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. Regulated exocytosis is the process by which stimulation of plasma membrane receptors on secretory cells results in the release of proteins and/or peptides from intracellular stores into the extracellular space (1). One common characteristic of regulated exocytosis, whether it be from neurons, cytotoxic lymphocytes, adipocytes, or mast cells, is that the cells response to this stimulus results in pre-formed intracellular granules moving toward and fusing with the plasma membrane. Secretory granule/plasma membrane fusion is the essence of regulated exocytosis, and there is an intense effort underway to identify the molecular mechanisms regulating this process in the hopes of identifying ways to modulate exocytosis.The RBL-2H3 1 mast cell line has been extensively studied as a model not only for mast cell biology but also as a paradigm for regulated exocytosis from non-neuronal cells (2). Stimulation of the high affinity IgE receptor, Fc⑀RI, on these cells by crosslinking initiates a signal transduction cascade that culminates in secretory granule fusion with the plasma membrane, thereby liberating a variety of inflammatory mediators (3, 4). Numerous proteins necessary for the tethering, docking, and fusion steps between various membrane compartments in eukaryotic cells have been described. Among those, SNAREs (soluble NSF-attachment protein receptors) are a large family of membrane-associated proteins essential for membrane-membrane fusion (5, 6). These proteins include members of the vesicle-associated synaptobrevin/VAMP family as well as members of the syntaxin and SNAP-23 families of "target" membrane SNAREs. The current model proposes that while vesicles are docked on the target membrane, SNAREs from the donor or vesicle membrane (v-SNAREs) form trans-SNARE complexes with their cognate SNARE partners on the opposing target membrane. Structurally, the exocytic SNARE complex is a trimolecular protein complex containing one member of the VAMP, syntaxin, and SNAP-23 family, each contributing to the formation of a four-helix coiled-coil bundle (7, 8) whose formation is sufficient for in vitro membrane fusion (9).Given that SNAREs play a central role in the membrane fusion process, it is likely that their function is modulated in vivo. In particular, protein kinases, which have been extensively associated with the regulation of exocytosis (10), could participate in SNARE function by phosphorylating residues essential in SNARE complex assembly or the binding of SNARE regulatory proteins (6,11,12). Members of the syntax...

We recently established that hybrid insulin peptides (HIPs) are present in human islets and that T cells reactive to HIPs are found in the residual islets of organ donors with type 1 diabetes (T1D). Here, we investigate whether HIP-reactive T cells are indicative of ongoing autoimmunity in patients with T1D. We used interferon-γ enzyme-linked immune absorbent spot analyses on peripheral blood mononuclear cells (PBMCs) to determine whether patients with new-onset T1D or control subjects displayed T-cell reactivity to a panel of 16 HIPs. We observed that nearly one-half of the patients responded to one or more HIPs. Responses to four HIPs were significantly elevated in patients with T1D but not in control subjects. To characterize the T cells reactive to HIPs, we used a carboxyfluorescein succinimidyl ester–based assay to clone T cells from PBMCs. We isolated six nonredundant, antigen-specific T-cell clones, most of which reacting to their target HIPs in the low nanomolar range. One T-cell clone was isolated from the same patient on two different blood draws, indicating persistence of this T-cell clone in the peripheral blood. This work suggests that HIPs are important target antigens in human subjects with T1D and may play a critical role in disease.

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