Overexpression of ERp29 in the thyrocytes of FRTL-5 cells

It was previously reported that the up-regulation of ERp29 mRNA depends on the levels of thyroid stimulating hormone (TSH) in the thyrocytes of FRTL-5 cells. In order to investigate the putative new function of ERp29 as an endoplasmic molecular (ER) chaperone, an ERp29-overexpressing FRTL-5 cell line was established. This cell line had approximately three times the levels of ERp29 protein and an enhanced level of thyroglobulin (Tg) secretion. The results showed both enhanced ERp29 expression and an interaction with the other ER chaperones such as GRP94, BiP, ERp72 and calnexin. In addition, ERp29 enhanced the expression of PKR-like ER kinase (PERK), which is a transmembrane protein located in the ER membrane. These findings suggest that ERp29 assists in protein folding as well as in the secretion of the secretory/plasma membrane proteins under close co-operation with other ER chaperones and the ER stress signaler, PERK.     

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated co-localization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.     

The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells

The protein ERp57/GRP58 is a member of the protein disulfide isomerase family and is also a glucose-regulated protein, which, together with the other GRPs, is induced by a variety of cellular stress conditions. ERp57/GRP58 is mainly located in the endoplasmic reticulum (ER), but has also been found in the cytoplasm and in the nucleus, where it can bind DNA. In order to identify a possible correlation between the stress-response and the nuclear location of ERp57/GRP58, its binding sites on DNA in HeLa cells have been searched by chromatin immunoprecipitation and cloning of the immunoprecipitated DNA fragments. Following sequencing of the cloned fragments, 10 DNA sequences have been securely identified as in vivo targets of ERp57/GRP58. Nine of them are present in the non-coding regions of identified genes, and seven of these in introns. The features of some of these DNA sequences, that is, DNase hypersensitivity, proximity of MAR regions, and homology to the non-coding regions of orthologue genes of mouse or rat, are compatible with a gene expression regulatory function. Considering the nature of the genes concerned, two of which code for DNA repair proteins, we would suggest that at least part of the mechanism of action of ERp57/GRP58 takes place through the regulation of these, and possibly other still unidentified, stress-response genes.     

Antigen retrieval to improve the immunocytochemistry detection of sigma-1 receptors and ER chaperones

Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris–HCl (pH 9.5) containing 6 M urea (80°C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 h effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures.     

The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion

Expression of the glucose-regulated proteins (GRPs), GRP78 and GRP94, is induced by a variety of stress conditions including treatment of cells with tunicamycin or the calcium ionophore A23187. The stimulus for induction of these resident endoplasmic reticulum (ER) proteins appears to be accumulation of misfolded or underglycosylated protein within the ER. We have studied the induction of mRNAs encoding two other resident ER proteins, ERp72 and protein disulfide isomerase (PDI), during the stress response in Chinese hamster ovary cells. ERp72 shares amino acid sequence homology with PDI within the presumed catalytic active sites. ERp72 mRNA and, to a lesser degree, PDI mRNA were induced by treatment of Chinese hamster ovary cells with tunicamycin or A23187. These results identify ERp72 as a member of the GRP family. Stable high level overproduction of ERp72 or PDI from recombinant expression vectors did not alter the constitutive or induced expression of other GRPs. High level overexpression resulted in secretion of the overproduced protein specifically but not other resident ER proteins. This suggests that the ER retention mechanism is mediated by more specific interactions than just KDEL sequence recognition.     

