Transfer of GRP94(Gp96)-associated peptides onto endosomal MHC class I molecules.

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the Kb-SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I.

To find the road traveled to tumor immunity: the trafficking itineraries of molecular chaperones in antigen-presenting cells.

Molecular chaperones, both endoplasmic reticulum and cytosol derived, have been identified as tumor rejection antigens; in animal models, they can elicit prophylactic and therapeutic immune responses against their tumor of origin. Chaperone immunogenic activity derives from three principal characteristics: they bind an array of immunogenic (poly)peptides, they can be efficiently internalized by professional antigen-presenting cells, and once internalized, they traffic to a subcellular compartment(s) where peptide release can occur. Within the antigen-presenting cell, chaperone-derived peptides can be assembled onto major histocompatibility class I molecules for presentation at the antigen-presenting cell surface, thereby yielding the requisite and specific CD8+ T-cell responses that contribute to the process of tumor rejection. Though it is clear that chaperones, in particular GRP94 (gp96), calreticulin and Hsp70, can elicit cellular immune responses, the subcellular basis of chaperone processing by antigen-presenting cells remains mysterious. In this review, we discuss recent reports describing the identification of a chaperone internalization receptor and the physiological release of chaperones from necrotic cells, and we present views on the trafficking pathways within antigen-presenting cells that may function to deliver the chaperone-associated peptides to subcellular organelles for their subsequent exchange onto major histocompatibility complex molecules.

Ribosome exchange revisited: a mechanism for translation-coupled ribosome detachment from the ER membrane.

In current models, ribosome release from the endoplasmic reticulum (ER) is coupled to the termination of protein translation. Thus, coincident with termination, membrane-bound ribosomes dissociate into their component subunits and are released into the cytosol. Here, we review past and current data and propose that the affinity of the ribosome for the ER membrane is decreased during translation, with ribosome release occurring when a membrane-bound ribosome is engaged in the synthesis of a protein lacking a signal sequence. Our model emphasizes a role for the conformation of the large ribosomal subunit in the regulation of membrane affinity and provides a mechanism for translation-coupled ribosome release.

Virally induced lytic cell death elicits the release of immunogenic GRP94/gp96.

Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8(+) T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90.

ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90. Thus, although both mature and nascent ErbB2 proteins are down-regulated by GA, only nascent ErbB1 is sensitive to the drug. To reveal the underlying mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is functional since its kinase activity was stimulated by epidermal growth factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2 and unlike that of ErbB1, indicating that the intracellular domain of the chimera confers GA sensitivity. This finding also suggests that the GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than Grp94, a homolog of Hsp90 that is restricted to the lumen of the endoplasmic reticulum, although both chaperones bind to and are inhibited by GA. Lack of Grp94 involvement in mediating ErbB2 sensitivity to GA is further suggested by the fact that a GA derivative with low affinity for Grp94 efficiently depleted ErbB2 protein in treated cells. To localize the specific region of ErbB2 that confers GA sensitivity, we made truncated receptors with progressive deletions of the cytoplasmic domain and tested the GA sensitivity of these molecules. We found that ErbB2 constructs containing an intact kinase domain retained GA sensitivity, whereas those lacking the kinase domain (ErbB2/DK) lost responsiveness to GA completely. Hsp90 co-immunoprecipitated with all ErbB2 constructs that were sensitive to GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely stabilizes ErbB2 via interaction with its kinase domain and that GA stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.

The fate of membrane-bound ribosomes following the termination of protein synthesis.

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane.

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.

Chaperone-mediated cross-priming: a hitchhiker's guide to vesicle transport (review).

The resident endoplasmic reticulum (ER) chaperone proteins GRP94 (gp96) and calreticulin can activate the immune system to slow or stop the progression of tumors by escorting tumor-derived peptides into the endogenous antigen presentation pathway of antigen presenting cells (APC). Although the phenomenology of cross-priming is well worked out, the mechanism(s) remains unclear. Continuing insights into cellular protein trafficking pathways suggest several means by which chaperones could travel from the extracellular space into the endosome, lysosome or ER of APC. In particular, proteins that cycle between two or more compartments and those that undergo and mediate retrograde flow offer models of how exogenous chaperones might travel in the APC. New insights into how non-chaperone proteins access the APC antigen presentation pathway also suggest several ways this process could occur.

A topological study of the human gamma-glutamyl carboxylase.

gamma-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. II. Ligand-mediated activation of GRP94 molecular chaperone and peptide binding activity.

