Synaptobrevin

Tetanus is a disease caused by the release of neurotoxins from the anaerobic, spore-forming rod Clostridium tetani. The clostridial protein, tetanus toxin, possesses a protease activity which selectively degrades the pre-synaptic vesicle protein synaptobrevin, resulting in a block of glycine and y-aminobutyric acid (GABA) release from presynaptic terminals. Consistent with the loss of neurogenic motor inhibition, symptoms of tetanus include muscular rigidity and hyperreflexia. The clinical course is characterized by increased muscle tone and spasms, which first affect the masseter muscle and the muscles of the throat, neck and shoulders. Death occurs by respiratory failure or heart failure. 

Much evidence supports a role for these proteins in exocytosis. For instance, injection of recombinant SNAP into the squid giant axon increases vesicular exocytosis. Also, membrane SNAP-25 and syntaxin are both targets for botulinum toxin while the vesicule protein, synaptobrevin, is a target for tetanus and botulinum toxins both these toxins are well known for disrupting transmitter release. 

Synaptobrevins (VAMPs) Synaptogyrin Synaptophysins PKA but diverge C-terminally. Synapsins Ia/b contain C-terminal phosphorylation sites for CaMKII and CDK 5. Interact with microfilaments, neurofilaments, microtubules, SH3 domains, calmodulin and annexin VI in vitro. Small-membrane proteins that are cleaved by tetanus toxin and by botulinum toxins B, D, F and G. Polytopic membrane protein that is tyrosine-phosphorylated. Function unknown. Polytopic membrane proteins, including synaptoporin, that are tyrosine-phosphorylated and bind to synaptobrevins. May regulate SNARE function... 

G. Schiavo, F. Benfenati, B. Poulain, O. Rossetto, P. Polverino de Laureto, B. R. Das-Gupta, C. Montecucco, Tetanus and Botulinum-B Neurotoxins Block Neurotransmitter Release by Proteolytic Cleavage of Synaptobrevin , Nature 1992a, 359, 832-835 G. Schiavo, O. Rossetto, A. Santucci, B. R. DasGupta, C. Montecucco, Botulinum Neurotoxins are Zinc Proteins , J. Biol. Chem. 1992b, 267, 23479-23483. 

All botulin neurotoxins act in a similar way. They only differ in the amino-acid sequence of some protein parts (Prabakaran et al., 2001). Botulism symptoms are provoked both by oral ingestion and parenteral injection. Botulin toxin is not inactivated by enzymes present in the gastrointestinal tracts. Foodborne BoNT penetrates the intestinal barrier, presumably due to transcytosis. It is then transported to neuromuscular junctions within the bloodstream and blocks the secretion of the neurotransmitter acetylcholine. This results in muscle limpness and palsy caused by selective hydrolysis of soluble A-ethylmalemide-sensitive factor activating (SNARE) proteins which participate in fusion of synaptic vesicles with presynaptic plasma membrane. SNARE proteins include vesicle-associated membrane protein (VAMP), synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP-25). Their degradation is responsible for neuromuscular palsy due to blocks in acetylcholine transmission from synaptic terminals. In humans, palsy caused by BoNT/A lasts four to six months. 

Schiavo, G., Malizio, C., Trimble, W.S., Polverino de Laureto, P., Milan, G., Sugiyama, H., Johnson, E.A. and Montecucco, C., Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala-Ala peptide bond, J. Biol. Chem., 269, 20213-20226, 1994. 

Schiavo, G., Shone, C.C., Rossetto, O., Alexander, F.C. and Montecucco, C., Botulinum neurotoxin serotype 1 is a zinc endopeptidase specific for VAMP/ synaptobrevin, J. Biol. Chern., 268, 11516-11519, 1993. 

The sequence of events that result in neurotransmission of information from one nerve cell to another across the s)mapses begins with a wave of depolarization which passes down the axon and results in the opening of the voltage-sensitive calcium charmels in the axonal terminal. These charmels are frequently concentrated in areas which correspond to the active sites of neurotransmitter release. A large (up to 100 M) but brief rise in the calcium concentration within the nerve terminal triggers the movement of the synaptic vesicles, which contain the neurotransmitter, towards the synaptic membrane. By means of specific membrane-bound proteins (such as synaptobrevin from the neuronal membrane and synaptotagrin from the vesicular membrane) the vesicles fuse with the neuronal membrane and release their contents into the synaptic gap by a process of exocytosis. Once released of their contents, the vesicle membrane is reformed and recycled within the neuronal terminal. This process is completed once the vesicles have accumulated more neurotransmitter by means of an energy-dependent transporter on the vesicle membrane (Table 2.3). 

The decisive element in exocytosis is the interaction between proteins known as SNAREs that are located on the vesicular membrane (v-SNAREs) and on the plasma membrane (t-SNAREs). In the resting state (1), the v-SNARE synaptobrevin is blocked by the vesicular protein synaptotagmin. When an action potential reaches the presynaptic membrane, voltage-gated Ca "" channels open (see p. 348). Ca "" flows in and triggers the machinery by conformational changes in proteins. Contact takes place between synaptobrevin and the t-SNARE synaptotaxin (2). Additional proteins known as SNAPs bind to the SNARE complex and allow fusion between the vesicle and the plasma membrane (3). The process is supported by the hydrolysis of GTP by the auxiliary protein Rab. 

Synaptobrevins (VAMPs)b Synaptophysins, synaptogyrin SV2 A, B, C SCAMPS 1 and 4 SVOP... 

Synaptic exocytosis involves three SNARE proteins the R-SNARE synaptobrevin/VAMP (isoforms 1 and 2) on the vesicle, and the Q-SNAREs syntaxin (isoforms 1 and 2) and SNAP-25 on the plasma membrane (Figure 4). Since SNAP-25 has two SNARE-motifs, synaptobrevin, syntaxin, and SNAP-25 together have four SNARE-motifs. Synaptobrevins and SNAP-25 are relatively simple SNARE proteins that are composed of little else besides SNARE motifs and membrane-attachment sequences (a transmembrane region for synaptobrevin, and a cysteine-rich palmitoylated sequence for SNAP-25). Syntaxins, in contrast, are complex proteins. The N-terminal two-thirds of syntaxins include a separate, autonomously folded domain (the so-called Habc-domain), while the C-terminal third is composed of a SNARE motif and transmembrane region just like synaptobrevin. 

Dissociated neuronal cultures provide a versatile system for analysis of mechanisms underlying neurotransmitter release. These cultures can be prepared from fetal or postnatal brain tissue. This preparation has been particularly instrumental in analysis of synapses deficient in key components of the release machinery. For instance, genetic deletion of synaptic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins such as synaptobrevin-2 and SNAP-25... 

The experiments using these optical tools consistently supported the premise that synaptic vesicle exocytosis and endocytosis are tightly coupled processes. This largely kinetic coupling is also backed up by recent molecular evidence that proteins critical for exocytosis such as synaptotagmin and synaptobrevin, are also essential for triggering endocytosis (Poskanzer et al., 2003 Deak et al., 2004 Nicholson-Tomishima and Ryan, 2004). 

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