The endoplasmic reticulum stress protein GRP94, in addition to BiP, associates with unassembled immunoglobulin chains.

The molecular chaperone BiP/GRP78 associates with various polypeptides in the endoplasmic reticulum, including immunoglobulin chains. We now show, using chemical cross-linking, that another endoplasmic reticulum stress protein, GRP94, associates with newly synthesized immunoglobulin light and heavy chains. We demonstrate the presence of ternary complexes composed of immunoglobulin chains, BiP and GRP94. Because both BiP and GRP94 associate far less with fully assembled immunoglobulin than with unassembled subunits, our data suggest that GRP94, like BiP, functions as a molecular chaperone. The presence of both BiP and GRP94 in the same complex further suggests that the two stress proteins work in concert during the folding and assembly of immunoglobulins.     

Human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly

The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.     

ADP-ribosylation of the molecular chaperone GRP78/BiP

Starvation of mouse hepatoma cells for essential amino acids or glucose results in the ADP-ribosylation of the molecular chaperone BiP/GRP78. Addition of the missing nutrient to the medium reverses the reaction. The signal mediating the response to environmental nutrients involves the translational efficiency. An inhibitor of proteins synthesis, cycloheximide, or reduced temperature, both of which reduce translational efficiency, stimulate the ADP-ribosylation of BiP/GRP78. Inhibition of N-linked glycosylation of proteins results in the overproduction of BiP/GRP78. The over produced protein is not ADP-ribosylated suggesting that this is the functional form of BiP/GRP78. The over produced BiP/GRP78 can, however, be ADP-ribosylated if the cells are starved for an essential amino acid. BiP/GRP78 resides in the lumen of the endoplasmic reticulum where it participates in the assembly of secretory and integral membrane proteins. ADP-ribosylation of BiP/GRP78 during starvation is probably part of a nutritional stress response which conserves limited nutrients by slowing flow through the secretory pathway.     

Lysozyme Mutants Accumulate in Cells while Associated at their N-terminal Alpha-domain with the Endoplasmic Reticulum Chaperone GRP78/BiP

Amyloidogenic human lysozyme variants deposit in cells and cause systemic amyloidosis. We recently observed that such lysozymes accumulate in the endoplasmic reticulum (ER) with the ER chaperone GRP78/BiP, accompanying the ER stress response. Here we investigated the region of lysozyme that is critical to its association with GRP78/BiP. In addition to the above-mentioned variants of lysozyme, we constructed lysozyme truncation or substitution mutants. These were co-expressed with GRP78/BiP (tagged with FLAG) in cultured human embryonic kidney cells, which were analyzed by western blotting and immunocytochemistry using anti-lysozyme and anti-FLAG antibodies. The amyloidogenic variants were confirmed to be strongly associated with GRP78/BiP as revealed by the co-immunoprecipitation assay, whereas N-terminal mutants pruned of 1-41 or 1-51 residues were found not to be associated with the chaperone. Single amino acid substitutions for the leucine array along the α-helices in the N-terminal region resulted in wild-type lysozyme remaining attached to GRP78/BiP. These mutations also tended to show lowered secretion ability. We conclude that the N-terminal α-helices region of the lysozyme is pivotal for its strong adhesion to GRP78/BiP. We suspect that wild-type lysozyme interacts with the GRP at this region as a step in the proper folding monitored by the ER chaperone.     

Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast

BiP/GRP78 is an essential member of the HSP70 family that resides in the lumen of the endoplasmic reticulum. In yeast, BiP/GRP78 is encoded by the KAR2 gene. A temperature sensitive mutation was isolated in KAR2 and found to cause a rapid block in protein secretion. Secretory precursors of a number of proteins (invertase, carboxypeptidase Y, alpha-factor, and BiP) accumulated that were characteristic of a block in translocation into the lumen of the ER. Protease protection experiments confirmed that the precursors accumulated on the cytoplasmic side of the ER membrane. Moreover, depletion of wild-type KAR2 protein also resulted in a block in translocation of secretory proteins. These results implicate BiP/GRP78 function in the continued translocation of proteins into the lumen of the ER.     

Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1

The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulates the ATPase activity of BiP/GRP78 at stoichiometric concentrations. The C-terminal tail of BiP/GRP78 is not required for the interaction with J-MTJ1, leaving the function of this portion of the molecule still unclear. Physical interactions between J-MTJ1 and BiP/GRP78 are stable and can be abolished by a single histidine --> glutamine substitution in the highly conserved HPD motif shared by all DnaJ-like proteins. The J-MTJ1 fragment, but not the mutant J-MTJ1:H89Q fragment, stimulates the ATPase activity of Escherichia coli DnaK, although at a higher concentration than its genuine partner DnaJ. Full-length DnaJ does not stimulate BiP over the range of concentrations investigated. These results indicate that the J domain of MTJ1 is sufficient for its interaction with BiP/GRP78 and cannot be substituted by E. coli DnaJ.     

