The oligomeric state of Derlin-1 is modulated by endoplasmic reticulum stress

The endoplasmic reticulum (ER) is a major site of protein synthesis in eukaryotes. Newly synthesized proteins are monitored by a process of quality control, which removes misfolded or unassembled polypeptides from the ER for degradation by the proteasome. This requires the retrotranslocation of the misfolded proteins from the ER lumen into the cytosol via a pathway that, for some substrates, involves members of the recently discovered Derlin family. The Derlin-1 isoform is present as a dimer in the ER, and we now show that its dimerization is modulated by ER stress. Three distinct types of chemically-induced ER stress substantially reduce the levels of Derlin-1 dimer as assayed by both cross-linking and co-immunoprecipitation. The potential function of the different Derlin-1 populations with respect to ER quality control is investigated by analysing their capacity to associate with a misfolded membrane protein fragment. We show for the first time that Derlin-1 can associate with an aberrant membrane protein fragment in the absence of the viral component US11, and conclude that it is the monomeric form of Derlin-1 that interacts with this potential ER-associated degradation substrate. On the basis of these data we propose a model where the pool of active Derlin-1 in the ER membrane can be modulated in response to ER stress.     

The retrotranslocation protein Derlin-1 binds peptide:N-glycanase to the endoplasmic reticulum

The deglycosylating enzyme, peptide:N-glycanase, acts on misfolded N-linked glycoproteins dislocated from the endoplasmic reticulum (ER) to the cytosol. Deglycosylation has been demonstrated to occur at the ER membrane and in the cytosol. However, the mechanism of PNGase association with the ER membrane was unclear, because PNGase lacked the necessary signal to facilitate its incorporation in the ER membrane, nor was it known to bind to an integral ER protein. Using HeLa cells, we have identified a membrane protein that associates with PNGase, thereby bringing it in close proximity to the ER and providing accessibility to dislocating glycoproteins. This protein, Derlin-1, has recently been shown to mediate retrotranslocation of misfolded glycoproteins. In this study we demonstrate that Derlin-1 interacts with the N-terminal domain of PNGase via its cytosolic C-terminus. Moreover, we find PNGase distributed in two populations; ER-associated and free in the cytosol, which suggests the deglycosylation process can proceed at either site depending on the glycoprotein substrate.     

Rosiglitazone ameliorates palmitic acid-induced endoplasmic reticulum stress and steroidogenic capacity in granulosa cells

Granulosa cells play essential roles in follicular development, oocyte maturation and sex hormone secretion. The exposure of granulosa cells to palmitic acid (PA), the main component of dietary saturated fat, inhibits cell viability. However, the mechanism underlying PA-induced cytotoxicity in granulosa cells has not been deeply investigated. Rosiglitazone (RSG) is a member of the thiazolidinedione family and is reported to protect cells from cytotoxicity and endoplasmic reticulum (ER) stress in other cell types, but whether RSG protects granulosa cells remain unknown. In this study, KGN cell line and primary granulosa cells were used as models of granulosa cells to explore the effects of PA and RSG and the underlying mechanisms. The results showed that PA inhibits cell viability and estradiol secretion through inducing ER stress and cAMP/PKA/CREB pathway. CCAAT/enhancer-binding protein homologous protein (CHOP), an ER stress marker, was demonstrated to participate in PA-induced cytotoxicity. RSG treatment rescued granulosa cells from PA-induced cell death and ER stress. Moreover, RSG was identified to ameliorate ER stress induced by tunicamycin in granulosa cells. In addition, RSG treatment rescued granulosa cells from PA-induced decrease of estrogen secretion by cAMP/PKA/CREB pathway. In conclusion, RSG can protect granulosa cells against PA-induced cytotoxicity by inhibiting ER stress, and can recover steroidogenic capacity, indicating a potential use of RSG in the treatment of granulosa cell dysfunction.     

AMPK activation prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 signaling and endoplasmic reticulum stress response

Lipid accumulation is a central event in the development of chronic metabolic diseases, including obesity and type 2 diabetes, but the mechanisms responsible for lipid accumulation are incompletely understood. This study was designed to investigate the mechanisms for excess nutrient-induced lipid accumulation and whether activation of AMP-activated protein kinase (AMPK) prevents the hepatic lipid accumulation in excess nutrient-treated HepG2 cells and high fat diet (HFD)-fed mice. Exposure of HepG2 cells to high levels of glucose or palmitate induced the endoplasmic reticulum (ER) stress response, activated sterol regulatory element-binding protein-1 (SREBP-1), and enhanced lipid accumulation, all of which were sensitive to ER stress inhibitor and gene silencing of eukaryotic initiation factor 2α. The increases in ER stress response and lipid accumulation were associated with activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Inhibition of mTORC1 signaling attenuated the ER stress response and lipid accumulation induced by high glucose or by deletion of tuberous sclerosis 2. In addition, AMPK activation prevented the mTORC1 activation, ER stress response, and lipid accumulation. This effect was mimicked or abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Finally, treatment of HFD-fed mice with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside inhibited the mTORC1 pathway, suppressed the ER stress response, and prevented insulin resistance and hepatic lipid accumulation. We conclude that activation of AMPK prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 and ER stress response.     

Murine polyomavirus requires the endoplasmic reticulum protein Derlin-2 to initiate infection

The pathways by which viruses enter cells are diverse, but in all cases, infection necessitates the transfer of the viral genome across a cellular membrane. Polyomavirus (Py) particles, after binding to glycolipid and glycoprotein receptors at the cell surface, are delivered to the lumen of the endoplasmic reticulum (ER). The nature and extent of virus disassembly in the ER, how the viral genome is transported to the cytosol and subsequently to the nucleus, and whether any cellular proteins are involved are not known. Here, we identify an ER-resident protein, Derlin-2, a factor implicated in the removal of misfolded proteins from the ER for cytosolic degradation, as a component of the machinery required for mouse Py to establish an infection. Inhibition of Derlin-2 function by expression of either a dominant-negative form of Derlin-2 or a short hairpin RNA that reduces Derlin-2 levels blocks Py infection by 50 to 75%. The block imposed by Derlin-2 inhibition occurs after the virus reaches the ER and can be bypassed by the introduction of Py DNA into the cytosol. These findings suggest a mode of Py entry that involves cytosolic access via the quality control machinery in the ER.     

Protective effect of catechin in type I Gaucher disease cells by reducing endoplasmic reticulum stress

Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) and is divided into three phenotypes, I, II, and III. Type I is the most prevalent form and has its onset in adulthood. The degree of endoplasmic reticulum (ER) stress is one of the factors that determine GD severity. It has recently been reported that antioxidants reduce ER stress and apoptosis by scavenging the oxidants that cause oxidative stress. For this report, we investigated the possibility that catechin can act on type I GD patient cells to alleviate the pathogenic conditions of GD. We treated GD cells with catechin and examined the expression level of GRP78/BiP (an ER stress marker) by western blots and fluorescence microscopy, the proliferation rate of GD cells, and scratch-induced wound healing activity. Our results show that catechin reduces the expression level of GRP78/BiP, leads to cell proliferation rates of GD cells similar levels to normal cells, and improves wound healing activity. We conclude that catechin protects against ER stress in GD cells and catechin-mediated reductions in ER stress may be associated with enhanced cell survival.     

Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.     

Conditions of endoplasmic reticulum stress favor the accumulation of cytosolic prion protein

After signal sequence-dependent targeting to the endoplasmic reticulum (ER), prion protein (PrP) undergoes several post-translational modifications, including glycosylation, disulfide bond formation, and the addition of a glycosylphosphatidylinositol anchor. As a result, multiple isoforms are generated. Because of the intrinsic weakness of the PrP signal sequence, a fraction of newly synthesized molecules fails to translocate and localizes to the cytosol. The physiopathologic role of this cytosolic isoform is still being debated. Here we have shown that, in both cultured cell lines and primary neurons, ER stress conditions weaken PrP co-translational translocation, favoring accumulation of aggregation-prone cytosolic species, which retain the signal sequence but lack N-glycans and disulfides. Inhibition of proteasomes further increases the levels of cytosolic PrP. Overexpression of spliced XBP1 facilitates ER translocation, suggesting that downstream elements of the Ire1-XBP1 pathway are involved in PrP targeting. These studies reveal a link between ER stress and the formation of cytosolic PrP isoforms potentially endowed with novel signaling or cytotoxic functions.     

