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Sample records for inhibits endoplasmic reticulum

  1. Paclitaxel inhibits selenoprotein S expression and attenuates endoplasmic reticulum stress.

    PubMed

    Qin, Hong-Shuang; Yu, Pei-Pei; Sun, Ying; Wang, Dan-Feng; Deng, Xiao-Fen; Bao, Yong-Li; Song, Jun; Sun, Lu-Guo; Song, Zhen-Bo; Li, Yu-Xin

    2016-06-01

    The primary effect of the endoplasmic reticulum (ER) stress response or unfolded protein response (UPR) is to reduce the load of unfolded protein and promote survival. However, prolonged and severe ER stress leads to tissue injury and serious diseases. Thus, it is important to identify drugs that can attenuate ER stress for the treatment of diseases. Natural products continue to provide lead compounds for drug discovery and front‑line pharmacotherapy for people worldwide. Previous studies have indicated that selenoprotein S (SelS) is a sensitive and ideal maker of ER stress. In the present study, a firefly luciferase reporter driven by the SelS gene promoter was used to screen for natural compounds capable of attenuating ER stress. From this, paclitaxel (PTX) was identified to efficiently inhibit the promoter activity of the SelS gene, and further results revealed that PTX significantly inhibited the tunicamycin‑induced upregulation of SelS at the mRNA and protein levels in HepG2 and HEK293T cells. In addition, PTX was able to efficiently inhibit the expression of the ER stress marker, glucose‑regulated protein 78, in ER stress, indicating that PTX may reverse ER stress. Taken together, these results suggest that PTX is able to inhibit SelS expression during ER stress and attenuate ER stress. PMID:27109260

  2. Paclitaxel inhibits selenoprotein S expression and attenuates endoplasmic reticulum stress

    PubMed Central

    QIN, HONG-SHUANG; YU, PEI-PEI; SUN, YING; WANG, DAN-FENG; DENG, XIAO-FEN; BAO, YONG-LI; SONG, JUN; SUN, LU-GUO; SONG, ZHEN-BO; LI, YU-XIN

    2016-01-01

    The primary effect of the endoplasmic reticulum (ER) stress response or unfolded protein response (UPR) is to reduce the load of unfolded protein and promote survival. However, prolonged and severe ER stress leads to tissue injury and serious diseases. Thus, it is important to identify drugs that can attenuate ER stress for the treatment of diseases. Natural products continue to provide lead compounds for drug discovery and front-line pharmacotherapy for people worldwide. Previous studies have indicated that selenoprotein S (SelS) is a sensitive and ideal maker of ER stress. In the present study, a firefly luciferase reporter driven by the SelS gene promoter was used to screen for natural compounds capable of attenuating ER stress. From this, paclitaxel (PTX) was identified to efficiently inhibit the promoter activity of the SelS gene, and further results revealed that PTX significantly inhibited the tunicamycin-induced upregulation of SelS at the mRNA and protein levels in HepG2 and HEK293T cells. In addition, PTX was able to efficiently inhibit the expression of the ER stress marker, glucose-regulated protein 78, in ER stress, indicating that PTX may reverse ER stress. Taken together, these results suggest that PTX is able to inhibit SelS expression during ER stress and attenuate ER stress. PMID:27109260

  3. Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress

    PubMed Central

    Yang, Rui; Teng, Xu; Li, Hui; Xue, Hong-Mei; Guo, Qi; Xiao, Lin; Wu, Yu-Ming

    2016-01-01

    In this study, the vitamin D3 plus nicotine (VDN) model of rats was used to prove that H2S alleviates vascular calcification (VC) and phenotype transformation of vascular smooth muscle cells (VSMC). Besides, H2S can also inhibit endoplasmic reticulum stress (ERS) of calcified aortic tissues. The effect of H2S on alleviating VC and phenotype transformation of VSMC can be blocked by TM, while PBA also alleviated VC and phenotype transformation of VSMC that was similar to the effect of H2S. These results suggest that H2S may alleviate rat aorta VC by inhibiting ERS, providing new target and perspective for prevention and treatment of VC. PMID:27022436

  4. Hydrogen Sulfide Improves Vascular Calcification in Rats by Inhibiting Endoplasmic Reticulum Stress.

    PubMed

    Yang, Rui; Teng, Xu; Li, Hui; Xue, Hong-Mei; Guo, Qi; Xiao, Lin; Wu, Yu-Ming

    2016-01-01

    In this study, the vitamin D3 plus nicotine (VDN) model of rats was used to prove that H2S alleviates vascular calcification (VC) and phenotype transformation of vascular smooth muscle cells (VSMC). Besides, H2S can also inhibit endoplasmic reticulum stress (ERS) of calcified aortic tissues. The effect of H2S on alleviating VC and phenotype transformation of VSMC can be blocked by TM, while PBA also alleviated VC and phenotype transformation of VSMC that was similar to the effect of H2S. These results suggest that H2S may alleviate rat aorta VC by inhibiting ERS, providing new target and perspective for prevention and treatment of VC. PMID:27022436

  5. Class A scavenger receptor activation inhibits endoplasmic reticulum stress-induced autophagy in macrophage.

    PubMed

    Huang, Hanpeng; Li, Xiaoyu; Zhuang, Yan; Li, Nan; Zhu, Xudong; Hu, Jin; Ben, Jingjing; Yang, Qing; Bai, Hui; Chen, Qi

    2014-05-01

    Macrophage death in advanced atherosclerosis promotes plaque necrosis and destabilization. Involvement of autophagy in bulk degradation of cellular components has been recognized recently as an important mechanism for cell survival under endoplasmic reticulum (ER) stress. We previously found that the engagement of class A scavenger receptor (SR-A) triggered JNK-dependent apoptosis in ER-stressed macrophages. However, pro-apoptotic mechanisms mediated by SR-A are not fully understood. Therefore, we sought to see if SR-A mediated apoptosis was associated with autophagy in macrophages. Here, we showed that fucoidan inhibited microtubule-associated protein light chain 3-phospholipid conjugates (LC3-II) formation as well as the number of autophagosomes under ER stress. The inhibition of LC3-II formation was paralleled by the activation of the mTOR pathway, and the inhibition of mTOR allowed LC3-II induction in macrophages treated with thapsigargin plus fucoidan. Furthermore, apoptosis induced by fucoidan was prevented under ER stress by the mTOR inhibitor. We propose that fucoidan, a SR-A agonist, may contribute to macrophage apoptosis during ER stress by inhibiting autophagy. PMID:25013404

  6. Autophagy inhibition facilitates erlotinib cytotoxicity in lung cancer cells through modulation of endoplasmic reticulum stress.

    PubMed

    Wang, Zhongliang; Du, Tingting; Dong, Xiaorong; Li, Zhenyu; Wu, Gang; Zhang, Ruiguang

    2016-06-01

    Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized the treatment for non-small cell lung cancer patients, but acquired resistance limit the efficiency of this treatment. As a homeostatic cellular recycling mechanism, autophagy has been proposed to participate in the EGFR-TKI resistance. However, the role of autophagy in lung cancer treatment and the underlying mechanisms have not been clarified. In this study, we found the sensitivity to erlotinib, a well-used EGFR-TKI, was correlated with basal autophagy level. Erlotinib was able to induce autophagy not only in TKI-sensitive, but also TKI-resistant cancer cells. Inhibition of autophagy significantly enhanced cytotoxicity of erlotinib in TKI-resistant cancer cells via modulation of endoplasmic reticulum (ER) stress induced apoptosis. In this process, CCAAT/enhancer binding protein homologous protein (CHOP) acted as an executioner. Downregulation of CHOP with siRNA blocked the autophagy inhibition and erlotinib co-treatment induced apoptosis and prevented cancer cells from this co-treatment-induced cell death. Our findings suggest that autophagy inhibition overcomes erlotinib resistance through modulation of ER stress mediated apoptosis. PMID:27035631

  7. Rapamycin Attenuates Mouse Liver Ischemia and Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress.

    PubMed

    Zhu, J; Hua, X; Li, D; Zhang, J; Xia, Q

    2015-01-01

    The roles of endoplasmic reticulum (ER) stress in liver ischemia and reperfusion injury (IRI) have been well recognized. However, the impact of rapamycin (Rapa), a broadly used immunosuppressive agent in human liver transplantation, on ER stress during IRI remains unclear. This study was designed to investigate the roles of Rapa in the regulation of ER stress in vivo and in vitro. In a mouse liver partial warm ischemia and reperfusion mode, we demonstrated that Rapa markedly protected livers from IRI, as evidenced by serum alanine aminotransferase (sALT) levels and liver histology. Then we also confirmed the protection of Rapa from thapsigargin (Tg)-induced cell death in primary hepatocytes. Both in vivo and in vitro experiments showed that the ER stress markers were markedly up-regulated by IRI and Tg treatment, whereas they were down-regulated by Rapa pretreatment, as monitored by Western blot at the protein levels and by quantitative reverse transcription polymerase chain reaction (RT-PCR) at the messenger RNA (mRNA) levels. In addition, it was also revealed that Rapa was able to remarkably inhibit the mammalian target of rapamycin (mTOR) pathway and enhance autophagy both in IR-stressed livers and Tg-treated primary hepatocytes. Thus, these results suggest that Rapa protects livers from IRI through inhibiting the ER stress pathway. PMID:26293028

  8. Inhibition of telomerase causes vulnerability to endoplasmic reticulum stress-induced neuronal cell death.

    PubMed

    Hosoi, Toru; Nakatsu, Kanako; Shimamoto, Akira; Tahara, Hidetoshi; Ozawa, Koichiro

    2016-08-26

    Endoplasmic reticulum (ER) stress is implicated in several diseases, such as cancer and neurodegenerative diseases. In the present study, we investigated the possible involvement of telomerase in ER stress-induced cell death. ER stress-induced cell death was ameliorated in telomerase reverse transcriptase (TERT) over-expressing MCF7 cells (MCF7-TERT cell). Telomerase specific inhibitor, BIBR1532, reversed the inhibitory effect of TERT on ER stress-induced cell death in MCF7-TERT cells. These findings suggest that BIBR1532 may specifically inhibit telomerase activity, thereby inducing cell death in ER stress-exposed cells. TERT was expressed in the SH-SY5Y neuroblastoma cell line. To analyze the possible involvement of telomerase in ER stress-induced neuronal cell death, we treated SH-SY5Y neuroblastoma cells with BIBR1532 and analyzed ER stress-induced cell death. We found that BIBR1532 significantly enhanced the ER stress-induced neuronal cell death. These findings suggest that inhibition of telomerase activity may enhance vulnerability to neuronal cell death caused by ER stress. PMID:27443785

  9. Protective Effects of Alisma orientale Extract against Hepatic Steatosis via Inhibition of Endoplasmic Reticulum Stress

    PubMed Central

    Jang, Min-Kyung; Han, Yu-Ran; Nam, Jeong Soo; Han, Chang Woo; Kim, Byung Joo; Jeong, Han-Sol; Ha, Ki-Tae; Jung, Myeong Ho

    2015-01-01

    Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion. PMID:26540043

  10. Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity.

    PubMed

    Yamauchi, Mika; Tsuruma, Kazuhiro; Imai, Shunsuke; Nakanishi, Tomohiro; Umigai, Naofumi; Shimazawa, Masamitsu; Hara, Hideaki

    2011-01-10

    Crocetin is a carotenoid that is the aglicone of crocin, which are found in saffron stigmas (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). In this study, we investigated the effects of crocetin on retinal damage. To examine whether crocetin affects stress pathways, we investigated intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, disruption of the mitochondrial membrane potential (ΔΨ(m)), and caspases activation. In vitro, we employed cultured retinal ganglion cells (RGC-5, a mouse ganglion cell-line transformed using E1A virus). Cell damage was induced by tunicamycin or hydrogen peroxide (H(2)O(2)) exposure. Crocetin at a concentration of 3μM showed the inhibitory effect of 50-60% against tunicamycin- and H(2)O(2)-induced cell death and inhibited increase in caspase-3 and -9 activity. Moreover, crocetin inhibited the enzymatic activity of caspase-9 in a cell-free system. In vivo, retinal damage in mice was induced by exposure to white light at 8000lx for 3h after dark adaptation. Photoreceptor damage was evaluated by measuring the outer nuclear layer thickness at 5days after light exposure and recording the electroretinogram (ERG). Retinal cell damage was also detected with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 48h after light exposure. Crocetin at 100mg/kg, p.o. significantly inhibited photoreceptor degeneration and retinal dysfunction and halved the expression of TUNEL-positive cells. These results indicate that crocetin has protective effects against retinal damage in vitro and in vivo, suggesting that the mechanism may inhibit increase in caspase-3 and -9 activities after retinal damage. PMID:20951131

  11. Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress.

    PubMed

    Li, Jian-Shuang; Wang, Wen-Jun; Sun, Yu; Zhang, Yu-Hao; Zheng, Ling

    2015-05-01

    Ursolic acid (UA) is a natural pentacyclic triterpenoid compound, which is enriched with many herbs and plants, such as apple, cranberry and olive. UA performs multiple biological activities including anti-oxidation, anti-inflammation, anti-cancer and hepatoprotection. However, the exact mechanism underlying the hepatoprotective activity of UA remains unclear. In this study, the effects of UA on the development of nonalcoholic fatty liver disease (NAFLD) were investigated. In vivo, UA treatment (0.14%, w/w) significantly decreased the liver weight, serum levels of ALT/AST and hepatic steatosis in db/db mice (a type 2 diabetic mouse model). In vitro, UA treatment (10-30 μg ml(-1)) significantly decreased palmitic acid induced intracellular lipid accumulation in L02 cells. Our results suggested that the beneficial effects of UA on NAFLD may be due to its ability to increase lipid β-oxidation and to inhibit the hepatic endoplasmic reticulum (ER) stress. Together, UA may be further considered as a natural compound for NAFLD treatment. PMID:25892149

  12. Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure

    PubMed Central

    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

    2013-01-01

    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. PMID:23479643

  13. Zebularine inhibits tumorigenesis and stemness of colorectal cancer via p53-dependent endoplasmic reticulum stress

    PubMed Central

    Yang, Pei-Ming; Lin, Yi-Ting; Shun, Chia-Tung; Lin, Shan-Hu; Wei, Tzu-Tang; Chuang, Shu-Hui; Wu, Ming-Shiang; Chen, Ching-Chow

    2013-01-01

    Aberrant DNA hypermethylation is frequently found in tumor cells and inhibition of DNA methylation is an effective anticancer strategy. In this study, the therapeutic effect of DNA methyltransferase (DNMT) inhibitor zebularine (Zeb) on colorectal cancer (CRC) was investigated. Zeb exhibited anticancer activity in cell cultures, tumor xenografts and mouse colitis-associated CRC model. It stabilizes p53 through ribosomal protein S7 (RPS7)/MDM2 pathways and DNA damage. Zeb-induced cell death was dependent on p53. Microarray analysis revealed that genes related to endoplasmic reticulum (ER) stress and unfolded protein response (UPR) were affected by Zeb. Zeb induced p53-dependent ER stress and autophagy. Pro-survival markers of ER stress/UPR (GRP78) and autophagy (p62) were increased in tumor tissues of CRC patients, AOM/DSS-induced CRC mice and HCT116-derived colonospheres. Zeb downregulates GRP78 and p62, and upregulates a pro-apoptotic CHOP. Our results reveal a novel mechanism for the anticancer activity of Zeb. PMID:24225777

  14. An initial phase of JNK activation inhibits cell death early in the endoplasmic reticulum stress response.

    PubMed

    Brown, Max; Strudwick, Natalie; Suwara, Monika; Sutcliffe, Louise K; Mihai, Adina D; Ali, Ahmed A; Watson, Jamie N; Schröder, Martin

    2016-06-15

    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). In mammalian cells, UPR signals generated by several ER-membrane-resident proteins, including the bifunctional protein kinase endoribonuclease IRE1α, control cell survival and the decision to execute apoptosis. Processing of XBP1 mRNA by the RNase domain of IRE1α promotes survival of ER stress, whereas activation of the mitogen-activated protein kinase JNK family by IRE1α late in the ER stress response promotes apoptosis. Here, we show that activation of JNK in the ER stress response precedes activation of XBP1. This activation of JNK is dependent on IRE1α and TRAF2 and coincides with JNK-dependent induction of expression of several antiapoptotic genes, including cIap1 (also known as Birc2), cIap2 (also known as Birc3), Xiap and Birc6 ER-stressed Jnk1(-/-) Jnk2(-/-) (Mapk8(-/-) Mapk9(-/-)) mouse embryonic fibroblasts (MEFs) display more pronounced mitochondrial permeability transition and increased caspase 3/7 activity compared to wild-type MEFs. Caspase 3/7 activity is also elevated in ER-stressed cIap1(-/-) cIap2(-/-) and Xiap(-/-) MEFs. These observations suggest that JNK-dependent transcriptional induction of several inhibitors of apoptosis contributes to inhibiting apoptosis early in the ER stress response. PMID:27122189

  15. Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

    PubMed

    Longo, Michele; Spinelli, Rosa; D'Esposito, Vittoria; Zatterale, Federica; Fiory, Francesca; Nigro, Cecilia; Raciti, Gregory A; Miele, Claudia; Formisano, Pietro; Beguinot, Francesco; Di Jeso, Bruno

    2016-06-01

    Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype. PMID:26940722

  16. Rapamycin attenuates visible light-induced injury in retinal photoreceptor cells via inhibiting endoplasmic reticulum stress.

    PubMed

    Li, Guang-Yu; Fan, Bin; Jiao, Ying-Ying

    2014-05-14

    An extended exposure of the retina to visible light may lead to photochemical damage in retinal photoreceptor cells. The exact mechanism of retinal light damage remains unknown, and an effective therapy is still unavailable. Here, we demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), markedly protected 661W photoreceptor cells from visible light exposure-induced damage at the nanomolar level. We also observed by transmission electron microscopy that light exposure led to severe endoplasmic reticulum (ER) stress in 661W cells as well as abnormal endomembranes and ER membranes. In addition, obvious upregulated ER stress markers were monitored by western blot at the protein level and by quantitative reverse transcription-polymerase chain reaction (RT-PCR) at the mRNA level. Interestingly, rapamycin pretreatment significantly suppressed light-induced ER stress and all three major branches of the unfolded protein response (UPR), including the RNA-dependent protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) pathways both at the protein and mRNA levels. Additionally, the inhibition of ER stress by rapamycin was further confirmed with a dithiothreitol (DTT; a classical ER stress inducer)-damaged 661W cell model. Meanwhile, our results also revealed that rapamycin was able to remarkably inhibit the activation of mTOR and its downstream factors eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), p-4EBP1, p70, p-p70, and phosphorylated ribosomal protein S6 kinase (p-S6K) in the light-injured 661W cells. Thus, these data indicate that visible light induces ER stress in 661W cells; whereas the mTOR inhibitor, rapamycin, effectively protects 661W cells from light injury through suppressing the ER stress pathway. PMID:24607296

  17. Melatonin inhibits autophagy and endoplasmic reticulum stress in mice with carbon tetrachloride-induced fibrosis.

    PubMed

    San-Miguel, Beatriz; Crespo, Irene; Sánchez, Diana I; González-Fernández, Bárbara; Ortiz de Urbina, Juan J; Tuñón, María J; González-Gallego, Javier

    2015-09-01

    This study aimed to investigate whether inhibition of autophagy and endoplasmic reticulum (ER stress) associates with the antifibrogenic effect of melatonin in mice treated with carbon tetrachloride (CCl4 ). Mice received CCl4 5 μL/g body wt i.p. twice a week for 4 wk or 6 wk. Melatonin was given at 5 or 10 mg/kg/day i.p, beginning 2 wk after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of α-smooth muscle actin (α-SMA)-positive cells. CCl4 induced an autophagic response measured as the presence of autophagic vesicles, protein 1 light chain 3 (LC3) staining, conversion of LC3-I to autophagosome-associated LC3-II, changes in expression of beclin-1, UV radiation resistance-associated gene (UVRAG), ubiquitin-like autophagy-related (Atg5), Atg12, Atg16L1, sequestosome 1 (p62/SQSTM1), and lysosome-associated membrane protein (LAMP)-2, and increased phosphorylation of the mammalian target of rapamycin (mTOR). There was an increase in the expression of the ER stress chaperones CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin-heavy-chain-binding protein (BiP/GRP78), and 94-kDa glucose-regulated protein (GRP94), and in the mRNA levels of pancreatic ER kinase (PERK), activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1 (IRE1), and spliced X-box-binding protein-1 (XBP1). Phospho-IRE1, ATF6, and phospho-PERK protein concentration also increased significantly. Immunohistochemical staining of α-SMA indicated an abrogation of hepatic stellate cells activation by melatonin. Furthermore, treatment with the indole resulted in significant inhibition of the autophagic flux and the unfolded protein response. Findings from this study give new insight into molecular pathways accounting for the protective effect of melatonin in fibrogenesis. PMID:25958928

  18. Oroxin A inhibits breast cancer cell growth by inducing robust endoplasmic reticulum stress and senescence.

    PubMed

    He, Jun; Du, Longsheng; Bao, Meimei; Zhang, Bin; Qian, Haixin; Zhou, Quansheng; Cao, Zhifei

    2016-03-01

    Breast cancer is a major cause of cancer death among women. Although various anticancer drugs have been used in clinics, drugs that are effective against advanced and metastatic breast cancer are still lacking and in great demand. In this study, we found that oroxin A, an active component isolated from the herb Oroxylum indicum (L.) Kurz, effectively inhibited the growth of human breast cancer cells MDA-MB-231 and MCF7 by inducing endoplasmic reticulum (ER) stress-mediated senescence. Oroxin A caused breast cancer cell cycle arrest at the G2/M stage, and reorganization of microtubules and actin cytoskeleton accompanied by a decrease in cellular mitosis. ER-specific probe ER-Tracker Red and confocal microscope imaging showed that ER-Tracker Red-positive cells increased in an oroxin A dosage-dependent manner. In addition, oroxin A increased cell population with high β-Gal activity and SAHF-positive staining; these data suggest that oroxin A induces breast cancer cell ER stress and senescence. Mechanistic studies showed that oroxin A led to a significant increase in intracellular reactive oxygen species levels, promoted expression of ER stress markers ATF4 and GRP78, and increased the phosphorylation of a key stress-response signaling protein p38, resulting in an ER stress-mediated senescence. Taken together, our data indicate that oroxin A exerts its antibreast cancer effects by inducing ER stress-mediated senescence, activating the key stress p38 signaling pathway, and increasing key ER stress genes ATF4 and GRP78 expression levels. PMID:26599214

  19. Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress

    PubMed Central

    LIN, PINGDONG; WENG, XIAPING; LIU, FAYUAN; MA, YUHUAN; CHEN, HOUHUANG; SHAO, XIANG; ZHENG, WENWEI; LIU, XIANXIANG; YE, HONGZHI; LI, XIHAI

    2015-01-01

    Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3

  20. Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition

    PubMed Central

    Mimura, Naoya; Hideshima, Teru; Shimomura, Toshiyasu; Suzuki, Rikio; Ohguchi, Hiroto; Rizq, Ola; Kikuchi, Shohei; Yoshida, Yasuhiro; Cottini, Francesca; Jakubikova, Jana; Cirstea, Diana; Gorgun, Gullu; Minami, Jiro; Tai, Yu-Tzu; Richardson, Paul G.; Utsugi, Teruhiro; Iwama, Atsushi; Anderson, Kenneth C.

    2014-01-01

    The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we therefore examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patients MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL-6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of CHOP, a fatal ER-stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. PMID:24934808

  1. Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

    PubMed

    Mimura, Naoya; Hideshima, Teru; Shimomura, Toshiyasu; Suzuki, Rikio; Ohguchi, Hiroto; Rizq, Ola; Kikuchi, Shohei; Yoshida, Yasuhiro; Cottini, Francesca; Jakubikova, Jana; Cirstea, Diana; Gorgun, Gullu; Minami, Jiro; Tai, Yu-Tzu; Richardson, Paul G; Utsugi, Teruhiro; Iwama, Atsushi; Anderson, Kenneth C

    2014-08-15

    The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. PMID:24934808

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

    PubMed Central

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

    2015-01-01

    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. PMID:26442866

  3. 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. PMID:26980729

  4. Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.

    PubMed

    Wang, Fan; Song, Wensi; Brancati, Giovanna; Segatori, Laura

    2011-12-16

    Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis. PMID:22006919

  5. 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

  6. Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT protein-dependent extraction of ceramides from the endoplasmic reticulum.

    PubMed

    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

    2012-07-13

    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

  7. Valproate attenuates diabetic nephropathy through inhibition of endoplasmic reticulum stress-induced apoptosis

    PubMed Central

    SUN, XIN-YI; QIN, HAN-JIAO; ZHANG, ZE; XU, YE; YANG, XIAO-CHUN; ZHAO, DONG-MING; LI, XIAO-NING; SUN, LIAN-KUN

    2016-01-01

    Previous studies have suggested that endoplasmic reticulum stress (ERS) is one of the mechanisms responsible for the pathogenesis of diabetic nephropathy (DN). Histone acetylation modification can regulate the transcription of genes and is involved in the regulation of ERS. Valproate (VPA), a nonselective histone deacetylase inhibitor, has been reported to have a protective role in kidney tissue injury, however, whether VPA can prevent DN remains to be elucidated. In the present study, it was found that VPA increases the expression of glucose-regulated protein (GRP78) and reduces the protein expression of C/EBP-homologous protein (CHOP), growth arrest and DNA-damage-inducible gene 153 and caspase-12 in a rat model of DN. VPA can reduce renal cell apoptosis and alleviate proteinuria and alterations in serum creatinine. VPA also upregulates the acetylation level of histone H4 in the promoter of GRP78 and downregulates the acetylation level of histone H4 in the promoter of CHOP. Collectively, the data indicate that VPA can relieve ERS and reduce renal cell apoptosis, and thus attenuate renal injury in a rat model of DN by regulating the acetylation level of histone H4 in ERS-associated protein promoters. PMID:26647757

  8. Inhibition of endoplasmic reticulum stress improves coronary artery function in the spontaneously hypertensive rats.

    PubMed

    Choi, Soo-Kyoung; Lim, Mihwa; Byeon, Seon-Hee; Lee, Young-Ho

    2016-01-01

    Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of cardiovascular complications. However, the role and mechanisms of ER stress in hypertension remain unclear. Thus, we hypothesized that enhanced ER stress contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). Sixteen-week old male SHRs and Wistar Kyoto Rats (WKYs) were used in this study. The SHRs were treated with ER stress inhibitor (Tauroursodeoxycholic acid; TUDCA, 100 mg/kg/day) for two weeks. There was a decrease in systolic blood pressure in SHR treated with TUDCA. The pressure-induced myogenic tone was significantly increased, whereas endothelium-dependent relaxation was significantly attenuated in SHR compared with WHY. Interestingly, treatment of ER stress inhibitor normalized myogenic responses and endothelium-dependent relaxation in SHR. These data were associated with an increase in expression or phosphorylation of ER stress markers (Bip, ATF6, CHOP, IRE1, XBP1, PERK, and eIF2α) in SHRs, which were reduced by TUDCA treatment. Furthermore, phosphorylation of MLC20 was increased in SHRs, which was reduced by the treatment of TUDCA. Therefore, our results suggest that ER stress could be a potential target for hypertension. PMID:27550383

  9. Inhibition of endoplasmic reticulum stress improves coronary artery function in the spontaneously hypertensive rats

    PubMed Central

    Choi, Soo-Kyoung; Lim, Mihwa; Byeon, Seon-Hee; Lee, Young-Ho

    2016-01-01

    Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of cardiovascular complications. However, the role and mechanisms of ER stress in hypertension remain unclear. Thus, we hypothesized that enhanced ER stress contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). Sixteen-week old male SHRs and Wistar Kyoto Rats (WKYs) were used in this study. The SHRs were treated with ER stress inhibitor (Tauroursodeoxycholic acid; TUDCA, 100 mg/kg/day) for two weeks. There was a decrease in systolic blood pressure in SHR treated with TUDCA. The pressure-induced myogenic tone was significantly increased, whereas endothelium-dependent relaxation was significantly attenuated in SHR compared with WHY. Interestingly, treatment of ER stress inhibitor normalized myogenic responses and endothelium-dependent relaxation in SHR. These data were associated with an increase in expression or phosphorylation of ER stress markers (Bip, ATF6, CHOP, IRE1, XBP1, PERK, and eIF2α) in SHRs, which were reduced by TUDCA treatment. Furthermore, phosphorylation of MLC20 was increased in SHRs, which was reduced by the treatment of TUDCA. Therefore, our results suggest that ER stress could be a potential target for hypertension. PMID:27550383

  10. Calcium binding chaperones of the endoplasmic reticulum.

    PubMed

    Coe, Helen; Michalak, Marek

    2009-01-01

    The endoplasmic reticulum is a major Ca(2+) store of the cell that impacts many cellular processes within the cell. The endoplasmic reticulum has roles in lipid and sterol synthesis, protein folding, post-translational modification and secretion and these functions are affected by intraluminal endoplasmic reticulum Ca(2+). In the endoplasmic reticulum there are several Ca(2+) buffering chaperones including calreticulin, Grp94, BiP and protein disulfide isomerase. Calreticulin is one of the major Ca(2+) binding/buffering chaperones in the endoplasmic reticulum. It has a critical role in Ca(2+) signalling in the endoplasmic reticulum lumen and this has significant impacts on many Ca(2+)-dependent pathways including control of transcription during embryonic development. In addition to Ca(2+) buffering, calreticulin plays important role in the correct folding and quality control of newly synthesized glycoproteins. PMID:20093733

  11. Heat shock inhibits. alpha. -amylase synthesis in barley aleurone without inhibiting the activity of endoplasmic reticulum marker enzymes

    SciTech Connect

    Sticher, L.; Biswas, A.K.; Bush, D.S.; Jones, R.L. )

    1990-02-01

    The effects of heat shock on the synthesis of {alpha}-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25{degree}C to 40{degree}C for 3 hours, inhibits the accumulation of {alpha}-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca{sup 2+}. When ER is isolated from heat-shocked aleurone layers, less newly synthesized {alpha}-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca{sup 2+} transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.