The N-terminal domain of eukaryotic Hsp90 proteins contains a conserved adenosine nucleotide binding pocket that also serves as the binding site for the Hsp90 inhibitors geldanamycin and radicicol. Although this domain is essential for Hsp90 function, the molecular basis for adenosine nucleotide-dependent regulation of GRP94, the endoplasmic reticulum paralog of Hsp90, remains to be established. We report that bis-ANS (1,1'-bis(4-anilino-5-napthalenesulfonic acid), an environment sensitive fluorophore known to interact with nucleotide-binding domains, binds to the adenosine nucleotide-binding domain of GRP94 and thereby activates its molecular chaperone and peptide binding activities. bis-ANS was observed to elicit a tertiary conformational change in GRP94 similar to that occurring upon heat shock, which also activates GRP94 function. bis-ANS activation of GRP94 function was efficiently blocked by radicicol, an established inhibitory ligand for the adenosine nucleotide binding pocket. Confirmation of the N-terminal nucleotide binding pocket as the bis-ANS-binding site was obtained following covalent incorporation of bis-ANS into GRP94, trypsinolysis, and sequencing of bis-ANS-labeled limit digestion products. These data identify a ligand dependent regulation of GRP94 function and suggest a model whereby GRP94 function is regulated through a ligand-dependent conversion of GRP94 from an inactive to an active conformation.

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding.

X-ray crystallographic studies of the N-terminal domain of Hsp90 have identified an unconventional ATP binding fold, thereby inferring a role for ATP in the regulation of the Hsp90 activity. In this report, N-ethylcarboxamidoadenosine (NECA) was used to investigate the nucleotide binding properties of GRP94, the endoplasmic reticulum paralog of Hsp90. Whereas Hsp90 did not bind NECA, GRP94 bound NECA in a saturable manner with a K(d) of 200 nm. NECA binding to GRP94 was efficiently blocked by geldanamycin and radicicol. Analysis of ligand binding stoichiometries by radioligand and calorimetric techniques indicated that GRP94 bound 1 mol of NECA/mol of GRP94 dimer. In contrast, GRP94 bound radicicol at a stoichiometry of 2 mol of radicicol/mol of GRP94 dimer. In [(3)H]NECA displacement assays, GRP94 displayed binding interactions with ATP, dATP, ADP, AMP, cAMP, and adenosine, but not GTP, CTP, or UTP. To accommodate the 0.5 mol of NECA:mol of GRP94 binding stoichiometry observed for the native GRP94 dimer, a model for allosteric regulation (negative cooperativity) of ligand binding is proposed. A hypothesis on the regulation of GRP94 conformation and activity by adenosine-based ligand(s) other than ATP and ADP is presented.

Ribosome-independent regulation of translocon composition and Sec61alpha conformation.

In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61alpha conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61alpha, ribophorin I, and TRAPalpha, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the ribosome does not define the composition of the digitonin-soluble translocon. Whereas in digitonin solution a highly electrostatic ribosome-translocon junction is observed, no stable interactions between ribosomes and Sec61alpha, ribophorin I, or TRAPalpha were observed following solubilization of RM with lipid-derived detergents at physiological salt concentrations. Sec61alpha was found to exist in at least two conformational states, as defined by mild proteolysis. A protease-resistant form was observed in RM and detergent-solubilized RM. Removal of peripheral proteins and ribosomes markedly enhanced the sensitivity of Sec61alpha to proteolysis, yet the readdition of inactive ribosomes to salt-washed membranes yielded only modest reductions in protease sensitivity. Addition of sublytic concentrations of detergents to salt-washed RM markedly decreased the protease sensitivity of Sec61alpha, indicating that a protease-resistant conformation of Sec61alpha can be conferred in a ribosome-independent manner.

The immunological properties of endoplasmic reticulum chaperones: a conflict of interest?

ER chaperones are abundant and highly conserved proteins that display both peptide binding and chaperone activity. Of the family of chaperones present in the mammalian ER, GRP94 and calreticulin are apparently unique in their ability to elicit CD8+ T-cell responses against components of their bound-peptide pools. The ability of GRP94 and calreticulin to elicit CD8+ T-cell responses indicates that both proteins bind peptides suitable for assembly on to MHC class-I molecules. The capacity to function as molecular chaperones and as peptide-binding proteins capable of transferring, directly or indirectly, peptides on to class-I molecules, indicates that GRP94 and calreticulin participate in the regulation of both peptide and polypeptide traffic in the ER. Perspectives on the regulation of and interplay between the peptide binding and chaperone activity of GRP94 and calreticulin are discussed.

Structural determinants for signal sequence function in the mammalian endoplasmic reticulum.

