Niti Puri

Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms

Mast cells degranulate and release the contents of intracellular secretory granules in response to the cross-linking of FcεRI by multivalent antigens. These granules contain a variety of biologically active inflammatory mediators; however, it is not clear whether these granules are homogenous or whether there is heterogeneity within the secretory granule population in mast cells. By using genetically altered mice lacking specific vesicle-associated SNARE membrane fusion proteins, we found that VAMP-8-deficient mast cells exhibited defects in FcεRI-regulated exocytosis, whereas synaptobrevin 2- or VAMP-3-deficient mast cells did not. Surprisingly, the defect in secretion in VAMP-8-deficient mice was limited to the subpopulation of mast cell secretory granules containing serotonin and cathepsin D, whereas regulated exocytosis of secretory granules containing histamine and TNF-α was normal. Confocal microscopy confirmed that serotonin and histamine were present in distinct intracellular granules and that most serotonin-containing granules were VAMP-8-positive. Thus, this study demonstrates that mast cells do indeed possess distinct subsets of secretory granules and that these subsets use different SNARE isoforms for exocytosis.

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis.

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule–plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP‐23 co‐localized with a lipid raft marker and was present in detergent‐insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule‐associated SNARE vesicle‐associated membrane protein (VAMP)‐2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP‐2 in these rafts were present in SNARE complexes containing SNAP‐23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP‐23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP‐23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP‐2 present in rafts increased twofold, and these proteins were now present in raft‐associated phospho‐SNAP‐23/syntaxin 4/VAMP‐2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.

Phosphorylation of SNAP-23 Regulates Exocytosis from Mast Cells

Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. The kinetics of SNAP-23 phosphorylation mirror the kinetics of exocytosis. We have identified amino acid residues Ser95 and Ser120 as the major phosphorylation sites in SNAP-23 in rodent mast cells. Quantitative analysis revealed that ∼10% of SNAP-23 was phosphorylated when mast cell degranulation was induced. These same residues were phosphorylated when mouse platelet degranulation was induced with thrombin, demonstrating that phosphorylation of SNAP-23 Ser95 and Ser120 is not restricted to mast cells. Although triggering exocytosis did not alter the absolute amount of SNAP-23 bound to SNAREs, after stimulation essentially all of the SNAP-23 bound to the plasma membrane SNARE syntaxin 4 and the vesicle SNARE VAMP-2 was phosphorylated. Regulated exocytosis studies revealed that overexpression of SNAP-23 phosphorylation mutants inhibited exocytosis from rat basophilic leukemia mast cells, demonstrating that phosphorylation of SNAP-23 on Ser120 and Ser95 modulates regulated exocytosis by mast cells.

Mast Cell Degranulation Requires N-Ethylmaleimide-Sensitive Factor-Mediated SNARE Disassembly

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.

The last exon of SNAP-23 regulates granule exocytosis from mast cells

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a “better” SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.

A Double in vivo Biotinylation Technique for Objective Assessment of Aging and Clearance of Mouse Erythrocytes in Blood Circulation

We have recently developed a new technique to objectively identify erythrocyte cohorts of defined age in mouse blood. The technique (termed double in vivo biotinylation, DIB) involves an initial biotinylation of all erythrocytes in circulation, followed after a few days by a second biotinylation, at a lower density, that labels the biotin-negative erythrocytes that have entered since the first biotinylation. The proportions of biotinhigh, biotinlow, and biotinnegative erythrocytes are enumerated by flow cytometry. The DIB technique allows us to track age-related changes on erythrocyte cohorts (Protocol A), and to simultaneously identify very young and older erythrocyte populations in the blood (Protocol B). Using this technique, we have reexamined: i) the relationship between age and buoyant density of erythrocytes, ii) erythrocyte destruction through a random removal mechanism, and iii) the expression of phosphatidylserine on aging erythrocytes. We have also used the DIB technique to study age-related changes in the expression of various markers like CD47 and CD147 and green autofluorescence in aging erythrocyte populations.

Interactions of polydispersed single-walled carbon nanotubes with T cells resulting in downregulation of allogeneic CTL responses in vitro and in vivo

Abstract Addition of polydispersed acid functionalised single-walled carbon nanotubes (AF-SWCNTs) significantly suppressed alloimmune cytotoxic T cell (CTL) response generated in a mixed lymphocyte reaction (MLR) between spleen cells from C57BL/6 (H-2b) and BALB/c (H-2d) mice. AF-SWCNTs treatment also decreased CD69 expression, enhanced apoptotic response in T cells and reduced significantly the recovery of live CD4+ and CD8+ T cells from MLR cultures. A two to threefold increase was noticed in the binding/uptake of AF-SWCNTs by T cells in MLR cultures as compared with control cultured T cells. Confocal microscopy confirmed the internalization of AF-SWCNTs by live CD8+ T cells in MLR cultures. Administration of AF-SWCNTs suppressed the generation of anti-P815 CTL response in C57BL/6 mice and the recovery of T-cell populations from the spleens. The results demonstrate a suppressive effect of AF-SWCNTs on CTL response and provide an insight into the mechanism of this suppression.

