HSPA5 gene encodes heat shock protein family A member 5 protein commonly known as GRP78 (glucose-regulated protein 78) or BiP (binding immunoglobulin protein) (“GRP78” will be used to refer to the protein throughout the remainder of this text for simplicity). It is an ER-resident molecular chaperone that belongs to the Hsp70 family of proteins.
GRP78 is ubiquitously expressed in mammalian cells, and, structurally, it is highly conserved across species. The cDNA clones encoding GRP78 were isolated in 1981 from the hamster mutant cell line k12 (Lee 1981). The gene locus was mapped to chromosome 9 in human-hamster somatic cell hybrids using a cross-reactive hamster cDNA probe (Law et al. 1984). In 1988, two different types of human GRP78 genes (functional and processed gene) were isolated after screening of a human genomic library with a full-length cDNA probe encoding the hamster GRP78 transcriptional unit (Ting and Lee 1988). The processed gene is an intronless pseudogene, consisting of a short repeat sequence in an AT-rich genomic region. The functional gene spans over 5 kb and contains eight exons. The active promoter contains two domains with different functions: the distal domain regulates the basal expression level, while the proximal domain is involved in the response to inducers of HSPA5 gene, such as calcium ionophores and temperature-sensitive mutations. Sequence comparison revealed high homology of protein-coding, 3′ untranslated regions and promoters of GRP78 gene in human, hamster, and rat. The position of the GRP78 locus was finally determined to be on the distal end of the long arm of chromosome 9 at band 9q34, in 1994 (Hendershot et al. 1994). More recently, Ni et al. identified a cytosolic GRP78 generated by alternative splicing of nuclear pre-mRNA (Ni et al. 2009).
GRP78 was initially found to assist in the formation of complete immunoglobulin (Ig) via binding to free heavy (H) chain in pre-B cell until a light (L) chain displaces it. The protein was also discovered as a protein involved in the synthesis of IgH chains in the absence of L chains in pre-B lymphocytes and some of their hybridomas (Wabl and Steinberg 1982; Haas and Wabl 1983). GRP78 associated non-covalently with the free nonsecreted IgH chains and transiently with Ig precursors in secreting hybridomas (Bole et al. 1986). Comparison of the sequences obtained for murine BiP and hamster GRP78 and examination of the induction of these proteins in a mutant fibroblast cell line demonstrated that BiP and GRP78 have the same NH2-terminal sequence as well as identical posttranslational modifications (Hendershot et al. 1988). Around the same time, Munro and Pelham discovered that an Hsp70 protein that is secreted into the ER is identical to the previously described GRP78 and BiP (Munro and Pelham 1986). Later, GRP78 was also found to play a role in protein translocation. For example, it was shown to bind unfolded molecules of hemagglutinin and block its transport across the ER membrane into the lumen (Gething et al. 1986). Furthermore, the degree of the association between GRP78 and secreted glycoproteins, such as factor VIII, von Willebrand Factor, and tissue plasminogen activator, correlated with the efficiency of secretion (Dorner et al. 1987)
The synthesis of GRP78 can be induced not only by depletion of glucose but also by different stimuli, such as physiological stress conditions, calcium ionophores, and reagents that block protein glycosylation (Lee 1987). It is mainly localized in the ER, where it assists and monitors the synthesis and processing of secretory proteins (Lindquist and Craig 1988). As a fundamental member of the translocation machinery, it binds transiently to newly synthesized proteins and is crucial for their folding and maturation in the ER. Upon induction of ER stress, GRP78 is upregulated and binds to permanently misfolded, underglycosylated, and unfolded proteins accumulated in the ER, preventing their aggregation, blocking the transport from the ER, and facilitating their degradation (Gething and Sambrook 1992; Gething 1999).
Furthermore, GRP78 plays a primary role in the signaling cascade resulting in the unfolded protein response (UPR). Under normal conditions, it binds the transmembrane ER stress sensors (PERK, IRE-1, ATF6), rendering them inactive. In addition, GRP78 protects cells from apoptosis by forming complexes with ER-associated pro-apoptotic signaling machineries, such as BIK, and blocking their activation. Under stress conditions, all these signaling pathways are activated following GRP78 release, impacting both cell survival and apoptosis. Although it is primarily localized in the ER, studies revealed GRP78 can also be found in the cytoplasm, nucleus, and mitochondria as well as on the cell surface. These findings have suggested new roles of GRP78 in signaling, proliferation, invasion, apoptosis, inflammation, and immunity control. Because of its conservation, ubiquitous expression, high cellular abundance, and central roles in different pathways, GRP78 could be targeted for therapeutic benefits (discussed below).
Structure and Function
As described earlier, GRP78 belongs to a highly conserved Hsp70 family of molecular chaperones. Humans have at least 13 isoforms of Hsp70s with different subcellular localizations, which have high sequence homology and share the same domain structure. The ER-resident GRP78 and the constitutively expressed cytosolic isoform, Hsc70, possess 66% sequence identity and 81% similarity. GRP78 has 654 amino acids, of which the N-terminal 18 amino acid residues function as an ER signal peptide targeting the protein to the ER and the KDEL at its C-terminus which serves as the ER retention sequence.
The intrinsic ATPase activity of Hsp70s, including GRP78, is very low (Kassenbrock and Kelly 1989; Chang et al. 2008) and regulated by co-chaperones under physiological conditions. J-domain proteins, one type of Hsp70 co-chaperones, bind substrate polypeptides and shuttle them to Hsp70s. They also bind Hsp70 themselves at the bottom of the NBD and stimulate ATP hydrolysis (Laufen et al. 1999). In mammals, there are seven ER-resident J-domain proteins including ERdj1–ERdj7. Each of these ER J-domain proteins has a unique domain structure and is thought to have various functions in protein folding and translocation in the ER (Otero et al. 2010). Another important class of co-chaperones are nucleotide exchange factors (NEF), which facilitate the release of ADP following ATP hydrolysis. For cytosolic Hsp70s, there are three classes of NEFs including Hsp110, HspBP1, and the BAG family of proteins; however, only two NEFs (Sil1 and GRP170) have been identified in the ER to date (Behnke et al. 2015). Sil1 shares structural similarity with HspBP1 and accelerates ADP release by binding to the top of IB/IIB subdomains and inducing a distortion within the NBD. Sil1 homologs have been identified in several species, but studies suggest they have varying functions in ER homeostasis. GRP170, along with its cytosolic counterpart Hsp110, belongs to the family of large Hsp70s, which are structurally similar to Hsp70 but differ in their functions. Although GRP170 is much larger than GRP78, its overall structure has high homology to GRP78 with major differences existing in the C-terminal region. Although no crystal structure of GRP78/GRP170 complex has been solved, studies in yeast suggest that the ATP-bound form of GRP170 NBD binds the ADP-bound form of GRP78 NBD, triggering the nucleotide release from the latter.
