A search for synthetic peptides that inhibit soluble N-ethylmaleimide sensitive-factor attachment receptor-mediated membrane fusion Chang H Jung1,*, Yoo-Soo Yang1,*, Jun-Seob Kim1, Jae-Il Shin1, Yong-Su Jin1, Jae Y Shin1, Jong H Lee2, Koo M Chung2, Jae S Hwang3, Jung M Oh1, Yeon-Kyun Shin4 and Dae-Hyuk Kweon1 School of Biotechnology and Bioengineering, Sungkyunkwan University, Gyeonggi-do, Korea School of Bioresource Sciences, Andong National University, Gyeongsangbuk-do, Korea Amorepacific R&D Center, Yongin, Korea Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA Keywords exocytosis; membrane fusion; neurotransmitter release; SNARE; SNAREpatterned peptide Correspondence D.-H Kweon, School of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, Korea Fax: +82 31 290 7870 Tel: +82 31 290 7869 E-mail: dhkweon@skku.edu *These authors contributed equally to this work (Received 28 December 2007, revised March 2008, accepted 10 April 2008) doi:10.1111/j.1742-4658.2008.06458.x 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 SNAREdriven 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 Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins have central roles in neurotransmission At the synapse, membrane fusion, which is required for neurotransmitter release, is mediated by SNAREs Vesicle-associated membrane protein (v)-SNARE synaptobrevin (VAMP2) associates with target membrane (t)-SNAREs syntaxin 1a and synaptosome-associated protein of 25 kDa (SNAP-25) [1–3] to form the highly stable ternary SNARE complex [4–6] Cumulative evidence has shown that the SNARE complex forms the core of the machine that generates the energy required for membrane fusion, while other accessory proteins are involved in docking, tethering, Ca2+-sensing and Abbreviations DOPS, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine; POPC, 1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphatidylcholine; SNAP-25, synaptosomeassociated protein of 25 kDa; SNARE, soluble N-ethylmaleimide sensitive factor attachment receptor; VAMP2, vesicle-associated membrane protein FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3051 Inhibition of neurotransmitter release C H Jung et al recycling A fusion pore is created as a result of membrane fusion and neurotransmitters are released through this pore [7–12] The SNARE complex is a parallel four-helical bundle [4,5] The four core helices are provided by the three SNARE proteins; one each from syntaxin and VAMP2, and two from SNAP-25 [4,5] Syntaxin and VAMP2 are transmembrane proteins with single transmembrane helices and are anchored to the presynaptic membrane and vesicular membrane, respectively SNAP-25 is peripherally attached to the presynaptic membrane Thus, formation of a parallel coiled-coil would result in close apposition of two membranes, and facilitate membrane fusion The core complex has been shown to be linked tightly to the membranes so that the force generated by SNARE complex formation can be faithfully transferred to the membranes [13–15] Impaired SNARE function is known to block neuronal exocytosis For example, synaptic SNARE proteins are the specific substrates of eight clostridial neurotoxins (one tetanus neurotoxin and seven botulinum neurotoxins; BoNT ⁄ A–G) [16,17] The neurotoxins bind specifically to the nerve terminals and deliver the N-terminal catalytic domain of the zinc-endopeptidase into the cytosol, where the catalytic domain specifically cleaves a SNARE protein at a single site within its cytosolic portion Such specific cleavage leads to an inhibition of neuroexocytosis and results in the paralytic syndromes of botulism and tetanus Low doses of BoNT ⁄ A are now widely used to alleviate the symptoms of various disorders, including paralytic strabismus, blephoraspasm, cervical dystonia and severe hyperhydrosis [18,19] The neurotoxin is also widely used for cosmetic purposes Clostridial toxins have proven very versatile with therapeutic and cosmetic uses Inhibition of neurotransmission might be achieved not only by the specific cleavage of SNARE proteins, but also by blocking SNARE complex formation, and several peptide inhibitors have been developed for this purpose [20–27] The peptides are mostly modeled on the sequences