Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein.

The COVID-19 pandemic has significantly impacted human health for three years. To mitigate the spread of SARS-CoV-2, the development of neutralizing antibodies has been accelerated, including the exploration of alternative antibody formats such as single-domain antibodies. In this study, we identified variable new antigen receptors (VNARs) specific for the receptor binding domain (RBD) of SARS-CoV-2 by immunizing a banded houndshark (Triakis scyllium) with recombinant wild-type RBD. Notably, the CoV2NAR-1 clone showed high binding affinities in the nanomolar range to various RBDs and demonstrated neutralizing activity against SARS-CoV-2 pseudoviruses. These results highlight the potential of the banded houndshark as an animal model for the development of VNAR-based therapeutics or diagnostics against future pandemics.

Bifunctional hetero di-disc for broad-spectrum influenza neutralization.

Nanodiscs containing sialic acid, which binds the hemagglutinin of the influenza virus, rupture the viral envelope and entrap viral ribonucleoproteins in the endolysosome. While nanodiscs are potent antiviral platforms, ganglioside GD1a containing α2,3-sialic acid does not cover all virus strains. When two nanodiscs containing different receptors 6'-sialyllactose and GD1a were mixed, one nanodisc inhibited the function of the other. A nanodisc loaded with two different receptors exhibited a biased activity toward only one receptor precluding the generation of a multifunctional nanodisc. Here, we suggest hetero di-disc, in which two nanodiscs loaded with each receptor were conjugated through protein trans-splicing for a broad-spectrum antiviral. The hetero di-disc showed strong antiviral activity in vitro and in vivo. Our results suggested that hetero di-discs not only expanded the inhibitory spectrum of nanodiscs but also enabled nanodisc-based delivery of multiple ligands without interference.

TNF-α Secreted from Macrophages Increases the Expression of Prometastatic Integrin αV in Gastric Cancer.

The tumor microenvironment comprising blood vessels, fibroblasts, immune cells, and the extracellular matrix surrounding cancer cells, has recently been targeted for research in cancer therapy. We aimed to investigate the effect of macrophages on the invasive ability of gastric cancer cells, and studied their potential mechanism. In transcriptome analysis, integrin αV was identified as a gene increased in AGS cells cocultured with RAW264.7 cells. AGS cells cocultured with RAW264.7 cells displayed increased adhesion to the extracellular matrix and greater invasiveness compared with AGS cells cultured alone. This increased invasion of AGS cells cocultured with RAW264.7 cells was inhibited by integrin αV knockdown. In addition, the increase in integrin αV expression induced by tumor necrosis factor-α (TNF-α) or by coculture with RAW264.7 cells was inhibited by TNF receptor 1 (TNFR1) knockdown. The increase in integrin αV expression induced by TNF-α was inhibited by both Mitogen-activated protein kinase (MEK) inhibitor and VGLL1 S84 peptide treatment. Finally, transcription of integrin αV was shown to be regulated through the binding of VGLL1 and TEAD4 to the promoter of integrin αV. In conclusion, our study demonstrated that TNFR1-ERK-VGLL1 signaling activated by TNF-α secreted from RAW264.7 cells increased integrin αV expression, thereby increasing the adhesion and invasive ability of gastric cancer cells.

Enhancing acid tolerance of Escherichia coli via viroporin-mediated export of protons and its application for efficient whole-cell biotransformation.

Escherichia coli-based whole-cell biocatalysts are widely used for the sustainable production of value-added chemicals. However, weak acids present as substrates and/or products obstruct the growth and fermentation capability of E. coli. Here, we show that a viroporin consisting of the influenza A matrix-2 (M2) protein, is activated by low pH and has proton channel activity in E. coli. The heterologous expression of the M2 protein in E. coli resulted in a significant increase in the intracellular pH and cell viability in the presence of various weak acids with different lengths of carbon chains. In addition, the feasibility of developing a robust and efficient E. coli-based whole-cell biocatalyst via introduction of the proton-selective viroporin was explored by employing (Z)-11-(heptanolyoxy)undec-9-enoic acid (ester) and 2-fucosyllactose (2'-FL) as model products, whose production is hampered by cytosolic acidification. The engineered E. coli strains containing the proton-selective viroporin exhibited approximately 80% and 230% higher concentrations of the ester and 2'-FL, respectively, than the control strains without the M2 protein. The simple and powerful strategy developed in this study can be applied to produce other valuable chemicals whose production involves substrates and/or products that cause cytosolic acidification.

Coptis chinensis Franch Directly Inhibits Proteolytic Activation of Kallikrein 5 and Cathelicidin Associated with Rosacea in Epidermal Keratinocytes.

Rosacea is a common and chronic inflammatory skin disease that is characterized by dysfunction of the immune and vascular system. The excessive production and activation of kallikerin 5 (KLK5) and cathelicidin have been implicated in the pathogenesis of rosacea. Coptis chinensis Franch (CC) has been used as a medicinal herb in traditional oriental medicine. However, little is known about the efficacy and mechanism of action of CC in rosacea. In this study, we evaluate the effect of CC and its molecular mechanism on rosacea in human epidermal keratinocytes. CC has the capacity to downregulate the expression of KLK5 and cathelicidin, and also inhibits KLK5 protease activity, which leads to reduced processing of inactive cathelicidin into active LL-37. It was determined that CC ameliorates the expression of pro-inflammatory cytokines through the inhibition of LL-37 processing. In addition, it was confirmed that chitin, an exoskeleton of Demodex mites, mediates an immune response through TLR2 activation, and CC inhibits TLR2 expression and downstream signal transduction. Furthermore, CC was shown to inhibit the proliferation of human microvascular endothelial cells induced by LL-37, the cause of erythematous rosacea. These results demonstrate that CC improved rosacea by regulating the immune response and angiogenesis, and revealed its mechanism of action, indicating that CC may be a useful therapeutic agent for rosacea.

Characterization of Ginkgo biloba Leaf Flavonoids as Neuroexocytosis Regulators.

Ginkgo biloba leaf (GBL) is known as a potential source of bioactive flavonoids, such as quercetin, arresting the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-zippering. Here, the GBL flavonoids were isolated in two different manners and then examined for their bioactivity, physicochemical stability, and biocompatibility. The majority of flavonoids in the non-hydrolyzed and acidolyzed isolates, termed non-hydrolyzed isolate (NI) and acidolyzed isolate (AI) hereafter, were rich in flavonol glycosides and aglycones, respectively. Glycosidic/aglyconic quercetin and kaempferol were abundant in both NI and AI, whereas a little of apigenin, luteolin, and isorhamnetin were found in AI. NI was more thermostable in all pH ranges than quercetin, kaempferol, and AI. NI and AI both inhibited neurotransmitter release from differentiated neuronal PC-12 cells. NI and AI showed 1/2-1/3 lower EC50/CC50 values than quercetin and kaempferol. The NI and AI exhibited no toxicity assessed by the tests on chorioallantoic membranes of hen's eggs, removing toxicological concerns of irritation potential. Moreover, GBL isolates, particularly AI, showed antioxidant and anti-inflammatory activities in the use below the CC50 levels. Taken together, these results suggest that GBL isolates that are rich in antioxidant flavonoids are effective anti-neuroexocytotic agents with high stability and low toxicity.

ATP reduction by MgtC and Mg2+ homeostasis by MgtA and MgtB enables Salmonella to accumulate RpoS upon low cytoplasmic Mg2+ stress.

RpoS is one of several alternative sigma factors known to alter gene expression profiles by RpoS-associated RNA polymerase in response to a variety of stresses. The enteric bacteria Salmonella enterica and Escherichia coli accumulate RpoS under low Mg2+ concentrations via a common mechanism in which the PhoP regulator activates expression of antiadaptor proteins that, by sequestering the adaptor RssB, prevent RpoS degradation by the protease ClpXP. Here, we demonstrate that this genetic program alone does not fully support RpoS accumulation when cytoplasmic Mg2+ concentration drops to levels that impair protein synthesis. Under these circumstances, only S. enterica continues RpoS accumulation in a manner dependent on other PhoP-activated programs (i.e. ATP reduction by the MgtC protein and Mg2+ import by the MgtA and MgtB transporters) that maintain translation homeostasis. Moreover, we provide evidence that the mgtC gene, which is present in S. enterica but not in E. coli, is responsible for the differences in RpoS accumulation between these two bacterial species. Our results suggest that bacteria possess a mechanism to control RpoS accumulation responding to cytoplasmic Mg2+ levels, the difference of which causes distinct RpoS accumulation in closely related bacterial species.

Envelope-deforming antiviral peptide derived from influenza virus M2 protein.