While the GRP78 chaperone complex recognizes unfolded/misfolded polypeptides and assists in folding/refolding, it also plays an important role in shuttling those that cannot be refolded to the proteasome for degradation through ER-associated degradation (ERAD) (Nishikawa et al. 2005). In this process, terminally misfolded proteins are retro-translocated to the cytosol, where they are ubiquitinated and degraded by the 26S proteasome. The exact mechanism of triage by GRP78 complex is unknown, but some studies suggest that distinct co-chaperones function as part of the folding machinery or degradation machinery. Thus, through this ATPase cycle and interactions with its host of co-chaperones, GRP78 plays a major role in maintaining protein homeostasis in the ER.
The importance of GRP78 chaperone machinery is further corroborated by genetic studies that have mostly been conducted in yeast. Null mutant of Kar2, a yeast homolog of GRP78, is inviable (Giaever et al. 2002). In addition, several proteins involved in folding and ER translocation complexes have been identified as synthetic lethal partners of Kar2. These include an ER molecular chaperone (ROT1), J-domain proteins (ERJ5, SCJ1, and SEC63), nucleotide exchange factors (SIL1 and LHS1/CER1/SSI1), and the catalytic subunit of the signal peptidase complex SEC11 (Scidmore et al. 1993; Schlenstedt et al. 1995; Craven et al. 1996; Hamilton and Flynn 1996; Kabani et al. 2000; Takeuchi et al. 2006; Carla Famá et al. 2007). In cells and mice, knockdown or heterozygous deletion of GRP78 has been shown to suppress tumor proliferation and increase chemosensitivity, suggesting the important role of GRP78 in cell survival under stress conditions (discussed below) (Luo et al. 2006; Pyrko et al. 2007; Dong et al. 2008; Wey et al. 2012b).
GRP78 in ER Protein Translocation
In eukaryotic cells, translocation into the ER is a critical step in the maturation of many proteins, including secreted proteins and those that reside in the secretary pathway. There are two ways proteins can be translocated through the ER membrane: co-translational and posttranslational translocation. In mammalian cells, co-translational pathway is thought to be predominant, while the posttranslational pathway appears to be utilized for smaller soluble polypeptides (Rapoport 2007; Zimmermann et al. 2011). In both scenarios, the translocation machinery involves a heteromeric channel complex as well as several associated proteins including GRP78 and its co-chaperones (Fig. 2). In fact, GRP78 and Sec63, an ER-membrane-bound J-protein, are required for both pathways in yeast (Brodsky et al. 1995; Panzner et al. 1995; Vembar et al. 2010).
In the co-translational pathway, the signal sequence of a nascent polypeptide chain is first recognized by the signal recognition particle (SRP). The SRP then binds to the SRP receptor on the ER membrane, delivering the ribosome-polypeptide complex to the heterotrimeric Sec61 channel composed of Sec61α, Sec61β, and Sec61γ. The polypeptide then passes through the channel as the ribosome complex elongates the chain and drives the translocation (Dudek et al. 2015). Inside the ER lumen, the signal sequence is cleaved by the signal peptidase, and the polypeptide is bound by the resident Hsp70 chaperone GRP78, which assists in proper folding and assembly. Sec63 and ERdj1 are two J-proteins identified in the ER membrane. Both of these proteins have J-domains exposed in the lumenal side, allowing them to recruit GRP78 to the membrane and facilitate its chaperone function by stimulating its ATPase activity (Nyathi et al. 2013). Sec63 stably interacts with the Sec61 complex and appears to be essential for co-translational translocation (Young et al. 2001; Lang et al. 2012). The cytosolic domain of ERdj1 is associated with translating ribosomes and is thought to be involved in the regulation of translation (Dudek et al. 2002, 2005; Blau et al. 2005). Thus, ERdj1 functions as a bridge between ribosome-nascent polypeptide complex and GRP78. Interestingly, the cytosolic domain of ERdj1 inhibits translation when not bound by GRP78, suggesting an additional role as a stress sensor.
The posttranslational translocation in mammalian cells involves another transmembrane complex Sec62/Sec63 in addition to Sec61 (Meyer et al. 2000; Dudek et al. 2015). In yeast, an additional complex Sec71p/Sec72p is also required. In this pathway, a fully translated substrate polypeptide bound by cytosolic molecular chaperones is transported to the channel on the ER membrane. As in the case of co-translational translocation, once the peptide is inserted into the channel and reaches the lumenal side, it is bound by GRP78, whose ATPase activity can be stimulated by the J-domain of Sec63 to assist in folding and maturation of the protein. As such, GRP78 and ATP are both required for this mode of translocation (Panzner et al. 1995).
In addition to assisting in folding of nascent proteins, GRP78 has also been suggested to play a key role in gating the Sec61 channel, i.e., the chaperone binds to the ER lumenal domain of Sec61α, alters the channel conformation, and facilitates insertion of precursor polypeptides (Schäuble et al. 2012). Once the N-terminus of a precursor polypeptide reaches the lumen, it is bound by GRP78, which acts as a molecular ratchet to mediate the completion of translocation and prevents the polypeptide from sliding back into the cytosol (Matlack et al. 1999; Tyedmers et al. 2010). Further, GRP78 appears to play a key role in closing the Sec61 gate and prevents leakage of Ca2+ (Schäuble et al. 2012). Thus, the ER-resident molecular chaperone GRP78, along with its co-chaperones, regulates the translocation of nascent polypeptides into the ER by participating in many of the key steps.
GRP78 in the Unfolded Protein Response (UPR)
In addition to being a component of the translocation machinery, GRP78 also plays an important role in the UPR (Fig. 3).
