Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Grp94 (HSP90B1)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_77

Synonyms

Historical Background

Grp94 is the endoplasmic reticulum (ER) paralog of heat shock protein (Hsp) 90. This chaperone/stress protein belongs to the protein family of glucose-regulated proteins (Grp), whose levels increase in mammalian cell cultures in the absence of glucose. Other ER stressors, such as calcium depletion, inhibition of glycosylation, and reducing agents, similarly affected Grp94 protein levels, making it one of the hallmarks of ER stress response (Lee 1987). Grp94 protein relative amount is increased also after exposure to low doses of endotoxin or curcumin (Vitadello et al. 2014a), to hypoxia or mild ischemia. The upregulation of Grp94 in several tumors explains the other widely used synonyms gp96 and TRA1 (Ansa-Addo et al. 2016).

The apparent Mr of Grp94 is around 94–100 kDa in most vertebrate species and is the product of a single gene. Whereas Grp94 orthologs are detected in metazoans, plants enclosed, no corresponding gene or protein has been so far identified in yeast and other monocellular eukaryotes, except for Leishmania (Marzec et al. 2012). The protein shows multiple Mr isoforms depending on the variable degree of glycosylation and on the presence of Ser/Thr- or Tyr-phosphorylation (Frasson et al. 2009 and references within). Whereas the functional significance of glycosylation remains still obscure, Ser/Thr-phosphorylation by Golgi casein kinase and Tyr-phosphorylation by Fyn Src-kinase are involved in Grp94 subcellular localization. Detailed analyses of Grp94 distribution in adult mammalian tissues and primary cells indicate relatively low expression levels of mRNA and protein in most tissues whereas several fold higher levels were observed in lung bronchial epithelium, pancreatic islets, kidney tubular cells, and dendritic cells (Mao et al. 2010).

Structurally, Grp94 contains four domains (Marzec et al. 2012 and Fig. 1a). The N-terminal (N) domain contains a nucleotide binding site, a peptide binding site, unmapped sites responsible for interaction with dendritic cells during antigen presentation (Jockheck-Clark et al. 2010), the binding site for the cochaperone Murine canopy 3 (CNPY3, Liu et al. 2010), and a putative transmembrane domain (Frasson et al. 2009 and references within). The second domain, as in Hsp90, is a charged linker between the N and middle (M) domains but it is longer in Grp94 and functionally important for binding of both nucleotide and calcium. Interestingly, the unique so far identified point mutation in human Grp94 hits proline 300 in this region and is responsible for reduced binding and folding of insulin-like growth factor (IGF) client protein (Marzec et al. 2016). The M-domain contains residues involved in ATP hydrolysis after interaction with the N-terminal domain, and the C-terminal domain mediates the dimerization of Grp94 and ER localization via the C-terminal KDEL peptide. Grp94 displays a “twisted V” conformation as crystal structure (Fig. 1b), whereas in solution it has an extended, “chair-like” conformation, which shifts toward less extended, more closed conformations after nucleotide binding.
Grp94 (HSP90B1), Fig. 1

Diagrams illustrate Grp94 structure and configuration. (a) Primary structure of Grp94 and domain boundaries identification. Numbers identify amino acids. Numbers 103 and 448 identify amino acid residues involved in ATP hydrolysis; number 217 indicates an identified site of glycosylation. Crosshatched regions identify functions specified below and described in the text. SP signal peptide, CHO oligosaccharide. (b) Schematic representations of domain interactions of Grp94 and Hsp90 proteins observed after binding with ATP or ADP. At variance with Hsp90, whose conformation is changed to a catalytically active one by nucleotide binding, Grp94 ground-state conformation is apparently not affected

Like other ER chaperones, such as Grp78 and calreticulin, Grp94 is a putative low affinity-high capacity Ca2+-binding protein. Grp94 binds about 16–28 moles of Ca2+ per mole of protein with 4 moderate-affinity (Kd 2 μM) binding sites and 11 low-affinity sites (Kd 600 μM). Thus, Grp94 may bind about 4 moles of calcium/mole of protein in the presence of 1 mmol/L calcium, namely the range of free calcium concentration considered to be present within the ER (Marzec et al. 2012).

