SNAP-25 a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinurn neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln'97-Arg'98 peptide bond, while serotype E cleaves the Arg'80-Ile'8' peptide linkage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.

In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca2+-signaled exocytosis is effectively blocked by antibodies and peptides to a Several lines of evidence have identified three synaptic proteins, syntaxin, VAMP, and SNAP-25, which together with synaptotagmin form the core complex postulated to coordinate regulated vesicular fusion for neurotransmitter release (see refs. 3-5). In vitro studies with solubilized and recombinant proteins have begun to define the molecular relationships between these proteins (6-9). In an initial 7S complex, SNAP-25 binds both syntaxin 1A and VAMP to increase the strength and specificity of their interaction (8, 9). Binding of a-SNAP to this complex displaces synaptotagmin from syntaxin and provides entry of N-ethylmaleimide-sensitive fusion protein (NSF) to form a 20S prefusion complex that upon ATP hydrolysis subsequently leads to its disassembly (6). These observations have led to the proposal that neurotransmission represents a specialization of general membrane trafficking in which recognition between neural-specific vesicle-and targetsoluble NSF attachment protein receptors (v-and t-SNAREs) targets synaptic vesicles to the plasma membrane and the ATP hydrolysis step mediated by NSF to drive exocytosis (10).These in vitro studies, however, have yet to resolve the full roles played by these synaptic vesicle and plasma membrane proteins in the cascade of events required for neurotransmitter release. For

The mouse mutant coloboma (Cm/+), which exhibits profound spontaneous hyperactivity and bears a deletion mutation on chromosome 2, including the gene encoding synaptosomal protein SNAP‐25, has been proposed to model aspects of attention‐deficit hyperactivity disorder. Increasing evidence suggests a crucial role for SNAP‐25 in the release of both classical neurotransmitters and neuropeptides. In the present study, we compared the release of specific neurotransmitters in vitro from synaptosomes and slices of selected brain regions from Cm/+ mice with that of +/+ mice. The release of dopamine (DA) and serotonin (5‐HT) from striatum, and of arginine vasopressin and corticotropin‐releasing factor from hypothalamus and amygdala is calcium‐dependent. Glutamate release from and content in cortical synaptosomes of Cm/+ mice are greatly reduced, which might contribute to the learning deficits in these mutants. In dorsal striatum of Cm/+ mutants, but not ventral striatum, KCI‐induced release of DA is completely blocked and that of 5‐HT is significantly attenuated, suggesting that striatal DA and 5‐HT deficiencies may be involved in hyperactivity. Further, although acetylcholine failed to induce hypothalamic corticotropin‐releasing factor release from Cm/+ slices, restraint stress increased plasma corticosterone levels in Cm/+ mice to a significantly higher level than in +/+ mice, suggesting an important role for arginine vasopressin in hypothalamic‐pituitary‐adrenal axis activation. These results suggest that reduced SNAP‐25 expression may contribute to a region‐specific and neurotransmitter‐specific deficiency in neurotransmitter release.

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

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