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.     

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.     

Molecular chaperone GRP78/BiP interacts with the large surface protein of hepatitis B virus in vitro and in vivo

The proper folding and assembly of viral envelope proteins are mediated by host chaperones. In this study, we demonstrated that an endoplasmic reticulum luminal chaperone GRP78/BiP bound specifically to the pre-S1 domain of the L protein in vitro and in vivo where complete viral particles were secreted, suggesting that GRP78/BiP plays an essential role in the proper folding of the L protein and/or assembly of viral envelope proteins.     

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.     

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.     

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.     

Facilitatory roles of novel compounds designed from cyclopentenone prostaglandins on neurite outgrowth-promoting activities of nerve growth factor

Cyclopentenone prostaglandins (PGs) are known to arrest the cell cycle at the G(1) phase in vitro and to suppress tumor growth in vivo. However, their effects on neurons are unclear. Here, we report that some cyclopentenone PGs function as neurite outgrowth-promoting factors. They promoted neurite outgrowth from PC12 cells and from dorsal root ganglion explants but only in the presence of nerve growth factor (NGF). We refer to these PGs as neurite outgrowth-promoting PGs (NEPPs). Through study of the structure-function relationship of NEPP1-10 and related compounds, we found that the cross-conjugated dienone moiety of NEPPs was essential for promoting neurite outgrowth, and NEPP10 was concluded to be the best candidate for drug development. We also investigated the intracellular mechanism of the promotion by NEPPs and obtained evidence that immunoglobulin heavy chain binding protein/glucose-regulated protein 78 (BiP/GRP78) plays a role in the promotion, based on the following observations: Antisense nucleotides for BiP/GRP78 gene blocked the promotion of neurite outgrowth; BiP/GRP78 protein level increased in response to NEPPs; and overexpression of BiP/GRP78 protein by adenoviral gene transfer promoted the neurite outgrowth by NGF.     

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.     

Bip/GRP78-induced production of cytokines and uptake of amyloid-beta(1-42) peptide in microglia

In the brains of Alzheimer's disease (AD) patients, fibrillar amyloid-beta peptides (Abeta) are markedly accumulated and the microglia associate with the amyloid plaques. However, the regulation of Abeta clearance is still unclear. In the present study, we examined the effect of a chaperone protein BiP/GRP78 on the microglial function. Exogenous addition of recombinant BiP/GRP78 induced the production of cytokines such as interleukin-6 and tumor necrosis factor-alpha, but heat treatment of this protein abolished the activity. Although Abeta(1-42) did not induce cytokine production, it was taken up by the microglia. In addition, the amount of Abeta(1-42) uptake and the number of microglia that phagocytosed Abeta(1-42) were markedly increased by BiP/GRP78. Exogenous BiP/GRP78 also translocated to the endoplasmic reticulum (ER). These results suggest that BiP/GRP78 stimulates Abeta clearance in the microglia, and that dysfunction in the ER may cause the accumulation of extracellular Abeta(1-42).     

Overexpression of the 78-kDa glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) inhibits tissue factor procoagulant activity

Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca(2+) and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells.     

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.     

La3+ binds to BiP/GRP78 and induces unfolded protein response in HepG2 cells

The effects of La3+ on the unfolded protein response signaling pathways were investigated in human hepatoblastoma HepG2 cells. Our data showed that La3+ could induce unfolded protein response in HepG2 cells, including a significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in a concentration and time-dependent manner, UPR transducer IRE1 phosphorylation and splicing activation IRE1 downstream substrate XBP1 mRNA. By using La3+-affinity chromatography, the possible cellular target of La3+ leading to UPR events was shown to be the ER residential chaperone BiP/GRP78. BiP/GRP78 was shown to be a La3+ binding protein and the interaction of La3+ with BiP/GRP78 resulted in dissociation of BiP-IRE1 complexes. La3+ induced dissociation of the BiP/GRP78-IRE1 complex was in a time and concentration manner. The apparent dissociation constant was estimated to be 4 nM. In addition, La3+ was observed to slightly stimulate the production of cellular ROS and cause alteration of intracellular Ca2+, indicating the possible involvement of ROS and Ca2+ alteration in La3+ induced UPR. The present work provides a new perspective for understanding the biological and toxicological effects of La3+.     

Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP)

We have characterized the association between the binding protein, BiP (also known as GRP 78), and misfolded forms of the influenza virus hemagglutinin precursor, HA0. BiP is a heat-shock-related protein that binds to unassembled immunoglobulin heavy chain and to a variety of misfolded proteins in the lumen of the ER. A small fraction (5-10%) of newly synthesized HA0 in CV-1 cells was found to be misfolded and retained in the ER. When glycosylation was blocked with tunicamycin, all of the HA0 produced was similarly misfolded. The misfolded HA0 was retained as relatively small (9-25-S) complexes associated with BiP. In these complexes the top domains of HA0 were correctly folded judging by their reactivity with monoclonal antibodies, but the polypeptides were cross-linked via anomalous interchain disulfides. The association with BiP was non-covalent and easily broken by warming to 37 degrees C or by adding ATP to the lysate. Pulse-chase experiments showed that HA0's self-association into complexes occurred immediately after synthesis and was followed rapidly by BiP association. The misfolded, BiP-associated HA0 was not transported to the plasma membrane but persisted as complexes in the ER for a long period of time before degradation (t1/2 = 6 h). The results suggested that BiP may be part of a quality control system in the ER and that one of its functions is to detect and retain misfolded proteins.     

Cloning and expression of glucose regulated protein 78 (GRP78) in<Emphasis Type="Italic"> Fenneropenaeus chinensis</Emphasis>

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35°C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25°C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

Chemical chaperone 4-phenylbutyric acid alleviates the aggregation of human familial pulmonary fibrosis-related mutant SP-A2 protein in part through effects on GRP78

G231V and F198S mutations in surfactant protein A2 (SP-A2) are associated with familial pulmonary fibrosis. These mutations cause defects in dimer/trimer assembly, trafficking, and secretion, as well as cause mutant protein aggregation. We investigated the effects and mechanisms of chemical chaperones on the cellular and biochemical properties of mutant SP-A2. Chemical chaperones, including 4-phenyl butyric acid (4-PBA), could enhance secretion and decrease intracellular aggregation of mutant SP-A2 in a dose-dependent manner. Interestingly, increased levels of aggregated mutant SP-A2, resulting from MG-132-mediated proteasome inhibition, could also be alleviated by 4-PBA. 4-PBA treatment reduced the degradation of mutant SP-A2 to chymotrypsin digestion in CHO-K1 cells and up-regulated GRP78 (BiP) expression. Overexpression of GRP78 in SP-A2 G231V- or F198S-expressing cells reduced, whereas shRNA-mediated knockdown of GRP78 enhanced aggregation of mutant SP-A2, suggesting that GRP78 regulates aggregation of mutant SP-A2. Together, these data indicate chemical chaperone 4-PBA and upregulation of GRP78 can alleviate aggregation to stabilize and facilitate secretion of mutant SP-A2. The up-regulation expression of GRP78 might partially contribute to the aggregate-alleviating effect of 4-PBA.     

Transient Association of Calnexin and Calreticulin with Newly Synthesized G1 and G2 Glycoproteins of Uukuniemi Virus (Family Bunyaviridae)

The membrane glycoproteins G1 and G2 of Uukuniemi virus, a member of the Bunyaviridae family, are cotranslationally cleaved from a common precursor in the endoplasmic reticulum (ER). Here, we show that newly made G1 and G2 associate transiently with calnexin and calreticulin, two lectins involved in glycoprotein folding in the ER. Stable complexes between G1-G2 and calnexin or calreticulin could be immunoprecipitated after solubilization of virus-infected BHK21 cells with the detergents digitonin or Triton X-100. In addition, G1-G2-calnexin complexes could be recovered after solubilization with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), while G1-G2-calreticulin complexes were not readily detected by using this detergent. Only endoglycosidase H-sensitive forms of G1 were found complexed with calnexin. Pulse-chase experiments showed that G1 and G2 associated with both chaperones transiently for up to 120 min. Sequential immunoprecipitations with anticalreticulin and anticalnexin antisera indicated that about 50% of newly synthesized G1 and G2 was associated with either calnexin or calreticulin. Our previous results have shown that newly synthesized G1 and G2 transiently interact also with the ER chaperone BiP and with protein disulfide isomerase (R. Persson and R. F. Pettersson, J. Cell Biol. 112:257-266, 1991). Taking all of this into consideration, we conclude that the folding of G1 and G2 in the ER is catalyzed by at least four different folding factors.     

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.     

Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress and leads to activation of the unfolded protein response, which reduces the stress and promotes cell survival at the early stage of stress, or triggers cell death and apoptosis when homeostasis is not restored under prolonged ER stress. Here, we report that Cab45S, a member of the CREC family, inhibits ER stress-induced apoptosis. Depletion of Cab45S increases inositol-requiring kinase 1 (IRE1) activity, thus producing more spliced forms of X-box-binding protein 1 mRNA at the early stage of stress and leads to phosphorylation of c-Jun N-terminal kinase, which finally induces apoptosis. Furthermore, we find that Cab45S specifically interacts with 78-kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP) on its nucleotide-binding domain. Cab45S enhances GRP78/BiP protein level and stabilizes the interaction of GRP78/BiP with IRE1 to inhibit ER stress-induced IRE1 activation and apoptosis. Together, Cab45S, a novel regulator of GRP78/BiP, suppresses ER stress-induced IRE1 activation and apoptosis by binding to and elevating GRP78/BiP, and has a role in the inhibition of ER stress-induced apoptosis.