Most secreted and transmembrane proteins initially fold and mature in the lumen of the ER. Since the flux of unfolded polypeptide chains that enter in the ER varies in response to different physiological and environmental conditions, the ER adjusts its protein-folding capacity according to their demands. In this way, it ensures the quality and fidelity of proteins and a homeostatic control of their maturation and secretion. Hypoxia, nutrient deprivation, acidosis, and certain chemical compounds (e.g., tunicamycin, thapsigargin, and brefeldin A) can induce ER stress, which leads to an imbalance between the load of unfolded proteins that are targeted to the ER and the capacity of the translocation machinery that handles this load (Urra et al. 2016). The consequent accumulation of unfolded proteins in the cytosol triggers the UPR, whose principles are still not well defined. What is known is that it is a cytoprotective response that allows cells to adapt to ER stress by activating intracellular signaling pathways, which involve ER transmembrane sensors and cytosolic effectors transmitting information about the protein-folding status in the ER to the cytosol and nucleus (Hetz 2012). As a result, cells transactivate UPR target genes, allowing them to restore homeostasis. Under ER stress, cells activate two temporally distinct types of responses: adaptive, which include immediate and long-term adaptation, and apoptotic (Hetz 2012). In the immediate adaptation, protein synthesis and translocation to the ER are attenuated by inhibition of translation as well as activation of regulated IRE1-dependent decay (RIDD), ER-associated degradation (ERAD), and autophagy. The long-term adaptation involves three resident ER transmembrane proteins, PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1), which trigger the activation of specific transcription factors and upregulation of UPR target genes. If ER stress is too severe and homeostasis cannot be reestablished, UPR induces apoptosis to eliminate irreversibly damaged cells (Sano and Reed 2013).
UPR sensors are ER transmembrane proteins with a lumenal domain, which senses the protein-folding environment in the ER, and a cytoplasmic effector domain, which interacts with the transcriptional or translational system. The cytoplasmic portions of PERK and IRE1 have kinase activity, and these sensors activate themselves by trans-autophosphorylation upon homodimerization. ATF6 is activated after translocation to the Golgi apparatus and cleavage by site-1 protease (S1P), allowing it to function as a transcription factor. Although these three sensors trigger independent signaling cascades, they are thought to work in concert and execute a complicated adaptive response. Importantly, all of these sensors appear to be regulated by one stress-sensing molecule: GRP78 (Bertolotti et al. 2000; Shen et al. 2002). Under normal conditions, GRP78 occupies the lumenal domains of the UPR sensors, blocking homodimerization of PERK and IRE1, as well as translocation of ATF6. Upon sensing ER stress and accumulation of unfolded proteins, GRP78 dissociates from the ER transmembrane proteins and preferentially binds to the unfolded proteins with higher affinity (Ron and Walter 2007). In this way, it acts as an important ER chaperone assisting the proper protein folding and allows the activation of IRE1, PERK, and ATF6 pathways. The three pathways of the UPR cumulatively aim to return the cell to homeostasis by upregulating resident ER chaperones, including GRP78, to assist in protein processing load in the ER, suppressing global protein synthesis, inducing protein degradation via ERAD, and organelle degradation through autophagy (Hetz 2012). Apart from the dissociation of GRP78 from the UPR sensors, ER stress also induces de novo synthesis of GRP78 to assist in protein folding in the ER. Thus, GRP78 induction is a well-established hallmark of ER stress and UPR activation.
IRE1 exists in two isoforms: IRE1α, which is ubiquitously expressed, and IRE1β, restricted to gastrointestinal epithelial cells. It is a bifunctional enzyme that has protein kinase and site-specific endoribonuclease activity. Under normal conditions, IRE1α is maintained in a repressed state through association with GRP78. Under ER stress, the release of IRE1α from GRP78 leads to partial IRE1α trans-autophosphorylation in its cytosolic kinase domain and dimerization in the plane of the ER membrane. This allows further IRE1α phosphorylation events, resulting in an increased affinity for nucleotides that allosterically activate the IRE1α RNase domain and unmask its dormant endoribonucleolytic activity (Hetz and Glimcher 2009). The activated RNase domain cleaves the mRNA transcript of X-box binding protein 1 (XBP1) by excision of a 26-nucleotide intron, and the mature spliced XBP1 mRNA is then translated into a potent transcriptional activator, spliced XBP1 (XBP1s). On the contrary, the unspliced XBP1 mRNA encoding XBP1u functions as a UPR inhibitor. XBP1s regulates a subset of UPR target genes involved in ER biogenesis and ERAD of unfolded proteins. Phosphorylated IRE1α can also act in alternative ways. It interacts with the tumor necrosis factor receptor-associated factor-2 (TRAF-2), which stimulates phosphorylation of c-Jun N-terminal kinase (JNK), activating the transcriptional activity of c-Jun and promoting autophagy (Urano et al. 2000; Ogata et al. 2006). It can also induce caspase-12 activation and apoptotic cell death. Under high stress conditions, IRE1α monomers form large clusters, which are probably optimal for RIDD of mRNA and high XBP1 mRNA splicing activity. After prolonged ER stress, IRE1α clusters dissociate and the activity of this stress sensor is attenuated. Recent studies showed that GRP78 dissociation is not sufficient for IRE1 activation, but other factors are also necessary (Gardner et al. 2013).
After dissociation from GRP78, PERK, similarly to IRE1, undergoes homodimerization and autophosphorylation. Activated PERK phosphorylates the α-subunit of the eukaryotic translation initiator factor-2 (eIF2α) at Ser51, inhibiting the guanine nucleotide exchange factor eIF2β, a pentameric complex that recycles eIF2 to its active GTP-bound form (Ron and Walter 2007). Lower levels of active eIF2 lead to a decrease in translational initiation and protein synthesis. In this way, PERK contributes to the immediate adaptation of UPR and globally reduces the load of unfolded proteins destined to enter the already stressed ER lumen. PERK-mediated eIF2α phosphorylation also contributes to transcriptional activation of the UPR target genes. For example, it promotes selective translation of the mRNA encoding ATF4, which controls the levels of pro-survival genes, and those related to redox balance, amino acid metabolism, protein folding, and autophagy. In addition, eIF2α phosphorylation can also activate nuclear factor-kB (NF- kB). Sustained PERK signaling upregulates the pro-apoptotic transcription factor C/EBP-homologous protein (CHOP) via transcriptional activation by ATF4 (Tabas and Ron 2011). Subsequently, CHOP upregulates growth arrest and DNA damage-inducible 34 (GADD34) and promotes further assembly of an active PP1C phosphatase complex, resulting in an attenuated PERK signaling through the dephosphorylation of eIF2α. In this way, GADD34 provides a negative feedback loop to terminate the stress response when homeostasis is restored (Hetz 2012). Phosphorylated eIF2α also inhibits cyclin D1 to halt the cell cycle, preventing Met-tRNA recruitment to the 40S ribosomal subunit for global suppression of protein synthesis.
