Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and transient association with GRP78-BiP in the endoplasmic reticulum and extensive internalization from the cell surface

The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is a type II integral membrane protein that is expressed at the infected cell surface. The intracellular assembly and transport of HN in CV1 cells was examined using conformation-specific HN mAbs and sucrose density sedimentation analysis. HN was found to oligomerize with a t1/2 of 25-30 min and these data suggest the oligomer is a tetramer consisting primarily of two noncovalently associated disulfide- linked dimers. As HN oligomers could be found that were sensitive to endoglycosidase H digestion and oligomers formed in the presence of the ER to the Golgi complex transport inhibitor, carbonylcyanide m- chlorophenylhydrazone (CCCP), these data are consistent with HN oligomerization occurring in the ER. Unfolded or immature HN molecules that could not be recognized by conformation-specific antibodies were found to specifically associate with the resident ER protein GRP78-BiP. Immunoprecipitation of BiP-HN complexes with an immunoglobulin heavy- chain binding protein (BiP) antibody indicated that newly synthesized HN associated and dissociated from GRP78-BiP (t1/2 20-25 min) in an inverse correlation with the gain in reactivity with a HN conformation- specific antibody, suggesting that the transient association of GRP78- BiP with immature HN is part of the normal HN maturation pathway. After pulse-labeling of HN in infected cells, it was found that HN is rapidly turned over in cells (t1/2 2-2.5 h). This led to the finding that the vast majority of HN expressed at the cell surface, rather than being incorporated into budding virions, is internalized and degraded after localization to endocytic vesicles and lysosomes.     

Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene.

The endoplasmic reticulum (ER)-localized chaperone protein, GRP78-BiP, is involved in the folding and oligomerization of secreted and membrane proteins, including the simian virus 5 hemagglutinin-neuraminidase (HN) glycoprotein. To understand this interaction better, we have constructed a series of HN mutants in which specific portions of the extracytoplasmic domain have been deleted. Analysis of these mutant polypeptides expressed in CV-1 cells have indicated that GRP78-BiP binds to selective sequences in HN and that there exists more than a single site of interaction. Mutant polypeptides have been characterized that are competent and incompetent for association with GRP78-BiP. These mutants have been used to show that the induction of GRP78-BiP synthesis due to the presence of nonnative protein molecules in the ER is dependent on GRP78-BiP complex formation with its substrates. These studies have implications for the function of the GRP78-BiP protein and the mechanism by which the gene is regulated.     

Down-regulation of paramyxovirus hemagglutinin-neuraminidase glycoprotein surface expression by a mutant fusion protein containing a retention signal for the endoplasmic reticulum.

The human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins are the principal components involved in virion receptor binding, membrane penetration, and ultimately, syncytium formation. While the requirement for both F and HN in this process has been determined from recombinant expression studies, stable physical association of these proteins in coimmunoprecipitation studies has not been observed. In addition, coexpression of other heterologous paramyxovirus F or HN glycoproteins with either HPIV3 F or HN does not result in the formation of syncytia, suggesting serotype-specific protein differences. In this study, we report that simian virus 5 and Sendai virus heterologous HN proteins and measles virus hemagglutinin (H) were found to be down-regulated when coexpressed with HPIV3 F. As an alternative to detecting physical associations of these proteins by coimmunoprecipitation, further studies were performed with a mutant HPIV3 F protein (F-KDEL) lacking a transmembrane anchor and cytoplasmic tail and containing a carboxyl-terminal retention signal for the endoplasmic reticulum (ER). F-KDEL was defective for transport to the cell surface and could down-regulate surface expression of HPIV3 HN and heterologous HN/H proteins from simian virus 5, Sendai virus, and measles virus in coexpression experiments. HN/H down-regulation appeared to result, in part, from an early block to HPIV3 HN synthesis, as well as an instability of the heterologous HN/H proteins within the ER. In contrast, coexpression of F-KDEL with HPIV3 wild-type F or the heterologous receptor-binding proteins, respiratory syncytial virus glycoprotein (G) and vesicular stomatitis virus glycoprotein (G), were not affected in transport to the cell surface. Together, these results support the notion that the reported serotype-specific restriction of syncytium formation may involve, in part, down-regulation of heterologous HN expression.     

GRP78: a chaperone with diverse roles beyond the endoplasmic reticulum

Glucose-regulated protein 78 (GRP78) is a well-characterized molecular chaperone that is ubiquitously expressed in mammalian cells. GRP78 is best known for binding to hydrophobic patches on nascent polypeptides within the endoplasmic reticulum (ER) and for its role in signaling the unfolded protein response. Structurally, GRP78 is highly conserved across species. The presence of GRP78 or a homologue in nearly every organism from bacteria to man, reflects the central roles it plays in cell survival. While the principal role of GRP78 as a molecular chaperone is a matter of continuing study, independent work demonstrates that like many other proteins with ancient origins, GRP78 plays more roles than originally appreciated. Studies have shown that GRP78 is expressed on the cell surface in many tissue types both in vitro and in vivo. Cell surface GRP78 is involved in transducing signals from ligands as disparate as activated alpha2-macroglobulin and antibodies. Plasmalemmar GRP78 also plays a role in viral entry of Coxsackie B, and Dengue Fever viruses. GRP78 disregulation is also implicated in atherosclerotic, thrombotic, and auto-immune disease. It is challenging to posit a hypothesis as to why an ER molecular chaperone, such as GRP78, plays such a variety of roles in cellular processes. An ancient and highly conserved protein such as GRP78, whose primary function is to bind to misfolded polypeptides, could be uniquely suited to bind a wide variety of ligands and thus, over time, could assume the wide variety of roles it now plays.     

GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis

The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.     

Transcriptional activation of endoplasmic reticulum chaperone GRP78 by HCMV IE1-72 protein

Glucose-regulated protein 78 (GRP78), a key regulator of endoplasmic reticulum (ER) stress, facilitates cancer cell growth and viral replication. The mechanism leading to grp78 gene activation during viral infection is largely unknown. In this study, we show that the immediate-early 1 (IE1-72) protein of the human cytomegalovirus (HCMV) is essential for HCMV-mediated GRP78 activation. IE1-72 upregulated grp78 gene expression depending on the ATP-binding site, the zinc-finger domain and the putative leucine-zipper motif of IE1-72, as well as the ER stress response elements (ERSEs) on the grp78 promoter. The purified IE1-72 protein bound to the CCAAT box within ERSE in vitro, whereas deletion mutants of IE1-72 deficient in grp78 promoter stimulation failed to do so. Moreover, IE1-72 binding to the grp78 promoter in infected cells accompanied the recruitment of TATA box-binding protein-associated factor 1 (TAF1), a histone acetyltransferase, and the increased level of acetylated histone H4, an indicator of active-state chromatin. These results provide evidence that HCMV IE1-72 activates grp78 gene expression through direct promoter binding and modulation of the local chromatin structure, indicating an active viral mechanism of cellular chaperone induction for viral growth.     

Beyond the endoplasmic reticulum: atypical GRP78 in cell viability, signalling and therapeutic targeting

GRP78 (glucose-regulated protein of 78 kDa) is traditionally regarded as a major ER (endoplasmic reticulum) chaperone facilitating protein folding and assembly, protein quality control, Ca(2+) binding and regulating ER stress signalling. It is a potent anti-apoptotic protein and plays a critical role in tumour cell survival, tumour progression and angiogenesis, metastasis and resistance to therapy. Recent evidence shows that GRP78 can also exist outside the ER. The finding that GRP78 is present on the surface of cancer but not normal cells in vivo represents a paradigm shift on how GRP78 controls cell homoeostasis and provides an opportunity for cancer-specific targeting. Cell-surface GRP78 has emerged as an important regulator of tumour cell signalling and viability as it forms complexes with a rapidly expanding repertoire of cell-surface protein partners, regulating proliferation, PI3K (phosphoinositide 3-kinase)/Akt signalling and cell viability. Evidence is also emerging that GRP78 serves as a receptor for viral entry into host cells. Additionally, a novel cytosolic form of GRP78 has been discovered prominently in leukaemia cells. These, coupled with reports of nucleus- and mitochondria-localized forms of GRP78, point to the previously unanticipated role of GRP78 beyond the ER that may be critical for cell viability and therapeutic targeting.     

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78

Alterations in Ca(2+) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.     

