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Cervical cancer is an important health issue for women worldwide, and the endoplasmic reticulum stress pathway is important for determining the chemotherapeutic response to cancer. However, the role of glucose-regulated protein 94 (GRP94) in taxane therapy for cervical cancer remains unclear. In this study, we generated GRP94 knockdown (GRP94-KD) Hela cells using short hairpin RNAs and found that GRP94-KD cells were resistant to taxane treatment in an MTT assay. Scrambled control cells demonstrated higher levels of apoptosis when treated with taxanes in comparison to GRP94-KD cells, as determined by cell cycle profiling, 4′,6-diamidino-2-phenylindole staining, and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. Caspase 3 and caspase 7 activity was also higher in scrambled control cells treated with taxane in comparison to GRP94-KD cells. Moreover, we found that depletion of GRP94 altered the levels of the apoptosis-related proteins Bcl2 and Bad, leading to sensitivity to taxane. Exposure to taxane also induced the expression of Bad in scrambled cells but not in GRP94-KD cells. In addition, the expression of Bcl2 was increased dramatically in GRP94-KD cells, whereas only a small increase was observed in scrambled cells. Therefore, we conclude that silencing GRP94 may increase resistance to taxane treatment in cervical cancer cells by altering the activation of the apoptosis pathway. In addition, GRP94 may represent a key biomarker for determining the therapeutic efficacy of taxane treatment in cervical cancer patients. [ABSTRACT FROM AUTHOR]

Objective To detect the protein markers related to lung adenocarcinoma for early diagnosis, monitoring and treatment of lung adenocarcinoma. Methods Soluble proteins extracted from lung adenocarcinoma and adjacent normal tissues were subjected to two-dimensional gel electrophoresis. Then the proteins over-expressed in lung adenocarcinoma were screened and idenified by matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF MS) and bioinformatic techniques. The expression of grp94, one of the over-expressed proteins, was confirmed by immunohistochemistry technique. Results Six proteins including grp94 were identified by proteomic technology. These proteins were significanlly over-expressed in lung adenocarcinoma issues compared with those in adjacent normal issues. The high-expresiion of protein grp94 was confirmed by immunohistochemistry technique. The functions of the identified proteins involved intracellular protein synthesis or assembly, removal of superoxide radicals in the body, and apoptosis, which are closely related to tumor biological characteristics. Conclusion Proteomic technology can effectively screen the protein markers of lung adenocarcinoma. These proteins include Cu, Zn superoxide dismutase; heat shock protein 27; chain B, triosephosphate isomerase; annexin I; 94 ku glucose-regulated protein; and heat shock protein 90-beta. They may be new targets for molecular diagnosis and treatment of lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Cholesteatoma is a destructive and expanding growth of keratinizing squamous epithelium in the middle ear or petrous apex. The molecular and cellular processes of the pathogenesis of acquired middle ear cholesteatoma have not been fully understood. In this study, comparative proteomic analysis was conducted to investigate the roles of specific proteins in the pathways regarding keratinocyte proliferation in cholesteatoma. The differential proteins were detected by comparing the two-dimension electrophoresis (2-DE) maps of the epithelial tissues of 12 attic cholesteatomas with those of retroauricular skins. There were 14 upregulated proteins in the epithelial tissues of cholesteatoma in comparison with retroauricular skin. The modulation of five crucial proteins, HSP27, PRDX2, GRP75, GRP78 and GRP94, was further determined by RT-PCR, Western blot and immunohistochemistry. Phosphorylation of HSP27 at Ser-82 was identified by mass spectroscopy. The results of this study suggested that phosphorylated HSP27 is the end expression of two potential signal-transduction pathways, and together with PRDX2, they are very likely involved in the proliferation of keratinocytes in cholesteatoma. Upregulations of GRP75, GRP78 and GRP94 in keratinocytes may be able to counter endoplasmic reticulum stress, to inhibit cell apoptosis, to prevent protein unfolding and to promote cholesteatoma growth. [ABSTRACT FROM AUTHOR]

Cisplatin is an essential chemotherapeutic drug in the treatment of many cancers. Its use, however, is limited by the development of resistance in many tumors. The ability to re-sensitize resistant tumors could significantly strengthen cisplatin therapy in patients. Caenorhabditis elegans is a suitable model for studying the cytoplasmic role of cisplatin in tumor cells. We have previously shown that the ATPase ASNA-1 has similar roles as a factor governing cisplatin sensitivity in mammalian tumor cells and C. elegans. Here we study the endoplasmic reticulum (ER) resident chaperone ENPL-1/ GRP94 and find that its depletion makes worms sensitive to cisplatin. Elevated ER stress levels in enpl-1 mutants is the likely cause of this sensitivity because a correlation can be made between cisplatin sensitivity and the high ER stress levels. We also find that asna-1 mutants have elevated unfolded protein response (UPR) activity and that the intrinsically cisplatin resistant wild-type worms become sensitive when ER stress is high. We conclude that enpl-1 is a cisplatin sensitizing factor and suggest that manipulation of its levels or of UPR activity will enhance the effects of cisplatin based cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract: The process of B-cell development is characterized by the activation of the unfolded protein response. Under certain circumstances, the unfolded protein response can be manipulated in a cell death-inducing way. Therefore, tackling the unfolded protein response might be an attractive strategy in the treatment of diffuse large B-cell lymphomas. Our research showed more basal unfolded protein response activity and differences in the inducibility of ER stress in activated B-cell versus germinal center cell lines. Moreover, the diffuse large B cell lymphoma patient data revealed that the glucose-regulated protein 94 is new potential discriminator for diffuse large B cell lymphoma. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to target ovarian cancer cells by coupling paclitaxel (Tx)-loaded nanoparticles (NPs-Tx) to antibodies against KDEL sequence, able to recognize GRP94 and GRP78 that are located at cell surface in cancer cells whereas they are in the endoplasmic reticulum in healthy cells. Tx-loaded poly (dl-lactic acid) nanoparticles coated with anti-KDEL antibodies (NPs-Tx-KDEL) were successfully prepared and characterized. Interaction between tumor cells and NPs-Tx or NPs-Tx-KDEL was observed by microscopy with fluorescently labeled NPs and the efficacy of the different formulations was compared by a viability assay. Particles functionalized with monoclonal antibodies (mAb) showed a higher binding to the cells even though the internalization rate appeared limited. The effect of NPs-Tx-KDEL on cell viability (proliferation) was compared to Tx, NPs, NPs-Tx, anti-KDEL mAb or anti-KDEL mAb in combination with NPs-Tx in Bg-1 ovarian cell line. Our data indicate that NPs-Tx-KDEL significantly increase sensitivity of Bg-1 cells to Tx compared to other treatments. This study confirms the interest of anti-cancer therapy by targeting cell surface GRP78 and GRP94 on cancer cells, and demonstrates the efficiency of coupling KDEL antibodies to NPs. [Copyright &y& Elsevier]