ATF6 is a member of a new class of ER stress transducers that exists in two isoforms, ATF6α and ATF6β. It has a basic leu zipper (bZIP) transcription factor in its cytosolic domain and a stress-sensing portion that projects into the ER lumen (Haze et al. 1999). In unstressed cells, ATF6 resides in the ER, and its trafficking is blocked by binding of GRP78 to its lumenal domain. ER stress disrupts the binding of GRP78 to ATF6, which is subsequently transported from the ER to the Golgi apparatus through interaction with the coat protein II (COPII). Here, ATF6 is cleaved by Golgi-resident proteases, first by site-1 protease (S1P) and then in an intramembrane region by site-2 protease (S2P), releasing the DNA-binding fragment (ATF6f) (Haze et al. 1999). ATF6f translocates to the nucleus and binds to the promoter of several ER chaperone genes, including GRP78, via cis-acting ER stress response element (ERSE), upregulating their transcription. It also controls the upregulation of genes encoding ERAD components and XBP1.
Although UPR aims to reestablish homeostasis in cells, prolonged ER stress and overloaded protein-folding environment are associated with enhanced cell death (Sano and Reed 2013). In this case, UPR can also trigger apoptosis, though its mechanism is poorly understood. Calcium from the ER might activate cytoplasmic proteases that contribute to cell death, even though it is unknown how ER stress promotes such a calcium leak. ER stress is also involved in the translocation of death effectors such as BAX and BAK to the mitochondria and caspase-2 that is activated in the cytoplasm (Heath-Engel et al. 2008). Furthermore, the activation of JNK by IRE1α might phosphorylate and inactivate the anti-apoptotic regulator BCL-2, contributing to cell death. BCL-2 levels might also be repressed by ATF4-mediated inhibition of the transcription factor CHOP. In addition, PERK-mediated eIF2α phosphorylation might contribute to cell death by inhibition of pro-survival protein synthesis.
Although our understanding of the steps involved in the activation of IRE1, PERK, and ATF6 effector functions is limited and further investigation is needed, it may be useful to consider implications of the UPR signaling in human diseases. As discussed above, the UPR enhances the capacity of the secretory apparatus and reduces ER load, protecting cells from high levels of ER stress. Cancer cells are constantly under stress conditions and critically depend on the UPR signaling to survive. Therefore, manipulation of UPR might represent a potential approach to treating cancer. In this scenario, GRP78 as a key regulator of the UPR sensors may also be considered a potential therapeutic target.
Cellular Localization of GRP78
Although GRP78 possesses ER signal peptide and retention sequence, and is primarily present in the ER, it has also been found in other cellular compartments (Ni et al. 2011). The mechanism of the translocation of GRP78 to other locations remains to be investigated, but these non-ER GRP78 appear to have important roles in cell proliferation and survival under stress conditions (Fig. 4).
Cell Surface GRP78
Overexpression of GRP78 can induce translocation to the cell surface, where it associates with different proteins and acts as a multifunctional receptor. Re-localization of GRP78 is associated with the development of chemoresistance and cell transformation; in fact, it was discovered on the cell surface of various types of cancer cells but not in normal tissues. Further studies showed the presence of cell surface GRP78 (sGRP78) in tumor and endothelial cells, promotes survival, proliferation, and apoptotic signaling. sGRP78 acts as a multifunctional receptor for several extracellular ligands and as a co-receptor for viral entry. In addition, it associates with cell-surface-anchored proteins and interacts with integral membrane proteins.
sGRP78 was isolated from 1-LN human prostate cancer cells and identified as a very high affinity receptor for activated α2-macroglobulin (α2-M*), a broad specificity proteinase inhibitor that binds to cell surface receptors. sGRP78 is essential for α2-M*-induced signaling transduction (Misra et al. 2002). sGRP78 also binds prostate apoptosis response protein-4 (Par-4), and their interaction was essential for apoptosis induced by Par-4 (Burikhanov et al. 2009). In another example, sGRP78 acts as a receptor for the human plasminogen kringle 5 (K5), which is known for inducing apoptosis in and inhibiting migration and proliferation of endothelial cells. K5 binds sGRP78 on endothelial cells with high affinity and specificity, and its antiangiogenic and pro-apoptotic activity requires GRP78 expression (Davidson et al. 2005).
sGRP78 was also found associated with different cell-surface-anchored proteins such as glycosylphosphatidylinositol (GPI) (Philippova et al. 2008), anchored T-cadherin, and teratocarcinoma-derived growth factor 1 (Cripto-1) (Kelber et al. 2009).
Finally, sGRP78 acts as endothelial receptor for Mucorales and plays a critical role in viral entry, such as Coxsackie virus A9 and dengue viruses, into host cells. sGRP78 was later found to act as a co-receptor for virus internalization by associating with MHC class I molecules on the cell surface (Ni et al. 2011).
These associations of sGRP78 with different proteins suggest a model for GRP78 translocation mechanisms from the ER to the cell surface and for a particular sGRP78 topography, compartmentalized to respond differently to agonists that bind N- or C-terminal domains.
GRP78 secretion is prevented by the C-terminal tetra-peptide KDEL. Possible mechanisms of translocation could be due to the fact that, under ER stress or pathological conditions, its intracellular levels are increased, exceeding the retention capacity of the KDEL retrieval system. Another possibility is that the activity of the various components of the KDEL system may be altered. Modifications such as glycosylation to the protein sequence adjacent to KDEL masking the KDEL motif could be an alternative mechanism of translocation. In fact, there are potential glycosylation sites in the C-terminus in close proximity to the KDEL motif, and a glycosylated form of GRP78 has been found (Ni et al. 2011).