ER localization of Grp94 is regulated, like other ER chaperones, through the C-terminal KDEL sequence. The KDEL sequence is recognized by a specific receptor, which is mainly localized in the cis-Golgi. Dimers-oligomers of Grp94 interacting with the aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1; previously known as p43) bind to the receptor and lead to its oligomerization and rapid transport out of cis-Golgi (Kim et al. 2010). The KDEL receptor-Grp94 complex returns to the ER where it dissociates, thus freeing the receptor for further cycling of transport.

In addition to the ER, Grp94 is detected at the cell surface of several tumors, immature cells, and, at very low levels, fully differentiated ones (Srivastava 2006; Gorza and Vitadello 2000; Patel et al. 2013; Vitadello et al. 2014b). The precise mechanism of anchorage of Grp94 molecules to the cell surface is presently unknown; however, data suggest that it occurs through noncovalent, nonionic binding with other proteins (Patel et al. 2013). Two signaling pathways responsible for Grp94 exit outside the ER have been so far identified (Fig. 2): (1) in differentiating myoblasts, the Tyr phosphorylation of Grp94 by Fyn Src-kinase is required for the chaperone translocation to Golgi and cell surface (Frasson et al. 2009) and (2) in LPS-stimulated monocytes and splenocytes, the Ser-phosphorylation of AIMP1 by c-Jun N-terminal kinase (JNK) releases the binding of the protein from Grp94 and the interaction with the KDEL receptor (Kim et al. 2010).
Grp94 (HSP90B1), Fig. 2

Signals responsible for Grp94 cell-surface localization. (a) Differentiation of skeletal myoblasts induced by low-serum culture medium activates an ER-resident Fyn, which interacts with and Tyr-phosphorylates Grp94, promoting its migration to the Golgi compartment and the cell surface. (b) Exposure of circulating or spleen macrophages to bacterial lipopolysaccharide activates TLR4 and JNK-mediated AIMP1 phosphorylation, which dissociates Grp94-KDEL receptor complex and favor Grp94 export to the cell surface

Another distinctive feature of Grp94 is the transmembrane configuration, which apparently involves a minor proportion of molecules and coexists in the ER compartment together with the luminal configuration (Vitadello et al. 2014b and references within).

Physiological Functions

Protein Chaperone

Grp94 exists as a homodimer or higher-order oligomer. Although it has been shown to bind to some nascent ER proteins, Grp94 participates to the second major ER chaperone system, which operates, in addition and/or in alternative to the lectin system, on unfolded regions of proteins containing hydrophobic residues and is involved in folding and assembly of multimeric proteins, whose first and most studied example is the immunoglobulin heavy chain (Marzec et al. 2012). A large ER-localized multiprotein complex is initiated by the ER chaperone Grp78 that recognizes protein hydrophobic residues and recruits Grp94 and other molecular chaperones, among which are protein-disulfide isomerase, cyclophilin B, and Grp170. As revealed by studies performed using chemical cross-linking, the complex forms also in the absence of protein synthesis (Ma and Hendershot 2004).

The mechanism through which Grp94 exerts its chaperone function is still obscure, due to controversial evidence concerning ATPase activity, to the lack of cochaperones and the paucity of client proteins. Conservation of amino acids involved in ATP binding and hydrolysis between Hsp90 and Grp94 would suggest a similar mechanism of nucleotide utilization. Grp94 binds adenosine nucleotides in vitro and mutations of either the ATP binding site, or the putative catalytic one, hamper chaperone activity in vivo (Randow and Seed 2001). However, structure analysis methods, including x-ray crystallography, mass spectrometry, and optical techniques showed that the ATP and ADP-bound forms of Grp94 are equivalent, that is, they display identical twisted “V”-shape conformation with closed C-domains and open N-domains in opposing orientation (Marzec et al. 2012; Fig. 1b). Therefore, ATP binding is not sufficient to drive Grp94 into a hydrolytically productive conformation: the open N-domain and the relaxed N–M orientation leave the catalytic residues too distant from the gamma phosphate of the ATP, at variance with Hsp90 and other proteins of the GHKL ATPase/kinase superfamily, whose ATPase activity results from the contact of residues localized in both the N- and M-domains with the bound nucleotide.

As argued by the structural analysis, cochaperone(s) would be required by Grp94 to promote ATP hydrolysis by rotating the catalytic loop and to regulate binding and release of client proteins.