  12. Inhibition of endoplasmic reticulum stress and atherosclerosis by 2-aminopurine in apolipoprotein e-deficient mice.

    PubMed

    Zhou, Lichun; Yang, Dezhi; Wu, Dong Fang; Guo, Zhong Mao; Okoro, Emmanuel; Yang, Hong

    2013-01-01

    We previously reported that the apolipoprotein (apo) B48-carrying lipoproteins obtained from apoE knockout (apoE (-/-) ) mice, so called E(-)/B48 lipoproteins, transformed mouse macrophages into foam cells and enhanced the phosphorylation of eukaryotic translation initiation factor 2 α (eIF-2 α ). Furthermore, the eIF-2 α phosphorylation inhibitor, 2-aminopurine (2-AP), attenuated E(-)/B48 lipoprotein-induced foam cell formation. The present report studied the effect of 2-AP on atherosclerosis in apoE (-/-) mice. Our results showed that the level of food intake, bodyweight, plasma cholesterol, and triglycerides was comparable in apoE (-/-) mice treated with or without 2-AP. However, the mean size of atherosclerotic lesions in the aorta sinus as well as the surface area of the entire aorta of 2-AP-treated apoE (-/-) mice were reduced by about 55% and 39%, respectively, compared to samples from untreated control apoE (-/-) mice. In addition, the 2-AP-treated apoE (-/-) mice showed a significant decrease in glucose-regulated protein 78 (GRP78) and phosphorylated eIF-2 α in their aortic samples as compared to levels in untreated control apoE (-/-) mice. These observations suggest that endoplasmic reticulum stress is a causal mechanism for the development of atherosclerosis in apoE (-/-) mice and that therapeutic strategies can be developed for using eIF-2 α phosphorylation inhibitors, such as 2-AP, to prevent or treat atherosclerosis. PMID:23984090

  13. Endoplasmic Reticulum Stress and Cancer

    PubMed Central

    Yadav, Raj Kumar; Chae, Soo-Wan; Kim, Hyung-Ryong; Chae, Han Jung

    2014-01-01

    The endoplasmic reticulum (ER) is the principal organelle responsible for multiple cellular functions including protein folding and maturation and the maintenance of cellular homeostasis. ER stress is activated by a variety of factors and triggers the unfolded protein response (UPR), which restores homeostasis or activates cell death. Multiple studies have clarified the link between ER stress and cancer, and particularly the involvement of the UPR. The UPR seems to adjust the paradoxical microenvironment of cancer and, as such, is one of resistance mechanisms against cancer therapy. This review describes the activity of different UPRs involved in tumorigenesis and resistance to cancer therapy. PMID:25337575

  14. Endoplasmic reticulum stress and atherosclerosis

    PubMed Central

    Hotamisligil, Gökhan S

    2010-01-01

    Atherosclerosis and related cardiovascular diseases represent one of the greatest threats to human health worldwide. Despite important progress in prevention and treatment, these conditions still account for one third of all deaths annually. Often presented together with obesity, insulin resistance and type 2 diabetes, these chronic diseases are strongly influenced by pathways that lie at the interface of chronic inflammation and nutrient metabolism. Here I discuss recent advances in the study of endoplasmic reticulum stress as one mechanism that links immune response with nutrient sensing in the pathogenesis of atherosclerosis and its complications. PMID:20376052

  15. 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. PMID:23353715

  16. Advanced glycation end products inhibit testosterone secretion by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

    PubMed

    Zhao, Yun-Tao; Qi, Ya-Wei; Hu, Chuan-Yin; Chen, Shao-Hong; Liu, You

    2016-08-01

    Diabetes severely impairs male reproduction. The present study assessed the effects and mechanisms of action of advanced glycation end products (AGEs), which play an important role in the development of diabetes complications, on testosterone secretion by rat Leydig cells. Primary rat Leydig cells were cultured and treated with AGEs (25, 50, 100 and 200 µg/ml). Testosterone production induced by human chorionic gonadotropin (hCG) was determined by ELISA. The mRNA and protein expression levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), which are involved in testosterone biosynthesis, were measured by reverse transcription-quantitative PCR and western blot analyssi, respectively. Reactive oxygen species (ROS) production in Leydig cells was measured using the dichlorofluorescein diacetate (DCFH-DA) probe. The expression levels of endoplasmic reticulum stress-related proteins [C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78)] in the Leydig cells were measured by western blot analysis. We found that the AGEs markedly suppressed testosterone production by rat Leydig cells which was induced by hCG in a concentration-dependent manner compared with the control (P<0.01). The mRNA and protein expression levels of StAR, 3β-HSD and P450scc were downregulated by the AGEs in a dose-dependent manner compared with the control (P<0.01). The antioxidant agent, N-acetyl‑L‑cysteine (NAC), and the endoplasmic reticulum stress inhibitor, tauroursodeoxycholic acid (TUDCA), reversed the inhibitory effects of AGEs. In addition, the content of ROS in Leydig cells treated with AGEs increased significantly. The expression levels of CHOP and GRP78 were markedly upregulated by the AGEs in the Leydig cells. From these findings, it can be concluded that AGEs inhibit testosterone production by rat Leydig cells by inducing oxidative stress and

  17. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increase sensitivity of osteosarcoma Saos-2 cells to cannabinoid receptor agonist WIN55,212-2.

    PubMed

    Zhang, Guodong; Bi, Haiyong; Gao, Ji; Lu, Xing; Zheng, Yanping

    2016-07-01

    WIN55,212-2, a cannabinoid receptor agonist, can activate cannabinoid receptors, which has proven anti-tumour effects in several tumour types. Studies showed that WIN can inhibit tumour cell proliferation and induce apoptosis in diverse cancers. However, the role and mechanism of WIN in osteosarcoma are still unclear. In this study, we examined the effect of WIN55,212-2 on osteosarcoma cell line Saos-2 in terms of cell viability and apoptosis. Meanwhile, we further explored the role of endoplasmic reticulum stress and autophagy in apoptosis induced by WIN55,212-2. Our results showed that the cell proliferation of Saos-2 was inhibited by WIN55,212-2 in a dose-dependent and time-dependent manner. WIN55,212-2-induced Saos-2 apoptosis through mitochondrial apoptosis pathway. Meanwhile, WIN55,212-2 can induce endoplasmic reticulum stress and autophagy in Saos-2 cells. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increased apoptosis induced by WIN55,212-2 in Saos-2 cells. These findings indicated that WIN55,212-2 in combination with autophagic inhibitor or endoplasmic reticulum stress activator may shed new light on osteosarcoma treatment. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27309350

  18. HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

    PubMed Central

    Jennelle, Lucas; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Brichacek, Beda; Bukrinsky, Michael

    2014-01-01

    HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism. PMID:25170080

  19. MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis.

    PubMed

    Li, Shengben; Liu, Lin; Zhuang, Xiaohong; Yu, Yu; Liu, Xigang; Cui, Xia; Ji, Lijuan; Pan, Zhiqiang; Cao, Xiaofeng; Mo, Beixin; Zhang, Fuchun; Raikhel, Natasha; Jiang, Liwen; Chen, Xuemei

    2013-04-25

    Translation inhibition is a major but poorly understood mode of action of microRNAs (miRNAs) in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here, we show that the translation inhibition, but not the mRNA cleavage activity, of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild-type and the amp1 mutant for membrane-bound, but not total, polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER. PMID:23622241

  20. 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

  1. Tunicamycin-induced inhibition of protein secretion into culture medium of Arabidopsis T87 suspension cells through mRNA degradation on the endoplasmic reticulum.

    PubMed

    Iwata, Yuji; Hayashi, Noriko; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

    2016-06-01

    The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants. PMID:26923805

  2. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    SciTech Connect

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-07-15

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

  3. Inhibition of translocation of nascent apolipoprotein B across the endoplasmic reticulum membrane is associated with selective inhibition of the synthesis of apolipoprotein B.

    PubMed

    Pan, M; Liang, J s; Fisher, E A; Ginsberg, H N

    2000-09-01

    In HepG2 cells, inhibition of apolipoprotein B100 (apoB) translocation across the endoplasmic reticulum by an microsomal triglyceride transfer protein (MTP) inhibitor (CP-10447) in the presence of N-acetyl-leucinyl-norleucinal, a proteasomal inhibitor, results in accumulation of newly synthesized apoB in the translocation channel. Here we demonstrated that such accumulation led to a specific reduction of apoB synthesis. ApoB mRNA levels remained unchanged, but we observed reduced rates of elongation of nascent apoB in puromycin-synchronized cells pretreated with MTP inhibitor. This observation was consistent with a longer half-ribosome transit time for the synthesis of apoB in MTP-inhibited cells. Initiation of translation of apoB mRNA was not impaired by MTP inhibition. Overall, these findings suggest that translocation arrest of apoB in the endoplasmic reticulum channel can exert a selective and negative effect on the synthesis of apoB at the stage of elongation. PMID:10846173

  4. Inhibition of homocysteine-induced endoplasmic reticulum stress and endothelial cell damage by l-serine and glycine.

    PubMed

    Sim, Woo-Cheol; Han, Inhoi; Lee, Wonseok; Choi, You-Jin; Lee, Kang-Yo; Kim, Dong Gwang; Jung, Seung-Hwan; Oh, Seon-Hee; Lee, Byung-Hoon

    2016-08-01

    Hyperhomocysteinemia is an independent risk factor for several cardiovascular diseases. The use of vitamins to modulate homocysteine metabolism substantially lowers the risk by reducing plasma homocysteine levels. In this study, we evaluated the effects of l-serine and related amino acids on homocysteine-induced endoplasmic reticulum (ER) stress and endothelial cell damage using EA.hy926 human endothelial cells. Homocysteine treatment decreased cell viability and increased apoptosis, which were reversed by cotreatment with l-serine. l-Serine inhibited homocysteine-induced ER stress as verified by decreased glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression as well as X-box binding protein 1 (xbp1) mRNA splicing. The effects of l-serine on homocysteine-induced ER stress are not attributed to intracellular homocysteine metabolism, but instead to decreased homocysteine uptake. Glycine exerted effects on homocysteine-induced ER stress, apoptosis, and cell viability that were comparable to those of l-serine. Although glycine did not affect homocysteine uptake or export, coincubation of homocysteine with glycine for 24h reduced the intracellular concentration of homocysteine. Taken together, l-serine and glycine cause homocysteine-induced endothelial cell damage by reducing the level of intracellular homocysteine. l-Serine acts by competitively inhibiting homocysteine uptake in the cells. However, the mechanism(s) by which glycine lowers homocysteine levels are unclear. PMID:27064126

  5. A proprotein convertase-inhibiting serpin with an endoplasmic reticulum targeting signal from Branchiostoma lanceolatum, a close relative of vertebrates

    PubMed Central

    Bentele, Caterina; Krüger, Olaf; Tödtmann, Ulf; Oley, Mareke; Ragg, Hermann

    2006-01-01

    Lancelets are considered to take a key position in the evolution of lineages leading to vertebrates. Herein, a serpin from the lancelet Branchiostoma lanceolatum, Bl-Spn1, was identified that inhibits the PCs (proprotein convertases) PC1/3 and furin. The inhibitor forms SDS-stable complexes with either of its targets. Analysis of the inhibitor/furin reaction products by mass spectroscopy assigns the enzyme's cleavage position C-terminally to Met-Met-Lys-Arg↓ in the reactive site loop of Spn1, in concordance with the classical recognition/cleavage site of the principal vertebrate PCs. The inhibitor is equipped with a canonical ER (endoplasmic reticulum) retrieval signal, Lys-Asp-Glu-Leu (KDEL), marking the inhibitor as a guardian of the cellular secretory routes. Deletion of the ER retrieval signal results in the export of the inhibitor into the medium of transfected COS-7 cells, consistent with the assigned intracellular location. These results identify Bl-Spn1 as the first serpin that may inhibit PC1/3-like subtilases at their natural sites of action. Phylogenetic comparisons support a concept implying a general role for ER-residing serpins in the surveillance of subtilase-like enzymes along the constitutive and regulated secretory pathways of metazoans including a role in the defence of intruders that turn PCs to their propagation. PMID:16445382

  6. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells.

    PubMed

    Seo, Jeong-Ah; Kim, Boyun; Dhanasekaran, Danny N; Tsang, Benjamin K; Song, Yong Sang

    2016-02-01

    Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells. PMID:26607901

  7. YiQiFuMai Powder Injection Ameliorates Cerebral Ischemia by Inhibiting Endoplasmic Reticulum Stress-Mediated Neuronal Apoptosis

    PubMed Central

    Hu, Yang

    2016-01-01

    YiQiFuMai (YQFM) powder injection as a modern preparation derived from Sheng Mai San, a traditional Chinese medicine, has been widely used in the treatment of cardiovascular and cerebrovascular diseases. However, its neuroprotective effect and underlying mechanism in cerebral ischemia remain to be explored. The present study was designed to investigate the neuroprotective effect of YQFM on endoplasmic reticulum (ER) stress-mediated neuronal apoptosis in the permanent middle cerebral artery occlusion- (MCAO-) injured mice and the oxygen-glucose deprivation- (OGD-) induced pheochromocytoma (PC12) cells. The results showed that single administration of YQFM (1.342 g/kg, i.p.) could reduce the brain infarction and improve the neurological deficits and the cerebral blood flow (CBF) after MCAO for 24 h in mice. Moreover, incubation with YQFM (100, 200, and 400 μg/mL) could increase the cell viability, decrease the caspase-3 activity, and inhibit the cell apoptosis in OGD-induced PC12 cells for 12 h. In addition, YQFM treatment could significantly modulate cleaved caspase-3 and Bcl-2 expressions and inhibit the expressions of ER stress-related marker proteins and signaling pathways in vivo and in vitro. In conclusion, our findings provide the first evidence that YQFM ameliorates cerebral ischemic injury linked with modulating ER stress-related signaling pathways, which provided some new insights for its prevention and treatment of cerebral ischemia diseases. PMID:27087890

  8. Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress.

    PubMed

    Zhai, Bo; Hu, Fengli; Yan, Haijiang; Zhao, Dali; Jin, Xin; Fang, Taishi; Pan, Shangha; Sun, Xueying; Xu, Lishan

    2015-01-01

    Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC. PMID:26381511

  9. Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

    PubMed Central

    Zhai, Bo; Hu, Fengli; Yan, Haijiang; Zhao, Dali; Jin, Xin; Fang, Taishi; Pan, Shangha; Sun, Xueying; Xu, Lishan

    2015-01-01

    Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC. PMID:26381511

  10. 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. PMID:26616142

  11. Mdivi-1 Protects Epileptic Hippocampal Neurons from Apoptosis via Inhibiting Oxidative Stress and Endoplasmic Reticulum Stress in Vitro.

    PubMed

    Xie, Nanchang; Wang, Cui; Wu, Chuanjie; Cheng, Xuan; Gao, Yanlun; Zhang, Haifeng; Zhang, Yi; Lian, Yajun

    2016-06-01

    Mitochondrial division inhibitor 1 (mdivi-1), a selective inhibitor of the mitochondrial fission protein dynamin-related protein 1, has been proposed to have a neuroprotective effect on hippocampal neurons in animal models of epilepsy. However, the effect of mdivi-1 on epileptic neuronal death in vitro remains unknown. Therefore, we investigated the effect of mdivi-1 and the underlying mechanisms in the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE) in vitro. We found that mitochondrial fission was increased in the HNC model of AE and inhibition of mitochondrial fission by mdivi-1 significantly decreased neuronal apoptosis induced by AE. In addition, mdivi-1 pretreatment significantly attenuated oxidative stress induced by AE characterized by decrease of reactive oxygen species (ROS) production and malondialdehyde level and by increase of superoxide dismutase activity. Moreover, mdivi-1 pretreatment significantly decreased endoplasmic reticulum (ER) stress markers glucose-regulated protein 78, C/EBP homologous protein expression and caspase-3 activation. Altogether, our findings suggest that mdivi-1 protected against AE-induced hippocampal neuronal apoptosis in vitro via decreasing ROS-mediated oxidative stress and ER stress. PMID:26801176

  12. Endoplasmic reticulum stress inhibits collagen synthesis independent of collagen-modifying enzymes in different chondrocyte populations and dermal fibroblasts.

    PubMed

    Vonk, Lucienne A; Doulabi, Behrouz Zandieh; Huang, Chun-Ling; Helder, Marco N; Everts, Vincent; Bank, Ruud A

    2010-06-01

    Chondrocytes respond to glucose deprivation with a decreased collagen synthesis due to disruption of a proper functioning of the endoplasmic reticulum (ER): ER stress. Since the mechanisms involved in the decreased synthesis are unknown, we have investigated whether chaperones and collagen-modifying enzymes are affected by glucose deprivation. Chondrocytes obtained from nucleus pulposus, annulus fibrosus, articular cartilage, and meniscus and dermal fibroblasts were cultured under control conditions or exposed to the ER stress-inducing treatments of tunicamycin addition or glucose withdrawal. Both treatments resulted in an up-regulation of the gene expression of the ER stress markers in all cell types, but dermal fibroblasts showed a delayed response to glucose deprivation. Collagen gene expression was down-regulated, and less collagen protein was present in the cells under both ER stress-inducing conditions. The expression levels of the prolyl 4-hydroxylases were either not affected (P4ha3) or increased (P4ha1 and P4ha2), the levels of the lysyl hydroxylases decreased, and the N-propeptidase Adamts2 decreased. Both treatments induced apoptosis. Chondrocytes respond more quickly to glucose deprivation, but it appears that chondrocytes can cope better with tunicamycin-induced ER stress than fibroblasts. Although collagen synthesis was inhibited by the treatments, some collagen-modifying enzymes and chaperones were up-regulated, suggesting that there is no causal relation between them. PMID:20555395

  13. Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis.

    PubMed Central

    Doedens, J R; Giddings, T H; Kirkegaard, K

    1997-01-01

    Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER. PMID:9371562

  14. Inhibition of 6-hydroxydopamine-induced endoplasmic reticulum stress by l-carnosine in SH-SY5Y cells.

    PubMed

    Oh, Yun-Mi; Jang, Eun-Hee; Ko, Jeong-Hyeon; Kang, Ju-Hee; Park, Chang-Shin; Han, Seung Baik; Kim, Jun Sig; Kim, Kyung Hwan; Pie, Jae-Eun; Shin, Dong Wun

    2009-07-31

    Conditions that cause endoplasmic reticulum malfunction (ER stress) play a key role in the development of various human diseases including neurodegenerative diseases. Carnosine is an endogenous peptide, present in excitable tissues such as brain and skeletal muscle. Although there are reports suggesting that carnosine has a biological role independent of its antioxidant activity, there have been no reports of the effects of carnosine on the ER stress response. We investigated the effects of carnosine on 6-hydroxydopamine (6-OHDA)-induced cell death and ER stress in SH-SY5Y cells. After assessing control cell viability in serum-free conditions for 24h (100% viability), we found that 50 microM 6-OHDA reduced cell viability to 76.4% of control values, whereas addition of 10mM carnosine significantly reduced cell death to 96.1% viability in a dose-dependent manner. Consistent with its cytoprotective action, carnosine markedly inhibited subsequent ER stress responses, including phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and c-jun, expression of glucose regulatory protein 78 and C/EBP homologous protein, and mRNA splicing of X-box protein 1. The measurement of reactive oxygen species (ROS) generation by 6-OHDA showed that addition of 10mM carnosine slightly but obviously inhibits the 6-OHDA-induced ROS production. In conclusion, our results show that carnosine almost completely inhibits 6-OHDA-induced ER stress responses and cytotoxicity, and that slight antioxidant activity of carnosine against 6-OHDA is observed. Further in vivo studies are needed to investigate clinical uses for carnosine. PMID:19394406

  15. Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice

    PubMed Central

    Choi, Yean-Jung; Kang, Min-Kyung; Kim, Yun-Ho; Kang, Young-Hee

    2015-01-01

    Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases. PMID:26599511

  16. Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells.

    PubMed

    Urresti, Jorge; Ruiz-Meana, Marisol; Coccia, Elena; Arévalo, Juan Carlos; Castellano, José; Fernández-Sanz, Celia; Galenkamp, Koen M O; Planells-Ferrer, Laura; Moubarak, Rana S; Llecha-Cano, Núria; Reix, Stéphanie; García-Dorado, David; Barneda-Zahonero, Bruna; Comella, Joan X

    2016-01-15

    Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG. PMID:26582200

  17. An endoplasmic reticulum-specific cyclophilin.

    PubMed Central

    Hasel, K W; Glass, J R; Godbout, M; Sutcliffe, J G

    1991-01-01

    Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression. Images PMID:1710767

  18. β-Asarone Inhibits IRE1/XBP1 Endoplasmic Reticulum Stress Pathway in 6-OHDA-Induced Parkinsonian Rats.

    PubMed

    Ning, Baile; Deng, Minzhen; Zhang, Qinxin; Wang, Nanbu; Fang, Yongqi

    2016-08-01

    Parkinson's disease (PD) is a neurodegenerative disease, with genetics and environment contributing to the disease onset. The limited pathological cognize of the disease restrained the approaches to improve the clinical treatment. Recently, studies showed that endoplasmic reticulum (ER) stress played an important role in the pathogenesis of PD. There was a neuroprotective effect partly mediated by modulating ER stress. β-Asarone is the essential constituent of Acorus tatarinowii Schott volatile oil. Our team observed that β-asarone could improve the behavior of parkinsonian rats; increase the HVA, Dopacl, and 5-HIAA levels; and reduce α-synuclein levels. Here we assumed that the protective role of β-asarone on parkinsonian rats was mediated via ER stress pathway. To prove the hypothesis we investigated the mRNA levels of glucose regulated protein 78 (GRP78) and C/EBP homologous binding protein (CHOP) in 6-hydroxy dopamine (6-OHDA) induced parkinsonian rats after β-asarone treatment. Furthermore, the inositol-requiring enzyme 1/X-Box Binding Protein 1 (IRE1/XBP1) ER stress pathway was also studied. The results showed that β-asarone inhibited the mRNA levels of GRP78 and CHOP, accompanied with the delined expressions of phosphorylated IER1 (p-IRE1) and XBP1. We deduced that β-asarone might have a protective effect on the 6-OHDA induced parkinsonian rats via IRE1/XBP1 Pathway. Collectively, all data indicated that β-asarone might be a potential candidate of medicine for clinical therapy of PD. PMID:27097550

  19. IL-15 expression increased in response to treadmill running and inhibited endoplasmic reticulum stress in skeletal muscle in rats.

    PubMed

    Yang, Hong-Tao; Luo, Li-Jie; Chen, Wen-Jia; Zhao, Lei; Tang, Chao-Shu; Qi, Yong-Fen; Zhang, Jing

    2015-02-01

    Interleukin 15 (IL-15) has recently been proposed as a circulating myokine involved in glucose uptake and utilization in skeletal muscle. However, the role and mechanism of IL-15 in exercise improving insulin resistance (IR) is unclear. Here, we investigated the alteration in expression of IL-15 and IL-15 receptor α (IL-15Rα) in skeletal muscle during treadmill running in rats with IR induced by a high-fat diet (HFD) and elucidated the mechanism of the anti-IR effects of IL-15. At 20 weeks of HFD, rats showed severe IR, with increased levels of fasting blood sugar and plasma insulin, impaired glucose tolerance, and reduced glucose transport activity. IL-15 immunoreactivity and mRNA level in gastrocnemius muscle were decreased markedly as compared with controls. IL-15Rα protein and mRNA levels in both soleus and gastrocnemius muscle were significantly decreased, which might attenuate the signaling or secretion of IL-15 in muscle. Eight-week treadmill running completely ameliorated HFD-induced IR and reversed the downregulated level of IL-15 and IL-15Rα in skeletal muscle of HFD-fed rats. To investigate whether IL-15 exerts its anti-IR effects directly in muscle, we pre-incubated muscle strips with the endoplasmic reticulum stress (ERS) inducer dithiothreitol (DTT) or tunicamycin (Tm); IL-15 treatment markedly decreased the protein expression of the ERS markers 78-kDa glucose-regulated protein, 94-kDa glucose-regulated protein and C/EBP homologous protein and inhibited ERS induced by DTT or Tm. Therefore, treadmill running promoted skeletal IL-15 and IL-15Rα expression in HFD-induced IR in rats. The inhibitory effect of IL-15 on ERS may be involved in improved insulin sensitivity with exercise training. PMID:24647688

  20. Hydrogen Sulfide Inhibits Formaldehyde-Induced Endoplasmic Reticulum Stress in PC12 Cells by Upregulation of SIRT-1

    PubMed Central

    Zhang, Ping; Chen, Li-Xun; Wang, Li; Xie, Ming; Wang, Chun-Yan; Tang, Xiao-Qing

    2014-01-01

    Background Formaldehyde (FA), a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S), the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER) stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1), a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action. Objective We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis. Principal Findings We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor) significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1. Conclusion/Significance These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity. PMID:24587076

  1. Combined inhibition of Hsp90 and heme oxygenase-1 induces apoptosis and endoplasmic reticulum stress in melanoma.

    PubMed

    Barbagallo, Ignazio; Parenti, Rosalba; Zappalà, Agata; Vanella, Luca; Tibullo, Daniele; Pepe, Francesco; Onni, Toniangelo; Li Volti, Giovanni

    2015-10-01

    Heat shock proteins are ubiquitous molecular chaperones involved in post-translational folding, stability, activation and maturation of many proteins that are essential mediators of signal transduction and cell cycle progression. Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment since it may act as a key regulator of various oncogene products and cell-signaling molecules. Heme oxygenase-1 (HO-1; also known as Hsp32) is an inducible enzyme participating in heme degradation and involved in oxidative stress resistance. Recent studies indicate that HO-1 activation may play a role in tumor development and progression. In the present study we investigated the chemotherapic effects of combining an Hsp90 inhibitor (NMS E973) and an HO-1 inhibitor (SnMP) on A375 melanoma cells. NMS E973 treatment was able to reduce cell viability and induce endoplasmic reticulum (ER) stress (i.e. Ire1α, ERO1, PDI, BIP and CHOP). Interestingly, no significant effect was observed in reactive oxygen species (ROS) formation. Finally, NMS E973 treatment resulted in a significant HO-1 overexpression, which in turn serves as a possible chemoresistance molecular mechanism. Interestingly, the combination of NMS E973 and SnMP produced an increase of ROS and reduced cell viability compared to NMS E973 treatment alone. The inhibitors combination exhibited higher ER stress, apoptosis as evidenced by bifunctional apoptosis regulator (BFAR) mRNA expression and lower phosphorylation of Akt when compared to NMS E973 alone. In conclusion, these data suggest that HO-1 inhibition potentiates NMS E973 toxicity and may be exploited as a strategy for melanoma treatment. PMID:26493719

  2. 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

    OBJECT 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. METHODS 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. RESULTS 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. CONCLUSIONS Docosahexaenoic acid therefore warrants further investigation as a potential therapeutic agent for the prevention of chronic traumatic encephalopathy. PMID

  3. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis.

    PubMed

    Dixon, Scott J; Patel, Darpan N; Welsch, Matthew; Skouta, Rachid; Lee, Eric D; Hayano, Miki; Thomas, Ajit G; Gleason, Caroline E; Tatonetti, Nicholas P; Slusher, Barbara S; Stockwell, Brent R

    2014-01-01

    Exchange of extracellular cystine for intracellular glutamate by the antiporter system xc (-) is implicated in numerous pathologies. Pharmacological agents that inhibit system xc (-) activity with high potency have long been sought, but have remained elusive. In this study, we report that the small molecule erastin is a potent, selective inhibitor of system xc (-). RNA sequencing revealed that inhibition of cystine-glutamate exchange leads to activation of an ER stress response and upregulation of CHAC1, providing a pharmacodynamic marker for system xc (-) inhibition. We also found that the clinically approved anti-cancer drug sorafenib, but not other kinase inhibitors, inhibits system xc (-) function and can trigger ER stress and ferroptosis. In an analysis of hospital records and adverse event reports, we found that patients treated with sorafenib exhibited unique metabolic and phenotypic alterations compared to patients treated with other kinase-inhibiting drugs. Finally, using a genetic approach, we identified new genes dramatically upregulated in cells resistant to ferroptosis.DOI: http://dx.doi.org/10.7554/eLife.02523.001. PMID:24844246

  4. Pharmacological inhibition of cystine–glutamate exchange induces endoplasmic reticulum stress and ferroptosis

    PubMed Central

    Dixon, Scott J; Patel, Darpan N; Welsch, Matthew; Skouta, Rachid; Lee, Eric D; Hayano, Miki; Thomas, Ajit G; Gleason, Caroline E; Tatonetti, Nicholas P; Slusher, Barbara S; Stockwell, Brent R

    2014-01-01

    Exchange of extracellular cystine for intracellular glutamate by the antiporter system xc− is implicated in numerous pathologies. Pharmacological agents that inhibit system xc− activity with high potency have long been sought, but have remained elusive. In this study, we report that the small molecule erastin is a potent, selective inhibitor of system xc−. RNA sequencing revealed that inhibition of cystine–glutamate exchange leads to activation of an ER stress response and upregulation of CHAC1, providing a pharmacodynamic marker for system xc− inhibition. We also found that the clinically approved anti-cancer drug sorafenib, but not other kinase inhibitors, inhibits system xc− function and can trigger ER stress and ferroptosis. In an analysis of hospital records and adverse event reports, we found that patients treated with sorafenib exhibited unique metabolic and phenotypic alterations compared to patients treated with other kinase-inhibiting drugs. Finally, using a genetic approach, we identified new genes dramatically upregulated in cells resistant to ferroptosis. DOI: http://dx.doi.org/10.7554/eLife.02523.001 PMID:24844246

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

    PubMed

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

    2014-07-01

    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

  6. Endoplasmic reticulum aminopeptidases: biochemistry, physiology and pathology.

    PubMed

    Hattori, Akira; Tsujimoto, Masafumi

    2013-09-01

    The human endoplasmic reticulum aminopeptidase (ERAP) 1 and 2 proteins were initially identified as homologues of human placental leucine aminopeptidase/insulin-regulated aminopeptidase. They are categorized as a unique class of proteases based on their subcellular localization on the luminal side of the endoplasmic reticulum. ERAPs play an important role in the N-terminal processing of the antigenic precursors that are presented on the major histocompatibility complex (MHC) class I molecules. ERAPs are also implicated in the regulation of a wide variety of physiological phenomena and pathogenic conditions. In this review, the current knowledge on ERAPs is summarized. PMID:23946506

  7. Inhibition of cardiac oxidative and endoplasmic reticulum stress-mediated apoptosis by curcumin treatment contributes to protection against acute myocarditis.

    PubMed

    Mito, Sayaka; Thandavarayan, Rajarajan A; Ma, Meilei; Lakshmanan, Arunprasath; Suzuki, Kenji; Kodama, Makoto; Watanabe, Kenichi

    2011-10-01

    Curcumin is used anecdotally as an herb in traditional Indian and Chinese medicine. In the present study, the effects and possible mechanism of curcumin in experimental autoimmune myocarditis (EAM) rats were further investigated. They were divided randomly into a treatment and vehicle group, and orally administrated curcumin (50 mg/kg/day) and 1% gum arabic, respectively, for 3 weeks after myosin injection. The results showed that curcumin significantly suppressed the myocardial protein expression of inducible nitric oxide synthase (iNOS) and the catalytic subunit of nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase. In addition, curcumin significantly decreased myocardial endoplasmic reticulum (ER) stress signaling proteins and improved cardiac function. Furthermore, curcumin significantly decreased the key regulators or inducers of apoptosis. In summary, our results indicate that curcumin has the potential to protect EAM by modulating cardiac oxidative and ER stress-mediated apoptosis, and provides a novel therapeutic strategy for autoimmune myocarditis. PMID:21781008

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

    PubMed Central

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

    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. PMID:26980943

  9. 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. PMID:26980943

  10. 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

  11. Piperine, an alkaloid from black pepper, inhibits growth of human colon cancer cells via G1 arrest and apoptosis triggered by endoplasmic reticulum stress.

    PubMed

    Yaffe, Paul B; Power Coombs, Melanie R; Doucette, Carolyn D; Walsh, Mark; Hoskin, David W

    2015-10-01

    Piperine, a piperidine alkaloid present in black pepper, inhibits the growth of cancer cells, although the mechanism of action is not well understood. In this study, we show that piperine (75-150 µM) inhibited the growth of several colon cancer cell lines but had little effect on the growth of normal fibroblasts and epithelial cells. Piperine inhibited HT-29 colon carcinoma cell proliferation by causing G1 phase cell cycle arrest that was associated with decreased expression of cyclins D1 and D3 and their activating partner cyclin-dependent kinases 4 and 6, as well as reduced phosphorylation of the retinoblastoma protein and up-regulation of p21/WAF1 and p27/KIP1 expression. In addition, piperine caused hydroxyl radical production and apoptosis that was partially dependent on the production of reactive oxygen species. Piperine-treated HT-29 cells showed loss of mitochondrial membrane integrity and cleavage of poly (ADP-ribose) polymerase-1, as well as caspase activation and reduced apoptosis in the presence of the pan-caspase inhibitor zVAD-FMK. Increased expression of the endoplasmic reticulum stress-associated proteins inositol-requiring 1α protein, C/EBP homologous protein, and binding immunoglobulin protein, and activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, as well as decreased phosphorylation of Akt and reduced survivin expression were also observed in piperine-treated HT-29 cells. Furthermore, piperine inhibited colony formation by HT-29 cells, as well as the growth of HT-29 spheroids. Cell cycle arrest and endoplasmic reticulum stress-associated apoptosis following piperine treatment of HT-29 cells provides the first evidence that piperine may be useful in the treatment of colon cancer. PMID:24819444

  12. Reduction in Embryonic Malformations and Alleviation of Endoplasmic Reticulum Stress by Nitric Oxide Synthase Inhibition in Diabetic Embryopathy

    PubMed Central

    Eckert, Richard L.; Reece, E. Albert

    2012-01-01

    Maternal diabetes-induced neural tube defects (NTDs) are associated with increased programmed cell death (apoptosis) in the neuroepithelium, which is related to intracellular nitrosative stress. To alleviate nitrosative stress, diabetic pregnant mice were fed via gavage an inhibitor of nitric oxide (NO) synthase (NOS) 2, L-N6-(1-iminoethyl)-lysine (L-NIL; 80 mg/kg), once a day from embryonic (E) day 7.5 to 9.5 during early stages of neurulation. The treatment significantly reduced NTD rate in the embryos, compared with that in vehicle (normal saline)-treated diabetic group. In addition to alleviation of nitrosative stress, endoplasmic reticulum (ER) stress was also ameliorated, assessed by quantification of associated factors. Apoptosis was reduced, indicated by caspase 8 activation. These results show that nitrosative stress is important in diabetes-induced NTDs via exacerbating ER stress, leading to increased apoptosis. Oral treatment with NOS-2 inhibitor alleviates nitrosative and ER stress, decreases apoptosis, and reduces NTDs in the embryos, providing information for further interventional studies to reduce diabetes-associated birth defects. PMID:22534324

  13. NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein.

    PubMed

    Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

    2016-05-13

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. PMID:27084451

  14. PI3K-Akt-mTOR signal inhibition affects expression of genes related to endoplasmic reticulum stress.

    PubMed

    Song, Q; Han, C C; Xiong, X P; He, F; Gan, W; Wei, S H; Liu, H H; Li, L; Xu, H Y

    2016-01-01

    PI3K-Akt-mTOR signaling pathway is associated with endoplasmic reticulum (ER) stress. However, it is not clear how this signaling pathway affects the ER stress. The present study aimed to determine whether the PI3K-Akt-mTOR signaling pathway regulates tunicamycin (TM)-induced increases in mRNA levels of genes involved in the ER stress, to help elucidate the mechanism by which this pathway affects the ER stress in primary goose hepatocytes. Primary hepatocytes were isolated from geese and cultured in vitro. After 12 h in a serum-free medium, the hepatocytes were incubated for 24 h in a medium with either no addition (control) or with supplementation of TM or TM together with PI3K-Akt-mTOR signaling pathway inhibitors (LY294002, rapamycin, NVP-BEZ235). Thereafter, the expression levels of genes involved in the ER stress (BIP, EIF2a, ATF6, and XBP1) were assessed. The results indicated that the mRNA level of BIP was up-regulated in 0.2, 2, and 20 μM TM treatment group (P < 0.05), whereas the mRNA levels of EIF2a, ATF6, and XBP1 were up-regulated in the 2 μM TM treatment group (P < 0.05). However, the TM mediated induction of mRNA levels of genes involved in the ER stress (BIP, EIF2a, ATF6, and XBP1) was down-regulated after the treatment with PI3K-Akt-mTOR pathway inhibitors (LY294002, NVP-BEZ235, and rapamycin). Therefore, our results strongly suggest that the PI3K-Akt-mTOR signaling pathway might be involved in the down-regulation of the TM-induced ER stress in primary goose hepatocytes. PMID:27525855

  15. Protein Translocation across the Rough Endoplasmic Reticulum

    PubMed Central

    Mandon, Elisabet C.; Trueman, Steven F.; Gilmore, Reid

    2013-01-01

    The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration. PMID:23251026

  16. Equine Viperin Restricts Equine Infectious Anemia Virus Replication by Inhibiting the Production and/or Release of Viral Gag, Env, and Receptor via Distortion of the Endoplasmic Reticulum

    PubMed Central

    Tang, Yan-Dong; Na, Lei; Zhu, Chun-Hui; Shen, Nan; Yang, Fei; Fu, Xian-Qiu; Wang, Yu-Hong; Fu, Li-Hua; Wang, Jia-Yi; Lin, Yue-Zhi; Wang, Xue-Feng; Wang, Xiaojun

    2014-01-01

    ABSTRACT Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the α-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin α-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the α-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level. IMPORTANCE In previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms

  17. Phenylbutyrate prevents disruption of blood-spinal cord barrier by inhibiting endoplasmic reticulum stress after spinal cord injury

    PubMed Central

    Zhou, Yulong; Ye, Libing; Zheng, Binbin; Zhu, Sipin; Shi, Hongxue; Zhang, Hongyu; Wang, Zhouguang; Wei, Xiaojie; Chen, Daqing; Li, Xiaokun; Xu, Huazi; Xiao, Jian

    2016-01-01

    This study aims to investigate the role of endocytoplasmic reticulum (ER) stress induced by spinal cord injury (SCI) in blood-spinal cord barrier (BSCB) disruption and the effect of phenylbutyrate (PBA) on BSCB disruption after SCI. After a moderate contusion injury at the T9 level of spinal cord with a vascular clip, PBA was immediately administered into injured rat via intraperitoneal injection (100 mg/kg) and then further treated once a day for 2 weeks for behavior test. Spinal cord was collected at 1 day post-injury for evaluation of the effects of ER stress and PBA on BSCB disruption after SCI. PBA significantly attenuated BSCB permeability and degradation of tight junction molecules such as P120, β-catenin, Occludin and Claudin5 at 1 day after injury and improved functional recovery in the rat model of trauma. The BSCB protective effect of PBA is related to the inhibition of ER stress induced by SCI. In addition, PBA significantly inhibited the increase of ER stress markers and prevents loss of tight junction and adherens junction proteins in TG-treated human brain microvascular endothelial cells (HBMEC). Taken together, our data demonstrate that therapeutic strategies targeting ER stress may be suitable for the therapy of preserving BSCB integrity after SCI. PBA may be a new candidate as a therapeutic agent for protecting SCI by a compromised BSCB. PMID:27186310

  18. Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress.

    PubMed

    Masud Alam, Md; Kariya, Ryusho; Kawaguchi, Azusa; Matsuda, Kouki; Kudo, Eriko; Okada, Seiji

    2016-10-01

    Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy. PMID:27484211

  19. Dehydroeffusol inhibits gastric cancer cell growth and tumorigenicity by selectively inducing tumor-suppressive endoplasmic reticulum stress and a moderate apoptosis.

    PubMed

    Zhang, Bin; Han, Hongyan; Fu, Shilong; Yang, Ping; Gu, Zhenlun; Zhou, Quansheng; Cao, Zhifei

    2016-03-15

    Gastric cancer is ranked as the third leading cause of cancer-related death in the world. Although extensive efforts have been made in recent decades to treat gastric cancer with various anticancer drugs, effective anti-gastric cancer therapeutics to cure the disease are still lacking in the clinics. Therefore, potent novel anti-gastric cancer drugs are greatly needed. In this study, we explored a novel anti-gastric cancer agent from a medicinal herb named Juncus effusus and found that the active component dehydroeffusol (DHE), a small molecular phenanthrene, effectively inhibited gastric cancer cell proliferation and tumorigenesis by inducing tumor suppressive endoplasmic reticulum (ER) stress and by triggering moderate apoptosis. Mechanistic studies revealed that DHE selectively activated the intracellular tumor suppressive stress response by promoting the overexpression of the key ER stress marker DNA damage-inducible transcript 3 (DDIT3), through upregulation of activating transcription factor 4 (ATF4). Concurrently, DHE suppressed the expression of the cell survival and ER stress marker glucose regulated protein of molecular mass 78 (GRP78) via downregulation of the transcription factor ATF6. In addition, DHE markedly activated the stress response signaling pathway MEKK4-MKK3/6-p38-DDIT3, but significantly inhibited ERK signaling. Our data suggest that DHE inhibits gastric cancer cell growth and tumorigenicity through selectively inducing a robust tumor suppressive ER stress response and a moderate apoptosis response. Therefore, DHE may provide a novel drug candidate for further development of potential anti-gastric cancer therapeutics. PMID:26774454

  20. Inhibition of Endoplasmic Reticulum Stress is Involved in the Neuroprotective Effect of bFGF in the 6-OHDA-Induced Parkinson’s Disease Model

    PubMed Central

    Cai, Pingtao; Ye, Jingjing; Zhu, Jingjing; Liu, Dan; Chen, Daqing; Wei, Xiaojie; Johnson, Noah R.; Wang, Zhouguang; Zhang, Hongyu; Cao, Guodong; Xiao, Jian; Ye, Junming; Lin, Li

    2016-01-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder with complicated pathophysiologic mechanisms. Endoplasmic reticulum (ER) stress appears to play a critical role in the progression of PD. We demonstrated that basic fibroblast growth factor (bFGF), as a neurotropic factor, inhibited ER stress-induced neuronal cell apoptosis and that 6-hydroxydopamine (6-OHDA)-induced ER stress was involved in the progression of PD in rats. bFGF administration improved motor function recovery, increased tyrosine hydroxylase (TH)-positive neuron survival, and upregulated the levels of neurotransmitters in PD rats. The 6-OHDA-induced ER stress response proteins were inhibited by bFGF treatment. Meanwhile, bFGF also increased expression of TH. The administration of bFGF activated the downstream signals PI3K/Akt and Erk1/2 in vivo and in vitro. Inhibition of the PI3K/Akt and Erk1/2 pathways by specific inhibitors partially reduced the protective effect of bFGF. This study provides new insight towards bFGF translational drug development for PD involving the regulation of ER stress. PMID:27493838

  1. Inhibition of Endoplasmic Reticulum Stress is Involved in the Neuroprotective Effect of bFGF in the 6-OHDA-Induced Parkinson's Disease Model.

    PubMed

    Cai, Pingtao; Ye, Jingjing; Zhu, Jingjing; Liu, Dan; Chen, Daqing; Wei, Xiaojie; Johnson, Noah R; Wang, Zhouguang; Zhang, Hongyu; Cao, Guodong; Xiao, Jian; Ye, Junming; Lin, Li

    2016-08-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder with complicated pathophysiologic mechanisms. Endoplasmic reticulum (ER) stress appears to play a critical role in the progression of PD. We demonstrated that basic fibroblast growth factor (bFGF), as a neurotropic factor, inhibited ER stress-induced neuronal cell apoptosis and that 6-hydroxydopamine (6-OHDA)-induced ER stress was involved in the progression of PD in rats. bFGF administration improved motor function recovery, increased tyrosine hydroxylase (TH)-positive neuron survival, and upregulated the levels of neurotransmitters in PD rats. The 6-OHDA-induced ER stress response proteins were inhibited by bFGF treatment. Meanwhile, bFGF also increased expression of TH. The administration of bFGF activated the downstream signals PI3K/Akt and Erk1/2 in vivo and in vitro. Inhibition of the PI3K/Akt and Erk1/2 pathways by specific inhibitors partially reduced the protective effect of bFGF. This study provides new insight towards bFGF translational drug development for PD involving the regulation of ER stress. PMID:27493838

  2. Nonvesicular lipid transfer from the endoplasmic reticulum.

    PubMed

    Lev, Sima

    2012-01-01

    The transport of lipids from their synthesis site at the endoplasmic reticulum (ER) to different target membranes could be mediated by both vesicular and nonvesicular transport mechanisms. Nonvesicular lipid transport appears to be the major transport route of certain lipid species, and could be mediated by either spontaneous lipid transport or by lipid-transfer proteins (LTPs). Although nonvesicular lipid transport has been extensively studied for more than four decades, its underlying mechanism, advantage and regulation, have not been fully explored. In particular, the function of LTPs and their involvement in intracellular lipid movement remain largely controversial. In this article, we describe the pathways by which lipids are synthesized at the ER and delivered to different cellular membranes, and discuss the role of LTPs in lipid transport both in vitro and in intact cells. PMID:23028121

  3. 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

  4. Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics.

    PubMed

    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

    2012-01-01

    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

  5. Membrane Protein Insertion at the Endoplasmic Reticulum

    PubMed Central

    Shao, Sichen; Hegde, Ramanujan S.

    2014-01-01

    Integral membrane proteins of the cell surface and most intracellular compartments of eukaryotic cells are assembled at the endoplasmic reticulum. Two highly conserved and parallel pathways mediate membrane protein targeting to and insertion into this organelle. The classical cotranslational pathway, utilized by most membrane proteins, involves targeting by the signal recognition particle followed by insertion via the Sec61 translocon. A more specialized posttranslational pathway, employed by many tail-anchored membrane proteins, is composed of entirely different factors centered around a cytosolic ATPase termed TRC40 or Get3. Both of these pathways overcome the same biophysical challenges of ferrying hydrophobic cargo through an aqueous milieu, selectively delivering it to one among several intracellular membranes and asymmetrically integrating its transmembrane domain(s) into the lipid bilayer. Here, we review the conceptual and mechanistic themes underlying these core membrane protein insertion pathways, the complexities that challenge our understanding, and future directions to over-come these obstacles. PMID:21801011

  6. 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

  7. Structural organization of the endoplasmic reticulum.

    PubMed

    Voeltz, Gia K; Rolls, Melissa M; Rapoport, Tom A

    2002-10-01

    The endoplasmic reticulum (ER) is a continuous membrane system but consists of various domains that perform different functions. Structurally distinct domains of this organelle include the nuclear envelope (NE), the rough and smooth ER, and the regions that contact other organelles. The establishment of these domains and the targeting of proteins to them are understood to varying degrees. Despite its complexity, the ER is a dynamic structure. In mitosis it must be divided between daughter cells and domains must be re-established, and even in interphase it is constantly rearranged as tubules extend along the cytoskeleton. Throughout these rearrangements the ER maintains its basic structure. How this is accomplished remains mysterious, but some insight has been gained from in vitro systems. PMID:12370207

  8. Barriers to uniformity within the endoplasmic reticulum.

    PubMed

    Wong, Andrew K O; Chao, Jesse T; Loewen, Christopher J R

    2014-08-01

    Differentiating the endoplasmic reticulum (ER) into different physical domains may help the ER spatially regulate its many functions. For example, ER sheets are highly decorated with ribosomes for protein synthesis, whereas tubules usually correspond to smooth ER. Hence, ER morphology may play direct roles in functional diversification within the ER. The ER also makes direct physical contacts with other organelles, called ER junctions, enabling further functional diversification through input from external sources. In yeast, an ER diffusion barrier has now been discovered at the bud neck that compartmentalizes the ER into bud and mother diffusion domains by restricting the lateral diffusion of ER membrane proteins. Therefore, diffusion barriers also likely contribute to functional diversification within the ER by creating suites of molecular factors within ER diffusion domains. PMID:24732434

  9. [Endoplasmic reticulum stress response in osteogenesis].

    PubMed

    Saito, Atsushi; Imaizumi, Kazunori

    2013-11-01

    Various cellular conditions such as synthesis of abundant proteins, expressions of mutant proteins and oxidative stress lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen. This type of stress is called ER stress. The excessive ER stress causes cellular damages followed by apoptosis. When ER stress occurs, cells are activated ER stress response (unfolded protein response) to avoid cellular damages. Recently, it has been clear that ER stress response plays crucial roles not only in cell survival after ER stress but also in regulating various cellular functions and tissue formations. In particular, ER stress and ER stress response regulate protein quality control, secretory protein production, and smooth secretion of proteins in the cells such as osteoblasts which synthesize and secrete enormous matrix proteins. PMID:24162596

  10. Protein Secretion and the Endoplasmic Reticulum

    PubMed Central

    Benham, Adam M.

    2012-01-01

    In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is controlled in the ER is now quite sophisticated. However, there remain gaps in our knowledge, particularly when applying insight gained from model systems to the more complex situations found in vivo. This article describes recent advances in our understanding of the ER and its role in preparing proteins for secretion, with an emphasis on glycoprotein quality control and pathways of disulfide bond formation. PMID:22700933

  11. Endoplasmic reticulum stress in brain ischemia.

    PubMed

    Su, Yingchao; Li, Feng

    2016-08-01

    Endoplasmic reticulum (ER) stress is an intricate mechanism that mediates numerous responses during brain ischemia, thus being essential to determine the fate of neurons. In recent years, studies of the mechanisms of brain ischemic injury have centered on ER stress, glutamate excitotoxicity, dysfunction of mitochondria, inflammatory reactions, calcium overload and death receptor pathways. The role of ER stress is highly important. In addition to resulting in neuronal cell death through calcium toxicity and apoptotic pathways, ER stress also triggers a series of adaptive responses including unfolded protein response (UPR), autophagy, the expression of pro-survival proteins and the enhancement of ER self-repair ability, leading to less ischemic brain damage. This paper provides an overview of recent advances in understanding of the relations between ER stress and brain ischemia. PMID:26289799

  12. Endoplasmic Reticulum Stress and Ethanol Neurotoxicity.

    PubMed

    Yang, Fanmuyi; Luo, Jia

    2015-01-01

    Ethanol abuse affects virtually all organ systems and the central nervous system (CNS) is particularly vulnerable to excessive ethanol exposure. Ethanol exposure causes profound damages to both the adult and developing brain. Prenatal ethanol exposure induces fetal alcohol spectrum disorders (FASD) which is associated with mental retardation and other behavioral deficits. A number of potential mechanisms have been proposed for ethanol-induced brain damage; these include the promotion of neuroinflammation, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, and thiamine deficiency. The endoplasmic reticulum (ER) regulates posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress and induces unfolded protein response (UPR) which are mediated by three transmembrane ER signaling proteins: pancreatic endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). UPR is initiated to protect cells from overwhelming ER protein loading. However, sustained ER stress may result in cell death. ER stress has been implied in various CNS injuries, including brain ischemia, traumatic brain injury, and aging-associated neurodegeneration, such as Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). However, effects of ethanol on ER stress in the CNS receive less attention. In this review, we discuss recent progress in the study of ER stress in ethanol-induced neurotoxicity. We also examine the potential mechanisms underlying ethanol-mediated ER stress and the interaction among ER stress, oxidative stress and autophagy in the context of ethanol neurotoxicity. PMID:26473940

  13. Proplatelet formation in megakaryocytes is associated with endoplasmic reticulum stress.

    PubMed

    Morishima, Nobuhiro; Nakanishi, Keiko

    2016-07-01

    Although previous studies suggest that proplatelet formation in megakaryocytes involves caspase-3, the mechanism underlying the activation of caspase-3 is unknown. Here, we analyzed caspase activation in a human megakaryoblastic cell line, MEG-01, which forms proplatelets spontaneously. Specific activation of caspase-3 and caspase-4 was found in proplatelets. Consistent with previous observations of caspase-4 autoactivation in response to endoplasmic reticulum (ER) stress, several ER stress marker proteins were expressed during proplatelet formation. A pharmacological ER stressor enhanced platelet production via proplatelet formation, whereas inhibition of caspase-4 caused suppression. These results suggest that ER stress is a mechanism underlying the maturation of megakaryocytes. PMID:27296088

  14. Unfolded protein stress in the endoplasmic reticulum and mitochondria: a role in neurodegeneration

    PubMed Central

    Bernales, Sebastián; Soto, Marisol Morales; McCullagh, Emma

    2012-01-01

    Protein-folding occurs in several intracellular locations including the endoplasmic reticulum and mitochondria. In normal conditions there is a balance between the levels of unfolded proteins and protein folding machinery. Disruption of homeostasis and an accumulation of unfolded proteins trigger stress responses, or unfolded protein responses (UPR), in these organelles. These pathways signal to increase the folding capacity, inhibit protein import or expression, increase protein degradation, and potentially trigger cell death. Many aging-related neurodegenerative diseases involve the accumulation of misfolded proteins in both the endoplasmic reticulum and mitochondria. The exact participation of the UPRs in the onset of neurodegeneration is unclear, but there is significant evidence for the alteration of these pathways in the endoplasmic reticulum and mitochondria. Here we will discuss the involvement of endoplasmic reticulum and mitochondrial stress and the possible contributions of the UPR in these organelles to the development of two neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer's disease (AD). PMID:22539924

  15. Deficiency of Rac1 Blocks NADPH Oxidase Activation, Inhibits Endoplasmic Reticulum Stress, and Reduces Myocardial Remodeling in a Mouse Model of Type 1 Diabetes

    PubMed Central

    Li, Jianmin; Zhu, Huaqing; Shen, E; Wan, Li; Arnold, J. Malcolm O.; Peng, Tianqing

    2010-01-01

    OBJECTIVE Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose. RESULTS In diabetic hearts, NADPH oxidase activation, its subunits' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose–induced ER stress was inhibited by blocking Rac1 or NADPH oxidase. CONCLUSIONS Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy. PMID:20522592

  16. Inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar UV-4.

    PubMed

    Warfield, Kelly L; Plummer, Emily M; Sayce, Andrew C; Alonzi, Dominic S; Tang, William; Tyrrell, Beatrice E; Hill, Michelle L; Caputo, Alessandro T; Killingbeck, Sarah S; Beatty, P Robert; Harris, Eva; Iwaki, Ren; Kinami, Kyoko; Ide, Daisuke; Kiappes, J L; Kato, Atsushi; Buck, Michael D; King, Kevin; Eddy, William; Khaliq, Mansoora; Sampath, Aruna; Treston, Anthony M; Dwek, Raymond A; Enterlein, Sven G; Miller, Joanna L; Zitzmann, Nicole; Ramstedt, Urban; Shresta, Sujan

    2016-05-01

    The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10-20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection. UV-4B also reduced infectious virus production in in vitro antiviral activity assays against all four DENV serotypes, including clinical isolates. A set of purified enzyme, in vitro, and in vivo studies demonstrated that inhibition of endoplasmic reticulum (ER) α-glucosidases and not the glycosphingolipid pathway appears to be responsible for the antiviral activity of UV-4B against DENV. Along with a comprehensive safety package, these and previously published data provided support for an Investigational New Drug (IND) filing and Phases 1 and 2 clinical trials for UV-4B with an indication of acute dengue disease. PMID:26946111

  17. Protective effect of S-allyl-L-cysteine against endoplasmic reticulum stress-induced neuronal death is mediated by inhibition of calpain.

