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.     

Cyclic structural changes in endoplasmic reticulum and Golgi apparatus in the hippocampal neurons of ground squirrels during hibernation

Repetitive remodeling and renewal of the cytoplasmic structures realizing synthesis of proteins accompanies the cycling of ground squirrels between torpor and arousal states during hibernation season. Earlier we have shown partial loss of ribosomes and nucleolus inactivation in CA3 hippocampal pyramidal neurons in each bout of torpor with rapid and full recovery after warming up. Here we describe reversible structural changes in endoplasmic reticulum (ER) and Golgi complex (G) in these neurons. Transformation of ER from mainly cysternal to tubular form and from mainly granular to smooth type occurs at every entrance in torpor, while the opposite change occurs at arousal. Torpor state is also associated with G fragmentation and loss of its flattened cisternae. Appearance in torpor of the autophagosomal vacuoles containing fragments of membrane structures and ribosomes is a sign of their partial destruction. Granular ER restoration, perhaps through assembly from the multilamellar membrane structures, whorls or bags, begins as early as in the middle of the torpor bout, while G flattened cisternae reappear only at warming. ER and G completely restore their structure 2-3 hours after the provoked arousal. Thus, hibernation represents and example of nerve cell structural adaptation to alterations in functional and metabolic activity through both active destruction and renewal of ribosomes, ER, and G. Perhaps, it is the incomplete ER autophagosomal degradation at torpor provides its rapid renewal at arousal by reassembly from the preserved fragments.     

Cyclic structural changes of endoplasmic reticulum and Golgi complex in hippocampal neurons of ground squirrels during hibernation

Repetitive remodeling and renewal of the cytoplasmic structures realizing protein synthesis accompanies the cycling of the ground squirrels between torpor and arousal states during hibernation season. Previously, we have shown the partial loss of ribosomes and inactivation of the nucleolus in pyramidal neurons in the hippocampus CA3 area at each bout of torpor with their rapid and full recovery after warming up. In the present paper, we describe reversible structural changes of the endoplasmic reticulum (ER) and Golgi complex (GC) in these neurons. The transformation of the ER form from mainly granular stacks of flattened cisternae to smooth tubules occurs at every entrance in torpor, while the reverse change happens at arousal. The torpor state is also associated with GC fragmentation and loss of their flattened cisternae, i.e., dictiosomes. In neurons, the appearance of the autophagosomal vacuoles containing fragments of membrane structures and ribosomes in torpor state is a sign of the partial destruction of ER and GC. Granular ER restoration, perhaps through assembly from the multilamellar membrane structures, bags or whorls begins in the middle of the torpor bout, while GC dictiosomes reappear only during warming. The ER and GC completely restore their structure 2–3 h after the beginning of arousal. Thus, hibernation represents an example of the structural adaptation of the nerve cell to deep changes in functional and metabolic activity through both the active destruction and renewal of ribosomes, ER, and GC. Perhaps, namely the incomplete ER autophagosomal degradation in torpor provides its rapid renewal at arousal through the reassembly from the preserved fragments.     

Myocyte enhancer factor-2 and cardiac muscle gene expression during hibernation in thirteen-lined ground squirrels

Long non-coding RNA urothelial carcinoma associated 1 (UCA1) promotes human bladder cancer cell proliferation, but the underlying mechanism remains unknown. After knocking down of UCA1 in BLZ-211 cells, several cell cycle-related genes (CDKN2B, EP300 and TGFβ-2) were screened by microarray assay and validated by real-time PCR. Interestingly, in western blot analysis, p300 (encoded by EP300) and its coactivator cAMP response element-binding protein (CREB) level were significantly down-regulated. Both suppression of UCA1 expression by shRNA in BLZ-211 cells and ectopic expression of UCA1 in UMUC-2 cells showed that UCA1 alteration paralleled to the expression and phosphorylation of CREB, and UCA1 obviously influenced AKT expression and activity. Furthermore, in BLZ-211 cells, cell cycle progression was greatly reduced after PI3-K pathway was blocked by LY294002, indicating that UCA1 affected cell cycle progression through CREB. Taken together, we concluded that UCA1 regulated cell cycle through CREB via PI3K-AKT dependent pathway in bladder cancer.     

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.     

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.     

Effects of hibernation on multicatalytic proteinase complex in thirteen-lined ground squirrels, Spermophilus tridecemlineatus

Multicatalytic proteinase complex (MCP) was studied in skeletal muscle of the hibernating ground squirrel, Spermophilus tridecemlineatus. MCP was partially purified using a S-400 gel filtration column and Centricon concentrating devices and assayed fluorometrically using three AMC-labeled substrates. K_m and V_max values were determined for each substrate with no significant differences between the enzyme from euthermic versus hibernating animals when assayed at 23 C. However, properties of MCP from euthermic and hibernating ground squirrels were differentially affected by low assay temperature (8–10 C) and also differed from the mouse enzyme, the data indicating that ground squirrel MCP is better suited for low temperature function. MCP preferentially degrades oxidatively-damaged proteins and quantification of protein carbonyl content showed that the level of oxidatively-damaged protein in skeletal muscle decreased by > 75% during hibernation suggesting a continuing role for the MCP in the torpid state. (Mol Cell Biochem 271: 205–213, 2005)     

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.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60–65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S_0.5 values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S_0.5 ATP increased by 330%. Assay at 10 °C also elevated S_0.5 ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60-65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S(0.5) values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S(0.5) ATP increased by 330%. Assay at 10 degrees C also elevated S(0.5) ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

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.     

Modulation of endoplasmic reticulum chaperone GRP78 by high glucose in hippocampus of streptozotocin-induced diabetic mice and C6 astrocytic cells

Diabetes mellitus is known to increase the risk of neurodegeneration, and both diseases are reported to be linked to dysfunction of endoplasmic reticulum (ER). Astrocytes are important in the defense mechanism of central nervous system (CNS), with great ability of tolerating accumulation of toxic substances and sensitivity in Ca²⁺ homeostasis which are two key functions of ER. Here, we investigated the modulation of the glucose-regulated protein 78 (GRP78) in streptozotocin (STZ)-induced diabetic mice and C6 cells cultured in high glucose condition. Our results showed that more reactive astrocytes were presented in the hippocampus of STZ-induced diabetic mice. Simultaneously, decrease of GRP78 expression was found in the astrocytes of diabetic mice hippocampus.