Abstract: Targeting the altered redox status of cancer cells is emerging as an interesting approach to potentiate chemotherapy. However, to maximize the effectiveness of this strategy and define the correct chemotherapeutic associations, it is important to understand the biological consequences of chronically exposing cancer cells to reactive oxygen species (ROS). Using an H2O2-generating system, we selected a ROS-resistant MCF-7 breast cancer cell line, namely Resox cells. By exploring different survival pathways that are usually induced during oxidative stress, we identified a constitutive overexpression of the endoplasmic reticulum chaperone, GRP94, in these cells, whereas levels of its cytoplasmic homolog HSP90, or GRP78, were not modified. This overexpression was not mediated by constitutive unfolded protein response (UPR) activation. The increase in GRP94 is tightly linked to an increase in cell proliferation and migration capacities, as shown by GRP94-silencing experiments. Interestingly, we also observed that GRP94 silencing inhibits migration and proliferation of the highly aggressive MDA-MB-231 cells. By immunohistochemistry, we showed that GRP94 expression was higher in recurrent human breast cancers than in their paired primary neoplasias. Similar to the situation in the Resox cells, this increase was not associated with an increase in UPR activation in recurrent tumors. In conclusion, this study suggests that GRP94 overexpression may be a hallmark of aggressiveness and recurrence in breast cancers. [Copyright &y& Elsevier]

The present work was aimed at studying the effects of a subchronic lithium treatment on rat liver and kidneys, paying attention to the relationship between lithium toxicity, oxidative stress, and stress protein expression. Male rats were submitted to lithium treatment by adding 2 g of lithium carbonate/kg of food for different durations up to 1 month. This treatment led to serum concentrations ranging from 0.5 mM (day 7) to 1.34 mM (day 28) and renal insufficiency highlighted by an increase of blood creatinine and urea levels and a decrease of urea excretion. Lithium treatment was found to trigger an oxidative stress both in kidney and liver, leading to an increase of lipid peroxidation level (TBARS) and of superoxide dismutase and catalase activities. Conversely, glutathione peroxidase activity was reduced. Constitutive HSP73 (heat shock protein 73) expression was not modified by lithium treatment, whereas inducible HSP72 was down-regulated in kidney. GRP94 (glucose regulated protein 94) appeared as two isoforms of 92 and 98 kDa: the 98-kDa protein being overexpressed in kidney by lithium treatment whereas 92-kDa protein was underexpressed both in kidney and liver. [ABSTRACT FROM AUTHOR]

Background: Retinol-binding protein 4 (RBP4) is an adipokine identified as a marker of insulin resistance in mice and humans. Protein tyrosine phosphatase 1B (PTP1B) expression levels as well as other genes involved in the endoplasmic reticulum (ER) stress response are increased in adipose tissue of obese, high-fat-diet-fed mice. In this study we investigated if serum and/ or adipose tissue RBP4 protein levels and expression levels of PTP1B and other ER stressresponse genes are altered in obese and obese/diabetic men resident in northeast Scotland. Methods: We studied three groups of male volunteers: (1) normal/overweight (body mass index [BMI] ,<30), (2) obese (BMI > 30), and (3) obese/diabetic (BMI ?30) controlling their diabetes either by diet or the antidiabetic drug metformin. We analyzed their serum and adipose tissue RBP4 protein levels as well as adipose tissue mRNA expression of PTP1B, binding immunoglobulin protein (BIP), activated transcription factor 4 (ATF4), and glucose-regulated protein 94 (GRP94) alongside other markers of adiposity (percentage body fat, leptin, cholesterol, triglycerides) and insulin resistance (oral glucose tolerance tests, insulin, homeostatic model assessment-insulin resistance, C-reactive protein, and adiponectin). Results: We found that obese Scottish subjects had significantly higher serum RBP4 protein levels in comparison to the normal/overweight subjects (P < 0.01). Serum RBP4 levels were normalized in obese/diabetic subjects treated with diet or metformin (P < 0.05). Adipose tissue RBP4 protein levels were comparable between all three groups of subjects as were serum and adipose transthyretin levels. Adipose tissue PTP1B mRNA levels were increased in obese subjects in comparison to normal/overweight subjects (P < 0.05); however diet and/or metformin treatment did not reverse this effect. Adipose tissue BIP, ATF4, and GRP94 expression levels were unchanged in obese and obese/diabetic subjects. Conclusions: Human obesity results in an increase in serum but not adipose tissue RBP4 protein levels, and these are normalized in obese/diabetic subjects, which exhibit improvements in insulin sensitivity through diet or metformin treatment. However, while adipose tissue PTP1B mRNA levels increase in obese Scottish subjects, these remain high in obese/diabetics on diet or metformin treatment. [ABSTRACT FROM AUTHOR]

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPK TKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) peptide-induced neurotoxicity is typically associated with cell death through mechanisms not entirely understood. Here, we investigated stress signaling events triggered by soluble Aβ in differentiated rat neuronal-like PC12 cells. Morphologic evaluation of apoptosis confirmed that Aβ induced nuclear fragmentation that was prevented by pre-treatment with the antiapoptotic bile acid tauroursodeoxycholic acid (TUDCA). In addition, Aβ exposure triggered an early signaling response by the endoplasmic reticulum (ER) and caspase-12-mediated apoptosis, which, however, was independent of the ER-stress pathway. Furthermore, ER stress markers, including GRP94, ATF-6α, CHOP, and eIF2α, were strongly downregulated by Aβ, independent of protein degradation, and partially restored by TUDCA. Calpain inhibition prevented caspase-12 activation and reduced levels of ATF-6α. Importantly, Aβ-induced GRP94 downregulation was related to protein secretion and partially rescued through inhibition of the secretory pathway by geldanamycin and brefeldin. In conclusion, we showed that the ER is a proximal stress sensor for soluble Aβ-induced toxicity, resulting in caspase-12 activation and cell death in PC12 neuronal cells. Moreover, ER chaperone GRP94 secretion was associated with Aβ-induced apoptotic signaling. These data provide new information linking apoptotic properties of Aβ peptide to distinct subcellular mechanisms of toxicity. Further characterization of this signaling pathway is likely to provide new perspectives for modulation of amyloid-induced apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Immune responses to ER stress have been closely related to the pathogenesis of autoimmune diseases. Using an immunoprecipitation assay, 24 (7.1%) of 336 MG serum samples immunoprecipitated a 90-kDa protein from the muscle cellular extracts, but none of the disease or healthy control sera. The 90-kDa protein was affinity-purified and found to match to ER chaperon GRP94 by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy analysis. The frequency of associated autoimmune diseases was much higher in the anti-GRP94-positive than the -negative patients (71% versus 11%, p<0.001). Autoimmunity to ER chaperone GRP94 is associated with a subset of MG patients who have additional autoimmune diseases. [Copyright &y& Elsevier]

Objectives. To investigate the presence of autoantibodies against mammalian chaperones of the endoplasmic reticulum (ER) in patients with RA and other immune-mediated diseases.Methods. Sera from healthy donors, from early RA patients with two follow-up samples, patients with SLE, SSc and IBD were collected and analysed for anti-ER chaperone antibodies. Detection of serum IgG antibodies against immunoglobulin heavy chain binding protein (BiP), glucose-regulated protein 94 (Grp94) and calnexin was carried out using ELISA. The specificity of sera positive for individual ER chaperones was confirmed by immunoblotting. Statistical analysis was performed using Welch’s t-test, Mann–Whitney U-test, partial correlation and Pearson’s correlation.Results. In patients with RA and SLE, autoantibody titres against BiP, Grp94 and calnexin were significantly higher than those in healthy controls. These autoantibodies were detectable in patients with early RA and titres remained stable for at least 6–12 months. Also several SSc and IBD patients exhibited autoantibodies against these ER chaperones; however, titres and frequencies were lower than in RA or SLE patients. Furthermore, anti-calnexin antibodies correlated significantly with the presence of BiP and Grp94 autoantibodies in patients with RA and SLE.Conclusion. Calnexin and Grp94 were identified as novel autoantigens in RA and calnexin in SLE. Since calnexin, Grp94 and BiP are ER-resident proteins of eukaryotic cells, our data suggest that autoantibody generation against ER chaperones is independent of initial exposure to the corresponding bacterial chaperones; rather, ER chaperones may represent genuine autoantigens. [ABSTRACT FROM PUBLISHER]

In the current study, we describe four novel members of the 90 kDa heat shock protein (HSP90) family expressed in Japanese quail, Coturnix japonica. The coding regions of the genes, CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1, exhibited more than 94% similarity to their related genes in chicken. The putative proteins encoded by these quail genes contained motifs considered essential for HSP90 gene function. In addition, the predicted proteins were more similar to HSP90AA1, HSP90AB1, HSP90B1 and TRAP1 proteins expressed in vertebrates than they were to other members of the HSP90 family. Exon numbers of CjHSP90AA1 (11), CjHSP90AB1 (12) or CjTRAP1 (18) are the same as the chicken and mammalian orthologs. Furthermore, gene order in the regions surrounding CjHSP90AB1 and CjTRAP1 has been preserved, providing evidence that the genomic regions were orthologous to HSP90-containing regions in the chicken genome. The promoter regions of the genes also contained conserved motifs identified in related genes of chicken. However, the nucleotide sequences of the 5′-flanking region of these genes were highly polymorphic. We also found that CjHSP90AA1 exhibited a robust response to heat shock treatment. Taken together, the data suggest that CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1 encode orthologs of HSP90AA1, HSP90AB1, HSP90B1 and TRAP1, respectively. [ABSTRACT FROM AUTHOR]

The envelope glycoprotein of vesicular stomatitis virus (VSV-G) enables viral entry into hosts as distant as insects and vertebrates. Because of its ability to support infection of most, if not all, human cell types VSV-G is used in viral vectors for gene therapy. However, neither the receptor nor any specific host factor for VSV-G has been identified. Here we demonstrate that infection with VSV and innate immunity via Toll-like receptors (TLRs) require a shared component, the endoplasmic reticulum chaperone gp96. Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. The ubiquitous. expression of gp96 is therefore essential for the remarkably broad tropism of VSV-G. Cells deficient in gp96 also lack functional TLRs, which suggests that pathogen-driven pressure for TLR-mediated immunity maintains the broad host range of VSV-G by positively selecting for the ubiquitous expression of gp96. [ABSTRACT FROM AUTHOR]

Lead, a ubiquitous heavy metal, is an important industrial and environmental pollutant that can target the vascular endothelium. To clarify the effects of lead on the unfolded protein response (UPR) and their significance in cytotoxicity, we examined the expression and function of endoplasmic reticulum (ER) chaperones glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94) in vascular endothelial cells. We used bovine aortic endothelial cells as an in vitro model of the vascular endothelium. Exposure of vascular endothelial cells to lead nitrate resulted in a marked induction of GRP78 and GRP94 messenger RNA levels. In response to lead, the expression of GRP78 and GRP94 proteins also significantly increased in a dose- and time-dependent manner. In addition, small interfering RNA (siRNA)-mediated knockdown of GRP78 significantly enhanced lead-induced cytotoxicity. Compared with other metal(loid)s, including cadmium chloride, zinc sulfate, copper sulfate, and sodium arsenite, lead nitrate was found to be the most potent metal to induce these chaperones in endothelial cells. In the examined UPR pathways, lead increased the phosphorylation of inositol-requiring enzyme 1 (IRE1) and c-jun N-terminal kinase (JNK). Interestingly, the lead-induced upregulation of GRP78 and GRP94 was almost completely blocked by the JNK inhibitor SP600125 or activator protein-1 (AP-1) inhibitor curcumin. Taken together, these results suggest that lead induces ER stress, but the induction of GRP78 and GRP94 expression via the JNK-AP-1 pathway functions as a defense mechanism against lead-induced cytotoxicity in vascular endothelial cells. [ABSTRACT FROM AUTHOR]

