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Molecular machinery driving vesicle fusion in neuromediator release. The core SNARE complex is formed by four α-helices contributed by synaptobrevin, syntaxin and SNAP-25, synaptotagmin serves as a calcium sensor and regulates intimately the SNARE zipping

SNARE proteins (an acronym derived from "SNAP (Soluble NSF Attachment Protein) REceptor") are a large protein superfamily consisting of more than 60 members in yeast and mammalian cells. The primary role of SNARE proteins is to mediate vesicle fusion, that is, the exocytosis of cellular transport vesicles with the cell membrane or with a target membrane bound compartment (such as a lysosome). The best studied SNAREs are those that mediate docking of synaptic vesicles with the presynaptic membrane in neurons. These SNAREs are the targets of the bacterial neurotoxins responsible for botulism and tetanus.

SNAREs can be divided into two categories: vesicle or v-SNAREs, which are incorporated into the membranes of transport vesicles during budding, and target or t-SNAREs, which are located in the membranes of target compartments. Evidence suggests that t-SNAREs form stable subcomplexes which serve as guides for v-SNARE binding to complete the formation of the SNARE complex . Several SNARE proteins are located on both vesicles and target membranes, therefore, a more recent classification scheme takes into account structural features of SNAREs, dividing them into R-SNAREs and Q-SNAREs. Often, R-SNAREs act as v-SNAREs and Q-SNAREs act as t-SNAREs. R-SNAREs are proteins that contribute an arginine (R) residue in the formation of the zero ionic layer in the assembled core SNARE complex. One particular R-SNARE is synaptobrevin, which is located in the synaptic vesicles. Q-SNAREs are proteins that contribute a glutamine (Q) residue in the formation of the zero ionic layer in the assembled core SNARE complex. Q-SNAREs include syntaxin and SNAP-25. Q-SNAREs are further classified as Qa, Qb, or Qc depending on their location in the four-helix bundle.

SNAREs are small, abundant, tail-anchored proteins which are often post-translationally inserted into membranes via a C-terminal transmembrane domain. Seven of the 38 known SNAREs, including SNAP-25, do not have a transmembrane domain and are instead attached to the membrane via lipid modifications such as palmitoylation . ). Tail-anchored proteins can be inserted into the plasma membrane, endoplasmic reticulum, mitochondria, and peroxisomes among other membranes, though any particular SNARE is targeted to a unique membrane. The targeting of SNAREs is accomplished by altering either the composition of the C-terminal flanking amino acid residues or the length of the transmembrane domain. Replacement of the transmembrane domain with lipid anchors leads to an intermediate stage of membrane fusion where only the two contacting leaflets fuse and not the two distal leaflets of the two membrane bilayer.

Although SNAREs vary considerably in structure and size, all share a segment in their cytosolic domain called a SNARE motif that consists of 60-70 amino acids and contains heptad repeats that have the ability to form coiled-coil structures. V- and t-SNAREs are capable of reversible assembly into tight, four-helix bundles called "trans"-SNARE complexes. In synaptic vesicles, the readily-formed metastable "trans" complexes are composed of three SNAREs: syntaxin 1 and SNAP-25 resident in cell membrane and synaptobrevin (also referred to as vesicle-associated membrane protein or VAMP) anchored in the vesicle membrane.

In neuronal exocytosis, syntaxin and synaptobrevin are anchored in respective membranes by their C-terminal domains, whereas SNAP-25 is tethered to the plasma membrane via several cysteine-linked palmitoyl chains. The core trans-SNARE complex is a four-$\alpha$-helix bundle, where one $\alpha$-helix is contributed by syntaxin 1, one $\alpha$-helix by synaptobrevin and two $\alpha$-helices are contributed by SNAP-25.

The plasma membrane-resident SNAREs have been shown to be present in distinct microdomains or clusters, the integrity of which is essential for the exocytotic competence of the cell.

SNARE Protein Complexes Mediate Membrane Fusion

Layering of the core SNARE complex. In the center is the zero hydrophilic ionic layer, flanked by hydrophobic leucine-zipper layers.

During membrane fusion, v-SNARE and t-SNARE proteins on separate membranes combine to form a trans-SNARE complex, also known as a “SNAREpin”. Depending on the stage of fusion of the membranes, these complexes may be referred to differently.

During fusion of trans-SNARE complexes, the membranes merge and SNARE proteins involved in complex formation after fusion are then referred to as a "cis"-SNARE complex, because they now reside in a single (or cis) resultant membrane. After fusion, the cis-SNARE complex is bound and disassembled by an adaptor protein, alphaSNAP. Then, the hexameric AAA-ATPase NSF catalyzes the ATP-dependent unfolding of the SNARE proteins and releases them into the cytosol for recycling.