The ER-resident protein CNPY3 has been recently identified as the first Grp94 cochaperone, specifically involved in Toll-like receptor (TLR) folding (Liu et al. 2010). CNPY3 binds Grp94 close to the ATP pocket and in competition with nucleotides, suggesting that it regulates the folding of conformational intermediates without nucleotides, as shown for some Hsp90 cochaperones (Marzec et al. 2012). Indeed, the same single point mutation at the highly conserved amino acid 103 (E103A) of Grp94, which was found to abolish ATPase activity and insulin-like growth factor (IGF) folding, hampered the interaction with CNPY3 and folding of TLR, but it did not affect the folding of other client proteins, such as integrins (Randow and Seed 2001).

Another recently identified Grp94 cochaperone is MZB1 (marginal zone B lymphocyte 1), an ER protein, which associates directly in an ATP-sensitive manner and is required for the interaction with immunoglobulin μHCs upon ER stress (Rosenbaum et al. 2014).

Grp94 also binds to MesD, mesoderm development, an ER chaperone required for LRP5 and LRP6 folding, and to LRP6 itself and plays a critical role in the maturation and surface expression of LRP6 and, therefore, in the regulation of canonical Wnt-signaling in intestinal epithelial cells (Liu et al. 2013).

Furthermore, the N-domain does not appear to be the unique protein region involved in client protein binding. A 27-aa C-terminal hydrophobic loop structure between aa 652–678 was demonstrated to be relevant for folding and interaction with several exclusive client proteins such as TLR, integrins, and PCSK-9 (Poirier et al. 2015).

At present, no recognition moiety has been identified for Grp94. Its small population of client proteins does not display common features other than the presence of disulfide bonds and, in the case of integrins and TLR, shows selectivity even among family members (Table 1; Marzec et al. 2012; Ansa-Addo et al. 2016 and references within).
Grp94 (HSP90B1), Table 1

List of proteins requiring Grp94 for folding, as shown by Grp94 knocking out or down experiments, or for holding and delivery to a precise subcellular localization, as shown by Grp94 coimmunoprecipitation and knocking down

Exclusive Grp94 foldase activity

Exclusive Grp94 holdase activity

Membrane receptors and interacting proteins

TLR (Toll-like receptor) (TLR1, TLR2, TLR4, TLR5, TRL6, TLR7, TLR9, TLR11)

HER-2 (human epidermal growth factor receptor 2)

LRP6 (low density lipoprotein-related protein receptor 6; Wnt coreceptor)

nNOS (neuronal nitric oxide synthase)

Integrins (α2, αL, αM, αV, αX, β5, β6, β7, β8)

GARP (glycoprotein A repetitions predominant; docking receptor for latent mTGF-β)

Platelet Glycoprotein IX (component of the von Willebrandt receptor)

Secreted enzymes and molecules

ADAMTS9 metalloprotease

PCSK-9 (proprotein convertase subtilisin kexin type 9)

Bile-salt-dependent lipase

IGFs (IGF-I, IGF-II)

In addition to play an exclusive role in folding selected substrates, an increasing body of evidence concerns the requirement of Grp94 for posttranslational interactions with nonclient proteins aimed to regulate their subcellular localization and function, i.e., revealing also for this Hsp90 paralog the exsitence of a specialized holdase role (Table 1).

The best holdase example is represented by Grp94 selective binding to proprotein convertase subtilisin kexin type 9, PCSK-9, a natural inducer of low-density lipoprotein receptor (LDLR) internalization and degradation. By binding the C-terminal region of Grp94, PCSK-9 is retained in the ER, whereas in the absence of interaction, the molecule is autocatalytically processed and secreted as heterodimer, which can recognize and interact with the LDLR promoting its catabolism (Poirier et al. 2015). In breast cancer cells overexpressing the HER2 receptor protein kinase, Grp94 interacts with plasmalemmal HER2 molecules and is required for their appropriate distribution at this site and functional recruitment (Patel et al. 2013). Comparably, Grp94 was demonstrated to coimmunoprecipitate with neuronal nitric oxide synthase (nNOS) and to be required in skeletal myofibers for the maintenance of the subsarcolemmal localization of nNOS molecules, which delocalize to sarcoplasm in muscle unloading conditions leading to nitrosative stress and proatrophic signaling (Vitadello et al. 2014a, b and Fig. 3).
Grp94 (HSP90B1), Fig. 3

Grp94 interacts with nNOS and protein levels influence its subcellular localization. (a) In physiologically loaded skeletal myofibers (ambulatory rat), nNOS localizes mainly at the subsarcolemma (dark border). Unloading (tail-suspended rat) causes decrease in myofiber cross-sectional area and Grp94 protein levels and delocalizes nNOS molecules to the sarcoplasm (dark spots). (b, c) Maintenance of physiological Grp94 protein levels, by means of myofiber cDNA transfection or treatment with curcumin, attenuates myofiber atrophy and preserves nNOS localization at the sarcolemma. (d) Selective decrease of Grp94 by means of antisense cDNA transfection, concomitantly to curcumin treatment, abolishes the attenuation of myofiber atrophy and the maintenance of nNOS localization at sarcolemma

Grp94 also participates to the ER quality control, that is, in targeting misfolded proteins to ER-associated degradation (ERAD). Misfolded and demannosylated protein substrates are bound by OS-9, a ER-lumenal lectin, which associates with Grp94 in order to present substrates to the ubiquitination and dislocation apparatus. The role of Grp94 is presumably to facilitate or mediate substrate recognition, since degradation of mutated glycosylated proteins is impaired in Grp94-depleted cells (Marzec et al. 2012).

Immune Regulator

Grp94 is involved in both innate and adaptive immune responses.

In addition to play a key role in the folding of several TLR, integrins and the docking receptor for latent membrane TGFβ GARP, Grp94 was proposed to activate professional antigen-presenting cells (pAPC) by direct interaction with TLR2 and  TLR4. By stringently controlling the level of endotoxin contaminant, this chaperone was demonstrated to activate human macrophages primarily through TLR2 and induced both IL-6 and TNF-α release (Huang et al. 2009; Fig. 3a). Some activation occurs through TLR4, concerning only IL-6 production. Besides, Grp94 appears to regulate the pathway activated by the TLR2 receptor downstream the mitogen extracellular kinase ( MEK)-ERK1/2. In kidney tubular cells, this chaperone binds and maintains in active conformation the serine-threonine protein phosphatase 5 (PP5), a negative modulator of  Raf-1 (Mkaddem et al. 2009).

In addition to initiate the innate immune response, Grp94 contributes to the adaptive one. It is fully recognized that Grp94 can direct the associated peptides to its N-terminal and charged linker domains into the major histocompatibility complex (MHC) class I cross-presentation pathway of pAPCs (Srivastava 2006; Jockheck-Clark et al. 2010). Peptides bound to Grp94 are internalized by pAPCs by endocytosis and trafficked to a post-ER endosomal compartment, where they are processed and loaded onto mature MHC class I molecules (Fig. 4b). The mechanism of endocytic uptake of the Grp94-peptide complex remains controversial. A large body of evidence indicates the requirement of receptor-mediated internalization, via the low-density lipoprotein receptor-related protein 1 ( CD91) and/or the scavenger receptors, but also the pathway of nonspecific fluid-phase uptake may take a prominent role (Jockheck-Clark et al. 2010). In both cases, the interaction of Grp94-peptide complex with pAPC elicits proinflammatory cytokine secretion and MHC class I/II upregulation, leading to subsequent priming and activation of peptide-specific CD8+ T lymphocytes. Recent data identify Grp94 among the more powerful chaperones involved in immune modulation against α-synuclein and Parkinson disease (Labrador-Garrido et al. 2016).

Calcium-Binding Protein

Although each Grp94 molecule can bind calcium by means of 15 calcium-binding sites, 1 or 2 of those with higher affinity has been mapped in the charged linker domain that follows the N-terminal ATPase domain (Marzec et al. 2012). Due to its relative abundance in the ER, Grp94 is expected to provide about 30 μM of Ca2+ storage capacity. This property, which is shared with other ER chaperones, like Grp78 and calreticulin, might imply the participation of Grp94 to the maintenance of calcium homeostasis. Furthermore, in vitro studies showed that Ca2+ binding at 100 nM changed Grp94 conformation and stimulated peptide binding.

In addition to protein folding, ER is deputed to the storage and utilization of calcium, which serves in many signaling pathways and physiological responses. Depletion of calcium from the stores induces an ER stress response. Besides, alterations in ER free calcium concentration strongly influence cell survival. If the ER has less free calcium to release upon stress, then cytosolic calcium levels will not rise to the critical levels needed for triggering deleterious downstream effectors. Therefore, the increased expression of ER chaperones with calcium-binding properties should protect cells by decreasing the amount of releasable calcium. Several studies performed on neuronal and myogenic cell lines showed that Grp94 overexpression counteracted the increase in intracellular Ca2+ evoked by exposure either to calcium ionophore or to oxidants, whereas decreased Grp94 expression appeared unable to cope with it (Pizzo et al. 2010 and references within). Passive calcium release from the stores, induced by means of sarcoendoplasmic calcium ATPase (SERCA) inhibition, showed that protein levels of Grp94 in myogenic cells inversely correlate to the rise in intracellular Ca2+ (Pizzo et al. 2010).

Physiological Function in Embryonic Development

Consistently with the observation that Grp94 is absent from most unicellular organisms, knockdown of Grp94 in different cell culture systems shows that low-basal expression levels of the protein are sufficient to support cell growth and proliferation (Marzec et al. 2012). Similarly, two Grp94 gene mutations causing knocking out of the protein (the Shepherd mutation in A. thaliana and the frameshifts terminating Grp94 prematurely in the pre-B lymphocyte 70Z/3 cell line) are not cell lethal but rather affect selected processes. Recent data demonstrate that cell polarity and intracellular transport are severely affected in both nonneoplastic and neoplastic cell lines lacking Grp94 (Ghosh et al. 2016). Although this body of evidence suggests that Grp94 is dispensable for growth of individual metazoan cells in culture, survival of murine grp94−/− embryonic stem cells (ES) remains strictly dependent from the presence of serum and IGFs (Marzec et al. 2012). In addition, a parallel independent study showed that grp94−/− ES cells display an increased, compensatory, expression of ER chaperones, whereas in the presence of a 50% reduction of Grp94 levels, as it occurs in hemizygosis, there is no compensatory response (Mao et al. 2010). Furthermore, grp94−/− ES cells show a decreased ability to activate the XBP-1 pathway of the ER stress response. This observation reveals the involvement of Grp94 in the ER stress signaling, a function up to now mainly attributed to Grp78 (Glembotski 2008). In grp94−/− ES cells, the decreased expression of the ER stress-induced transcription factor XBP-1 might be explained with the misfolding of the Grp94 client proteins TLR4 and TLR2, which in macrophages are known to activate the ER-stress sensor, inositol-requiring enzyme 1a (IRE 1), and its downstream target XBP-1 (Mao et al. 2010).

Conversely, Grp94 absence is responsible for the occurrence of lethal phenotypes in plants, nematodes, fruit flies, and mice. In mammals, Grp94 transcripts are found in oocytes and in 5–8.5-d embryos protein expression is highest in the embryonic and extraembryonic ectoderm and lower in the visceral endoderm. Grp94 is also detected in mesoderm cells emerging from the primitive streak. At later stages during organogenesis (E9.5–13.5), Grp94 is expressed within the developing heart, neuroepithelium, and branchial arches (our unpublished observations). Despite of the widespread and early expression of Grp94 during embryonic development, its requirement during differentiation and organogenesis differs depending on the pattern of expression and the essential function of the client proteins (Zhu and Lee 2015).

