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Regulation via SNAP-25 palmitoylation
The Q-SNARE protein Synaptosomal-associated protein 25 (SNAP-25) is composed of two α-helical domains connected by a random coil linker. The random coil linker region is most notable for its four cysteine residues. The α-helical domains combine with those of both syntaxin and synaptobrevin (also known as vesicle associated membrane protein or VAMP) to form the 4-α-helix coiled-coil SNARE complex critical to efficient exocytosis.

While syntaxin and synaptobrevin both contain transmembrane domains which allow for docking with target and vesicle membranes respectively, SNAP-25 relies on the palmitoylation of cysteine residues found in its random coil region for docking to the target membrane. Some studies have suggested that association with syntaxin via SNARE interactions precludes the need for such docking mechanisms. Syntaxin knockdown studies however, failed to show a decrease in membrane bound SNAP-25 suggesting alternate docking means exist. The covalent bonding of fatty acid chains to SNAP-25 via thioester linkages with one or more cysteine residues therefore, provides for regulation of docking and ultimately SNARE mediated exocytosis. This process is mediated by a specialized enzyme called DHHC palmitoyl transferase. The cysteine rich domain of SNAP-25 has also been shown to weakly associate with the plasma membrane possibly allowing it to be localized near the enzyme for subsequent palmitoylation. The reverse of this process is carried out by another enzyme called palmitoyl protein thioesterase (see figure).



The availability of SNAP-25 in the SNARE complex is also theorized to possibly be spatially regulated via localization of lipid microdomains in the target membrane. Palmitoylated cysteine residues could be localized to the desired target membrane region via a favorable lipid environment (possibly cholesterol rich) complimentary to the fatty acid chains bonded to the cysteine residues of SNAP-25.

SNAP-25 regulation of voltage-gated Ca2+ channels in neuronal axon terminals
As an action potential reaches the axon terminal, depolarization events stimulate the opening of voltage-gated calcium channels (VGCCs) allowing the rapid influx of calcium down its electrochemical gradient. Calcium goes on to stimulate exocytosis via binding with synaptotagmin 1. SNAP-25 however, has been shown to negatively regulate VGCC function in glutamatergic neuronal cells<ous]] SNAP-25 leads to a reduction of current density through VGCC's and therefore a decrease in the amount of calcium that is binding the synaptotagmin, causing a decrease in neuronal glutamatergic exocytosis. Conversely, underexpression of SNAP-25 allows for an increase in VGCC current density and increase in exocytosis. Further investigation has suggested possible relationships between SNAP-25 over/underexpression and a variety of brain diseases. In Attention-Deficit/Hyperactivity Disorder or ADHD, polymorphisms at the SNAP-25 gene locus in humans have been linked to the disease suggesting a potential role in its manifestation. This is further suggested by heterogeneous SNAP-25 knockout studies performed on coloboma mutant mice, which led to phenotypic characteristics of ADHD. Studies have also shown a correlation of SNAP-25 over/underexpression and the onset of schizophrenia.

Syntaxin and the Habc Domain
Syntaxin consists of a transmembrane domain (TMD), alpha-helical SNARE domain, a short linker region, and the Habc domain which consists of three alpha-helical regions. The SNARE domain in syntaxin serves as a target site for docking of SNAP-25 and synaptobrevin in order to form the four helix bundle requisite to the SNARE complex and subsequent fusion. The Habc domain, however, serves as an autoinhibitory domain in syntaxin. It has been shown to fold over and associate with the SNARE domain of syntaxin inducing a “closed” state, creating a physical barrier to the formation of the SNARE motif. Conversely, the Habc domain can again disassociate with the SNARE domain leaving syntaxin free to associate with both SNAP-25 and synaptobrevin.

Syntaxin 1B and readily releasable pool of vesicles
There is an immense diversity of syntaxin subtypes, with 15 varieties in the human genome. It has been suggested that syntaxin1B has a role in regulating number of synaptic vesicles ready for exocytosis in the axon terminal. This is also called the readily releasable pool (RRP) of vesicles. A knock out study in 2014 showed that the lack of syntaxin1B led to a significant decrease in RRP size.