SNARE proteins syntaxin

In another application, STED microscopy also revealed the ring-like structure of the protein hruchpilot at synaptic active zones in the drosophila neuromuscular junction [90], Further studies included the visualization of the spatial distribution of the SNARE protein syntaxin [91], the nuclear protein SC35 [85], and the nicotinic acetylcholine receptor [92]. IsoSTED microscopy resolved the tube-like 3D-distribution of the TOM20 protein complex in the mitochondria in a mammalian cell [87]. 

A second mechanism that impinges on the localization of transporters is through the association with proteins, the most prominent example being syntaxin. Syntaxin is a t-SNARE protein necessary for the fusion of vesicles with the plasma membrane (see the chapter on exocytosis). On the cell surface syntaxin consistently stabilizes the localization of GABA, noradrenaline, glycine, and 5HT transporters the PKCa isoform can sever the interaction with syntaxin suggesting a general mechanism for transporter internalization. 

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. 

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. 

Fig. 1 Structure of the neuronal SNAREs. Upper panel domain structure of the three neuronal SNARE proteins involved in synaptic vesicle fusion. Syntaxin 1A and SNAP-25 (contains two SNARE motifs) are associated with the presynaptic membrane, whereas synaptobrevin 2 is synaptic vesicle associated. The SNARE motifs form a stable complex (core complex) whose crystal structure has been analyzed (lower panel). In the complex, each of the SNARE motifs adopts an alpha-helical structure, and the four alpha-helices are aligned in parallel forming a twisted bundle (modified from Sutton et al. 1998). Stability of the complex is mediated by layers of interaction (—7 to +8) in which amino acids from each of the four alpha-helices participate (see text).

Fig. 5 GPCR regulation of exocytosis downstream of Ca2+-entry. (a) Sequence of steps leading from recruitment to maturation of synaptic vesicles from a reserve pool (RP) to a readily-releasable pool (RRP) displaying slow (asynchronous) and fast (synchronous highly Ca2+-sensitive pool, HCSP synaptotagmin 1 (SYT 1) supported) components, (b) Protein-protein interactions of SNARES (SYX, syntaxin SYB, synaptobrevin and SNAP-2s-7S complex) and major putative regulatory proteins. Phosphoproteins are shown in shaded boxes (phosphorylation sites for PKA and PKC are indicated where known) with phosphorylation-dependent interactions depicted by arrows (increase indicated by filled arrows decrease indicated by open arrows). Circle-end connectors indicate a phosphorylation-independent or as yet unspecified interaction. Potential effects of interactions at various points of the sequence in A are discussed in the text.

Blackmer T, Larsen EC, Bartleson C et al (2005) G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nat Neurosci 8 421-5 Boczan J, Leenders AG, Sheng ZH (2004) Phosphorylation of syntaphilin by cAMP-dependent protein kinase modulates its interaction with syntaxin-1 and annuls its inhibitory effect on vesicle exocytosis. J Biol Chem 279 18911-19... 

Glutamate synapses are specialized for rapid vesicle fusion/neurotransmitter release. Fusion is initiated by SNARE proteins such as syntaxin-1, synaptobrevin-2, and SNAP-25 (see Chapter 2.4 this volume). Presynaptic gene expression, in general, is widely disturbed in schizophrenia (Mimics et al., 2000 Mimics et al., 2001 Hemby et al., 2002), including reduced expression of SNAP-25 RNA(Hemby et al., 2002) and... 

In neurons, the SNARE complex consists of three main proteins the v-SNARE synaptobrevin or VAMP (vesicle-associated membrane protein), and two t-SNAREs, syntaxin and SNAP-25 (synaptosomal associated protein of 25 kD). Synaptobrevins traverse the synaptic vesicle membrane in an asymmetric manner a few amino acids are found inside the vesicle, but most of the molecule lies outside the vesicle, within the cytoplasm. Synaptobrevin makes contact with another protein anchored to the plasma membrane of the presynaptic neuron, syntaxin, which is associated with SNAP-25. Via these interactions, the SNARE proteins play a role in the docking and fusion of synaptic vesicles to the active zone. 