Molecules interfering with lipid bilayer function exhibit strong antiviral activity against a broad range of enveloped viruses, with a lower risk of resistance development than that for viral protein-targeting drugs. Amphipathic peptides are rich sources of such membrane-interacting antivirals. Here, we report that influenza viruses were effectively inactivated by M2 AH, an amphipathic peptide derived from the M2 protein of the influenza virus. Although overall hydrophobicity () of M2 AH was not related to antiviral activity, modification of the hydrophobic moment (<µH>) of M2 AH dramatically altered the antiviral activity of this peptide. M2 MH, a derivative of M2 AH with a <µH> of 0.874, showed a half maximal inhibitory concentration (IC50) of 53.3 nM against the A/PR/8/34 strain (H1N1), which is 16-times lower than that of M2 AH. The selectivity index (IC50/CC50), where CC50 is the half maximal cytotoxic concentration, was 360 for M2 MH and 81 for M2 AH. Dynamic light scattering spectroscopy and electron microscopy revealed that M2 AH-derived peptides did not disrupt liposomes but altered the shape of viruses. This result suggests that the shape of virus envelope was closely related to its activity. Thus, we propose that deforming without rupturing the membranes may achieve a high selectivity index for peptide antivirals.

Development of a quantitative assay for 2´-fucosyllactose via one-pot reaction with α1,2-fucosidase and l-fucose dehydrogenase.

2'-Fucosyllactose (2'-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2'-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2'-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni2+ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2'-FL in a large number of samples simultaneously. This method can be used to quantify 2'-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2'-FL.

Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis.

Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6 Synaptotagmin 1 (Syt1), a Ca(2+) sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7 Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6 In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca(2+) levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca(2+) These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

Engineering of α-1,3-fucosyltransferases for production of 3-fucosyllactose in Escherichia coli.

Fucosyllactoses (FLs), present in human breast milk, have been reported to benefit human health immensely. Especially, 3-fucosyllactose (3-FL) has numerous benefits associated with a healthy gut ecosystem. Metabolic engineering of microorganisms is thought to be currently the only option to provide an economically feasible route for large-scale production of 3-FL. However, engineering principles for α-1,3-fucosyltransferases (1,3-FTs) are not well-known, resulting in the lower productivity of 3-FL than that of 2'-fucosyllactose (2'-FL), although both 2'-FL and 3-FL follow a common pathway to produce GDP-L-fucose. The C-terminus of 1,3-FTs is composed of heptad repeats, responsible for dimerization of the enzymes, and a peripheral membrane anchoring region. It has long been thought that truncation of most heptad repeats, retaining just 1 or 2, helps the soluble expression of 1,3-FTs. However, whether the introduction of truncated version of 1,3-FTs enhances the production of 3-FL in a metabolically engineered strain, is yet to be tested. In this study, the effect of these structural components on the production of 3-FL in Escherichia coli was evaluated through systematic truncation and elongation of the C-terminal regions of three 1,3-FTs from Helicobacter pylori. Although these three 1,3-FTs contained heptad repeats and membrane-anchoring regions of varying lengths, they commonly exhibited an optimal performance when the number of heptad repeats was increased, and membrane-binding region was removed. The production of 3-FL could be increased 10-20-fold through this simple strategy.

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii.

The probiotic yeast Saccharomyces boulardii has been extensively studied for the prevention and treatment of diarrheal diseases, and it is now commercially available in some countries. S. boulardii displays notable phenotypic characteristics, such as a high optimal growth temperature, high tolerance against acidic conditions, and the inability to form ascospores, which differentiate S. boulardii from Saccharomyces cerevisiae The majority of prior studies stated that S. boulardii exhibits sluggish or halted galactose utilization. Nonetheless, the molecular mechanisms underlying inefficient galactose uptake have yet to be elucidated. When the galactose utilization of a widely used S. boulardii strain, ATCC MYA-796, was examined under various culture conditions, the S. boulardii strain could consume galactose, but at a much lower rate than that of S. cerevisiae While all GAL genes were present in the S. boulardii genome, according to analysis of genomic sequencing data in a previous study, a point mutation (G1278A) in PGM2, which codes for phosphoglucomutase, was identified in the genome of the S. boulardii strain. As the point mutation resulted in the truncation of the Pgm2 protein, which is known to play a pivotal role in galactose utilization, we hypothesized that the truncated Pgm2 might be associated with inefficient galactose metabolism. Indeed, complementation of S. cerevisiaePGM2 in S. boulardii restored galactose utilization. After reverting the point mutation to a full-length PGM2 in S. boulardii by Cas9-based genome editing, the growth rates of wild-type (with a truncated PGM2 gene) and mutant (with a full-length PGM2) strains with glucose or galactose as the carbon source were examined. As expected, the mutant (with a full-length PGM2) was able to ferment galactose faster than the wild-type strain. Interestingly, the mutant showed a lower growth rate than that of the wild-type strain on glucose at 37°C. Also, the wild-type strain was enriched in the mixed culture of wild-type and mutant strains on glucose at 37°C, suggesting that the truncated PGM2 might offer better growth on glucose at a higher temperature in return for inefficient galactose utilization. Our results suggest that the point mutation in PGM2 might be involved in multiple phenotypes with different effects.IMPORTANCESaccharomyces boulardii is a probiotic yeast strain capable of preventing and treating diarrheal diseases. However, the genetics and metabolism of this yeast are largely unexplored. In particular, molecular mechanisms underlying the inefficient galactose metabolism of S. boulardii remain unknown. Our study reports that a point mutation in PGM2, which codes for phosphoglucomutase, is responsible for inferior galactose utilization by S. boulardii After correction of the mutated PGM2 via genome editing, the resulting strain was able to use galactose faster than a parental strain. While the PGM2 mutation made the yeast use galactose slowly, investigation of the genomic sequencing data of other S. boulardii strains revealed that the PGM2 mutation is evolutionarily conserved. Interestingly, the PGM2 mutation was beneficial for growth at a higher temperature on glucose. We speculate that the PGM2 mutation was enriched due to selection of S. boulardii in the natural habitat (sugar-rich fruits in tropical areas).

Improved squalene production through increasing lipid contents in Saccharomyces cerevisiae.

Squalene, a valuable acyclic triterpene, can be used as a chemical commodity for pharmacology, flavor, and biofuel industries. Microbial production of squalene has been of great interest due to its limited availability, and increasing prices extracted from animal and plant tissues. Here we report genetic perturbations that synergistically improve squalene production in Saccharomyces cerevisiae. As reported previously, overexpression of a truncated HMG-CoA reductase 1 (tHMG1) led to the accumulation 20-fold higher squalene than a parental strain. In order to further increase squalene accumulation in the tHMG1 overexpressing yeast, we introduced genetic perturbations-known to increase lipid contents in yeast-to enhance squalene accumulation as lipid body is a potential storage of squalene. Specifically, DGA1 coding for diacylglycerol acyltranferase was overexpressed to enhance lipid biosynthesis, and POX1 and PXA2 coding for acyl-CoA oxidase and a subunit of peroxisomal ABC transporter were deleted to reduce lipid ß-oxidation. Simultaneous overexpression of tHMG1 and DGA1 coding for rate-limiting enzymes in the mevalonate and lipid biosynthesis pathways led to over 250-fold higher squalene accumulation than a control strain. However, deletion of POX1 and PXA2 in the tHMG1 overexpressing yeast did not improve squalene accumulation additionally. Fed-batch fermentation of the tHMG1 and DGA1 co-overexpressing yeast strain resulted in the production of squalene at a titer of 445.6 mg/L in a nitrogen-limited minimal medium. This report demonstrates that increasing storage capacity for hydrophobic compounds can enhance squalene production, suggesting that increasing lipid content is an effective strategy to overproduce a hydrophobic molecule in yeast.

Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regarding the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening.

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 µM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

Fumarate-Mediated Persistence of Escherichia coli against Antibiotics.

Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking.

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.

Dynamic light scattering analysis of SNARE-driven membrane fusion and the effects of SNARE-binding flavonoids.

Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.

Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states.

SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized, pre-fusion conformations have been elusive. We used single-molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent pre-fusion intermediates. Although the N-terminal half above the conserved ionic layer maintains a robust helical bundle structure, the membrane-proximal C-terminal half shows high FRET, representing a helical bundle (45%), low FRET, reflecting a frayed conformation (39%) or mid FRET revealing an as-yet unidentified structure (16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion state, and complete SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) assembly happens concomitantly with membrane fusion. However, our results show that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as complexins and synaptotagmin 1.

SNARE zippering is hindered by polyphenols in the neuron.

Fusion of synaptic vesicles with the presynaptic plasma membrane in the neuron is mediated by soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) proteins. SNARE complex formation is a zippering-like process which initiates at the N-terminus and proceeds to the C-terminal membrane-proximal region. Previously, we showed that this zippering-like process is regulated by several polyphenols, leading to the arrest of membrane fusion and the inhibition of neuroexocytosis. In vitro studies using purified SNARE proteins reconstituted in liposomes revealed that each polyphenol uniquely regulates SNARE zippering. However, the unique regulatory effect of each polyphenol in cells has not yet been examined. In the present study, we observed SNARE zippering in neuronal PC12 cells by measuring the fluorescence resonance energy transfer (FRET) changes of a cyan fluorescence protein (CFP) and a yellow fluorescence protein (YFP) fused to the N-termini or C-termini of SNARE proteins. We show that delphinidin and cyanidin inhibit the initial N-terminal nucleation of SNARE complex formation in a Ca(2+)-independent manner, while myricetin inhibits Ca(2+)-dependent transmembrane domain association of the SNARE complex in the cell. This result explains how polyphenols exhibit botulinum neurotoxin-like activity in vivo.