Expression of the endoplasmic reticulum chaperone GRP94 gene in ischemic gerbil brain

GRP94 (glucose regulated protein 94) gene expression in the ischemic-hippocampus of gerbils, which was induced by a temporary occlusion of the bilateral common carotid arteries (CCAs), was tested by Northern blot analysis. The maximum GRP94 gene expression level was detected at the occipital lobe 10 min after the induction of ischemia. In the hippocampus, GRP94 gene expression reached a maximum 15 min after inducing ischemia. Following reperfusion, the maximum expression level was shown at 12 h and continuing thereafter.     

Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase

Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the beta-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a beta-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases.     

Temperature-sensitive Chinese hamster cell mutant with a defect in glycoprotein synthesis: accumulation of the EGF receptor in the endoplasmic reticulum and the role of the glucose-regulated protein GRP78

A temperature-sensitive mutant of Chinese hamster fibroblasts with a defect in glycoprotein synthesis is investigated after transfection and amplification of the gene for the human EGF receptor. We demonstrate that at the nonpermissive temperature a partially glycosylated species of the receptor accumulates in the endoplasmic reticulum. The oligosaccharides present are the high mannose types, since they can be removed completely by treatment with endoglycosidase H. Pulse-chase experiments show that the abnormal species of the receptor cannot be chased to a form that is either resistant to endoglycosidase H, or altered in its mobility on SDS polyacrylamide gels. The abnormal species of the receptor appears within the first hour of a shift to the nonpermissive temperature, and no further changes are observed upon prolonged incubation of cells at 40 degrees C. However, after 3-4 hours immunoprecipitations of the receptor yield another protein, which has properties very similar, if not identical, to the glucose-regulated protein GRP78. The induction of this protein at 40 degrees C can be suppressed completely with an inhibitor of RNA synthesis, without any effect on the glycosylation defect, or on the accumulation of the EGF receptor in the endoplasmic reticulum.     

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.     

黄芪注射液对网腔钙结合蛋白基因沉默阿霉素损伤心肌细胞内质网应激伴侣蛋白GRP78,GRP94 mRNA的作用

目的：研究黄芪注射液对网腔钙结合蛋白（calumenin）基因沉默阿霉素损伤心肌细胞内质网应激伴侣蛋白GRP78,GRP94 mRNA的作用。方法：实验将体外培养的1~3 d乳鼠心肌细胞分为5组：对照组、模型组（正常细胞+3 mg/L阿霉素）、calumenin基因沉默模型组（慢病毒感染细胞+3 mg/L阿霉素）、黄芪组1（正常细胞+3 mg/L阿霉素+200 g/L黄芪）、黄芪组2（慢病毒感染细胞+3 mg/L阿霉素+200 g/L黄芪）。构建慢病毒-calumenin质粒,转染乳鼠培养心肌细胞,采用实时荧光定量分析（real-time PCR）检测各组心肌细胞calumenin及内质网应激伴侣蛋白GRP78、GRP94 mRNA表达。结果：①与对照组比较,模型组心肌细胞calumenin mRNA表达减少（P<0.05）,而calumenin基因沉默模型组及黄芪组2心肌细胞calumenin mRNA表达明显减少（P<0.01）;与模型组比较,黄芪组1心肌细胞calumenin mRNA表达增加（P<0.05）;与calumenin基因沉默模型组比较,黄芪组2心肌细胞calumenin mRNA表达明显增加（P<0.01）。②与对照组相比较,模型组及calumenin基因沉默模型组心肌细胞内质网应激伴侣蛋白GRP78、GRP94 mRNA表达明显增多（P<0.01）;与模型组比较,黄芪组1心肌细胞GRP78、GRP94 mRNA表达明显减少（P<0.01）;与calumenin基因沉默模型组比较,黄芪组2心肌细胞内质网应激伴侣蛋白GRP78、GRP94 mRNA表达明显减少（P<0.01）。结论：①阿霉素损伤可引起心肌细胞calumenin表达减少。②Calumenin可缓解阿霉素损伤所诱导心肌细胞内质网应激。黄芪注射液可抑制阿霉素损伤所诱导心肌细胞内质网应激,这种作用可能系通过calumenin介导实现的。