, Guang

    2017-01-01

    Diabetic nephropathy (DN) is a progressive kidney disease due to glomerular capillary damage in diabetic patients. Endoplasmic reticulum (ER) stress caused by reactive oxygen species (ROS) is associated with DN progression. Epidermal growth factor receptor (EGFR) mediates oxidative stress and damage of cardiomyocytes in diabetic mice. Here we demonstrated that AG1478, a specific inhibitor of EGFR, blocked EGFR and AKT phosphorylation in diabetic mice. Oxidative stress and ER stress markers were eliminated after AG1478 administration. AG1478 decreased pro-fibrotic genes TGF-β and collagen IV. Furthermore, we found that high glucose (HG) induced oxidative stress and ER stress, and subsequently increased ATF4 and CHOP. These changes were eliminated by either AG1478 or ROS scavenger N-acetyl-L-cysteine (NAC) administration. These results were confirmed by knock-down approaches in renal mesangial SV40 cells. However, AG1478, not NAC, reversed HG induced EGFR and AKT phosphorylation. These results suggest that EGFR/AKT/ROS/ER stress signaling plays an essential role in DN development and inhibiting EGFR may serve as a potential therapeutic strategy in diabetic kidney diseases. PMID:28427241

  14. NELL2 function in the protection of cells against endoplasmic reticulum stress.

    PubMed

    Kim, Dong Yeol; Kim, Han Rae; Kim, Kwang Kon; Park, Jeong Woo; Lee, Byung Ju

    2015-01-01

    Continuous intra- and extracellular stresses induce disorder of Ca(2+) homeostasis and accumulation of unfolded protein in the endoplasmic reticulum (ER), which results in ER stress. Severe long-term ER stress triggers apoptosis signaling pathways, resulting in cell death. Neural epidermal growth factor-like like protein 2 (NELL2) has been reported to be important in protection of cells from cell death-inducing environments. In this study, we investigated the cytoprotective effect of NELL2 in the context of ER stress induced by thapsigargin, a strong ER stress inducer, in Cos7 cells. Overexpression of NELL2 prevented ER stress-mediated apoptosis by decreasing expression of ER stress-induced C/EBP homologous protein (CHOP) and increasing ER chaperones. In this context, expression of anti-apoptotic Bcl-xL was increased by NELL2, whereas NELL2 decreased expression of pro-apoptotic proteins, such as cleaved caspases 3 and 7. This anti-apoptotic effect of NELL2 is likely mediated by extracellular signal-regulated kinase (ERK) signaling, because its inhibitor, U0126, inhibited effects of NELL2 on the expression of anti- and pro-apoptotic proteins and on the protection from ER stress-induced cell death.

  15. Endoplasmic reticulum stress related molecular mechanisms in nonalcoholic fatty liver disease (NAFLD).

    PubMed

    Wang, Lifeng; Chen, J; Ning, C; Lei, D; Ren, Jun

    2018-05-16

    Non-alcoholic fatty liver disease (NAFLD) has emerged as a common public health problem and a common cause of chronic liver diseases. However, the underlying mechanisms leading to the development and progression of NAFLD remain elusive. Accumulating evidence has depicted an essential role for endoplasmic reticulum (ER) stress in the development of steatosis and later progression into nonalcoholic steatohepatitis and hepatocarcinoma. With the accumulation of unfolded and misfolded proteins in the ER lumen, ER stress is provoked to turn on the unfolded protein response (UPR). ER stress triggers a cascade reaction of transcriptional and translational events that restore ER homeostasis, promoting cell survival and adaptation. However, prolonged ER stress may be transit physiological mechanisms to pathological consequences, including insulin resistance, fat accumulation, inflammation, apoptosis, and autophagy, all of which with important roles in the development of NAFLD. Therefore, understanding the role of ER stress in the onset and pathogenesis of NAFLD is pertinent to the management of this devastating metabolic disease. Here we will summarize available information on recent findings linking ER stress to the pathogenesis of NAFLD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. The Role of p58IPK in Protecting the Stressed Endoplasmic Reticulum

    PubMed Central

    Rutkowski, D. Thomas; Kang, Sang-Wook; Goodman, Alan G.; Garrison, Jennifer L.; Taunton, Jack; Katze, Michael G.

    2007-01-01

    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 p58IPK 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 p58IPK was found to normally reside in association with BiP. ER lumenal p58IPK 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 p58IPK in the ER lumen where its putative function as a cochaperone explains the stress-sensitivity phenotype of knockout cells and mice. PMID:17567950

  17. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    PubMed Central

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal MD; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-01-01

    Impaired homeostasis of lysosomal Ca2+ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca2+ are not known. We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channels to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca2+ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+for the lysosome. DOI: http://dx.doi.org/10.7554/eLife.15887.001 PMID:27213518

  18. Stress-induced self-cannibalism: on the regulation of autophagy by endoplasmic reticulum stress.

    PubMed

    Deegan, Shane; Saveljeva, Svetlana; Gorman, Adrienne M; Samali, Afshin

    2013-07-01

    Macroautophagy (autophagy) is a cellular catabolic process which can be described as a self-cannibalism. It serves as an essential protective response during conditions of endoplasmic reticulum (ER) stress through the bulk removal and degradation of unfolded proteins and damaged organelles; in particular, mitochondria (mitophagy) and ER (reticulophagy). Autophagy is genetically regulated and the autophagic machinery facilitates removal of damaged cell components and proteins; however, if the cell stress is acute or irreversible, cell death ensues. Despite these advances in the field, very little is known about how autophagy is initiated and how the autophagy machinery is transcriptionally regulated in response to ER stress. Some three dozen autophagy genes have been shown to be required for the correct assembly and function of the autophagic machinery; however; very little is known about how these genes are regulated by cellular stress. Here, we will review current knowledge regarding how ER stress and the unfolded protein response (UPR) induce autophagy, including description of the different autophagy-related genes which are regulated by the UPR.

  19. Chronic Intermittent Hypobaric Hypoxia Improves Cardiac Function through Inhibition of Endoplasmic Reticulum Stress.

    PubMed

    Yuan, Fang; Zhang, Li; Li, Yan-Qing; Teng, Xu; Tian, Si-Yu; Wang, Xiao-Ran; Zhang, Yi

    2017-08-11

    We investigated the role of endoplasmic reticulum stress (ERS) in chronic intermittent hypobaric hypoxia (CIHH)-induced cardiac protection. Adult male Sprague-Dawley rats were exposed to CIHH treatment simulating 5000 m altitude for 28 days, 6 hours per day. The heart was isolated and perfused with Langendorff apparatus and subjected to 30-min ischemia followed by 60-min reperfusion. Cardiac function, infarct size, and lactate dehydrogenase (LDH) activity were assessed. Expression of ERS molecular chaperones (GRP78, CHOP and caspase-12) was assayed by western blot analysis. CIHH treatment improved the recovery of left ventricular function and decreased cardiac infarct size and activity of LDH after I/R compared to control rats. Furthermore, CIHH treatment inhibited over-expression of ERS-related factors including GRP78, CHOP and caspase-12. CIHH-induced cardioprotection and inhibition of ERS were eliminated by application of dithiothreitol, an ERS inducer, and chelerythrine, a protein kinase C (PKC) inhibitor. In conclusion CIHH treatment exerts cardiac protection against I/R injury through inhibition of ERS via PKC signaling pathway.

  20. Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure.

    PubMed

    Beetz, Christian; Johnson, Adam; Schuh, Amber L; Thakur, Seema; Varga, Rita-Eva; Fothergill, Thomas; Hertel, Nicole; Bomba-Warczak, Ewa; Thiele, Holger; Nürnberg, Gudrun; Altmüller, Janine; Saxena, Renu; Chapman, Edwin R; Dent, Erik W; Nürnberg, Peter; Audhya, Anjon

    2013-03-26

    Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of gait disorders. Their pathological hallmark is a length-dependent distal axonopathy of nerve fibers in the corticospinal tract. Involvement of other neurons can cause additional neurological symptoms, which define a diverse set of complex hereditary spastic paraplegias. We present two siblings who have the unusual combination of early-onset spastic paraplegia, optic atrophy, and neuropathy. Genome-wide SNP-typing, linkage analysis, and exome sequencing revealed a homozygous c.316C>T (p.R106C) variant in the Trk-fused gene (TFG) as the only plausible mutation. Biochemical characterization of the mutant protein demonstrated a defect in its ability to self-assemble into an oligomeric complex, which is critical for normal TFG function. In cell lines, TFG inhibition slows protein secretion from the endoplasmic reticulum (ER) and alters ER morphology, disrupting organization of peripheral ER tubules and causing collapse of the ER network onto the underlying microtubule cytoskeleton. The present study provides a unique link between altered ER architecture and neurodegeneration.

  1. Neuronal dystonin isoform 2 is a mediator of endoplasmic reticulum structure and function.

    PubMed

    Ryan, Scott D; Ferrier, Andrew; Sato, Tadasu; O'Meara, Ryan W; De Repentigny, Yves; Jiang, Susan X; Hou, Sheng T; Kothary, Rashmi

    2012-02-01

    Dystonin/Bpag1 is a cytoskeletal linker protein whose loss of function in dystonia musculorum (dt) mice results in hereditary sensory neuropathy. Although loss of expression of neuronal dystonin isoforms (dystonin-a1/dystonin-a2) is sufficient to cause dt pathogenesis, the diverging function of each isoform and what pathological mechanisms are activated upon their loss remains unclear. Here we show that dt(27) mice manifest ultrastructural defects at the endoplasmic reticulum (ER) in sensory neurons corresponding to in vivo induction of ER stress proteins. ER stress subsequently leads to sensory neurodegeneration through induction of a proapoptotic caspase cascade. dt sensory neurons display neurodegenerative pathologies, including Ca(2+) dyshomeostasis, unfolded protein response (UPR) induction, caspase activation, and apoptosis. Isoform-specific loss-of-function analysis attributes these neurodegenerative pathologies to specific loss of dystonin-a2. Inhibition of either UPR or caspase signaling promotes the viability of cells deficient in dystonin. This study provides insight into the mechanism of dt neuropathology and proposes a role for dystonin-a2 as a mediator of normal ER structure and function.

  2. Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites.

    PubMed

    Di Mattia, Thomas; Wilhelm, Léa P; Ikhlef, Souade; Wendling, Corinne; Spehner, Danièle; Nominé, Yves; Giordano, Francesca; Mathelin, Carole; Drin, Guillaume; Tomasetto, Catherine; Alpy, Fabien

    2018-06-01

    Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP-A and VAP-B, interact with proteins from other organelles that possess a small VAP-interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain-containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro MOSPD2 is an ER-anchored protein, and it interacts with several FFAT-containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle-bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  3. Thiamine deficiency induces endoplasmic reticulum stress and oxidative stress in human neurons derived from induced pluripotent stem cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Xin; Xu, Mei; Frank, Jacqueline A.

    Thiamine (vitamin B1) deficiency (TD) plays a major role in the etiology of Wernicke's encephalopathy (WE) which is a severe neurological disorder. TD induces selective neuronal cell death, neuroinflammation, endoplasmic reticulum (ER) stress and oxidative stress in the brain which are commonly observed in many aging-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and progressive supranuclear palsy (PSP). However, the underlying cellular and molecular mechanisms remain unclear. The progress in this line of research is hindered due to the lack of appropriate in vitro models. The neurons derived for the human induced pluripotent stemmore » cells (hiPSCs) provide a relevant and powerful tool for the research in pharmaceutical and environmental neurotoxicity. In this study, we for the first time used human induced pluripotent stem cells (hiPSCs)-derived neurons (iCell neurons) to investigate the mechanisms of TD-induced neurodegeneration. We showed that TD caused a concentration- and duration-dependent death of iCell neurons. TD induced ER stress which was evident by the increase in ER stress markers, such as GRP78, XBP-1, CHOP, ATF-6, phosphorylated eIF2α, and cleaved caspase-12. TD also triggered oxidative stress which was shown by the increase in the expression 2,4-dinitrophenyl (DNP) and 4-hydroxynonenal (HNE). ER stress inhibitors (STF-083010 and salubrinal) and antioxidant N-acetyl cysteine (NAC) were effective in alleviating TD-induced death of iCell neurons, supporting the involvement of ER stress and oxidative stress. It establishes that the iCell neurons are a novel tool to investigate cellular and molecular mechanisms for TD-induced neurodegeneration. - Highlights: • Thiamine deficiency (TD) causes death of human neurons in culture. • TD induces both endoplasmic reticulum (ER) stress and oxidative stress. • Alleviating ER stress and oxidative stress reduces TD

  4. Inhibition of glioma growth by minocycline is mediated through endoplasmic reticulum stress-induced apoptosis and autophagic cell death

    PubMed Central

    Liu, Wei-Ting; Huang, Chih-Yuan; Lu, I-Chen; Gean, Po-Wu

    2013-01-01

    Background We have reported that minocycline (Mino) induced autophagic death in glioma cells. In the present study, we characterize the upstream regulators that control autophagy and switch cell death from autophagic to apoptotic. Methods Western blotting and immunofluorescence were used to detect the expressions of eukaryotic translation initiation factor 2α (eIF2α), transcription factor GADD153 (CHOP), and glucose-regulated protein 78 (GRP78). Short hairpin (sh)RNA was used to knock down eIF2α or CHOP expression. Autophagy was assessed by the conversion of light chain (LC)3-I to LC3-II and green fluorescent protein puncta formation. An intracranial mouse model and bioluminescent imaging were used to assess the effect of Mino on tumor growth and survival time of mice. Results The expression of GRP78 in glioma was high, whereas in normal glia it was low. Mino treatment increased GRP78 expression and reduced binding of GRP78 with protein kinase-like endoplasmic reticulum kinase. Subsequently, Mino increased eIF2α phosphorylation and CHOP expression. Knockdown of eIF2α or CHOP reduced Mino-induced LC3-II conversion and glioma cell death. When autophagy was inhibited, Mino induced cell death in a caspase-dependent manner. Rapamycin in combination with Mino produced synergistic effects on LC3 conversion, reduction of the Akt/mTOR/p70S6K pathway, and glioma cell death. Bioluminescent imaging showed that Mino inhibited the growth of glioma and prolonged survival time and that these effects were blocked by shCHOP. Conclusions Mino induced autophagy by eliciting endoplasmic reticulum stress response and switched cell death from autophagy to apoptosis when autophagy was blocked. These results coupled with clinical availability and a safe track record make Mino a promising agent for the treatment of malignant gliomas. PMID:23787763

  5. A Systems Biological View of Life-and-Death Decision with Respect to Endoplasmic Reticulum Stress-The Role of PERK Pathway.

    PubMed

    Márton, Margita; Kurucz, Anita; Lizák, Beáta; Margittai, Éva; Bánhegyi, Gábor; Kapuy, Orsolya

    2017-01-05

    Accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER) leads to the activation of three branches (Protein kinase (RNA)-like endoplasmic reticulum kinase [PERK], Inositol requiring protein 1 [IRE-1] and Activating trascription factor 6 [ATF6], respectively) of unfolded protein response (UPR). The primary role of UPR is to try to drive back the system to the former or a new homeostatic state by self-eating dependent autophagy, while excessive level of ER stress results in apoptotic cell death. Our study focuses on the role of PERK- and IRE-1-induced arms of UPR in life-or-death decision. Here we confirm that silencing of PERK extends autophagy-dependent survival, whereas the IRE-1-controlled apoptosis inducer is downregulated during ER stress. We also claim that the proper order of surviving and self-killing mechanisms is controlled by a positive feedback loop between PERK and IRE-1 branches. This regulatory network makes possible a smooth, continuous activation of autophagy with respect to ER stress, while the induction of apoptosis is irreversible and switch-like. Using our knowledge of molecular biological techniques and systems biological tools we give a qualitative description about the dynamical behavior of PERK- and IRE-1-controlled life-or-death decision. Our model claims that the two arms of UPR accomplish an altered upregulation of autophagy and apoptosis inducers during ER stress. Since ER stress is tightly connected to aging and age-related degenerative disorders, studying the signaling pathways of UPR and their role in maintaining ER proteostasis have medical importance.

  6. Ribosomal protein L19 overexpression activates the unfolded protein response and sensitizes MCF7 breast cancer cells to endoplasmic reticulum stress-induced cell death.

    PubMed

    Hong, Mina; Kim, HyungRyong; Kim, Inki

    2014-07-18

    Although first identified for their roles in protein synthesis, certain ribosomal proteins exert pleiotropic physiological functions in the cell. Ribosomal protein L19 is overexpressed in breast cancer cells by amplification and copy number variation. In this study, we examined the novel pro-apoptotic role of ribosomal protein L19 in the breast cancer cell line MCF7. Overexpression of RPL19 sensitized MCF7 cells to endoplasmic reticulum stress-induced cell death. RPL19 overexpression itself was not cytotoxic; however, cell death induction was enhanced when RPL19 overexpressing cells were incubated with endoplasmic reticulum stress-inducing agents, and this sensitizing effect was specific to MCF7 cells. Examination of the cell signaling pathways that mediate the unfolded protein response (UPR) revealed that overexpression of RPL19 induced pre-activation of the UPR, including phosphorylation of pERK-like ER kinase (PERK), phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), and activation of p38 MAPK-associated stress signaling. Our findings suggest that upregulation of RPL19 induces ER stress, resulting in increased sensitivity to ER stress and enhanced cell death in MCF7 breast cancer cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Supplementing dietary sugar promotes endoplasmic reticulum stress-independent insulin resistance and fatty liver in goose.

    PubMed

    Geng, Tuoyu; Zhao, Xing; Xia, Lili; Liu, Long; Li, Fuyuan; Yang, Biao; Wang, Qianqian; Montgomery, Sean; Cui, Hengmi; Gong, Daoqing

    2016-08-05

    It is known that endoplasmic reticulum stress (ERS) contributes to insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD) in mammals. However, we recently demonstrated that overfeeding with a traditional diet (mainly consisting of cooked maize) does not induce ERS in goose. As cellular studies show that high glucose and palmitate can trigger ERS in mammalian cells, we hypothesized that supplementing sugar to the traditional diet could induce ERS, thus promoting insulin resistance and fatty liver. To test the hypothesis, we first treated goose primary hepatocytes with high glucose (25 mM and 50 mM) and palmitate (0.5 mM) supplemented with or without 0.25 mM oleate. Data indicated that, as in mammalian cells, high glucose and palmitate indeed induced ERS in goose primary hepatocytes, and palmitate-induced ERS was suppressed by supplemental 0.25 mM oleate. We then tested the hypothesis with an in vivo study, in which Landes geese overfed with traditional or novel diets (i.e., the traditional diet supplemented with sugar) were compared with control geese (normally fed with cooked maize) for ERS, IR and fatty liver. The differences in glucose tolerance, insulin tolerance and postprandial blood glucose between the geese overfed with traditional and novel diets suggested that supplementing dietary sugar promoted IR. This promotion was accompanied with an increasing trend of liver weight and abdominal fat weight relative to body weight. Surprisingly, compared to overfeeding with the traditional diet, overfeeding with the novel diet did not induce ERS, even further suppressed ERS in goose fatty liver. Together, our findings suggest that supplementing dietary sugar promotes ERS-independent IR and fatty liver in goose. It is intriguing to discover the factor(s) protecting goose liver from ERS as well as the non-ERS mechanism underlying IR. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Soluble forms of polyQ-expanded huntingtin rather than large aggregates cause endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Leitman, Julia; Ulrich Hartl, F.; Lederkremer, Gerardo Z.

    2013-11-01

    In Huntington’s disease, as in other neurodegenerative diseases, it was initially thought that insoluble protein aggregates are the toxic species. However, growing evidence implicates soluble oligomeric polyglutamine-expanded huntingtin in cytotoxicity. Here we show that pathogenic huntingtin inhibits endoplasmic reticulum (ER)-associated degradation and induces ER stress before its aggregation into visible inclusions. All three branches of the unfolded protein response are activated. ER stress can be compensated by overexpression of p97/VCP, suggesting its sequestration by pathogenic huntingtin as a main cause. Stress correlates with the presence of huntingtin oligomers and is independent of continual huntingtin synthesis. Stress levels, measured in striatal neurons, are stabilized but only slowly subside on huntingtin aggregation into inclusions. Our results can be explained by the constant conversion of huntingtin monomers to toxic oligomers; large aggregates sequester the former, precluding further conversion, whereas pre-existing toxic oligomers are only gradually depleted.

  9. Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baumann, O.; Walz, B.; Somlyo, A.V.

    Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental compositon of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 {plus minus} 1.1 mmol/kg (dry weight). During a 3-sec nonsaturating light stimulus, {approximately}50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was {approximately}0.7. Our results show unambiguouslymore » that the ER is the source of Ca{sup 2+} release during cell stimulation and suggest the Mg{sup 2+} can nearly balance the charge movement of Ca{sup 2+}.« less

  10. Neuroprotective effect of S-allyl-l-cysteine derivatives against endoplasmic reticulum stress-induced cytotoxicity is independent of calpain inhibition.

    PubMed

    Imai, Toru; Kosuge, Yasuhiro; Saito, Hiroaki; Uchiyama, Taketo; Wada, Taira; Shimba, Shigeki; Ishige, Kumiko; Miyairi, Shinichi; Makishima, Makoto; Ito, Yoshihisa

    2016-03-01

    S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on μ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC. Copyright © 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  11. Fenofibrate Does Not Affect Burn-Induced Hepatic Endoplasmic Reticulum Stress

    PubMed Central

    Hiyama, Yaeko; Marshall, Alexandra H.; Kraft, Robert; Arno, Anna; Jeschke, Marc G.

    2013-01-01

    Background Burn injury causes major metabolic derangements such as hypermetabolism, hyperlipidemia, and insulin resistance and is associated with liver damage, hepatomegaly, and hepatic endoplasmic reticulum (ER) stress. Although the physiological consequences of such derangements have been delineated, the underlying molecular mechanisms remain unknown. Previously, it was shown that fenofibrate improves patient outcome by attenuating post-burn stress responses. Methods Fenofibrate, a peroxisome proliferator-activated receptor (PPAR)-α agonist, regulates liver lipid metabolism and has been used to treat hypertriglyceridemia and hypercholesterolemia for many years. The aim of the present study is to determine the effects of fenofibrate on burn-induced hepatic morphologic and metabolic changes. We randomized rats to sham, burn injury, and burn injury plus fenofibrate. Animals were sacrificed and livers were assessed at 24 or 48 hours post-burn. Results Burn injury decreased albumin and increased alanine transaminase (p = 0.1 vs. sham), indicating liver injury. Fenofibrate administration did not restore albumin or decrease alanine transaminase. In addition, ER stress was significantly increased after burn injury both with and without fenofibrate (p < 0.05 vs. sham). Burn injury increased fatty acid metabolism gene expression (p < 0.05 vs. sham), downstream of PPARα. Fenofibrate treatment increased fatty acid metabolism further, which reduced post-burn hepatic steatosis (burn vs. sham p < 0.05, burn+fenofibrate vs. sham not significant). Conclusions Fenofibrate did not alleviate thermal injury induced hepatic ER stress and dysfunction but reduced hepatic steatosis by modulating hepatic genes related to fat metabolism. PMID:23866789

  12. A molecular web: endoplasmic reticulum stress, inflammation, and oxidative stress.

    PubMed

    Chaudhari, Namrata; Talwar, Priti; Parimisetty, Avinash; Lefebvre d'Hellencourt, Christian; Ravanan, Palaniyandi

    2014-01-01

    Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress.

