Search

The 94 kDa molecular chaperone, glucose‐regulated protein 94 (Grp94), has garnered interest during the last decade due to its direct association with endoplasmic reticulum (ER) stress and disease. Grp94 belongs to the Hsp90 family of molecular chaperones and is a master regulator of ER homeostasis due to its ability to fold and stabilize proteins/receptors, and to chaperone misfolded proteins for degradation. Multiple studies have demonstrated that Grp94 knockdown or inhibition leads to the degradation of client protein substrates, which leads to disruption of disease‐dependent signaling pathways. As a result, small molecule inhibitors of Grp94 have become a promising therapeutic approach to target a variety of disease states. Specifically, Grp94 has proven to be a promising target for cancer, glaucoma, immune‐mediated inflammation, and viral infection. Moreover, Grp94‐peptide complexes have been utilized effectively as adjuvants for vaccines against a variety of disease states. This work highlights the significance of Grp94 biology and the development of therapeutics that target this molecular chaperone in multiple disease states. [ABSTRACT FROM AUTHOR]

GRP94 (glucose‐regulated protein 94) is a well‐studied chaperone with a lysine, aspartic acid, glutamic acid and leucine (KDEL) motif at its C‐terminal, which is responsible for GRP94 localization in the endoplasmic reticulum (ER). GRP94 is upregulated during ER stress to help fold unfolded proteins or direct proteins to ER‐associated degradation. In a previous study, engineered GRP94 without the KDEL motif stimulated a powerful immune response in vaccine cells. In this report, we show that endogenous GRP94 is naturally secreted into the medium in a truncated form that lacks the KDEL motif in Chinese hamster ovary cells. The secretion of the truncated form of GRP94 was stimulated by the induction of ER stress. These truncations prevent GRP94 recognition by KDEL receptors and retention inside the cell. This study sheds light on a potential trafficking phenomenon during the unfolded protein response that may help understand the functional role of GRP94 as a trafficking molecule. [ABSTRACT FROM AUTHOR]

Background: Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle‐specific integrin‐associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes. Methods: Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail‐suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno‐associated virus infection. In vivo interference of exogenous melusin with dominant‐negative kinases and other atrophy attenuators (Grp94 cDNA; 7‐nitroindazole) on size of unloaded rat myofibers was also explored. Results: Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail‐suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of β1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (−31%), compared with controls (−48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin‐expressing adeno‐associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (−33%) by reducing Atrogin‐1 and MurF‐1 transcripts (P ≤ 0.002), despite of a two‐fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co‐transfection with dominant‐negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7‐nitroindazole treatment or with cDNA transfection of the nNOS‐interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy. Conclusions: Disuse/unloading‐induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) has been reported during normal development of cortical neurons and cerebellar white matter and may also contribute to the pathogenesis of neurological conditions, such as Marinesco‐Sjogren syndrome and Borna virus infection, which result in cerebellar defects. The UPR is initiated when the processing capacity of the endoplasmic reticulum (ER) is overwhelmed. Misfolded proteins accumulate and can activate ER stress sensors; PKR‐like endoplasmic reticulum kinase (PERK), inositol‐requiring enzyme 1 (IRE1), activated transcription factor 6 (ATF6) and their downstream targets glucose‐regulated protein 78 (GRP78), glucose‐regulated protein 94 (GRP94) and protein disulfide isomerase (PDI). In order to provide a fuller appreciation of the possible importance of ER stress‐associated proteins in the context of cerebellar disease, we have profiled the expression of ER stress sensors and their downstream targets in the developing cerebellar cortex in postnatal rat. Activation of PERK and IRE1 stress sensors was observed for the first time in normally developing granule cell precursors. A second proliferative pPERK‐positive population was also detected in the internal granular layer (IGL). In general, the density of UPR protein‐positive cells was found to decrease significantly when profiles in early and late postnatal ages were compared. These data may be relevant to studies of medulloblastoma and warrant further investigation. [ABSTRACT FROM AUTHOR]

Skeletal muscle atrophy following unloading or immobilization represents a major invalidating event in bedridden patients. Among mechanisms involved in atrophy development, a controversial role is played by neuronal NOS (nNOS; NOS1), whose dysregulation at the protein level and/or subcellular distribution also characterizes other neuromuscular disorders. This study aimed to investigate unloading‐induced changes in nNOS before any evidence of myofiber atrophy, using vastus lateralis biopsies obtained from young healthy subjects after a short bed‐rest and rat soleus muscles after exposure to short unloading periods. Our results showed that (1) changes in nNOS subcellular distribution using NADPH‐diaphorase histochemistry to detect enzyme activity were observed earlier than using immunofluorescence to visualize the protein; (2) loss of active nNOS from the physiological subsarcolemmal localization occurred before myofiber atrophy, i.e. in 8‐day bed‐rest biopsies and in 6 h‐unloaded rat soleus, and was accompanied by increased nNOS activity in the sarcoplasm; (3) nNOS (Nos1) transcript and protein levels decreased significantly in the rat soleus after 6 h and 1 day unloading, respectively, to return to ambulatory levels after 4 and 7 days of unloading, respectively; (4) unloading‐induced nNOS redistribution appeared dependent on mitochondrial‐derived oxidant species, indirectly measured by tropomyosin disulfide bonds which had increased significantly in the rat soleus already after a 6 h‐unloading bout; (5) activity of displaced nNOS molecules is required for translocation of the FoxO3 transcription factor to myofiber nuclei. FoxO3 nuclear localization in rat soleus increased after 6 h unloading (about four‐fold the ambulatory level), whereas it did not when nNOS expression and activity were inhibited in vivo before and during 6 h unloading. In conclusion, this study demonstrates that the redistribution of active nNOS molecules from sarcolemma to sarcoplasm not only is ahead of the atrophy of unloaded myofibers, and is induced by increased production of mitochondrial superoxide anion, but also drives FoxO3 activation to initiate muscle atrophy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure-activity relationship studies led to the discovery of compound 30, which exhibits 540 n m affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cell signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers. [ABSTRACT FROM AUTHOR]