A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins

We demonstrate the existence of a large endoplasmic reticulum (ER)-localized multiprotein complex that is comprised of the molecular chaperones BiP; GRP94; CaBP1; protein disulfide isomerase (PDI); ERdj3, a recently identified ER Hsp40 cochaperone; cyclophilin B; ERp72; GRP170; UDP-glucosyltransferase; and SDF2-L1. This complex is associated with unassembled, incompletely folded immunoglobulin heavy chains. Except for ERdj3, and to a lesser extent PDI, this complex also forms in the absence of nascent protein synthesis and is found in a variety of cell types. Cross-linking studies reveal that the majority of these chaperones are included in the complex. Our data suggest that this subset of ER chaperones forms an ER network that can bind to unfolded protein substrates instead of existing as free pools that assembled onto substrate proteins. It is noticeable that most of the components of the calnexin/calreticulin system, which include some of the most abundant chaperones inside the ER, are either not detected in this complex or only very poorly represented. This study demonstrates an organization of ER chaperones and folding enzymes that has not been previously appreciated and suggests a spatial separation of the two chaperone systems that may account for the temporal interactions observed in other studies.     

Nascent lipidated apolipoprotein B is transported to the Golgi as an incompletely folded intermediate as probed by its association with network of endoplasmic reticulum molecular chaperones,GRP94, ERp72, BiP,calreticulin, and cyclophilin B

We have previously demonstrated that endoplasmic reticulum (ER)-resident molecular chaperones interact with apolipoprotein B-100 (apoB) during its maturation. The initial stages of apoB folding occur while it is bound to the ER membrane, where it becomes partially lipidated to form a primordial intermediate. We determined whether this intermediate is dependent on the assistance of molecular chaperones for its subsequent folding steps. To that end, microsomes were prepared from HepG2 cells and luminal contents were subjected to KBr density gradient centrifugation. Immunoprecipitation of apoB followed by Western blotting showed that the luminal pool floated at a density of 1.12 g/ml and, like the membrane-bound pool, was associated with GRP94, ERp72, BiP, calreticulin, and cyclophilin B. Except for calreticulin, chaperone/apoB ratio in the lumen was severalfold higher than that in the membrane, suggesting a role for these chaperones both in facilitating the release of the primordial intermediate into the ER lumen and in providing stability. Subcellular fractionation on sucrose gradients showed that apoB in the Golgi was associated with the same array of chaperones as the pool of apoB recovered from heavy microsomes containing the ER, except that chaperone/apoB ratio was lower. KBr density gradient fractionation showed that the major pool of luminal apoB in the Golgi was recovered from 1.02 < d < 1.08 g/ml, whereas apoB in ER was recovered primarily from 1.08 < d < 1.2 g/ml. Both fractions were associated with the same spectrum of chaperones. Together with the finding that GRP94 was found associated with sialylated apoB, we conclude that correct folding of apoB is dependent on the assistance of molecular chaperone, which play multiple roles in its maturation throughout the secretory pathway including distal compartments such as the trans-Golgi network.     

Mixed-disulfide folding intermediates between thyroglobulin and endoplasmic reticulum resident oxidoreductases ERp57 and protein disulfide isomerase

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.     

Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress (ERS), and triggers the unfolded protein response (UPR) that consequently reduces accumulation of unfolded proteins by increasing the quantity of ER chaperones. Calumenin, a Ca²⁺-binding protein with multiple EF hand motifs, which is located in the ER/SR, is highly expressed during the early developmental stage of the heart, similar to other ER-resident chaperones. The aim of this study was to investigate the functional role of calumenin during ERS in the heart. Like other chaperones (e.g., GRP94 and GRP78), calumenin expression was highly upregulated during ERS induced by 10 μg/ml tunicamycin, but attenuated in the presence of 500 μM PBA, the chemical chaperone in neonatal rat ventricular cardiomyocytes (NRVCs). Upon 7.5-fold overexpression of calumenin using a recombinant adenovirus system, the expression levels of ERS markers (GRP78, p-PERK, and p-elF2α) and ER-initiated apoptosis markers (CHOP and p-JNK) were reduced, whereas the survival protein BCL-2 was upregulated during ERS compared to the control. Evaluation of cell viability by TUNEL assay showed that apoptosis was also significantly reduced by calumenin overexpression in ERS-induced cells. Taken together, our results suggest that calumenin plays an essential role in the alleviation of ERS in neonatal rat cardiomyocytes.     

Increased expression of ERp57/GRP58 is protective against pancreatic beta cell death caused by autophagic failure

Autophagy is a tightly regulated self-digestion system. As in other cell types, autophagy plays an essential role in the homeostasis of pancreatic beta cells. However, the mechanisms involved in the deterioration of beta cell function caused by autophagic failure have not yet been fully elucidated. To gain insight into its mechanisms, we compared the protein expression of islets from beta cell-specific Atg7-deficient mice (Atg7^Δβ-cell mice) and their controls (Atg7^f/f mice). Liquid chromatography/mass spectrometry after 1-dimensional electrophoresis identified the increased expression of ERp57/GRP58 in islets isolated from Atg7^Δβ-cell mice compared with those from Atg7^f/f mice. The expression level of ERp57 was also elevated in rat insulinoma INS-1 cells by inducible knock-down of the atg7-gene. In Atg7 knock-down INS-1 cells, the suppression of ERp57 expression by siRNA resulted in an increase in the level of cleaved Caspase-3 protein and a decrease in the number of live cells. Furthermore, cell cycle analyses demonstrated that the suppressed expression of ERp57 increased the sub-G1 population. These data reveal that increased expression of ERp57 may contribute to the protection from beta cell death caused by autophagic failure.     

ERO1α-dependent endoplasmic reticulum–mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1)

Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.     

The unfolded protein response to endoplasmic reticulum stress in cultured astrocytes and rat brain during experimental diabetes

Oxidative-nitrosative stress and inflammatory responses are associated with endoplasmic reticulum (ER) stress in diabetic retinopathy, raising the possibility that disturbances in ER protein processing may contribute to CNS dysfunction in diabetics. Upregulation of the unfolded protein response (UPR) is a homeostatic response to accumulation of abnormal proteins in the ER, and the present study tested the hypothesis that the UPR is upregulated in two models for diabetes, cultured astrocytes grown in 25 mmol/L glucose for up to 4 weeks and brain of streptozotocin (STZ)-treated rats with diabetes for 1–7 months. Markers associated with translational blockade (phospho-eIF2α and apoptosis (CHOP), inflammatory response (inducible nitric oxide synthase, iNOS), and nitrosative stress (nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) were not detected in either model. Nrf2 was present in nuclei of low- and high-glucose cultures, consistent with oxidative stress. Astrocytic ATF4 expression was not altered by culture glucose concentration, whereas phospho-IRE and ATF6 levels were higher in low- compared with high-glucose cultures. The glucose-regulated chaperones, GRP78 and GRP94, were also expressed at higher levels in low- than high-glucose cultures, probably due to recurrent glucose depletion between feeding cycles. In STZ-rat cerebral cortex, ATF4 level was transiently reduced at 4 months, and p-IRE levels were transiently elevated at 3 months. However, GRP78 and GRP94 expression was not upregulated, and iNOS, amyloid-β, and nuclear accumulation of GAPDH were not evident in STZ-diabetic brain. High-glucose cultured astrocytes and STZ-diabetic brain are relatively resistant to diabetes-induced ER stress, in sharp contrast with cultured retinal Müller cells and diabetic rodent retina.     

Bosellia serrata-induced apoptosis is related with ER stress and calcium release

It has been reported that the gum resin of Boswellia serrata (BS), which has been shown to have antiinflammatory properties, might also have anticancer effects. This study examined the potential of BS as an anticancer agent. The BS extract induces apoptosis in HeLa human cervical carcinoma cells, as confirmed by two apoptosis analyses, Hoechst staining and Annexin V/PI assay. Among the apoptosis pathways, the ER stress-associated mechanism was examined to determine its role in BS-induced apoptosis. The expression of GRP78 and CHOP, which are representatives of the ER stress proteins, and the calcium-binding protein-calpain were determined. The results showed significantly higher levels of both GRP78 and CHOP, and stronger calpain activity in the BS-treated cells than in the control cells. This shows that there is a correlation between ER stress signaling and apoptosis, which suggests the possibility of the BS-ER stress initiator as an anticancer therapeutic agent in human cervical carcinoma.     

Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells evidence for a unique role in secretory-protein synthesis.

Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.