It is also possible that GRP78 translocation may be due to specific GRP78-interacting proteins. For example, ERdj1 binds GRP78, and silencing ERdj1 suppresses GRP78 expression in macrophages; also Par-4 is required for translocation of GRP78 from the ER to the cell surface in PC3 cells. The translocation can also occur regardless of the presence of ER stress, as in the ectopic expression of GRP78. In another case, the KDEL retrieval system appears to regulate the extent of GRP78 translocating from the ER: when the KDEL motif is deleted, more surface localization of GRP78 was observed (Ni et al. 2011).
The topography of sGRP78 is critical for its activity as a multifunctional receptor. The N-terminal, C-terminal, and middle portion of GRP78 are all found on the cell surface and identified by flow cytometry assays, using different epitopes at these segments of GRP78. The existence of an extracellular N-terminal region of cell surface GRP78 is also supported by the blockade of Cripto, GPI-anchored T-cadherin, and Par-4 functions by an antibody against the N-terminus of GRP78. Similarly, the existence of the exposed C-terminus of GRP78 on the cell surface is found using an antibody against the C-terminal region. Finally, although GRP78 is a hydrophilic protein, a predicted structural model of sGRP78, presented as a transmembrane protein with the C-terminus exposed extracellularly, showed several potential transmembrane segments. Moreover, a subpopulation of GRP78 is found to be an ER transmembrane protein with the N-terminus protruding into the cell cytosol, using biochemical analysis of microsomal fractions, such as limited trypsin digestion and cell-membrane separation by sodium carbonate extraction (Gonzalez-Gronow et al. 2009). A more recent study confirms that GRP78 is mainly localized within the ER, where it can be present as a soluble ER lumenal protein or an ER membrane-embedded protein.
Previously, Ni et al. identified a cytosolic form of GRP78 called GRP78va (Ni et al. 2009). GRP78va is produced by an alternative splicing pattern. The mRNA of GRP78 retains intron 1, which results in alternative translation initiation. Thus, the protein encoded by this gene lacks the N-terminal ER signal peptide and is not targeted to the ER. The tissue expression profile of GRP78va was similar to that of canonical GRP78 but was found particularly elevated in leukemic cells and leukemia patients. Interestingly, Grp78va transcript was not detected in control patients. Further, the expression of GRP78va was upregulated by ER stress inducers, such as thapsigargin and tunicamycin, in a variety of human cell lines, suggesting that both canonical and cytosolic forms of GRP78 are regulated in a similar manner. Unlike its ER counterpart, GRP78va activated PERK and increased phosphorylation of eIF2α. It was suggested that GRP78va binds P58IPK, an inhibitor of PERK, allowing constant activation of PERK. Overexpression of GRP78va did not affect ATF6 activation and slightly decreased Xbp1 splicing by IRE1. Knockdown of GRP78va resulted in caspase-3 activation and reduced cell viability.
GMBP1 is a peptide that binds the surface of gastric cancer multidrug-resistant (MDR) cells and can reverse their MDR phenotype. Using proteomic methods, the receptor of GMBP1 was identified to be GRP78. In these MDR cells, GRP78 was primarily localized in the cytoplasm demonstrated by immunostaining (Kang et al. 2013; Wang et al. 2015).
In CHO cells, overexpressed GRP78 was found in both ER and nucleus (Morris et al. 1997). GRP78 in the nucleus was observed as puncta in fluorescence microscopy. In addition, electron microscopy demonstrated membrane-bound vesicle formation within the nucleus. It is not understood whether nuclear GRP78 has unique functions. In another example, Matsumoto and Hanawalt reported that in human fibroblast treated with gilvocarcin V, a compound that promotes DNA-protein cross-linking, GRP78 was found to be cross-linked to DNA, suggesting that GRP78 exists in the nucleus (Matsumoto and Hanawalt 2000). Interestingly, GRP78 protein that is found cross-linked to DNA lacks the ER signal peptide. It is unclear whether this GRP78 is identical to GRP78va or another isoform that is yet to be identified.
While GRP78 residing in the ER plays an important role in the regulation of UPR, it has also been found in the mitochondria under stress conditions. In rat brain tumor cells treated with the ER stress inducer thapsigargin, immunostaining signal for GRP78 was diffused throughout the cytoplasm and also co-localized with the mitochondrial marker MitoTracker (Sun et al. 2006). A combination of cellular fractionation and proteinase digestion also demonstrated that a subpopulation of GRP78 resided inside mitochondria derived from cells undergoing ER stress. Within the mitochondria, GRP78 was localized in the intermembrane space, inner membrane, and matrix but was not associated with the outer membrane. GRP78 has been found associated with Raf-1 on the outer membrane of mitochondria. Raf-1 is a serine/threonine-protein kinase that is upstream of MAPK/ERK cascade and functions as a regulatory link controlling proliferation, differentiation, apoptosis, survival, and oncogenic transformation (Shu et al. 2008). These seemingly contradicting reports might suggest that submitochondrial localization of GRP78 is determined by its interaction with certain molecules or by specific ER stress inducers. The exact role of mitochondrial GRP78 is not fully understood.
In early 1990s, it was discovered that GRP78 was secreted in rat exocrine pancreatic cells (Takemoto et al. 1992). Although GRP78 as an ER molecular chaperone has been well studied, it was not until recently that secreted GRP78 has gained more attention. In 2006, Kern et al. reported that proteasome inhibitor bortezomib-resistant solid tumor cell lines PC-3 and HRT-18 secreted GRP78 and that GRP78 was responsible for bortezomib resistance (Kern et al. 2009). Interestingly, not all the cells tested able to secrete a high amount of GRP78, suggesting that secretion of the ER molecular chaperone is cell-type specific. Treating cells with bortezomib upregulated GRP78 and increased its secretion, likely as a cytoprotective mechanism. More recently, it was found that colon cancer cells release a large amount of GRP78 into tumor microenvironment (Peng and Li 2013; Fu et al. 2014). Following these observations, Li et al. investigated the mechanism of GRP78 secretion and discovered that acetylation plays an important role in regulating GRP78 trafficking (Li et al. 2016). GRP78 was found in an exosome fraction from colon cancer cells, indicating that it is secreted via membrane vesicles. Histone deacetylase (HDAC) inhibitors caused GRP78 aggregation in the ER and prevented its release. Specifically, HDAC6, an isoform upregulated in colon cancer cells, was responsible for regulating GRP78 secretion. The importance of acetylation on GRP78 secretion was further confirmed by acetylation-mimicking mutants of GRP78, which indicated that K633 in the C-terminal region is likely the site of acetylation affecting its export from the ER. Furthermore, acetylation of K633 resulted in slow growth of tumor cells, suggesting a possible therapeutic approach targeting the secretion of GRP78.