Hydroxytyrosol ameliorates insulin resistance by modulating endoplasmic reticulum stress and prevents hepatic steatosis in diet-induced obesity mice

Endoplasmic reticulum (ER) is a principal organelle responsible for energy and nutrient management. Its dysfunction has been viewed in the context of obesity and related glucolipid metabolic disorders. However, therapeutic approaches to improve ER adaptation and systemic energy balance in obesity are limited. Thus, we examined whether hydroxytyrosol (HT), an important polyphenolic compound found in virgin olive oil, could correct the metabolic impairments in diet-induced obesity (DIO) mice. Here, we found that HT gavage for 10 weeks significantly ameliorated glucose homeostasis and chronic inflammation and decreased hepatic steatosis in DIO mice. At the molecular level, ER stress indicators, inflammatory and insulin signaling markers demonstrated that high-fat diet (HFD)-induced ER stress and insulin resistance (IR) in insulin sensitive tissue were corrected by HT. In vitro studies confirmed that HT supplementation (100 μM) attenuated palmitate-evoked ER stress, thus rescuing the downstream JNK/IRS pathway. As a result from suppression of ER stress in the liver, HT further decreased hepatic sterol regulatory element-binding protein-1 expression (SREBP1). Additionally, aberrant expression of genes involved in hepatic lipogenesis (SREBP1, ACC, FAS, SCD1) caused by HFD was restored by HT. These findings suggested that HT ameliorated chronic inflammation and IR and decreased hepatic steatosis in obesity by beneficial modulation of ER stress.     

疱疹病毒与内质网应激

内质网是分泌型蛋白和膜蛋白折叠及翻译后修饰的主要场所.病毒感染所引起的宿主细胞内环境的改变可使细胞或病毒的未折叠和/或错误折叠蛋白在内质网中大量聚集,使内质网处于生理功能紊乱的应激状态.为了缓解这种应激压力,细胞会启动未折叠蛋白反应(UPR),并通过一系列分子的信号转导维持内质网稳态;同时病毒也会通过对UPR的精密调控...     

ATM blocks tunicamycin-induced endoplasmic reticulum stress

Endoplasmic reticulum stress (ER-stress) is associated with ataxia telangiectasia mutated (ATM) gene. We present here conclusive data showing that ATM blocks ER-stress induced by tunicamycin or ionizing radiation (IR). X-box protein-1 (XBP-1) splicing, GRP78 expression and caspase-12 activation were increased by tunicamycin or IR in Atm-deficient AT5BIVA fibroblasts. Activation of caspase-12 and caspase-3 by tunicamycin was significantly reduced in cells transfected with wild-type Atm (AT5BIVA/wtATM). Atm knockdown by siRNA, however, noticeably elevated ER-stress and chemosensitivity to tunicamycin. In summary, we present substantial data demonstrating that ATM blocks the ER stress signaling associated with cancer cell proliferation.     

Effect of overexpression of kinase- or RNase-deficient OsIRE1 on the endoplasmic reticulum stress response in transgenic rice plants

IRE1 is an endoplasmic reticulum (ER) stress sensor protein in eukaryotes. In this study, we generated transgenic rice plants overexpressing three types of OsIRE1, including wild-type OsIRE1 (IRE1-OE) and two disrupted-IRE1s deficient in either kinase activity (K519A-OE) or RNase activity (K833A-OE), under the control of a constitutive promoter. Overexpression of wild-type IRE1 induced the ER stress response in transgenic rice even under non-stress conditions, whereas K519A-OE and K833A-OE had dominant negative effects on endogenous OsIRE1 expression in these transgenic plants. These lines exhibited phenotypes that were quite similar to those of OsIRE1 knock-down rice. These observations suggest that the two types of functionally disrupted OsIRE1 proteins behave as competitive inhibitors toward the ER stress response in transgenic rice plants. Furthermore, OsIRE1 may have a vital, as yet unidentified function, as determined through the characterization of the transgenic plants generated in this study.     

Monitoring endoplasmic reticulum stress responsive mRNAs by RNA sequencing

Gene expression profile upon endoplasmic reticulum (ER) stress was analyzed by deep shotgun sequencing of mRNAs (DSSR) using RNAs from polysomes or cytoplasm of the HT29 cell. Two time points, 4h after tunicamycin treatment when IRE1α signaling pathway is active and 16h after the treatment when it is inactive, were used. There was a transient decrease in the proportion of shorter mRNA species (<1000bp) in polysome, while it increased transiently in the cytoplasm. Despite such an overall change and decrease in total amount of polysomes, the majority of the 6966 genes analyzed had less than 2 fold change in their expressions. We searched for the genes whose expression was elevated by 2 folds or more in both polysome and cytoplasm and confirmed the results with RT-PCR. There were 7 genes elevated only at 4h (Group I), 20 genes only at 16h (Group II) and 7 genes both at 4 and 16h (Group III). There were 3 genes involved in ribosomal RNA biogenesis in Group I and 2 genes involved mTOR control in Group III. This was consistent with the concept that the ribosome is the essential site for managing ER stress. DSSR is a useful tool for the search of candidates of ER stress responsive genes.