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′- N-ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N-terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition. [ABSTRACT FROM AUTHOR]

This review discusses the potential use of intracellular tumor antigens as targets of antibody-based immunotherapy for the treatment of solid tumors. In addition, it describes the characteristics of the intracellular tumor antigens targeted with antibodies which have been described in the literature and have been identified in the authors' laboratory. Finally, the mechanism underlying the trafficking of the intracellular tumor antigens to the plasma membrane of tumor cells are reviewed. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was to investigate the expression of Hsp90β and GRP94, and elucidate the clinical significance of their expression, in patients with resectable non-small-cell lung cancer ( NSCLC). Methods and results Surgical tissue specimens were obtained from 208 patients with NSCLC who underwent surgical resection. The expression levels of Hsp90β and GRP94 were assessed with tissue microarrays and immunohistochemistry. No correlations were observed between Hsp90β or GRP94 expression and several clinicopathological factors. The high-Hsp90β group [median overall survival ( OS) 20.4 months; 95% confidence interval ( CI) 0.000-40.864] showed a significant decrease in OS as compared with the low-Hsp90β group (median OS not reached; P = 0.003). In contrast to the Hsp90β analysis, the GRP94 analysis did not show a difference in OS. Moreover, in subgroup analyses of patients with squamous cell carcinoma histology, OS ( P = 0.012) and relapse-free survival ( P = 0.044) were significantly worse in the high-Hsp90β group than in the low-Hsp90β group. Multivariate analysis suggested that old age [hazard ratio ( HR) 1.568; 95% CI 1.019-2.412; P = 0.041], advanced disease ( HR 2.066; 95% CI 1.218-3.502; P = 0.007) and high Hsp90β expression ( HR 1.802; 95% CI 1.061-3.060; P = 0.029) were independent poor prognostic factors for OS. Conclusions Hsp90β expression might be a useful marker of poor OS, although further large prospective studies are warranted to validate our findings. [ABSTRACT FROM AUTHOR]

HER2 receptor dimerization is a critical step in the HER2 activation process. Here, we demonstrated that heat shock protein gp96 on cell membrane interacts with HER2, facilitates HER2 dimerization and promotes cell proliferation. Cell membrane gp96 levels were observed to correlate with HER2 phosphorylation in primary breast tumors. Finally, we provide evidence that targeting gp96 with a specific monoclonal antibody led to decreased cell growth and increased apoptosis in vitro, and suppression of tumor growth in vivo. Our work represents a new therapeutic strategy for inhibiting HER2 signaling in cancer. [ABSTRACT FROM AUTHOR]

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]

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]

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 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]

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]

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: Hypoxic environment in solid tumor is known to favor cell survival and to initiate the formation of new capillaries. In this work, we identified by 2D gel analysis 94-kDa glucose-regulated protein (GRP94) as being upregulated in human endothelial cells in response to hypoxia. Three putative hypoxia responsive elements (HRE) were found in the GRP94 promoter. Competition experiments of HIF-1 DNA binding using specific probes containing each HRE sequence of the GRP94 promoter clearly evidenced that HIF-1 binds these sequences with high affinity. The human GRP94 promoter was then cloned upstream of the luciferase gene and showed enhanced activity in hypoxic conditions. Mutation of two of the three HREs present in this promoter completely inhibited the hypoxia-induced increase in luciferase activity. [Copyright &y& Elsevier]

N-linked glycans with complex structure have a major role in the biological activity of a wide variety of cell surface and secreted glycoproteins. Here, we show that geldanamycin, an inhibitor of Hsp90, interferes with the formation of complex glycosylated mammalian prion protein (PrPC ). Similarly to inhibitors of α-mannosidases, geldanamycin stabilized a high mannose PrPC glycoform and prevented the subsequent processing into complex structures. Moreover, a PrP/Grp94 complex could be isolated from geldanamycin-treated cells, suggesting that Grp94 might play a role in the processing of PrPC in the endoplasmic reticulum. Inhibition of complex glycosylation did not interfere with the glycosylphosphatidylinositol (GPI) anchor attachment and cellular trafficking of high mannose PrPC to the outer leaflet of the plasma membrane. In scrapie-infected neuroblastoma cells, however, high mannose PrPC glycoforms were preferred substrates for the formation of PrP-scrapie (PrPSc ). Our study reveals that complex glycosylation is dispensable for the cellular trafficking of PrPC , but modulates the formation of PrPSc . [ABSTRACT FROM AUTHOR]