One relevant example is given by skeletal muscle progenitors where Grp94 expression is required for differentiation and maturation (Gorza and Vitadello 2000; Wanderling et al. 2007). The phenotype of grp94−/− mice is lethal at E7.5, and embryo bodies generated by grp94−/− ES cells show any type of tissue differentiation, except the formation of striated and smooth muscles. The loss of ability to give origin to muscle is due to the absolute requirement of Grp94 in folding of IGFs. When supplemented with IGF, grp94−/− ES cells are able to differentiate and express myogenin and contractile proteins; they can also fuse into myotube-like syncitia, although with far less efficiency of wild-type ES cells. Since Grp94 depletion does not affect myoD levels, the chaperone is not required for the commitment to myogenesis. Conversely, Grp94 appears to be involved in myogenesis initiation. Few hours after switching the myogenic cell line C2C12 to differentiation medium, the protein translocates from the ER to the Golgi compartment and the cell surface, after interacting with and being Tyr-phosphorylated by the Src-kinase Fyn (Frasson et al. 2009 and Fig. 2a). It is presently unknown whether this relocalization is related to delivery of specific protein cargo; an identified relevant client is IGF-II, whose secretion and autocrine interaction with IGF-I and IGF-II receptors (IGF-R1) is required to proceed along the differentiation program. Eventually, Grp94 appears to be involved in myotube formation. Knocking down Grp94 protein levels to 40% of wild-type C2C12 cells is permissive for myogenic differentiation but not for myotube formation (Gorza and Vitadello 2000). Since IGF supplementation does not fully rescue the myotube phenotype, it is likely that other Grp94 client proteins play a relevant role at this later stage. Muscle-specific Grp94 KO with late embryonic onset to allow muscle specification and avoid interference with lethal embryonic events led to smaller skeletal muscles with decreased IGF contents and growth defects associating with diminished circulating IGF-1 (Barton et al. 2012).

Another example of the tissue specificity of Grp94 involvement in tissue differentiation and maturation has been recently provided by the study of murine hematopoietic and lymphopoietic precursors after conditional knock out of Grp94 (Staron et al. 2010). Despite the essential role of the chaperone in the folding of most TLRs and integrins, only lymphopoiesis, that is, maturation of pro-B lymphocytes and development of thymocytes, is severely affected. Furthermore, this defect cannot be rescued by wild-type precursors, indicating that the chaperone regulates B- and T-lymphopoiesis in a cell-intrinsic fashion. In particular, Grp94 is required for Treg maintenance and function, being an exclusive chaperone of both GARP and integrins (Zhang et al. 2015).

Conditional depletion of GRP94 in the liver deeply affected hepatocyte survival, severely perturbing fat metabolism and favoring proliferation of conditional KO-resistant cells, which expressed Grp94 and showed neoplastic derangement (Rachidi et al. 2015; Chen et al. 2014).

Conditional knockout of GRP94 in enterocytes caused loss of intestinal barrier function, decreased number of villi, and significant reduction of crypt and postnatal death, due to occurrence of Wnt signaling defect (Ansa-Addo et al. 2016).

Pathophysiological Functions

Cancer

Whereas GRP94 is required to maintain cell adhesion and promote differentiation, which are tumor suppressing factors, it can also support tumorigenesis as a stress chaperone and regulator of pro-oncogenic signaling pathways. Most neoplastic cells express high levels of chaperone/stress proteins. ER chaperones, and especially Grp94, are upregulated probably because of hypoxia and hypoglycemia in the tumor environment. A direct consequence of this overexpression is the enhancement of neoplastic cell survival, despite controversial evidence indicated Grp94 to be required in breast cancer cells only for migration and metastasis (Dejeans et al. 2012). The opposite has been described for HER2-overexpressing breast cancer cells, where Grp94 level and function appear crucial determinants of cell viability (Patel et al. 2013). Such a variability among cancer cell subpopulations might reflect the presence or the absence of selective protein interactions requiring Grp94 holdase activity (Table 1).

Furthermore, since Grp94 plays an exclusive role in folding of multiple strategically important oncogenic clienteles, among which are integrins, Wnt coreceptor, IGF1, and TLR, this chaperone has been suggested to be pro-oncogenic. As examples, the findings that liver cancer originates from hepatocytes escaped from conditional Grp94 KO (Chen et al. 2014; Rachidi et al. 2015) and that occurrence of multiple myeloma and colitis-associated colon cancer are attenuated when Grp94 is deleted in B lymphocytes and macrophages, respectively (Ansa-Addo et al. 2016).

Also, Grp94 expression in cancer cells increases the binding and the extracellular release of tumor-specific peptides. The biological relevance of increased peptide binding by Grp94 remains controversial, since this complex might stimulate immunosurveillance as well as deceive it. The finding that specific antitumor immunity, raised after purification of the Grp94-peptide complex, suppressed tumor metastasis favored the former hypothesis. Furthermore, vaccination with Grp94-peptide complexes isolated from different tumors elicited antitumor response against all the tumors (Srivastava 2006). Unfortunately, these results were only partially repeated in clinical trials concerning early stage melanoma and kidney cancer patients (Randazzo et al. 2012).

More promising appears the goal to disrupt Grp94-oncogenic client protein interaction, such as anticancer therapies targeting the Hsp90 chaperone. Highly selective ligands for Grp94 have been identified and demonstrated to destabilize Grp94-HER2 interaction at the plasmalemma and, thus, decrease viability of HER2 overexpressing breast cancer cells (Patel et al. 2013; Ansa-Addo et al. 2016).

Autoimmune Diseases

Grp94 displays a double-faceted role in pathogenesis of idiopathic inflammatory and/or autoimmune diseases.

The chaperone is required for the proper maintenance and function of Treg lymphocytes, as loss of Grp94 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo, i.e., severe systemic inflammation secondary to accumulation of pathogenic IFN-γ- and IL-17-producing T cells (Zhang et al. 2015).

On the other hand, overexpression of Grp94 and of other ER chaperones, which are upregulated by ER stress in inflammatory lesions, might lead to inflammation and autoimmunity (Morito and Nagata 2012). In particular, released or cell-surface Grp94 molecules may play a relevant role. Spontaneous systemic lupus erythematosus-like autoimmune phenotype was experimentally obtained by knocking out AIMP1, which allows the interaction of Grp94 with the KDEL receptor and its retention in the ER (Kim et al. 2010). Released Grp94 is increased in synovial fluid from the joints of human rheumatoid arthritis patients, where it interacts with the extracellular domains of both TLR2 and TLR4, promoting the self-perpetuating activation of synovial macrophages (Huang et al. 2009) and in plasma of type I diabetic patients, where it forms complexes with circulating immunoglobulins (Pagetta et al. 2014). Anti-GRP94 autoantibodies have been also observed in patients affected with systemic lupus erythematosus (SLE), rheumatoid arthritis, and myasthenia gravis (Morito and Nagata 2012).

Ischemia-Reperfusion

Ischemia-reperfusion represents a strong inducer of the ER stress response. Several studies have been performed in the brain, where the activation of the ER stress response upregulates ER chaperone expression and is protective. Such an issue is far more relevant for mammalian cardiomyocytes, where control of intracellular calcium concentration [Ca2+]i becomes critical during ischemia. Although evidence obtained in vitro and in vivo confirmed the activation of the ER stress response in ischemic-reperfused cardiomyocytes, it is less clear what function the ER stress response serves in the cardiac context. Several studies show that ER stress response contributes to ischemic damage, by inducing both apoptosis and autophagy-mediated cell death; conversely, others support the protective role of the ER stress response and of ER chaperones, among which is Grp94, in decreasing size of infarct or maintaining cardiomyocyte viability (Glembotski 2008). Such a discrepancy might depend upon the context of the study, in that protective aspects of the ER stress response predominate early after the ischemic injury, whereas pro-apoptotic features occur later. Although the mechanism through which Grp94 exerts cardioprotection remains to be determined, a body of evidence point to its role in the maintenance of calcium homeostasis. Grp94 overexpression improves myocyte survival after exposure to oxidative stress, a usual consequence of ischemia-reperfusion, because it reduces the degree of protein oxidation (Pizzo et al. 2010). Calcium dyshomeostasis is both an effect and a cause of oxidative stress. Grp94 overexpression, which can also be pharmacologically upregulated, appears to disrupt this pathway by reducing the amount of releasable calcium from the stores. Ischemia/reperfusion injury also induces an innate immune response, leading to an inflammatory reaction and tissue damage that have been attributed to engagement of TLR2 and 4 (Fig. 4). In the ischemic kidney, Grp94 contributes to the activation of ERK1/2 downstream pathway by dissociating from PP5 and thus inactivating this negative modulator. Although multiple pathway activated by ischemia-reperfusion act upon the ERK1/2 cascade of renal tubular cells, the dissociation of Grp94 from PP5 is dependent only from TLR2 activation (Mkaddem et al. 2009).
Grp94 (HSP90B1), Fig. 4

Schematic representations of the pathways activated by exposure of pAPCs to Grp94 in innate and adaptive immune responses. (a) Interaction of soluble Grp94 with TLR2 induces IL-6 and TNF-α secretion in human macrophages through ERK1/2 pathway activation. (b) Peptides complexed to Grp94 are internalized by pAPCs after either receptor-mediated or fluid-phase endocytosis, and loaded onto mature MHC class I molecules in a post-ER compartment. HSPG heparan sulfate proteoglycan

Inhibitors and Expression Regulators

The small number of client proteins and the involvement in specific cellular events make this chaperone an interesting target to be inhibited or increased, depending of the desired outcome.