  13. A Molecular Web: Endoplasmic Reticulum Stress, Inflammation, and Oxidative Stress

    PubMed Central

    Chaudhari, Namrata; Talwar, Priti; Parimisetty, Avinash; Lefebvre d’Hellencourt, Christian; Ravanan, Palaniyandi

    2014-01-01

    Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress. PMID:25120434

  14. Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum

    DOE PAGES

    Saberianfar, Reza; Sattarzadeh, Amirali; Joensuu, Jussi J.; ...

    2016-05-23

    Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). Here in this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered intomore » all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.« less

  15. Endoplasmic reticulum-plasma membrane junctions: structure, function and dynamics.

    PubMed

    Okeke, Emmanuel; Dingsdale, Hayley; Parker, Tony; Voronina, Svetlana; Tepikin, Alexei V

    2016-06-01

    Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are contact sites between the ER and the PM; the distance between the two organelles in the junctions is below 40 nm and the membranes are connected by protein tethers. A number of molecular tools and technical approaches have been recently developed to visualise, modify and characterise properties of ER-PM junctions. The junctions serve as the platforms for lipid exchange between the organelles and for cell signalling, notably Ca(2+) and cAMP signalling. Vice versa, signalling events regulate the development and properties of the junctions. Two Ca(2+) -dependent mechanisms of de novo formation of ER-PM junctions have been recently described and characterised. The junction-forming proteins and lipids are currently the focus of vigorous investigation. Junctions can be relatively short-lived and simple structures, forming and dissolving on the time scale of a few minutes. However, complex, sophisticated and multifunctional ER-PM junctions, capable of attracting numerous protein residents and other cellular organelles, have been described in some cell types. The road from simplicity to complexity, i.e. the transformation from simple 'nascent' ER-PM junctions to advanced stable multiorganellar complexes, is likely to become an attractive research avenue for current and future junctologists. Another area of considerable research interest is the downstream cellular processes that can be activated by specific local signalling events in the ER-PM junctions. Studies of the cell physiology and indeed pathophysiology of ER-PM junctions have already produced some surprising discoveries, likely to expand with advances in our understanding of these remarkable organellar contact sites. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  16. Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction.

    PubMed

    Fu, Hai Ying; Sanada, Shoji; Matsuzaki, Takashi; Liao, Yulin; Okuda, Keiji; Yamato, Masaki; Tsuchida, Shota; Araki, Ryo; Asano, Yoshihiro; Asanuma, Hiroshi; Asakura, Masanori; French, Brent A; Sakata, Yasushi; Kitakaze, Masafumi; Minamino, Tetsuo

    2016-03-04

    Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment. © 2016 American Heart Association, Inc.

  17. The Role of Endoplasmic Reticulum Stress in Diabetic Nephropathy.

    PubMed

    Fan, Ying; Lee, Kyung; Wang, Niansong; He, John Cijiang

    2017-03-01

    Diabetic nephropathy (DN) has become the leading cause of end-stage renal disease (ESRD) worldwide. Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development and progression of DN. Recent findings suggested that many attributes of DN, such as hyperglycemia, proteinuria, and increased advanced glycation end products and free fatty acids, can all trigger unfolded protein response (UPR) in kidney cells. Herein, we review the current knowledge on the role of ER stress in the setting of kidney injury with a specific emphasis on DN. As maladaptive ER stress response caused by excessively prolonged UPR will eventually cause cell death and increase kidney injury, several ER stress inhibitors have been shown to improve DN in animal models, albeit blocking both adaptive and maladaptive UPR. More recently, reticulon-1A (RTN1A), an ER-associated protein, was shown to be increased in both human and mouse diabetic kidneys. Its expression correlates with the progression of DN, and its polymorphisms are associated with kidney disease in people with diabetes. Increased RTN1A expression heightened the ER stress response and renal cell apoptosis, and conversely reduced RTN1A in renal cells decreased apoptosis and ameliorated kidney injury and DN progression, suggesting that RTN1A may be a novel target to specifically restrain the maladaptive UPR. These findings suggest that ER stress response in renal cells is a key driver of progression of DN and that the inhibition of the unchecked ER stress response in DN, such as by inhibition of RTN1A function, may be a promising therapeutic approach against DN.

  18. Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

    PubMed

    Guiliano, David B; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J; Campbell, Elaine C; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J; Kellam, Paul; Hebert, Daniel N; Gould, Keith G; Powis, Simon J; Antoniou, Antony N

    2014-11-01

    HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease. Copyright © 2014 by the American College of Rheumatology.

  19. MicroRNA-29a mitigation of endoplasmic reticulum and autophagy aberrance counteracts in obstructive jaundice-induced fibrosis in mice.

    PubMed

    Huang, Ying-Hsien; Yang, Ya-Ling; Huang, Fu-Chen; Tiao, Mao-Meng; Lin, Yen-Cheng; Tsai, Ming-Horng; Wang, Feng-Sheng

    2018-01-01

    Hepatic fibrosis was caused by a number of signaling pathways that damage liver integrity. We have previously shown that microRNA-29a (miR-29a) protects against liver fibrosis. Aberrant endoplasmic reticulum (ER) and autophagy function reportedly exaggerate hepatic disorders. The aim of this study was to characterize the biological influence of miR-29a on ER function in injured livers with bile duct ligation (BDL). We performed BDL on miR-29a transgenic mice (miR-29aTg) and wild-type mice to induce cholestatic liver injury. Rat T6 cells were transfected with miR-29a mimic and tunicamycin. Compared to the wild-type mice, the BDL deterioration of liver function in terms of total bilirubin, alanine transaminase, and aspartate transaminase activity in the miR-29aTg mice was significantly reduced. Affected livers in the miR-29aTg mice demonstrated a slight fibrotic matrix formation. miR-29a over-expression reduced the BDL disturbance of the expressions of inositol-requiring kinase 1alpha, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase, spliced-X-box binding protein 1 (sXBP1), CCAAT/enhancer-binding protein homologous protein (CHOP), ULK, LC3BII, p62, and cleaved caspase-8, 9 and 3. In vitro, T6 cells exposed to tunicamycin by increasing abundances of CHOP, sXBP1, cleaved caspase-3, and LC3BII were diminished in the cell cultures transfected with the miR-29a mimic. On the other hand, we observed that miR-29a signaling protected liver tissues from BDL-mediated metabolic dysfunction and excessive fibrosis histopathology. This study provides new molecular insight into the miR-29a stabilization of ER integrity that slows the progression of cholestatic liver deterioration. Impact statement Long-term hepatic damage caused by hepatitis and cholestasis can accelerate fibrosis matrix over-production, which is a harmful process attributed to the dysregulation of a number of cellular and molecular events. The purpose of this study is to characterize

  20. Critical Role of Endoplasmic Reticulum Stress in Chronic Intermittent Hypoxia-Induced Deficits in Synaptic Plasticity and Long-Term Memory

    PubMed Central

    Xu, Lin-Hao; Xie, Hui; Shi, Zhi-Hui; Du, Li-Da; Wing, Yun-Kwok; Li, Albert M.

    2015-01-01

    Abstract Aims: This study examined the role of endoplasmic reticulum (ER) stress in mediating chronic intermittent hypoxia (IH)-induced neurocognitive deficits. We designed experiments to demonstrate that ER stress is initiated in the hippocampus under chronic IH and determined its role in apoptotic cell death, impaired synaptic structure and plasticity, and memory deficits. Results: Two weeks of IH disrupted ER fine structure and upregulated ER stress markers, glucose-regulated protein 78, caspase-12, and C/EBP homologous protein, in the hippocampus, which could be suppressed by ER stress inhibitors, tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid. Meanwhile, ER stress induced apoptosis via decreased Bcl-2, promoted reactive oxygen species production, and increased malondialdehyde formation and protein carbonyl, as well as suppressed mitochondrial function. These effects were largely prevented by ER stress inhibitors. On the other hand, suppression of oxidative stress could reduce ER stress. In addition, the length of the synaptic active zone and number of mature spines were reduced by IH. Long-term recognition memory and spatial memory were also impaired, which was accompanied by reduced long-term potentiation in the Schaffer collateral pathway. These effects were prevented by coadministration of the TUDCA. Innovation and Conclusion: These results show that ER stress plays a critical role in underlying memory deficits in obstructive sleep apnea (OSA)-associated IH. Attenuators of ER stress may serve as novel adjunct therapeutic agents for ameliorating OSA-induced neurocognitive impairment. Antioxid. Redox Signal. 23, 695–710. PMID:25843188

  1. Juvenile Traumatic Brain Injury Results in Cognitive Deficits Associated with Impaired Endoplasmic Reticulum Stress and Early Tauopathy.

    PubMed

    Hylin, Michael J; Holden, Ryan C; Smith, Aidan C; Logsdon, Aric F; Qaiser, Rabia; Lucke-Wold, Brandon P

    2018-05-22

    The leading cause of death in the juvenile population is trauma, and in particular neurotrauma. The juvenile brain response to neurotrauma is not completely understood. Endoplasmic reticulum (ER) stress has been shown to contribute to injury expansion and behavioral deficits in adult rodents and furthermore has been seen in adult postmortem human brains diagnosed with chronic traumatic encephalopathy. Whether endoplasmic reticulum stress is increased in juveniles with traumatic brain injury (TBI) is poorly delineated. We investigated this important topic using a juvenile rat controlled cortical impact (CCI) model. We proposed that ER stress would be significantly increased in juvenile rats following TBI and that this would correlate with behavioral deficits using a juvenile rat model. A juvenile rat (postnatal day 28) CCI model was used. Binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) were measured at 4 h in the ipsilateral pericontusion cortex. Hypoxia-inducible factor (HIF)-1α was measured at 48 h and tau kinase measured at 1 week and 30 days. At 4 h following injury, BiP and CHOP (markers of ER stress) were significantly elevated in rats exposed to TBI. We also found that HIF-1α was significantly upregulated 48 h following TBI showing delayed hypoxia. The early ER stress activation was additionally asso-ciated with the activation of a known tau kinase, glycogen synthase kinase-3β (GSK-3β), by 1 week. Tau oligomers measured by R23 were significantly increased by 30 days following TBI. The biochemical changes following TBI were associated with increased impulsive-like or anti-anxiety behavior measured with the elevated plus maze, deficits in short-term memory measured with novel object recognition, and deficits in spatial memory measured with the Morris water maze in juvenile rats exposed to TBI. These results show that ER stress was increased early in juvenile rats exposed to TBI, that these rats developed tau oligomers over the

  2. The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation.

    PubMed

    Preston, G Michael; Brodsky, Jeffrey L

    2017-02-15

    The endoplasmic reticulum (ER) serves as a warehouse for factors that augment and control the biogenesis of nascent proteins entering the secretory pathway. In turn, this compartment also harbors the machinery that responds to the presence of misfolded proteins by targeting them for proteolysis via a process known as ER-associated degradation (ERAD). During ERAD, substrates are selected, modified with ubiquitin, removed from the ER, and then degraded by the cytoplasmic 26S proteasome. While integral membrane proteins can directly access the ubiquitination machinery that resides in the cytoplasm or on the cytoplasmic face of the ER membrane, soluble ERAD substrates within the lumen must be retrotranslocated from this compartment. In either case, nearly all ERAD substrates are tagged with a polyubiquitin chain, a modification that represents a commitment step to degrade aberrant proteins. However, increasing evidence indicates that the polyubiquitin chain on ERAD substrates can be further modified, serves to recruit ERAD-requiring factors, and may regulate the ERAD machinery. Amino acid side chains other than lysine on ERAD substrates can also be modified with ubiquitin, and post-translational modifications that affect substrate ubiquitination have been observed. Here, we summarize these data and provide an overview of questions driving this field of research. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  3. Erlotinib promotes endoplasmic reticulum stress-mediated injury in the intestinal epithelium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fan, Lu; Hu, Lingna; Yang, Baofang

    Erlotinib, a popular drug for treating non-small cell lung cancer (NSCLC), causes diarrhea in approximately 55% of patients receiving this drug. In the present study, we found that erlotinib induced barrier dysfunction in rat small intestine epithelial cells (IEC-6) by increasing epithelial permeability and down-regulating E-cadherin. The mRNA levels of various pro-inflammatory cytokines (Il-6, Il-25 and Il-17f) were increased after erlotinib treatment in IEC-6 cells. Erlotinib concentration- and time-dependently induced apoptosis and endoplasmic reticulum (ER) stress in both IEC-6 and human colon epithelial cells (CCD 841 CoN). Intestinal epithelial injury was also observed in male C57BL/6J mice administrated with erlotinib.more » Knockdown of C/EBP homologous protein (CHOP) with small interference RNA partially reversed erlotinib-induced apoptosis, production of IL-6 and down-regulation of E-cadherin in cultured intestinal epithelial cells. In conclusion, erlotinib caused ER stress-mediated injury in the intestinal epithelium, contributing to its side effects of diarrhea in patients. - Highlights: • Erlotinib destroyed barrier integrity both in vitro and in vivo. • Erlotinib induced inflammation both in vitro and in vivo. • Erlotinib induced apoptosis both in vitro and in vivo. • ER stress contributed to erlotinib-induced barrier dysfunction.« less

  4. Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy.

    PubMed

    Paran, Christopher W; Zou, Kai; Ferrara, Patrick J; Song, Haowei; Turk, John; Funai, Katsuhiko

    2015-12-01

    Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that contributes to abnormal Ca(2+) homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition. Copyright © 2015. Published by Elsevier B.V.

  5. Efficient IgM assembly and secretion require the plasma cell induced endoplasmic reticulum protein pERp1

    PubMed Central

    van Anken, Eelco; Pena, Florentina; Hafkemeijer, Nicole; Christis, Chantal; Romijn, Edwin P.; Grauschopf, Ulla; Oorschot, Viola M. J.; Pertel, Thomas; Engels, Sander; Ora, Ari; Lástun, Viorica; Glockshuber, Rudi; Klumperman, Judith; Heck, Albert J. R.; Luban, Jeremy; Braakman, Ineke

    2009-01-01

    Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C(X)6C motif, and pERp1 displays only modest oxidoreductase activity. pERp1 emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM. PMID:19805154

  6. Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems☆

    PubMed Central

    Wlcek, Katrin; Hofstetter, Lia; Stieger, Bruno

    2014-01-01

    Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR− rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane. PMID:24406246

  7. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation

    PubMed Central

    Obata, Yuuki; Toyoshima, Shota; Wakamatsu, Ei; Suzuki, Shunichi; Ogawa, Shuhei; Esumi, Hiroyasu; Abe, Ryo

    2014-01-01

    Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation. PMID:25493654

  9. A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol

    PubMed Central

    Inoue, Takamasa; Tsai, Billy

    2011-01-01

    Non-enveloped viruses penetrate host membranes to infect cells. A cell-based assay was used to probe the endoplasmic reticulum (ER)-to-cytosol membrane transport of the non-enveloped SV40. We found that, upon ER arrival, SV40 is released into the lumen and undergoes sequential disulfide bond disruptions to reach the cytosol. However, despite these ER-dependent conformational changes, SV40 crosses the ER membrane as a large and intact particle consisting of the VP1 coat, the internal components VP2, VP3, and the genome. This large particle subsequently disassembles in the cytosol. Mutant virus and inhibitor studies demonstrate VP3 and likely the viral genome, as well as cellular proteasome, control ER-to-cytosol transport. Our results identify the sequence of events, as well as virus and host components, that regulate ER membrane penetration. They also suggest that the ER membrane supports passage of a large particle, potentially through either a sizeable protein-conducting channel or the lipid bilayer. PMID:21589906

  10. Attenuation of endoplasmic reticulum stress by caffeine ameliorates hyperoxia-induced lung injury

    PubMed Central

    Jing, Xigang; Michalkiewicz, Teresa; Afolayan, Adeleye J.; Wu, Tzong-Jin; Konduri, Girija G.

    2017-01-01

    Rodent pups exposed to hyperoxia develop lung changes similar to bronchopulmonary dysplasia (BPD) in extremely premature infants. Oxidative stress from hyperoxia can injure developing lungs through endoplasmic reticulum (ER) stress. Early caffeine treatment decreases the rate of BPD, but the mechanisms remain unclear. We hypothesized that caffeine attenuates hyperoxia-induced lung injury through its chemical chaperone property. Sprague-Dawley rat pups were raised either in 90 (hyperoxia) or 21% (normoxia) oxygen from postnatal day 1 (P1) to postnatal day 10 (P10) and then recovered in 21% oxygen until P21. Caffeine (20 mg/kg) or normal saline (control) was administered intraperitoneally daily starting from P2. Lungs were inflation-fixed for histology or snap-frozen for immunoblots. Blood caffeine levels were measured in treated pups at euthanasia and were found to be 18.4 ± 4.9 μg/ml. Hyperoxia impaired alveolar formation and increased ER stress markers and downstream effectors; caffeine treatment attenuated these changes at P10. Caffeine also attenuated the hyperoxia-induced activation of cyclooxygenase-2 and markers of apoptosis. In conclusion, hyperoxia-induced alveolar growth impairment is mediated, in part, by ER stress. Early caffeine treatment protects developing lungs from hyperoxia-induced injury by attenuating ER stress. PMID:28213471

  11. Astrocytes and endoplasmic reticulum stress: A bridge between obesity and neurodegenerative diseases.

    PubMed

    Martin-Jiménez, Cynthia A; García-Vega, Ángela; Cabezas, Ricardo; Aliev, Gjumrakch; Echeverria, Valentina; González, Janneth; Barreto, George E

    2017-11-01

    Endoplasmic reticulum (ER) is a subcellular organelle involved in protein folding and processing. ER stress constitutes a cellular process characterized by accumulation of misfolded proteins, impaired lipid metabolism and induction of inflammatory responses. ER stress has been suggested to be involved in several human pathologies, including neurodegenerative diseases and obesity. Different studies have shown that both neurodegenerative diseases and obesity trigger similar cellular responses to ER stress. Moreover, both diseases are assessed in astrocytes as evidences suggest these cells as key regulators of brain homeostasis. However, the exact contributions to the effects of ER stress in astrocytes in the various neurodegenerative diseases and its relation with obesity are not well known. Here, we discuss recent advances in the understanding of molecular mechanisms that regulate ER stress-related disorders in astrocytes such as obesity and neurodegeneration. Moreover, we outline the correlation between the activated proteins of the unfolded protein response (UPR) in these pathological conditions in order to identify possible therapeutic targets for ER stress in astrocytes. We show that ER stress in astrocytes shares UPR activation pathways during both obesity and neurodegenerative diseases, demonstrating that UPR related proteins like ER chaperone GRP 78/Bip, PERK pathway and other exogenous molecules ameliorate UPR response and promote neuroprotection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells

    PubMed Central

    Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

    2015-01-01

    Background This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. Material/Methods HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. Results The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. Conclusions TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway. PMID:26551326

  13. Endoplasmic reticulum stress in amelogenesis imperfecta and phenotypic rescue using 4-phenylbutyrate

    PubMed Central

    Brookes, Steven J.; Barron, Martin J.; Boot-Handford, Ray; Kirkham, Jennifer; Dixon, Michael J.

    2014-01-01

    Inherited diseases caused by genetic mutations can arise due to loss of protein function. Alternatively, mutated proteins may mis-fold, impairing endoplasmic reticulum (ER) trafficking, causing ER stress and triggering the unfolded protein response (UPR). The UPR attempts to restore proteostasis but if unsuccessful drives affected cells towards apoptosis. Previously, we reported that in mice, the p.Tyr64His mutation in the enamel extracellular matrix (EEM) protein amelogenin disrupts the secretory pathway in the enamel-forming ameloblasts, resulting in eruption of malformed tooth enamel that phenocopies human amelogenesis imperfecta (AI). Defective amelogenin post-secretory self-assembly and processing within the developing EEM has been suggested to underlie the pathogenesis of X chromosome-linked AI. Here, we challenge this concept by showing that AI pathogenesis associated with the p.Tyr64His amelogenin mutation involves ameloblast apoptosis induced by ER stress. Furthermore, we show that 4-phenylbutyrate can rescue the enamel phenotype in affected female mice by promoting cell survival over apoptosis such that they are able to complete enamel formation despite the presence of the mutation, offering a potential therapeutic option for patients with this form of AI and emphasizing the importance of ER stress in the pathogenesis of this inherited conformational disease. PMID:24362885

  14. Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

    PubMed

    Lee, Ji Eun; Oney, McKenna; Frizzell, Kimberly; Phadnis, Nitin; Hollien, Julie

    2015-02-13

    Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response, which plays important roles in development and disease. Here we show that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the tricarboxylic acid cycle and respiratory chain complexes were down-regulated. The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells. Copyright © 2015 Lee et al.

  15. Drosophila melanogaster Activating Transcription Factor 4 Regulates Glycolysis During Endoplasmic Reticulum Stress

    PubMed Central

    Lee, Ji Eun; Oney, McKenna; Frizzell, Kimberly; Phadnis, Nitin; Hollien, Julie

    2015-01-01

    Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response, which plays important roles in development and disease. Here we show that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the tricarboxylic acid cycle and respiratory chain complexes were down-regulated. The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells. PMID:25681259

  16. Endoplasmic reticulum stress in amelogenesis imperfecta and phenotypic rescue using 4-phenylbutyrate.

    PubMed

    Brookes, Steven J; Barron, Martin J; Boot-Handford, Ray; Kirkham, Jennifer; Dixon, Michael J

    2014-05-01

    Inherited diseases caused by genetic mutations can arise due to loss of protein function. Alternatively, mutated proteins may mis-fold, impairing endoplasmic reticulum (ER) trafficking, causing ER stress and triggering the unfolded protein response (UPR). The UPR attempts to restore proteostasis but if unsuccessful drives affected cells towards apoptosis. Previously, we reported that in mice, the p.Tyr64His mutation in the enamel extracellular matrix (EEM) protein amelogenin disrupts the secretory pathway in the enamel-forming ameloblasts, resulting in eruption of malformed tooth enamel that phenocopies human amelogenesis imperfecta (AI). Defective amelogenin post-secretory self-assembly and processing within the developing EEM has been suggested to underlie the pathogenesis of X chromosome-linked AI. Here, we challenge this concept by showing that AI pathogenesis associated with the p.Tyr64His amelogenin mutation involves ameloblast apoptosis induced by ER stress. Furthermore, we show that 4-phenylbutyrate can rescue the enamel phenotype in affected female mice by promoting cell survival over apoptosis such that they are able to complete enamel formation despite the presence of the mutation, offering a potential therapeutic option for patients with this form of AI and emphasizing the importance of ER stress in the pathogenesis of this inherited conformational disease.

  17. Unfolded protein response activation compensates endoplasmic reticulum-associated degradation deficiency in Arabidopsis.

    PubMed

    Li, Qingliang; Wei, Hai; Liu, Lijing; Yang, Xiaoyuan; Zhang, Xiansheng; Xie, Qi

    2017-07-01

    Abiotic stresses often disrupt protein folding and induce endoplasmic reticulum (ER) stress. There is a sophisticated ER quality control (ERQC) system to mitigate the effects of malfunctioning proteins and maintain ER homeostasis. The accumulation of misfolded proteins in the ER activates the unfolded protein response (UPR) to enhance ER protein folding and the degradation of misfolded proteins mediate by ER-associated degradation (ERAD). That ERQC reduces abiotic stress damage has been well studied in mammals and yeast. However, in plants, both ERAD and UPR have been studied separately and found to be critical for plant abiotic stress tolerance. In this study, we discovered that UPR-associated transcription factors AtbZIP17, AtbZIP28 and AtbZIP60 responded to tunicamycin (TM) and NaCl induced ER stress and subsequently enhanced Arabidopsis thaliana abiotic stress tolerance. They regulated the expression level of ER chaperones and the HRD1-complex components. Moreover, overexpression of AtbZIP17, AtbZIP28 and AtbZIP60 could restore stress tolerance via ERAD in the HRD1-complex mutant hrd3a-2, which suggested that UPR and ERAD have an interactive mechanism in Arabidopsis. © 2017 Institute of Botany, Chinese Academy of Sciences.

  18. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  19. Endoplasmic reticulum stress preconditioning attenuates methylmercury-induced cellular damage by inducing favorable stress responses

    PubMed Central

    Usuki, Fusako; Fujimura, Masatake; Yamashita, Akio

    2013-01-01

    We demonstrate that methylmercury (MeHg)-susceptible cells preconditioned with an inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase, thapsigargin, showed resistance to MeHg cytotoxicity through favorable stress responses, which included phosphorylation of eukaryotic initiation factor 2 alpha (Eif2α), accumulation of activating transcription factor 4 (Atf4), upregulation of stress-related proteins, and activation of extracellular signal regulated kinase pathway. In addition, ER stress preconditioning induced suppression of nonsense-mediated mRNA decay (NMD) mainly through the phospho-Eif2α-mediated general suppression of translation initiation and possible combined effects of decreased several NMD components expression. Atf4 accumulation was not mediated by NMD inhibition but translation inhibition of its upstream open reading frame (uORF) and translation facilitation of its protein-coding ORF by the phospho-Eif2α. These results suggested that ER stress plays an important role in MeHg cytotoxicity and that the modulation of ER stress has therapeutic potential to attenuate MeHg cytotoxicity, the underlying mechanism being the induction of integrated stress responses. PMID:23907635

  20. A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

    PubMed Central

    van Lith, Marcel; Karala, Anna-Riikka; Bown, Dave; Gatehouse, John A.; Ruddock, Lloyd W.; Saunders, Philippa T.K.

    2007-01-01

    Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility. PMID:17507649

  1. Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis

    PubMed Central

    Villeneuve, Julien; Duran, Juan; Scarpa, Margherita; Bassaganyas, Laia; Van Galen, Josse; Malhotra, Vivek

    2017-01-01

    Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi–cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor–mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain–containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions. PMID:27807044

  2. Characterization of wheat endoplasmic reticulum oxidoreductin 1 and its application in Chinese steamed bread.

    PubMed

    Liu, Guang; Wang, JingJing; Hou, Yi; Huang, Yan-Bo; Wang, JiaJia; Li, Cunzhi; Guo, ShiJun; Li, Lin; Hu, Song-Qing

    2018-08-01

    This study investigated characteristics of recombinant wheat Endoplasmic Reticulum Oxidoreductin 1 (wEro1) and its influence on Chinese steamed bread (CSB) qualities. The purified wEro1 monomer, which contained two conserved redox active motif sites, bound to flavin adenine dinucleotide (FAD) cofactor with a molecular weight of ∼47 kDa. wEro1 catalyzed the reduction of both bound and free FAD, and its reduction activity of free FAD reached 7.8 U/mg. Moreover, wEro1 catalyzed the oxidation of dithiothreitol and wheat protein disulfide isomerase (wPDI). Both glutathione and the reduced ribonuclease could work as electron donors for wEro1 in catalyzing the oxidation of wPDI. Additionally, wEro1 supplementation improved the CSB qualities with an increased specific volume of CSB and decreased crumb hardness, which was attributed to water-insoluble wheat proteins increasing and gluten network strengthening. The results give an understanding of the properties and function of wEro1 to facilitate its application especially in the flour-processing industry. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Decreased MORF leads to prolonged endoplasmic reticulum stress in periodontitis-associated chronic inflammation.

    PubMed

    Xue, Peng; Li, Bei; An, Ying; Sun, Jin; He, Xiaoning; Hou, Rui; Dong, Guangying; Fei, Dongdong; Jin, Fang; Wang, Qintao; Jin, Yan

    2016-11-01

    The association between inflammation and endoplasmic reticulum (ER) stress has been described in many diseases. However, if and how chronic inflammation governs the unfolded protein response (UPR) and promotes ER homeostasis of chronic inflammatory disease remains elusive. In this study, chronic inflammation resulted in ER stress in mesenchymal stem cells in the setting of periodontitis. Long-term proinflammatory cytokines induced prolonged ER stress and decreased the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Interestingly, we showed that chronic inflammation decreases the expression of lysine acetyltransferase 6B (KAT6B, also called MORF), a histone acetyltransferase, and causes the upregulation of a key UPR sensor, PERK, which lead to the persistent activation of the UPR in PDLSCs. Furthermore, we found that the activation of UPR mediated by MORF in chronic inflammation contributes to the PERK-related deterioration of the osteogenic differentiation of PDLSCs both in vivo and in vitro. Taken together, our results suggest that chronic inflammation compromises UPR function through MORF-mediated-PERK transcription, which is a previously unrecognized mechanism that contributes to impaired ER function, prolonged ER stress and defective osteogenic differentiation of PDLSCs in periodontitis.

  4. Cadmium supplement triggers endoplasmic reticulum stress response and cytotoxicity in primary chicken hepatocytes.

    PubMed

    Shao, Cheng-Cheng; Li, Nan; Zhang, Zi-Wei; Su, Jian; Li, Shu; Li, Jin-Long; Xu, Shi-Wen

    2014-08-01

    Cadmium (Cd), a potent hepatotoxin, has been reported to induce endoplasmic reticulum (ER) stress in various cell types. However, whether such effect exists in bird is still unclear. To delineate the effects of Cd exposure on ER stress response, we examined the expression of 78-kDa glucose-regulated protein (GRP78) and alteration in calcium homeostasis in primary chicken hepatocytes treated with 2-22 µM Cd for 24 h. A significant decrease of cell viability was observed in chicken hepatocytes following Cd administration. In cells treated with Cd, GRP78 protein levels increased in a dose-dependent manner. In addition, GRP78 and GRP94mRNA levels were elevated in response to Cd exposure. The increase of the intracellular Ca(2+) concentration in chicken hepatocytes was found during Cd exposure. Cd significantly decreased the CaM mRNA levels in hepatocytes. These results show that Cd regulates the expression of GRP78 and calcium homeostasis in chicken hepatocytes, suggesting that ER stress induced by Cd plays an important role in the mechanisms of Cd cytotoxicity to the bird hepatocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. [Effect of endoplasmic reticulum stress on the expression and osteogenic differentiation of periodontal ligament stem cells].

    PubMed

    Xue, Peng; Li, Bei; Tan, Jun; An, Ying; Jin, Yan; Wang, Qintao

    2015-09-01

    To determine the activity of endoplasmic reticulum stress (ERS) and its effect on osteogenic differentiation of periodontal ligament stem cells (PDLSC) in inflammatory microenvironment. PDLSC were obtained from the primary culture of the human tooth and cloned with limited diluted method. Real-time reverse transcription (RT)-PCR was used to examine the different expression of thapsigargin (TG) treated PDLSC and lipopolysaccharide (LPS) treated PDLSC. Real-time RT-PCR, alizarin red staining and cetyl pyridine chloride quantitative analyze were used to examine the osteogenic differentiation of PDLSC, TG + PDLSC, LPS + PDLSC and LPS + PDLSC + 4-PBA. Protein kinase receptor like endoplasmic reticulum kinase (PERK), glucose regulated protein 78 (GRP78), transcription activation factor 4(ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) mRNA expression in group PDLSC + TG in 6 h were respectively 1.49 ± 0.24, 2.77 ± 0.60, 1.75 ± 0.16, 2.16 ± 0.32, which were all greater than that in group PDLSC (P < 0.05). PERK, CHOP mRNA expression reached the peak at 6 h (1.76 ± 0.08, 2.31 ± 0.17) and were greater than group PDLSC (P < 0.05). ERS could suppress osteogenic differentiation of TG + PDLSC and LPS + PDLSC. The runt-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN) mRNA expression of group TG + PDLSC was respectively 0.73 ± 0.06, 0.01 ± 0.00, 0.20 ± 0.06 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group LPS + PDLSC was respectively 0.80 ± 0.06, 0.48 ± 0.05, 0.29 ± 0.04 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group PDLSC + TG + 4-PBA was respectively 1.10 ± 0.09, 0.74 ± 0.05, 0.67 ± 0.13, which were greater higher than that of group LPS + PDLSC (P < 0.05). ERS was activated in PDLSC and suppressed osteogenic differentiation of PDLSC, which can simulate inflammatory microenvironment in vitro. This effect can be recovered by using ERS inhibitor 4-PBA.

  6. PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

    PubMed

    van Lith, Marcel; Hartigan, Nichola; Hatch, Jennifer; Benham, Adam M

    2005-01-14

    Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

  7. Severe Burn-Induced Intestinal Epithelial Barrier Dysfunction Is Associated With Endoplasmic Reticulum Stress and Autophagy in Mice

    PubMed Central

    Huang, Yalan; Feng, Yanhai; Wang, Yu; Wang, Pei; Wang, Fengjun; Ren, Hui

    2018-01-01

    The disruption of intestinal barrier plays a vital role in the pathophysiological changes after severe burn injury, however, the underlying mechanisms are poorly understood. Severe burn causes the disruption of intestinal tight junction (TJ) barrier. Previous studies have shown that endoplasmic reticulum (ER) stress and autophagy are closely associated with the impairment of intestinal mucosa. Thus, we hypothesize that ER stress and autophagy are likely involved in burn injury-induced intestinal epithelial barrier dysfunction. Mice received a 30% total body surface area (TBSA) full-thickness burn, and were sacrificed at 0, 1, 2, 6, 12 and 24 h postburn. The results showed that intestinal permeability was increased significantly after burn injury, accompanied by the damage of mucosa and the alteration of TJ proteins. Severe burn induced ER stress, as indicated by increased intraluminal chaperone binding protein (BIP), CCAAT/enhancer-binding protein homologous protein (CHOP) and inositol-requiring enzyme 1(IRE1)/X-box binding protein 1 splicing (XBP1). Autophagy was activated after burn injury, as evidenced by the increase of autophagy related protein 5 (ATG5), Beclin 1 and LC3II/LC3I ratio and the decrease of p62. Besides, the number of autophagosomes was also increased after burn injury. The levels of p-PI3K(Ser191), p-PI3K(Ser262), p-AKT(Ser473), and p-mTOR were decreased postburn, suggesting that autophagy-related PI3K/AKT/mTOR pathway is involved in the intestinal epithelial barrier dysfunction following severe burn. In summary, severe burn injury induces the ER stress and autophagy in intestinal epithelia, leading to the disruption of intestinal barrier. PMID:29740349

  8. Involvement of endoplasmic reticulum in hepatitis B virus replication.

    PubMed

    Xia, Weiliang; Shen, Yan; Xie, Haiyang; Zheng, Shusen

    2006-11-01

    The mitochondrial calcium and downstream proline-rich tyrosine kinase-2 (PyK2) signaling pathway are critical to hepatitis B virus (HBV) replication, and the endoplasmic reticulum (ER) plays an important role in intracellular calcium regulation. To investigate the role of ER in HBV replication, the HBV genome transfected HepG2.2.15 cells were treated by cyclosporine A (CsA), cyclopiazonic acid (CPA), ryanodine and U73122, which are all specific blockers of calcium channels located in either ER or mitochondria. The HBV replication level was evaluated by two methods: slot blot hybridization analysis of intracellular HBV DNA and real-time polymerase chain reaction (PCR) analysis of secreted HBV DNA in supernatant; the activation of PyK2 kinase was detected by Western blot analysis. Results indicated that the HBV replication was inhibited when mitochondrial permeability transition pore, ER Ca2+ -ATPase and ER inositol 1,4,5-trisphosphate receptor (IP3R) were blocked by CsA, CPA and U73122, respectively; but not inhibited when ER ryanodine receptor was blocked by ryanodine. The PyK2 phosphorylation level declined after treatment of 2 microg/ml CsA, 5 microM CPA and 25 microM U73122, but not changed apparently after 50 microM ryanodine treatment. Compared with monotreatment, a more powerful inhibitory effect was achieved when the CsA, CPA and U73122 were combined used in twosome or triple manner, while the HBV replication level did not change apparently when ryanodine combined with CsA, CPA or U73122. In conclusion, besides the mitochondria, the ER also participates in the HBV replication through calcium-PyK2 signaling pathway; the calcium channels of ER Ca2+ -ATPase and ER IP3R are responsible for this role; during this complicated process, an interaction between ER and mitochondria maybe involved.

  9. Preconditioning with Endoplasmic Reticulum Stress Ameliorates Endothelial Cell Inflammation

    PubMed Central

    Leonard, Antony; Paton, Adrienne W.; El-Quadi, Monaliza; Paton, James C.; Fazal, Fabeha

    2014-01-01

    Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability. PMID:25356743

  10. Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

    PubMed

    Leonard, Antony; Paton, Adrienne W; El-Quadi, Monaliza; Paton, James C; Fazal, Fabeha

    2014-01-01

    Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

  11. Bi-polarized translation of ascidian maternal mRNA determinant pem-1 associated with regulators of the translation machinery on cortical Endoplasmic Reticulum (cER).

    PubMed

    Paix, Alexandre; Le Nguyen, Phuong Ngan; Sardet, Christian

    2011-09-01

    Polarized cortical mRNA determinants such as maternal macho-1 and pem-1 in ascidians, like budding yeast mating factor ASH1 reside on the cER-mRNA domain a subdomain of cortical Endoplasmic Reticulum(ER) and are translated in its vicinity. Using high resolution imaging and isolated cortical fragments prepared from eggs and embryos we now find that macho-1 and pem-1 RNAs co-localize with phospho-protein regulators of translation initiation (MnK/4EBP/S6K). Translation of cortical pem-1 RNA follows its bi-polarized relocalization. About 10 min after fertilization or artificial activation with a calcium ionophore, PEM1 protein is detected in the vegetal cortex in the vicinity of pem-1 RNA. About 40 min after fertilization-when pem-1 RNA and P-MnK move to the posterior pole-PEM1 protein remains in place forming a network of cortical patches anchored at the level of the zygote plasma membrane before disappearing. Cortical PEM1 protein is detected again at the 4 cell stage in the posterior centrosome attracting body (CAB) region where the cER-mRNA domain harboring pem-1/P-MnK/P-4EBP/P-S6K is concentrated. Bi-polarized PEM1 protein signals are not detected when pem-1 morpholinos are injected into eggs or zygotes or when MnK is inhibited. We propose that localized translation of the pem-1 RNA determinant is triggered by the fertilization/calcium wave and that the process is controlled by phospho-protein regulators of translation initiation co-localized with the RNA determinant on a sub-domain of the cortical Endoplasmic Reticulum. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

    PubMed

    Shih, Ying-Chun; Chen, Chao-Ling; Zhang, Yan; Mellor, Rebecca L; Kanter, Evelyn M; Fang, Yun; Wang, Hua-Chi; Hung, Chen-Ting; Nong, Jing-Yi; Chen, Hui-Ju; Lee, Tzu-Han; Tseng, Yi-Shuan; Chen, Chiung-Nien; Wu, Chau-Chung; Lin, Shuei-Liong; Yamada, Kathryn A; Nerbonne, Jeanne M; Yang, Kai-Chien

    2018-04-13

    Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor β1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor β1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4

  13. Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.

    PubMed

    Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

    2012-01-01

    Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

  14. Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

    2014-12-01

    Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung

  15. The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

    PubMed Central

    Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

    2016-01-01

    The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

  16. Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems.

    PubMed

    Wlcek, Katrin; Hofstetter, Lia; Stieger, Bruno

    2014-03-01

    Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR(-) rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Adipocyte Fatty Acid Binding Protein Potentiates Toxic Lipids-Induced Endoplasmic Reticulum Stress in Macrophages via Inhibition of Janus Kinase 2-dependent Autophagy

    PubMed Central

    Hoo, Ruby L. C.; Shu, Lingling; Cheng, Kenneth K. Y.; Wu, Xiaoping; Liao, Boya; Wu, Donghai; Zhou, Zhiguang; Xu, Aimin

    2017-01-01

    Lipotoxicity is implicated in the pathogenesis of obesity-related inflammatory complications by promoting macrophage infiltration and activation. Endoplasmic reticulum (ER) stress and adipocyte fatty acid binding protein (A-FABP) play key roles in obesity and mediate inflammatory activity through similar signaling pathways. However, little is known about their interplay in lipid-induced inflammatory responses. Here, we showed that prolonged treatment of palmitic acid (PA) increased ER stress and expression of A-FABP, which was accompanied by reduced autophagic flux in macrophages. Over-expression of A-FABP impaired PA-induced autophagy associating with enhanced ER stress and pro-inflammatory cytokine production, while genetic ablation or pharmacological inhibition of A-FABP reversed the conditions. PA-induced expression of autophagy-related protein (Atg)7 was attenuated in A-FABP over-expressed macrophages, but was elevated in A-FABP-deficient macrophages. Mechanistically, A-FABP potentiated the effects of PA by inhibition of Janus Kinase (JAK)2 activity, thus diminished PA-induced Atg7 expression contributing to impaired autophagy and further augmentation of ER stress. These findings suggest that A-FABP acts as autophagy inhibitor to instigate toxic lipids-induced ER stress through inhibition of JAK2-dependent autophagy, which in turn triggers inflammatory responses in macrophages. A-FABP-JAK2 axis may represent an important pathological pathway contributing to obesity-related inflammatory diseases. PMID:28094778

  18. [Role of PI3K/Akt pathway in endoplasmic reticulum stress and apoptosis induced by saturated fatty acid in human steatotic hepatocytes].

    PubMed

    Qu, Mei; Shen, Wei

    2015-03-01

    To investigate the roles of PI3K/Akt signaling in the unfolded protein response (UPR) and non-UPR signaling pathways of endoplasmic reticulum stress and apoptosis in hepatocytes under conditions of saturated fatty acid-induced steatosis. A steatosis model of hepatocytes (L02 cell and HepG2 cell line) was induced by palmitate sodium saturated fatty acids.The hepatocytes were divided into normal control group,experimental group (treated with palmitate sodium) and intervention group (treated with palmitate sodium and LY294002, a PI3K/Akt inhibitor). Cell apoptosis was detected by flow cytometry with Annexin V/PI double-staining.Western blot analysis was used to examine the protein expression of GRP78, PI3K, P-PI3K,Akt, P-Akt, CHOP and Bax.The F test and t-test were used in statistical analyses. Flow cytometry showed that palmitate sodium induced cell apoptosis in steatotic hepatocytes;moreover, a significant increase in cell apoptosis was observed in the palmitate sodium-induced steatotic hepatocytes in the presence of LY294002.For the normal control group, the experimental group and the intervention group, the apoptosis ratios of L02 cells were 4.41 ± 0.78% vs. 6.01 ± 1.49% vs. 19.50 ± 2.53% after 24 hours of treatment,and 12.56 ± 2.78% vs. 29.72 ± 6.39% vs. 44.60 ± 4.17% after 48 hours of treatment in respectively (all P < 0.05),and of HepG2 cells were 11.16 ± 1.15% vs. 17.50 ± 6.83% vs. 30.41 ± 3.62% after 24 hours of treatment, and 22.37 ± 1.24% vs. 33.85 ± 5.79% vs. 48.56 ± 4.21% after 48 hours of treatment (all P < 0.05). Western blot analysis showed that expression of GRP78 was significantly upregulated in the palmitate sodium-induced steatosis hepatocytes, indicating activation of endoplasmic reticulum stress. In addition, the palmitate sodium treatment also activated the PI3K/Akt pathway,induced expression of CHOP and Bax of the UPR and non-UPR signaling pathways respectively. Moreover, Pretreatment with LY294002 inhibited the palmitate sodium

  19. A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.

    PubMed

    Henderson, Mark J; Baldwin, Heather A; Werley, Christopher A; Boccardo, Stefano; Whitaker, Leslie R; Yan, Xiaokang; Holt, Graham T; Schreiter, Eric R; Looger, Loren L; Cohen, Adam E; Kim, Douglas S; Harvey, Brandon K

    2015-01-01

    Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

  20. Dissecting Endoplasmic Reticulum Unfolded Protein Response (UPRER) in Managing Clandestine Modus Operandi of Alzheimer’s Disease

    PubMed Central

    Rahman, Safikur; Archana, Ayyagari; Jan, Arif Tasleem; Minakshi, Rinki

    2018-01-01

    Alzheimer’s disease (AD), a neurodegenerative disorder, is most common cause of dementia witnessed among aged people. The pathophysiology of AD develops as a consequence of neurofibrillary tangle formation which consists of hyperphosphorylated microtubule associated tau protein and senile plaques of amyloid-β (Aβ) peptide in specific brain regions that result in synaptic loss and neuronal death. The feeble buffering capacity of endoplasmic reticulum (ER) proteostasis in AD is evident through alteration in unfolded protein response (UPR), where UPR markers express invariably in AD patient’s brain samples. Aging weakens UPRER causing neuropathology and memory loss in AD. This review highlights molecular signatures of UPRER and its key molecular alliance that are affected in aging leading to the development of intriguing neuropathologies in AD. We present a summary of recent studies reporting usage of small molecules as inhibitors or activators of UPRER sensors/effectors in AD that showcase avenues for therapeutic interventions. PMID:29467648

  1. Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

    PubMed Central

    Weis, MaryAnn; Rai, Jyoti; Hudson, David M.; Dimori, Milena; Zimmerman, Sarah M.; Hogue, William R.; Swain, Frances L.; Burdine, Marie S.; Mackintosh, Samuel G.; Tackett, Alan J.; Suva, Larry J.; Eyre, David R.