Cytokine regulation of expression of class I MHC antigens

The major histocompatibility complex (MHC) is a chromosomal region that has been extensively characterized in several mammals, particularly man and mouse (Flavell et al., 1986 and Ploegh et al ., 1981). The region which represents about 0.1% of the total genome in mouse, comprises a large number of genes classified into class I and class II MHC genes encoding a large number of polymorphic class I or class II MHC molecules respectively. Class I genes include HLA-A, B, C genes in man and H2D, K, L in the mouse. Class II MHC genes include HLADR, DP, DQ in human and H-2IA and IE genes in the mouse. Class I MHC molecules are made of a highly polymorphic glycosylated transmembrane α-chain of about 45 kDa, associated with a non-polymorphic, nonglycosylated, 12-kDa chain, called β2-microglobulin, not encoded within the MHC locus. Class II molecules encoded entirely within the MHC locus are made of two transmembrane polymorphic chains, α and β, which associate to form heterodimer. Class I heavy and light chains comprise three and one extracellular domains respectively, whereas both α and β chains of class II molecules have two extracellular domains each. Both class I and class II MHC antigens show very high polymorphism and two of the extracellular domains, α1 and α2 in class I and α1 a n d β1 in class II heterodimers display much more variability within different alleles than the other so called constant domains (α3 and β2 in class I and α2 and β2 in class II molecules). Constant domains resemble immunoglobulin domain, which make MHC molecules members of the immunoglobulin super family (Williams et al., 1988). Class I molecules are constitutively expressed on most types of nucleated cells. Class II molecules are expressed largely by cells of immune lineage, particularly B cells, macrophages, and dendritic cells. MHC class I molecules present antigenic peptides to CD8+ T cells. The majority of class I-binding peptides are derived from nuclear and cytosolic proteins. MHC class II molecules present peptides from antigens degraded by the endosomal/ lysosomal pathway to CD4+ T cells (Benham et al., 1995). Antigen receptor on T-cells (T-cell receptor or TCR) can recognize antigenic peptides only when the latter are presented associated with class I or class II MHC antigens. In addition, during T-cell differentiation, only those Tc e l l s are permitted to develop which can recognize antigen associated with self MHC molecules, a phenomenon which is referred to as MHC restriction (Zinkernagel et al., 1979). While T-cells can only be activated in a MHC restricted manner, a class of non-T cytotoxic lymphocytes called natural killer (NK) cells, can spontaneously kill target cells in a MHC non-restricted manner. Levels of expression of class I MHC antigens on tumor cells however determine the susceptibility of tumor cells to NK cells (Haridas and Saxena, 1995a, 1995b, Saxena et al., 1996). Expression of class I MHC molecules thus play a crucial role in determining the susceptibility of target cells to both T cells and NK cells.

Transcription regulation of nuclear receptor PXR: Role of SUMO-1 modification and NDSM in receptor function

Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the nuclear receptor superfamily of ligand-modulated transcription factors. PXR plays an important role in metabolism and elimination of diverse noxious endobiotics and xenobiotics. Like in case of some nuclear receptors its function may also be differentially altered, positively or negatively, by various post-translational modifications. In this context, regulation of PXR function by SUMOylation is the subject of present investigation. Here, we report that human PXR is modified by SUMO-1 resulting in its enhanced transcriptional activity. RT-PCR analysis showed that PXR SUMOylation in presence of rifampicin also enhances the endogenous expression levels of key PXR-regulated genes like CYP3A4, CYP2C9, MDR1 and UGT1A1. In addition, mammalian two-hybrid assay exhibited enhanced interaction between PXR and co-activator SRC-1. EMSA results revealed that SUMOylation has no influence on the DNA binding ability of PXR. In silico analysis suggested that PXR protein contains four putative SUMOylation sites, centered at K108, K129, K160 and K170. In addition to this, we identified the presence of NDSM (Negative charge amino acid Dependent SUMOylation Motif) in PXR. Substitution of all its four putative lysine residues along with NDSM abolished the effect of SUMO-1-mediated transactivation function of PXR. Furthermore, we show that interaction between PXR and E2-conjugation enzyme UBCh9, an important step for implementation of SUMOylation event, was reduced in case of NDSM mutant PXRD115A. Overall, our results suggest that SUMOylation at specific sites on PXR protein are involved in enhancement of transcription function of this receptor.

Phosphorylation of SNAP-23 regulates its dynamic membrane association during mast cell exocytosis

ABSTRACT Upon allergen challenge, mast cells (MCs) respond by releasing pre-stored mediators from their secretory granules by the transient mechanism of porosome-mediated cell secretion. The target SNARE SNAP-23 has been shown to be important for MC exocytosis, and our previous studies revealed the presence of one basal (Thr102) and two induced (Ser95 and Ser120) phosphorylation sites in its linker region. To study the role of SNAP-23 phosphorylation in the regulation of exocytosis, green fluorescence protein-tagged wild-type SNAP-23 (GFP-SNAP-23) and its phosphorylation mutants were transfected into rat basophilic leukemia (RBL-2H3) MCs. Studies on GFP-SNAP-23 transfected MCs revealed some dynamic changes in SNAP-23 membrane association. SNAP-23 was associated with plasma membrane in resting MCs, however, on activation a portion of it translocated to cytosol and internal membranes. These internal locations were secretory granule membranes. This dynamic change in the membrane association of SNAP-23 in MCs may be important for mediating internal granule-granule fusions in compound exocytosis. Further studies with SNAP-23 phosphorylation mutants revealed an important role for the phosphorylation at Thr102 in its initial membrane association, and of induced phosphorylation at Ser95 and Ser120 in its internal membrane association, during MC exocytosis. Summary: The current study has revealed the phosphorylation-dependent dynamic nature of membrane association of SNAP-23 for mediation of different fusion steps in compound exocytosis from mast cells during allergen challenge.