    PubMed

    Imai, Toru; Kosuge, Yasuhiro; Endo-Umeda, Kaori; Miyagishi, Hiroko; Ishige, Kumiko; Makishima, Makoto; Ito, Yoshihisa

    2014-02-01

    Endoplasmic reticulum (ER) stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca(2+) and leads to activation of calpain, a Ca(2+)-dependent cysteine protease. We have shown previously that S-allyl-L-cysteine (SAC) in aged garlic extracts significantly protects cultured rat hippocampal neurons (HPNs) against ER stress-induced neurotoxicity. The neuroprotective effect of SAC was compared with those of the related antioxidant compounds, L-cysteine (CYS) and N-acetylcysteine (NAC), on calpain activity in HPNs and also in vitro. SAC, but not CYS or NAC, reversibly restored the survival of HPNs and increased the degradation of α-spectrin, a substrate for calpain, induced by tunicamycin, a typical ER stress inducer. Activities of μ- and m-calpains in vitro were also concentration dependently suppressed by SAC, but not by CYS or NAC. At submaximal concentration, although ALLN (5 pM), which blocks the active site of calpain, and calpastatin (100 pM), an endogenous calpain-inhibitor protein, additively inhibited μ-calpain activity in vitro in combination with SAC, the effect of PD150606 (25 μM), which prevents interaction of Ca(2+) with the Ca(2+)-binding site of calpain, was unaffected by SAC. In contrast, SAC (1 mM) significantly reversed the effect of PD150606 at a concentration that elicited supramaximal inhibition (100 μM), but did not affect ALLN (1 nM)- and calpastatin (100 nM)-induced inhibition of μ-calpain activity. These results suggest that the protective effects of SAC against ER stress-induced neuronal cell death are not attributable to antioxidant activity, but to suppression of calpain through interaction with its Ca(2+)-binding site. PMID:24287800

  18. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

  19. Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition.

    PubMed

    Ram, Babul Moni; Ramakrishna, Gayatri

    2014-11-01

    Cyclosporine A (CsA), a widely used immunosuppressant shows cytotoxic effects by either inducing apoptosis or redirecting the cell towards non-apoptotic cell death. However, there still remains a lacuna in understanding the mechanism of CsA induced non-apoptotic cell death. In the present study we investigated calcineurin dependent or independent cytotoxic effects of CsA, a calcineurin inhibitor, in cervical cancerous SiHa cells. Decreased cell viability and massive cytoplasmic vacuolations were observed in CsA treated SiHa cells, having increased calcineurin activity. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR), accompanied by a decrease in cyclophilin B (ER resident PPIase), preceded the formation of the vacuoles. These vacuoles stained positive for many ER resident markers confirming their ER origin; but the absence of autophagosomal marker, LC3II, ruled out autophagy. Extensively vacuolated cells eventually undergo cell death which lacked the typical apoptotic features, but showed significant decrease in AIP (ALG2 interacting protein) as seen in paraptosis. ER-vacuolation was prevented by cycloheximide and salubrinal thereby indicating requirement of active protein synthesis. Inhibiting calcineurin activity by either Tacrolimus (FK506) or by knockdown of calcineurin B subunit did not result in either ER-stress or cellular vacuolation. However, knockdown of cyclophilin B by siRNA resulted in increased expression of Bip and IRE1α, together with cytoplasmic vacuolation. In conclusion, we report that persistent ER stress due to cyclophilin B inhibition in CsA treated cervical cancer cells caused cellular vacuolation which culminated in a non-apoptotic cell death response similar to paraptosis. Additionally, the paraptotic effects of CsA are independent of calcineurin inhibition. PMID:25003316

  20. Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis

    PubMed Central

    Chen, Liye; Ridley, Anna; Hammitzsch, Ariane; Al-Mossawi, Mohammad Hussein; Bunting, Helen; Georgiadis, Dimitris; Chan, Antoni; Kollnberger, Simon; Bowness, Paul

    2016-01-01

    Objective Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. Methods Flow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3ε-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition. Results The AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3ε-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion. Conclusions ERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment. PMID:26130142

  1. [The progress of study about endoplasmic reticulum stress in glaucoma].

    PubMed

    Hu, J; Jiang, B

    2016-03-01

    In eukaryotic cells, the most secreted proteins and membrane proteins are compounded, modified and folded into the correct structure in the endoplasmic reticulum. Only correctly folded proteins can be transported to the golgi apparatus for further processing. If the endoplasmic reticulum is insufficient to deal with the accumulation of unfolded or misfolded proteins, balance will be broken, and endoplasmic reticulum stress (ERS) will be started. To eliminate the unfolded proteins, cells will activate unfolded protein response (UPR) immediately for self-protection. If the induced ERS is strong or persistent, the UPR could not maintain the balance of homeostasis in endoplasmic reticulum. Then the ERS will lead to C/EBP homologous protein activation and initiate cell apoptosis. The continuous ERS may participate in the occurrence and development of many diseases, such as neurodegenerative diseases and type 2 diabetes. In this article, the research progress of ERS and its relationship with glaucoma is reviewed. PMID:26979122

  2. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  3. Endoplasmic Reticulum Stress, Genome Damage, and Cancer

    PubMed Central

    Dicks, Naomi; Gutierrez, Karina; Michalak, Marek; Bordignon, Vilceu; Agellon, Luis B.

    2015-01-01

    Endoplasmic reticulum (ER) stress has been linked to many diseases, including cancer. A large body of work has focused on the activation of the ER stress response in cancer cells to facilitate their survival and tumor growth; however, there are some studies suggesting that the ER stress response can also mitigate cancer progression. Despite these contradictions, it is clear that the ER stress response is closely associated with cancer biology. The ER stress response classically encompasses activation of three separate pathways, which are collectively categorized the unfolded protein response (UPR). The UPR has been extensively studied in various cancers and appears to confer a selective advantage to tumor cells to facilitate their enhanced growth and resistance to anti-cancer agents. It has also been shown that ER stress induces chromatin changes, which can also facilitate cell survival. Chromatin remodeling has been linked with many cancers through repression of tumor suppressor and apoptosis genes. Interplay between the classic UPR and genome damage repair mechanisms may have important implications in the transformation process of normal cells into cancer cells. PMID:25692096

  4. Endoplasmic Reticulum Stress in Endometrial Cancer

    PubMed Central

    Ulianich, Luca; Insabato, Luigi

    2014-01-01

    Endometrial cancer (EC) is a common gynecologic malignancy often diagnosed at early stage. In spite of a huge advance in our understanding of EC biology, therapeutic modalities do not have significantly changed over the past 40 years. A restricted number of genes have been reported to be mutated in EC, mediating cell proliferation and invasiveness. However, besides these alterations, few other groups and ourselves recently identified the activation of the unfolded protein response (UPR) and GRP78 increase following endoplasmic reticulum (ER) stress as mechanisms favoring growth and invasion of EC cells. Here, a concise update on currently available data in the field is presented, analyzing the crosstalk between the UPR and the main signaling pathways regulating EC cell proliferation and survival. It is evident that this is a rapidly expanding and promising issue. However, more data are very likely to yield a better understanding on the mechanisms through which EC cells can survive the low oxygen and glucose tumor microenvironment. In this perspective, the UPR and, particularly, GRP78 might constitute a novel target for the treatment of EC in combination with traditional adjuvant therapy. PMID:25593927

  5. Endoplasmic reticulum dysfunction in Alzheimer's disease.

    PubMed

    Li, Jie-Qiong; Yu, Jin-Tai; Jiang, Teng; Tan, Lan

    2015-02-01

    The endoplasmic reticulum (ER) serves many crucial cellular functions. However, when misfolded or unfolded proteins accumulated in the ER, the stress of ER will be induced. Meanwhile, the intracellular signaling network, which is called unfolded protein response, will also be activated to cope with. Those unfolded proteins can be recognized by three kinds of stress sensors which are IRE1, PERK, and ATF6. Based on lots of medical reports, ER stress in postmortem brains from Alzheimer's disease (AD) patients, animals, and vitro models have indicated that ER dysfunction might work as an important part in causing AD. In this review, we demonstrated that the effect of ER stress contributed to the pathogenesis of AD. ER stress associates almost the whole brain pathology processes which can be observed in AD, such as gene mutation of presenilin1, the abnormal clipped mRNA of presenilin2, β-amyloid production, tau phosphorylation, and cell death. The status of ER stress and unfolded protein response in the pathogenesis of AD also suggests they can be used as potential therapeutic agents. PMID:24715417

  6. Shaping the endoplasmic reticulum in vitro.

    PubMed

    Ferencz, Csilla-Maria; Guigas, Gernot; Veres, Andreas; Neumann, Brigitte; Stemmann, Olaf; Weiss, Matthias

    2016-09-01

    Organelles in eukaryotic cells often have complex shapes that deviate significantly from simple spheres. A prime example is the endoplasmic reticulum (ER) that forms an extensive network of membrane tubules in many mammalian cell types and in reconstitution assays in vitro. Despite the successful hunt for molecular determinants of ER shape we are still far from having a comprehensive understanding of ER network morphogenesis. Here, we have studied the hitherto neglected influence of the host substrate when reconstituting ER networks in vitro as compared to ER networks in vivo. In culture cells we observed cytoplasm-spanning ER networks with tubules being connected almost exclusively by three-way junctions and segment lengths being narrowly distributed around a mean length of about 1μm. In contrast, networks reconstituted from purified ER microsomes on flat glass or gel substrates of varying stiffness showed significantly broader length distributions with an up to fourfold larger mean length. Self-assembly of ER microsomes on small oil droplets, however, yielded networks that resembled more closely the native ER network of mammalian cells. We conclude from these observations that the ER microsomes' inherent self-assembly capacity is sufficient to support network formation with a native geometry if the influence of the host substrate's surface chemistry becomes negligible. We hypothesize that under these conditions the networks' preference for three-way junctions follows from creating 'starfish-shaped' vesicles when ER microsomes with a protein-induced spontaneous curvature undergo fusion. PMID:27287725

  7. Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

    PubMed

    Sbiera, Silviu; Leich, Ellen; Liebisch, Gerhard; Sbiera, Iuliu; Schirbel, Andreas; Wiemer, Laura; Matysik, Silke; Eckhardt, Carolin; Gardill, Felix; Gehl, Annemarie; Kendl, Sabine; Weigand, Isabel; Bala, Margarita; Ronchi, Cristina L; Deutschbein, Timo; Schmitz, Gerd; Rosenwald, Andreas; Allolio, Bruno; Fassnacht, Martin; Kroiss, Matthias

    2015-11-01

    Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer. PMID:26305886

  8. Ginsenoside Rg1 ameliorates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress-induced apoptosis in a streptozotocin-induced diabetes rat model.

    PubMed

    Yu, Haitao; Zhen, Juan; Yang, Yang; Gu, Jinning; Wu, Suisheng; Liu, Quan

    2016-04-01

    Ginsenoside Rg1 has been demonstrated to have cardiovascular protective effects. However, whether the cardioprotective effects of ginsenoside Rg1 are mediated by endoplasmic reticulum (ER) stress-induced apoptosis remain unclear. In this study, among 80 male Wistar rats, 15 rats were randomly selected as controls; the remaining 65 rats received a diet rich in fat and sugar content for 4 weeks, followed by intraperitoneal injection of streptozotocin (STZ, 40 mg/kg) to establish a diabetes model. Seven days after STZ injection, 10 rats were randomly selected as diabetic model (DM) controls, 45 eligible diabetic rats were randomized to three treatment groups and administered ginsenoside Rg1 in a dosage of 10, 15 or 20 mg/kg/day, respectively. After 12 weeks of treatment, rats were killed and serum samples obtained to determine cardiac troponin (cTn)-I. Myocardial tissues were harvested for morphological analysis to detect myocardial cell apoptosis, and to analyse protein expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and Caspase-12. Treatment with ginsenoside Rg1 (10-20 mg/kg) significantly reduced serum cTnI levels compared with DM control group (all P < 0.01). Ginsenoside Rg1 (15 and 20 mg/kg) significantly reduced the percentage of apoptotic myocardial cells and improved the parameters of cardiac function. Haematoxylin and eosin and Masson staining indicated that ginsenoside Rg1 could attenuate myocardial lesions and myocardial collagen volume fraction. Additionally, ginsenoside Rg1 significantly reduced GRP78, CHOP, and cleaved Caspase-12 protein expression in a dose-dependent manner. These findings suggest that ginsenoside Rg1 appeared to ameliorate diabetic cardiomyopathy by inhibiting ER stress-induced apoptosis in diabetic rats. PMID:26869403

  9. Sialic Acid Rescues Repurified Lipopolysaccharide-Induced Acute Renal Failure via Inhibiting TLR4/PKC/gp91-Mediated Endoplasmic Reticulum Stress, Apoptosis, Autophagy, and Pyroptosis Signaling

    PubMed Central

    Yang, Chih-Ching; Yao, Chien-An; Chien, Chiang-Ting

    2014-01-01

    Lipopolysaccharides (LPS) through Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) activation induce systemic inflammation where oxidative damage plays a key role in multiple organ failure. Because of the neutralization of LPS toxicity by sialic acid (SA), we determined its effect and mechanisms on repurified LPS (rLPS)-evoked acute renal failure. We assessed the effect of intravenous SA (10 mg/kg body weight) on rLPS-induced renal injury in female Wistar rats by evaluating blood and kidney reactive oxygen species (ROS) responses, renal and systemic hemodynamics, renal function, histopathology, and molecular mechanisms. SA can interact with rLPS through a high binding affinity. rLPS dose- and time-dependently reduced arterial blood pressure, renal microcirculation and blood flow, and increased vascular resistance in the rats. rLPS enhanced monocyte/macrophage (ED-1) infiltration and ROS production and impaired kidneys by triggering p-IRE1α/p-JNK/CHOP/GRP78/ATF4-mediated endoplasmic reticulum (ER) stress, Bax/PARP-mediated apoptosis, Beclin-1/Atg5-Atg12/LC3-II-mediated autophagy, and caspase 1/IL-1β-mediated pyroptosis in the kidneys. SA treatment at 30 min, but not 60 min after rLPS stimulation, gp91 siRNA and protein kinase C-α (PKC) inhibitor efficiently rescued rLPS-induced acute renal failure via inhibition of TLR4/PKC/NADPH oxidase gp91-mediated ER stress, apoptosis, autophagy and pyroptosis in renal proximal tubular cells, and rat kidneys. In response to rLPS or IFNγ, the enhanced Atg5, FADD, LC3-II, and PARP expression can be inhibited by Atg5 siRNA. Albumin (10 mg/kg body weight) did not rescue rLPS-induced injury. In conclusion, early treatment (within 30 min) of SA attenuates rLPS-induced renal failure via the reduction in LPS toxicity and subsequently inhibiting rLPS-activated TLR4/PKC/gp91/ER stress/apoptosis/autophagy/pyroptosis signaling. PMID:24973090

  10. Endoplasmic reticulum stress in liver disease.

    PubMed

    Malhi, Harmeet; Kaufman, Randal J

    2011-04-01

    The unfolded protein response (UPR) is activated upon the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that are sensed by the binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78). The accumulation of unfolded proteins sequesters BiP so it dissociates from three ER-transmembrane transducers leading to their activation. These transducers are inositol requiring (IRE) 1α, PKR-like ER kinase (PERK), and activating transcription factor (ATF) 6α. PERK phosphorylates eukaryotic initiation factor 2 alpha (eIF2α) resulting in global mRNA translation attenuation, and concurrently selectively increases the translation of several mRNAs, including the transcription factor ATF4, and its downstream target CHOP. IRE1α has kinase and endoribonuclease (RNase) activities. IRE1α autophosphorylation activates the RNase activity to splice XBP1 mRNA, to produce the active transcription factor sXBP1. IRE1α activation also recruits and activates the stress kinase JNK. ATF6α transits to the Golgi compartment where it is cleaved by intramembrane proteolysis to generate a soluble active transcription factor. These UPR pathways act in concert to increase ER content, expand the ER protein folding capacity, degrade misfolded proteins, and reduce the load of new proteins entering the ER. All of these are geared toward adaptation to resolve the protein folding defect. Faced with persistent ER stress, adaptation starts to fail and apoptosis occurs, possibly mediated through calcium perturbations, reactive oxygen species, and the proapoptotic transcription factor CHOP. The UPR is activated in several liver diseases; including obesity associated fatty liver disease, viral hepatitis, and alcohol-induced liver injury, all of which are associated with steatosis, raising the possibility that ER stress-dependent alteration in lipid homeostasis is the mechanism that underlies the steatosis. Hepatocyte apoptosis is a pathogenic event in several liver

  11. Endoplasmic Reticulum-Associated Degradation and Lipid Homeostasis.

    PubMed

    Stevenson, Julian; Huang, Edmond Y; Olzmann, James A

    2016-07-17

    The endoplasmic reticulum is the port of entry for proteins into the secretory pathway and the site of synthesis for several important lipids, including cholesterol, triacylglycerol, and phospholipids. Protein production within the endoplasmic reticulum is tightly regulated by a cohort of resident machinery that coordinates the folding, modification, and deployment of secreted and integral membrane proteins. Proteins failing to attain their native conformation are degraded through the endoplasmic reticulum-associated degradation (ERAD) pathway via a series of tightly coupled steps: substrate recognition, dislocation, and ubiquitin-dependent proteasomal destruction. The same ERAD machinery also controls the flux through various metabolic pathways by coupling the turnover of metabolic enzymes to the levels of key metabolites. We review the current understanding and biological significance of ERAD-mediated regulation of lipid metabolism in mammalian cells. PMID:27296502

  12. α-Solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway.

    PubMed

    Hasanain, M; Bhattacharjee, A; Pandey, P; Ashraf, R; Singh, N; Sharma, S; Vishwakarma, A L; Datta, D; Mitra, K; Sarkar, J

    2015-01-01

    α-Solanine is a glycoalkaloid found in species of the nightshade family including potato. It was primarily reported to have toxic effects in humans. However, there is a growing body of literature demonstrating in vitro and in vivo anticancer activity of α-solanine. Most of these studies have shown activation of apoptosis as the underlying mechanism in antitumor activity of α-solanine. In this study, we report α-solanine as a potential inducer of autophagy, which may act synergistically or in parallel with apoptosis to exert its cytotoxic effect. Induction of autophagy was demonstrated by several assays including electron microscopy, immunoblotting of autophagy markers and immunofluorescence for LC3 (microtubule-associated protein 1 (MAP1) light chain-3) puncta. α-Solanine-induced autophagic flux was demonstrated by additionally enhanced--turnover of LC3-II and--accumulation of LC3-specific puncta after co-incubation of cells with either of the autophagolysosome inhibitors--chloroquine and--bafilomycin A1. We also demonstrated α-solanine-induced oxidative damage in regulating autophagy where pre-incubation of cells with reactive oxygen species (ROS) scavenger resulted in suppression of CM-H2DCFDA (5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester) fluorescence as well as decrease in LC3-II turnover. α-Solanine treatment caused an increase in the expression of endoplasmic reticulum (ER) stress proteins (BiP, activating transcription factor 6 (ATF6), X-box-binding protein 1, PERK, inositol-requiring transmembrane kinase/endonuclease 1, ATF4 and CCAAT-enhancer-binding protein (C/EBP)-homologous protein) suggesting activation of unfolded protein response pathway. Moreover, we found downregulation of phosphorylated Akt (Thr308 and Ser473), mammalian target of rapamycin (mTOR; Ser2448 and Ser2481) and 4E-BP1 (Thr37/46) by α-solanine implying suppression of the Akt/mTOR pathway. Collectively, our results signify that α-solanine induces

  13. α-Solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway

    PubMed Central

    Hasanain, M; Bhattacharjee, A; Pandey, P; Ashraf, R; Singh, N; Sharma, S; Vishwakarma, A L; Datta, D; Mitra, K; Sarkar, J

    2015-01-01

    α-Solanine is a glycoalkaloid found in species of the nightshade family including potato. It was primarily reported to have toxic effects in humans. However, there is a growing body of literature demonstrating in vitro and in vivo anticancer activity of α-solanine. Most of these studies have shown activation of apoptosis as the underlying mechanism in antitumor activity of α-solanine. In this study, we report α-solanine as a potential inducer of autophagy, which may act synergistically or in parallel with apoptosis to exert its cytotoxic effect. Induction of autophagy was demonstrated by several assays including electron microscopy, immunoblotting of autophagy markers and immunofluorescence for LC3 (microtubule-associated protein 1 (MAP1) light chain-3) puncta. α-Solanine-induced autophagic flux was demonstrated by additionally enhanced – turnover of LC3-II and – accumulation of LC3-specific puncta after co-incubation of cells with either of the autophagolysosome inhibitors – chloroquine and – bafilomycin A1. We also demonstrated α-solanine-induced oxidative damage in regulating autophagy where pre-incubation of cells with reactive oxygen species (ROS) scavenger resulted in suppression of CM-H2DCFDA (5 (and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester) fluorescence as well as decrease in LC3-II turnover. α-Solanine treatment caused an increase in the expression of endoplasmic reticulum (ER) stress proteins (BiP, activating transcription factor 6 (ATF6), X-box-binding protein 1, PERK, inositol-requiring transmembrane kinase/endonuclease 1, ATF4 and CCAAT-enhancer-binding protein (C/EBP)-homologous protein) suggesting activation of unfolded protein response pathway. Moreover, we found downregulation of phosphorylated Akt (Thr308 and Ser473), mammalian target of rapamycin (mTOR; Ser2448 and Ser2481) and 4E-BP1 (Thr37/46) by α-solanine implying suppression of the Akt/mTOR pathway. Collectively, our results signify that

  14. The protein translocation machinery of the endoplasmic reticulum.

    PubMed

    Walter, P; Gilmore, R; Müller, M; Blobel, G

    1982-12-24

    The rough endoplasmic reticulum (r.e.r.) has been postulated to possess a single translation-coupled translocation system (in multiple copies) that effects signal sequence-mediated translocation of all secretory and lysosomal proteins and integration of all integral membrane proteins whose port of entry is the rough endoplasmic reticulum (G. Blobel 1980 Proc. natn. Acad. Sci. U.S.A. 77, 1496-1500). Two proteins have been isolated that are components of the r.e.r. translocation system. Their properties and function in protein translocation across and integration into membranes are discussed. PMID:6131460

  15. Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

    PubMed Central

    Laplante, J M; O'Rourke, F; Lu, X; Fein, A; Olsen, A; Feinstein, M B

    2000-01-01

    A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation. PMID:10794731

  16. Continuous network of endoplasmic reticulum in cerebellar Purkinje neurons.

    PubMed Central

    Terasaki, M; Slater, N T; Fein, A; Schmidek, A; Reese, T S

    1994-01-01

    Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions. Images PMID:7519781

  17. Stressed-Out Endoplasmic Reticulum Inflames the Mitochondria.

    PubMed

    Shin, Sunny; Argon, Yair

    2015-09-15

    Bacterial infection induces inflammasome activation and release of interleukin-1 (IL-1) cytokines. Bronner et al. (2015) show that during Brucella abortus infection, an endoplasmic reticulum stress sensor, IRE1α, initiates NLRP3- and caspase-2-mediated mitochondrial damage that potentiates NLRP3 inflammasome assembly. PMID:26377891

  18. 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. PMID:27048289

  19. Inhibition of calcium uptake via the sarco/endoplasmic reticulum Ca2+-ATPase in a mouse model of Sandhoff disease and prevention by treatment with N-butyldeoxynojirimycin.

    PubMed

    Pelled, Dori; Lloyd-Evans, Emyr; Riebeling, Christian; Jeyakumar, Mylvaganam; Platt, Frances M; Futerman, Anthony H

    2003-08-01

    Gangliosides are found at high levels in neuronal tissues where they play a variety of important functions. In the gangliosidoses, gangliosides accumulate because of defective activity of the lysosomal proteins responsible for their degradation, usually resulting in a rapidly progressive neurodegenerative disease. However, the molecular mechanism(s) leading from ganglioside accumulation to neurodegeneration is not known. We now examine the effect of ganglioside GM2 accumulation in a mouse model of Sandhoff disease (one of the GM2 gangliosidoses), the Hexb-/- mouse. Microsomes from Hexb-/- mouse brain showed a significant reduction in the rate of Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which was prevented by feeding Hexb-/- mice with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycolipid synthesis that reduces GM2 storage. Changes in SERCA activity were not due to transcriptional regulation but rather because of a decrease in Vmax. Moreover, exogenously added GM2 had a similar effect on SERCA activity. The functional significance of these findings was established by the enhanced sensitivity of neurons cultured from embryonic Hexb-/- mice to cell death induced by thapsigargin, a specific SERCA inhibitor, and by the enhanced sensitivity of Hexb-/- microsomes to calcium-induced calcium release. This study suggests a mechanistic link among GM2 accumulation, reduced SERCA activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease. PMID:12756243

  20. Autophagy is activated for cell survival after endoplasmic reticulum stress.

    PubMed

    Ogata, Maiko; Hino, Shin-ichiro; Saito, Atsushi; Morikawa, Keisuke; Kondo, Shinichi; Kanemoto, Soshi; Murakami, Tomohiko; Taniguchi, Manabu; Tanii, Ichiro; Yoshinaga, Kazuya; Shiosaka, Sadao; Hammarback, James A; Urano, Fumihiko; Imaizumi, Kazunori

    2006-12-01

    Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress. PMID:17030611

  1. Autophagy Is Activated for Cell Survival after Endoplasmic Reticulum Stress▿

    PubMed Central

    Ogata, Maiko; Hino, Shin-ichiro; Saito, Atsushi; Morikawa, Keisuke; Kondo, Shinichi; Kanemoto, Soshi; Murakami, Tomohiko; Taniguchi, Manabu; Tanii, Ichiro; Yoshinaga, Kazuya; Shiosaka, Sadao; Hammarback, James A.; Urano, Fumihiko; Imaizumi, Kazunori

    2006-01-01

    Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 “dots”), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress. PMID:17030611

  2. Endoplasmic reticulum stress in mouse decidua during early pregnancy.

    PubMed

    Gu, Xiao-Wei; Yan, Jia-Qi; Dou, Hai-Ting; Liu, Jie; Liu, Li; Zhao, Meng-Long; Liang, Xiao-Huan; Yang, Zeng-Ming

    2016-10-15

    Unfolded or misfolded protein accumulation in the endoplasmic reticulum lumen leads to endoplasmic reticulum stress (ER stress). Although it is known that ER stress is crucial for mammalian reproduction, little is known about its physiological significance and underlying mechanism during decidualization. Here we show that Ire-Xbp1 signal transduction pathway of unfolded protein response (UPR) is activated in decidual cells. The process of decidualization is compromised by ER stress inhibitor tauroursodeoxycholic acid sodium (TUDCA) and Ire specific inhibitor STF-083010 both in vivo and in vitro. A high concentration of ER stress inducer tunicamycin (TM) suppresses stromal cells proliferation and decidualization, while a lower concentration is beneficial. We further show that ER stress induces DNA damage and polyploidization in stromal cells. In conclusion, our data suggest that the GRP78/Ire1/Xbp1 signaling pathway of ER stress-UPR is activated and involved in mouse decidualization. PMID:27283502

  3. [The biological effects of liposome interactions with the endoplasmic reticulum].

    PubMed

    Foia, L; Costuleanu, N; Pavel, M

    1998-01-01

    Liposome research is a thriving field at the confluence of biophysics, cell biology and medicine. The principal medical application of liposomes is based on their potential to act as carriers for a broad spectrum of drugs and other agents, including antigens with or without immunomodulators in vaccination. Treatment of peritoneal macrophages of rats with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV) potentiated their activation for tumor cell lysis by endotoxins. The measurement of the fluorescence anisotropy of diphenylhexatriene showed a phase transition. No phase transition was observed in the rough endoplasmic reticulum membranes of macrophages either treated or not treated with cholesterol/DPPC SUV. The synergistic effect of DPPC SUV on the tumoricidal activity of macrophages induced by endotoxins appears to be correlated with the changes in the properties of the rough endoplasmic reticulum membranes. Both effects were transient; they had the same kinetics of induction and reversion. PMID:10756813

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

    SciTech Connect

    Grondin, Melanie; Marion, Michel; Denizeau, Francine; Averill-Bates, Diana A. . E-mail: averill.diana@uqam.ca

    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 from 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.

  5. Endoplasmic reticulum stress: implications for inflammatory bowel disease pathogenesis

    PubMed Central

    Kaser, Arthur; Martínez-Naves, Eduardo; Blumberg, Richard S.

    2015-01-01

    Purpose of review To provide an overview of the emerging role of cellular stress responses in inflammatory bowel disease (IBD). Recent findings The unfolded protein response (UPR) is a primitive cellular pathway that is engaged when responding to endoplasmic reticulum stress and regulates autophagy. Highly secretory cells such as Paneth cells and goblet cells in the intestines are particularly susceptible to endoplasmic reticulum stress and are exceedingly dependent upon a properly functioning UPR to maintain cellular viability and homeostasis. Primary genetic abnormalities within the components of the UPR (e.g. XBP1, ARG2, ORMDL3), genes that encode proteins reliant upon a robust secretory pathway (e.g. MUC2, HLAB27) and environmental factors that create disturbances in the UPR (e.g. microbial products and inflammatory cytokines) are important factors in the primary development and/or perpetuation of intestinal inflammation. Summary Endoplasmic reticulum stress is an important new pathway involved in the development of intestinal inflammation associated with IBD and likely other intestinal inflammatory disorders. PMID:20495455

  6. Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells.

    PubMed

    Phang, Chung-Weng; Karsani, Saiful Anuar; Sethi, Gautam; Abd Malek, Sri Nurestri

    2016-01-01

    Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb. PMID:26859847

  7. Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells

    PubMed Central

    Phang, Chung-Weng; Karsani, Saiful Anuar; Sethi, Gautam; Abd Malek, Sri Nurestri

    2016-01-01

    Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb. PMID:26859847

  8. C1q/TNF-Related Protein 9 (CTRP9) attenuates hepatic steatosis via the autophagy-mediated inhibition of endoplasmic reticulum stress.

    PubMed

    Jung, Tae Woo; Hong, Ho Cheol; Hwang, Hwan-Jin; Yoo, Hye Jin; Baik, Sei Hyun; Choi, Kyung Mook

    2015-12-01

    C1q/TNF-Related Protein (CTRP) 9, the closest paralog of adiponectin, has been reported to protect against diet-induced obesity and non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism has not been fully elucidated. We explored the protective effect of CTRP9 against hepatic steatosis and apoptosis, and identified the mechanisms through autophagy and endoplasmic reticulum (ER) stress using in vitro and in vivo experiments. Treating HepG2 cells with human recombinant CTRP9 significantly ameliorated palmitate- or tunicamycin-induced dysregulation of lipid metabolism, caspase 3 activity and chromatin condensation, which lead to reduction of hepatic triglyceride (TG) accumulation. CTRP9 treatment induced autophagy markers including LC3 conversion, P62 degradation, Beclin1 and ATG7 through AMPK phosphorylation in human primary hepatocytes. Furthermore, CTRP9 decreased palmitate- or tunicamycin-induced ER stress markers, such as eIF2α, CHOP and IRE-1, in HepG2 cells. Compound C, an AMPK inhibitor, and 3 methyladenine (3 MA), an autophagy inhibitor, canceled the effects of CTRP9 on ER stress, apoptosis and hepatic steatosis. In the livers of HFD-fed mice, adenovirus-mediated CTRP9 overexpression significantly induced AMPK phosphorylation and autophagy, whereas suppressed ER stress markers. In addition, both SREBP1-mediated lipogenic gene expression and apoptosis were significantly attenuated, which result in improvement in hepatic steatosis by overexpression of CTRP9. These results demonstrate that CTRP9 alleviates hepatic steatosis through relief of ER stress via the AMPK-mediated induction of autophagy. PMID:26419929

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

    PubMed Central

    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

    Purpose 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. Methods 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. Results 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

  10. Protein misfolding in the endoplasmic reticulum as a conduit to human disease.

    PubMed

    Wang, Miao; Kaufman, Randal J

    2016-01-21

    In eukaryotic cells, the endoplasmic reticulum is essential for the folding and trafficking of proteins that enter the secretory pathway. Environmental insults or increased protein synthesis often lead to protein misfolding in the organelle, the accumulation of misfolded or unfolded proteins - known as endoplasmic reticulum stress - and the activation of the adaptive unfolded protein response to restore homeostasis. If protein misfolding is not resolved, cells die. Endoplasmic reticulum stress and activation of the unfolded protein response help to determine cell fate and function. Furthermore, endoplasmic reticulum stress contributes to the aetiology of many human diseases. PMID:26791723

  11. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

    PubMed Central

    LEE, JIN-HAENG; JEONG, JAEHO; JEONG, EUI MAN; CHO, SUNG-YUP; KANG, JEONG WOOK; LIM, JISUN; HEO, JINBEOM; KANG, HYUNSOOK; KIM, IN-GYU; SHIN, DONG-MYUNG

    2014-01-01

    Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases. PMID:24481335

  12. Disulfide Mispairing During Proinsulin Folding in the Endoplasmic Reticulum.

    PubMed

    Haataja, Leena; Manickam, Nandini; Soliman, Ann; Tsai, Billy; Liu, Ming; Arvan, Peter

    2016-04-01

    Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene-induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, β-cell ER stress, and onset of diabetes. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond formation, using bioengineered proinsulins that can form only two (or even only one) of the native proinsulin disulfide bonds. In the absence of preexisting proinsulin disulfide pairing, Cys(B19)-Cys(A20) (a major determinant of ER stress response activation and proinsulin stability) preferentially initiates B-A chain disulfide bond formation, whereas Cys(B7)-Cys(A7) can initiate only under oxidizing conditions beyond that existing within the ER of β-cells. Interestingly, formation of these two "interchain" disulfide bonds demonstrates cooperativity, and together, they are sufficient to confer intracellular transport competence to proinsulin. The three most common proinsulin disulfide mispairings in the ER appear to involve Cys(A11)-Cys(A20), Cys(A7)-Cys(A20), and Cys(B19)-Cys(A11), each disrupting the critical Cys(B19)-Cys(A20) pairing. MIDY mutations inhibit Cys(B19)-Cys(A20) formation, but treatment to force oxidation of this disulfide bond improves folding and results in a small but detectable increase of proinsulin export. These data suggest possible therapeutic avenues to ameliorate ER stress and diabetes. PMID:26822090

  13. Pael receptor, endoplasmic reticulum stress, and Parkinson's disease.

    PubMed

    Takahashi, Ryosuke; Imai, Yuzuru

    2003-10-01

    Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations of the parkin gene. Parkin is an E3 ubiquitin ligase that specifically recognizes its substrate protein, promoting its ubiquitination and subsequent degradation. Accordingly, we hypothesized that AR-JP may be caused by accumulation of an unidentified neurotoxic protein, which is a substrate of parkin. Based on this hypothesis, we cloned parkin-binding protein using a yeast two-hybrid system and identified a putative G protein-coupled receptor protein,which we named the Pael receptor (Pael-R). When overexpressed in cells, this receptor became unfolded, insoluble, and ubiquitinated. Accumulation of the insoluble Pael-R subsequently led to endoplasmic reticulum (ER) stress-induced cell death. Parkin specifically ubiquitinates the unfolded Pael-R and promotes its degradation, resulting in suppression of cell death induced by the accumulation of unfolded Pael-R. Moreover, insoluble Pael-R accumulates in the brains of AR-JP patients. It is highly expressed by the dopaminergic neurons of the substantia nigra, strongly suggesting that accumulation of unfolded Pael-R may lead to selective death of dopaminergic neurons in AR-JP.Recently, we identified Hsp70 and its co-chaperone CHIP as novel parkin-binding partners. We found that CHIP enhanced parkinmediated ubiquitination of Pael-R. In concert with Hsp70, CHIP also enhanced the ability of parkin to inhibit cell death induced by Pael-R, indicating that CHIP and Hsp70 are both co-factors of parkin. PMID:14579121

  14. Endoplasmic Reticulum Stress and the Inflammatory Basis of Metabolic Disease

    PubMed Central

    Hotamisligil, Gökhan S.

    2010-01-01

    The endoplasmic reticulum (ER) is the major site in the cell for protein folding and trafficking and is central to many cellular functions. Failure of the ER's adaptive capacity results in activation of the unfolded protein response (UPR), which intersects with many different inflammatory and stress signaling pathways. These pathways are also critical in chronic metabolic diseases such as obesity, insulin resistance, and type 2 diabetes. The ER and related signaling networks are emerging as a potential site for the intersection of inflammation and metabolic disease. PMID:20303879

  15. One step at a time: endoplasmic reticulum-associated degradation

    PubMed Central

    Vembar, Shruthi S.; Brodsky, Jeffrey L.

    2009-01-01

    Protein folding in the endoplasmic reticulum (ER) is monitored by ER quality control (ERQC) mechanisms. Proteins that pass ERQC criteria traffic to their final destinations through the secretory pathway, whereas non-native and unassembled subunits of multimeric proteins are degraded by the ER-associated degradation (ERAD) pathway. During ERAD, molecular chaperones and associated factors recognize and target substrates for retrotranslocation to the cytoplasm, where they are degraded by the ubiquitin–proteasome machinery. The discovery of diseases that are associated with ERAD substrates highlights the importance of this pathway. Here, we summarize our current understanding of each step during ERAD, with emphasis on the factors that catalyse distinct activities. PMID:19002207

  16. Endoplasmic reticulum stress: an unrecognized actor in solid organ transplantation.

    PubMed

    Pallet, Nicolas; Fougeray, Sophie; Beaune, Philippe; Legendre, Christophe; Thervet, Eric; Anglicheau, Dany

    2009-09-15

    Endoplasmic reticulum (ER) stress is an adaptive response to the accumulation of misfolded proteins within the ER, which can trigger cell dedifferentiation and cell suicide. Increasing evidences suggest its implication in mediating allograft injury. Herein, we summarize the mechanisms of ER stress and discuss its implication in allograft injury. Increasing our understanding of the cellular and molecular mechanisms of acute and chronic allograft damages could lead to the development of new biomarkers and to the discovery of new therapeutic strategies to prevent the initiation of graft dysfunction or to promote the tissue regeneration after injury. PMID:19741454

  17. Identification of 1,2,3-triazole derivatives that protect pancreatic β cells against endoplasmic reticulum stress-mediated dysfunction and death through the inhibition of C/EBP-homologous protein expression.

    PubMed

    Duan, Hongliang; Arora, Daleep; Li, Yu; Setiadi, Hendra; Xu, Depeng; Lim, Hui-Ying; Wang, Weidong

    2016-06-15

    The C/EBP-homologous protein (CHOP) acts as a mediator of endoplasmic reticulum (ER) stress-induced pancreatic insulin-producing β cell death, a key element in the pathogenesis of diabetes. Chemicals that inhibit the expression of CHOP might therefore protect β cells from ER stress-induced apoptosis and prevent or ameliorate diabetes. Here, we used high-throughput screening to identify a series of 1,2,3-triazole amide derivatives that inhibit ER stress-induced CHOP-luciferase reporter activity. Our SAR studies indicate that compounds with an N,1-diphenyl-5-methyl-1H-1,2,3-triazole-4-carboxamide backbone potently protect β cell against ER stress. Several representative compounds inhibit ER stress-induced up-regulation of CHOP mRNA and protein, without affecting the basal level of CHOP expression. We further show that a 1,2,3-triazole derivative 4e protects β cell function and survival against ER stress in a CHOP-dependent fashion, as it is inactive in CHOP-deficient β cells. Finally, we show that 4e significantly lowers blood glucose levels and increases concomitant β cell survival and number in a streptozotocin-induced diabetic mouse model. Identification of small molecule inhibitors of CHOP expression that prevent ER stress-induced β cell dysfunction and death may provide a new modality for the treatment of diabetes. PMID:27157393

  18. [Involvement of endoplasmic reticulum stress in solid organ transplantation].

    PubMed

    Pallet, Nicolas; Bouvier, Nicolas; Beaune, Philippe; Legendre, Christophe; Anglicheau, Dany; Thervet, Eric

    2010-04-01

    Endoplasmic reticulum (ER) stress is a situation caused by the accumulation of unfolded proteins in the endoplasmic reticulum, triggering an evolutionary conserved adaptive response termed the unfolded protein response. When adaptation fails, excessive and prolonged ER stress triggers cell suicide. Important roles for ER-initiated cell death pathways have been recognized for several diseases, including diabetes, hypoxia, ischemia/reperfusion injury, neurodegenerative and heart diseases. The implication of the ER stress is not well recognized in solid organ transplantation, but increasing evidence suggests its implication in mediating allograft injury. The purpose of this review is to summarize the mechanisms of ER stress and to discuss its implication during tissue injury in solid organ transplantation. The possible implications of the ER stress in the modifications of cell functional properties and phenotypic changes are also discussed beyond the scope of adaptation and cell death. Increasing the understanding of the cellular and molecular mechanisms of acute and chronic allograft damages could lead to the development of new biomarkers and to the discovery of new therapeutic strategies to prevent the initiation of graft dysfunction or to promote the tissue regeneration after injury. PMID:20412745

  19. The Cdc48 machine in endoplasmic reticulum associated protein degradation.

    PubMed

    Wolf, Dieter H; Stolz, Alexandra

    2012-01-01

    The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in all eukaryotic cells that transforms ATP hydrolysis into mechanic power to unfold and pull proteins against physical forces, which make up a protein's structure and hold it in place. From the many cellular processes, Cdc48 is involved in, its function in endoplasmic reticulum associated protein degradation (ERAD) is understood best. This quality control process for proteins of the secretory pathway scans protein folding and discovers misfolded proteins in the endoplasmic reticulum (ER), the organelle, destined for folding of these proteins and their further delivery to their site of action. Misfolded lumenal and membrane proteins of the ER are detected by chaperones and lectins and retro-translocated out of the ER for degradation. Here the Cdc48 machinery, recruited to the ER membrane, takes over. After polyubiquitylation of the protein substrate, Cdc48 together with its dimeric co-factor complex Ufd1-Npl4 pulls the misfolded protein out and away from the ER membrane and delivers it to down-stream components for degradation by a cytosolic proteinase machine, the proteasome. The known details of the Cdc48-Ufd1-Npl4 motor complex triggered process are subject of this review article. PMID:21945179

  20. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    SciTech Connect

    Liu Qiong; Zhan Jinbiao . E-mail: jzhan2k@zju.edu.cn; Chen Xinhong; Zheng Shu

    2006-05-12

    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 purified 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.

  1. Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis.

    PubMed

    Manfredi, Giovanni; Kawamata, Hibiki

    2016-06-01

    Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS. PMID:26282323

  2. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori

    PubMed Central

    Seo, Minchul; Ryou, Hee-Joo; Yun, Eun-Young; Goo, Tae-Won

    2015-01-01

    We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis. PMID:26556347

  3. Molecular Characterization of the Endoplasmic Reticulum: insights from proteomic studies

    PubMed Central

    Chen, Xuequn; Karnovsky, Alla; Sans, Maria Dolors; Andrews, Philip C.; Williams, John A.

    2012-01-01

    The endoplasmic reticulum (ER) is a multifunctional intracellular organelle responsible for the synthesis, processing and trafficking of a wide variety of proteins essential for cell growth and survival. Thesefore, comprehensive characterization of the ER proteome is of great importance to the understanding of its functions and has been actively pursued in the past decade by scientists in the proteomics field. This review summarizes major proteomic studies published in the past decade that focused on the ER proteome. We evaluate the data sets obtained from two different organs, liver and pancreas each of which contains a primary cell type (hepatocyte and acinar cell) with specialized functions. We also discuss how the nature of the proteins uncovered is related to the methods of organelle purification, organelle purity and the techniques used for protein separation prior to mass spectrometry. In addition, this review also puts emphasis on the biological insights gained from these studies regarding to the molecular functions of the endoplasmic reticulum including protein synthesis and translocation, protein folding and quality control, ER-associated degradation and ER stress, ER export and membrane trafficking, calcium homeostasis, and detoxification and drug metabolism. PMID:21080494

  4. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori.

    PubMed

    Seo, Minchul; Ryou, Hee-Joo; Yun, Eun-Young; Goo, Tae-Won

    2015-01-01

    We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H₂O₂, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis. PMID:26556347

  5. Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis

    PubMed Central

    Bravo-Sagua, R.; Rodriguez, A.E.; Kuzmicic, J.; Gutierrez, T.; Lopez-Crisosto, C.; Quiroga, C.; Díaz-Elizondo, J.; Chiong, M.; Gillette, T.G.; Rothermel, B.A.; Lavandero, S.

    2014-01-01

    The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial–associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate. PMID:23228132

  6. Cell death and survival through the endoplasmic reticulum-mitochondrial axis.

    PubMed

    Bravo-Sagua, R; Rodriguez, A E; Kuzmicic, J; Gutierrez, T; Lopez-Crisosto, C; Quiroga, C; Díaz-Elizondo, J; Chiong, M; Gillette, T G; Rothermel, B A; Lavandero, S

    2013-02-01

    The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate. PMID:23228132

  7. Crude Saponins of Panax notoginseng Have Neuroprotective Effects To Inhibit Palmitate-Triggered Endoplasmic Reticulum Stress-Associated Apoptosis and Loss of Postsynaptic Proteins in Staurosporine Differentiated RGC-5 Retinal Ganglion Cells.

    PubMed

    Wang, Dan-dan; Zhu, Hua-zhang; Li, Shi-wei; Yang, Jia-ming; Xiao, Yang; Kang, Qiang-rong; Li, Chen-yang; Zhao, Yun-shi; Zeng, Yong; Li, Yan; Zhang, Jian; He, Zhen-dan; Ying, Ying

    2016-02-24

    Increased apoptosis of retinal ganglion cells (RGCs) contributes to the gradual loss of retinal neurons at the early phase of diabetic retinopathy (DR). There is an urgent need to search for drugs with neuroprotective effects against apoptosis of RGCs for the early treatment of DR. This study aimed to investigate the neuroprotective effects of saponins extracted from Panax notoginseng, a traditional Chinese medicine, on apoptosis of RGCs stimulated by palmitate, a metabolic factor for the development of diabetes and its complications, and to explore the potential molecular mechanism. We showed that crude saponins of P. notoginseng (CSPN) inhibited the increased apoptosis and loss of postsynaptic protein PSD-95 by palmitate in staurosporine-differentiated RGC-5 cells. Moreover, CSPN suppressed palmitate-induced reactive oxygen species generation and endoplasmic reticulum stress-associated eIF2α/ATF4/CHOP and caspase 12 pathways. Thus, our findings address the potential therapeutic significance of CSPN for the early stage of DR. PMID:26832452

  8. Stress Responses from the Endoplasmic Reticulum in Cancer

    PubMed Central

    Kato, Hironori; Nishitoh, Hideki

    2015-01-01

    The endoplasmic reticulum (ER) is a dynamic organelle that is essential for multiple cellular functions. During cellular stress conditions, including nutrient deprivation and dysregulation of protein synthesis, unfolded/misfolded proteins accumulate in the ER lumen, resulting in activation of the unfolded protein response (UPR). The UPR also contributes to the regulation of various intracellular signaling pathways such as calcium signaling and lipid signaling. More recently, the mitochondria-associated ER membrane (MAM), which is a site of close contact between the ER and mitochondria, has been shown to function as a platform for various intracellular stress responses including apoptotic signaling, inflammatory signaling, the autophagic response, and the UPR. Interestingly, in cancer, these signaling pathways from the ER are often dysregulated, contributing to cancer cell metabolism. Thus, the signaling pathway from the ER may be a novel therapeutic target for various cancers. In this review, we discuss recent research on the roles of stress responses from the ER, including the MAM. PMID:25941664

  9. Pharmacological Modulators of Endoplasmic Reticulum Stress in Metabolic Diseases

    PubMed Central

    Jung, Tae Woo; Choi, Kyung Mook

    2016-01-01

    The endoplasmic reticulum (ER) is the principal organelle responsible for correct protein folding, a step in protein synthesis that is critical for the functional conformation of proteins. ER stress is a primary feature of secretory cells and is involved in the pathogenesis of numerous human diseases, such as certain neurodegenerative and cardiometabolic disorders. The unfolded protein response (UPR) is a defense mechanism to attenuate ER stress and maintain the homeostasis of the organism. Two major degradation systems, including the proteasome and autophagy, are involved in this defense system. If ER stress overwhelms the capacity of the cell’s defense mechanisms, apoptotic death may result. This review is focused on the various pharmacological modulators that can protect cells from damage induced by ER stress. The possible mechanisms for cytoprotection are also discussed. PMID:26840310

  10. Endoplasmic Reticulum Stress Sensing in the Unfolded Protein Response

    PubMed Central

    Gardner, Brooke M.; Pincus, David; Gotthardt, Katja; Gallagher, Ciara M.; Walter, Peter

    2013-01-01

    Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1’s sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review. PMID:23388626

  11. Endoplasmic reticulum aminopeptidases in the pathogenesis of ankylosing spondylitis.

    PubMed

    Kenna, Tony J; Robinson, Philip C; Haroon, Nigil

    2015-09-01

    There has been significant progress in our understanding of the pathogenesis of AS. The advent of genome-wide association studies has increased the known loci associated with AS to more than 40. The endoplasmic reticulum resident aminopeptidases (ERAP) 1 and 2 were identified in this manner and are of particular interest. There appears to be a genetic as well as a functional interaction of ERAP1 and 2 with HLA-B27 based on the known functions of these molecules. Recent studies on the structure, immunological effects and the peptide-trimming properties of ERAP 1 and 2 have helped to provide insight into their pathogenic potential in AS. In this review, we explore the role of ERAP 1 and 2 in the pathogenesis of AS. PMID:26070942

  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. The Gp78 ubiquitin ligase: probing endoplasmic reticulum complexity.

    PubMed

    St Pierre, Pascal; Nabi, Ivan R

    2012-02-01

    The endoplasmic reticulum (ER) has been classically divided, based on electron microscopy analysis, into parallel ribosome-studded rough ER sheets and a tubular smooth ER network. Recent studies have identified molecular constituents of the ER, the reticulons and DP1, that drive ER tubule formation and whose expression determines expression of ER sheets and tubules and thereby rough and smooth ER. However, segregation of the ER into only two domains remains simplistic and multiple functionally distinct ER domains necessarily exist. In this review, we will discuss the sub-organization of the ER in different domains focusing on the localization and role of the gp78 ubiquitin ligase in the mitochondria-associated smooth ER and on the evidence for a quality control ERAD domain. PMID:22045301

  14. Terasaki spiral ramps in the rough endoplasmic reticulum.

    PubMed

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

    2014-10-31

    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. PMID:25396396

  15. Cytoprotective small molecule modulators of endoplasmic reticulum stress.

    PubMed

    Munshi, Soumyabrata; Dahl, Russell

    2016-06-01

    Cellular health depends on the normal function of the endoplasmic reticulum (ER) to fold, assemble, and modify critical proteins to maintain viability. When the ER cannot process proteins effectively, a condition known as ER stress ensues. When this stress is excessive or prolonged, cell death via apoptotic pathways is triggered. Interestingly, most major diseases have been shown to be intimately linked to ER stress, including diabetes, stroke, neurodegeneration, and many cancers. Thus, controlling ER stress presents a significant strategy for drug development for these diseases. The goal of this review is to present various small molecules that alleviate ER stress with the intention that they may serve as useful starting points for therapeutic agent development. PMID:27091069

  16. Endoplasmic reticulum stress in myotonic dystrophy type 1 muscle.

    PubMed

    Ikezoe, Koji; Nakamori, Masayuki; Furuya, Hirokazu; Arahata, Hajime; Kanemoto, Soshi; Kimura, Takashi; Imaizumi, Kazunori; Takahashi, Masanori P; Sakoda, Saburo; Fujii, Naoki; Kira, Jun-ichi

    2007-11-01

    In myotonic dystrophy type 1 (DM1), alternative splicing of ryanodine receptor 1 (RyR1) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) genes has been reported. These proteins are essential for maintaining intracellular Ca2+ in skeletal muscle. To clarify involvement of endoplasmic reticulum (ER) stress in DM1 muscles, we examined the activation of ER stress-related proteins by immunohistochemistry, western blot analysis and RT-PCR. In four of five DM1 muscle biopsies, except for a muscle biopsy from a patient with the shortest CTG expansion and no myotonia, increased expression of GRP78 and calnexin, and phosphorylation of PERK and eIF-2 alpha were revealed in fibers with sarcoplasmic masses and in highly atrophic fibers with pyknotic nuclear clumps. Caspase-3 and -7 were also expressed in these fibers. Increased expression of GRP78 in these DM1 muscles was confirmed by western blot analysis. GRP78 mRNA and spliced isoform of XBP1 mRNA were also increased in DM1 muscle biopsies. Furthermore, we demonstrated increased expression of GRP78 in highly atrophic fibers with pyknotic nuclear clumps in all three muscle biopsies from neurogenic muscular atrophies. However, five muscle biopsies from central core disease presumably with disturbed intracellular Ca2+ homeostasis and a muscle biopsy from paramyotonia congenita with myotonia showed no activation of these proteins. Taken together, ER stress is involved in muscle wasting in DM1. However, it seems to be evoked not only by disrupted intracellular Ca2+ homeostasis. PMID:17661063

  17. Maternal obesity alters endoplasmic reticulum homeostasis in offspring pancreas.

    PubMed

    Soeda, Jumpei; Mouralidarane, Angelina; Cordero, Paul; Li, Jiawei; Nguyen, Vi; Carter, Rebeca; Kapur, Sabrina R; Pombo, Joaquim; Poston, Lucilla; Taylor, Paul D; Vinciguerra, Manlio; Oben, Jude A

    2016-06-01

    The prevalence of non-alcoholic fatty pancreas disease (NAFPD) is increasing in parallel with obesity rates. Stress-related alterations in endoplasmic reticulum (ER), such as the unfolded protein response (UPR), are associated with obesity. The aim of this study was to investigate ER imbalance in the pancreas of a mice model of adult and perinatal diet-induced obesity. Twenty female C57BL/6J mice were assigned to control (Con) or obesogenic (Ob) diets prior to and during pregnancy and lactation. Their offspring were weaned onto Con or Ob diets up to 6 months post-partum. Then, after sacrifice, plasma biochemical analyses, gene expression, and protein concentrations were measured in pancreata. Offspring of Ob-fed mice had significantly increased body weight (p < 0.001) and plasma leptin (p < 0.001) and decreased insulin (p < 0.01) levels. Maternal obesogenic diet decreased the total and phosphorylated Eif2α and increased spliced X-box binding protein 1 (XBP1). Pancreatic gene expression of downstream regulators of UPR (EDEM, homocysteine-responsive endoplasmic reticulum-resident (HERP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP)) and autophagy-related proteins (LC3BI/LC3BII) were differently disrupted by obesogenic feeding in both mothers and offspring (from p < 0.1 to p < 0.001). Maternal obesity and Ob feeding in their offspring alter UPR in NAFPD, with involvement of proapoptotic and autophagy-related markers. Upstream and downstream regulators of PERK, IRE1α, and ATF6 pathways were affected differently following the obesogenic insults. PMID:26979740

  18. Peptidyl prolyl cis-trans-isomerase activity associated with the lumen of the endoplasmic reticulum.

    PubMed Central

    Bose, S; Freedman, R B

    1994-01-01

    Peptidyl prolyl cis-trans-isomerase (PPI) activity was detected in microsomal fractions from bovine and rat liver. Extensive washing, proteinase and sonication treatments indicated that although some of this activity was due to adsorbed cytosolic enzymes, there was also an active but latent microsomal PPI activity. Density-gradient subfractionation indicated that activity was associated with vesicles derived from both the rough and the smooth endoplasmic reticulum (ER), suggesting that the activity was located within the ER lumen. The luminal PPI activity was inhibited by cyclosporin A and was active towards an unfolded protein substrate as well as towards the standard peptide substrate. PMID:8010971

  19. 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

  20. Endoplasmic Reticulum-associated Degradation (ERAD) and Autophagy Cooperate to Degrade Polymerogenic Mutant Serpins*

    PubMed Central

    Kroeger, Heike; Miranda, Elena; MacLeod, Ian; Pérez, Juan; Crowther, Damian C.; Marciniak, Stefan J.; Lomas, David A.

    2009-01-01

    The serpinopathies are a family of diseases characterized by the accumulation of ordered polymers of mutant protein within the endoplasmic reticulum. They are a diverse group including α1-antitrypsin deficiency and the inherited dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. We have used transient transfection of COS7 cells and mouse embryonic fibroblasts, PC12 cell lines that conditionally express wild type and mutant neuroserpin and fly models of FENIB to assess the cellular handling of wild type and mutant serpins. By using a polymer-specific monoclonal antibody, we show that mutant neuroserpin forms polymers after a delay of at least 30 min and that polymers can be cleared in PC12 cell lines and from the brain in a fly model of FENIB. At steady state, the fractions of intracellular polymerogenic G392E mutant neuroserpin in the monomeric and polymeric states are comparable. Inhibition of the proteasome with MG132 reveals that both mutant neuroserpin and α1-antitrypsin are degraded predominantly by endoplasmic reticulum-associated degradation (ERAD). Pharmacological and genetic inhibitions demonstrate that autophagy is responsible for bulk turnover of wild type and mutant serpins, but can be stimulated by rapamycin to compensate for proteasome inhibition. The significance of these findings to the treatment of serpinopathies is discussed. PMID:19549782

  1. Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

    PubMed Central

    Suresh, Arvind; Subedi, Kalpana; Kyathanahalli, Chandrashekara; Jeyasuria, Pancharatnam; Condon, Jennifer C.

    2013-01-01

    We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner. PMID:24058658

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. ...

  3. Inhibition of endoplasmic reticulum stress-activated IRE1α-TRAF2-caspase-12 apoptotic pathway is involved in the neuroprotective effects of telmisartan in the rotenone rat model of Parkinson's disease.

    PubMed

    Tong, Qiang; Wu, Liang; Jiang, Teng; Ou, Zhou; Zhang, Yingdong; Zhu, Dongya

    2016-04-01

    Telmisartan, one unique angiotensin II type 1 receptor blocker, has been attracting attention due to its putative peroxisome proliferator-activated receptor (PPAR)-γ or β/δ actions. Recently, telmisartan has been reported to exert neuroprotective effects in animal models of Parkinson's disease (PD). However, the underlying mechanisms have not been fully clarified. Recently, accumulating evidence has shown that endoplasmic reticulum (ER) stress plays a crucial role in rotenone-induced neuronal apoptosis. Additionally, studies have revealed that inositol-requiring enzyme/endonuclease 1α (IRE1α) is necessary and sufficient to trigger ER stress. In the present study, we aimed to determine whether ER stress-activated IRE1α-mediated apoptotic pathway is involved in the neuroprotection of telmisartan in the rotenone rats of PD and explore the possible involvement of PPAR-β/δ activation. The catalepsy tests were performed to test the catalepsy symptom. The dopamine content and α-synuclein expression were ascertained through high-performance liquid chromatography and immunohistochemistry, respectively. The expression of IRE1α, TNF receptor associated factor 2 (TRAF2), caspase-12 and PPAR-β/δ was detected by western blot. Neuronal apoptosis was assessed by TUNEL and immunohistochemistry. Our results show that telmisartan ameliorated the catalepsy symptom and attenuated dopamine depletion as well as α-synuclein accumulation. Moreover, telmisartan decreased ER stress-mediated neuronal apoptosis. Furthermore, telmisartan inhibited IRE1α-TRAF2-caspase-12 apoptotic signaling pathway. Additionally, telmisartan activated PPAR β/δ, implying that PPAR-β/δ activation properties of telmisartan are possibly or partially involved in the neuroprotective effects. In conclusion, our findings suggest that suppressing ER stress-activated IRE1α-TRAF2-caspase-12 apoptotic pathway is involved in the neuroprotective effects of telmisartan in the rotenone rats of PD. PMID

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

    SciTech Connect

    Luo, Ying; Li, Shu-Jun; Yang, Jian; Qiu, Yuan-Zhen; Chen, Fang-Ping

    2013-09-06

    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 reticulum 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.

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

    PubMed Central

    Qian, Dandan; Tian, Lihong; Qu, Leqing

    2015-01-01

    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. PMID:26395408

  6. Periostin promotes secretion of fibronectin from the endoplasmic reticulum.

    PubMed

    Kii, Isao; Nishiyama, Takashi; Kudo, Akira

    2016-02-19

    Extracellular matrix (ECM) proteins are synthesized in the endoplasmic reticulum (ER), transported to the extracellular milieu through the secretory pathway, and assembled into an extracellular architecture. A previous study of ours showed that periostin, a secretory protein, interacts with fibronectin and is involved in ECM remodeling. Here we show that periostin played a role in fibronectin secretion from the ER. Co-immunoprecipitation and in situ proximity ligation assays revealed an interaction between periostin and fibronectin in the ER. Although accumulation of fibronectin was detected in the ER of fibroblastic C3H10T1/2 cells, forced expression of periostin in those cells decreased the accumulation of fibronectin in the ER, suggesting that periostin promoted the secretion of fibronectin. A substitution mutant of tryptophan at the position 65 to alanine in the EMI domain of periostin, which caused periostin to lose its ability to interact with fibronectin, did not decrease the accumulation. Furthermore, targeted disruption of periostin in mice caused the non-fibrillar and ectopic deposition of fibronectin in the periodontal ligament. Thus, these results demonstrate a subcellular role of periostin in promotion of fibronectin secretion from the ER. PMID:26820539

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

    PubMed

    Qian, Dandan; Tian, Lihong; Qu, Leqing

    2015-01-01

    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. PMID:26395408

  8. Supramolecular architecture of endoplasmic reticulum-plasma membrane contact sites.

    PubMed

    Fernández-Busnadiego, Rubén

    2016-04-15

    The endoplasmic reticulum (ER) forms membrane contact sites (MCS) with most other cellular organelles and the plasma membrane (PM). These ER-PM MCS, where the membranes of the ER and PM are closely apposed, were discovered in the early days of electron microscopy (EM), but only recently are we starting to understand their functional and structural diversity. ER-PM MCS are nowadays known to mediate excitation-contraction coupling (ECC) in striated muscle cells and to play crucial roles in Ca(2+)and lipid homoeostasis in all metazoan cells. A common feature across ER-PM MCS specialized in different functions is the preponderance of cooperative phenomena that result in the formation of large supramolecular assemblies. Therefore, characterizing the supramolecular architecture of ER-PM MCS is critical to understand their mechanisms of function. Cryo-electron tomography (cryo-ET) is a powerful EM technique uniquely positioned to address this issue, as it allows 3D imaging of fully hydrated, unstained cellular structures at molecular resolution. In this review I summarize our current structural knowledge on the molecular organization of ER-PM MCS and its functional implications, with special emphasis on the emerging contributions of cryo-ET. PMID:27068966

  9. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    PubMed

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized. PMID:26139824

  10. Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum

    PubMed Central

    Saberianfar, Reza; Sattarzadeh, Amirali; Joensuu, Jussi J.; Kohalmi, Susanne E.; Menassa, Rima

    2016-01-01

    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). 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 into 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. PMID:27242885

  11. Heme oxygenase-1 comes back to endoplasmic reticulum

    SciTech Connect

    Kim, Hong Pyo; Pae, Hyun-Ock; Back, Sung Hun; Chung, Su Wol; Woo, Je Moon; Son, Yong; Chung, Hun-Taeg

    2011-01-07

    Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

  12. Endoplasmic reticulum stress regulation in hematopoietic stem cells.

    PubMed

    Miharada, Kenichi

    2016-08-01

    Adult hematopoietic stem cells (HSCs) reside in bone marrow and are maintained in a dormant state within a special microenvironment, their so-called "niche". Detaching from the niche induces cell cycle progression, resulting in a reduction of the reconstitution capacity of HSCs. In contrast, fetal liver HSCs actively divide without losing their stem cell potentials. Thus, it has been unclear what types of cellular responses and metabolic changes occur in growing HSCs. We previously discovered that HSCs express relatively low levels of endoplasmic reticulum (ER) chaperone proteins governing protein folding, making HSCs vulnerable to an elevation of stress signals caused by accumulation of un-/misfolded proteins (ER stress) upon in vitro culture. Interestingly, fetal liver HSCs do not show ER stress elevation despite unchanged levels of chaperone proteins. Our latest studies utilizing multiple mouse models revealed that in the fetal liver bile acids as chemical chaperones play a key role supporting the protein folding which results in the suppression of ER stress induction. These findings highlight the importance of ER stress regulations in hematopoiesis. PMID:27599423

  13. Involvement of Endoplasmic Reticulum Stress Response in Orofacial Inflammatory Pain

    PubMed Central

    Yang, Eun Sun; Bae, Jin Young; Kim, Tae Heon; Kim, Yun Sook; Suk, Kyoungho

    2014-01-01

    Endoplasmic reticulum (ER) stress is involved in many neurological diseases and inflammatory responses. Inflammatory mediators induce neuronal damage and trigger the neuropathic or inflammatory pain. But there is very little data on the role of the ER stress response in pain mechanisms. In this study, we explored whether the ER stress response is involved in orofacial inflammatory pain by using a complete Freund's adjuvant (CFA)-injected rat model. The thermal pain hypersensitivity increased significantly after CFA injection. We found that the protein and mRNA levels of ER stress response genes, GRP78/Bip and p-eIF2α, increased significantly in trigeminal ganglion (TG) of CFA-injected rats compared to control animals. In immunofluorescence analysis, a significant increase of GRP78 and p-eIF2α immunopositive neurons was observed in CFA-injected TG compared to control TG. When we administered an ER stress modulator, salubrinal, CFA-induced thermal pain hypersensitivity was temporally reduced. Thus, our study suggests that ER stress responses in TG neurons contribute to CFA-induced inflammatory pain, and may comprise an important molecular mechanism underlying the orofacial inflammatory pain pathway. PMID:25548537

  14. The antioxidant machinery of the endoplasmic reticulum: Protection and signaling.

    PubMed

    Delaunay-Moisan, Agnès; Appenzeller-Herzog, Christian

    2015-06-01

    Cellular metabolism is inherently linked to the production of oxidizing by-products, including reactive oxygen species (ROS) hydrogen peroxide (H2O2). When present in excess, H2O2 can damage cellular biomolecules, but when produced in coordinated fashion, it typically serves as a mobile signaling messenger. It is therefore not surprising that cell health critically relies on both low-molecular-weight and enzymatic antioxidant components, which protect from ROS-mediated damage and shape the propagation and duration of ROS signals. This review focuses on H2O2-antioxidant cross talk in the endoplasmic reticulum (ER), which is intimately linked to the process of oxidative protein folding. ER-resident or ER-regulated sources of H2O2 and other ROS, which are subgrouped into constitutive and stimulated sources, are discussed and set into context with the diverse antioxidant mechanisms in the organelle. These include two types of peroxide-reducing enzymes, a high concentration of glutathione derived from the cytosol, and feedback-regulated thiol-disulfide switches, which negatively control the major ER oxidase ER oxidoreductin-1. Finally, new evidence highlighting emerging principles of H2O2-based cues at the ER will likely set a basis for establishing ER redox processes as a major line of future signaling research. A fundamental problem that remains to be solved is the specific, quantitative, time resolved, and targeted detection of H2O2 in the ER and in specialized ER subdomains. PMID:25744411

  15. Endoplasmic reticulum: Where nucleotide sugar transport meets cytokinin control mechanisms

    PubMed Central

    Niemann, Michael CE; Werner, Tomáš

    2015-01-01

    The endoplasmic reticulum (ER) is a multifunctional eukaryotic organelle where the vast majority of secretory proteins are folded and assembled to achieve their correct tertiary structures. The lumen of the ER and Golgi apparatus also provides an environment for numerous glycosylation reactions essential for modifications of proteins and lipids, and for cell wall biosynthesis. These glycosylation reactions require a constant supply of cytosolically synthesized substrate precursors, nucleotide sugars, which are transported by a group of dedicated nucleotide sugar transporters (NST). Recently, we have reported on the identification of a novel ER-localized NST protein, ROCK1, which mediates the transport of UDP-linked acetylated hexosamines across the ER membrane in Arabidopsis. Interestingly, it has been demonstrated that the activity of ROCK1 is important for the regulation of cytokinin-degrading enzymes, cytokinin oxidases/dehydrogenases (CKX), in the ER and, thus, for cytokinin responses. In this addendum we will address the biochemical and cellular activity of the ROCK1 transporter and its phylogenetic relation to other NST proteins. PMID:26418963

  16. 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

  17. Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques

    PubMed Central

    Costantini, Lindsey; Snapp, Erik

    2013-01-01

    This UNIT describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using commercially available widefield and confocal laser scanning microscopes (CLSM). It has been long appreciated that the ER plays a number of key roles in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has been often restricted to biochemical assays that average the behaviors of millions of lysed cells or to imaging static fixed cells. Now, with new fluorescent protein reporter tools, highly sensitive commercial microscopes, and photobleaching techniques, it is possible to interrogate the behaviors of ER proteins, membranes, and stress pathways in single cells with exquisite spatial and temporal resolution. The ER presents a unique set of imaging challenges including the high mobility of ER membranes, a diverse range of dynamic ER structures, and the influence of post-translational modifications on fluorescent protein reporters. Solutions to these challenges are described and considerations for performing photobleaching assays, especially Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) for ER proteins will be discussed. In addition, ER reporters and ER-specific pharmacologic compounds are presented with a focus on misfolded secretory protein stress and the Unfolded Protein Response (UPR). PMID:24510787

  18. Endoplasmic Reticulum Stress Interacts With Inflammation in Human Diseases

    PubMed Central

    Cao, Stewart Siyan; Luo, Katherine L.; Shi, Lynn

    2015-01-01

    The endoplasmic reticulum is a critical organelle for normal cell function and homeostasis. Disturbed protein folding process in the ER, termed ER stress, leads to the activation of unfolded protein response (UPR) that encompasses a complex network of intracellular signaling pathways. The UPR can either restore ER homeostasis or activate pro-apoptotic pathways depending on specific insults, intensity and duration of the stress, and cell types. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune diseases, infection, neurodegenerative disease, and metabolic disorders. In the cell, ER stress and inflammatory signaling share extensive regulators and effectors in a broad spectrum of biological processes. In spite of different etiologies, the two signaling pathways were shown to form a vicious cycle in exacerbating cellular dysfunction and causing apoptosis in many cells and tissues. However, the interaction between ER stress and inflammation in many of these diseases remains elusive. Further understanding of those issues may enable the development of novel therapies that spontaneously target these pathogenic pathways. PMID:26201832

  19. Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum.

    PubMed

    Saberianfar, Reza; Sattarzadeh, Amirali; Joensuu, Jussi J; Kohalmi, Susanne E; Menassa, Rima

    2016-01-01

    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). 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 into 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. PMID:27242885

  20. Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules.

    PubMed

    Smyth, Jeremy T; Schoborg, Todd A; Bergman, Zane J; Riggs, Blake; Rusan, Nasser M

    2015-08-01

    Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. We have addressed this by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. We show that in Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, we show that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species. PMID:26289801

  1. Quantitative proteomic survey of endoplasmic reticulum in mouse liver.

    PubMed

    Song, Yanping; Jiang, Ying; Ying, Wantao; Gong, Yan; Yan, Yujuan; Yang, Dong; Ma, Jie; Xue, Xiaofang; Zhong, Fan; Wu, Songfeng; Hao, Yunwei; Sun, Aihua; Li, Tao; Sun, Wei; Wei, Handong; Zhu, Yunping; Qian, Xiaohong; He, Fuchu

    2010-03-01

    To gain a better understanding of the critical function of the endoplasmic reticulum (ER) in liver, we carried out a proteomic survey of mouse liver ER. The ER proteome was profiled with a new three-dimensional, gel-based strategy. From 6152 and 6935 MS spectra, 903 and 1042 proteins were identified with at least two peptides matches at 95% confidence in the rough (r) and smooth (s) ER, respectively. Comparison of the rER and sER proteomes showed that calcium-binding proteins are significantly enriched in the sER suggesting that the ion-binding function of the ER is compartmentalized. Comparison of the rat and mouse ER proteomes showed that 662 proteins were common to both, comprising 53.5% and 49.3% of those proteomes, respectively. We proposed that these proteins were stably expressed proteins that were essential for the maintenance of ER function. GO annotation with a hypergeometric model proved this hypothesis. Unexpectedly, 210 unknown proteins and some proteins previously reported to occur in the cytosol were highly enriched in the ER. This study provides a reference map for the ER proteome of liver. Identification of new ER proteins will enhance our current understanding of the ER and also suggest new functions for this organelle. PMID:20073521

  2. The endoplasmic reticulum: a social network in plant cells.

    PubMed

    Chen, Jun; Doyle, Caitlin; Qi, Xingyun; Zheng, Huanquan

    2012-11-01

    The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules. The ER plays crucial roles in the biosynthesis and transport of proteins and lipids, and in calcium (Ca(2+) ) regulation in compartmentalized eukaryotic cells including plant cells. To support its well-segregated functions, the shape of the ER undergoes notable changes in response to both developmental cues and outside influences. In this review, we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER, and the importance of the interconnected ER network in cell polarity. In animal and yeast cells, two family proteins, the reticulons and DP1/Yop1, are required for shaping high-curvature ER tubules, while members of the atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network. In plant cells, recent data also indicate that the reticulons are involved in shaping ER tubules, while RHD3, a plant member of the atlastin GTPases, is required for the generation of an interconnected ER network. We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles, with a focus on how the ER and Golgi interplay in plant cells. PMID:23046093

  3. N-Linked Protein Glycosylation in the Endoplasmic Reticulum

    PubMed Central

    Breitling, Jörg; Aebi, Markus

    2013-01-01

    The attachment of glycans to asparagine residues of proteins is an abundant and highly conserved essential modification in eukaryotes. The N-glycosylation process includes two principal phases: the assembly of a lipid-linked oligosaccharide (LLO) and the transfer of the oligosaccharide to selected asparagine residues of polypeptide chains. Biosynthesis of the LLO takes place at both sides of the endoplasmic reticulum (ER) membrane and it involves a series of specific glycosyltransferases that catalyze the assembly of the branched oligosaccharide in a highly defined way. Oligosaccharyltransferase (OST) selects the Asn-X-Ser/Thr consensus sequence on polypeptide chains and generates the N-glycosidic linkage between the side-chain amide of asparagine and the oligosaccharide. This ER-localized pathway results in a systemic modification of the proteome, the basis for the Golgi-catalyzed modification of the N-linked glycans, generating the large diversity of N-glycoproteome in eukaryotic cells. This article focuses on the processes in the ER. Based on the highly conserved nature of this pathway we concentrate on the mechanisms in the eukaryotic model organism Saccharomyces cerevisiae. PMID:23751184

  4. Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules

    PubMed Central

    Smyth, Jeremy T.; Schoborg, Todd A.; Bergman, Zane J.; Riggs, Blake; Rusan, Nasser M.

    2015-01-01

    Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. We have addressed this by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. We show that in Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, we show that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species. PMID:26289801

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

    SciTech Connect

    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 presence 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.

  6. 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.

  7. Role of endoplasmic reticulum stress in drug-induced toxicity.

    PubMed

    Foufelle, Fabienne; Fromenty, Bernard

    2016-02-01

    Drug-induced toxicity is a key issue for public health because some side effects can be severe and life-threatening. These adverse effects can also be a major concern for the pharmaceutical companies since significant toxicity can lead to the interruption of clinical trials, or the withdrawal of the incriminated drugs from the market. Recent studies suggested that endoplasmic reticulum (ER) stress could be an important event involved in drug liability, in addition to other key mechanisms such as mitochondrial dysfunction and oxidative stress. Indeed, drug-induced ER stress could lead to several deleterious effects within cells and tissues including accumulation of lipids, cell death, cytolysis, and inflammation. After recalling important information regarding drug-induced adverse reactions and ER stress in diverse pathophysiological situations, this review summarizes the main data pertaining to drug-induced ER stress and its potential involvement in different adverse effects. Drugs presented in this review are for instance acetaminophen (APAP), arsenic trioxide and other anticancer drugs, diclofenac, and different antiretroviral compounds. We also included data on tunicamycin (an antibiotic not used in human medicine because of its toxicity) and thapsigargin (a toxic compound of the Mediterranean plant Thapsia garganica) since both molecules are commonly used as prototypical toxins to induce ER stress in cellular and animal models. PMID:26977301

  8. LDL–cholesterol transport to the endoplasmic reticulum: current concepts

    PubMed Central

    Pfisterer, Simon G.; Peränen, Johan; Ikonen, Elina

    2016-01-01

    Purpose of review In this article, we summarize the present information related to the export of LDL-derived cholesterol from late endosomes, with a focus on Nieman-Pick disease, type C1 (NPC1) cholesterol delivery toward the endoplasmic reticulum (ER). We review data suggesting that several pathways may operate in parallel, including membrane transport routes and membrane contact sites (MCSs). Recent findings There is increasing appreciation that MCSs provide an important mechanism for intermembrane lipid transfer. In late endosome–ER contacts, three protein bridges involving oxysterol binding protein related protein (ORP)1L-vesicle associated membrane protein-associated protein (VAP), steroidogenic acute regulatory protein (StAR)D3-VAP and ORP5-NPC1 proteins have been reported. How much they contribute to the flux of LDL–cholesterol to the ER is currently open. Studies for lipid transfer via MCSs have been most advanced in Saccharomyces cerevisiae. Recently, a new sterol-binding protein family conserved between yeast and man was identified. Its members localize at MCSs and were named lipid transfer protein anchored at membrane contact sites (Lam) proteins. In yeast, sterol transfer between the ER and the yeast lysosome may be facilitated by a Lam protein. Summary Increasing insights into the role of MCSs in directional sterol delivery between membranes propose that they might provide routes for LDL–cholesterol transfer to the ER. Future work should reveal which specific contacts may operate for this, and how they are controlled by cholesterol homeostatic machineries. PMID:27054443

  9. PERK-opathies: An Endoplasmic Reticulum Stress Mechanism Underlying Neurodegeneration.

    PubMed

    Bell, Michelle C; Meier, Shelby E; Ingram, Alexandria L; Abisambra, Jose F

    2016-01-01

    The unfolded protein response (UPR) plays a vital role in maintaining cell homeostasis as a consequence of endoplasmic reticulum (ER) stress. However, prolonged UPR activity leads to cell death. This time-dependent dual functionality of the UPR represents the adaptive and cytotoxic pathways that result from ER stress. Chronic UPR activation in systemic and neurodegenerative diseases has been identified as an early sign of cellular dyshomeostasis. The Protein Kinase R-like ER Kinase (PERK) pathway is one of three major branches in the UPR, and it is the only one to modulate protein synthesis as an adaptive response. The specific identification of prolonged PERK activity has been correlated with the progression of disorders such as diabetes, Alzheimer's disease, and cancer, suggesting that PERK plays a role in the pathology of these disorders. For the first time, the term "PERK-opathies" is used to group these diseases in which PERK mediates detriment to the cell culminating in chronic disorders. This article reviews the literature documenting links between systemic disorders with the UPR, but with a specific emphasis on the PERK pathway. Then, articles reporting links between the UPR, and more specifically PERK, and neurodegenerative disorders are presented. Finally, a therapeutic perspective is discussed, where PERK interventions could be potential remedies for cellular dysfunction in chronic neurodegenerative disorders. PMID:26679859

  10. STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions

    PubMed Central

    Carrasco, Silvia; Meyer, Tobias

    2013-01-01

    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 Ca2+ levels and directly activate Orai PM Ca2+ channels across the junction space. In an inverse process, a voltage-gated PM Ca2+ channel can directly open ER ryanodine-receptor Ca2+ channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca2+ 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. PMID:21548779

  11. The protective effect of the earthworm active ingredients on hepatocellular injury induced by endoplasmic reticulum stress.

    PubMed

    Wang, Qi; Duan, Leng-Xin; Xu, Zheng-Shun; Wang, Jian-Gang; Xi, Shou-Min

    2016-08-01

    The earthworm is a widely used Chinese herbal medicine. There are more than 40 prescriptions including earthworms in the "Compendium of Materia Medica". TCM theory holds that earthworms exert antispasmodic and antipyretic effects through the liver meridian to calm the liver. However, the clinical effect of earthworms on liver injury has not been clearly demonstrated. We have previously established a method to extract the active ingredients from earthworms (hereinafter referred to as EWAs) [1]. In the present study, we observed protective effect of the EWAs on tunicamycin-induced ERS (endoplasmic reticulum stress) model in human hepatic L02 cells. The results showed that the EWAs promote proliferation and reduced apoptosis of ERS model in L02 cells (P<0.01). The up-regulation of ERS-related proteins, including PERK (protein kinase RNA-like endoplasmic reticulum kinase), eIF2a (eukaryotic translation initiation factor 2a), ATF4 (activating transcription factor 4) and CHOP (CCAAT/enhancer binding protein homologous protein), in L02 cell under ERS was inhibited by treatment of the EWAs (P<0.01). In summary, our data suggest the EWAs can significant attenuate ERS-induced hepatocyte injury via PERK-eIF2a-ATF4 pathway. PMID:27470367

  12. Effects of a Sublethal and Transient Stress of the Endoplasmic Reticulum on the Mitochondrial Population.

    PubMed

    Vannuvel, Kayleen; Van Steenbrugge, Martine; Demazy, Catherine; Ninane, Noëlle; Fattaccioli, Antoine; Fransolet, Maude; Renard, Patricia; Raes, Martine; Arnould, Thierry

    2016-09-01

    Endoplasmic reticulum (ER) and mitochondria are not discrete intracellular organelles but establish close physical and functional interactions involved in several biological processes including mitochondrial bioenergetics, calcium homeostasis, lipid synthesis, and the regulation of apoptotic cell death pathways. As many cell types might face a transient and sublethal ER stress during their lifetime, it is thus likely that the adaptive UPR response might affect the mitochondrial population. The aim of this work was to study the putative effects of a non-lethal and transient endoplasmic reticulum stress on the mitochondrial population in HepG2 cells. The results show that thapsigargin and brefeldin A, used to induce a transient and sublethal ER stress, rapidly lead to the fragmentation of the mitochondrial network associated with a decrease in mitochondrial membrane potential, O2 (•-) production and less efficient respiration. These changes in mitochondrial function are transient and preceded by the phosphorylation of JNK. Inhibition of JNK activation by SP600125 prevents the decrease in O2 (•-) production and the mitochondrial network fragmentation observed in cells exposed to the ER stress but has no impact on the reduction of the mitochondrial membrane potential. In conclusion, our data show that a non-lethal and transient ER stress triggers a rapid activation of JNK without inducing apoptosis, leading to the fragmentation of the mitochondrial network and a reduction of O2 (•-) production. J. Cell. Physiol. 231: 1913-1931, 2016. © 2015 Wiley Periodicals, Inc. PMID:26680008

  13. Role of p97 and Syntaxin 5 in the Assembly of Transitional Endoplasmic Reticulum

    PubMed Central

    Roy, Line; Bergeron, John J.M.; Lavoie, Christine; Hendriks, Rob; Gushue, Jennifer; Fazel, Ali; Pelletier, Amélie; Morré, D. James; Subramaniam, V. Nathan; Hong, Wanjin; Paiement, Jacques

    2000-01-01

    Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc−1) isolated from rat liver homogenates reconstitute tER by Mg2+GTP- and Mg2+ATP-hydrolysis–dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway. PMID:10930451

  14. Gelatin Nanostructured Lipid Carriers Incorporating Nerve Growth Factor Inhibit Endoplasmic Reticulum Stress-Induced Apoptosis and Improve Recovery in Spinal Cord Injury.

    PubMed

    Zhu, Si-Pin; Wang, Zhou-Guang; Zhao, Ying-Zheng; Wu, Jiang; Shi, Hong-Xue; Ye, Li-Bing; Wu, Fen-Zan; Cheng, Yi; Zhang, Hong-Yu; He, Songbin; Wei, Xiaojie; Fu, Xiao-Bing; Li, Xiao-Kun; Xu, Hua-Zi; Xiao, Jian

    2016-09-01

    Clinical translation of growth factor therapies faces multiple challenges; the most significant one is the short half-life of the naked protein. Gelatin nanostructured lipid carriers (GNLs) had previously been used to encapsulate the basic fibroblast growth factor to enhance the functional recovery in hemiparkinsonian rats. In this research, we comparatively study the enhanced therapy between nerve growth factor (NGF) loaded GNLs (NGF-GNLs) and NGF only in spinal cord injury (SCI). The effects of NGF-GNLs and NGF only were tested by the Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test, and footprint analysis. Western blot analysis and immunofluorescent staining were further performed to identify the expression of ER stress-related proteins, neuron-specific marker neuronal nuclei (NeuN), and growth-associated protein 43 (GAP43). Correlated downstream signals Akt/GSK-3β and ERK1/2 were also analyzed with or without inhibitors. Results showed that NGF-GNLs, compared to NGF only, enhanced the neuroprotection effect in SCI rats. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 inhibited by NGF-GNL treatment were more obvious. Meanwhile, NGF-GNLs in the recovery of SCI are related to the inhibition of ER stress-induced cell death via the activation of downstream signals PI3K/Akt/GSK-3β and ERK1/2. PMID:26232067

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

    SciTech Connect

    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 viruses 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.

  16. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    PubMed Central

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  17. Diverse roles of endoplasmic reticulum stress sensors in bacterial infection.

    PubMed

    Pillich, Helena; Loose, Maria; Zimmer, Klaus-Peter; Chakraborty, Trinad

    2016-12-01

    Bacterial infection often leads to cellular damage, primarily marked by loss of cellular integrity and cell death. However, in recent years, it is being increasingly recognized that, in individual cells, there are graded responses collectively termed cell-autonomous defense mechanisms that induce cellular processes designed to limit cell damage, enable repair, and eliminate bacteria. Many of these responses are triggered not by detection of a particular bacterial effector or ligand but rather by their effects on key cellular processes and changes in homeostasis induced by microbial effectors when recognized. These in turn lead to a decrease in essential cellular functions such as protein translation or mitochondrial respiration and the induction of innate immune responses that may be specific to the cellular deficit induced. These processes are often associated with specific cell compartments, e.g., the endoplasmic reticulum (ER). Under non-infection conditions, these systems are generally involved in sensing cellular stress and in inducing and orchestrating the subsequent cellular response. Thus, perturbations of ER homeostasis result in accumulation of unfolded proteins which are detected by ER stress sensors in order to restore the normal condition. The ER is also important during bacterial infection, and bacterial effectors that activate the ER stress sensors have been discovered. Increasing evidence now indicate that bacteria have evolved strategies to differentially activate different arms of ER stress sensors resulting in specific host cell response. In this review, we will describe the mechanisms used by bacteria to activate the ER stress sensors and discuss their role during infection. PMID:26883353

  18. Topography of glycosylation reactions in the rough endoplasmic reticulum membrane

    SciTech Connect

    Perez, M.; Hirschberg, C.B.

    1986-05-25

    The translocation of UDP-glucose and GDP-mannose from an external to a luminal compartment has been examined in rat liver vesicles derived from the rough endoplasmic reticulum (RER). RER vesicles with the same topographical orientation as in vivo were incubated with a mixture of (/sup 3/H)UDP-glucose and UDP-(/sup 14/C)glucose to demonstrate that the intact sugar nucleotide was translocated into the lumen of the vesicles. The translocation of UDP-glucose was dependent on temperature and was saturable at high concentrations of the sugar nucleotide. The transfer of glucose to endogenous acceptors was dependent on the translocation of UDP-glucose into the lumen of the RER since leaky vesicles resulted in both a decrease in transport and transfer of glucose to endogenous acceptors. Preliminary results suggest that the mechanism of UDP-glucose transport into RER-derived vesicles is via a coupled exchange with luminal UMP. Using the same experimental approach to detect translocation of UDP-glucose into the lumen of RER vesicles, we were unable to detect transport of GDP-mannose. Incubation of leaky vesicles with GDP-mannose resulted in stimulation of the amount of mannose transferred to endogenous acceptors, in marked contrast to that observed for UDP-glucose and UDP-N-acetylglucosamine. These results suggest that whereas UDP-glucose is translocated across the RER membrane in vitro, GDP-mannose is not transported. In addition, these results tentatively suggest that there is asymmetric synthesis of the lipid-linked oligosaccharides within the membrane of the RER.

  19. Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs

    PubMed Central

    Wakai, Takuya; Zhang, Nan; Vangheluwe, Peter; Fissore, Rafael A.

    2013-01-01

    Summary Changes in the intracellular concentration of free calcium ([Ca2+]i) regulate diverse cellular processes including fertilization. In mammalian eggs, the [Ca2+]i changes induced by the sperm unfold in a pattern of periodical rises, also known as [Ca2+]i oscillations. The source of Ca2+ during oscillations is the endoplasmic reticulum ([Ca2+]ER), but it is presently unknown how [Ca2+]ER is regulated. Here, we show using mouse eggs that [Ca2+]i oscillations induced by a variety of agonists, including PLCζ, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [Ca2+]ER and cause differential effects on the refilling and overall load of [Ca2+]ER. We also found that Ca2+ influx is required to refill [Ca2+]ER, because the loss of [Ca2+]ER was accelerated in medium devoid of Ca2+. Pharmacological inactivation of the function of the mitochondria and of the Ca2+-ATPase pumps PMCA and SERCA altered the pattern of oscillations and abruptly reduced [Ca2+]ER, especially after inactivation of mitochondria and SERCA functions. We also examined the expression of SERCA2b protein and found that it was expressed throughout oocyte maturation and attained a conspicuous cortical cluster organization in mature eggs. We show that its overexpression reduces the duration of inositol-1,4,5-trisphosphate-induced [Ca2+]i rises, promotes initiation of oscillations and enhances refilling of [Ca2+]ER. Collectively, our results provide novel insights on the regulation of [Ca2+]ER oscillations, which underlie the unique Ca2+-signalling system that activates the developmental program in mammalian eggs. PMID:24101727

  20. Targeted induction of endoplasmic reticulum stress induces cartilage pathology.

    PubMed

    Rajpar, M Helen; McDermott, Ben; Kung, Louise; Eardley, Rachel; Knowles, Lynette; Heeran, Mel; Thornton, David J; Wilson, Richard; Bateman, John F; Poulsom, Richard; Arvan, Peter; Kadler, Karl E; Briggs, Michael D; Boot-Handford, Raymond P

    2009-10-01

    Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS), a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS-causing mutation (COL10A1 p.Asn617Lys) to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER) stress and a robust unfolded protein response (UPR) due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF-mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress-inducing protein (the cog mutant of thyroglobulin) in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant extracellular matrix

  1. Endoplasmic reticulum stress: key promoter of rosacea pathogenesis.

    PubMed

    Melnik, Bodo C

    2014-12-01

    Recent scientific interest in the pathogenesis of rosacea focuses on abnormally high facial skin levels of cathelicidin and the trypsin-like serine protease kallikrein 5 (KLK5) that cleaves the cathelicidin precursor protein into the bioactive fragment LL-37, which exerts crucial proinflammatory, angiogenic and antimicrobial activities. Furthermore, increased expression of toll-like receptor 2 (TLR2) has been identified in rosacea skin supporting the participation of the innate immune system. Notably, TLRs are expressed on sensory neurons and increase neuronal excitability linking TLR signalling to the transmission of neuroinflammatory responses. It is the intention of this viewpoint to present a unifying concept that links all known clinical trigger factors of rosacea such as UV irradiation, heat, skin irritants and special foods to one converging point: enhanced endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR). ER stress via upregulation of transcription factor ATF4 increases TLR2 expression, resulting in enhanced production of cathelicidin and KLK5 mediating downstream proinflammatory, angiogenic and antimicrobial signalling. The presented concept identifies rosacea trigger factors as environmental stressors that enhance the skin's ER stress response. Exaggerated cutaneous ER stress that stimulates the TLR2-driven inflammatory response may involve sebocytes, keratinocytes, monocyte-macrophages and sensory cutaneous neurons. Finally, all antirosacea drugs are proposed to attenuate the ER stress signalling cascade at some point. Overstimulated ER stress signalling may have evolutionarily evolved as a compensatory mechanism to balance impaired vitamin D-driven LL-37-mediated antimicrobial defenses due to lower exposure of UV-B irradiation of the northern Celtic population. PMID:25047092

  2. Endoplasmic reticulum aminopeptidase 1 and rheumatic disease: functional variation

    PubMed Central

    Tran, Tri M.; Colbert, Robert A.

    2015-01-01

    Purpose of review To review recent developments in our understanding of endoplasmic reticulum (ER) aminopeptidase-1 (ERAP1) function in relation to its role in MHC class I peptide presentation and HLA class I-associated diseases. Recent findings ERAP1 polymorphisms exhibiting loss-of-function have been associated with protection from ankylosing spondylitis (AS). The aminopeptidase function of ERAP1 optimizes peptides for binding and presentation by MHC class I. Most studies have revealed reduced MHC class I expression in situations of reduced ERAP1 function. Under these circumstances the presented peptides are often N-terminally extended, and cell surface complexes are unstable and fall apart more readily. In contrast, peptides presented by HLA-B*27:05 when ERAP1 is silenced are frequently extended on the C-terminus. Recent work has emphasized the importance of assessing the function of allotypes encoded by ERAP1 haplotypes, rather than effects of single amino acid substitutions. The allotypes found in a series of AS patients were poorer at restoring HLA-B27 expression than allotypes found in unaffected controls, which may seem contrary to the genetic data linking loss-of-function to protection. Summary More work is needed to understand how ERAP1 variants associated with risk and protection influence the quality and quantity of peptides available for binding to HLA class I molecules in the ER. Moreover, we need to determine allele-specific effects of ERAP1 variants in the context of HLA-B*51 and HLA-Cw*6, which are associated with Behçet’s disease and psoriasis, respectively. PMID:26002027

  3. 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. PMID:26775176

  4. Ethanol Induces Endoplasmic Reticulum Stress in the Developing Brain

    PubMed Central

    Ke, Zunji; Wang, Xin; Liu, Ying; Fan, Zhiqin; Chen, Gang; Xu, Mei; Bower, Kimberley A.; Frank, Jacqueline A.; Li, Mingtao; Fang, Shengyun; Shi, Xianglin; Luo, Jia

    2016-01-01

    Background Ethanol exposure during brain development causes profound damages to the central nervous system (CNS). The underlying cellular/molecular mechanisms remain unclear. The endoplasmic reticulum (ER) is involved in posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress, which is characterized by translational attenuation, synthesis of ER chaperone proteins, and activation of transcription factors. Sustained ER stress ultimately leads to cell death. ER stress is implicated in various neurodegenerative processes. Methods Using a third trimester equivalent mouse model of ethanol exposure, we tested the hypothesis that ethanol induces ER stress in the developing brain. Seven-day-old C57BL/6 mice were acutely exposed to ethanol by subcutaneous injection and the expression of ER stress-inducible proteins (ERSIPs) and signaling pathways associated with ER stress were examined. Results Ethanol exposure significantly increased the expression of ERSIPs and activated signaling pathways associated with ER stress; these include ATF6, CHOP/GADD153, GRP78, and mesencephalic astrocyte-derived neurotrophic factor as well as the phosphorylation of IRE1α, eIF2α, PERK, and PKR. The ethanol-induced increase in ERSIPs occurred within 4 hours of ethanol injection, and levels of some ERSIPs remained elevated after 24 hours of ethanol exposure. Ethanol-induced increase in phosphorylated eIF2α, caspase-12, and CHOP was distributed in neurons of specific areas of the cerebral cortex, hippocampus, and thalamus. Conclusions Our finding indicates that ethanol induces ER stress in immature neurons, providing novel insight into ethanol’s detrimental effect on the developing CNS. PMID:21599712

  5. Ca2+-Dependent Endoplasmic Reticulum Stress Regulates Mechanical Stress-Mediated Cartilage Thinning.

    PubMed

    Zhu, M; Zhou, S; Huang, Z; Wen, J; Li, H

    2016-07-01

    Our previous study identified that endoplasmic reticulum stress (ERS) plays a critical role in chondrocyte apoptosis and mandibular cartilage thinning in response to compressive mechanical force, although the underlying mechanisms remain elusive. Because the endoplasmic reticulum (ER) is a primary site of intracellular Ca(2+) storage, we hypothesized that Ca(2+)-dependent ERS might be involved in mechanical stress-mediated mandibular cartilage thinning. In this study, we used in vitro and in vivo models to determine Ca(2+) concentrations, histological changes, subcellular changes, apoptosis, and the expression of ERS markers in mandibular cartilage and chondrocytes. The results showed that in chondrocytes, cytosolic Ca(2+) ([Ca(2+)]i) was dramatically increased by compressive mechanical force. Interestingly, the inhibition of Ca(2+) channels by ryanodine and 2-aminoethoxydiphenyl borate, inhibitors of ryanodine receptors and inositol trisphosphate receptors, respectively, partially rescued mechanical force-mediated mandibular cartilage thinning. Furthermore, chondrocyte apoptosis was also compromised by inhibiting the increase in [Ca(2+)]i that occurred in response to compressive mechanical force. Mechanistically, the ERS induced by compressive mechanical force was also repressed by [Ca(2+)]i inhibition, as demonstrated by a decrease in the expression of the ER stress markers 78 kDa glucose-regulated protein (GRP78) and 94 kDa glucose-regulated protein (GRP94) at both the mRNA and protein levels. Collectively, these data identified [Ca(2+)]i as a critical mediator of the pathological changes that occur in mandibular cartilage under compressive mechanical force and shed light on the treatment of mechanical stress-mediated cartilage degradation. PMID:27053115

  6. 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.

  7. The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells

    PubMed Central

    Johnson, Guyla G.; White, Misti C.; Wu, Jian-He; Vallejo, Matthew; Grimaldi, Maurizio

    2014-01-01

    Background The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein processing. Accumulation of unfolded proteins following ER Ca2+ depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR. Methods Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription–PCR, viability assays, and single cell Ca2+ imaging. Results ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca2+, activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca2+. Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca2+ permeability causes opposite effects on ERSR deployment and death of malignant glioma cells. Conclusions Excessive ER Ca2+ loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies. PMID:24569545

  8. PARM-1 Is an Endoplasmic Reticulum Molecule Involved in Endoplasmic Reticulum Stress-Induced Apoptosis in Rat Cardiac Myocytes

    PubMed Central

    Isodono, Koji; Takahashi, Tomosaburo; Imoto, Hiroko; Nakanishi, Naohiko; Ogata, Takehiro; Asada, Satoshi; Adachi, Atsuo; Ueyama, Tomomi; Oh, Hidemasa; Matsubara, Hiroaki

    2010-01-01

    To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER). In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease. PMID:20305782

  9. 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. PMID:26711579

  10. Possible Pharmacological Approach Targeting Endoplasmic Reticulum Stress to Ameliorate Leptin Resistance in Obesity

    PubMed Central

    Hosoi, Toru; Ozawa, Koichiro

    2016-01-01

    Obesity is associated with metabolic syndrome, such as diabetes, hypertension, and hyperlipidemia. Therefore, drug development for the treatment of obesity is needed. Leptin is an anti-obesity hormone that inhibits food intake and increases energy metabolism, and, as such, treatments involving leptin were expected to be beneficial for obesity; however, since most obese patients are in a state of leptin resistance, these treatments may not be useful. Therefore, the amelioration of leptin resistance has recently been attracting interest as a treatment for obesity. The mechanisms underlying the development of leptin resistance need to be elucidated in more detail. Endoplasmic reticulum (ER) stress was recently suggested to be involved in the pathogenesis of leptin resistance. The molecular mechanisms responsible for leptin resistance and possible pharmacological treatments for obesity have been discussed herein, with a focus on ER stress. PMID:27375555

  11. Endoplasmic reticulum stress-mediated induction of SESTRIN 2 potentiates cell survival

    PubMed Central

    Ayo, Abiodun; Pakos-Zebrucka, Karolina; Patterson, John B

    2016-01-01

    Upregulation of SESTRIN 2 (SESN2) has been reported in response to diverse cellular stresses. In this study we demonstrate SESTRIN 2 induction following endoplasmic reticulum (ER) stress. ER stress-induced increases in SESTRIN 2 expression were dependent on both PERK and IRE1/XBP1 arms of the unfolded protein response (UPR). SESTRIN 2 induction, post ER stress, was responsible for mTORC1 inactivation and contributed to autophagy induction. Conversely, knockdown of SESTRIN 2 prolonged mTORC1 signaling, repressed autophagy and increased ER stress-induced cell death. Unexpectedly, the increase in ER stress-induced cell death was not linked to autophagy inhibition. Analysis of UPR pathways identified prolonged eIF2α, ATF4 and CHOP signaling in SESTRIN 2 knockdown cells following ER stress. SESTRIN 2 regulation enables UPR derived signals to indirectly control mTORC1 activity shutting down protein translation thus preventing further exacerbation of ER stress. PMID:26930721

  12. A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins

    PubMed Central

    Merianda, Tanuja T.; Lin, Andrew C.; Lam, Joyce S.Y.; Vuppalanchi, Deepika; Willis, Dianna E.; Karin, Norman; Holt, Christine E.; Twiss, Jeffery L.

    2013-01-01

    Subcellular localization of protein synthesis provides a means to regulate the protein composition in far reaches of a cell. This localized protein synthesis gives neuronal processes autonomy to rapidly respond to extracellular stimuli. Locally synthesized axonal proteins enable neurons to respond to guidance cues and can help to initiate regeneration after injury. Most studies of axonal mRNA translation have concentrated on cytoplasmic proteins. While ultrastructural studies suggest that axons do not have rough endoplasmic reticulum or Golgi apparatus, mRNAs for transmembrane and secreted proteins localize to axons. Here, we show that growing axons with protein synthetic activity contain ER and Golgi components needed for classical protein synthesis and secretion. Isolated axons have the capacity to traffic locally synthesized proteins into secretory pathways and inhibition of Golgi function attenuates translation-dependent axonal growth responses. Finally, the capacity for secreting locally synthesized proteins in axons appears to be increased by injury. PMID:19022387

  13. 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.

  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. Rg1 exhibits neuroprotective effects by inhibiting the endoplasmic reticulum stress-mediated c-Jun N-terminal protein kinase apoptotic pathway in a rat model of Alzheimer's disease.

    PubMed

    Mu, Jun-Shan; Lin, Hang; Ye, Jian-Xin; Lin, Min; Cui, Xiao-Ping

    2015-09-01

    The neuroprotective agents currently used to treat Alzheimer's disease (AD) often only target one aspect of the disease process. Therefore, identifying effective drug targets associated with the pathogenesis of AD is critical for the production of novel AD therapeutic strategies. The present study aimed to investigate the underlying mechanisms of the neuroprotective effects of Rg1 on a rat model of AD. A double transgenic β‑amyloid (Aβ) precursor protein/PS1 rat model was established, which co‑expressed mutations associated with AD. Aβ plaques and neurofibrillary tangles (NFTs) were detected by immunohistochemistry. The detection of the protein expression levels of caspase‑3 and terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling (TUNEL) staining were used to determine the level of apoptosis in the brain tissue. The expression levels of the endoplasmic reticulum (ER) stress biomarker, glucose‑regulated protein 78 (Grp78), and the mitochondrial apoptosis biomarkers, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax), were analyzed by western blotting. Furthermore, the expression of the proteins associated with the ER stress unfolded protein response (UPR) was determined, in order to examine the levels of ER stress. The mRNA expression of downstream genes of UPR were also detected by reverse transcription‑polymerase chain reaction. The protein expression levels of the apoptosis‑associated phosphorylated‑c‑Jun N‑terminal protein kinase (p‑JNK), caspase‑12 and cAMP response element‑binding transcription factor homologous protein were determined by western blotting. The results of the present study indicated that the accumulation of NFTs and Aβ plaques was significantly decreased in the Rg1‑treated AD rats, compared with untreated AD rats. The expression of caspase‑3 and the number of TUNEL‑positive cells were also significantly decreased in the Rg1‑treated rats, as compared with the AD rats

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

    SciTech Connect

    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{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 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{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+}.

  17. 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

  18. [Role of endoplasmic reticulum-plasma membrane junctions in intracellular calcium homeostasis and cardiovascular disease].

    PubMed

    Zhao, Ming; Jia, Hang-Huan; Xu, Man; Yu, Xiao-Jiang; Liu, Long-Zhu; Zang, Wei-Jin

    2016-08-25

    Calcium overload is one of the important mechanisms of cardiovascular disease. Endoplasmic reticulum is an important organelle which regulates intracellular calcium homeostasis by uptake, storage and mobilization of calcium. So it plays a critical role in regulation of intracellular calcium homeostasis. Endoplasmic reticulum, which is widely distributed in cytoplasm, has a large number of membrane junction sites. Recent studies have reported that these junction sites are distributed on plasma membrane and organelle membranes (mitochondria, lysosomes, Golgi apparatus, etc.), separately. They could form complexes to regulate calcium transport. In this review, we briefly outlined the recent research progresses of endoplasmic reticulum-plasma membrane junctions in intracellular calcium homeostasis and cardiovascular disease, which may offer a new strategy for prevention and treatment of cardiovascular disease. PMID:27546511

  19. The Role of Endoplasmic Reticulum Stress and Unfolded Protein Response in Atherosclerosis

    PubMed Central

    Ivanova, Ekaterina A.; Orekhov, Alexander N.

    2016-01-01

    Pathogenesis of atherosclerosis is a complex process involving several metabolic and signalling pathways. Accumulating evidence demonstrates that endoplasmic reticulum stress and associated apoptosis can be induced in the pathological conditions of atherosclerotic lesions and contribute to the disease progression. Notably, they may play a role in the development of vulnerable plaques that induce thrombosis and are therefore especially dangerous. Endoplasmic reticulum stress response is regulated by several signaling mechanisms that involve protein kinases and transcription factors. Some of these molecules can be regarded as potential therapeutic targets to improve treatment of atherosclerosis. In this review we will discuss the role of endoplasmic reticulum stress and apoptosis in atherosclerosis development in different cell types and summarize the current knowledge on potential therapeutic agents targeting molecules regulating these pathways and their possible use for anti-atherosclerotic therapy. PMID:26840309

  20. Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis.