Abstract: Heat shock proteins (HSPs) are induced not only under heat stress conditions but also under other environmental stresses such as water stress. In plants, HSPs families are larger than those of other eukaryotes. In order to elucidate a possible connection between HSP expression and photosynthetic acclimation or conditioning, we conducted a water stress experiment in loblolly pine (Pinus taeda L.) seedlings involving progressive treatment consisting of one cycle of mild stress (−1 MPa) followed by two cycles of severe stress (−1.7 MPa). Net photosynthesis was measured at each stress level. Photosynthetic acclimation occurred in the progressive treatment after the first cycle, but not in the severe treatment, suggesting that a cycle of mild stress conditioned the trees to adapt to a more severe stress. Real time results indicated specific patterns in needles in the expression of HSP70, HSP90 and sHSP genes for each treatment, both at maximum stress and at recovery. We identified a pine homolog to GRP94 (ER resident HSP90) that was induced after rehydration coincident with acclimation. Further analysis of the promoter region of the pine GRP94 showed putative cis-elements associated with water stress and rehydration, corresponding to the expression pattern observed in our experiment. [Copyright &y& Elsevier]

Curcumin is a non-toxic polyphenol with pleiotropic activities and limited bioavailability. We investigated whether a brief exposure to low doses of curcumin would induce in the myogenic C2C12 cell line an endoplasmic reticulum (ER) stress response and protect against oxidative stress. A 3-hr curcumin administration (5–10 μM) increased protein levels of the ER chaperone Grp94, without affecting those of Grp78, calreticulin and haeme-oxygenase-1 (HO-1). Exposure of cells to hydrogen peroxide 24 hrs after the curcumin treatment decreased caspase-12 activation, total protein oxidation and translocation of NF-κB to the nucleus, compared with untreated cells. Grp94 overexpression, achieved by means of either stable or transient trasfection, induced comparable cytoprotective effects to hydrogen peroxide. The delayed cytoprotection induced by curcumin acted through Grp94, because the curcumin-induced increase in Grp94 expression was hampered by either stable or transient transfection with antisense cDNA; in these latter cells, the extent of total protein oxidation, as well as the translocation of NF-κB to the nucleus, and the percentage of apoptotic cells were comparable to those observed in both curcumin-untreated wild-type and empty vector transfected cells. Defining the mechanism(s) by which Grp94 exerts its antioxidant defence, the determination of cytosolic calcium levels in C2C12 cells by fura-2 showed a significantly reduced amount of releasable calcium from intracellular stores, both in conditions of Grp94 overexpression and after curcumin pre-treatment. Therefore, a brief exposure to curcumin induces a delayed cytoprotection against oxidative stress in myogenic cells by increasing Grp94 protein level, which acts as a regulator of calcium homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: GRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. This dichotomy suggests that GRP94 is required for the functional expression of secretory and/or membrane proteins that enable the integration of cells into tissues. To explore this hypothesis, we have identified the Drosophila ortholog of GRP94, Gp93, and report that Gp93 is an essential gene in Drosophila. Loss of zygotic Gp93 expression is late larval-lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. Gp93 mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of Gp93-expression are profound; Gp93 mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of Gp93 expression resemble those reported for mutations in labial, an endodermal homeotic gene required for copper cell specification, and α-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding lab protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton to confer epithelial membrane specialization. [Copyright &y& Elsevier]

Abstract: The endoplasmic reticulum chaperone GRP94 is essential for early embryonic development and in particular affects differentiation of muscle lineages. To determine why an ubiquitously expressed protein has such a specific effect, we investigated the function of GRP94 in the differentiation of established myogenic cell lines in culture. Using both genetic suppression of expression, via RNA interference, and inhibition of function, via specific chemical inhibitors, we show that GRP94 expression and activity are needed for the in vitro fusion of myoblasts precursors into myotubes and the expression of contractile proteins that mark terminal differentiation. The inhibition can be complemented by addition of insulin-like growth factors to the cultures. GRP94 is not needed for the initial steps of myogenesis, only for the steps downstream of MyoD up-regulation, coinciding with the known need for synergistic input from growth factor signaling. Indeed, GRP94 is needed for the production of insulin-like growth factors I and II (IGF-I and IGF-II) by the differentiating cells. Moreover, the depletion of the chaperone does not increase the rate of apoptosis that always accompanies myogenic differentiation. Thus, the major effect of GRP94 on muscle differentiation is mediated by its regulation of IGF production. [Copyright &y& Elsevier]

Abstract: The 94-kDa glucose-regulated protein (GRP94) is an endoplasmic reticulum (ER) chaperone. We cloned the first mollusk grp94 from a cDNA library of the Pacific oyster Crassostrea gigas. Analysis of C. gigas grp94 (cggrp94) clone containing 3212 bp DNA revealed that the cDNA contains a 2391 bp open reading frame that encodes a 797 amino acid protein of 91.6 kDa. The deduced amino acid sequence of cgGRP94 is 67%, 68%, and 67% homologous to the GRP94 of Homo sapiens, GP96 of Strongylocentrotus purpuratus, and GP96 of Xenopus laevis, respectively. CgGRP94 contains an N-terminal 22 amino acid sequence, which is characteristic of a signal sequence. It also contains a HATPase_c domain. In addition, it contains the KDEL (-Lys-Asp-Glu-Leu) peptide motif at the C-terminus, which suggests that cgGRP94 localizes in the ER. Northern blot analysis showed that cggrp94 mRNA is expressed at high levels in the gill which cggrp94 mRNA is induced during air exposure condition. Expression patterns of cggrp94 mRNA differed between gill and mantle, and cggrp94 mRNA was induced at high temperature during air exposure condition. These indicate that cggrp94 mRNA is induced by hypoxia and heat shock stress, and there are different strategies for air exposure condition between gill and mantle. [Copyright &y& Elsevier]