The changes in SNARE protein expression do not appear to be due to the effects of antipsychotic treatment. In rats, haloperidol and chlorpromazine increased SNAP-25 protein levels in the hippocampus (Barr et al., 2006), while in the postmortem brains of individuals with schizophrenia, SNAP-25 levels are lower in the hippocampus (Young et al., 1998 Fatemi et al., 2001 Thompson et al., 2003a). Likewise, no changes in the level of mRNA encoding SNAP-25, syntaxin or synaptobrevin were observed in the prefrontal cortex of rats chronically treated with haloperidol (Nakahara et al., 1998). 

Stimulus-evoked, calcium-dependent release of acetylcholine (ACh) from the cholinergic synapse normally occurs through the formation of a fusion complex between ACh-containing vesicles and the intracellular leaflet of the nerve terminal membrane (Amon et al., 2001). This synaptic vesicle fusion complex consists of several proteins of the SNARE family, including a 25 kDa synaptosomal associated protein (SNAP-25), vesicle-associated membrane protein (VAMP, or synaptobrevin), and the synaptic membrane protein syntaxin. Other SNARE proteins have been identified as components of membrane transport systems in yeast and mammals but have not been implicated as targets for BoNTs. Meanwhile, type A and E neurotoxins cleave SNAP-25 while types B, D, F, and G act on VAMP and type C1 toxin cleaves both syntaxin and SNAP-25. Neurotoxin-mediated cleavage of any of these substrates disrupts the processes involved in the exocytotic release of ACh and leads to flaccid paralysis of the affected skeletal muscles. 

SNARE proteins assemble into a heterotrimeric SNARE complex (or core complex). The crystal structure of the core complex revealed a bundle of four a-helices, one each contributed by synaptobrevin and syntaxin and two contributed by SNAP-25 (28). The process of SNARE complex assembly proceeds from N-terminal to C-terminal direction in what is commonly referred to as a zippering action, which brings the C-terminal membrane anchors of the transSNAREs together (29). This action is proposed to force the closely apposed membranes together to initiate fusion. 

The other t-SNARE candidate protein, syntaxin, is also expressed in islet cells (Jacobsson ef al., 1994). Treatment of digitonin-... 

Serotypes B, D, E, and G cleave different sites on the synaptic vesicle protein, synaptobrevin (VAMP), whereas serotypes A and E cleave the presynaptic membrane-associated protein SNAP-25 (Schiavo et al., 2000 Simpson, 2004). Serotype Cl is unique in that it cleaves two cytoplasmic proteins, syntaxin and SNAP-25 (Wiltiamson et al., 1996). Interaction of these SNAREs on the surface of synaptic vesicles and active zone membranes is required for voltage- and Ca " -dependent release of neurotransmitter cleavage by BoNT inhibits this process, leading to muscle weakness and paralysis (Sutton et al., 1998 Schiavo et al., 2000). Cleavage of SNARE proteins appears to be sufficient to account for all actions of the BoNTs, and the SNARE hypothesis has received near universal acceptance since its introduction in the early 1990s. 

Although metalloprotease inhibitors are not constrained by a brief therapeutic window, recovery from BoNT intoxication may stiU be delayed because of the time required to replace cleaved SNARE proteins with intact ones. Estimates from pulse-chase experiments suggest that a halftime of 1 day would be required to replace SNAP-25, 4—5 days to replace synaptobrevin, and 6 days to replace syntaxin, even if no further cleavage were to occur (Foran et al., 2003). Examination of the fraction of total SNARE proteins cleaved by BoNT reveals that a relatively smaU fraction is cleaved at the neuromuscular junction at the time of total muscle paralysis. From local injections of BoNT/A in vivo, Jurasinski et al. (2001) estimated that paralysis requires cleavage of less than 35% of the total SNAP-25. Meunier et al. (2003) reported that isolated diaphragm muscles exposed to 2 nM BoNT/A have only 6.5% of their SNAP-25 in the cleaved form, and suggested that the SNAP-25 relevant to transmitter release must exist in a smaU specialized pool. It is likely that the critical pool of SNAP-25 is even lower than that found by Meunier et al. (2003), since the... 

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