    PubMed

    Bustamante, Juanita; Bersier, Geraldine; Badin, Romina Aron; Cymeryng, Cora; Parodi, Armando; Boveris, Alberto

    2002-05-01

    Early stages of rat thymocyte apoptosis measured as annexin-V positive events and induced by methylprednisolone (MPS), etoposide, and thapsigargin, showed a sequential increase in nitric oxide (NO) production by mitochondrial and endoplasmic reticulum membranes. Thapsigargin induced the highest NO production, a sevenfold increase as compared with untreated thymocytes, in mitochondrial and microsomal membranes. MPS and etoposide were equally effective in increasing NO production by mitochondrial membranes by a factor of 4-5, with only a slight increase in NO production by endoplasmic reticulum membranes. Western blot analysis of both types of membrane indicated that a nitric oxide synthase (NOS) isoenzyme is present in mitochondrial membranes and reacts with antibodies to i-NOS (type II), while reactivity to antibodies to e-NOS (type III) was restricted to endoplasmic reticulum. The participation of endoplasmic reticulum during apoptosis was further determined by alterations in UDP-Glucosyltransferase (UDP-GT) and NADPH cytochrome P450 reductase. Increased UDP-GT activity was observed after thapsigargin treatment, and no changes were found after treatment with etoposide or MPS. NADPH cytochrome P450 reductase activity markedly decreased during apoptosis, being stronger after thapsigargin treatment. The latest stage of the apoptotic process was measured by caspase activities. Caspase 3 activity was markedly increased by the three apoptosis inducers; caspase 6 was only activated by MPS and etoposide, while caspase 8 was not activated by any of these inducers. It is clear that mitochondria and endoplasmic reticulum are involved in thapsigargin induced thymocyte apoptosis. Meanwhile, other thymocyte apoptotic pathways, such as those induced by MPS or etoposide, seem to centrally involve mitochondria but not endoplasmic reticulum. PMID:12009851

  1. Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy.

    PubMed

    Wang, Hao; Sun, Ruo-Qiong; Camera, Daria; Zeng, Xiao-Yi; Jo, Eunjung; Chan, Stanley M H; Herbert, Terence P; Molero, Juan C; Ye, Ji-Ming

    2016-07-01

    The accumulation of unfolded proteins within the endoplasmic reticulum (ER) causes ER stress and activation of unfolded protein response (UPR). This response can trigger ER-associated degradation and autophagy, which clear unfolded proteins and restore protein homeostasis. Recently, it has become clear that ubiquitination plays an important role in the regulation of autophagy. In the present study, we investigated how the E3 ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 (Nedd4-2) interacts with ER stress and autophagy. In mice, we found that an increase in the expression of Nedd4-2, which was concomitant with the activation of the UPR and autophagy, was caused by a prolonged high-fructose and high-fat diet that induces ER stress in the liver. Pharmacologic induction of ER stress also led to an increase in Nedd4-2 expression in cultured cells, which was coincident with UPR and autophagy activation. The inhibition of inositol-requiring enzyme 1 significantly suppressed Nedd4-2 expression. Moreover, increased Nedd4-2 expression in vivo was closely associated with the activation of inositol-requiring enzyme 1 and increased expression of the spliced form of X-box binding protein 1. Furthermore, knockdown of Nedd4-2 in cultured cells suppressed both basal autophagy and ER stress-induced autophagy, whereas overexpression of Nedd4-2-induced autophagy. Taken together, our findings provide evidence that Nedd4-2 is up-regulated in response to ER stress by the spliced form of X-box binding protein 1 and that this is important in the induction of an appropriate autophagic response.-Wang, H. Sun, R.-Q., Camera, D., Zeng, X.-Y., Jo, E., Chan, S. M. H., Herbert, T. P., Molero, J. C., Ye, J.-M. Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy. PMID:27022162

  2. Inhibitors of Endoplasmic Reticulum α-Glucosidases Potently Suppress Hepatitis C Virus Virion Assembly and Release▿

    PubMed Central

    Qu, Xiaowang; Pan, Xiaoben; Weidner, Jessica; Yu, Wenquan; Alonzi, Dominic; Xu, Xiaodong; Butters, Terry; Block, Timothy; Guo, Ju-Tao; Chang, Jinhong

    2011-01-01

    α-Glucosidases I and II are endoplasmic reticulum-resident enzymes that are essential for N-linked glycan processing and subsequent proper folding of glycoproteins. In this report, we first demonstrate that downregulation of the expression of α-glucosidase I, II, or both in Huh7.5 cells by small hairpin RNA technology inhibited the production of hepatitis C virus (HCV). In agreement with the essential role of α-glucosidases in HCV envelope glycoprotein processing and folding, treatment of HCV-infected cells with a panel of imino sugar derivatives, which are competitive inhibitors of α-glucosidases, did not affect intracellular HCV RNA replication and nonstructural protein expression but resulted in the inhibition of glycan processing and subsequent degradation of HCV E2 glycoprotein. As a consequence, HCV virion assembly and secretion were inhibited. In searching for imino sugars with better antiviral activity, we found that a novel imino sugar, PBDNJ0804, had a superior ability to inhibit HCV virion assembly and secretion. In summary, we demonstrated that glucosidases are important host factor-based antiviral targets for HCV infection. The low likelihood of drug-resistant virus emergence and potent antiviral efficacy of the novel glucosidase inhibitor hold promise for its development as a therapeutic agent for the treatment of chronic hepatitis C. PMID:21173177

  3. 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

  4. ALUMINUM ALTERS CALCIUM TRANSPORT IN PLASMA MEMBRANE AND ENDOPLASMIC RETICULUM FROM RAT BRAIN

    EPA Science Inventory

    Calcium is actively transported into intracellular organelles and out of the cytoplasm by Ca2+/Mg2+-ATPases located in the endoplasmic reticulum and plasma membranes. he effects of aluminum on calcium transport were examined in the adult rat brain. 5Ca-uptake was examined in micr...

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

    SciTech Connect

    Faria, Gisele; Cardoso, Cristina R.B.; Larson, Roy E.; Silva, Joao S.; Rossi, Marcos A.

    2009-01-15

    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 of 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.

  6. Bright fluorogenic squaraines with tuned cell entry for selective imaging of plasma membrane vs. endoplasmic reticulum.

    PubMed

    Collot, Mayeul; Kreder, Rémy; Tatarets, Anatoliy L; Patsenker, Leonid D; Mely, Yves; Klymchenko, Andrey S

    2015-12-14

    A rational design of squaraine dyes with lipophilic and zwitterionic groups tunes cell entry, allowing for selective far-red/near-infrared imaging of plasma membrane vs. endoplasmic reticulum. They exhibit up to 110-fold fluorescence enhancement in biomembranes and enable cellular imaging at 1 nM concentration, which make them the brightest membrane probes to date. PMID:26455447

  7. The endoplasmic reticulum protein folding factory and its chaperones: new targets for drug discovery?

    PubMed Central

    McLaughlin, Martin; Vandenbroeck, Koen

    2011-01-01

    Cytosolic heat shock proteins have received significant attention as emerging therapeutic targets. Much of this excitement has been triggered by the discovery that HSP90 plays a central role in the maintenance and stability of multifarious oncogenic membrane receptors and their resultant tyrosine kinase activity. Numerous studies have dealt with the effects of small molecules on chaperone- and stress-related pathways of the endoplasmic reticulum (ER). However, unlike cytosolic chaperones, relatively little emphasis has been placed upon translational avenues towards targeting of the ER for inhibition of folding/secretion of disease-promoting proteins. Here, we summarise existing small molecule inhibitors and potential future targets of ER chaperone-mediated inhibition. Client proteins of translational relevance in disease treatment are outlined, alongside putative future disease treatment modalities based on ER-centric targeted therapies. Particular attention is paid to cancer and autoimmune disorders via the effects of the GRP94 inhibitor geldanamycin and its population of client proteins, overloading of the unfolded protein response, and inhibition of members of the IL-12 family of cytokines by celecoxib and non-coxib analogues. PMID:20942857

  8. 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. PMID:21266191

  9. Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress

    PubMed Central

    Nakato, Ryosuke; Ohkubo, Yu; Konishi, Akari; Shibata, Mari; Kaneko, Yuki; Iwawaki, Takao; Nakamura, Tomohiro; Lipton, Stuart A.; Uehara, Takashi

    2015-01-01

    Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson’s disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death. PMID:26446798

  10. Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins.

    PubMed

    Kim, P K; Hollerbach, C; Trimble, W S; Leber, B; Andrews, D W

    1999-12-24

    The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane. PMID:10601239

  11. The combination of 1α,25dihydroxyvitaminD3 with resveratrol improves neuronal degeneration by regulating endoplasmic reticulum stress, insulin signaling and inhibiting tau hyperphosphorylation in SH-SY5Y cells.

    PubMed

    Cheng, Jinbo; Xia, Xianghou; Rui, Yehua; Zhang, Zengli; Qin, Liqiang; Han, Shufen; Wan, Zhongxiao

    2016-07-01

    Endoplasmic reticulum (ER) stress is a critical factor involved in the pathogenesis of Alzheimer's disease (AD). Vitamin D and resveratrol are two nutritional factors that have reported neuroprotective effects, and findings from cellular models suggest that resveratrol could potentiate vitamin D's effects. We aimed to determine the effects of vitamin D & resveratrol on ER stress mediated neurodegeneration and whether synergistic effects existed. Tunicamycin and Aβ25-35 was utilized to induce ER stress in SH-SY5Y cells, cells were then incubated with vitamin D and resveratrol. The combination of vitamin D & resveratrol completely reversed tunicamycin and Aβ25-35 induced cytotoxicity in SH-SY5Y cells, as well as elevation in ER stress markers (i.e.GRP78, p-eIF2α and CHOP), insulin signaling disruption (i.e. elevation in p-IRS-1serine307 and reduction in p-Akt serine473) and tau phosphorylation (i.e. reduction in p-GSK3β serine9, and elevation in p-Tau serine396 &404). Further studies are required to clarify whether the observed synergistic effects in the present study would also existed in vivo, this will lay scientific foundation whether the combination of vitamin D with resveratrol might be an effective maneuver in the treatment of AD in human subjects. PMID:27133915

  12. Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress.

    PubMed

    Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

    2015-01-14

    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. PMID:25429417

  13. Ascorbic acid protects against cadmium-induced endoplasmic reticulum stress and germ cell apoptosis in testes.

    PubMed

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

    2012-11-01

    Cadmium (Cd) is a testicular toxicant which induces endoplasmic reticulum (ER) stress and germ cell apoptosis in testes. This study investigated the effects of ascorbic acid on Cd-evoked ER stress and germ cell apoptosis in testes. Male mice were intraperitoneally injected with CdCl(2) (2.0 mg/kg). As expected, a single dose of Cd induced testicular germ cell apoptosis. Interestingly, Cd-triggered testicular germ cell apoptosis was almost completely inhibited in mice treated with ascorbic acid. Interestingly, ascorbic acid significantly attenuated Cd-induced upregulation of GRP78 in testes. In addition, ascorbic acid significantly attenuated Cd-triggered testicular IRE1α and eIF2α phosphorylation and XBP-1 activation, indicating that this antioxidant counteracts Cd-induced unfolded protein response (UPR) in testes. Finally, ascorbic acid significantly attenuated Cd-evoked upregulation of CHOP and JNK phosphorylation, two components in ER stress-mediated apoptotic pathway. In conclusion, ascorbic acid protects mice from Cd-triggered germ cell apoptosis via inhibiting ER stress and UPR in testes. PMID:22569276

  14. Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

    PubMed Central

    Leichner, Gil S.; Avner, Rachel; Harats, Dror; Roitelman, Joseph

    2011-01-01

    In mammalian cells, the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), which catalyzes the rate-limiting step in the mevalonate pathway, is ubiquitylated and degraded by the 26 S proteasome when mevalonate-derived metabolites accumulate, representing a case of metabolically regulated endoplasmic reticulum-associated degradation (ERAD). Here, we studied which mevalonate-derived metabolites signal for HMGR degradation and the ERAD step(s) in which these metabolites are required. In HMGR-deficient UT-2 cells that stably express HMGal, a chimeric protein between β-galactosidase and the membrane region of HMGR, which is necessary and sufficient for the regulated ERAD, we tested inhibitors specific to different steps in the mevalonate pathway. We found that metabolites downstream of farnesyl pyrophosphate but upstream to lanosterol were highly effective in initiating ubiquitylation, dislocation, and degradation of HMGal. Similar results were observed for endogenous HMGR in cells that express this protein. Ubiquitylation, dislocation, and proteasomal degradation of HMGal were severely hampered when production of geranylgeranyl pyrophosphate was inhibited. Importantly, inhibition of protein geranylgeranylation markedly attenuated ubiquitylation and dislocation, implicating for the first time a geranylgeranylated protein(s) in the metabolically regulated ERAD of HMGR. PMID:21778231

  15. Endoplasmic reticulum stress-regulated CXCR3 pathway mediates inflammation and neuronal injury in acute glaucoma

    PubMed Central

    Ha, Y; Liu, H; Xu, Z; Yokota, H; Narayanan, S P; Lemtalsi, T; Smith, S B; Caldwell, R W; Caldwell, R B; Zhang, W

    2015-01-01

    Acute glaucoma is a leading cause of irreversible blindness in East Asia. The mechanisms underlying retinal neuronal injury induced by a sudden rise in intraocular pressure (IOP) remain obscure. Here we demonstrate that the activation of CXCL10/CXCR3 axis, which mediates the recruitment and activation of inflammatory cells, has a critical role in a mouse model of acute glaucoma. The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. Blockade of the CXCR3 pathway by deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1β and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia. PMID:26448323

  16. The antitumor natural compound falcarindiol promotes cancer cell death by inducing endoplasmic reticulum stress

    PubMed Central

    Jin, H R; Zhao, J; Zhang, Z; Liao, Y; Wang, C-Z; Huang, W-H; Li, S-P; He, T-C; Yuan, C-S; Du, W

    2012-01-01

    Falcarindiol (FAD) is a natural polyyne with various beneficial biological activities. We show here that FAD preferentially kills colon cancer cells but not normal colon epithelial cells. Furthermore, FAD inhibits tumor growth in a xenograft tumor model and exhibits strong synergistic killing of cancer cells with 5-fluorouracil, an approved cancer chemotherapeutic drug. We demonstrate that FAD-induced cell death is mediated by induction of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Decreasing the level of ER stress, either by overexpressing the ER chaperone protein glucose-regulated protein 78 (GRP78) or by knockout of components of the UPR pathway, reduces FAD-induced apoptosis. In contrast, increasing the level of ER stress by knocking down GRP78 potentiates FAD-induced apoptosis. Finally, FAD-induced ER stress and apoptosis is correlated with the accumulation of ubiquitinated proteins, suggesting that FAD functions at least in part by interfering with proteasome function, leading to the accumulation of unfolded protein and induction of ER stress. Consistent with this, inhibition of protein synthesis by cycloheximide significantly decreases the accumulation of ubiquitinated proteins and blocks FAD-induced ER stress and cell death. Taken together, our study shows that FAD is a potential new anticancer agent that exerts its activity through inducing ER stress and apoptosis. PMID:22914324

  17. Endoplasmic reticulum stress induces ligand-independent TNFR1-mediated necroptosis in L929 cells

    PubMed Central

    Saveljeva, S; Mc Laughlin, S L; Vandenabeele, P; Samali, A; Bertrand, M J M

    2015-01-01

    Endoplasmic reticulum (ER) stress-induced cellular dysfunction and death is associated with several human diseases. It has been widely reported that ER stress kills through activation of the intrinsic mitochondrial apoptotic pathway. Here we demonstrate that ER stress can also induce necroptosis, an receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL)-dependent form of necrosis. Remarkably, we observed that necroptosis induced by various ER stressors in L929 cells is dependent on tumor necrosis factor receptor 1 (TNFR1), but occurs independently of autocrine TNF or lymphotoxin α production. Moreover, we found that repression of either TNFR1, RIPK1 or MLKL did not protect the cells from death but instead allowed a switch to ER stress-induced apoptosis. Interestingly, while caspase inhibition was sufficient to protect TNFR1- or MLKL-deficient cells from death, rescue of the RIPK1-deficient cells additionally required RIPK3 depletion, indicating a switch back to RIPK3-dependent necroptosis in caspase-inhibited conditions. The finding that ER stress also induces necroptosis may open new therapeutic opportunities for the treatment of pathologies resulting from unresolved ER stress. PMID:25569104

  18. Thioredoxin-1 Increases Survival in Sepsis by Inflammatory Response Through Suppressing Endoplasmic Reticulum Stress.

    PubMed

    Chen, Guobing; Li, Xiang; Huang, Mengbing; Li, Mei; Zhou, Xiaoshuang; Li, Ye; Bai, Jie

    2016-07-01

    Sepsis is the main cause of death in critically ill patients, pathogenesis of which is still unclear. The nuclear factor κB (NF-κB) inflammatory signal pathway mediated by endoplasmic reticulum stress is involved in sepsis. Thioredoxin-1 (Trx-1) is an important protein of regulating oxidative stress. It plays a crucial role in the anti-oxidation, anti-apoptosis, and anti-inflammation. However, the role and the mechanism of Trx-1 in sepsis have not been extensively studied. In the present study, we showed that the survival was longer in sepsis induced by cecal ligation and puncture in Trx-1 overexpression transgenic (Tg) mice compared with wild-type mice. Wet/dry lung weight ratio was decreased in Trx-1 Tg mice. The levels of TNF-α and IL-1β in plasma and lung tissue were inhibited in Tg mice. The expressions of glucose-regulated protein 78, inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2, C/EBP homologous protein, NF-κB, and inhibitors of NF-κBα were increased in lung tissue. More importantly, the overexpression of Trx-1 in transgenic mice suppressed NF-κB inflammatory signal pathway by inhibiting the activation of molecules involved in ER stress. Our results suggest that Trx-1 may play protective role in extending survival in sepsis by regulating inflammatory response through suppressing ER stress. PMID:27299588

  19. Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress.

    PubMed

    Tanaka, Ken-ichiro; Yamaguchi, Toru; Kaji, Hiroshi; Kanazawa, Ippei; Sugimoto, Toshitsugu

    2013-08-30

    Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus. PMID:23933252

  20. Stop-and-move of a marginally hydrophobic segment translocating across the endoplasmic reticulum membrane.

    PubMed

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

    2013-09-01

    Many membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane via the protein-conducting channel, the so-called translocon. The hydrophobic transmembrane segment of the translocating nascent polypeptide chain stops at the translocon and then moves laterally into the membrane. Partitioning of the hydrophobic segment into the membrane is the primary determinant for membrane insertion. Here, we examined the behavior of a marginally hydrophobic segment at the translocon and found that its stop-translocation was greatly affected by the C-terminally attached ribosomes. The marginally hydrophobic segment first stops at the membrane and then moves into the lumen as long as the nascent chain is attached to translating ribosomes. When it is released from the ribosome by the termination codon, the marginally hydrophobic segment does not move. Puromycin or RNase treatment also suppressed movement. The movement was reversibly inhibited by high-salt conditions and irreversibly inhibited by ethylenediaminetetraacetic acid. There is an unstable state prior to the stable membrane insertion of the transmembrane segment. This characteristic state is maintained by the synthesizing ribosome. PMID:23747484

  1. Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum.

    PubMed Central

    Lanoix, J; Roy, L; Paiement, J

    1989-01-01

    As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2508629

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

    SciTech Connect

    Naranmandura, Hua; Xu, Shi; Koike, Shota; Pan, Li Qiang; Chen, Bin; Wang, Yan Wei; Rehman, Kanwal; Wu, Bin; Chen, Zhe; Suzuki, Noriyuki

    2012-05-01

    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 reticulum (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 in

  3. Evidence of endoplasmic reticulum-related Ca sup 2+ ATPase in human microvascular endothelial cells

    SciTech Connect

    Bikfalvi, A.; Enouf, J.; Bredoux, R.; Dupuy, E.; Bourdeau, N.; Levy-Toledano, S.; Tobelem, G. ); Lompre, A. )

    1989-09-01

    The authors demonstrated by immunological and molecular methods the presence of a reticulum endoplasmic-related Ca{sup 2+}-ATPase in human omental microvascular endothelial cells (HOME cells). HOME cells reacted positively with a previously characterized sarcoplasmic reticulum Ca{sup 2+}-ATPase antibody as demonstrated by indirect immunofluorescence. Western blotting revealed that the antibody recognized a 95-100 kDa protein. {sup 35}S-Metabolic labeling led to the detection of a similar protein with which the purified sarcoplasmic reticulum Ca{sup 2+}-ATPase compete. Dot-blotting experiments indicated that a substantial amount of Ca{sup 2+}-ATPase was present in HOME cell membranes. In addition, Northern blot analysis using a cDNA probe from cardiac sarcoplasmic reticulum showed the presence of mRNA species of 4 kb. As these experiments were conducted in comparison with cell types with well-defined Ca{sup 2+}-ATPases, the results suggest the presence of a endoplasmic reticulum-related Ca{sup 2+}-ATPase in HOME cells.

  4. The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum.

    PubMed

    Lipschutz, Joshua H; Lingappa, Vishwanath R; Mostov, Keith E

    2003-06-01

    We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins. Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P., and Mostov, K. E. (2000) Mol. Biol. Cell 11, 4259-4275). Here, using an exogenously transfected basolateral protein, the polymeric immunoglobulin receptor (pIgR), and a secretory protein, gp80, we show that pIgR and gp80 protein synthesis and delivery are increased in cells overexpressing Sec10 despite the fact that mRNA levels are unchanged, which is highly indicative of post-transcriptional regulation. To test specificity, we also examined the synthesis and delivery of an exogenous apical protein, CNT1 (concentrative nucleoside transporter 1), and found no increase in CNT1 protein synthesis, delivery, or mRNA levels in cells overexpressing Sec10. Sec10-GFP-overexpressing cell lines were created, and staining was seen in the endoplasmic reticulum. It was demonstrated previously in yeast that high levels of expression of SEB1, the Sec61beta homologue, suppressed sec15-1, an exocyst mutant (Toikkanen, J., Gatti, E., Takei, K., Saloheimo, M., Olkkonen, V. M., Soderlund, H., De Camilli, P., and Keranen, S. (1996) Yeast 12, 425-438). Sec61beta is a member of the Sec61 heterotrimer, which is the main component of the endoplasmic reticulum translocon. By co-immunoprecipitation we show that Sec10, which forms an exocyst subcomplex with Sec15, specifically associates with the Sec61beta component of the translocon and that Sec10 overexpression increases the association of other exocyst complex members with Sec61beta. Proteosome inhibition does not appear to be the mechanism by which increased protein synthesis occurs in the face of equivalent amounts of m

  5. Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

    PubMed Central

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

    2014-01-01

    ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

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

    SciTech Connect

    Pan, Mu-Yun; Shen, Yuh-Chiang; Lu, Chien-Hsing; Yang, Shu-Yi; Ho, Tsing-Fen; Peng, Yu-Ta; Chang, Chia-Che

    2012-12-15

    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 ER 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 as an

  7. Minimally modified low-density lipoprotein induces macrophage endoplasmic reticulum stress via toll-like receptor 4.

    PubMed

    Yao, Shutong; Yang, Nana; Song, Guohua; Sang, Hui; Tian, Hua; Miao, Cheng; Zhang, Ying; Qin, Shucun

    2012-07-01

    Minimally modified low-density lipoprotein (mm-LDL) induces intimal foam cell formation, which is promoted by endoplasmic reticulum stress (ERS), a cross-point to link cellular processes with multiple risk factors that exist in all stages of atherosclerosis. However, it remains unclear whether mm-LDL-induced lipid accumulation in macrophages involves ERS and its underlying mechanisms. We showed that mm-LDL induced the accumulation of lipid droplets in RAW264.7 macrophages with increased free cholesterol in the endoplasmic reticulum, which was markedly attenuated by pretreatment with an antibody against toll-like receptor 4 (TLR4). Additionally, mm-LDL stimulated the transport of Cy3-labeled activating transcription factor 6 (ATF6), a key sensor to the unfolded protein response (UPR), from cytoplasm into nucleus. The expression of phosphorylated inositol-requiring enzyme 1 (p-IRE1), another sensor to the UPR, and its two downstream molecules, X box binding protein 1 and glucose-regulated protein 78 (GRP78), were significantly upregulated by mm-LDL. The alterations induced by mm-LDL were all significantly inhibited by antibodies against TLR4 or CD36. In addition, the upregulation of p-IRE1 and GRP78 and the nuclear translocation of ATF6 induced by mm-LDL were significantly attenuated by TLR4 siRNA. These results suggest that mm-LDL may induce free cholesterol accumulation in the endoplasmic reticulum and subsequently stimulate ERS and activate the UPR signaling pathway mediated by ATF6 and IRE1 in macrophages, a process that is potentially mediated by TLR4. PMID:22480542

  8. TRAIL death receptors, Bcl-2 protein family, and endoplasmic reticulum calcium pool.

    PubMed

    Sheikh, M Saeed; Huang, Ying

    2004-01-01

    Calcium (Ca(2+)) is one of the highly versatile second messengers critical in cellular pathophysiology. Alterations in Ca(2+) homeostasis affect many cellular processes, including apoptosis. Recent studies have started to unravel the molecular mechanisms of apoptosis regulation in context to intracellular Ca(2+) pools. In this regard, Bcl-2 has been reported to mediate its anti-apoptotic effects, partly, by lowering the endoplasmic reticulum (ER) Ca(2+) load and by inhibiting the mitochondrial uptake of Ca(2+). However, the opposite is true for Bax and Bak that promote apoptosis, in part, by increasing the ER Ca(2+) load and Ca(2+) transfer from the ER to mitochondria. Massive ER Ca(2+) depletion coupled with upregulation of DR5 has also been reported to induce apoptosis. The mechanistic details of how some of these molecules affect intracellular Ca(2+) contents and sense perturbations in Ca(2+) homeostasis remain to be elucidated. The recent explosion of information in the fields of cell signaling and apoptosis is likely to facilitate the future investigations aiming to explore these issues. PMID:15110177

  9. Unexpected blockade of adipocyte differentiation by K-7174: Implication for endoplasmic reticulum stress

    SciTech Connect

    Shimada, Tsuyoshi; Hiramatsu, Nobuhiko; Okamura, Maro; Hayakawa, Kunihiro; Kasai, Ayumi; Yao, Jian; Kitamura, Masanori

    2007-11-16

    Preadipocytes constitutively express GATA-2 and GATA-3 that are required to halt the cells at the undifferentiated stage. However, we unexpectedly found that K-7174, a GATA-specific inhibitor, did not induce but rather inhibited differentiation of 3T3-L1 preadipocytes. It was associated with lack of lipid accumulation, blunted expression of adipocyte markers including adiponectin and peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), and sustained expression of a preadipocyte marker monocyte chemoattractant protein 1 (MCP-1). Subsequent experiments revealed that K-7174 had the potential to induce endoplasmic reticulum (ER) stress evidenced by induction of GRP78 and CHOP. Other inducers of ER stress completely reproduced the effects of K-7174 including suppression of lipid accumulation, blockade of induction of adiponection and PPAR{gamma} and maintenance of MCP-1 expression. These results indicated a possibility that ER stress suppresses adipocyte differentiation and that GATA inhibitor K-7174 has the potential for interfering with adipogenesis through induction of ER stress.

  10. Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate

    PubMed Central

    Cottam, Eleanor M; Maier, Helena J; Manifava, Maria; Vaux, Laura C; Chandra-Schoenfelder, Priya; Gerner, Wilhelm; Britton, Paul; Ktistakis, Nick T

    2011-01-01

    Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defense against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, infectious bronchitis virus (IBV), activates autophagy. A screen of individual IBV nonstructural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses mouse hepatitis virus, and severe acute respiratory syndrome virus, and the equivalent nsp5–7 of the arterivirus porcine reproductive and respiratory syndrome virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II -positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double-FYVE-domain containing protein (DFCP) indicating localized concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signaling, activation of sirtuin 1 or induction of ER stress. PMID:21799305