Posttranslational folding of vesicular stomatitis virus G protein in the ER: involvement of noncovalent and covalent complexes

In this study, we show that posttranslational folding of Vesicular Stomatitis virus G protein subunits can involve noncovalent, multimeric complexes as transient intermediates. The complexes are heterogeneous in size (4-21S20,W), contain several G glycopolypeptides, and are associated with BiP/GRP78. The newly synthesized, partially intrachain disulfide-bonded G proteins enter these complexes immediately after chain termination, and are released 1-4 min later as fully oxidized, trimerization-competent monomers. These monomers are properly folded, judging by their binding of conformation-specific mAbs. When the G protein is translated in the presence of DTT, it remains reduced, largely unfolded and aggregated in the ER, but it can fold successfully when the DTT is removed. In this case, contrary to normal folding, the aggregates become transiently disulfide cross-linked. We also demonstrated that the fidelity of the folding process is dependent on metabolic energy. Finally, we established that the G protein of the folding mutant of the Vesicular Stomatitis virus, ts045, is blocked at a relatively late step in the folding pathway and remains associated with oligomeric, BiP/GRP78-containing folding complexes.     

Folding of hepatitis C virus E1 glycoprotein in a cell-free system

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.     

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.     

Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein

To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a panel of well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations: 1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant G proteins which do not form correct intrachain disulfide bonds.     

Chaperones involved in hepatitis B virus morphogenesis

Little is known about host cell factors necessary for hepatitis B virus (HBV) assembly which involves envelopment of cytosolic nucleocapsids by the S, M and L transmembrane viral envelope proteins and subsequent budding into intraluminal cisternae. Central to virogenesis is the L protein that mediates hepatocyte receptor binding and envelopment of capsids. To serve these topologically conflicting roles, L protein exhibits an unusual dual membrane topology, disposing its N-terminal preS domain inside and outside of the virion lipid envelope. The mixed topology is achieved by posttranslational preS translocation of about half of the L protein molecules across a post-endoplasmic reticulum membrane. Here we identify and characterize a preS-specific sequence that confers the suppression of cotranslational translocation even of a model reporter. This cytosolic anchorage sequence specifically binds the cognate heat shock protein Hsc70, thus indicating chaperone participitation in HBV morphogenesis. Conversely, the M envelope protein needs the assistance of the chaperone calnexin for proper folding and trafficking. Calnexin selectively binds to the N-glycan, specific for M, rather than to the N-glycan, common to all three envelope proteins. As inhibition of the calnexin-M interaction blocks the secretion of viral envelopes, we propose an essential role for calnexin, as well as for Hsc70, in chaperoning HBV assembly.     

Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.     

Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.     

Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.

The 78,000-dalton glucose-regulated protein (GRP78) and the immunoglobulin heavy-chain-binding protein (BiP) were shown to be the same protein by NH2-terminal sequence comparison. Immunoprecipitation of GRP78-BiP induced by glucose starvation and a temperature-sensitive mutation in a hamster fibroblast cell line demonstrated the association of GRP78-BiP with other cellular proteins. In both fibroblasts and lymphoid cells, GRP78-BiP was found to label with 32Pi and [3H]adenosine. Phosphoamino acid analysis demonstrated that GRP78-BiP is phosphorylated on serine and threonine residues. Conditions which induce increased production of GRP78-BiP resulted in decreased incorporation of 32Pi and [3H]adenosine into GRP78-BiP. Furthermore, we report here that the phosphorylated form of BiP resides in the endoplasmic reticulum and that BiP which is associated with heavy chains is not phosphorylated or labeled with [3H]adenosine, whereas free BiP is. This suggests that posttranslational modifications may be important in regulating the synthesis and binding of BiP.     

Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.     

Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells

Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.     

Sleep deprivation induces the unfolded protein response in mouse cerebral cortex

Little is known about the molecular mechanisms underlying sleep. We show the induction of key regulatory proteins in a cellular protective pathway, the unfolded protein response (UPR), following 6 h of induced wakefulness. Using C57/B6 male mice maintained on a 12:12 light/dark cycle, we examined, in cerebral cortex, the effect of different durations of prolonged wakefulness (0, 3, 6, 9 and 12 h) from the beginning of the lights-on inactivity period, on the protein expression of BiP/GRP78, a chaperone and classical UPR marker. BiP/GRP78 expression is increased with increasing durations of sleep deprivation (6, 9 and 12 h). There is no change in BiP/GRP78 levels in handling control experiments carried out during the lights-off period. PERK, the transmembrane kinase responsible for attenuating protein synthesis, which is negatively regulated by binding to BiP/GRP78, is activated by dissociation from BiP/GRP78 and by autophosphorylation. There is phosphorylation of the elongation initiation factor 2alpha and alteration in ribosomal function. These changes are first observed after 6 h of induced wakefulness. Thus, prolonging wakefulness beyond a certain duration induces the UPR indicating a physiological limit to wakefulness.     

Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus

Proteins synthesized in the ER are generally transported to the Golgi complex and beyond only when they have reached a fully folded and assembled conformation. To analyze how the selective retention of misfolded proteins works, we monitored the long-term fate of a membrane glycoprotein with a temperature-dependent folding defect, the G protein of tsO45 vesicular stomatitis virus. We used indirect immunofluorescence, immunoelectron microscopy, and a novel Nycodenz gradient centrifugation procedure for separating the ER, the intermediate compartment, and the Golgi complex. We also employed the folding and recycling inhibitors dithiothreitol and AIF4-, and coimmunoprecipitation with calnexin antibodies. The results showed that the misfolded G protein is not retained in the ER alone; it can move to the intermediate compartment and to the cis-Golgi network but is then recycled back to the ER. In the ER it is associated with calnexin and BiP/GRP78. Of these two chaperones, only BiP/GRP78 seems to accompany it through the recycling circuit. Thus, the retention of this misfolded glycoprotein is the result of multiple mechanisms including calnexin binding in the ER and selective retrieval from the intermediate compartment and the cis-Golgi network.