GRP78 in Cancer
As a molecular chaperone, GRP78’s primary function is to help cells cope with stress conditions and promote survival. While this is beneficial to the host organism under normal conditions, the pro-survival nature of GRP78 could present potential problems in malignant tumors. It has been shown that GRP78 is upregulated in a wide variety of cancers and its expression correlates with aggressiveness (Lee 2014). Recent studies suggest that GRP78 contributes to tumorigenesis and cancer progression through several different mechanisms (Li and Li 2012; Luo and Lee 2013; Lee 2014).
Proliferation and Apoptosis
Recent studies suggest that GRP78 is crucial for cell proliferation (Fig. 5). It was found to be upregulated in human glioma cell lines, and its expression level correlated with the rate of proliferation in these cells. Further, knocking down GRP78 slowed down the growth of the cells, indicating that GRP78 plays a critical role in cell proliferation (Pyrko et al. 2007). In addition, suppression of GRP78 had a negative effect on tumor growth in a mouse model of breast cancer. Importantly, GRP78 suppression did not affect the normal organs. This suggests that cancer cells, which are considered to be under high ER stress conditions, may be more dependent on this molecular chaperone for their survival and proliferation (Dong et al. 2008). GRP78 has also been shown to bind Wnt and assists in its proper processing (Verras et al. 2008). Thus, it is essential for the Wnt/β-catenin pathway, which constitutes an important part of the proliferation signaling. As a primary member of the ER translocation and folding complex, GRP78 regulates maturation of many molecules involved in proliferation signaling. As such, it may contribute to cancer cell proliferation through maintaining the signaling network (Luo and Lee 2013).
As discussed above, GRP78 is an essential regulator of UPR and is considered anti-apoptotic in general. It was shown that a subpopulation of GRP78 can exist as an ER transmembrane protein and interact with caspase-7, preventing its activation and inhibiting apoptosis (Reddy et al. 2003). BCL-2-interacting killer (BIK) is a BH3-only protein that resides in ER and induces apoptosis via BAX/BAK activation. It also binds pro-survival BCL-2 family of proteins such as BCL-2 and BCL-XL and inhibits their activities. GRP78 interacts with BIK in a BH3-domain independent manner, and increased expression of GRP78 sequesters BIK, preventing its apoptotic activity and its binding to pro-survival factors (Zhou et al. 2011). GRP78 was also found to co-localize with Raf-1 on the outer membrane of mitochondria and protect cells from apoptosis through preventing cytochrome c release (Shu et al. 2008).
Adequate nutrients and oxygen supply are necessary for the growth of tumor cells. The tumor vasculature is responsible for providing sufficient nutrients and oxygen; therefore, angiogenesis is critical for tumor progression and metastasis. Several lines of evidence suggest that GRP78 has an important role in angiogenesis.
In a mouse model of breast cancer, GRP78 heterozygosity significantly reduced tumor microvessel density, while no effect on vasculature of normal organs was observed, suggesting that GRP78 is critical for angiogenesis (Dong et al. 2008). Furthermore, it was shown that in Grp78+/− mice, angiogenesis was suppressed during the early phase of tumor growth, and reduced metastasis was observed. Knockdown of GRP78 suppressed proliferation and cell migration in immortalized endothelial cells, which is consistent with the proposed role of GRP78 in angiogenesis (Dong et al. 2011). Vascular endothelial growth factor (VEGF) is an angiogenic factor that is essential for the growth of vascular endothelial cells. Recently, it was shown that GRP78 knockdown suppresses VEGF-induced angiogenesis in human umbilical vein endothelial cells (HUVECs) (Katanasaka et al. 2010). In VEGF-activated HUVECs, an elevated level of cell surface GRP78 was also observed. These studies indicate that GRP78 supports angiogenesis in the tumor microenvironment and promotes tumor growth.
Invasion and Metastasis
One of the hallmarks of cancer is tissue invasion and metastasis, which typically accompany with poor prognosis (Hanahan and Weinberg 2011). GRP78, both the ER lumenal and the cell surface form, has been shown to be upregulated in metastatic cancer cell lines, and its level correlates with lymph node metastases (Fu and Lee 2006; Zhang et al. 2006). GRP78 expression level is also associated with metastasis in non-small cell lung cancer (Sun et al. 2012). It was recently shown that sGRP78 promotes migration of colorectal cancer cells through interaction with cell matrix adhesion molecule β1-integrin and degradation of extracellular matrix (Li et al. 2013). In addition, it was suggested that sGRP78 is involved in regulating focal adhesion kinase (FAK), which is important for cell migration, and also interacts with urokinase-type plasminogen activator (uPA) and uPA receptor system, which has been implicated in invasion and metastasis. Another ligand for sGRP78 is α2-macroglobulin (α2M*), an inhibitor of proteinases. Binding of α2M* to GRP78 activates 21-kDa-activated kinase 2 (PAK-2) and promotes metastasis of 1-LN prostate cancer cells (Misra et al. 2005). Finally, GRP78 heterozygosity severely suppressed metastasis in mice, indicating a critical role of GRP78 in tumor metastasis (Dong et al. 2011).
Inflammation and Immunity
Inflammation and tumorigenesis/tumor progression have been closely linked in the past decade (Grivennikov et al. 2010). Inflammatory responses precede tumor development in certain cases and also play important roles throughout the progression of the tumor. As a major player in the ER chaperone complex that maintains protein homeostasis, GRP78 is involved in the regulation of multiple pro-inflammatory cytokines (Li and Li 2012). It was shown that deletion of GRP78 in chimeric mice results in differential expression of cytokines and chemokines such as interleukin-1 alpha (IL-1α), interleukin-7 (IL-7), and tumor necrosis factor alpha (TNF-α) (Wey et al. 2012a).
Evading the immune response has been considered another characteristic of malignant tumors (Hanahan and Weinberg 2011). Several studies suggest that GRP78 is also involved in assisting cancer cells in escaping the immune system. In T-cells, cell surface GRP78 forms a complex with transforming growth factor-β (TGF-β)/latency-associated peptide (LAP), and knockdown of GRP78 results in reduced expression of cell surface TGF-β/LAP, indicating that GRP78 is involved in TGF-β-mediated immune regulation (Oida and Weiner 2010). Since TGF-β acts as an immunosuppressant and its overexpression can promote cancer progression, stabilization of cell surface TGF-β by GRP78 may also contribute to cancer development. Further, inhibition of GRP78 induction in the fibrosarcoma B/C10ME resulted in an increase in apoptosis. When B/C10ME cells incapable of inducing GRP78 expression were injected into mice, the tumor did not progress with apparent activation of the cytotoxic T-cell response (Jamora et al. 1996).
GRP78 in Neurological Disorders
Although most studies on ER stress and the UPR have been focused on tumor development and progression, new evidence suggests their involvement in other human diseases, in particular neurological disorders (Roussel et al. 2013).
ER stress and upregulation of the UPR have been observed in neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease, amyotrophic lateral sclerosis, and prion disease (Roussel et al. 2013). These diseases show loss of proper protein homeostasis, an increase in and accumulation of “toxic” proteins that are misfolded in the cell prior to neuronal loss. In neurons, the elaborate network of chaperone and co-chaperone proteins mediates the correct folding and re-folding of proteins. They interact with protein degradation pathways, such as the ubiquitin-proteasome system or autophagy, to ensure the effective removal of irreversibly misfolded and potentially pathogenic proteins (Arndt et al. 2007). For this reason, several recent studies have evaluated the involvement of chaperone proteins in neurodegeneration. GRP78 is upregulated during the UPR, but its role in neurodegeneration is not well understood.
AD is characterized by the accumulation of amyloid-β and formation of neurofibrillary tangles, which are composed of hyperphosphorylated tau (p-tau) protein. The amyloid-β precursor protein (APP) generates amyloid-β that forms plaques in the brain, leading to a progressive loss of memory. Activation of the UPR was demonstrated in AD patients, through the investigation of expression and localization of GRP78 and phosphorylated PERK (Hoozemans et al. 2005). Increased expression of GRP78 was found in AD temporal cortex and hippocampus, compared to non-demented control patients. Furthermore, cellular localization of GRP78 was assessed in both the temporal cortex and hippocampus of AD and non-demented control cases, and GRP78 was found in neurons in all investigated cases, whereas very low signals were seen in glial cells. In addition, a substantial number of neurons in AD hippocampus were positive for p-PERK, indicating the activation of the UPR, while its expression was occasionally observed in neurons in AD temporal cortex. The increased presence of GRP78 in AD neurons and the presence of p-PERK only in AD cases confirm activation of UPR in AD.
Interestingly, both GRP78- and p-PERK-positive neurons were negative for p-tau, whereas p-tau-positive tangles were not GRP78- and p-PERK-positive. This indicates that increased GRP78 expression in the temporal cortex and hippocampus and activation of the UPR might precede tangle formation and postpone neurodegeneration, suggesting an initial neuroprotective role of the UPR in AD pathogenesis. Further studies are needed to demonstrate whether a sustained UPR might initiate or accelerate neurodegeneration (Hoozemans et al. 2005).
PD is characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta. PD neurons show increased levels of ubiquitinated protein deposits in the neuronal cytoplasm (Lewy bodies), which are mainly composed of α-synuclein, and protein inclusions in the neurites. In vitro and in vivo PD models showed that α-synuclein binds GRP78 in cells bearing α-synuclein aggregates and subsequently activates the UPR pathway. Furthermore, the accumulation of α-synuclein in dopaminergic cells, subjected to glucose deprivation, induces the expression of GRP78- and UPR-related transcription factor ATF4/CREB2. In these cells, cytochrome c is also released from the mitochondria, indicating apoptosis. Thus, α-synuclein aggregation may play a role in the induction of apoptotic changes, through the activation of the UPR pathway (Bellucci et al. 2011). In a rat model of PD, the complex α-synuclein-GRP78 was also found. GRP78 may prevent the neurotoxicity caused by α-synuclein. In fact, overexpression of GRP78 may have a neuroprotective effect in α-synuclein-induced Parkinson-like neurodegeneration. It appears to prevent the loss of dopaminergic neurons and dopamine in the nervous system by downregulation of UPR target genes and the level of apoptosis (Gorbatyuk et al. 2012).
Compounds Targeting GRP78
As discussed above, GRP78 is involved in tumor proliferation, survival, and invasion, and its overexpression is correlated with the development of resistance to various chemotherapeutic agents in cancer cell lines. Thus, GRP78 is an attractive target for therapeutic intervention for the treatment of cancer.
Suppressor of GRP78 Expression
Versipelostatin (VST) is a macrocyclic polyketide isolated from Streptomyces versipellis and was identified as a downregulator of the GRP78 gene. It was shown to inhibit upregulation of GRP78 stimulated by ER stress stimuli such as tunicamycin and glucose deprivation (Park et al. 2002). Later, VST was also found to repress the production of XBP1 and ATF4 under glucose starvation. VST selectively killed glucose-deprived cancer cells and synergistically acted with cisplatin, inhibiting tumor growth of MKN74 xenografts (Park et al. 2004).
Genistein is an active phytoestrogen present in soy that inhibits cell growth and angiogenesis via inhibition of tyrosine kinases. It was found that genistein prevents the accumulation of GRP78 mRNA induced by thapsigargin while having no effects on GRP78 levels on its own (Price et al. 1992; Cao et al. 1995). Later, genistein was found to antagonize the binding of the nuclear factor-Y/CCAAT-binding factor (NF-Y/CBF) to the CCAAT sequence element of the transcription start sites in the GRP78 and Hsp70 promoters, inhibiting the expression of stress response-related genes (Zhou and Lee 1998).
Molecules That Bind GRP78
(-)-Epigallocatechin gallate (EGCG) is the main polyphenol present in green tea. EGCG showed various activities such as chemopreventive, antitumor, and antioxidant and interact with several protein targets including GRP78. The direct interaction between EGCG and GRP78 was confirmed in both in vitro and in vivo binding assays. EGCG was also shown to bind the NBD of GRP78 and inhibits its ATPase activity. Furthermore, EGCG inhibits the formation of caspase-7/GRP78 complex and suppresses the anti-apoptotic function of GRP78. It was shown that the binding sites of caspase-7 and EGCG on GRP78 overlap within the N-terminal region (Ermakova et al. 2006).
Salicylic acid (SA) is a natural signaling molecule of plants that shows anti-inflammatory, analgesic, and antineoplastic effects in human. A 78 kDa SA-binding protein was purified from human fibroblasts and identified as GRP78. SA was shown to displace a heptapeptide that binds the substrate-binding pocket within the SBD of GRP78 without inhibiting ATP binding, indicating that SA binds the SBD of GRP78. SA did not induce GRP78 expression (Deng et al. 2001).
The bacterial cytotoxin SubAB has also been found to bind GRP78. SubAB consists of an enzymatic “A” subunit similar to the protein-digesting enzyme subtilisin and five “B” subunits which bind GRP78. SubAB specifically cleaves the bond between Leu416 and Leu417 of GRP78 within the linker connecting the NBD and SBD. Treatment of cells with SubAB upregulates CHOP, likely causing apoptotic cell death (Paton et al. 2006).
A cyclic 13-mer peptide Pep42 (CTVALPGGYVRVC), which is preferentially internalized into melanoma cells, was found to target sGRP78 on these cells. In this case, sGRP78 acts as a receptor for Pep42 and aids in its internalization. The peptide selectively targeted GRP78-expressing cancer cells, and anti-GRP78 antibody blocked the uptake of Pep42 (Kim et al. 2006; Yoneda et al. 2008). The interaction between sGRP78-Pep42 was later exploited for the synthesis of Pep42-drug conjugates to improve selective targeting of tumor cells. In cell proliferation assays, these conjugates showed three- to fourfold improvement in IC50 values (Yoneda et al. 2008).
Hsp70 inhibitors and their site of action
Site of interaction
Fatty acid acyl benzamides
VER-155008 is an adenosine-derived Hsp70 inhibitor developed through structure-based design (Williamson et al. 2009) (Fig. 6). The nucleoside inhibitor binds to the ATPase pocket of Hsp70/Hsc70 and blocks its ATPase activity. It also inhibits cell proliferation of various tumor cell lines, induces apoptosis, and potentiates the apoptotic effect of Hsp90 inhibitors in HCT116 colon carcinoma cells (Massey et al. 2010). GRP78 binding and isomer selectivity of VER-155008 were evaluated by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The combination of these studies revealed a slight selectivity of the nucleoside analog toward GRP78 in vitro. Furthermore, comparison of the X-ray crystallographic structures of GRP78 and the cytosolic Hsp70 showed a key structural difference in the ATPase domain. While GRP78 has a nonpolar residue (Ile61), Hsp70 has a polar Thr37. The increased hydrophobicity in the ATP-binding pocket of GRP78 could explain the observed modest selectivity. Furthermore, this structural difference could be exploited to develop more selective and potent GRP78 inhibitors (Macias et al. 2011).
Recently, a thiazole benzenesulfonamide derivative HA15 was found to be a selective GRP78 inhibitor (Cerezo et al. 2016). HA15 demonstrated both in vitro and in vivo cytotoxity against melanoma cells, including BRAF inhibitor resistant cell lines, while showing minimal toxicity in normal cells. The IC50 value of HA15 in A375 melanoma cells was in the low micromolar range. HA15 induced an early ER stress with increased expression of UPR components and led to cell death through equal contribution of apoptotic and autophagic mechanisms. Using biochemical assays and proteomic/transcriptomic analysis, GRP78 was identified as specific target of HA15. HA15 did not bind the cytosolic Hsp70 in cell lysates or recombinant Hsp70, suggesting this compound is specific for GRP78. Although the site of interaction of HA15 has not been determined, these data suggest that development of more potent and selective inhibitors of GRP78 is possible.
Cells are constantly exposed to a variety of stress stimuli, which could cause severe damages if not mitigated. Molecular chaperones, many of which are stress-inducible, have evolved to protect host organisms from these stress inducers by preventing protein aggregation or removing aggregates that cannot be salvaged. The ER is a critical organelle for the maturation of extracellular proteins and those that reside in the secretory pathway. The ER also provides a unique oxidizing environment, which allows disulfide formation required for the functional protein structure. Because of this essential function, ER is prone to oxidative stress. As one of the major molecular chaperones in the ER, GRP78 plays an important role in maintaining ER homeostasis through assisting proper protein translocation and folding and coordinating the UPR under stress conditions. In protein translocation, newly translated polypeptides are bound by the GRP78 chaperone complex once they reach the lumen. This allows the polypeptides to fold properly and to be further processed for complete maturation. In this scenario, GRP78 itself is also tightly regulated by a host of co-chaperones such as J-domain proteins and NEFs, which are thought to bring GRP78 near the translocon and to stimulate its ATPase activity. GRP78 also functions as a sensor for ER stress and protects cells from damages by upregulating the UPR. The UPR activates three downstream sensors (i.e., IRE1α, ATF6, and PERK), all of which are sequestered by GRP78 binding under normal conditions. Upon ER stress, GRP78 releases these sensors, activates the UPR, and plays a key role in returning the cell to normal conditions. In case of severe stress, the UPR triggers apoptosis to prevent further damage to the host organism. Thus, GRP78 is essential for the survival of cells.
Recent studies suggested that GRP78 has “pro-cancer” properties. It is involved in many aspects of cancer progression and has been found upregulated in many types of cancer. In addition, cell surface GRP78 has been identified in cancer cells but not in normal cells. Although the mechanism of its translocation to cell surface is not understood, its selective cell surface expression in cancer suggests an additional role of GRP78 in cancer progression. Although knockdown of GRP78 was shown to inhibit cancer cell growth, its potential as an anticancer therapeutic target has not been established due to lack of specific and potent GRP78 inhibitors. Macias et al. suggested the possibility of developing GRP78 specific inhibitor based on a small difference in the nucleotide-binding site between GRP78 and the cytosolic Hsc70 (Macias et al. 2011). More recently, a thiazole benzenesulfonamide HA15 was shown to bind GRP78 but not Hsp70 and suppress tumor growth in mouse xenograft with melanoma cells (Cerezo et al. 2016). Importantly, the compound induced ER stress and upregulated autophagic and apoptotic cell death, supporting previous studies using GRP78 siRNA. Although additional studies are needed to establish the druggability of GRP78 for cancer therapeutics, recent promising results warrant further investigation.
We acknowledge financial support from the DoD (OCRP: W81XWH-14-1-0172).
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