    2016-01-01

    Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis. PMID:27119146

  2. Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

    PubMed Central

    Wang, Jie; Pareja, Kristeen A; Kaiser, Chris A; Sevier, Carolyn S

    2014-01-01

    Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP's ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress. DOI: http://dx.doi.org/10.7554/eLife.03496.001 PMID:25053742

  3. Obesity-induced endoplasmic reticulum stress suppresses nuclear factor-Y expression.

    PubMed

    Liu, Yulan; Zhang, Yuwei; Zhang, Yanjie; Zhang, Jinlong; Liu, Yin; Feng, Peiqun; Su, Zhiguang

    2017-02-01

    Nuclear transcription factor Y (NF-Y) is an evolutionarily conserved transcription factor composed of three subunits, NF-YA, NF-YB, and NF-YC. NF-Y plays crucial roles in pre-adipocyte maintenance and/or commitment to adipogenesis. NF-YA dysfunction in adipocyte resulted in an age-dependent progressive loss of adipose tissue associated with metabolic complications. Endoplasmic reticulum (ER) stress has emerged as an important mediator in the pathogenesis of obesity. However, it is not known if NF-YA is involved in the ER stress-mediated pathogenesis of obesity. We first examined the effects of ER stress on the NF-YA expression in cultured 3T3-L1 adipocytes; then in ob/ob genetic obesity mice, we tested the effect of chemical chaperones alleviating ER stress on the expression levels of NF-YA. Subsequently, we inhibited the new mRNA synthesis using actinomycin D in 3T3-L1 cells to explore the mechanism modulating NF-YA expression. Finally, we evaluated the involvement of PPARg in the regulation of NF-YA expression by ER stress. We demonstrated that both obesity- and chemical chaperone -induced ER stress suppressed NF-YA expression and alleviation of ER stress by chemical chaperone could recover NF-YA expression in ob/ob mice. Moreover, we showed that ER stress suppressed NF-YA mRNA transcription through the involvement of peroxisome proliferator-activated receptor gamma (PPARg). Activation of PPARg ameliorates the ER stress-induced NF-YA suppression. Our findings may point to a possible role of NF-YA in stress conditions that occur in chronic obesity, ER stress might be involved in the pathogenesis of obesity through NF-YA depletion.

  4. Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

    PubMed

    Szczesna-Skorupa, E; Chen, C D; Rogers, S; Kemper, B

    1998-12-08

    Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

  5. Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.

    PubMed

    Hirayama, Hiroto; Seino, Junichi; Kitajima, Toshihiko; Jigami, Yoshifumi; Suzuki, Tadashi

    2010-04-16

    In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.

  6. Endoplasmic Reticulum Ca2+ Handling in Excitable Cells in Health and Disease

    PubMed Central

    Mattson, Mark P.

    2011-01-01

    The endoplasmic reticulum (ER) is a morphologically and functionally diverse organelle capable of integrating multiple extracellular and internal signals and generating adaptive cellular responses. It plays fundamental roles in protein synthesis and folding and in cellular responses to metabolic and proteotoxic stress. In addition, the ER stores and releases Ca2+ in sophisticated scenarios that regulate a range of processes in excitable cells throughout the body, including muscle contraction and relaxation, endocrine regulation of metabolism, learning and memory, and cell death. One or more Ca2+ ATPases and two types of ER membrane Ca2+ channels (inositol trisphosphate and ryanodine receptors) are the major proteins involved in ER Ca2+ uptake and release, respectively. There are also direct and indirect interactions of ER Ca2+ stores with plasma membrane and mitochondrial Ca2+-regulating systems. Pharmacological agents that selectively modify ER Ca2+ release or uptake have enabled studies that revealed many different physiological roles for ER Ca2+ signaling. Several inherited diseases are caused by mutations in ER Ca2+-regulating proteins, and perturbed ER Ca2+ homeostasis is implicated in a range of acquired disorders. Preclinical investigations suggest a therapeutic potential for use of agents that target ER Ca2+ handling systems of excitable cells in disorders ranging from cardiac arrhythmias and skeletal muscle myopathies to Alzheimer disease. PMID:21737534

  7. The reticulons: Guardians of the structure and function of the endoplasmic reticulum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Di Sano, Federica; Bernardoni, Paolo; Piacentini, Mauro, E-mail: mauro.piacentini@uniroma2.it

    2012-07-01

    The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by a specific class of curvature stabilizing proteins, the reticulons and DP1; however it is still unclear how the sheets are assembled. The ER is the cellular compartment responsible for secretory and membrane protein synthesis. The reducing conditions of ER lead to the intra/inter-chain formation of new disulphide bonds into polypeptides during protein folding assessed by enzymatic or spontaneous reactions. Moreover, ER represents the main intracellular calcium storage site and it plays an important role in calcium signalingmore » that impacts many cellular processes. Accordingly, the maintenance of ER function represents an essential condition for the cell, and ER morphology constitutes an important prerogative of it. Furthermore, it is well known that ER undergoes prominent shape transitions during events such as cell division and differentiation. Thus, maintaining the correct ER structure is an essential feature for cellular physiology. Now, it is known that proper ER-associated proteins play a fundamental role in ER tubules formation. Among these ER-shaping proteins are the reticulons (RTN), which are acquiring a relevant position. In fact, beyond the structural role of reticulons, in very recent years new and deeper functional implications of these proteins are emerging in relation to their involvement in several cellular processes.« less

  8. Protein disulfide isomerases: Redox connections in and out of the endoplasmic reticulum.

    PubMed

    Soares Moretti, Ana Iochabel; Martins Laurindo, Francisco Rafael

    2017-03-01

    Protein disulfide isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily. As redox folding catalysts from the endoplasmic reticulum (ER), their roles in ER-related redox homeostasis and signaling are well-studied. PDIA1 exerts thiol oxidation/reduction and isomerization, plus chaperone effects. Also, substantial evidence indicates that PDIs regulate thiol-disulfide switches in other cell locations such as cell surface and possibly cytosol. Subcellular PDI translocation routes remain unclear and seem Golgi-independent. The list of signaling and structural proteins reportedly regulated by PDIs keeps growing, via thiol switches involving oxidation, reduction and isomerization, S-(de)nytrosylation, (de)glutathyonylation and protein oligomerization. PDIA1 is required for agonist-triggered Nox NADPH oxidase activation and cell migration in vascular cells and macrophages, while PDIA1-dependent cytoskeletal regulation appears a converging pathway. Extracellularly, PDIs crucially regulate thiol redox signaling of thrombosis/platelet activation, e.g., integrins, and PDIA1 supports expansive caliber remodeling during injury repair via matrix/cytoskeletal organization. Some proteins display regulatory PDI-like motifs. PDI effects are orchestrated by expression levels or post-translational modifications. PDI is redox-sensitive, although probably not a mass-effect redox sensor due to kinetic constraints. Rather, the "all-in-one" organization of its peculiar redox/chaperone properties likely provide PDIs with precision and versatility in redox signaling, making them promising therapeutic targets. Copyright © 2016. Published by Elsevier Inc.

  9. Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases

    PubMed Central

    Araki, Kazutaka; Iemura, Shun-ichiro; Kamiya, Yukiko; Ron, David; Kato, Koichi; Natsume, Tohru

    2013-01-01

    Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α–associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a′ domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells. PMID:24043701

  10. Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways.

    PubMed

    Ying, Jinhe; Xu, Huan; Wu, Dhua; Wu, Xiaoguang

    2015-01-01

    Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis.

  11. A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

    PubMed

    Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F

    2008-11-01

    The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

  12. Endoplasmic Reticulum Transport of Glutathione by Sec61 Is Regulated by Ero1 and Bip.

    PubMed

    Ponsero, Alise J; Igbaria, Aeid; Darch, Maxwell A; Miled, Samia; Outten, Caryn E; Winther, Jakob R; Palais, Gael; D'Autréaux, Benoit; Delaunay-Moisan, Agnès; Toledano, Michel B

    2017-09-21

    In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H 2 O 2 -dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. YD277 Suppresses Triple-Negative Breast Cancer Partially Through Activating the Endoplasmic Reticulum Stress Pathway

    PubMed Central

    Chen, Zekun; Wu, Qiuju; Ding, Ye; Zhou, Wenhui; Liu, Rong; Chen, Haiying; Zhou, Jia; Feng, Jing; Chen, Ceshi

    2017-01-01

    Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcomes. YD277 is a novel small molecule derived from ML264, a KLF5 inhibitor that elicits cytotoxic effects in colon cancer cell lines. Our previous studies suggest that Krüpple-like factor 5 (KLF5) is a promising therapeutic target for TNBC. In this study, we demonstrated that YD277 significantly induced G1 cell cycle arrest and apoptosis in MDA-MB-231 and MDA-MB-468 TNBC cells, independent of KLF5 inhibition. YD277 also reduced the protein expression levels of Cyclin D1, Bcl2 and Bclxl and promoted the expression of p21 and p27. Moreover, the pro-apoptotic activity of YD277 in TNBC was mediated by the transcription of IRE1α, a key molecule in the endoplasmic reticulum (ER) stress pathway. Finally, YD277 (15 mg/kg) significantly suppressed the growth of MDA-MB-231 tumor xenografts in nude mice. These findings indicate that YD277 is a promising chemotherapeutic candidate for TNBC. PMID:28740556

  14. Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage.

    PubMed

    Bronner, Denise N; Abuaita, Basel H; Chen, Xiaoyun; Fitzgerald, Katherine A; Nuñez, Gabriel; He, Yongqun; Yin, Xiao-Ming; O'Riordan, Mary X D

    2015-09-15

    Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Diet-induced obesity induces endoplasmic reticulum stress and insulin resistance in the amygdala of rats☆

    PubMed Central

    Castro, Gisele; C. Areias, Maria Fernanda; Weissmann, Lais; Quaresma, Paula G.F.; Katashima, Carlos K.; Saad, Mario J.A.; Prada, Patricia O.

    2013-01-01

    Insulin acts in the hypothalamus, decreasing food intake (FI) by the IR/PI3K/Akt pathway. This pathway is impaired in obese animals and endoplasmic reticulum (ER) stress and low-grade inflammation are possible mechanisms involved in this impairment. Here, we highlighted the amygdala as an important brain region for FI regulation in response to insulin. This regulation was dependent on PI3K/AKT pathway similar to the hypothalamus. Insulin was able to decrease neuropeptide Y (NPY) and increase oxytocin mRNA levels in the amygdala via PI3K, which may contribute to hypophagia. Additionally, obese rats did not reduce FI in response to insulin and AKT phosphorylation was decreased in the amygdala, suggesting insulin resistance. Insulin resistance was associated with ER stress and low-grade inflammation in this brain region. The inhibition of ER stress with PBA reverses insulin action/signaling, decreases NPY and increases oxytocin mRNA levels in the amygdala from obese rats, suggesting that ER stress is probably one of the mechanisms that induce insulin resistance in the amygdala. PMID:24251109

  16. Endoplasmic reticulum and mitochondria interplay mediates apoptotic cell death: relevance to Parkinson's disease.

    PubMed

    Arduíno, Daniela Moniz; Esteves, A Raquel; Cardoso, Sandra M; Oliveira, Catarina R

    2009-09-01

    Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Many cellular mechanisms are thought to be involved in the death of these specific neurons in PD, including oxidative stress, changes of intracellular calcium homeostasis, and mitochondrial dysfunction. Since recent studies have revealed that also endoplasmic reticulum (ER) stress in conjunction with abnormal protein degradation can contribute to the PD pathophysiology, we investigated here the molecular mechanisms underlying the interplay between ER and mitochondria and its relevance in the control of neuronal cell death in PD. We observed that MPP+ induced changes in the mitochondrial function, affecting mitochondrial membrane potential and electron transport chain function. Likewise, it was also evident the unfolded protein response activation by an overexpression of GRP78 protein. Moreover, stress stimuli caused the release of Ca2+ from the ER that consistently induced mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Besides, Ca2+ release inhibition prevented MPP+ mediated mitochondria-dependent caspases activation. Our findings show that ER and mitochondria are in a close communication, establishing a dynamic ER-Ca2+-mitochondria interconnection that can play a prominent role in the neuronal cell death induction under particular stressful circumstances of PD pathology.

  17. Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

    PubMed Central

    Casey, Amanda K.; Chen, Shuliang; Novick, Peter; Ferro-Novick, Susan; Wente, Susan R.

    2015-01-01

    The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions. PMID:26041935

  18. Role of Endoplasmic Reticulum Aminopeptidases in Health and Disease: from Infection to Cancer

    PubMed Central

    Cifaldi, Loredana; Romania, Paolo; Lorenzi, Silvia; Locatelli, Franco; Fruci, Doriana

    2012-01-01

    Endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 (ERAPs) are essential for the maturation of a wide spectrum of proteins involved in various biological processes. In the ER, these enzymes work in concert to trim peptides for presentation on MHC class I molecules. Loss of ERAPs function substantially alters the repertoire of peptides presented by MHC class I molecules, critically affecting recognition of both NK and CD8+ T cells. In addition, these enzymes are involved in the modulation of inflammatory responses by promoting the shedding of several cytokine receptors, and in the regulation of both blood pressure and angiogenesis. Recent genome-wide association studies have identified common variants of ERAP1 and ERAP2 linked to several human diseases, ranging from viral infections to autoimmunity and cancer. More recently, inhibition of ER peptide trimming has been shown to play a key role in stimulating innate and adaptive anti-tumor immune responses, suggesting that inhibition of ERAPs might be exploited for the establishment of innovative therapeutic approaches against cancer. This review summarizes data currently available for ERAP enzymes in ER peptide trimming and in other immunological and non-immunological functions, paying attention to the emerging role played by these enzymes in human diseases. PMID:22942706

  19. Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes*

    PubMed Central

    Iracheta-Vellve, Arvin; Petrasek, Jan; Gyongyosi, Benedek; Satishchandran, Abhishek; Lowe, Patrick; Kodys, Karen; Catalano, Donna; Calenda, Charles D.; Kurt-Jones, Evelyn A.; Fitzgerald, Katherine A.; Szabo, Gyongyi

    2016-01-01

    Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-β (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4. Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4. In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver. PMID:27810900

  20. Evidence that endoplasmic reticulum (ER) stress and caspase-4 activation occur in human neutrophils

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Binet, Francois; Chiasson, Sonia; Girard, Denis, E-mail: denis.girard@iaf.inrs.ca

    2010-01-01

    Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenicmore » trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2{alpha} are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.« less

  1. Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores.

    PubMed

    Gómez-Sánchez, Rubén; Rose, Jaqueline; Guimarães, Rodrigo; Mari, Muriel; Papinski, Daniel; Rieter, Ester; Geerts, Willie J; Hardenberg, Ralph; Kraft, Claudine; Ungermann, Christian; Reggiori, Fulvio

    2018-05-30

    The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion. © 2018 Gómez-Sánchez et al.

  2. Angiogenin Mediates Cell-Autonomous Translational Control under Endoplasmic Reticulum Stress and Attenuates Kidney Injury

    PubMed Central

    Mami, Iadh; Bouvier, Nicolas; El Karoui, Khalil; Gallazzini, Morgan; Rabant, Marion; Laurent-Puig, Pierre; Li, Shuping; Tharaux, Pierre-Louis; Beaune, Philippe; Thervet, Eric; Chevet, Eric; Hu, Guo-Fu

    2016-01-01

    Endoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but the molecular bases underlying the biologic outcomes on the evolution of renal disease remain mostly unknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but its contribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediated contribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression was significantly higher in samples from injured human kidneys than in samples from normal human kidneys, and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renal epithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcription factor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules and inhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramatically greater in ANG knockout mice (Ang−/−) mice than in wild-type mice. These results indicate that ANG is a critical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism. PMID:26195817

  3. Endoplasmic Reticulum Stress in the Diabetic Kidney, the Good, the Bad and the Ugly.

    PubMed

    Cunard, Robyn

    2015-04-20

    Diabetic kidney disease is the leading worldwide cause of end stage kidney disease and a growing public health challenge. The diabetic kidney is exposed to many environmental stressors and each cell type has developed intricate signaling systems designed to restore optimal cellular function. The unfolded protein response (UPR) is a homeostatic pathway that regulates endoplasmic reticulum (ER) membrane structure and secretory function. Studies suggest that the UPR is activated in the diabetic kidney to restore normal ER function and viability. However, when the cell is continuously stressed in an environment that lies outside of its normal physiological range, then the UPR is known as the ER stress response. The UPR reduces protein synthesis, augments the ER folding capacity and downregulates mRNA expression of genes by multiple pathways. Aberrant activation of ER stress can also induce inflammation and cellular apoptosis, and modify signaling of protective processes such as autophagy and mTORC activation. The following review will discuss our current understanding of ER stress in the diabetic kidney and explore novel means of modulating ER stress and its interacting signaling cascades with the overall goal of identifying therapeutic strategies that will improve outcomes in diabetic nephropathy.

  4. Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

    PubMed Central

    Risco, Cristina; Rodríguez, Juan R.; López-Iglesias, Carmen; Carrascosa, José L.; Esteban, Mariano; Rodríguez, Dolores

    2002-01-01

    Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements. PMID:11799179

  5. Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer.

    PubMed

    Cheng, Xi; Feng, Haoran; Wu, Haoxuan; Jin, Zhijian; Shen, Xiaonan; Kuang, Jie; Huo, Zhen; Chen, Xianze; Gao, Haoji; Ye, Feng; Ji, Xiaopin; Jing, Xiaoqian; Zhang, Yaqi; Zhang, Tao; Qiu, Weihua; Zhao, Ren

    2018-05-30

    Apatinib, a novel tyrosine kinase inhibitor (TKI), has been confirmed for its efficacy and safety in the treatment of advanced gastric carcinoma and some other solid tumors. However, the direct functional mechanisms of tumor lethality mediated by apatinib have not yet been fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, in this study, we demonstrated that apatinib could induce both apoptosis and autophagy in human colorectal cancer (CRC) via a mechanism that involved endoplasmic reticulum (ER) stress. Moreover, activation of the IRE1α pathway from apatinib-induced ER stress is responsible for the induction of autophagy; however, blocking autophagy could enhance the apoptosis in apatinib-treated human CRC cell lines. Furthermore, the combination of apatinib with autophagy inhibitor chloroquine (CQ) tends to have the most significant anti-tumor effect of CRC both in vitro and in vivo. Overall, our data show that because apatinib treatment could induce ER stress-related apoptosis and protective autophagy in human CRC cell lines, targeting autophagy is a promising therapeutic strategy to relieve apatinib drug resistance in CRC. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

  6. Endoplasmic reticulum stress in vasopressin neurons of familial diabetes insipidus model mice: aggregate formation and mRNA poly(A) tail shortening.

    PubMed

    Arima, Hiroshi; Morishita, Yoshiaki; Hagiwara, Daisuke; Hayashi, Masayuki; Oiso, Yutaka

    2014-01-01

    The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone, which binds to newly synthesized secretory and transmembrane proteins to facilitate protein folding. BiP mRNA is expressed in the arginine vasopressin (AVP) neurons in the supraoptic nucleus of wild-type mice even in basal conditions, and the expression levels increase in response to dehydration. These data suggest that AVP neurons are subjected to ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is caused by mutations in the gene locus of AVP. The mutant proteins could accumulate in the ER and possibly increase ER stress in the AVP neurons. We bred mice possessing a mutation causing FNDI, which manifested progressive polyuria, as do the patients with FNDI. Electron microscopic analyses demonstrated that aggregates accumulated in the ER of AVP neurons in FNDI mice. Despite polyuria, which could potentially induce dehydration, AVP mRNA expression was decreased in the supraoptic nucleus, and the AVP mRNA poly(A) tail length was shortened in FNDI mice compared with wild-type mice. Incubation of hypothalamic explants of wild-type mice with ER stressors caused shortening of the poly(A) tail length of AVP mRNA, accompanied by decreases in the expression. These data revealed a mechanism by which ER stress decreases poly(A) tail length of AVP mRNA, and this reduces the load of unfolded proteins that form the aggregates in ER of the AVP neurons in FNDI mice.

  7. Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum.

    PubMed

    Ishikawa, Yoshihiro; Holden, Paul; Bächinger, Hans Peter

    2017-10-20

    Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/ SERPINH1 ) and 65-kDa FK506-binding protein (FKBP65/ FKBP10 ), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Effect of the low- versus high-intensity exercise training on endoplasmic reticulum stress and GLP-1 in adolescents with type 2 diabetes mellitus.

    PubMed

    Lee, Sung Soo; Yoo, Jae Ho; So, Yong Seok

    2015-10-01

    [Purpose] The primary objective of this study was to investigate the effect of low-intensity exercise training compare with high-intensity exercise training on endoplasmic reticulum stress and glucagon-like peptide-1 in adolescents with type 2 diabetes mellitus. [Subjects and Methods] The low-intensity exercise training group performed aerobic exercise training at an intensity of ≤ 45% of the heart rate reserve. The high-intensity interval exercise training group performed interval exercise training at an intensity of ≥ 80% of the heart rate reserve. The exercise-related energy consumption was determined for both groups on a per-week basis (1,200 kcal/week). [Results] Both groups showed improvement in the glucose-regulated protein 78 and dipeptidyl peptidase-4, but the size of the between-group effect was not statistically significant. The high-intensity interval exercise training group showed a significant reduction in percentage body fat. The C-peptide level increased after the 12-weeks programs and was significantly different, between the groups. Fasting glucose, insulin resistance in the fasting state according to homeostasis model assessment, and leptin decreased after the 12-weeks exercise program and were significantly different between the groups, and glucagon-like peptide-1 increased after the 12-week exercise programs and was significantly different between the groups. [Conclusion] In conclusion high-intensity interval exercise training, as defined in this study, may lead to improvements in body composition, glycemic control, endoplasmic reticulum stress, and the glucagon-like peptide-1 in adolescents with type 2 diabetes mellitus.

  9. Mutations in the Polybasic Juxtamembrane Sequence of Both Plasma Membrane- and Endoplasmic Reticulum-localized Epidermal Growth Factor Receptors Confer Ligand-independent Cell Transformation*

    PubMed Central

    Bryant, Kirsten L.; Antonyak, Marc A.; Cerione, Richard A.; Baird, Barbara; Holowka, David

    2013-01-01

    Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling. PMID:24142702

  10. Effect of the low- versus high-intensity exercise training on endoplasmic reticulum stress and GLP-1 in adolescents with type 2 diabetes mellitus

    PubMed Central

    Lee, Sung Soo; Yoo, Jae Ho; So, Yong Seok

    2015-01-01

    [Purpose] The primary objective of this study was to investigate the effect of low-intensity exercise training compare with high-intensity exercise training on endoplasmic reticulum stress and glucagon-like peptide-1 in adolescents with type 2 diabetes mellitus. [Subjects and Methods] The low-intensity exercise training group performed aerobic exercise training at an intensity of ≤ 45% of the heart rate reserve. The high-intensity interval exercise training group performed interval exercise training at an intensity of ≥ 80% of the heart rate reserve. The exercise-related energy consumption was determined for both groups on a per-week basis (1,200 kcal/week). [Results] Both groups showed improvement in the glucose-regulated protein 78 and dipeptidyl peptidase-4, but the size of the between-group effect was not statistically significant. The high-intensity interval exercise training group showed a significant reduction in percentage body fat. The C-peptide level increased after the 12-weeks programs and was significantly different, between the groups. Fasting glucose, insulin resistance in the fasting state according to homeostasis model assessment, and leptin decreased after the 12-weeks exercise program and were significantly different between the groups, and glucagon-like peptide-1 increased after the 12-week exercise programs and was significantly different between the groups. [Conclusion] In conclusion high-intensity interval exercise training, as defined in this study, may lead to improvements in body composition, glycemic control, endoplasmic reticulum stress, and the glucagon-like peptide-1 in adolescents with type 2 diabetes mellitus. PMID:26644644

  11. Traffic of Human α-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex1[W

    PubMed Central

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2013-01-01

    The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins. PMID:23449646

  12. Curcumin enhances the effects of irinotecan on colorectal cancer cells through the generation of reactive oxygen species and activation of the endoplasmic reticulum stress pathway.

    PubMed

    Huang, Yan-Feng; Zhu, Da-Jian; Chen, Xiao-Wu; Chen, Qi-Kang; Luo, Zhen-Tao; Liu, Chang-Chun; Wang, Guo-Xin; Zhang, Wei-Jie; Liao, Nv-Zhu

    2017-06-20

    Although initially effective against metastatic colorectal cancer (CRC), irinotecan-based chemotherapy leads to resistance and adverse toxicity. Curcumin is well known for its anti-cancer effects in many cancers, including CRC. Here, we describe reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress as important mechanisms by which curcumin enhances irinotecan's effects on CRC cells. CRC cell lines were treated with curcumin and/or irinotecan for 24 h, and then evaluated using cell proliferation assays, cell apoptosis assays, cell cycle analysis, intracellular Ca2+ measurements, ROS measurements and immunoblotting for key ER stress-related proteins. We found that cell viability was inhibited and apoptosis was increased, accompanied by ROS generation and ER stress activation in CRC cells treated with curcumin alone or in combination with irinotecan. Blocking ROS production attenuated the expression of two markers of ER stress: binding of immunoglobulin protein (BIP) and CCAAT/enhancer-binding protein homologous protein (CHOP). Blocking CHOP expression using RNA interference also inhibited ROS generation. These results demonstrated that curcumin could enhance the effects of irinotecan on CRC cells by inhibiting cell viability and inducing cell cycle arrest and apoptosis, and that these effects may be mediated, in part, by ROS generation and activation of the ER stress pathway.

  13. A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum

    PubMed Central

    1987-01-01

    We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation. PMID:3305520

  14. Tributyltin-induced endoplasmic reticulum stress and its Ca(2+)-mediated mechanism.

    PubMed

    Isomura, Midori; Kotake, Yaichiro; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

    2013-10-01

    Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca(2+) signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca(2+) homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca(2+) depletion, and to test this idea, we examined the effect of TBT on intracellular Ca(2+) concentration using fura-2 AM, a Ca(2+) fluorescent probe. TBT increased intracellular Ca(2+) concentration in a TBT-concentration-dependent manner, and Ca(2+) increase in 700nM TBT was mainly blocked by 50μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca(2+) concentration by releasing Ca(2+) from ER, thereby causing ER stress. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X-box binding protein 1 transcription factor.

    PubMed

    Dias-Teixeira, Karina Luiza; Calegari-Silva, Teresa Cristina; dos Santos, Guilherme R R M; Vitorino Dos Santos, José; Lima, Carolina; Medina, Jorge Mansur; Aktas, Bertal Huseyin; Lopes, Ulisses G

    2016-04-01

    Endoplasmic reticulum (ER) stress triggers the integrated ER-stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR duringLeishmania amazonensisinfection. Treatment of RAW 264.7 infected macrophages with the ER stress-inducing agent thapsigargin (TG; 1 μM) increasedL. amazonensisinfectivity in an IFN1-α receptor (IFNAR)-dependent manner. In Western blot assays, we showed thatL. amazonensisactivates the inositol-requiring enzyme (IRE1)/ X-box binding protein (XBP)-1-splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for theIfnbgene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)-3 to the nucleus and a decrease in IFN1-β expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)-1, that protect parasites from oxidative stress. We conclude thatL. amazonensisactivation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1-β expression.-Dias-Teixeira, K. L., Calegari-Silva, T. C., Dos Santos, G. R. R. M., Vitorino dos Santos, J., Lima, C., Medina, J. M., Aktas, B. H., Lopes, U. G. The integrated endoplasmic reticulum stress response inLeishmania amazonensismacrophage infection: the role of X-box binding protein 1 transcription factor. © FASEB.

  16. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    PubMed

    Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

    2012-05-01

    3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in Saccharomyces cerevisiae.

    PubMed

    Tian, Siqi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-01-01

    To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.

  18. Resveratrol protects against early polymicrobial sepsis-induced acute kidney injury through inhibiting endoplasmic reticulum stress-activated NF-κB pathway

    PubMed Central

    Wang, Nian; Mao, Li; Yang, Liu; Zou, Jiang; Liu, Ke; Liu, Meidong; Zhang, Huali; Xiao, Xianzhong; Wang, Kangkai

    2017-01-01

    Resveratrol, a polyphenol compound derived from various edible plants, protects against sepsis-induced acute kidney injury (AKI) via its anti-inflammatory activity, but the underlying mechanisms remain largely unknown. In this study, a rat model of sepsis was established by cecal ligation and puncture (CLP), 30 mg/kg resveratrol was intraperitoneally administrated immediately after the CLP operation. HK-2 cells treated by 1 μg/ml lipopolysaccharide, 0.2 μM tunicamycin, 2.5 mM irestatin 9389 and 20 μM resveratrol were used for in vitro study. The results demonstrated that resveratrol significantly improved the renal function and tubular epithelial cell injury and enhanced the survival rate of CLP-induced rat model of sepsis, which was accompanied by a substantial decrease of the serum content and renal mRNA expressions of TNF-α, IL-1β and IL-6. In addition, resveratrol obviously relieved the endoplasmic reticulum stress, inhibited the phosphorylation of inositol-requiring enzyme 1(IRE1) and nuclear factor-κB (NF-κB) in the kidney. In vitro studies showed that resveratrol enhanced the cell viability, reduced the phosphorylation of NF-κB and production of inflammatory factors in lipopolysaccharide and tunicamycin-induced HK-2 cells through inhibiting IRE1 activation. Taken together, administration of resveratrol as soon as possible after the onset of sepsis could protect against septic AKI mainly through inhibiting IRE1-NF-κB pathway-triggered inflammatory response in the kidney. Resveratrol might be a readily translatable option to improve the prognosis of sepsis. PMID:28430592

  19. 6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress.

    PubMed

    Hu, Rong; Zhou, Ping; Peng, Yong-Bo; Xu, Xiaojun; Ma, Jiang; Liu, Qun; Zhang, Lei; Wen, Xiao-Dong; Qi, Lian-Wen; Gao, Ning; Li, Ping

    2012-01-01

    6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.

  20. 6-Shogaol Induces Apoptosis in Human Hepatocellular Carcinoma Cells and Exhibits Anti-Tumor Activity In Vivo through Endoplasmic Reticulum Stress

    PubMed Central

    Peng, Yong-Bo; Xu, Xiaojun; Ma, Jiang; Liu, Qun; Zhang, Lei; Wen, Xiao-Dong; Qi, Lian-Wen; Gao, Ning; Li, Ping

    2012-01-01

    6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo. PMID:22768104

  1. Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

    PubMed

    Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; Plaza-Davila, María; Martinez-Ruiz, Antonio; Fernandez-Bermejo, Miguel; Mateos-Rodriguez, Jose M; Salido, Gines M; Gonzalez, Antonio

    2018-01-01

    Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca 2+ concentration ([Ca 2+ ] c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca 2+ ] c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells. © 2017 Wiley Periodicals, Inc.

  2. Sigma Receptor 1 Modulates Endoplasmic Reticulum Stress in Retinal Neurons

    PubMed Central

    Ha, Yonju; Dun, Ying; Thangaraju, Muthusamy; Duplantier, Jennifer; Dong, Zheng; Liu, Kebin; Ganapathy, Vadivel

    2011-01-01

    Purpose. To investigate the mechanism of σ receptor 1 (σR1) neuroprotection in retinal neurons. Methods. Oxidative stress, which is implicated in diabetic retinopathy, was induced in mouse primary ganglion cells (GCs) and RGC-5 cells, and the effect of the σR1 ligand (+)-pentazocine on pro- and anti-apoptotic and endoplasmic reticulum (ER) stress gene expression was examined. Binding of σR1 to BiP, an ER chaperone protein, and σR1 phosphorylation status were examined by immunoprecipitation. Retinas were harvested from Ins2Akita/+ diabetic mice treated with (+)-pentazocine, and the expression of ER stress genes and of the retinal transcriptome was evaluated. Results. Oxidative stress induced the death of primary GCs and RGC-5 cells. The effect was decreased by the application of (+)-pentazocine. Stress increased σR1 binding to BiP and enhanced σR1 phosphorylation in RGC-5 cells. BiP binding was prevented, and σR1 phosphorylation decreased in the presence of (+)-pentazocine. The ER stress proteins PERK, ATF4, ATF6, IRE1α, and CHOP were upregulated in RGC-5 cells during oxidative stress, but decreased in the presence of (+)-pentazocine. A similar phenomenon was observed in retinas of Ins2Akita/+ diabetic mice. Retinal transcriptome analysis of Ins2Akita/+ mice compared with wild-type revealed differential expression of the genes critically involved in oxidative stress, differentiation, and cell death. The expression profile of those genes was reversed when the Ins2Akita/+ mice were treated with (+)-pentazocine. Conclusions. In retinal neurons, the molecular chaperone σR1 binds BiP under stressful conditions; (+)-pentazocine may exert its effects by dissociating σR1 from BiP. As stress in retinal cells increases, phosphorylation of σR1 is increased, which is attenuated when agonists bind to the receptor. PMID:20811050

  3. Sigma receptor 1 modulates endoplasmic reticulum stress in retinal neurons.

    PubMed

    Ha, Yonju; Dun, Ying; Thangaraju, Muthusamy; Duplantier, Jennifer; Dong, Zheng; Liu, Kebin; Ganapathy, Vadivel; Smith, Sylvia B

    2011-01-01

    To investigate the mechanism of σ receptor 1 (σR1) neuroprotection in retinal neurons. Oxidative stress, which is implicated in diabetic retinopathy, was induced in mouse primary ganglion cells (GCs) and RGC-5 cells, and the effect of the σR1 ligand (+)-pentazocine on pro- and anti-apoptotic and endoplasmic reticulum (ER) stress gene expression was examined. Binding of σR1 to BiP, an ER chaperone protein, and σR1 phosphorylation status were examined by immunoprecipitation. Retinas were harvested from Ins2Akita/+ diabetic mice treated with (+)-pentazocine, and the expression of ER stress genes and of the retinal transcriptome was evaluated. Oxidative stress induced the death of primary GCs and RGC-5 cells. The effect was decreased by the application of (+)-pentazocine. Stress increased σR1 binding to BiP and enhanced σR1 phosphorylation in RGC-5 cells. BiP binding was prevented, and σR1 phosphorylation decreased in the presence of (+)-pentazocine. The ER stress proteins PERK, ATF4, ATF6, IRE1α, and CHOP were upregulated in RGC-5 cells during oxidative stress, but decreased in the presence of (+)-pentazocine. A similar phenomenon was observed in retinas of Ins2Akita/+ diabetic mice. Retinal transcriptome analysis of Ins2Akita/+ mice compared with wild-type revealed differential expression of the genes critically involved in oxidative stress, differentiation, and cell death. The expression profile of those genes was reversed when the Ins2Akita/+ mice were treated with (+)-pentazocine. In retinal neurons, the molecular chaperone σR1 binds BiP under stressful conditions; (+)-pentazocine may exert its effects by dissociating σR1 from BiP. As stress in retinal cells increases, phosphorylation of σR1 is increased, which is attenuated when agonists bind to the receptor.

  4. The role of endoplasmic reticulum stress in hippocampal insulin resistance.

    PubMed

    Sims-Robinson, Catrina; Bakeman, Anna; Glasser, Rebecca; Boggs, Janet; Pacut, Crystal; Feldman, Eva L

    2016-03-01

    Metabolic syndrome, which includes hypertension, hyperglycemia, obesity, insulin resistance, and dyslipidemia, has a negative impact on cognitive health. Endoplasmic reticulum (ER) stress is activated during metabolic syndrome, however it is not known which factor associated with metabolic syndrome contributes to this stress. ER stress has been reported to play a role in the development of insulin resistance in peripheral tissues. The role of ER stress in the development of insulin resistance in hippocampal neurons is not known. In the current study, we investigated ER stress in the hippocampus of 3 different mouse models of metabolic syndrome: the C57BL6 mouse on a high fat (HF) diet; apolipoprotein E, leptin, and apolipoprotein B-48 deficient (ApoE 3KO) mice; and the low density lipoprotein receptor, leptin, and apolipoprotein B-48 deficient (LDLR 3KO) mice. We demonstrate that ER stress is activated in the hippocampus of HF mice, and for the first time, in ApoE 3KO mice, but not LDLR 3KO mice. The HF and ApoE 3KO mice are hyperglycemic; however, the LDLR 3KO mice have normal glycemia. This suggests that hyperglycemia may play a role in the activation of ER stress in the hippocampus. Similarly, we also demonstrate that impaired insulin signaling is only present in the HF and ApoE 3KO mice, which suggests that ER stress may play a role in insulin resistance in the hippocampus. To confirm this we pharmacologically induced ER stress with thapsigargin in human hippocampal neurons. We demonstrate for the first time that thapsigargin leads to ER stress and impaired insulin signaling in human hippocampal neurons. Our results may provide a potential mechanism that links metabolic syndrome and cognitive health. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response.

    PubMed

    Kilaparty, Surya P; Agarwal, Rakhee; Singh, Pooja; Kannan, Krishnaswamy; Ali, Nawab

    2016-07-01

    Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.

  6. Amplified RLR signaling activation through an interferon-stimulated gene-endoplasmic reticulum stress-mitochondrial calcium uniporter protein loop

    PubMed Central

    Cheng, Jinbo; Liao, Yajin; Zhou, Lujun; Peng, Shengyi; Chen, Hong; Yuan, Zengqiang

    2016-01-01

    Type I interferon (IFN-I) is critical for a host against viral and bacterial infections via induction of hundreds of interferon-stimulated genes (ISGs), but the mechanism underlying the regulation of IFN-I remains largely unknown. In this study, we first demonstrate that ISG expression is required for optimal IFN-β levels, an effect that is further enhanced by endoplasmic reticulum (ER) stress. Furthermore, we identify mitochondrial calcium uniporter protein (MCU) as a mitochondrial antiviral signaling protein (MAVS)-interacting protein that is important for ER stress induction and amplified MAVS signaling activation. In addition, by performing an ectopic expression assay to screen a library of 117 human ISGs for effects on IFN-β levels, we found that tumor necrosis factor receptor 1 (TNFR1) significantly increases IFN-β levels independent of ER stress. Altogether, our findings suggest that MCU and TNFR1 are involved in the regulation of RIG-I-like receptors (RLR) signaling. PMID:26892273

  7. 4-Phenylbutyrate protects rat skin flaps against ischemia-reperfusion injury and apoptosis by inhibiting endoplasmic reticulum stress

    PubMed Central

    YUE, ZHEN-SHUANG; ZENG, LIN-RU; QUAN, REN-FU; TANG, YANG-HUA; ZHENG, WEN-JIE; QU, GANG; XU, CAN-DA; ZHU, FANG-BING; HUANG, ZHONG-MING

    2016-01-01

    4-phenylbutyrate (4-PBA) is a low molecular weight fatty acid, which has been demonstrated to regulate endoplasmic reticulum (ER) stress. ER stress-induced cell apoptosis has an important role in skin flap ischemia; however, a pharmacological approach for treating ischemia-induced ER dysfunction has yet to be reported. In the present study, the effects of 4-PBA-induced ER stress inhibition on ischemia-reperfusion injury were investigated in the skin flap of rats, and transcriptional regulation was examined. 4-PBA attenuated ischemia-reperfusion injury and inhibited cell apoptosis in the skin flap. Furthermore, 4-PBA reversed the increased expression levels of two ER stress markers: CCAAT/enhancer-binding protein-homologous protein and glucose-regulated protein 78. These results suggested that 4-PBA was able to protect rat skin flaps against ischemia-reperfusion injury and apoptosis by inhibiting ER stress marker expression and ER stress-mediated apoptosis. The beneficial effects of 4-PBA may prove useful in the treatment of skin flap ischemia-reperfusion injury. PMID:26648447

  8. Protein Kinase A Activity Is Necessary for Fission and Fusion of Golgi to Endoplasmic Reticulum Retrograde Tubules

    PubMed Central

    Tenorio, María J.; Luchsinger, Charlotte; Mardones, Gonzalo A.

    2015-01-01

    It is becoming increasingly accepted that together with vesicles, tubules play a major role in the transfer of cargo between different cellular compartments. In contrast to our understanding of the molecular mechanisms of vesicular transport, little is known about tubular transport. How signal transduction molecules regulate these two modes of membrane transport processes is also poorly understood. In this study we investigated whether protein kinase A (PKA) activity regulates the retrograde, tubular transport of Golgi matrix proteins from the Golgi to the endoplasmic reticulum (ER). We found that Golgi-to-ER retrograde transport of the Golgi matrix proteins giantin, GM130, GRASP55, GRASP65, and p115 was impaired in the presence of PKA inhibitors. In addition, we unexpectedly found accumulation of tubules containing both Golgi matrix proteins and resident Golgi transmembrane proteins. These tubules were still attached to the Golgi and were highly dynamic. Our data suggest that both fission and fusion of retrograde tubules are mechanisms regulated by PKA activity. PMID:26258546

  9. Free Oligosaccharides to Monitor Glycoprotein Endoplasmic Reticulum-associated Degradation in Saccharomyces cerevisiae*

    PubMed Central

    Hirayama, Hiroto; Seino, Junichi; Kitajima, Toshihiko; Jigami, Yoshifumi; Suzuki, Tadashi

    2010-01-01

    In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. “Free” forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-β-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole α-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae. PMID:20150426

  10. Characterization of Russell bodies accumulating mutant antithrombin derived from the endoplasmic reticulum.

    PubMed

    Kimura, Koji; Kawaguchi, Kosuke; Ueda, Yumiko; Arai, Seisuke; Morita, Masashi; Imanaka, Tsuneo; Wada, Ikuo

    2015-01-01

    The endoplasmic reticulum (ER) adjusts its size and architecture to adapt to change in the surrounding environment. Russell bodies (RBs) were originally described as dilated structures of the ER cisternae containing large amounts of mutant immunoglobulin. Similar structures are observed in a wide variety of mutant proteins accumulated in the ER. We previously prepared Chinese hamster ovary (CHO) cells in which the expression of mutant antithrombin (AT) (C95R) was controlled with a Tet-On system and showed that RBs can be conditionally formed. However the precise architecture and intracellular behavior of RBs have been as yet only poorly characterized. To characterize the properties of RB, we prepared the same system using a green fluorescent protein (GFP)-fused mutant and measured the dynamics and architecture of RBs. We observed the mobile nature of the molecule in the RB lumen and RBs were separated from the rest of the ER network by narrow tubes. Furthermore, we found that the RBs were not simply expanded ER membranes. The RB lumen is filled with misfolded proteins that are surrounded by ER membranes. In addition, RBs mostly maintain their structure during cell division, possess ribosomes on their membranes and synthesize AT(C95R)-GFP. Based on the characterization of the hydrodynamic radius of AT(C95R)-GFP and the effect of DP1, an ER-shaping protein, we propose that RBs are spontaneously formed as a result of the partitioning of the misfolded AT with the shaping protein.

  11. [Over-expressed Bax inhibitor 1 (BI-1) inhibits apoptosis of hippocampal neurons via endoplasmic reticulum IRE1-JNK pathway in rats with subarachnoid hemorrhage].

    PubMed

    Liu, Jiaxin; Zhou, Shuai; Qian, Xiying; Zhang, Yueting; Zhao, Jianhua

    2017-10-01

    Objective To investigate the protective effect of lentivirus-mediated BI-1 overexpression on hippocampal neurons in rats with subarachnoid hemorrhage (SAH) and the relationship with endoplasmic reticulum IRE1-JNK signaling pathway. Methods The lentivirus solution of BI-1 over-expression was injected into the brain of rats 24 hours before SAH rat model was established by intravascular puncture method. At 24 hours after modeling, the brain water content and neurological score of the rats were measured. The apoptosis of hippocampal neurons was detected by TUNEL assay. Western blotting was used to detect the expressions of BI-1 protein and endoplasmic reticulum stress (ERS) marker proteins GRP78 and IRE1. ERS in hippocampal neurons of the rats with SAH was intervened by IRE1α-specific inhibitor KIRA6, and then the protein expressions of p-IRE1, p-JNK, Bax, Bcl2 and caspase-3 were detected by Western blotting. Results BI-1 over-expression improved neurobehavioral score, decreased brain water content and hippocampal neuron apoptosis rate, and also down-regulated GRP78 and IRE1 protein levels in the rats with SAH. Both the interference of KIRA6 and the over-expression of BI-1 inhibited the expressions of p-IRE1, p-JNK, Bax and caspase-3, and promoted the expression of anti-apoptotic protein Bcl2. Conclusion Over-expression of BI-1 can inhibit the apoptosis of hippocampal neurons in rats with SAH by inhibiting the activation of ERS-mediated IRE1-JNK signaling pathway, thus ultimately attenuating the early brain injury following SAH.

  12. Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Regulates Steroidogenic Activity via Steroidogenic Acute Regulatory Protein (StAR)-Voltage-dependent Anion Channel 2 (VDAC2) Interaction*

    PubMed Central

    Prasad, Manoj; Kaur, Jasmeet; Pawlak, Kevin J.; Bose, Mahuya; Whittal, Randy M.; Bose, Himangshu S.

    2015-01-01

    Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221–229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis. PMID:25505173

  13. Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity.

    PubMed

    Yuen, Christen Y L; Shek, Roger; Kang, Byung-Ho; Matsumoto, Kristie; Cho, Eun Ju; Christopher, David A

    2016-08-22

    In eukaryotes, classical protein disulfide isomerases (PDIs) facilitate the oxidative folding of nascent secretory proteins in the endoplasmic reticulum by catalyzing the formation, breakage, and rearrangement of disulfide bonds. Terrestrial plants encode six structurally distinct subfamilies of PDIs. The novel PDI-B subfamily is unique to terrestrial plants, and in Arabidopsis is represented by a single member, PDI8. Unlike classical PDIs, which lack transmembrane domains (TMDs), PDI8 is unique in that it has a C-terminal TMD and a single N-terminal thioredoxin domain (instead of two). No PDI8 isoforms have been experimentally characterized to date. Here we describe the characterization of the membrane orientation, expression, sub-cellular localization, and biochemical function of this novel member of the PDI family. Histochemical staining of plants harboring a PDI8 promoter:β-glucuronidase (GUS) fusion revealed that the PDI8 promoter is highly active in young, expanding leaves, the guard cells of cotyledons, and in the vasculature of several organs, including roots, leaves, cotyledons, and flowers. Immunoelectron microscopy studies using a PDI8-specific antibody on root and shoot apical cells revealed that PDI8 localizes to the endoplasmic reticulum (ER). Transient expression of two PDI8 fusions to green fluorescent protein (spGFP-PDI8 and PDI8-GFP-KKED) in leaf mesophyll protoplasts also resulted in labeling of the ER. Protease-protection immunoblot analysis indicated that PDI8 is a type I membrane protein, with its catalytic domain facing the ER lumen. The lumenal portion of PDI8 was able to functionally complement the loss of the prokaryotic protein foldase, disulfide oxidase (DsbA), as demonstrated by the reconstitution of periplasmic alkaline phosphatase in Escherichia coli. The results indicate that PDI8 is a type I transmembrane protein with its catalytic domain facing the lumen of the ER and functions in the oxidation of cysteines to produce disulfide

  14. Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress.

    PubMed

    Kim, Seong Hun; Kim, Kook Hwan; Kim, Hyoung-Kyu; Kim, Mi-Jeong; Back, Sung Hoon; Konishi, Morichika; Itoh, Nobuyuki; Lee, Myung-Shik

    2015-04-01

    Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-diabetic and anti-obesity activity. FGF21 expression is increased in patients with and mouse models of obesity or nonalcoholic fatty liver disease (NAFLD). However, the functional role and molecular mechanism of FGF21 induction in obesity or NAFLD are not clear. As endoplasmic reticulum (ER) stress is triggered in obesity and NAFLD, we investigated whether ER stress affects FGF21 expression or whether FGF21 induction acts as a mechanism of the unfolded protein response (UPR) adaptation to ER stress induced by chemical stressors or obesity. Hepatocytes or mouse embryonic fibroblasts deficient in UPR signalling pathways and liver-specific eIF2α mutant mice were employed to investigate the in vitro and in vivo effects of ER stress on FGF21 expression, respectively. The in vivo importance of FGF21 induction by ER stress and obesity was determined using inducible Fgf21-transgenic mice and Fgf21-null mice with or without leptin deficiency. We found that ER stressors induced FGF21 expression, which was dependent on a PKR-like ER kinase-eukaryotic translation factor 2α-activating transcription factor 4 pathway both in vitro and in vivo. Fgf21-null mice exhibited increased expression of ER stress marker genes and augmented hepatic lipid accumulation after tunicamycin treatment. However, these changes were attenuated in inducible Fgf21-transgenic mice. We also observed that Fgf21-null mice with leptin deficiency displayed increased hepatic ER stress response and liver injury, accompanied by deteriorated metabolic variables. Our results suggest that FGF21 plays an important role in the adaptive response to ER stress- or obesity-induced hepatic metabolic stress.

  15. Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum

    PubMed Central

    Mitsuda, Satoshi; Yokomichi, Tomonobu; Yokoigawa, Junpei; Kataoka, Takao

    2014-01-01

    Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum. PMID:24649404

  16. 3-Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines.

    PubMed

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Kwak, Byung Kook; Loffroy, Romaric; Vali, Mustafa

    2010-03-01

    Autophagy, a cellular response to stress, plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). Recently, we reported that the pyruvate analog 3-bromopyruvate (3-BrPA) promoted tumor cell death by targeting GAPDH. In continuance, we investigated the intracellular response of two human HCC cell lines (Hep3B and SK-Hep1) that differ in their status of key apoptotic regulators, p53 and Fas. 3-BrPA treatment induced endoplasmic reticulum (ER) stress, translation inhibition and apoptosis based on Western blot and qPCR, pulse labeling, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 in both the cell lines. However, electron microscopy revealed that 3-BrPA treated SK-Hep1 cells underwent classical apoptotic cell death while Hep3B cells initially responded with the protective autophagy that failed to prevent eventual apoptosis. 3-BrPA treatment promotes apoptosis in human HCC cell lines, irrespective of the intracellular response.

  17. SGK3 sustains ERα signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis.

    PubMed

    Wang, Yuanzhong; Zhou, Dujin; Phung, Sheryl; Warden, Charles; Rashid, Rumana; Chan, Nymph; Chen, Shiuan

    2017-02-21

    Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.

  18. Endoplasmic Reticulum Stress Caused by Lipoprotein Accumulation Suppresses Immunity against Bacterial Pathogens and Contributes to Immunosenescence

    PubMed Central

    Singh, Jogender

    2017-01-01

    ABSTRACT The unfolded protein response (UPR) is a stress response pathway that is activated upon increased unfolded and/or misfolded proteins in the endoplasmic reticulum (ER), and enhanced ER stress response prolongs life span and improves immunity. However, the mechanism by which ER stress affects immunity remains poorly understood. Using the nematode Caenorhabditis elegans, we show that mutations in the lipoproteins vitellogenins, which are homologs of human apolipoprotein B-100, resulted in upregulation of the UPR. Lipoprotein accumulation in the intestine adversely affects the immune response and the life span of the organism, suggesting that it could be a contributing factor to immunosenescence. We show that lipoprotein accumulation inhibited the expression of several immune genes encoding proteins secreted by the intestinal cells in an IRE-1-independent manner. Our studies provide a mechanistic explanation for adverse effects caused by protein aggregation and ER stress on immunity and highlight the role of an IRE-1-independent pathway in the suppression of the expression of genes encoding secreted proteins. PMID:28559483

  19. Tauroursodeoxycholic acid dampens oncogenic apoptosis induced by endoplasmic reticulum stress during hepatocarcinogen exposure

    PubMed Central

    Vandewynckel, Yves-Paul; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Verhelst, Xavier; Van den Bussche, Anja; Raevens, Sarah; Van Steenkiste, Christophe; Van Troys, Marleen; Ampe, Christophe; Descamps, Benedicte; Vanhove, Chris; Govaere, Olivier; Geerts, Anja; Van Vlierberghe, Hans

    2015-01-01

    Hepatocellular carcinoma (HCC) is characterized by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which activates the unfolded protein response (UPR). However, the role of ER stress in tumor initiation and progression is controversial. To determine the impact of ER stress, we applied tauroursodeoxycholic acid (TUDCA), a bile acid with chaperone properties. The effects of TUDCA were assessed using a diethylnitrosamine-induced mouse HCC model in preventive and therapeutic settings. Cell metabolic activity, proliferation and invasion were investigated in vitro. Tumor progression was assessed in the HepG2 xenograft model. Administration of TUDCA in the preventive setting reduced carcinogen-induced elevation of alanine and aspartate aminotransferase levels, apoptosis of hepatocytes and tumor burden. TUDCA also reduced eukaryotic initiation factor 2α (eIf2α) phosphorylation, C/EBP homologous protein expression and caspase-12 processing. Thus, TUDCA suppresses carcinogen-induced pro-apoptotic UPR. TUDCA alleviated hepatic inflammation by increasing NF-κB inhibitor IκBα. Furthermore, TUDCA altered the invasive phenotype and enhanced metabolic activity but not proliferation in HCC cells. TUDCA administration after tumor development did not alter orthotopic tumor or xenograft growth. Taken together, TUDCA attenuates hepatocarcinogenesis by suppressing carcinogen-induced ER stress-mediated cell death and inflammation without stimulating tumor progression. Therefore, this chemical chaperone could represent a novel chemopreventive agent. PMID:26293671

  20. ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells.

    PubMed

    Baudier, Jacques

    2018-05-01

    In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)-mitochondria organizing network (ERMIONE) controls mitochondria-ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear-encoded ATPases associated with diverse cellular activities (AAA+-ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain-containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial-ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE-like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial-ER interaction and recognizable neurological syndromes. The primate-specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties. © 2017 Cambridge Philosophical Society.

  1. 2,4,6-Trichlorophenol cytotoxicity involves oxidative stress, endoplasmic reticulum stress, and apoptosis.

    PubMed

    Zhang, Xiaoning; Zhang, Xiaona; Niu, Zhidan; Qi, Yongmei; Huang, Dejun; Zhang, Yingmei

    2014-01-01

    This study aims to evaluate the cytotoxicity and potential mechanisms of 2,4,6-trichlorophenol (2,4,6-TCP) in mouse embryonic fibroblasts. Our results show that 2,4,6-TCP causes morphological changes and reduces cell viability. The overproduction of reactive oxygen species, the upregulation of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX1) messenger RNA (mRNA) expressions, and the nuclear translocation of Nrf2 protein demonstrate that 2,4,6-TCP induces oxidative stress, and the Nrf2/HMOX1 pathway might be involved in 2,4,6-TCP-induced antioxidative response. Simultaneously, our data also demonstrate that 2,4,6-TCP upregulates the expressions of binding immunoglobulin protein, inositol-requiring enzyme/endonuclease 1α, and C/EBP homologous protein; stimulates α subunit of eukaryotic translation initiation factor 2 phosphorylation; and induces the splicing of Xbp1 mRNA, suggesting that endoplasmic reticulum (ER) stress is triggered. Moreover, 2,4,6-TCP alters the mitochondrial membrane potential and increases the apoptosis rate, the caspase 3 activity, and the Bax/Bcl-2 ratio, demonstrating that the mitochondrial pathway is involved in the 2,4,6-TCP-induced apoptosis. Thus, these results show that 2,4,6-TCP induces oxidative stress, ER stress, and apoptosis, which together contribute to its cytotoxicity in vitro. © The Author(s) 2014.

  2. Curcumin inhibits endoplasmic reticulum stress induced by cerebral ischemia-reperfusion injury in rats

    PubMed Central

    Zhu, Haiying; Fan, Yanxia; Sun, Hongyu; Chen, Liyan; Man, Xiao

    2017-01-01

    The aim of the present study was to observe the dynamic changes of the growth arrest and DNA damage-inducible 153 (GADD153) gene and caspase-12 in the brain tissue of rats with cerebral ischemia-reperfusion injury (CIRI) and the impact of curcumin pretreatment. A total of 60 rats were randomly divided into the normal group (N), the sham operation group (S), the dimethyl sulfoxide control group (D) and the curcumin treatment group (C). For group D and C, 12 (T1), 24 (T2) and 72 h (T3) of reperfusion were performed after 2 h ischemia. The expression levels of GADD153 and caspase-12 in the brain tissue were detected and compared among the groups by immunohistochemistry, immunofluorescence double staining and western blotting. The expression levels of GADD153 and caspase-12 were increased at T1compared with groups N and S, and the expression of caspase-12 peaked at T2 in group D, while GADD153 was increased until T3 in group D. Compared with group D, the expression levels of GADD153 and caspase-12 in group C at T2 and T3 were significantly decreased (P<0.05). Endoplasmic reticulum stress is involved in the pathological process of CIRI. Curcumin may decrease the expression levels of the above two factors, thus exhibiting protective effects against CIRI in rats. PMID:29067098

  3. Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney

    PubMed Central

    De Miguel, Carmen; Hamrick, William C.; Hobbs, Janet L.; Pollock, David M.; Carmines, Pamela K.; Pollock, Jennifer S.

    2017-01-01

    Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14–24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury. PMID:28230089

  4. Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney.

    PubMed

    De Miguel, Carmen; Hamrick, William C; Hobbs, Janet L; Pollock, David M; Carmines, Pamela K; Pollock, Jennifer S

    2017-02-23

    Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ET B deficient (ET B def) or transgenic control (TG-con) rats were used in the presence or absence of ET A receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ET B def rats showed a 14-24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ET A blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ET B def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ET A receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ET B receptor has protective effects. These results highlight targeting the ET A receptor as a therapeutic approach against ER stress-induced kidney injury.

  5. Exercise ameliorates endoplasmic reticulum stress-mediated vascular dysfunction in mesenteric arteries in atherosclerosis.

    PubMed

    Hong, Junyoung; Kim, Kwangchan; Park, Eunkyung; Lee, Jonghae; Markofski, Melissa M; Marrelli, Sean P; Park, Yoonjung

    2018-05-21

    Endoplasmic reticulum (ER) stress is closely associated with atherosclerosis, but the effects of exercise on ER stress-mediated endothelial dysfunction in atherosclerosis is not yet fully understood. We assessed endothelium-dependent vasodilation in isolated mesenteric arteries from wild type (WT), WT with exercise (WT-EX), ApoE knockout (ApoE KO), and ApoE KO mice with exercise (ApoE KO-EX). Vasodilation to acetylcholine (ACh) was elicited in the presence of inhibitors of ER stress, eNOS, caspase-1, and UCP-2 (Tudca, L-NAME, AC-YVARD-cmk, and Genipin, respectively) and the ER stress inducer (Tunicamycin). Immunofluorescence was used to visualize the expression of CHOP, as an indicator of ER stress, in superior mesenteric arteries (SMA). Dilation to ACh was attenuated in ApoE KO but was improved in ApoE KO-EX. Incubation of Tudca and AC-YVARD-cmk improved ACh-induced vasodilation in ApoE KO. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reversed the increase in CHOP expression in the endothelium of SMA of ApoE KO mice. We conclude that ER stress plays a significant role in endothelial dysfunction of resistance arteries in atherosclerosis and that exercise attenuates ER stress and regulates its critical downstream signaling pathways including eNOS, UCP-2 and caspase-1.

  6. Endoplasmic reticulum stress mediates withaferin A-induced apoptosis in human renal carcinoma cells.

    PubMed

    Choi, Min Jung; Park, Eun Jung; Min, Kyoung Jin; Park, Jong-Wook; Kwon, Taeg Kyu

    2011-04-01

    The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) results in cellular stress that initiates a specialized response designated as the unfolded protein response. ER stress has been implicated in a variety of common diseases, such as diabetes, ischemia and neurodegenerative disorders. Withaferin A, a major chemical constituent of Withania somnifera, has been reported to inhibit tumor cell growth. We show that withaferin A induced a dose-dependent apoptotic cell death in several types of human cancer cells, as measured by FACS analysis and PARP cleavage. Treatment of Caki cells with withaferin A induced a number of signature ER stress markers, including phosphorylation of eukaryotic initiation factor-2α (eIF-2 α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78. In addition, withaferin A caused up-regulation of CAAT/enhancer-binding protein-homologous protein (CHOP), suggesting the induction of ER stress. Pretreatment with N-acetyl cysteine (NAC) significantly inhibited withaferin A-mediated ER stress proteins and cell death, suggesting that reactive oxygen species (ROS) mediate withaferin A-induced ER stress. Furthermore, CHOP siRNA or inhibition of caspase-4 activity attenuated withaferin A-induced apoptosis. Taken together, the present study provides strong evidence supporting an important role of the ER stress response in mediating withaferin A-induced apoptosis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

    PubMed Central

    Ferris, Sean P.; Jaber, Nikita S.; Molinari, Maurizio; Arvan, Peter; Kaufman, Randal J.

    2013-01-01

    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. PMID:23864712

  8. Chemical chaperone 4-phenylbutyric acid protects H9c2 cardiomyocytes from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis.

    PubMed

    Jian, Lian; Lu, Yuan; Lu, Shan; Lu, Chengzhi

    2016-05-01

    Myocardial ischemia/reperfusion (I/R) is a potential contributor to high rates of mortality in several cardiovascular diseases. I/R initiates the unfolded protein response and endoplasmic reticulum (ER) stress, which may lead to apoptotic pathways and exaggerate I/R injury. 4‑phenylbutyric acid (4‑PBA), a low molecular weight, terminal aromatic substituted fatty acid, has been reported to function as an ER chaperone. The aim of the present study was to investigate whether 4‑PBA is able to reduce ER stress‑induced apoptosis and prevent cardiomyocyte damage during the process of I/R in vitro. Accordingly, the rat cardiomyocyte line, H9c2, was treated with hypoxia/reoxygenation as an I/R model in vitro. Myocardium apoptosis was determined with TUNEL staining. The expression of ER stress‑related proteins were examined by western blotting. The resulting data showed that I/R activates the ER stress proteins, glucose‑regulated protein 78, activating transcription factor 6 and protein kinase RNA‑like endoplasmic reticulum kinase, which were all reduced by pretreatment with 4‑PBA. In addition, pretreatment with 4‑PBA significantly inhibited the expression levels of pro‑apoptotic proteins, C/EBP homologous protein, B cell lymphoma (Bcl‑2)‑associated X protein and phosphorylated c‑Jun N‑terminal kinase, and enhanced the expression of the anti‑apoptotic protein Bcl‑2 (n=3; P<0.05). The data demonstrated that I/R initiates ER stress‑associated apoptotic pathways, and 4‑PBA pretreatment protected the cardiomyocytes from I/R‑induced cell death. To the best of our knowledge, the present study is the first to report on the cell repair mechanism of 4‑PBA against I/R damage in cardiomyocytes based on ER stress‑associated apoptotic pathways.

  9. Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes

    PubMed Central

    Kajimoto, Kazuaki; Minami, Yoshitaka; Harashima, Hideyoshi

    2014-01-01

    The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor α (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. PMID:25161868

  10. Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways

    PubMed Central

    Ying, Jinhe; Xu, Huan; Wu, Dhua; Wu, Xiaoguang

    2015-01-01

    Aim: Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Methods: Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Results: Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Conclusions: Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis. PMID:26722474

  11. Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells

    PubMed Central

    Caballano-Infantes, Estefania; Terron-Bautista, José; Beltrán-Povea, Amparo; Cahuana, Gladys M; Soria, Bernat; Nabil, Hajji; Bedoya, Francisco J; Tejedo, Juan R

    2017-01-01

    Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies. PMID:28289506

  12. Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding.

    PubMed

    Cha-Molstad, Hyunjoo; Sung, Ki Sa; Hwang, Joonsung; Kim, Kyoung A; Yu, Ji Eun; Yoo, Young Dong; Jang, Jun Min; Han, Dong Hoon; Molstad, Michael; Kim, Jung Gi; Lee, Yoon Jee; Zakrzewska, Adriana; Kim, Su-Hyeon; Kim, Sung Tae; Kim, Sun Yong; Lee, Hee Gu; Soung, Nak Kyun; Ahn, Jong Seog; Ciechanover, Aaron; Kim, Bo Yeon; Kwon, Yong Tae

    2015-07-01

    We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.

  13. Regulation of diacylglycerol acyltransferase 2 protein stability by gp78-associated endoplasmic-reticulum-associated degradation.

    PubMed

    Choi, Kwangman; Kim, Hyeongki; Kang, Hyunju; Lee, So-Young; Lee, Sang Jun; Back, Sung Hoon; Lee, Seo Hyun; Kim, M Sun; Lee, Jeong Eun; Park, Ju Young; Kim, Jiye; Kim, Sunhong; Song, Jae-Hyung; Choi, Yura; Lee, Suui; Lee, Hyun-Jun; Kim, Jong Heon; Cho, Sungchan

    2014-07-01

    Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by diacylglycerol acyltransferase (DGAT) in the endoplasmic reticulum (ER). DGAT2, one of the two DGAT enzymes, is barely detectable in cells, even though its mRNA transcripts are maintained at considerable levels. However, little is known about how DGAT2 expression is altered by protein stability. DGAT2 was highly unstable in cells and was rapidly degraded by proteasomes in an ubiquitin-dependent manner. Deletion mutation analysis identified transmembrane domain 1 (TMD1) as a protein degradation signal. TMD1 is also important for ER localization of DGAT2. Moreover, DGAT2 interacted with p97/VCP, a crucial component of the ER-associated degradation (ERAD) pathway, and polyubiquitinated DGAT2 accumulated following treatment with an ERAD inhibitor. Furthermore, gp78, an E3 ligase involved in ERAD, regulates the degradation of DGAT2 through direct interactions and ubiquitination. Consequently, the stabilization of DGAT2 increased the number of lipid droplets in hepatic cells. Therefore, DGAT2 is regulated by gp78-associated ERAD at the post-translational level. © 2014 FEBS.

  14. Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells.

    PubMed

    Caballano-Infantes, Estefania; Terron-Bautista, José; Beltrán-Povea, Amparo; Cahuana, Gladys M; Soria, Bernat; Nabil, Hajji; Bedoya, Francisco J; Tejedo, Juan R

    2017-02-26

    Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca 2+ flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.

  15. Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ueda, Masashi; Li, Shimo; Itoh, Masanori

    The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ERmore » luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.« less

  16. Estrogen reduces endoplasmic reticulum stress to protect against glucotoxicity induced-pancreatic β-cell death.

    PubMed

    Kooptiwut, Suwattanee; Mahawong, Pitchnischa; Hanchang, Wanthanee; Semprasert, Namoiy; Kaewin, Suchada; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-Thai

    2014-01-01

    Estrogen can improve glucose homeostasis not only in diabetic rodents but also in humans. However, the molecular mechanism by which estrogen prevents pancreatic β-cell death remains unclear. To investigate this issue, INS-1 cells, a rat insulinoma cell line, were cultured in medium with either 11.1mM or 40mM glucose in the presence or the absence of estrogen. Estrogen significantly reduced apoptotic β-cell death by decreasing nitrogen-induced oxidative stress and the expression of the ER stress markers GRP 78, ATF6, P-PERK, PERK, uXBP1, sXBP1, and CHOP in INS-1 cells after prolonged culture in medium with 40mM glucose. In contrast, estrogen increased the expression of survival proteins, including sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA-2), Bcl-2, and P-p38, in INS-1 cells after prolonged culture in medium with 40mM glucose. The cytoprotective effect of estrogen was attenuated by addition of the estrogen receptor (ERα and ERβ) antagonist ICI 182,780 and the estrogen membrane receptor inhibitor G15. We showed that estrogen decreases not only oxidative stress but also ER stress to protect against 40mM glucose-induced pancreatic β-cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Endoplasmic reticulum stress regulates inflammation and insulin resistance in skeletal muscle from pregnant women.

    PubMed

    Liong, Stella; Lappas, Martha

    2016-04-15

    Sterile inflammation and infection are key mediators of inflammation and peripheral insulin resistance associated with gestational diabetes mellitus (GDM). Studies have shown endoplasmic reticulum (ER) stress to induce inflammation and insulin resistance associated with obesity and type 2 diabetes, however is paucity of studies investigating the effects of ER stress in skeletal muscle on inflammation and insulin resistance associated with GDM. ER stress proteins IRE1α, GRP78 and XBP-1s were upregulated in skeletal muscle of obese pregnant women, whereas IRE1α was increased in GDM women. Suppression of ER stress, using ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or siRNA knockdown of IRE1α and GRP78, significantly downregulated LPS-, poly(I:C)- or IL-1β-induced production of IL-6, IL-8, IL-1β and MCP-1. Furthermore, LPS-, poly(I:C)- or TNF-α-induced insulin resistance was improved following suppression of ER stress, by increasing insulin-stimulated phosphorylation of IR-β, IRS-1, GLUT-4 expression and glucose uptake. In summary, our inducible obesity and GDM-like models suggests that the development of GDM may be involved in activating ER stress-induced inflammation and insulin resistance in human skeletal muscle. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos*

    PubMed Central

    Cui, Jingqiu; Chen, Wei; Sun, Jinhong; Guo, Huan; Madley, Rachel; Xiong, Yi; Pan, Xingyi; Wang, Hongliang; Tai, Andrew W.; Weiss, Michael A.; Arvan, Peter; Liu, Ming

    2015-01-01

    Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design. PMID:26446786

  19. From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

    PubMed

    Farinha, Carlos M; Canato, Sara

    2017-01-01

    CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

  20. Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Young, Kevin G.; University of Ottawa Center for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario; Kothary, Rashmi

    2008-09-10

    Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages precedingmore » the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3{alpha}, but not nesprin-3{beta}. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.« less

  1. The Ca2+-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

    PubMed

    Espinoza-Fonseca, L Michel

    2017-03-28

    Ca 2+ transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca 2+ homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca 2+ uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca 2+ and other ions across the SR. During Ca 2+ uptake by the SR Ca 2+ -ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca 2+ transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca 2+ release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca 2+ transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

  2. Characterization of aromatic residue-controlled protein retention in the endoplasmic reticulum of Saccharomyces cerevisiae.