The molecular chaperone, Hsp90, is an essential eukaryotic protein that assists in the maturation and activation of client proteins. Hsp90 function depends upon the binding and hydrolysis of ATP, which causes large conformational rearrangements in the chaperone. Hsp90 is highly conserved from bacteria to eukaryotes, and similar nucleotide-dependent conformations have been demonstrated for the bacterial, yeast, and human proteins. There are, however, important species-specific differences in the ability of nucleotide to shift the conformation from one state to another. Although the role of nucleotide in conformation has been well studied for the cytosolic yeast and human proteins, the conformations found in the absence of nucleotide are less well understood. In contrast to cytosolic Hsp90, crystal structures of the endoplasmic reticulum homolog, Grp94, show the same conformation in the presence of both ADP and AMPPNP. This conformation differs from the yeast AMPPNP-bound crystal state, suggesting that Grp94 may have a different conformational cycle. In this study, we use small angle X-ray scattering and rigid body modeling to study the nucleotide free states of cytosolic yeast and human Hsp90s, as well as mouse Grp94. We show that all three proteins adopt an extended, chair-like conformation distinct from the extended conformation observed for the bacterial Hsp90. For Grp94, we also show that nucleotide causes a small shift toward the crystal state, although the extended state persists as the major population. These results provide the first evidence that Grp94 shares a conformational state with other Hsp90 homologs. [ABSTRACT FROM AUTHOR]

The upregulation of GRP78 and GRP94 under the induction of A23187 and its function in drug resistance to etoposide (VP-16) was investigated in human lung cancer cell line SK-MES-1. The expression of GRP78 and GRP94 induced by A23187 at different concentrations was analyzed by RT-PCR and Western blotting. Cell survival to VP-16 was determined using a colony-formation assay. The expression of GRP78 and GRP94 in the cells was found to correspond well with the cell survival to VP-16. The results showed that upregulation of GRP78 and GRP94 can significantly confer the chemoresistance to VP-16 in human lung cancer cell line SK-MES-1. [ABSTRACT FROM AUTHOR]

Abstract: Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian Hsp90 paralogs, which are cytoplasmic Hsp90α and β, endoplasmic reticulum GRP94, and mitochondrial Trap-1. Given that each of the Hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one Hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each Hsp90 paralog. Here, we present side-by-side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-Hsp90 inhibitors geldanamycin (Gdm) and radamide. These structures reveal paralog-specific differences in the Hsp90 and GRP94 conformations in response to Gdm binding. We also report significant variation in the pose and disparate binding affinities for the Gdm–radicicol chimera radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors. [Copyright &y& Elsevier]

GRP94, a member of HSP90 family, is involved in folding and degradation of endoplasmic reticulum proteins. The proteome analysis of Setaria cervi , a bovine filarial parasite showed that a 91 kDa protein was over expressed, after the parasites were maintained in glucose deprived medium. The MALDI- LC/MS analysis of the 91 kDa band confirmed it as endoplasmin precursor (GRP94). Amino acid sequence alignment of S.cervi GRP94 exhibited maximum similarity with human filarial parasite Wuchereria bancrofti , Brugia malayi and Loa loa GRP94. Tunicamycin treatment of S. cervi worms revealed that the expression of GRP94 is associated with ER stress. Transcription of S. cervi grp94 as well as igf is regulated by transcription factors ATF-6 and XBP-1S which was confirmed by Real Time PCR. Moreover, marked alteration in the expression of igf after 3 h and 6 h of drug treatment suggested propagation of survival pathway under ER stress. The activities of ER stress markers protein disulphide isomerase and glycosyltransferase were significantly reduced after 6 h of tunicamycin treatment. The present findings thus indicate that the expression of GRP94 and regulation of its expression is under ER stress in Setaria cervi. To our knowledge this is the first report of identification of GRP94, in any filarial parasite till date. [Display omitted] • A 91 kDa GRP94 has been identified in S.cervi under glucose starvation. • Tunicamycin induced ER stress in filarial S.cervi. • Activated ER stress leads to upregulated expression of grp94. • Transcription of grp94 is regulated by ATF-6 and XBP-1 s. • Upregulated expression of IGF under UPR suggests antiapoptosis in S.cervi. [ABSTRACT FROM AUTHOR]

The presence of circulating tumor cells (CTC) is common in prostate cancer patients, however until recently their clinical significance was unknown. The CTC stage is essential for the formation of distant metastases, and their continuing presence after radical prostatectomy has been shown to predict recurrent or latent disease. Despite their mechanistic and prognostic importance, due both to their scarcity and difficulties in their isolation, little is known about the characteristics that enable their production and survival. The aim of this study was to investigate the molecular mechanisms underlying the survival of CTC cells. A novel CTC cell line from the bloodstream of an orthotopic mouse model of castration-resistant prostate cancer was established and compared with the primary tumor using attachment assays, detachment culture, Western blot, flow cytometry and 2D gel electrophoresis. Decreased adhesiveness and expression of adhesion molecules E-cadherin, β4-integrin and γ-catenin, together with resistance to detachment and drug-induced apoptosis and upregulation of Bcl-2 were integral to the development of CTC and their survival. Using proteomic studies, we observed that the GRP94 glycoprotein was suppressed in CTC. GRP94 was also shown to be suppressed in a tissue microarray study of 79 prostate cancer patients, indicating its possible role in prostate cancer progression. Overall, this study suggests molecular alterations accounting for the release and survival of CTC, which may be used as drug targets for either anti-metastatic therapy or the suppression of latent disease. We also indicate the novel involvement of GRP94 suppression in prostate cancer metastasis. [ABSTRACT FROM AUTHOR]