The major recent advance has been the identification of selective Grp94 inhibitors/inducers. Molecules such as radicicol, geldanamycin, and analogues inhibit Hsp90 ATP hydrolysis and peptide binding too. The nucleotide analog 5- N-ethylcarboxamidoadenosine (NECA), a broad-spectrum adenosine A2 receptor antagonist, which binds Grp94 and not Hsp90, is presently used only in vitro (Marzec et al. 2012). The molecule S-methyl 2-(4,6-dimethoxypyrimidine-2-yloxy)-3-methylbutanoate (GPM1) has been shown to directly interact with hydrophobic residues of Grp94 within the dimerization domain (Fig. 1a), facilitating its oligomerization and retrograde transport to endoplasmic reticulum via the KDEL receptor. In vivo administration of this compound reduced maturation of pAPCs and activation of B and T cells, alleviating the symptoms associated to an experimental model of systemic lupus erythematosus in mice (AIMP1−/−) (Han et al. 2010). A recent report identified a number of purine-scaffold compounds which bind Grp94 with high-affinity and influence and change the conformation of the chaperone by stabilizing it. This ligand-protein crosstalk accounts for the Hsp90 paralog selectivity of the compounds, which is also supported by the inhibition of secretion and trafficking of known Grp94 client proteins (Patel et al. 2013). Furthermore, these compounds represent highly promising drugs against HER2-overexpressing breast cancer, because they disrupt Grp94 holdase function on HER-2.

In other contexts where Grp94 protein levels are decreased, such as muscle disuse atrophy, upregulation of Grp94 to physiological levels might be wanted to redistribute nNOS molecules to myofiber sarcolemma and attenuate muscle atrophy (Vitadello et al. 2014b). Very low dosage of the vegetal polyphenol curcumin appears to selectively upregulate Grp94 expression in muscle cells both in vitro and in vivo and attenuate disuse muscle atrophy by favoring Grp94 holdase function on nNOS (Pizzo et al. 2010; Vitadello et al. 2014a). Circulating recombinant Grp94 may also be useful in decreasing LDL receptor turnover and risk of atherosclerosis, by promoting LDL uptake (Poirier et al. 2015).

Summary

Grp94 is a chaperone/stress protein of the ER and is expressed only in multicellular organisms. At variance of the paralog Hsp90, Grp94 is involved in the folding of a limited population of client proteins and is assisted by very few cochaperones. Grp94 absolute requirement for embryonic development derives from the specific role of the client proteins played in critical contexts, like IGFs for differentiation of muscle tissues, and TLR, integrins, and GARP for lymphopoiesis and immune tolerance. Furthermore, since Grp94 plays an exclusive role in folding of multiple strategically important oncogenic clienteles, among which are integrins, Wnt coreceptor, IGF1, and TLR, this chaperone has been suggested to be pro-oncogenic.

Recent investigations showed that also this Hsp90 paralog displays relevant holdase function with selected, nonclient proteins. By means of these novel interactions, Grp94 has been demonstrated (i) to negatively regulate LDL receptor turnover, and, therefore, to increase circulating LDL clearance; (ii) to positively influence survival of HER-2 overexpressing breast cancer cells; (iii) to be required to maintain nNOS sarcolemmal localization and myofiber trophism in disused skeletal muscle.

New pharmacological/biotechnological approaches show that each of these holdase functions may be successfully maintained or disrupted, by means of circulating recombinant Grp94 to compete with LDL-R degradation, purine-scaffold compounds to selectively inhibit Grp94-HER2 binding, vegetal polyphenol curcumin to maintain physiological levels of muscle Grp94 expression and attenuate muscle atrophy.

Further studies will clarify whether other Grp94-mediated effects, such as cytoprotection, participation to calcium homeostasis and cell differentiation, originate from holdase partnership with yet unidentified proteins.

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Biomedical SciencesUniversity of PadovaPadovaItaly
  2. 2.CNR-Institute of Neuroscience, Padova sectionPadovaItaly