Abstract: Shrutee JAKHANWAL

Regulation of the Neuronal SNARE Complex by Accessory Proteins

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Shrutee Jakhanwal, Reinhard Jahn

Department of Neurobiology, Max- Planck Institute of Biophysical Chemistry, Göttingen, Germany

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Neuronal exocytosis lies at the heart of the process of synaptic neurotransmission. The process of neuronal exocytosis is mediated by a highly conserved family of proteins called SNAREs (Soluble N-ethylmaleimide Attachment Receptor Proteins). There are three kinds of neuronal SNAREs: Syntaxin, SNAP-25 and Synaptobrevin. Syntaxin (with one SNARE motif) and SNAP-25 (with two SNARE motifs) are present on the plasma membrane, whereas Synaptobrevin is present on the synaptic vesicles. Years of work in this field have established that the SNAREs assemble into a highly stable four-helix bundle called the SNARE complex through their SNARE motifs, which brings the membranes together to mediate fusion. Although it is very clear that the four-helix bundle is extremely critical for fusion to occur; speculations over the starting point of the process, the exact steps leading to the formation of the neuronal complex and the mechanism of regulation of the process of neuronal exocytosis is highly unclear. The proteins that have been speculated to have a regulatory role in this process are Munc-18, Munc-13, Synaptotagmin and Complexin. My project aims at deciphering the precise roles of the regulatory proteins and to speculate the interplay between the regulatory proteins and the SNARE proteins that underlies the secret of the exquisitely regulated process of neuronal exocytosis.

The initial focus of my project lies on speculating the role of the regulatory proteins Munc-18 and Munc-13. Munc-18 is a member of SM-family of proteins. Munc-18 forms a very tight complex with Syntaxin, which forces Syntaxin into a closed conformation. In this closed conformation, the SNARE motif of Syntaxin is no longer available to mediate SNARE-complex formation. During the priming step, Syntaxin-1 transitions from a closed conformation (that binds Munc18-1 tightly) to an open conformation (that becomes accessible for SNARE-complex formation). Munc-13 has been hypothesized to promote the transition from closed-Syntaxin to open-Syntaxin during the priming step of SNARE-mediated fusion. The mechanism of action of Munc-13 however, is highly unclear. Interestingly, the Syntaxin-1-Munc18 complex is resistant to the action of NSF-?SNAP and, is speculated to be the starting point for the exquisitely regulated process of neurotransmitter release.

My project aims at deciphering the molecular details of these processes using in-vitro techniques such as liposome preparation, incorporation of purified SNARE-proteins and accessory proteins into these liposomes to produce a mimic for the synaptic vesicles in the neurons and to study the interaction and effect of different proteins under different conditions using in-vitro techniques such as FRET, Stopped-flow measurements, Anisotropy measurements, and a few others. These investigations would help us achieve a better vision of the intricate steps that contribute to the beauty of the exquisitely regulated process of neurotransmitter release.