Summary: Glucose-related proteins (GRPs) are ubiquitously expressed in endoplasmic reticulum and able to assist in protein folding and assembly; consequently, they are considered as molecular chaperones. GRP78 and GRP94 expression was induced by glucose starvation and up-regulated in the malignancies. To clarify the roles of both molecules in tumorigenesis and progression of gastric carcinomas, immunohistochemistry was used on tissue microarray containing gastric carcinomas, adenomas, and nonneoplastic mucosa using the antibodies against GRP78 and GRP94, with a comparison of their expression with clinicopathological parameters of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III, and HGC-27) were studied for both proteins by immunohistochemistry and Western blot. There was more expression of both proteins in gastric carcinoma and adenoma than in nonneoplastic mucosas (P < .05). All gastric carcinoma cell lines showed their expression at different levels. They were positively correlated with tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis, and Union Internationale Contre le Cancer staging (P < .05), with positive relationship between both proteins (P < .05). Univariate analysis indicated the postsurgical cumulative survival rate of patients with positive GRP78 or GRP94 expression to be lower than that in those without GRP78 or GRP94 expression (P < .05), but the close link disappeared if stratified according to depth of invasion (P > .05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, Union Internationale Contre le Cancer staging, and Lauren classification (P < .05), but not GRP78 and GRP94 expression, were independent prognostic factors for carcinomas (P > .05). Up-regulated expression of GRP78 and GRP94 was possibly involved in pathogenesis, growth, invasion, and metastasis of gastric carcinomas. They were considered objective and effective markers for the aggressive behavior and poor prognosis in gastric carcinomas. [Copyright &y& Elsevier]

Almost 60 years ago, the pioneering work of George Klein and others showed that cancers could be made targets for the immune system. Identification of the tumor targets, known as tumor antigens, became a focus in cancer biology that led to the discovery of the immunological properties of heat-shock proteins (HSPs) in 1986 by Pramod Srivastava and colleagues. Since then, the use of HSPs in the therapeutics of cancer and infectious disease in several clinical trials has been guided by our understanding of the role and effects of HSPs in adaptive and innate immune responses, investigated primarily in mice. This review will highlight the immunological properties of HSPs as we understand them today and review the clinical work on human cancers. Several Phase I and II clinical trials in different types of cancer that have been completed, as well as ongoing Phase III trials, will be summarized. [ABSTRACT FROM AUTHOR]

Abstract: The evolving realization that stress proteins, which have for many years been considered to be exclusively intracellular molecules under normal conditions, can be released from viable cells via a number of potential routes/pathways has prompted interest into their extracellular biology and intercellular signaling properties. That the stress proteins Hsp60, Hsp70 and gp96 can elicit both pro- and anti-inflammatory effects suggests that these molecules play a key role in the maintenance of immunological homeostasis, and a better understanding of the immunobiology of extracellular stress proteins might reveal new and more effective approaches for controlling and managing infectious disease, inflammatory disease and cancer. A number of cell surface receptors for stress proteins have been identified, and the intracellular consequences of these cell surface receptor–ligand interactions have been characterized. To date, studies into the intercellular signaling properties of stress proteins and their interactions with antigen presenting cells have focused on specific receptor-mediated uptake, and have not considered the fact that such cells can also take up proteins via non-specific endocytosis/pinocytosis. Herein we present a methodological approach for assessing receptor-mediated and non-receptor-mediated uptake of gp96 by rat bone marrow-derived dendritic cells. [Copyright &y& Elsevier]

Abstract: Neurodegenerative diseases are often associated with the occurrence of misfolded proteins preceding neuronal cell death. Accumulation of misfolded proteins in the endoplasmic reticulum induces ER stress, which in consequence enhances chaperone expression to restore protein homeostasis. Here we used organotypic hippocampal slice cultures to analyze the time course of chaperone expression and neuronal death after induction of ER stress by tunicamycin treatment. Shortly after explantation many cells stain positive for Fluoro Jade B demonstrating neuronal cell death. While in control cultures the number of Fluoro Jade B labeled cells remarkably decrease over the total period of cultivation, neuronal death remains elevated in ER-stressed slice cultures. Caspase-3 staining revealed that neuronal death is primarily due to apoptosis in tunicamycin-treated slice cultures. The chaperone GRP78/BiP is expressed at low levels in control sections. Its expression is largely restricted to hippocampal neurons. Tunicamycin treatment resulted in upregulation of GRP78/BiP in the neuronal cells. Double-immunolabeling for GFAP shows a concomitant de novo expression of GRP78/BiP in astrocytes. The astrocytic GRP78/BiP upregulation might reflect an early, neuroprotective response. The increase of GRP78/BiP in neurons and astrocytes show successful induction of the ER stress response. The hippocampal slice cultures are, thus, a useful tool to examine the process of neurodegeneration and to investigate neuroprotective devices in an ER stress paradigm. [Copyright &y& Elsevier]

Abstract: Ecotropic Virus Integration site 12 (Evi12) is a common virus insertion site (cVIS) in retrovirally induced murine models of leukemia and lymphoma, suggesting an important role for this locus in these hematopoietic disorders. Evi12 is located near the promoter of the ER chaperone protein and Hsp90 family member Grp94. Here we show that viral insertion in Evi12 results in the expression of aberrant Grp94 transcripts in Cas-Br-MuLV as well as in AKXD induced hematopoietic tumors, demonstrating that Grp94 is a common viral target gene. While most transcripts encode for truncated forms of Grp94, transcripts containing viral gag sequences were detected in the leukemia cell line NFS107. Interestingly, these fusion transcripts encode for myristylated viral–Grp94 fusion proteins that localize to the plasma membrane. Combined with recent evidence that myristylated forms of Hsp90 transform cells, our data suggest that myristylation of target genes may be an important mechanism in retrovirally mediated oncogenesis. Since retroviral insertion in Evi12 also affects the expression of a recently identified novel gene Grp94 neighboring nucleotidase (Gnn), located at the other side of Evi12, it appears that proviral insertion can lead to deregulation of two genes present in the same locus. [Copyright &y& Elsevier]

Abstract: Phosphorylated double-stranded RNA-dependent protein kinase (PKR) is thought to play an important role during ER stress induced cell death, but its molecular mechanism of action has not yet been clarified completely. To resolve this issue, we employed a PKR inhibitor together with ER stress inducers (tunicamycin, thapsigargin, and 2-deoxyglucose) and found that this treatment applied to SK-N-SH and HepG2 cells suppressed the expressional induction of 94kDa glucose regulated protein (GRP94) but not GRP78 proteins at both protein and mRNA levels. Although GRP94 mRNA increased, no significant difference was observed in the mRNA level of spliced X box binding protein 1 (XBP1) and reporter gene assay using GRP78 and GRP94 promoter with an ER stress response element (ERSE) showed that PKR inhibitor did not affect their activity. These results suggest that a novel mechanism other than ERSE-dependent mRNA transcription is required for the induction of GRP94 and phosphorylation of PKR contributes to the induction of GRP94 under ER stress. [Copyright &y& Elsevier]

Abstract: To unveil some molecular mechanisms underlying lithium toxicity, expression changes of stress-related genes or proteins were analysed in A549 cells, cultured for 3 days in presence of lithium. A dose-dependant cell-growth inhibition was found for concentrations ranging from 2 (toxicity threshold) to 12 mM (lethality threshold). cDNA arrays technology was used to analyse effects of 5 and 10 mM lithium. Among genes involved in cell cycle regulation, proliferating cell nuclear antigen (PCNA) was down-regulated and cyclin kinase inhibitor p21 (CDKN1A), up-regulated. Genes of paraoxonase 2, known to prevent LDL lipid peroxidation, and of catalase and SOD were found to be down-regulated whereas genes of cytochrome P450 (CYP2F1, CYP2E1) were up-regulated. This probably results in higher intracellular levels of reactive oxygen species and account for increased levels of lipid peroxidation commonly associated with lithium exposure. Moreover, lithium was found to down-regulate genes coding for anti-apoptotic gene BAG-1 and for most of the molecular chaperones (HSP, GRP). This might account for lithium toxicity since these proteins are critical for cell survival. At translational level, a 105 kDa protein was found to be over-expressed. This protein was recognized by the anti-GRP94, anti-KDEL and anti-phosphoserine monoclonal antibodies suggesting that, lithium could induce post-translational modifications of GRP94 phosphorylation. Using tunicamycin and thapsigargin, it was concluded that lithium effects are not related to defect in N-linked glycosylation and/or to changes in calcium homeostasis. [Copyright &y& Elsevier]

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90. [ABSTRACT FROM AUTHOR]

Abstract: CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells. [Copyright &y& Elsevier]

Abstract: The mood stabilizing drug lithium is a highly effective treatment for bipolar disorder. Previous studies in our laboratory found that chronic treatment with the mood stabilizing drug valproate in rat brain increased the expression of endoplasmic reticulum (ER) stress proteins GRP78, GRP94 and calreticulin. We report here that in primary cultured rat cerebral cortical cells, expression of GRP78, GRP94 and calreticulin are increased not only by valproate, but also by lithium after chronic treatment for 1 week at therapeutically relevant concentrations. However, two other mood stabilizing drugs carbamazepine and lamotrigine had no effect on expression of GRP78, GRP94 or calreticulin. Chronic treatment with lithium for 1 week increased both mRNA and protein levels of ER stress proteins. In contrast to a classic GRP78 inducer thapsigargin, an inhibitor of the ER Ca2+-ATPase, chronic treatment with lithium or valproate for 1 week modestly increased GRP78 expression in neuronal cells, had no effect on basal intracellular free Ca2+ concentration and does not induce cell death. These results indicate that lithium and valproate may increase expression of GRP78, GRP94 and calreticulin in primary cultured rat cerebral cortical cells without causing cell damage. These results also suggest that the mechanism of GRP78 increase induced by lithium and valproate may be different from that of thapsigargin. [Copyright &y& Elsevier]

The molecular chaperone Grp94 (gp96) of the endoplasmic reticulum (ER) lumen plays an essential role in the structural maturation and/or secretion of proteins destined for transport to the cell surface. Its proposed role in binding and transferring peptides for immune recognition is, however, controversial. Using SPR spectroscopy, we studied the interaction of native glycosylated Grp94 at neutral pH and 25 and 37 °C with the viral immunogenic octapeptide RGYVYQGL (VSV8), derived from vesicular stomatitis virus nucleoprotein (52–59). The peptide binds reversibly with low affinity ([ A]0.5 ≈ 640 µm) and a hyperbolic binding isotherm, and the binding is partially inhibited by ATP and Ca2+ at concentrations that are present in the ER lumen, and the effects are explained by conformational changes in the native chaperone induced by these ligands. Our data present experimental support for the recent proposal that, under native conditions, VSV8 binds to Grp94 by an adsorptive, rather than a bioselective, mechanism, and thus further challenge the proposed in vivo peptide acceptor–donor function of the chaperone in the context of antigen-presenting cell activation. [ABSTRACT FROM AUTHOR]

The molecular chaperone Grp94 (gp96) of the endoplasmic reticulum (ER) lumen plays an essential role in the structural maturation and/or secretion of proteins destined for transport to the cell surface. Its proposed role in binding and transferring peptides for immune recognition is, however, controversial. Using SPR spectroscopy, we studied the interaction of native glycosylated Grp94 at neutral pH and 25 and 37 °C with the viral immunogenic octapeptide RGYVYQGL (VSV8), derived from vesicular stomatitis virus nucleoprotein (52–59). The peptide binds reversibly with low affinity ([ A]0.5 ≈ 640 µm) and a hyperbolic binding isotherm, and the binding is partially inhibited by ATP and Ca2+ at concentrations that are present in the ER lumen, and the effects are explained by conformational changes in the native chaperone induced by these ligands. Our data present experimental support for the recent proposal that, under native conditions, VSV8 binds to Grp94 by an adsorptive, rather than a bioselective, mechanism, and thus further challenge the proposed in vivo peptide acceptor–donor function of the chaperone in the context of antigen-presenting cell activation. [ABSTRACT FROM AUTHOR]

N-acetylglucosaminyltransferase V (GnT-V) is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line (GnT-V-AS/7721) was constructed from 7721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7721 and parental 7721 cells. The data indicated that GnT-V-AS/7721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7721 by the analysis on key molecules in both two unfolded protein response (UPR) pathways. For ATF6 and Ire1/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form of XBP-1. As for PERK/eIF2α pathway, the activation of ER eIF2α kinase PERK was observed. To confirm the results from GnT-V-AS/7721 cells, the key molecules in the UPR were examined again in 7721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7721 cells. Rate of 3H-Man incorporation corrected with rate of 3H-Leu incorporation in GnT-V-AS/7721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.Cell Research (2006) 16: 82–92 doi:10.1038/sj.cr.7310011; published online 16 January 2006 [ABSTRACT FROM AUTHOR]

Abstract: Prior induction of an endoplasmic reticulum stress response results in protection against reactive cytotoxins in the LLC-PK1 cell line. The purpose of this investigation was to determine therefore if the endoplasmic reticulum was disrupted by iodoacetamide, tert-butylhydroperoxide or sulfamethoxazole hydroxylamine. Toxic concentrations of the three toxins caused a dramatic loss of GRP94 protein within 3–8h of exposure, while induction of GRP78 and calreticulin occurred at 8 and 24h following exposure. There was no evidence of cytosolic elevation of calcium and neither dantrolene nor xestospongin were able to block the cytotoxicity of IDAM and TBHP. Exposure to the toxins led to DNA degradation and cleavage of procaspase-12. There was only evidence of procaspase-3 cleavage after TBHP exposure. These results demonstrate that the ER is disrupted by the reactive cytotoxins examined in LLC-PK1cells and suggest that the cytoprotection against low to moderate concentrations of cytotoxins observed following endoplasmic reticulum stress protein induction is likely due to a mechanism other than maintenance of calcium homeostasis. [Copyright &y& Elsevier]

Abstract: Background: To investigate the relationship between the expression of glucose-regulated protein94 (GRP94) and GRP78 at the level of mRNA and protein in vivo and in human lung cancer. Methods: RT-PCR, real-time PCR, immunohistochemistry and/or Western blot were used in 54 cases of lung cancer and corresponding normal lung tissue. Results: The expression pattern of GRP94 and GRP78 was similar. There was a significant overexpression of GRP94 and GRP78 at both mRNA and protein levels in cancer tissues as compared to normal tissues. The relative levels of GRP94 and GRP78 mRNA evaluated by RT-PCR in cancer and normal lung tissue were: GRP94: 3.48±2.06 versus 2.01±1.83; GRP78: 3.64±1.87 versus 2.21±1.54; by real-time PCR were: GRP94: 2.89±0.64 versus 1.12±0.54; GRP78: 2.56±0.82 versus 0.96±0.42. The relative level of GRP94 and GRP78 protein by Western blot in cancer and normal lung tissue were: GRP94: 3.46±1.72 versus 1.81±0.92; GRP78: 4.84±2.55 versus 1.91±1.15, indicating an approximate 2-fold and a 3-fold increase in GRP94 and GRP78 protein in cancer tissue as compared with normal tissue. Immunohistochemistry result for GRP94 and GRP78 in cancer and normal tissue was similar, that is: a stronger stain was observed in cancer tissue (main intensity of staining ++ to +++) compared to normal tissue (main intensity of staining + to ++). All the difference for GRP94 and GRP78 between the two tissues were significant (p <0.05). Furthermore, the overexpression of GRP94 and GRP78 in the cancer tissue correlated with grade of differentiation and stage of tumors. There was stronger expression in poorly differentiated tumors than in well-moderately differentiated tumors (p <0.05). There was also stronger expression in stage III than in stages I and II tumors (p <0.05). No statistically significant differences were found among various pathologic types of tumors. Correlation analysis showed that there is a positive correlation between GRP94 and GRP78. Conclusion: The expression pattern of GRP94 and GRP78 was similar in human lung cancer. They both were related with the differentiation and progression of the cancer. The expression at mRNA and protein level may be valuable in evaluating the grade of differentiation and clinical stage of human lung cancer. [Copyright &y& Elsevier]

By comparative genomics, we have identified a gene of the intracellular pathogen Listeria monocytogenes that encodes an LPXTG surface protein absent from nonpathogenic Listeria species. This gene, vip, is positively regulated by PrfA, the transcriptional activator of the major Listeria virulence factors. Vip is anchored to the Listeria cell wall by sortase A and is required for entry into some mammalian cells. Using a ligand overlay approach, we identified a cellular receptor for Vip, the endoplasmic reticulum (ER) resident chaperone Gp96 recently shown to interact with TLRs. The Vip–Gp96 interaction is critical for bacterial entry into some cells. Comparative infection studies using oral and intravenous inoculation of nontransgenic and transgenic mice expressing human E-cadherin demonstrated a role for Vip in Listeria virulence, not only at the intestine level but also in late stages of the infectious process. Vip thus appears as a new virulence factor exploiting Gp96 as a receptor for cell invasion and/or signalling events that may interfere with the host immune response in the course of the infection. [ABSTRACT FROM AUTHOR]

Abstract: TRAP1 (tumor necrosis factor receptor-associated protein 1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. In this study, we mainly examined the behavior of Dictyostelium TRAP1 homologue, Dd-TRAP1, during Dictyostelium development by immunoelectron microscopy. In vegetatively growing D. discoideum Ax-2 cells, Dd-TRAP1 locates in nucleolus and vesicles in addition to the cell cortex including cell membrane. Many of Dd-TRAP1 molecules moved to the mitochondrial matrix in response to differentiation, although Dd-TRAP1 on the cell membrane seems to be retained. Some Dd-TRAP1 was also found to be secreted to locate outside the cell membrane in Ax-2 cells starved for 6 h. At the multicellular slug stage, Dd-TRAP1 was primarily located in mitochondria and cell membrane in both prestalk and prespore cells. More importantly, in differentiating prespore cells, a significant number of Dd-TRAP1 locates in the PSV (prespore-specific vacuole) that is a sole cell type-specific organelle and essential for spore wall formation, whereas some Dd-TRAP1 in the cell cortical region of prestalk cells. These findings strongly suggest the importance of Dd-TRAP1 regulated temporally and spatially during Dictyostelium development. Incidentally, we also have certified that the glucose-regulated protein 94 (Dd-GRP94) is predominantly located in Golgi vesicles and cisternae, followed by its colocalization with Dd-TRAP1 in the PSV. [Copyright &y& Elsevier]