Signal sequences function in protein targeting to and translocation across the endoplasmic reticulum membrane. To investigate the structural requirements for signal sequence function, chimeras of the Escherichia coli LamB signal peptide and prolactin were prepared. The LamB signal peptide was chosen by virtue of the extensive biophysical and biological characterization of its activity. In vitro, nascent prolactin chains bearing the LamB signal peptide (LamB) were targeted in a signal recognition particle (SRP)-dependent manner to rough microsomes but remained protease- and salt-sensitive and translocated at low efficiency. Full translocation activity was obtained in a gain of function mutant (LamB*) in which three hydrophobic residues in the LamB hydrophobic core were converted to leucine residues. Cross-linking studies demonstrated that the LamB* signal sequence displayed markedly enhanced interactions with SRP and integral membrane proteins. In contrast, chemically denatured LamB and LamB*-precursors bound with identical efficiencies and in a salt-resistant manner to rough microsomes, suggesting that during de novo synthesis the signal sequence of LamB-bearing precursors assumes a conformation refractory to translocation. These data indicate that a leucine-rich signal sequence is necessary for optimal interaction with SRP and suggest that SRP, by maintaining the signal sequence in a conformation suitable for membrane binding, performs a chaperone function.

Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages.

Immunization of mice with GRP94, the endoplasmic reticulum (ER) Hsp90, elicits cytotoxic T lymphocyte (CTL) responses to chaperone-bound, source cell-derived peptides. Elicitation of a CTL response requires that GRP94-associated peptides be transferred onto major histocompatability complex (MHC) class I molecules, a process that is postulated to accompany GRP94 internalization by antigen presenting cells, such as macrophages (Mphi) and dendritic cells (DC). In studies of GRP94 uptake in elicited Mphi, we report that Mphi display specific cell surface binding of GRP94, and that surface-bound GRP94 can be internalized via receptor mediated endocytosis. GRP94 internalized by this pathway co-localized predominately with transferrin-positive early endosomes. At time periods of up to 20 minutes, little trafficking of GRP94 to the lysosomal compartment was observed. When GRP94 was present in the medium, and thus accessible to both receptor-mediated and fluid phase internalization pathways, internalization was modestly inhibited in the presence of yeast mannan, a competitive inhibitor of mannose/fucose receptor activity, and substantially inhibited by dimethylamiloride, an inhibitor of macropinocytosis. GRP94 internalized via macropinocytosis did not display prominent co-staining with the lysosomal marker LAMP-2. These data identify multiple pathways of GRP94 internalization and indicate that receptor-dependent uptake of GRP94 is not dependent upon its high mannose oligosaccharide moiety. Most significantly, these data demonstrate the existence of cell surface receptor(s), apparently unique to antigen presenting cells, that function in the binding and internalization of the ER chaperone GRP94.

Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides.

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.

Structural transitions accompanying the activation of peptide binding to the endoplasmic reticulum Hsp90 chaperone GRP94.

GRP94, the endoplasmic reticulum Hsp90 paralog, binds a diverse array of peptides, a subset of which are suitable for assembly onto nascent MHC class I molecules. At present, the mechanism, site, and regulation of peptide binding to GRP94 are unknown. Using VSV8, the immunodominant peptide epitope of the vesicular stomatitis virus, and native, purified GRP94, we have investigated GRP94-peptide complex formation. The formation of stable GRP94-VSV8 complexes was slow; competition studies demonstrated that peptide binding to GRP94 was specific. VSV8 binding to GRP94 was stimulated 2-fold or 4-fold, respectively, following chemical denaturation/renaturation or transient heat shock. The activation of GRP94-peptide binding occurred coincident with a stable, tertiary conformational change, as identified by tryptophan fluorescence and proteolysis studies. Analysis of GRP94 secondary structure by circular dichroism spectroscopy indicated an identical alpha-helical content for the native, chemically denatured/renatured, and heat-shocked forms of GRP94. Through use of the environment-sensitive fluorophores acrylodan and Nile Red, it was observed that the activation of peptide binding was accompanied by enhanced peptide and solvent accessibility to a hydrophobic binding site(s). Peptide binding to native or activated GRP94 was identical in the presence or absence of ATP or ADP. These results are discussed with respect to a model in which peptide binding to GRP94 occurs within a hydrophobic binding pocket whose accessibility is conformationally regulated in an adenine nucleotide-independent manner.

Biochemical, cell biological and immunological issues surrounding the endoplasmic reticulum chaperone GRP94/gp96.

The past year has born witness to compelling demonstrations of the utility of peptide complexes with glucose regulated protein 94 (GRP94, also known as gp96) in cancer immunotherapy. Insights into the structural basis of peptide binding to GRP94 have been obtained and the role of the transporter for antigen presentation in defining the GRP94-bound peptide composition has been determined.

Structure and folding of nascent polypeptide chains during protein translocation in the endoplasmic reticulum.

To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo and in vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed.