N-ethylmaleimide Sensitive Factor Attachment Protein Research Articles

Developing an Intensity-Based Conformational Sensor for Snares

Vesicle trafficking, a process essential to organelle biogenesis and secretion, requires the fusion of membranes. Most cellular membrane fusion occurs via the machinery of soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) proteins, whose zippering catalyze the fusion of two opposing membranes. After the fusion event, the zippered (cis-)SNAREs are then disassembled by NSF in a step requiring ATP hydrolysis. To gain insights into the regulatory mechanism of SNARE assembly and disassembly, we engineered an intensity-based conformational sensor for SNAREs (icsenSNARE) by linking the structural transition of the SNARE complex to a fluorescence signal of cpGFP.

Role of Munc18-1 in the biological functions and pathogenesis of neurological disorders (Review).

The release of neurotransmitters following the fusion of synaptic vesicles and the presynaptic membrane is an important process in the transmission of neuronal information. Syntaxin-binding protein 1 (Munc18-1) is a synaptic fusion protein binding protein, which mainly regulates synaptic vesicle fusion and neurotransmitter release by interacting with soluble N-ethylmaleimide sensitive factor attachment protein receptor. In addition to affecting neurotransmitter transmission, Munc18-1 is also involved in regulating neurosynaptic plasticity, neurodevelopment and neuroendocrine cell release functions (including thyroxine and insulin release). A number of previous studies have demonstrated that Munc18-1 has diverse and vital biological functions, and that its abnormal expression serves an important role in the pathogenesis of a variety of neurological diseases, including epileptic encephalopathy, schizophrenia, autism, Parkinsons disease, Alzheimers disease, multiple sclerosis, Duchennes muscular dystrophy and neuronal ceroid lipofuscinosis. The present review summarizes the function of Munc18-1 and its possible relationship to the pathogenesis of various neurological diseases.

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

The Arabidopsis R-SNARE protein YKT61 is essential for gametophyte development.

Gametophyte development is a pre-requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss-of-function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three-base-pair deletion in YKT61 in the ykt61-3 mutant resulted in a single-amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N-terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9-mediated editing and expression from a Cas9-resistant transgene.

Requirements of Qa-SNARE LjSYP132s for Nodulation and Seed Development in Lotus japonicus

SNAREs (soluble N-ethyl maleimide-sensitive factor attachment protein receptors) mediate membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s are the members of Qa-SNAREs in Lotus japonicus. Two isoforms, LjSYP132a and LjSYP132b, are generated by alternative splicing. Immunoblot analysis detected strong expression of LjSYP132s in infected root nodules and seeds by posttranscriptional modification. In either LjSYP132a or LjSYP132b silenced roots (RNAi-LjSYP132a, RNAi-LjSYP132b), the infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR showing that both genes were expressed at the early stage of infection. However, during the nodulation, only LjSYP132a was induced. LjSYP132s protein was observed in the Mesorhizobium loti-inoculated roots of mutants, nfr1, castor and pollux, suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the peribacteroid membrane suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b, R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting that LjSYP132b has a role in pollen tube growth and nutrient transport in the plasma membrane of seeds.

Dynamic Capture and Release of Endoplasmic Reticulum Exit Sites by Golgi Stacks in Arabidopsis.

SummaryProtein transport from the endoplasmic reticulum (ER) to Golgi stacks is mediated by the coat protein complex COPII, which is assembled at an ER subdomain called ER exit site (ERES). However, the dynamic relationship between ERESs and Golgi stacks is unknown. Here, we propose a dynamic capture-and-release model of ERESs by Golgi stacks in Arabidopsis thaliana. Using variable-angle epifluorescence microscopy with high-temporal-resolution imaging, COPII-component-bound ERESs were detected as punctate structures with sizes of 300–500 nm. Some punctate ERESs are distributed on ER tubules and sheet rims, whereas others gather around a Golgi stack in an ER-network cavity to form a beaded-ring structure. Free ERESs that wander into an ER cavity are captured by a Golgi stack in a cytoskeleton-independent manner. Then, they are released by the Golgi stack for recycling. The dynamic ERES cycling might contribute to efficient transfer of de novo synthesized cargo proteins from the ER to Golgi stacks.

GPA5 Encodes a Rab5a Effector Required for Post-Golgi Trafficking of Rice Storage Proteins.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

SUMMARYIntracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

Role of SNAREs in Atopic Dermatitis–Related Cytokine Secretion and Skin-Nerve Communication

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in atopic dermatitis (AD) is unknown. This study identifies the function of soluble N-ethylmaleimide sensitive factor attachment protein receptor in AD-related cytokine secretion and epidermis-nerve communication. Herein, we report that various cytokines were simultaneously upregulated and coreleased in innate immunity-activated primary human keratinocytes. AD-related cytokines thymic stromal lymphopoietin, endothelin-1, and inflammatory tumor necrosis factor-α activated distinct but overlapping sensory neurons. Tumor necrosis factor-α potentiated thymic stromal lymphopoietin-induced Ca2+-influx, whereas endothelin-1 caused itch-selective B-type natriuretic peptide release. In primary human keratinocytes, B-type natriuretic peptide upregulated genes promoting dermatological and neuroinflammatory diseases and conditions. VAMP3, SNAP-29, and syntaxin 4 proved important in driving cytokine release from primary human keratinocytes. Depletion of VAMP3 inhibited nearly all the cytokine release including thymic stromal lymphopoietin and endothelin-1. Accordingly, VAMP3 co-occurred with endothelin-1 in the skins of patients with AD. Our study pinpoints the pivotal role of soluble N-ethylmaleimide sensitive factor attachment protein receptors in mediating cytokine secretion related to AD. VAMP3 is identified as a suitable target for developing broad-spectrum anticytokine therapeutics for controlling itch and atopic skin inflammation.

Retrograde trafficking and quality control of yeast synaptobrevin, Snc1, are conferred by its transmembrane domain.

Synaptobrevin/vesicle-associated membrane protein 2 (VAMP2) is an essential soluble N-ethyl maleimide–sensitive factor attachment protein receptor (SNARE) protein that has been extensively studied in its role in synaptic vesicle fusion. However, sorting and trafficking of VAMP2 within the endosomal system is not well understood. Here, we use the yeast VAMP2 homologue Snc1 to investigate the pathways and signals required for endocytic trafficking. We identify two genetically distinct retrieval pathways from the endosomal system: a plasma membrane recycling pathway that requires the Rcy1 F-box protein and a retrograde pathway originating from the multivesicular/prevacuole endosome dependent on the Snx4-Atg20 sorting nexin complex. Lysine residues within the transmembrane domain of Snc1 are necessary for presentation of a Snx4-Atg20–dependent sorting signal located within its juxtamembrane region. Mutations of the transmembrane lysine residues ablate retrograde sorting and subject Snc1 to quality control via sorting into the degradative multivesicular endosome pathway. Degradative sorting requires lysine residues in the juxtamembrane region of Snc1 and is mediated by the Rsp5 ubiquitin ligase and its transmembrane adapters, Ear1 and Ssh4, which localize to endosome and vacuole membranes. This study shows that Snc1 is trafficked between the endosomal system and the Golgi apparatus via multiple pathways and provides evidence for protein quality control surveillance of a SNARE protein in the endo-vacuolar system.

SNARE proteins SYP22 and VAMP727 negatively regulate plant defense

ABSTRACTSoluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are the key regulators control trafficking of cargo proteins to their final destinations and plays key role in plant development; however, their roles in plant defense remain largely unknown. R-SNARE VAMP727 and Qa-SNARE SYP22 were previously reported to associate with vacuolar protein deposition and brassinosteroids (BRs) receptor BRI1 plasma membrane targeting. Here, we identified that VAMP727 and SYP22 are induced by infection of root-knot nematode (RKN), a plant pathogen, which cause severe growth defect and yield loss. Furthermore, decreased root-knot nematode (RKN) invasion, growth and disease index were observed in bri1-5 and SYP22ND, a SYP22 negative dominant mutants when compared to control plants. Overall, our results suggest that VAMP727-SYP22 SNARE complexes regulate plant defense might be via control of abundances of BRI1 on the plasma membrane.

Molecular interplay of autophagy and endocytosis in human health and diseases.

Autophagy, an evolutionarily conserved process for maintaining the physio-metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras-like GTPases (Rabs), soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal-associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane-bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy-endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ–activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane–localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane–localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

Structural Characterization of Full-Length Synaptotagmin-1 to Cis or Trans Membranes

Neurotransmission involves Ca2+ dependent fusion of synaptic vesicles (SV) to the presynaptic plasma membrane (PM), thereby releasing neurotransmitter into the synaptic cleft. Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARES) drive membrane fusion, but SNAREs are not calcium sensitive. The Ca2+ sensor, Synaptotagmin-1 (Syt1), thus couples neuron depolarization to synchronous neurotransmitter release. Syt1 is a vesicular-tethered protein, with two homologous C2A and C2B domains attached through a juxtamembrane linker. Upon Ca2+ influx, loops on the two domains bind Ca2+ and insert into charged membrane. The C2B domain contains other regions capable of membrane interaction, including the lysine rich polybasic face. Syt1 can interact with either bilayer surface, binding cis- to the SV membrane, or trans- to the PM. Cis-binding likely plays an inhibitory role by back-binding both domains to the SV preventing membrane fusion. Trans-binding then permits fusion, docking the SV to the PM surface. These membrane interactions are lipid specific in order to drive membrane bridging. On the synaptic vesicle surface, only phosphatidylserine (PS) contributes negative charge, while on the PM both PS and phosphatidylinositol-4,5-bisphosphate (PIP2) contribute to the charge density. Through various EPR techniques, we explored cis- and trans-binding under various physiologically relevant lipid, salt, and ionic compositions, to determine if Syt1 may act as a distance regulator between the SV and PM. A series of methods were developed to determine and differentiate membrane insertion of the domains in the full-length protein. With this, we characterized an ATP and PIP2 competition to the polybasic face which blocks or promotes trans-binding. We also characterized the juxtamembrane linker and the preferential binding of C2A to PS and C2B's to PIP2 in the presence of SV or PM mimicking membranes with and without Ca2+.

Bradyrhizobium diazoefficiens USDA 110- Glycine max Interactome Provides Candidate Proteins Associated with Symbiosis.

Although the legume-rhizobium symbiosis is a most-important biological process, there is a limited knowledge about the protein interaction network between host and symbiont. Using interolog- and domain-based approaches, we constructed an interspecies protein interactome containing 5115 protein-protein interactions between 2291 Glycine max and 290 Bradyrhizobium diazoefficiens USDA 110 proteins. The interactome was further validated by the expression pattern analysis in nodules, gene ontology term semantic similarity, co-expression analysis, and luciferase complementation image assay. In the G. max-B. diazoefficiens interactome, bacterial proteins are mainly ion channel and transporters of carbohydrates and cations, while G. max proteins are mainly involved in the processes of metabolism, signal transduction, and transport. We also identified the top 10 highly interacting proteins (hubs) for each species. Kyoto Encyclopedia of Genes and Genomes pathway analysis for each hub showed that a pair of 14-3-3 proteins (SGF14g and SGF14k) and 5 heat shock proteins in G. max are possibly involved in symbiosis, and 10 hubs in B. diazoefficiens may be important symbiotic effectors. Subnetwork analysis showed that 18 symbiosis-related soluble N-ethylmaleimide sensitive factor attachment protein receptor proteins may play roles in regulating bacterial ion channels, and SGF14g and SGF14k possibly regulate the rhizobium dicarboxylate transport protein DctA. The predicted interactome provide a valuable basis for understanding the molecular mechanism of nodulation in soybean.

Emerging evidence for the modulation of exocytosis by signalling lipids.

Membrane fusion is a key event in exocytosis of neurotransmitters and hormones stored in intracellular vesicles. In this process, soluble N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are essential components of the exocytotic molecular machinery, while lipids have been seen traditionally as structural elements. However, the so‐called signalling lipids, such as sphingosine and arachidonic acid, interact with SNAREs and directly modulate the frequency and mode of fusion events. Interestingly, recent work has proved that the sphingosine analogue FTY‐720, used in the treatment of multiple sclerosis, mimics the effects of signalling lipids. In the present Review, we discuss recent investigations suggesting that endogenous signalling lipids and synthetic analogues can modulate important physiological aspects of secretion, such as quantal release, vesicle recruitment into active sites, vesicle transport and even organelle fusion in the cytosol. Therefore, these compounds are far from being merely structural components of cellular membranes.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

Molecular Mechanisms of Fast Neurotransmitter Release.

This review summarizes current knowledge of synaptic proteins that are central to synaptic vesicle fusion in presynaptic active zones, including SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), synaptotagmin, complexin, Munc18 (mammalian uncoordinated-18), and Munc13 (mammalian uncoordinated-13), and highlights recent insights in the cooperation of these proteins for neurotransmitter release. Structural and functional studies of the synaptic fusion machinery suggest new molecular models of synaptic vesicle priming and Ca2+-triggered fusion. These studies will be a stepping-stone toward answering the question of how the synaptic vesicle fusion machinery achieves such high speed and sensitivity.

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D–overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ–activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.