    PubMed

    Mei, Meng; Zhai, Chao; Li, Xinzhi; Zhou, Yu; Peng, Wenfang; Ma, Lixin; Wang, Qinhong; Iverson, Brent L; Zhang, Guimin; Yi, Li

    2017-12-15

    An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion.

    PubMed

    Wei, Wei; Gao, Yanhui; Wang, Cheng; Zhao, Lijun; Sun, Dianjun

    2013-06-01

    Protein retention in the enamel layer during tooth formation is well known to be associated with dental fluorosis but the underlying mechanism is unclear. The functions of the endoplasmic reticulum (ER) correlate directly with secreted protein metabolism. We used an ameloblast-derived cell line to determine whether excessive amounts of fluoride cause ER stress, and whether this interferes with the secretion of enamel matrix proteinases. ER stress activates a signaling network called the unfolded protein response (UPR). Here, we used real-time RT-PCR and immunofluorescence to study the effect of fluoride on the expression, translation, and secretion of UPR transcription factors in ameloblast-like cells. Measurement of both the gene and protein expression of UPR transcription factors indicated that high-dose fluoride increases the expression of UPR transcription factors in a dose-dependent manner. We also used ELISA to detect and quantify the enamel proteinases secreted by ameloblasts. We found a corresponding decrease in extracellular secretion of the enamel proteinases matrix metalloproteinase-20 and kallikrein-4, after exposure to fluoride. Furthermore, correlation analysis indicated that the expression of UPR transcription factors showed a strong inverse correlation with that of enamel proteinases. The results suggest that high-dose fluoride initiates an ER stress response in ameloblasts and induces the UPR, which interferes with the synthesis and secretion of enamel proteinases. Taken together, these results suggest that excessive ingestion of fluoride during tooth formation can decrease the secretion of proteinases, thus causing protein retention in the enamel layer, indicating that the ER stress response may be responsible for dental fluorosis. Copyright © 2011 Wiley Periodicals, Inc.

  4. Fluoride induces endoplasmic reticulum stress and inhibits protein synthesis and secretion.

    PubMed

    Sharma, Ramaswamy; Tsuchiya, Masahiro; Bartlett, John D

    2008-09-01

    Exposure to excessive amounts of fluoride (F(-)) causes dental fluorosis in susceptible individuals; however, the mechanism of F(-)-induced toxicity is unclear. Previously, we have shown that high-dose F(-) activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells. In this study we determined if low-dose F(-) causes ER stress and activates the UPR, and we also determined whether F(-) interferes with the secretion of proteins from the ER. We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F(-)-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F(-) were examined for eucaryotic initiation factor-2, subunit alpha (eIF2alpha) phosphorylation. We found that F(-) decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F(-). These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2alpha. Importantly, F(-)-induced phosphorylation of eIF2alphawas confirmed in vivo. These data suggest that F(-) initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis.

  5. Fluoride Induces Endoplasmic Reticulum Stress and Inhibits Protein Synthesis and Secretion

    PubMed Central

    Sharma, Ramaswamy; Tsuchiya, Masahiro; Bartlett, John D.

    2008-01-01

    Background Exposure to excessive amounts of fluoride (F−) causes dental fluorosis in susceptible individuals; however, the mechanism of F−-induced toxicity is unclear. Previously, we have shown that high-dose F− activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells. Objectives In this study we determined if low-dose F− causes ER stress and activates the UPR, and we also determined whether F− interferes with the secretion of proteins from the ER. Methods We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F−-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F− were examined for eucaryotic initiation factor-2, subunit alpha (eIF2α) phosphorylation. Results We found that F− decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F−. These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2α. Importantly, F−-induced phosphorylation of eIF2αwas confirmed in vivo. Conclusions These data suggest that F− initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis. PMID:18795154

  6. Interaction surface and topology of Get3-Get4-Get5 protein complex, involved in targeting tail-anchored proteins to endoplasmic reticulum.

    PubMed

    Chang, Yi-Wei; Lin, Tai-Wen; Li, Yi-Chuan; Huang, Yu-Shan; Sun, Yuh-Ju; Hsiao, Chwan-Deng

    2012-02-10

    Recent work has uncovered the "GET system," which is responsible for endoplasmic reticulum targeting of tail-anchored proteins. Although structural information and the individual roles of most components of this system have been defined, the interactions and interplay between them remain to be elucidated. Here, we investigated the interactions between Get3 and the Get4-Get5 complex from Saccharomyces cerevisiae. We show that Get3 interacts with Get4-Get5 via an interface dominated by electrostatic forces. Using isothermal titration calorimetry and small-angle x-ray scattering, we further demonstrate that the Get3 homodimer interacts with two copies of the Get4-Get5 complex to form an extended conformation in solution.

  7. Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury

    PubMed Central

    Barone, Sharon; Destefano-Shields, Christina; Brooks, Marybeth; Murray-Stewart, Tracy; Dunworth, Matthew; Li, Weimin; Doherty, Joanne R.; Hall, Mark A.; Smith, Roger D.; Cleveland, John L.; Casero, Robert A.; Soleimani, Manoocher

    2017-01-01

    Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX) increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI). Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H2O2 and aminopropanal, significantly diminished the severity of cisplatin AKI. In vitro studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and enhances the expression of binding immunoglobulin protein BiP/GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153). The increased expression of these endoplasmic reticulum stress response (ERSR) markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP) and apoptosis (e.g. reduced activated caspase-3). These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI. PMID:28886181

  8. Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury.

    PubMed

    Zahedi, Kamyar; Barone, Sharon; Destefano-Shields, Christina; Brooks, Marybeth; Murray-Stewart, Tracy; Dunworth, Matthew; Li, Weimin; Doherty, Joanne R; Hall, Mark A; Smith, Roger D; Cleveland, John L; Casero, Robert A; Soleimani, Manoocher

    2017-01-01

    Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX) increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI). Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H2O2 and aminopropanal, significantly diminished the severity of cisplatin AKI. In vitro studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and enhances the expression of binding immunoglobulin protein BiP/GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153). The increased expression of these endoplasmic reticulum stress response (ERSR) markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP) and apoptosis (e.g. reduced activated caspase-3). These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI.

  9. Vildagliptin preserves the mass and function of pancreatic β cells via the developmental regulation and suppression of oxidative and endoplasmic reticulum stress in a mouse model of diabetes

    PubMed Central

    Hamamoto, S; Kanda, Y; Shimoda, M; Tatsumi, F; Kohara, K; Tawaramoto, K; Hashiramoto, M; Kaku, K

    2013-01-01

    Aim We investigated the molecular mechanisms by which vildagliptin preserved pancreatic β cell mass and function. Methods Morphological, biochemical and gene expression profiles of the pancreatic islets were investigated in male KK-Ay-TaJcl(KK-Ay) and C57BL/6JJcl (B6) mice aged 8 weeks which received either vildagliptin or a vehicle for 4 weeks. Results Body weight, food intake, fasting blood glucose, plasma insulin and active glucagon-like peptide-1 were unchanged with vildagliptin treatment in both mice. In KK-Ay mice treated with vildagliptin, increased plasma triglyceride (TG) level and islet TG content were decreased, insulin sensitivity significantly improved, and the glucose tolerance ameliorated with increases in plasma insulin levels. Furthermore, vildagliptin increased glucose-stimulated insulin secretion, islet insulin content and pancreatic β cell mass in both strains. By vildagliptin, the expression of genes involved in cell differentiation/proliferation was upregulated in both strains, those related to apoptosis, endoplasmic reticulum stress and lipid synthesis was decreased and those related to anti-apoptosis and anti-oxidative stress was upregulated, in KK-Ay mice. The morphological results were consistent with the gene expression profiles. Conclusion Vildagliptin increases β cell mass by not only directly affecting cell kinetics but also by indirectly reducing cell apoptosis, oxidative stress and endoplasmic reticulum stress in diabetic mice. PMID:22950702

  10. Curcuminoid EF24 enhances the anti-tumour activity of Akt inhibitor MK-2206 through ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction in gastric cancer.

    PubMed

    Chen, Xi; Dai, Xuanxuan; Zou, Peng; Chen, Weiqian; Rajamanickam, Vinothkumar; Feng, Chen; Zhuge, Weishan; Qiu, Chenyu; Ye, Qingqing; Zhang, Xiaohua; Liang, Guang

    2017-05-01

    Gastric cancer is one of the leading causes of morbidity and mortality worldwide. Akt is an anti-apoptotic kinase that plays a dynamic role in cell survival and is implicated in the pathogenesis of gastric cancer. MK-2206, the first allosteric inhibitor of Akt, is in clinical trials for a number of cancers. Although preclinical studies showed promise, clinical trials reported it had no effect when given alone at tolerated doses. The aim of our study was to delineate the effects of MK-2206 on gastric cancer cells and explore the ability of combination treatments to enhance the anti-tumour activity of MK-2206. SGC-7901, BGC-823 cells and immunodeficient mice were chosen as a model to study the treatment effects. Changes in cell viability, apoptosis and ROS, endoplasmic reticulum stress and mitochondrial dysfunction in the cells were analysed by MTT assays, ROS imaging and FACSCalibur, electron microscopy, JC-1 staining and western blotting. MK-2206 induced apoptotic cell death through the generation of ROS. We utilized ROS production to target gastric cancer cells by combining MK-2206 and an ROS inducer EF24. Our in vitro and in vivo xenograft studies showed that combined treatment with MK-2206 and EF24 synergistically induced apoptosis in gastric cancer cells and caused cell cycle arrest. These activities were mediated through ROS generation and the induction of endoplasmic reticulum stress and mitochondrial dysfunction. Targeting ROS generation by using a combination of an Akt inhibitor and EF24 could have potential as a therapy for gastric cancer. © 2017 The British Pharmacological Society.

  11. Do inositol supplements enhance phosphatidylinositol supply and thus support endoplasmic reticulum function?

    PubMed

    Michell, Robert H

    2018-06-03

    This review attempts to explain why consuming extra myoinositol (Ins), an essential component of membrane phospholipids, is often beneficial for patients with conditions characterised by insulin resistance, non-alcoholic fatty liver disease and endoplasmic reticulum (ER) stress. For decades we assumed that most human diets provide an adequate Ins supply, but newer evidence suggests that increasing Ins intake ameliorates several disorders, including polycystic ovary syndrome, gestational diabetes, metabolic syndrome, poor sperm development and retinopathy of prematurity. Proposed explanations often suggest functional enhancement of minor facets of Ins Biology such as insulin signalling through putative inositol-containing 'mediators', but offer no explanation for this selectivity. It is more likely that eating extra Ins corrects a deficiency of an abundant Ins-containing cell constituent, probably phosphatidylinositol (PtdIns). Much of a cell's PtdIns is in ER membranes, and an increase in ER membrane synthesis, enhancing the ER's functional capacity, is often an important part of cell responses to ER stress. This review: (a) reinterprets historical information on Ins deficiency as describing a set of events involving a failure of cells adequately to adapt to ER stress; (b) proposes that in the conditions that respond to dietary Ins there is an overstretching of Ins reserves that limits the stressed ER's ability to make the 'extra' PtdIns needed for ER membrane expansion; and (c) suggests that eating Ins supplements increases the Ins supply to Ins-deficient and ER-stressed cells, allowing them to make more PtdIns and to expand the ER membrane system and sustain ER functions.

  12. Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation.

    PubMed

    de Almeida, Sérgio F; Picarote, Gonçalo; Fleming, John V; Carmo-Fonseca, Maria; Azevedo, Jorge E; de Sousa, Maria

    2007-09-21

    HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

  13. Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope

    PubMed Central

    Zattas, Dimitrios; Hochstrasser, Mark

    2014-01-01

    The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

  14. Mitochondria-associated endoplasmic reticulum membrane (MAM) regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltage-dependent anion channel 2 (VDAC2) interaction.

    PubMed

    Prasad, Manoj; Kaur, Jasmeet; Pawlak, Kevin J; Bose, Mahuya; Whittal, Randy M; Bose, Himangshu S

    2015-01-30

    Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221-229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Komoike, Yuta, E-mail: komoike@research.twmu.ac.jp

    Acrylamide (AA) is a well-known neurotoxic compound in humans and experimental animals. However, intracellular stress signaling pathways responsible for the neurotoxicity of AA are still not clear. In this study, we explored the involvement of the endoplasmic reticulum (ER) stress response in AA-induced neuronal damage in vitro and in vivo. Exposure of SH-SY5Y human neuroblastoma cells to AA increased the levels of phosphorylated form of eukaryotic translation initiation factor 2α (eIF2α) and its downstream effector, activating transcription factor 4 (ATF4), indicating the induction of the unfolded protein response (UPR) by AA exposure. Furthermore, AA exposure increased the mRNA level ofmore » c/EBP homologous protein (CHOP), the ER stress-dependent apoptotic factor, and caused the accumulation of reactive oxygen species (ROS) in SH-SY5Y cells. Treatments of SH-SY5Y cells with the chemical chaperone, 4-phenylbutyric acid and the ROS scavenger, N-acetyl-cysteine reduced the AA-induced expression of ATF4 protein and CHOP mRNA, and resulted in the suppression of apoptosis. In addition, AA-induced eIF2α phosphorylation was also suppressed by NAC treatment. In consistent with in vitro study, exposure of zebrafish larvae at 6-day post fertilization to AA induced the expression of chop mRNA and apoptotic cell death in the brain, and also caused the disruption of brain structure. These findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress and subsequent eIF2α–ATF4–CHOP signaling cascade. The accumulation of ROS by AA exposure appears to be responsible for this ER stress-mediated apoptotic pathway. - Highlights: • Exposure of SH-SY5Y cells to AA activates the eIF2α–ATF4 pathway of the UPR. • Exposure of SH-SY5Y cells to AA induces the CHOP expression and apoptosis. • Exposure of zebrafish to AA induces the chop expression and apoptosis in the brain. • AA possibly induces apoptotic neuronal cell death through the

  16. Alteration of the proteostasis network of plant cells promotes the post-endoplasmic reticulum trafficking of recombinant mutant (L444P) human β-glucocerebrosidase

    PubMed Central

    Babajani, Gholamreza; Kermode, Allison R

    2014-01-01

    Gaucher disease is a prevalent lysosomal storage disease characterized by a deficiency in the activity of lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a L444P missense mutation in the GCase protein, which results in its disrupted folding in the endoplasmic reticulum (ER) and impaired post-ER trafficking. To determine whether the post-ER trafficking of this severely malfolded protein can be restored, we expressed the mutant L444P GCase as a recombinant protein in transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, in which the GCase variant was equipped with a plant signal peptide to allow for secretion upon rescued trafficking out of the ER. The recombinant L444P mutant GCase was retained in the plant endoplasmic reticulum (ER). Kifunensine and Eeyarestatin I, both inhibitors of ER-associated degradation (ERAD), and the proteostasis regulators, celastrol and MG-132, increased the steady-state levels of the mutant protein inside the plant cells and further promoted the post-ER trafficking of L444P GCase, as indicated by endoglycosidase-H sensitivity- and secretion- analyses. Transcript profiling of genes encoding ER-molecular chaperones, ER stress responsive proteins, and cytoplasmic heat shock response proteins, revealed insignificant or only very modest changes in response to the ERAD inhibitors and proteostasis regulators. An exception was the marked response to celastrol which reduced the steady-state levels of cytoplasmic HSP90 transcripts and protein. As HSP90 participates in the targeting of misfolded proteins to the proteasome pathway, its down-modulation in response to celastrol may partly account for the mechanism of improved homeostasis of L444P GCase mediated by this triterpene. PMID:24713615

  17. Glutathione S-Transferase P-Mediated Protein S-Glutathionylation of Resident Endoplasmic Reticulum Proteins Influences Sensitivity to Drug-Induced Unfolded Protein Response

    PubMed Central

    Ye, Zhi-Wei; Zhang, Jie; Ancrum, Tiffany; Manevich, Yefim; Townsend, Danyelle M.

    2017-01-01

    Abstract Aims: S-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs. Results: We identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a cluster of proteins cooperatively involved in the regulation of ER stress (immunoglobulin heavy chain-binding protein [BiP], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca2+-ATPase [SERCA]) that individually co-immunoprecipitated with GSTP (implying protein complex formation) and were subject to reactive oxygen species (ROS) induced S-glutathionylation. S-glutathionylation of each of these six proteins was attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP (Gstp1/p2−/−) compared to wild type (Gstp1/p2+/+). Moreover, Gstp1/p2−/− cells were significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (2-fold). Innovation: Within the family of GST isozymes, GSTP has been ascribed the broadest range of catalytic and chaperone functions. Now, for the first time, we identify it as an ER resident protein that catalyzes S-glutathionylation of critical ER proteins within this organelle. Of note, this can provide a nexus for linkage of redox based signaling and pathways that regulate the unfolded protein response (UPR). This has novel importance in determining how some drugs kill cancer cells. Conclusions: Contextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the oxidative ER environment and indicate that, within the ER, GSTP influences the cellular consequences of the UPR

  18. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase C674 promotes ischemia- and hypoxia-induced angiogenesis via coordinated endothelial cell and macrophage function.

    PubMed

    Mei, Yu; Thompson, Melissa D; Shiraishi, Yasunaga; Cohen, Richard A; Tong, Xiaoyong

    2014-11-01

    Ischemia is a complex phenomenon modulated by the concerted action of several cell types. We have identified that sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2 (SERCA 2) cysteine 674 (C674) S-glutathiolation is essential for ischemic angiogenesis, vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) migration and network formation. A heterozygote SERCA 2 C674S knockin (SKI) mouse shows impaired ischemic blood flow recovery after femoral artery ligation, and its ECs show depleted endoplasmic reticulum (ER) Ca(2+) stores and impaired angiogenic behavior. Here we studied the role of SERCA 2 C674 in the interaction between ECs and macrophages in the context of ischemia and discovered the involvement of the ER stress response protein, ER oxidoreductin-1α (ERO1). In wild type (WT) mice, expression of ERO1 was increased in the ischemic hind limb in vivo, as well as in ECs and macrophages exposed to hypoxia in vitro. The increase in ERO1 to ischemia/hypoxia was less in SKI mice. In WT ECs, both vascular cell adhesion molecule 1 (VCAM1) expression and bone marrow-derived macrophage adhesion to ECs were increased by hypoxia, and both were attenuated in SKI ECs. In WT ECs, ERO1 siRNA blocked hypoxia-induced VCAM1 expression and macrophage adhesion. In WT macrophages, hypoxia also stimulated both ERO1 and VEGF expression, and both were less in SKI macrophages. Compared with conditioned media of hypoxic SKI macrophages, conditioned media from WT macrophages had a greater effect on EC angiogenic behavior, and were blocked by VEGF neutralizing antibody. Taken together, under hypoxic conditions, SERCA 2 C674 and ERO1 enable increased VCAM1 expression and macrophage adhesion to ECs, as well as macrophage VEGF production that, in turn, promote angiogenesis. This study highlights the hitherto unrecognized interaction of two ER proteins, SERCA 2 C674 and ERO1, which mediate the EC and macrophage angiogenic response to ischemia/hypoxia. Copyright © 2014

  19. Calneuron 1 Increased Ca2+ in the Endoplasmic Reticulum and Aldosterone Production in Aldosterone-Producing Adenoma.

    PubMed

    Kobuke, Kazuhiro; Oki, Kenji; Gomez-Sanchez, Celso E; Gomez-Sanchez, Elise P; Ohno, Haruya; Itcho, Kiyotaka; Yoshii, Yoko; Yoneda, Masayasu; Hattori, Noboru

    2018-01-01

    Aldosterone production is initiated by angiotensin II stimulation and activation of intracellular Ca 2+ signaling. In aldosterone-producing adenoma (APA) cells, the activation of intracellular Ca 2+ signaling is independent of the renin-angiotensin-aldosterone systems. The purpose of our study was to clarify molecular mechanisms of aldosterone production related to Ca 2+ signaling. Transcriptome analysis revealed that the CALN1 gene encoding calneuron 1 had the strongest correlation with CYP11B2 (aldosterone synthase) among genes encoding Ca 2+ -binding proteins in APA. CALN1 modulation and synthetic or fluorescent compounds were used for functional studies in human adrenocortical carcinoma (HAC15) cells. CALN1 expression was 4.4-fold higher in APAs than nonfunctioning adrenocortical adenomas. CALN1 expression colocalized with CYP11B2 expression as investigated using immunohistochemistry in APA and zona glomerulosa of male rats fed by a low-salt diet. CALN1 expression was detected in the endoplasmic reticulum (ER) by using GFP-fused CALN1, CellLight ER-RFP, and the corresponding antibodies. CALN1 -overexpressing HAC15 cells showed increased Ca 2+ in the ER and cytosol fluorescence-based studies. Aldosterone production was potentiated in HAC15 cells by CALN1 expression, and dose-responsive inhibition with TMB-8 showed that CALN1-mediated Ca 2+ storage in ER involved sarcoendoplasmic reticulum calcium transport ATPase. The silencing of CALN1 decreased Ca 2+ in ER, and abrogated angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells. Increased CALN1 expression in APA was associated with elevated Ca 2+ storage in ER and aldosterone overproduction. Suppression of CALN1 expression prevented angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells, suggesting that CALN1 is a potential therapeutic target for excess aldosterone production. © 2017 American Heart Association, Inc.

