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.     

GRP78 from grass carp (Ctenopharyngodon idella) provides cytoplasm protection against thermal and Pb2+ stress

Glucose regulated protein (GRP) located in endoplasmic reticulum (ER) was a member of heat shock protein (Hsp) family. The protective mechanism adapted to ER stimuli was closely related to GRP. GRP78, known as BiP, was one of central regulator responded to stress in ER. Grass carp (Ctenopharyngodon idella) GRP78 (CiGRP78) was up-regulated in almost tissues, especially in liver, under heat shock (34 °C), cold stress (4 °C) or lead nitrate (0.25 mmol/L) stress. In order to understand the function of CiGRP78 in cellular protection, CiGRP78 ORF cDNA was inserted into the plasmid of pET-32a(+) or pEGFP-C1 respectively, then the recombinant plasmids were transformed or transfected into Escherichia coli cells, mouse myeloma cells (SP2/0) or grass carp kidney cells (CIK). In the cells, CiGRP78 was over-expressed following thermal, cold or Pb²⁺ stress. Results showed that CiGRP78 not only contributed to protecting prokaryotic cells against thermal or cold extremes, but also played the same role in SP2/0 and CIK cells. After treatment with heat stress at 42 °C for 1 h, although the viability of the cells declined a lot, CIK cells with pEGFP-C1/CiGRP78 exhibited a higher survival rate (28%) than wild-type cells (7%) or cells with only pEGFP-C1 (5.1%). When the time lag extended to 2.5 h, the survival rates were 19%, 5.7%, 4.8% respectively. In addition, CiGRP78 would also provide a transient cytoplasm protection against Pb²⁺ stress in a dose- and time-dependent manner. After treatment with lead nitrate at concentration of 10 μmol/L for 12 h, 24 h or 36 h, the survival rates of cells with pEGFP-C1 or wild-type cells were 46.7% or 46.7% (12 h), 25% or 22% (24 h), 10% or 11% (36 h) respectively. When the cells were treated with lead nitrate at the concentration of 25 μmol/L, the survival rates of cells with pEGFP-C1 or wild-type cells were 45.5% or 30% (12 h), 16.7% or 25% (24 h), 6.5% or 8% (36 h), respectively. CiGRP78 provided a distinct protection in CIK cells at the low concentration for 24 h. The survival rates of CIK cells with pEGFP-C1/CiGRP78 treated with lead nitrate at concentration of 10 μmol/L or 25 μmol/L were 65.9% or 58.8% respectively. When the cells were treated with lead nitrate at concentration of 50 μmol/L for 24 h, the survival rate of the CIK cells was only about 30%. If the process-time was extended to 36 h, CiGRP78 could not provide any cytoplasm protection for CIK cells.     

Molecular identification of glucose-regulated protein 78 (grp78) gene from the Indian meal moth,Plodia interpunctella,and its regulation by nutrient uptake

Glucose-regulated protein 78 (GRP78) is a member of the HSP70 family of proteins and is localized in the endoplasmic reticulum (ER) within cells. GRP78 and its gene has been identified in only a few species of insects, and its role is not clear. Here, we identified full-length grp78 cDNA from the Indian meal moth, Plodia interpunctella, and demonstrated the role of grp78 in developmental and physiological processes of the insect. The deduced amino acid sequence of GRP78 contained highly conserved functional motifs of the HSP70 family and the C-terminal motif of KDEL, which is characteristic of ER-localized HSP70. It also showed high identity (93–94%) with GRP78 and related HSP70 proteins of lepidopteran species. Gene expression analysis showed that grp78 mRNA levels were high in the egg, feeding larval, and adult stages, but low in the molting, wandering larval, and pupal stages of development. In a tissue comparison of fifth instar P. interpunctella, grp78 level was higher in the gut than in the integument or fat bodies. Grp78 level decreased greatly when fifth-instar larvae were starved for 48 h, but recovered within 3–6 h after re-feeding. Our data suggest that grp78 is highly associated with dietary energy conditions during development and may play an important role in the nutritional physiology of insects.     

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.     

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.     

Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP_4.96, GP_4.94 and GP_4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP_4.96and GP_4.94in immature (testicular) sperm. In mature human sperm GP_5.04, GP_4.96, and GP_4.94were the 3 phosphoforms observed. GP_4.94[P = 0.014]andGP_5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.     

Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

Chinese hamster ovary (CHO) cells are regarded as one of the most commonly used mammalian hosts, which decreases the productivity due to loss in culture viability. Overexpressing antiapoptosis genes in CHO cells was developed as a means of limiting cell death upon exposure to environmental insults. Glucose‐regulated protein 78 (GRP78) is traditionally regarded as a major ER chaperone that participates in protein folding and other cell processes. It is also a potent antiapoptotic protein and plays a critical role in cell survival, proliferation, and metastasis. In this study, the impact of GRP78 on CHO cells in response to environmental insults such as serum deprivation and oxidative stress was investigated. First, it was confirmed that CHO cells were very sensitive to environmental insults. Then, GRP78 overexpressing CHO cell line was established and exposed to serum deprivation and H₂O₂. Results showed that GRP78 engineering increased the viability and decreased the apoptosis of CHO cells. The survival advantage due to GRP78 engineering could be mediated by suppression of caspase‐3 involved in cell death pathways in stressed cells. Besides, GRP78 engineering also enhanced yields of antibody against transferrin receptor in CHO cells. GRP78 should be a potential application in the biopharmaceutical industries.     

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.     

Roles of GRP78 in physiology and cancer

As one member of 70 kDa heat shock proteins, glucose‐regulated protein 78 (GRP78) participates in protein folding, transportation and degradation. This sort of capacity can be enhanced by stresses under which GRP78 is induced rapidly. Unlike its homologues, GRP78 presents multifaceted subcellular position: When ER retention, it serves as the switch of unfolded protein response; When mitochondrial binding, it directly interacts with apoptotic executors; When cell surface residing, it recognizes extracellular ligands, transducing proliferative signals, especially in certain tumors. The close correlation between GRP78 and neoplasm provides us further insight into the event of carcinogenesis and cancer cell chemoresistance, indicating its prognostic predicting significance and validating potential therapeutics for clinical usage, especially because its small molecular inhibitors are emerging quickly these years. What's more, GRP78‐related signaling may be helpful for clearer understanding of its biological mechanisms. J. Cell. Biochem. 110: 1299–1305, 2010. © 2010 Wiley‐Liss, Inc.