Regulated secretion of neurotransmitters and hormones controls every aspect of the human body. Hormones and transmitters are stored in secretory vesicles which must fuse with the cell plasma membrane for release to occur (Fig. 1). During vesicle fusion, a set of three SNARE proteins plays the central role: syntaxin, SNAP-25 and synaptobrevin. These proteins, normally resident in the two opposing membranes, engage each other and form an extremely tight SNARE complex. Formation of this complex drives fusion of two membranes, leading to release of the vesicular cargo into the extracellular space (exocytosis). Comprehensive description of the protein-protein and protein-lipid interactions governing the SNARE assembly pathway will advance our understanding of the regulation of neurotransmitter and hormone release.

By investigating SNARE proteins, both in solution and in membranes, we uncovered vesicular restriction of synaptobrevin as a likely basis for the tight control of neurotransmitter and hormone release (Hu et al., 2002). Our further studies revealed that a common lipid - sphingosine - can activate synaptobrevin and facilitate vesicle fusion (Darios et al., 2009). We also uncovered a positive role for omega-3 and omega-6 fatty acids in promoting formation of the syntaxin/SNAP-25 heterodimer on the plasma membrane (Darios et al., 2006). Our additional studies demonstrated that a vesicular protein, synaptotagmin, interacts in a structurally-defined way with the syntaxin/SNAP-25 heterodimer and then rapidly cross-links two membranes following a rise in calcium concentration (Rickman et al., 2006; Connell et al., 2008). Together, our molecular studies have allowed us to propose a working model for vesicle fusion (Fig. 2).

Having understood the precise mechanics of vesicle fusion, our focus moved towards developing tools that will help us to manipulate the level of secretion of neurotransmitters or hormones. The best starting point for this endeavour is to understand how we can improve botulinum neurotoxins. These powerful toxins enter neurons to cleave a single SNARE protein leading to a long-lasting blockade of neurotransmission. In our previous work, we shed light on the molecular mechanism underlying the difference in duration of neuromuscular paralysis caused by botulinum toxins, types A and E, both of which cleave SNAP-25 (Bajohrs et al., 2004). Recently, we developed a new, SNARE-based protein clipping technique which allows assembly of functional recombinant units into new products. One fascinating feature of the neuronal SNARE complex is its stability and resistance to harsh treatments, including acid, alkali and SDS. The unique properties of the SNARE coiled-coil bundle have not yet been considered for exploitation in medicine and biotechnology. We are currently using SNARE-derived polypeptides to engineer new versions of botulinum neurotoxins which will allow silencing of select subtypes of neurons and endocrine cells. Together, these studies should provide novel insights into the working of the secretory machinery in both health and disease, and may lead to novel therapeutic approaches.