  20. Endoplasmic reticulum stress involved in high-fat diet and palmitic acid-induced vascular damages and fenofibrate intervention

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lu, Yunxia, E-mail: wwwdluyx@sina.com; The Comprehensive Laboratory, Anhui Medical University, Hefei, Anhui 230032; Cheng, Jingjing

    Fenofibrate (FF) is widely used to lower blood lipids in clinical practice, but whether its protective effect on endothelium-dependent vasodilatation (EDV) in thoracic aorta is related with endoplasmic reticulum (ER) stress remains unknown. In this study, female Sprauge Dawley rats were divided into standard chow diets (SCD), high-fat diets (HFD) and HFD plus FF treatment group (HFD + FF) randomly. The rats of latter two groups were given HFD feeding for 5 months, then HFD + FF rats were treated with FF (30 mg/kg, once daily) via gavage for another 2 months. The pathological and tensional changes, protein expression of eNOS, and ER stress relatedmore » genes in thoracic aorta were measured. Then impacts of palmitic acid (PA) and FF on EDV of thoracic aorta from normal female SD rats were observed. Ultimately the expression of ER stress related genes were assessed in primary mouse aortic endothelial cells (MAEC) treated by fenofibric acid (FA) and PA. We found that FF treatment improved serum lipid levels and pathological changes in thoracic aorta, accompanied with decreased ER stress and increased phosphorylation of eNOS. FF pretreatment also improved EDV impaired by different concentrations of PA treatment. The dose- and time-dependent inhibition of cell proliferation by PA were inverted by FA pretreatment. Phosphorylation of eNOS and expression of ER stress related genes were all inverted by FA pretreatment in PA-treated MAEC. Our findings show that fenofibrate recovers damaged EDV by chronic HFD feeding and acute stimulation of PA, this effect is related with decreased ER stress and increased phosphorylation of eNOS. - Highlights: • Fenofibrate treatment improved pathological changes in thoracic aorta by chronic high-fat-diet feeding. • Fenofibrate pretreatment improved endothelium-dependent vasodilation impaired by different concentrations of palmitic acid. • The inhibition of proliferation in endothelial cells by palmitic acid were inverted by

  1. Inhibition of Nogo-B promotes cardiac hypertrophy via endoplasmic reticulum stress.

    PubMed

    Li, Junli; Wu, Wenchao; Xin, Yanguo; Zhao, Mingyue; Liu, Xiaojing

    2018-05-14

    Nogo-B is a key endoplasmic reticulum (ER) protein that regulates ER stress signaling. However, its role in cardiac hypertrophy remains poorly understood. ER stress is interrelated with autophagy in the process of cardiac hypertrophy. Therefore, we aimed to test the hypothesis that both ER stress and autophagy signaling mediate the function of Nogo-B in cardiac hypertrophy. Rat models of transverse aortic constriction (TAC), neonatal rat cardiomyocytes (NRCMs) stimulated with norepinephrine (Ne) and primary cardiac fibroblasts treated with transforming growth factor β1 (TGF-β1) were used in this study. The expression of Nogo-B and markers of ER stress were determined by quantitative RT-PCR, western blotting and immunofluorescence. Autophagy was measured by monitoring autophagic flux. Specific small interfering RNA (siRNA) of Nogo-B was transfected to investigate the role of Nogo-B in regulating cardiac hypertrophy. In TAC-induced hypertrophic heart tissues, Ne-treated hypertrophic cardiomyocytes and TGF-β1-stimulated cardiac fibroblasts, the expression of Nogo-B, and markers of ER stress were significantly elevated. Impairment of autophagic flux was observed in the activated cardiac fibroblasts. Down-regulation of Nogo-B by siRNA further exacerbated Ne-induced cardiomyocyte hypertrophy and TGF-β1-induced cardiac fibroblast activation. Gene silencing of Nogo-B promoted the activation of the ER stress pathway and the impairment of autophagic flux. Moreover, inhibition of Nogo-B activated the protein kinase RNA-like ER kinase (PERK)/activating transcriptional factor 4 (ATF4) and activating transcriptional factor 6 (ATF6) branches of ER stress pathways. These findings suggest that inhibition of Nogo-B promotes cardiomyocyte hypertrophy and cardiac fibroblast activation by activating the PERK/ATF4 signaling pathway and defects of autophagic flux. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  2. Secretagogues differentially activate endoplasmic reticulum stress responses in pancreatic acinar cells.

    PubMed

    Kubisch, Constanze H; Logsdon, Craig D

    2007-06-01

    Endoplasmic reticulum (ER) stress leads to the accumulation of misfolded proteins in the ER lumen and initiates the unfolded protein response (UPR). Components of the UPR are important in pancreatic development, and recent studies have indicated that the UPR is activated in the arginine model of acute pancreatitis. However, the effects of secretagogues on UPR components in the pancreas are unknown. The present study aimed to examine the effects of different types and concentrations of secretagogues on acinar cell function and specific components of the UPR. Rat pancreatic acini were stimulated with the CCK analogs CCK8 (10 pM-10 nM) or JMV-180 (10 nM-10 microM) or with bombesin (1-100 nM). Components of the UPR, including chaperone BiP expression, PKR-like ER kinase (PERK) phosphorylation, X box-binding protein 1 (XBP1) splicing, and CCAAT/enhancer binding protein homologous protein (CHOP) expression, were measured, as were effects on amylase secretion and intracellular trypsin activation. CCK8 generated a biphasic secretion dose-response curve, and high concentrations increased intracellular active trypsin levels. In contrast, JMV-180 and bombesin secretion dose-response curves were monophasic, and high concentrations did not increase intracellular trypsin activity. All three secretagogues increased BiP levels and XBP1 splicing. However, only supraphysiological levels of CCK8 associated with inhibited amylase secretion and trypsin activation stimulated PERK phosphorylation and expression of CHOP. The effects of CCK8 on UPR components were rapid, occurring within 5-20 min. In conclusion, ER stress response mechanisms appear to be involved in both pancreatic physiology and pathophysiology, and future efforts should be directed at understanding the roles of these mechanisms in the pancreas.

  3. NETWORKED 3B: a novel protein in the actin cytoskeleton-endoplasmic reticulum interaction.

    PubMed

    Wang, Pengwei; Hussey, Patrick J

    2017-03-01

    In plants movement of the endoplasmic reticulum (ER) is dependent on the actin cytoskeleton. However little is known about proteins that link the ER membrane and the actin cytoskeleton. Here we identified a novel protein, NETWORKED 3B (NET3B), which is associated with the ER and actin cytoskeleton in vivo. NET3B belongs to a superfamily of plant specific actin binding proteins, the NETWORKED family. NET3B associates with the actin cytoskeleton in vivo through an N-terminal NET actin binding (NAB) domain, which has been well-characterized in other members of the NET family. A three amino acid insertion, Val-Glu-Asp, in the NAB domain of NET3B appears to lower its ability to localize to the actin cytoskeleton compared with NET1A, the founding member of the NET family. The C-terminal domain of NET3B links the protein to the ER. Overexpression of NET3B enhanced the association between the ER and the actin cytoskeleton, and the extent of this association was dependent on the amount of NET3B available. Another effect of NET3B overexpression was a reduction in ER membrane diffusion. In conclusion, our results revealed that NET3B modulates ER and actin cytoskeleton interactions in higher plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  4. γ-Oryzanol protects pancreatic β-cells against endoplasmic reticulum stress in male mice.

    PubMed

    Kozuka, Chisayo; Sunagawa, Sumito; Ueda, Rei; Higa, Moritake; Tanaka, Hideaki; Shimizu-Okabe, Chigusa; Ishiuchi, Shogo; Takayama, Chitoshi; Matsushita, Masayuki; Tsutsui, Masato; Miyazaki, Jun-ichi; Oyadomari, Seiichi; Shimabukuro, Michio; Masuzaki, Hiroaki

    2015-04-01

    Endoplasmic reticulum (ER) stress is profoundly involved in dysfunction of β-cells under high-fat diet and hyperglycemia. Our recent study in mice showed that γ-oryzanol, a unique component of brown rice, acts as a chemical chaperone in the hypothalamus and improves feeding behavior and diet-induced dysmetabolism. However, the entire mechanism whereby γ-oryzanol improves glucose metabolism throughout the body still remains unclear. In this context, we tested whether γ-oryzanol reduces ER stress and improves function and survival of pancreatic β-cells using murine β-cell line MIN6. In MIN6 cells with augmented ER stress by tunicamycin, γ-oryzanol decreased exaggerated expression of ER stress-related genes and phosphorylation of eukaryotic initiation factor-2α, resulting in restoration of glucose-stimulated insulin secretion and prevention of apoptosis. In islets from high-fat diet-fed diabetic mice, oral administration of γ-oryzanol improved glucose-stimulated insulin secretion on following reduction of exaggerated ER stress and apoptosis. Furthermore, we examined the impact of γ-oryzanol on low-dose streptozotocin-induced diabetic mice, where exaggerated ER stress and resultant apoptosis in β-cells were observed. Also in this model, γ-oryzanol attenuated mRNA level of genes involved in ER stress and apoptotic signaling in islets, leading to amelioration of glucose dysmetabolism. Taken together, our findings demonstrate that γ-oryzanol directly ameliorates ER stress-induced β-cell dysfunction and subsequent apoptosis, highlighting usefulness of γ-oryzanol for the treatment of diabetes mellitus.

  5. Acid Sphingomyelinase Mediates Oxidized-LDL Induced Apoptosis in Macrophage via Endoplasmic Reticulum Stress

    PubMed Central

    Zhao, Min; Pan, Wei; Shi, Rui-zheng; Bai, Yong-ping; You, Bo-yang; Zhang, Kai; Fu, Qiong-mei; Schuchman, Edward H.

    2016-01-01

    Aim: Macrophage apoptosis is a vital event in advanced atherosclerosis, and oxidized low-density lipoprotein (ox-LDL) is a major contributor to this process. Acid sphingomyelinase (ASM) and ceramide are also involved in the induction of apoptosis, particularly in macrophages. Our current study focuses on ASM and investigates its role in ox-LDL-induced macrophage apoptosis. Methods: Human THP-1 and mouse peritoneal macrophages were cultured in vitro and treated with ox-LDL. ASM activity and ceramide levels were quantified using ultra performance liquid chromatography. Protein and mRNA levels were analyzed using Western blot analysis and quantitative realtime PCR, respectively. Cell apoptosis was determined using Hoechst staining and flow cytometry. Results: Ox-LDL-induced macrophage apoptosis was triggered by profound endoplasmic reticulum (ER) stress, leading to an upregulation of ASM activity and ceramide levels at an early stage. ASM was inhibited by siRNA or desipramine (DES), and/or ceramide was degraded by recombinant acid ceramidase (AC). These events attenuated the effect of ox-LDL on ER stress. In contrast, recombinant ASM upregulated ceramide and ER stress. ASM siRNA, DES, recombinant AC, and ER stress inhibitor 4-phenylbutyric acid were blocked by elevated levels of C/EBP homologous protein (CHOP); ox-LDL induced elevated levels of CHOP. These events attenuated macrophage apoptosis. Conclusion: These results indicate that ASM/ceramide signaling pathway is involved in ox-LDL-induced macrophage apoptosis via ER stress pathway. PMID:26923251

  6. Myricetin induces apoptosis via endoplasmic reticulum stress and DNA double-strand breaks in human ovarian cancer cells

    PubMed Central

    XU, YE; XIE, QI; WU, SHAOHUA; YI, DAN; YU, YANG; LIU, SHIBING; LI, SONGYAN; LI, ZHIXIN

    2016-01-01

    The mechanisms underlying myricetin-induced cancer cell apoptosis remain to be elucidated. Certain previous studies have shown that myricetin induces apoptosis through the mitochondrial pathway. Apoptosis, however, can also be induced by other classical pathways, including endoplasmic reticulum (ER) stress and DNA double-strand breaks (DSBs). The aim of the present study was to assess whether these two apoptotic pathways are involved in myricetin-induced cell death in SKOV3 ovarian cancer cells. The results revealed that treatment with myricetin inhibited viability of SKOV3 cells in a dose-dependent manner. Myricetin induced nuclear chromatin condensation and fragmentation, and also upregulated the protein levels of active caspase 3 in a time-dependent manner. In addition, myricetin upregulated ER stress-associated proteins, glucose-regulated protein-78 and C/EBP homologous protein in SKOV3 cells. Phosphorylation of H2AX, a marker of DNA DSBs, was revealed to be upregulated in myricetin-treated cells. The data indicated that myricetin induces DNA DSBs and ER stress, which leads to apoptosis in SKOV3 cells. PMID:26782830

  7. HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis

    PubMed Central

    Kennedy, Donna; Mnich, Katarzyna; Oommen, Deepu; Chakravarthy, Reka; Almeida-Souza, Leonardo; Krols, Michiel; Saveljeva, Svetlana; Doyle, Karen; Gupta, Sanjeev; Timmerman, Vincent; Janssens, Sophie; Gorman, Adrienne M; Samali, Afshin

    2017-01-01

    BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants. PMID:29048431

  8. HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis.

    PubMed

    Kennedy, Donna; Mnich, Katarzyna; Oommen, Deepu; Chakravarthy, Reka; Almeida-Souza, Leonardo; Krols, Michiel; Saveljeva, Svetlana; Doyle, Karen; Gupta, Sanjeev; Timmerman, Vincent; Janssens, Sophie; Gorman, Adrienne M; Samali, Afshin

    2017-08-31

    BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants.

  9. SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

    PubMed Central

    Nogueira, Cristina; Erlmann, Patrik; Villeneuve, Julien; Santos, António JM; Martínez-Alonso, Emma; Martínez-Menárguez, José Ángel; Malhotra, Vivek

    2014-01-01

    TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18. DOI: http://dx.doi.org/10.7554/eLife.02784.001 PMID:24842878

  10. Sec13 safeguards the integrity of the endoplasmic reticulum and organogenesis of the digestive system in zebrafish.

    PubMed

    Niu, Xubo; Gao, Chuan; Jan Lo, Li; Luo, Yue; Meng, Chunmei; Hong, Jian; Hong, Wanjin; Peng, Jinrong

    2012-07-15

    The Sec13-Sec31 heterotetramer serves as the outer coat in the COPII complex, which mediates protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Although it has been studied in depth in yeast and cultured cells, the role of COPII in organogenesis in a multicellular organism has not. We report here that a zebrafish sec13(sq198) mutant, which exhibits a phenotype of hypoplastic digestive organs, has a mutation in the sec13 gene. The mutant gene encodes a carboxyl-terminus-truncated Sec13 that loses its affinity to Sec31a, which leads to disintegration of the ER structure in various differentiated cells in sec13(sq198), including chondrocytes, intestinal epithelial cells and hepatocytes. Disruption of the ER structure activates an unfolded protein response that eventually causes the cells to undergo cell-cycle arrest and cell apoptosis, which arrest the growth of developing digestive organs in the mutant. Our data provide the first direct genetic evidence that COPII function is essential for the organogenesis of the digestive system. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol

    PubMed Central

    Infante, Rodney Elwood; Radhakrishnan, Arun

    2017-01-01

    Cells employ regulated transport mechanisms to ensure that their plasma membranes (PMs) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins (LDL) and synthesis. To date, all inhibitors of cholesterol transport block steps in lysosomes, limiting our understanding of post-lysosomal transport steps. Here, we establish the cholesterol-binding domain 4 of anthrolysin O (ALOD4) as a reversible inhibitor of cholesterol transport from PM to endoplasmic reticulum (ER). Using ALOD4, we: (1) deplete ER cholesterol without altering PM or overall cellular cholesterol levels; (2) demonstrate that LDL-derived cholesterol travels from lysosomes first to PM to meet cholesterol needs, and subsequently from PM to regulatory domains of ER to suppress activation of SREBPs, halting cholesterol uptake and synthesis; and (3) determine that continuous PM-to-ER cholesterol transport allows ER to constantly monitor PM cholesterol levels, and respond rapidly to small declines in cellular cholesterol by activating SREBPs, increasing cholesterol uptake and synthesis. DOI: http://dx.doi.org/10.7554/eLife.25466.001 PMID:28414269

  12. The α-helical structure of prodomains promotes translocation of intrinsically disordered neuropeptide hormones into the endoplasmic reticulum.

    PubMed

    Dirndorfer, Daniela; Seidel, Ralf P; Nimrod, Guy; Miesbauer, Margit; Ben-Tal, Nir; Engelhard, Martin; Zimmermann, Richard; Winklhofer, Konstanze F; Tatzelt, Jörg

    2013-05-17

    Different neuropeptide hormones, which are either too small to adopt a stable conformation or are predicted to be intrinsically disordered, are synthesized as larger precursors containing a prodomain in addition to an N-terminal signal peptide. We analyzed the biogenesis of three unstructured neuropeptide hormones and observed that translocation of these precursors into the lumen of the endoplasmic reticulum (ER) is critically dependent on the presence of the prodomain. The hormone domains could be deleted from the precursors without interfering with ER import and secretion, whereas constructs lacking the prodomain remained in the cytosol. Domain-swapping experiments revealed that the activity of the prodomains to promote productive ER import resides in their ability to adopt an α-helical structure. Removal of the prodomain from the precursor did not interfere with co-translational targeting of the nascent chain to the Sec61 translocon but with its subsequent productive translocation into the ER lumen. Our study reveals a novel function of prodomains to enable import of small or intrinsically disordered secretory proteins into the ER based on their ability to adopt an α-helical conformation.

  13. Houttuynia cordata Thunb fraction induces human leukemic Molt-4 cell apoptosis through the endoplasmic reticulum stress pathway.

    PubMed

    Prommaban, Adchara; Kodchakorn, Kanchanok; Kongtawelert, Prachya; Banjerdpongchai, Ratana

    2012-01-01

    Houttuynia cordata Thunb (HCT) is a native herb found in Southeast Asia which features various pharmacological activities against allergy, inflammation, viral and bacterial infection, and cancer. The aims of this study were to determine the cytotoxic effect of 6 fractions obtained from silica gel column chromatography of alcoholic HCT extract on human leukemic Molt-4 cells and demonstrate mechanisms of cell death. Six HCT fractions were cytotoxic to human lymphoblastic leukemic Molt-4 cells in a dose-dependent manner by MTT assay, fraction 4 exerting the greatest effects. Treatment with IC50 of HCT fraction 4 significantly induced Molt-4 apoptosis detected by annexinV-FITC/propidium iodide for externalization of phosphatidylserine to the outer layer of cell membrane. The mitochondrial transmembrane potential was reduced in HCT fraction 4-treated Molt-4 cells. Moreover, decreased expression of Bcl-xl and increased levels of Smac/Diablo, Bax and GRP78 proteins were noted on immunoblotting. In conclusion, HCT fraction 4 induces Molt-4 apoptosis cell through an endoplasmic reticulum stress pathway.

  14. Endoplasmic Reticulum Stress and Disrupted Neurogenesis in the Brain Are Associated with Cognitive Impairment and Depressive-Like Behavior after Spinal Cord Injury.

    PubMed

    Wu, Junfang; Zhao, Zaorui; Kumar, Alok; Lipinski, Marta M; Loane, David J; Stoica, Bogdan A; Faden, Alan I

    2016-11-01

    Clinical and experimental studies show that spinal cord injury (SCI) can cause cognitive impairment and depression that can significantly impact outcomes. Thus, identifying mechanisms responsible for these less well-examined, important SCI consequences may provide targets for more effective therapeutic intervention. To determine whether cognitive and depressive-like changes correlate with injury severity, we exposed mice to sham, mild, moderate, or severe SCI using the Infinite Horizon Spinal Cord Impactor and evaluated performance on a variety of neurobehavioral tests that are less dependent on locomotion. Cognitive impairment in Y-maze, novel objective recognition, and step-down fear conditioning tasks were increased in moderate- and severe-injury mice that also displayed depressive-like behavior as quantified in the sucrose preference, tail suspension, and forced swim tests. Bromo-deoxyuridine incorporation with immunohistochemistry revealed that SCI led to a long-term reduction in the number of newly-generated immature neurons in the hippocampal dentate gyrus, accompanied by evidence of greater neuronal endoplasmic reticulum (ER) stress. Stereological analysis demonstrated that moderate/severe SCI reduced neuronal survival and increased the number of activated microglia chronically in the cerebral cortex and hippocampus. The potent microglial activator cysteine-cysteine chemokine ligand 21 (CCL21) was elevated in the brain sites after SCI in association with increased microglial activation. These findings indicate that SCI causes chronic neuroinflammation that contributes to neuronal loss, impaired hippocampal neurogenesis and increased neuronal ER stress in important brain regions associated with cognitive decline and physiological depression. Accumulation of CCL21 in brain may subserve a pathophysiological role in cognitive changes and depression after SCI.

  15. Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells

    PubMed Central

    Montagnani Marelli, Marina; Marzagalli, Monica; Moretti, Roberta M.; Beretta, Giangiacomo; Casati, Lavinia; Comitato, Raffaella; Gravina, Giovanni L.; Festuccia, Claudio; Limonta, Patrizia

    2016-01-01

    Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma. PMID:27461002

  16. Astaxanthin attenuates glutamate-induced apoptosis via inhibition of calcium influx and endoplasmic reticulum stress.

    PubMed

    Lin, Xiaotong; Zhao, Yan; Li, Shanhe

    2017-07-05

    Astaxanthin (AST) is a carotenoid that has been shown to have neuroprotective effects. In this study, it was found that AST significantly inhibited glutamate-induced loss of cell viability and apoptosis. AST pretreatment attenuated glutamate-induced activation of caspase-3, reduction of anti-apoptotic protein Bcl-2, and increase of pro-apoptotic protein Bak. In addition, AST pretreatment suppressed the production of intracellular reactive oxygen species. AST treatment also prevented glutamate-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK), which has been shown to promote apoptotic events. Furthermore, AST treatment greatly reduced the elevation of intracellular calcium level induced by glutamate and inhibited the activity of calpain, a calcium-dependent protease that plays an important role in mediating apoptosis stimulated by calcium overload in cytoplasm. Both oxidative stress and calcium overload can lead to endoplasmic reticulum (ER) stress. C/EBP-homologous protein (CHOP) is a bZIP transcription factor that can be activated by ER stress and promotes apoptosis. Here we found that AST attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78). Overall, these results suggested that AST might protect cells against glutamate-induced apoptosis through maintaining redox balance and inhibiting glutamate-induced calcium influx and ER stress. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Docosahexaenoic acid inhibits monocrotaline-induced pulmonary hypertension via attenuating endoplasmic reticulum stress and inflammation.

    PubMed

    Chen, Rui; Zhong, Wei; Shao, Chen; Liu, Peijing; Wang, Cuiping; Wang, Zhongqun; Jiang, Meiping; Lu, Yi; Yan, Jinchuan

    2018-02-01

    Endoplasmic reticulum (ER) stress and inflammation contribute to pulmonary hypertension (PH) pathogenesis. Previously, we confirmed that docosahexaenoic acid (DHA) could improve hypoxia-induced PH. However, little is known about the link between DHA and monocrotaline (MCT)-induced PH. Our aims were, therefore, to evaluate the effects and molecular mechanisms of DHA on MCT-induced PH in rats. Rat PH was induced by MCT. Rats were treated with DHA daily in the prevention group (following MCT injection) and the reversal group (after MCT injection for 2 wk) by gavage. After 4 wk, mean pulmonary arterial pressure (mPAP), right ventricular (RV) hypertrophy index, and morphological and immunohistochemical analyses were evaluated. Rat pulmonary artery smooth muscle cells (PASMCs) were used to investigate the effects of DHA on cell proliferation stimulated by platelet-derived growth factor (PDGF)-BB. DHA decreased mPAP and attenuated pulmonary vascular remodeling and RV hypertrophy, which were associated with suppressed ER stress. DHA blocked the mitogenic effect of PDGF-BB on PASMCs and arrested the cell cycle via inhibiting nuclear factor of activated T cells-1 (NFATc1) expression and activation and regulating cell cycle-related proteins. Moreover, DHA ameliorated inflammation in lung and suppressed macrophage and T lymphocyte accumulation in lung and adventitia of resistance pulmonary arteries. These findings suggest that DHA could protect against MCT-induced PH by reducing ER stress, suppressing cell proliferation and inflammation.

  18. Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

    PubMed

    Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

    2003-07-30

    Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd.

  19. Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant.

    PubMed

    Wang, Liang; Liu, Lei; Qin, Lingsong; Luo, Qingming; Zhang, Zhihong

    2017-04-01

    Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum (ER) fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells. However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3- or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.

  20. Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins*

    PubMed Central

    Crowder, Justin J.; Geigges, Marco; Gibson, Ryan T.; Fults, Eric S.; Buchanan, Bryce W.; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G.; Rubenstein, Eric M.

    2015-01-01

    Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane. PMID:26055716