SNAREs are thought to be the core required components of the fusion machinery and can function independently of additional cytosolic accessory proteins. This was demonstrated by engineering “flipped” SNAREs, where the SNARE domains face the extracellular space rather than the cytosol. When cells containing v-SNAREs contact cells containing t-SNAREs, trans-SNARE complexes form and cell-cell fusion ensues.

Formation of SNARE Complexes

The core SNARE complex is a 4-$\alpha$-helix bundle. Synaptobrevin and syntaxin contribute one $\alpha$-helix each, while SNAP-25 participates with two $\alpha$-helices (abbreviated as Sn1 and Sn2). The interacting amino acid residues that zip the SNARE complex can be grouped into layers. Each layer has 4 amino acid residues - one residue per each of the 4 $\alpha$-helices. In the center of the complex is the zero ionic layer composed of one arginine (R) and three glutamine (Q) residues, and it is flanked by leucine zippering. Layers '-1', '+1' and '+2' at the centre of the complex most closely follow ideal leucine-zipper geometry and aminoacid composition.

The zero ionic layer is composed of R56 from VAMP-2, Q226 from syntaxin-1A, Q53 from Sn1 and Q174 from Sn2, and is completely buried within the leucine-zipper layers. The positively charged guanidino group of the arginine (R) residue interact with the carboxyl groups of each of the three glutamine (Q) residues.

The flanking leucine-zipper layers act as a water-tight seal to shield the ionic interactions from the surrounding solvent. Exposure of the zero ionic layer to the water solvent by breaking the flanking leucine zipper leads to instability of the SNARE complex and is the putative mechanism by which $\alpha$-SNAP and NSF recycle the SNARE complexes after the completion of synaptic vesicle exocytosis.

Proposed mechanism of membrane fusion

Assembly of the SNAREs into the "trans" complexes likely bridges the opposing lipid bilayers of membranes belonging to cell and secretory granule, bringing them in proximity and inducing their fusion. The influx of calcium into the cell triggers the completion of the assembly reaction, which is mediated by an interaction between the putative calcium sensor, synaptotagmin, with membrane lipids and/or the partially assembled SNARE complex.

According to the "zipper" hypothesis, the complex assembly starts at the N-terminal parts of SNARE motifs and proceeds towards the C-termini that anchor interacting proteins in membranes. Formation of the "trans"-SNARE complex proceeds through an intermediate complex composed of SNAP-25 and syntaxin-1, which later accommodates synaptobrevin-2 (the quoted syntaxin and synaptobrevin isotypes participate in neuronal neuromediator release).

Based on the stability of the resultant cis-SNARE complex, it has been postulated that energy released during the assembly process serves as a means for overcoming the repulsive forces between the membranes. There are several models that propose explanation of a subsequent step – the formation of stalk and fusion pore, but the exact nature of these processes remains debated. A recent in vitro single-molecule content-mixing study showed that yeast SNARE complex is enough to expand fusion pores.

According to the "clamp" hypothesis, a reversible clamping protein (known as complexin), inhibits synaptic vesicle fusion. When calcium binds to the calcium sensor synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators like synaptotagmin and complexin, act as markers on the cell membrane. Cells expressing such “flipped” synaptic SNARE switches fuse constitutionally. However, when the expression of complexin blocked fusion alone. When calcium was added back, the cell began to exhibit fusion. This suggests that synaptotagmin and complexin may be co-regulators in synaptic vesicle fusion.

However, recent evidence including detailed structural and functional studies have proposed that SNAREs mostly function in accord with the "zipper" model. Nevertheless, it remains unclear whether SNARE assembly between membranes directly leads to the merger of lipid bilayers.

Toxins

Many neurotoxins directly affect SNARE complexes. Such toxins as the botulinum and tetanus toxins work by targeting the SNARE components. These toxins prevent proper vesicle recycling and result in poor muscle control, spasms, paralysis, and even death.

Specifically, the botulinum toxin attacks the SNAP-25 protein of the SNARE complex. The botulinum toxin degrades and cleaves the SNAP-25 protein, a protein that is required for vesicle fusion that releases neurotransmitters. Botulinum toxin essentially cleaves these SNARE proteins, and in doing so, prevents synaptic vesicles from fusing with the cellular synaptic membrane and releasing their neurotransmitters. The tetanus toxin follows a similar pathway, but instead attacks the protein synaptobrevin on the synaptic vesicle.

These toxins result in acquiring tetanus, a medical condition characterized by a prolonged contraction of skeletal muscle fibers, and botulism, a type of food poisoning that can lead to muscle paralysis including breathing muscles, causing respiratory failure.

Botulinum Neurotoxin (BoNT)

Botulinum Toxin (BoNT) is one of the most potent toxins to have ever been discovered. It is a proteolytic enzyme that cleaves SNARE proteins in neurons. It's structure is composed of two peptide subunits, a Heavy Chain (100kDas) and a Light Chain (50kDas), which are held together by a disulfide bond. The action of BoNT follows a 4-step mechanism including binding to the neuronal membrane, endocytosis, membrane translocation, and proteolytic cleavage of SNARE proteins.

Target SNARE proteins of Botulinum Neurotoxin (BoNT) and Tetanus Neurotoxin (TeNT) inside the axon terminal.

In its mechanism of action, the Heavy Chain of BoNT is first used to find its neuronal targets and bind to the gangliosides and membrane proteins of presynaptic neurons. Next, the toxin is then endocytosed into the cell membrane. The Heavy Chain undergoes a conformational change important for translocating the Light Chain into the cytosol of the neuron. Finally, after the Light Chain of BoNT is brought into the cytosol of the targeted neuron, it is released from the Heavy Chain so that it can reach its active cleavage sites on the SNARE proteins. The Light Chain is released from the Heavy Chain by the reduction of the disulfide bond holding the two together. The reduction of this disulfide bond is mediated by the NADPH-thioredoxin reductase-thioredoxin system. The Light Chain of BoNT acts as a metalloprotease on SNARE proteins that is dependent on Zn(II) ions, cleaving them and eliminating their function in exocytosis.

There are 8 known isotypes of BoNT, named BoNT/A - BoNT/H, with different specific cleavage sites on SNARE proteins. SNAP25 is a member of the SNARE protein family located in the membrane of cells. BoNT isotypes A, C, and E target SNAP-25 proteins in neuronal membranes and cleave them. BoNT/C also targets Syntaxin-1, another SNARE protein located in the membrane of cells, and degenerates these proteins with a similar outcome as with SNAP-25. A third SNARE protein, Synaptobrevin (VAMP), is located on cell vesicles. VAMP2 is targeted and cleaved by BoNT isotypes B, D, F in synaptic neurons.

In each case, Botuliunum Neurotoxins cause functional damage to SNARE proteins. By doing so, this prevents synaptic vesicles from fusing with the cellular synaptic membrane and releasing their neurotransmitters into the synaptic cleft. This results in acquiring botulism, which can lead to muscle paralysis including breathing muscles, causing respiratory failure.

Tetanus Neurotoxin (TeNT)

The breakdown of responsibilities and mechanisms of the heavy (HC) and light chain (LC) of tetanus neurotoxin: The HC assists in binding of TeNT to both the ganglioside receptor and the final receptor. Once TeNT is in the vesicle in the inhibitory interneuron space the HC assists in translocation of the LC into the cytoplasm. Then the LC, characterized by zinc endopeptidase activity, inhibits neurotransmission by cleavage of synaptobrevin 1.

Tetanus toxin, or TeNT, is composed of a heavy chain (100KDa) and a light chain (50kDa) connected by a disulfide bond. The heavy chain is responsible for neurospecific binding of TeNT to the nerve terminal membrane, endocytosis of the toxin, and translocation of the light chain into the cytosol. The light chain has zinc-dependent endopepdtidase or more specifically matrix metalloproteinase (MMP) activity through which cleaveage of synaptobrevin or VAMP is carried out.

For the light chain of TeNT to be activated one atom of zinc must be bound to every molecule of toxin. When zinc is bound reduction of the disulfide bond will be carried out primarily via the NADPH-thioredoxin reductase-thioredoxin redox system. Then the light chain is free to cleave the Gln76-Phe77 bond of synaptobrevin. Cleavage of synaptobrevin affects the stability of the SNARE core by restricting it from entering the low energy conformation which is the target for NSF binding. This cleavage of synaptobrevin is the final target of TeNT and even in low doses the neurotoxin will inhibit neurotransmitter exocytosis.

External links

• SNARE+Proteins at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
• SNARE+Complex at the U.S. National Library of Medicine Medical Subject Headings (MeSH)

Category:Transmembrane proteins Category:Protein families Category:Neurophysiology

• Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses.