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.

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion.

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

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.

CAPS (mammalian UNC-31) protein localizes to membranes involved in dense-core vesicle exocytosis.

CAPS is a neural/endocrine-specific protein discovered as a cytosolic factor required for Ca2+-activated dense-core vesicle (DCV) exocytosis in permeable neuroendocrine cells. We report that CAPS is also a membrane-associated, peripherally bound protein in brain homogenates that localizes Selectively to plasma membranes and to DCVs but not to small clear synaptic vesicles (SVs). CAPS exhibits high affinity and saturable binding to DCVs by interaction with bilayer phospholipids. Specific CAPS antibodies inhibit Ca2+-activated norepinephrine release from lysed synaptosomes that contain membrane-associated CAPS, indicating that membrane-bound CAPS is essential for neural DCV exocytosis. CAPS is a functional component of the exocytotic machinery that localizes selectively to DCVs, and it may confer distinct regulatory features on neuropeptide and biogenic amine transmitter secretion.

Stem cell factor binding to retrovirus primer binding site silencers.

Using modified nuclear lysis and binding conditions, we have examined the binding of an embryonal carcinoma (EC) cell factor, binding factor A, to a stem cell-specific silencer which acts at the DNA level and overlaps the Moloney murine leukemia virus (M-MuLV) proline primer binding site (PBS). Following our protocol, we found that in vitro binding of factor A correlated with the in vivo activity of the M-MuLV silencer. Factor A bound specifically to the wild-type silencer element at room temperature and 30 degrees C, but not at 4 degrees C, and bound 10-fold better to the full-length silencer than to a minimal silencer core element. The factor was enriched in nuclear compared with cytosolic extracts and in undifferentiated EC cells compared with differentiated cells in which the silencer is nonfunctional. Salt and ion requirements for factor A binding were investigated, and partial purification steps indicated the factor to be a heparin-Sepharose-binding moiety of greater than 100 kDa. To examine possible relationships between silencer and PBS activities, sequences representing phenylalanine, isoleucine, lysine-1,2, lysine-3, methionine, and tryptophan PBS DNA fragments were tested in vivo for stem cell-specific repression of M-MuLV expression and in vitro in DNA binding assays. Of these PBS elements, only the lysine-1,2 PBS DNA fragment showed consistently high levels of repression. Interestingly, the lysine-1,2 PBS DNA fragment also formed a complex with an EC cell factor with characteristics similar to those of factor A. However, the two factors did not cross-compete in binding studies, suggesting that they may be different but related factors. Our results suggest that expression of Mason-Pfizer monkey virus, visna virus, and spumavirus, which use the lysine-1,2 PBS, may be inhibited in undifferentiated stem cells.

Retrovirus-mediated insertion of expressed and non-expressed genes at identical chromosomal locations.

During retrovirus replication, a cellularly derived tRNA is annealed to the viral RNA at the primer binding site (PBS) to prime reverse transcription, and both the tRNA and the PBS become copied and matched together on complementary proviral DNA strands prior to integration. Using a viral PBS single base pair mutant which affects provirus expression in undifferentiated cells, we show that reversion to wild type (wt) occurs at a frequency of approximately 50%. Daughter cell lines containing wt or mutant proviruses at identical chromosomal sites have been isolated, supporting a model where an integrated PBS-mismatched provirus was copied before mismatch correction could occur. Virus expression in daughter cells containing the mutant provirus was 100-fold higher than in cells bearing the wt counterpart. Additionally, proviral 5' DNA and cellular 5' flanking DNA became methylated in daughter cells containing wt but not mutant integrants. These results strongly support the current model of retrovirus reverse transcription, and indicate that the wt PBS region contains an element which suppresses virus expression and directs the methylation of viral and neighboring cellular DNA.

Characterization of the Moloney murine leukemia virus stem cell-specific repressor binding site.

The Moloney murine leukemia virus (M-MuLV) repressor binding site (RBS) mediates cell-type-specific repression in embryonal carcinoma (EC) cells of expression from several different promoters, including the M-MuLV long terminal repeat promoter. Silencing has been shown to depend on an element normally located in the proviral 5' noncoding region and occurs at the DNA level in the absence of retroviral proteins. Using fragments of the RBS region, we now show that the minimal size of the silencer corresponds to M-MuLV nt 147-163 and overlaps with the retroviral primer binding site region by 17 of its 18 bp. A panel of point mutations within the RBS has been examined to yield a consensus RBS sequence which is consistent with the notion that a previously identified nuclear factor (binding factor A) mediates RBS repression. Viral vectors using neomycin, beta-galactosidase, and luciferase reporters have been employed to show that RBS-mediated repression occurs in EC and embryonal stem, but not in other tested cell types. Repression was observed to occur within 48 hr of infection, prior to when global methylation of proviruses has been reported to occur. Repression also occurred after azacytidine treatment of EC cells, supporting the notion that the RBS functions independently of provirus methylation. However, levels of provirus methylation in selected cells were increased in the presence of a wild-type RBS, and methylation correlated with a secondary stage of virus repression. Thus, the M-MuLV RBS acts directly to control expression in EC cells and also appears to trigger a secondary level of repression which is coincident with provirus methylation.