Inhibition of neurotransmitter release by peptides that mimic the N-terminal domain of SNAP-25

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal–truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.     

How botulinum and tetanus neurotoxins block neurotransmitter release

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.     

Specificity of botulinum protease for human VAMP family proteins

The botulinum neurotoxin light chain (BoNT-LC) is a zinc-dependent metalloprotease that cleaves neuronal SNARE proteins such as SNAP-25, VAMP2, and Syntaxin1. This cleavage interferes with the neurotransmitter release of peripheral neurons and results in flaccid paralysis. SNAP, VAMP, and Syntaxin are representative of large families of proteins that mediate most membrane fusion reactions, as well as both neuronal and non-neuronal exocytotic events in eukaryotic cells. Neuron-specific SNARE proteins, which are target substrates of BoNT, have been well studied; however, it is unclear whether other SNARE proteins are also proteolyzed by BoNT. Herein, we define the substrate specificity of BoNT-LC/B, /D, and /F towards recombinant human VAMP family proteins. We demonstrate that LC/B, /D, and /F are able to cleave VAMP1, 2, and 3, but no other VAMP family proteins. Kinetic analysis revealed that all LC have higher affinity and catalytic activity for the non-neuronal SNARE isoform VAMP3 than for the neuronal VAMP1 and 2 isoforms. LC/D in particular exhibited extremely low catalytic activity towards VAMP1 relative to other interactions, which we determined through point mutation analysis to be a result of the Ile present at residue 48 of VAMP1. We also identified the VAMP3 cleavage sites to be at the Gln 59-Phe 60 (LC/B), Lys 42-Leu 43 (LC/D), and Gln 41-Lys 42 (LC/F) peptide bonds, which correspond to those of VAMP1 or 2. Understanding the substrate specificity and kinetic characteristics of BoNT towards human SNARE proteins may aid in the development of novel therapeutic uses for BoNT.     

神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

A search for synthetic peptides that inhibit soluble N-ethylmaleimide sensitive-factor attachment receptor-mediated membrane fusion

Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.     

Human synaptotagmin-II is not a high affinity receptor for botulinum neurotoxin B and G: increased therapeutic dosage and immunogenicity

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins essential for exocytosis. The synaptic vesicle protein synaptotagmin-II of rat and mouse acts as neuronal high affinity receptor for BoNT/B and BoNT/G. Here, we show that human synaptotagmin-II is not a high affinity receptor for BoNT/B and G due to a phenylalanine to leucine mutation in its luminal domain present only in humans and chimpanzees. It eliminates one of three major interactions between synaptotagmin-II and BoNT/B and hereby explains the disparity in potency of BoNT/B in humans and mice as well as the 40-fold higher dosage of rimabotulinumtoxinB versus onabotulinumtoxinA.     

SNARE complex regulation by phosphorylation

SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors) are ubiquitous proteins that direct vesicular trafficking and exocytosis. In neurons, SNAREs act to mediate release of neurotransmitters, which is a carefully regulated process. Calcium influx has long been shown to be the key trigger of release. However, calcium alone cannot regulate the degree of vesicle content release. For example, only a limited number of docked vesicles releases neurotransmitters when calcium entry occurs; this suggests that exocytosis is regulated by other factors besides calcium influx. Regulation of the degree of release is best explained by looking at the many enzymatic proteins that interact with the SNARE complex. These proteins have been hypothesized to regulate the formation, stability, or disassembly of the SNARE complex and therefore may regulate neurotransmitter release. One group of enzymatic regulators is the protein kinases. These proteins phosphorylate sites on both SNARE proteins and proteins that interact with SNARE proteins. Recent research has identified some of the specific effects that phosphorylation (or dephosphorylation) at these sites can produce. Additionally, palmitoylation of SNAP-25, regulates the localization, and hence activity of this key SNARE protein. This review focuses on the location and effects of phosphorylation on SNARE regulation.     

SNARE Zippering and Synaptic Strength

Synapses vary widely in the probability of neurotransmitter release. We tested the hypothesis that the zippered state of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex determines initial release probability. We tested this hypothesis at phasic and tonic synapses which differ by 100-1000-fold in neurotransmitter release probability. We injected, presynaptically, three Clostridial neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by the zippering of the SNARE complex or open to proteolytic attack. Under low stimulation conditions, the catalytic light-chain fragment of botulinum B (BoNT/B-LC) inhibited evoked release at both phasic and tonic synapses and cleaved VAMP; however, neither BoNT/D-LC nor tetanus neurotoxin (TeNT-LC) were effective in these conditions. The susceptibility of VAMP to only BoNT/B-LC indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end to approximately the zero-layer with the C-terminal end exposed under resting state. Therefore, the existence of the same partially zippered state of the trans-SNARE complex at both phasic and tonic synapses indicates that release probability is not determined solely by the zippered state of the trans-SNARE complex at least to the zero-layer.     

Association of botulinum neurotoxin serotype A light chain with plasma membrane-bound SNAP-25

The Clostridium botulinum neurotoxins (BoNTs) cleave SNARE proteins, which inhibit binding and thus fusion of neurotransmitter vesicles to the plasma membrane of peripheral neurons. BoNTs comprise an N-terminal light chain (LC) and C-terminal heavy chain, which are linked by a disulfide bond. There are seven serotypes (A-G) of BoNTs based upon immunological neutralization. Although the binding and entry of BoNT/A into neurons has been subjected to considerable investigation, the intracellular events that allow BoNT/A to efficiently cleave SNAP-25 within neurons is less well understood. Earlier studies showed that intracellular LC/A bound to the plasma membrane of neurons. In this study, intracellular LC/A is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of LC/A bound residues 80-110 of SNAP-25, which was also observed in cultured neurons. Association of the N-terminal 8 amino acids of LC/A and residues 80-110 of SNAP-25 also enhanced substrate cleavage. These findings explain how LC/A associates with SNAP-25 on the plasma membrane and provide a basis for LC/A cleavage of SNAP-25 within the SNARE complex.     

Traffic of botulinum toxins A and E in excitatory and inhibitory neurons

Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gamma-aminobutyric acid (GABA)-ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations.     

New insights into clostridial neurotoxin-SNARE interactions

Botulinum neurotoxin serotype A (BoNT/A) has achieved a dichotomous status in modern medicine; it is both a versatile treatment for several neurological disorders and a lethal poison responsible for causing the neuroparalytic syndrome botulism. The extent of paralysis largely depends on the dosage of toxin received. The toxins block neurotransmitter release by delivering their Zn(2+)-dependent protease components to the presynaptic side of chemical synapses. These highly specialized enzymes exclusively hydrolyze peptide bonds within SNARE (soluble N-ethylmaleiamide-sensitive factor attachment protein receptor) proteins. Recently, the structural basis for the highly specific interaction between BoNT/A and its target SNARE, SNAP-25 (synaptosomal-associated protein of 25kDa), was elucidated. New details regarding the nature of the toxin-SNARE interactions could be exploited for novel inhibitor design.     

Regulated exocytosis and SNARE function (Review)

The pairing of cognate v- and t-SNAREs between two opposing lipid bilayers drives spontaneous membrane fusion and confers specificity to intracellular membrane trafficking. These fusion events are regulated by a cascade of protein-protein interactions that locally control SNARE activity and complex assembly, determining when and where fusion occurs with high efficiency in vivo. This basic regulation occurs at all transport steps and is mediated by conserved protein families such as Rab proteins and their effectors and Sec1/unc18 proteins. Regulated exocytosis employs auxiliary components that couple the signal (which triggers exocytosis) to the fusion machinery. At the neuronal synapse, munc13 as well as munc18 control SNARE complex assembly. Synaptotagmin and complexin ensure fast synchronous calcium-evoked neurotransmitter release.     

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.     

Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). To understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 A resolution. The structure of BoNT/G-LC reveals a C-terminal beta-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1' residue. In addition, structural and sequence analyses have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP.     

Mechanism of neurotransmitter release coming into focus

Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca²⁺‐triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca²⁺‐dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N‐ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin‐1, SNAP‐25, and synaptobrevin‐2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N‐ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18‐1 and Munc13‐1 orchestrate SNARE complex formation in an NSF‐SNAP‐resistant manner by a mechanism whereby Munc18‐1 binds to synaptobrevin and to a self‐inhibited “closed” conformation of syntaxin‐1, thus forming a template to assemble the SNARE complex, and Munc13‐1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of syntaxin‐1. Synaptotagmin‐1 functions as the major Ca²⁺ sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.     

Mechanism of substrate recognition by botulinum neurotoxin serotype A

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting neurotransmitter-carrying vesicle fusion to the plasma membrane of peripheral neurons. Unlike other zinc proteases, BoNTs recognize extended regions of SNAP25 for cleavage; however, the molecular basis for this extended substrate recognition is unclear. Here, we define a multistep mechanism for recognition and cleavage of SNAP25 by BoNT/A. SNAP25 initially binds along the belt region of BoNT/A, which aligns the P5 residue to the S5 pocket at the periphery of the active site. Although the exact order of each step of recognition of SNAP25 by BoNT/A at the active site is not clear, the initial binding could subsequently orient the P4'-residue of SNAP25 to form a salt bridge with the S4'-residue, which opens the active site allowing the P1'-residue access to the S1'-pocket. Subsequent hydrophobic interactions between the P3 residue of SNAP25 and the S3 pocket optimize alignment of the scissile bond for cleavage. This explains how the BoNTs recognize and cleave specific coiled SNARE substrates and provides insight into the development of inhibitors to prevent botulism.     

Membrane fusion: structure snared at last

The structure of the core of the neuronal 'SNARE complex', involved in neurotransmitter release, has been determined recently. Its topological similarity to viral fusion proteins suggests how the SNARE complex might facilitate membrane fusion.     

A second SNARE role for exocytic SNAP25 in endosome fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle-plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.     

The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.