N-linked glycans with complex structure have a major role in the biological activity of a wide variety of cell surface and secreted glycoproteins. Here, we show that geldanamycin, an inhibitor of Hsp90, interferes with the formation of complex glycosylated mammalian prion protein (PrPC ). Similarly to inhibitors of α-mannosidases, geldanamycin stabilized a high mannose PrPC glycoform and prevented the subsequent processing into complex structures. Moreover, a PrP/Grp94 complex could be isolated from geldanamycin-treated cells, suggesting that Grp94 might play a role in the processing of PrPC in the endoplasmic reticulum. Inhibition of complex glycosylation did not interfere with the glycosylphosphatidylinositol (GPI) anchor attachment and cellular trafficking of high mannose PrPC to the outer leaflet of the plasma membrane. In scrapie-infected neuroblastoma cells, however, high mannose PrPC glycoforms were preferred substrates for the formation of PrP-scrapie (PrPSc ). Our study reveals that complex glycosylation is dispensable for the cellular trafficking of PrPC , but modulates the formation of PrPSc . [ABSTRACT FROM AUTHOR]

The α isoform of human 90-kDa heat shock protein (HSP90α) is composed of three domains: the N-terminal (residues 1–400); middle (residues 401–615) and C-terminal (residues 621–732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650–697 of HSP90α were essential for the binding to the middle domain. An almost identical region (residues 657–720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662–678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90α and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins. [ABSTRACT FROM AUTHOR]

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the Kb -SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I. [ABSTRACT FROM AUTHOR]

Objectives: This paper reviews results of our studies examining the regulation of endoplasmic reticulum (ER) stress proteins by valproate (VPA), and discusses the possible implications in bipolar disorder. Methods: Our previous studies in the field are reviewed along with relevant literature. Results: Using differential display PCR, we identified GRP78 as a VPA-regulated gene in rat cerebral cortex. We also showed that other members of the ER stress proteins family, GRP94 and calreticulin, are also upregulated by VPA. Immunohistochemistry identified that ER stress proteins are increased in frontal and parietal cortex, as well as regions of the hippocampus in rat brain following chronic treatment with VPA. Conclusions: Regulation of ER stress proteins by VPA may prove to be important to the mechanism of action of the drug. The neuroprotective role of these proteins may also prove to be involved in the pathophysiology of bipolar disorder. [ABSTRACT FROM AUTHOR]

The Arabidopsis shepherd (shd) mutant shows expanded shoot apical meristems (SAM) and floral meristems (FM), disorganized root apical meristems, and defects in pollen tube elongation. We have discovered that SHD encodes an ortholog of GRP94, an ER-resident HSP90-like protein. Since the shd phenotypes in SAM and FM are similar to those of the clavata (clv) mutants, we have explored the possibility that CLV complex members could be SHD targets. The SAM and FM morphology of shd clv double mutants are indistinguishable from those of clv single mutants, and the wuschel (wus) mutation is completely epistatic to the shd mutation, indicating that SHD and CLV act in the same genetic pathway to suppress WUS function. Moreover, the effects of CLV3 overexpression that result in the elimination of SAM activity were abolished in the shd mutant, indicating that CLV function is dependent on SHD function. Therefore, we conclude that the SHD protein is required for the correct folding and/or complex formation of CLV proteins. [ABSTRACT FROM AUTHOR]

Molecular chaperones, both endoplasmic reticulum and cytosol derived, have been identified as tumor rejection antigens; in animal models, they can elicit prophylactic and therapeutic immune responses against their tumor of origin. Chaperone immunogenic activity derives from three principal characteristics: they bind an array of immunogenic (poly)peptides, they can be efficiently internalized by professional antigen-presenting cells, and once internalized, they traffic to a subcellular compartment(s) where peptide release can occur. Within the antigen-presenting cell, chaperone-derived peptides can be assembled onto major histocompatibility class I molecules for presentation at the antigen-presenting cell surface, thereby yielding the requisite and specific CD8+ T-cell responses that contribute to the process of tumor rejection. Though it is clear that chaperones, in particular GRP94 (gp96), calreticulin and Hsp70, can elicit cellular immune responses, the subcellular basis of chaperone processing by antigen-presenting cells remains mysterious. In this review, we discuss recent reports describing the identification of a chaperone internalization receptor and the physiological release of chaperones from necrotic cells, and we present views on the trafficking pathways within antigen-presenting cells that may function to deliver the chaperone-associated peptides to subcellular organelles for their subsequent exchange onto major histocompatibility complex molecules. [ABSTRACT FROM AUTHOR]