of the SNARE motifs in SNAP-25 These peptides are thought to competitively inhibit SNARE complex formation by interfering with interactions between parental SNARE proteins However, no systematic study has evaluated the efficacy of SNARE-patterned sequences from all four SNARE motifs As such, there is no design principle to guide the development of potent peptide SNARE inhibitors Because individual sequences modeled on the SNARE motifs are expected to inhibit SNARE complex formation to differing degrees, careful assessment of the effect of SNARE-patterned peptides on SNARE 3052 complex formation might help us better understand SNARE-driven neuronal exocytosis Such efforts will also help us identify the potent peptide sequences that interfere with neurotransmission In this study, we designed and synthesized small peptides derived from SNARE motifs We assessed their inhibitory activities on membrane fusion, neuroexocytosis of digitoninpermeabilized PC12 cells and SNARE complex formation in PC12 cells We found that the peptide sequences derived from the N-terminal regions of SNARE motifs show high inhibitory activity Results Design of peptides modeled on the SNARE motifs Peptide sequences modeled on SNARE motifs are most likely to compete with parental SNARE proteins for SNARE complex formation, inhibiting SNARE assembly In an initial attempt to search for the potent peptide sequences that inhibit SNARE complex formation and neurotransmitter release, we synthesized thirteen 17-mer peptides representing the various regions in the SNARE motifs of individual SNAREs Three peptide sequences were derived from each SNARE motif, giving 12 from four SNARE motifs Three sequences from each SNARE motif represent the N-terminal, middle and C-terminal regions (Fig 1A) Sequences representing the middle regions contained the amino acid Q or R that is present in the zero-layer of the SNARE complex (bold italics in Table 1) [5] Individual peptide sequences are shown in Table One additional 17-mer peptide (designated as LIB), which was previously selected as a SNARE inhibitor from an a-helix-constrained combinatorial peptide library [24], was prepared as a positive control Inhibition of SNARE-driven membrane fusion by synthetic peptides In order to measure the efficacy of 13 synthetic peptides, a fluorescence lipid-mixing assay was performed using SNARE-reconstituted liposomes (see Experimental procedures) [3,28] The lipid-mixing assay is a well-established method that has frequently been used to show various features of SNARE-driven membrane fusion [7,12,28–33] For the lipid-mixing assay, full-length t-SNARE complex was reconstituted into large unilamellar vesicles (100 nm diameter) composed of 1-palmitoyl-2dioleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) in FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS C H Jung et al Inhibition of neurotransmitter release A B kDa 37 25 20 15 P- 1a VA M in ax nt Sy SN A P- 25 10 Fig Design of SNARE motif-patterned peptides and purification of full-length SNARE proteins (A) Synthetic peptides patterned after the a-helical regions of SNARE motifs Peptide names are designated in the gray box Hexamer peptides were also synthesized patterned after VpN, VpM and VpC sequences The amino acid sequences of synthetic peptides are shown in Table (B) SDS ⁄ PAGE analysis of purified recombinant SNARE proteins used in this study a molar ratio of 65 : 35 Full-length v-SNARE was reconstituted into a separate population of the same POPC ⁄ DOPS vesicles containing 1.5 mol% each of fluorescence lipids When the t-SNARE and v-SNARE vesicles were mixed without adding the synthetic peptides there was an increase in the fluorescence signal (Fig 2A), indicating that lipid mixing had occurred Several synthetic peptides showed rather poor solubility in water, whereas others had good solubility in buffer Therefore, dimethylsulfoxide was used to dissolve peptides with poor water solubility and peptide ⁄ dimethylsulfoxide solutions were injected directly into the fusion reaction The added dimethylsulfoxide influenced lipid mixing somewhat (Fig 2A); therefore, the inhibition efficacies of the peptides were tested in two different batches depending on the solvent conditions (Fig 2) Some synthetic peptides had profound effects on membrane fusion when tested at a peptide concentration of 200 lm (Fig 2A,B) Peptides representing the N-terminal region of the SNARE motifs SCN (from SNAP-25C), SynN (from Syntaxin 1a) and VpN (from VAMP2) reduced membrane fusion significantly, whereas the other peptides were much less effective The most effective peptides (VpN and SynN) decreased membrane fusion by as much as 60–70% of the control (Fig 2B) The amphipathic character of the SNARE-patterned peptides may make them fusogenic [34–36] In order to rule out this possibility, the fusion activities of the peptides were tested in the absence of SNAREs (Fig 2B, gray bar) None of the 17-mer peptides had significant membrane fusion activity without SNAREs The cytoplasmic domain of VAMP2 (VpS, amino acids 1–96) has been used frequently as an inhibitor for SNAREdriven membrane fusion [3] VpS suppressed membrane fusion very effectively at lower concentrations (20 lm) (Fig 2A,B) and served as a good positive control to compare the inhibitory effect of the synthetic peptides Table Amino acid sequences of synthetic peptides Glutamine and arginine residues in the zero layer are shown in bold italics SNN (7–23) MRNELEEMQRRADQLAD SCN (140–156) DARENEMDENLEQVSGI SynN (192–208) LSEIETRHSEIIKLENS VpN (30–46) RRLQQTQAQVDEVVDIM LIB SAAEAFAKLYAEAFAKG VpN1 RRLQQT VpM1 VNVDKV VpC1 QFETSA SNM (45–61) RTLVMLDEQGEQLERIE SCM (166–182) DMGNEIDTQNRQIDRIM SynM (218–224) DMAMLVESQGEMIDRIE VpM (48–64) VNVDKVLERDQKLSELD Argireline EEMQRR VpN2 QAQVDE VpM2 LERDQK VpC2 KLKRKY FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS SNC (65–71) DQINKDMKEAEKNLTDL SCC (190–206) TRIDEANQRATKMLGSG SynC (245–261) ERAVSDTKKAVKYQSKA VpC (76–92) QFETSAAKLKRKYWWKN VpN3 EVVDIM VpM3 KLSELD VpC3 KYWWKN 3053 Inhibition of neurotransmitter release C H Jung et al A B C Fig Inhibitory effects of the synthetic peptides on SNARE-driven membrane fusion Peptides were added at 200 lM (A) Percent of maximum fluorescence intensity was plotted as a function of time in the presence or absence of the peptides The maximum fluorescence intensity was obtained by adding 0.1% Triton X-100 (upper) for peptides dissolved in dimethylsulfoxide (DMSO) and (lower) for peptides dissolved in distilled water (DW) VpS, soluble domain of VAMP2 (amino acids 1–96) (B) The inhibitory effect of synthetic peptides was converted to ‘% of control’ (black bar) Percentage of maximum fluorescence intensity in the presence of synthetic peptides was divided by that of dimethylsulfoxide or distilled water (DW) depending on the dissolving solvents Each bar represents mean ± SD of three independent experiments *P < 0.05 versus control SNARE-independent membrane fusion was shown as ‘% of maximum fluorescence intensity’ (gray bar) (C) Western blot analysis of SNARE complex formation was performed after the membrane fusion assay in order to confirm that the N-terminal-mimicking peptides inhibited SNARE complex formation Numbers below the figure are relative band intensities Asterisks denote peptides dissolved in dimethylsulfoxide and should be compared with dimethylsulfoxide as the control; others were dissolved in water and should be compared with distilled water 3054 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS C H Jung et al Inhibition of neurotransmitter release In order to verify whether the reduced membrane fusion was caused by inhibition of SNARE complex formation by the peptides, western blot analysis was performed for six peptides after membrane fusion The SDS-resistant SNARE complex was detected using anti-(SNAP-25) IgG SCN, SynN and VpN reduced SNARE complex formation, whereas SNN, SCM and LIB were not effective (Fig 2C), supporting the idea that the inhibitory activities were due to inhibition of SNARE complex formation by the peptides Interestingly, SCM increased membrane fusion by  70% (Fig 2A,B), although it did not enhance SNARE complex formation (Fig 2C) It has previously been shown that a synthetic peptide from the C-terminal region of VAMP2 promoted SNARE-mediated fusion [37] Pobbati et al [37] showed that this stimulatory effect might be due to its role in refolding the t-SNARE complexes into an active conformation We wonder whether SCM has a similar effect in SNARE-mediated membrane fusion, warranting further investigation A Inhibition of neurotransmitter release from PC12 cells by the synthetic peptides C In order to measure the effects of synthetic peptides on neuronal exocytosis, we prepared permeabilized PC12 cells loaded with [3H]-noradrenaline Depolarization of these cells by high K+ concentrations (50 mm KCl) results in the release of neurotransmitters such as noradrenaline, acetylcholine and arachidonic acid [38,39] Inhibition of this release by synthetic peptides could be assessed by measuring [3H]-noradrenaline release after stimulation with high concentrations of K+, in the presence or absence of synthetic peptides In the permeabilized PC12 cells, stimulation with high concentrations of K+ significantly increased [3H]noradrenaline release, compared with the basal levels We tested the effects of the synthetic peptides on the high K+ concentration-stimulated release of [3H]-noradrenaline Addition of SCN, SynN and VpN efficiently inhibited neuroexocytosis even at a final concentration of 10 lm, whereas other peptides showed no significant effect (Fig 3A), consistent with results from the lipid-mixing assay Inhibition of [3H]-noradrenaline release by the N-terminal-mimicking peptides SCN, SynN and VpN was as much as 20–30%, when compared with controls (Fig 3A) Verapamil, an L-type calcium-channel blocker, inhibited neurotransmitter release by  70% at the same concentration (10 lm) [40,41] Thus, N-terminalmimicking peptides serve as good SNARE-targeting neuroexocytosis inhibitors We were not able to test B Fig Inhibitory effects of the synthetic peptide on [3H]-noradrenaline release in high K+-stimulated PC12 cells (A) The high K+-stimulated [3H]-noradrenaline release over 15 was measured in the presence or absence of synthetic peptides The amount of [3H]-noradrenaline released measured in the presence of an inhibitory peptide was divided by that measured in the absence of the peptide, where the amount of [3H]-noradrenaline release is (cpm of high K+-stimulated sample – cpm of basal level release) per mg of protein Each bar represents mean ± SD of 5–7 observations *P < 0.05 versus K+-evoked control (B, C) Inhibition of SNARE complex formation by the synthetic peptides in K+-stimulated PC12 cells SNARE complex was detected by western blotting using an anti-(SNAP-25) IgG (B) and relative band densities are represented by the bar graph (C) Cells were pretreated with synthetic peptides for h prior to stimulation with K+ (50 mM KCl), and then further incubated for 15 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3055 Inhibition of neurotransmitter release C H Jung et al higher peptide concentrations because some peptides were dissolved in dimethylsulfoxide which killed PC12 cells at higher concentrations In order to verify whether the peptides inhibited SNARE assembly in PC12 cells, membrane proteins were extracted from the cells and analyzed by western blotting using anti-(SNAP-25) IgG (Fig 3B,C) Consistent with other results, SCN, SynN and VpN inhibited SNARE complex formation significantly (Fig 3B,C) VpN inhibited SNARE complex formation with a similar efficiency to verapamil N-Terminal-mimicking peptides inhibited SNARE complex formation by directly interacting with native SNARE proteins, whereas verapamil did the same by blocking calcium entry By contrast, other peptides did not have noticeable inhibitory effects on SNARE complex formation Therefore, the results show that the reduction in neuroexocytosis by SCN, VpN and SynN was a direct consequence of the inhibition of SNARE complex formation Smaller peptides might be beneficial in terms of synthesis costs, as well as membrane penetration Therefore, we synthesized nine additional 6-residue peptides derived from v-SNARE VAMP2 (Fig 1A and Table 1) We also made the well-known Argireline [26,42] We did not observe any measurable inhibitory effects with the 6-mer peptides when tested using the lipid-mixing assay (data not shown) and noradrenaline release assay (Fig 3) Time-dependent effects of inhibitory peptides on membrane fusion and neurotransmitter release N-Terminal zippering may be the rate-limiting step in SNARE complex formation The N-terminal-mimicking peptides may have inhibited this N-terminal zippering, resulting in the most significant inhibition of membrane fusion and neurotransmitter release Conversely, N-terminal-mimicking peptides might not inhibit SNARE complex formation and membrane fusion when partial N-terminal zippering has occurred In order to test this, five selected synthetic peptides were added to the membrane fusion reaction mixture before or after preincubation of t- and v-SNARE vesicles at °C overnight [3] SNN and SCC were selected as negative controls because they did not inhibit membrane fusion and neurotransmitter release (Figs and 3) Preincubation of t- and v-SNARE vesicle mixture at °C overnight accelerated the membrane fusion reaction by  40% when the reaction was induced by increasing the temperature to 37 °C, consistent with previous results (Fig 4A; upper left) [3] When SCN, SynN and VpN were added before preincubation, membrane fusion was consistently inhibited However, the inhibitory effect of those peptides was much lower when they were added after preincubation SNN and SCC did not inhibit membrane fusion, regardless of preincubation Thus, N-terminal-mimicking peptides seem to inhibit membrane fusion by hindering N-terminal zippering When membrane fusion was complete, the amount of SNARE complex was measured by western blotting (Fig 4B) SNN and SCC did not change the amount of SNARE complex formed, regardless of preincubation However, there was a dramatic change in the amount of SNARE complex depending on the addition time of SCN, SynN and VpN When SCN, SynN and VpN were added after preincubation they did not reduce SNARE complex formation much By contrast, these peptides inhibited SNARE complex formation very efficiently when added before preincubation Therefore, N-terminal-mimicking peptides inhibited SNARE complex formation by hindering N-terminal zippering, which is consistent with the membrane fusion result These experiments show that N-terminal-mimicking peptides not inhibit membrane fusion if the N-terminals of t- and v-SNARE proteins are already zipped together, suggesting that the target of the peptides is newly forming SNARE complex In order to confirm this in PC12 cells, already-loaded neurotransmitters were depleted by pretreating with high concentrations of K+ before the addition of inhibitory peptides After pretreatment with high concentrations of K+, inhibitory peptides were added and cells were cultured for 24 h to regenerate synaptic vesicles By doing so, we are able to measure noradrenaline release from the newly docking vesicles only In Fig 4C, it is clearly shown that the noradrenaline release is dramatically Fig Time-dependent effects of inhibitory peptides on membrane fusion and neurotransmitter release (A) Synthetic peptides were added to the membrane fusion reaction mixture before or after preincubation of t- and v-SNARE vesicles at °C overnight (B) After completion of the membrane fusion reaction in (A), western blot analysis was performed to measure the amount of SNARE complex formation +, peptide added after preincubation; ), peptide added before preincubation (C) Neurotransmitter release from PC12 cells was measured after depleting already-docked synaptic vesicles Already-docked synaptic vesicles were depleted by pretreating with a high K+ concentration before the addition of synthetic peptides After addition of the peptides, PC12 cells were cultured for an additional 24 h for regeneration of the SNARE complex and neurotransmitter release was measured again (D) PC12 cells of (C) were subjected to western blot analysis to measure the amount of SNARE complex formation 3056 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS C H Jung et al Inhibition of neurotransmitter release A B C D FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3057 Inhibition of neurotransmitter release C H Jung et al reduced by N-terminal-mimicking peptides after pretreatment with high K+ concentrations SCN, SynN and VpN reduced noradrenaline release to the level of verapamil, which may represent the minimum level of stimulated neuroexocytosis SNN and SCC did not inhibit noradrenaline release regardless of pretreatment with high K+ concentrations Verapamil, a calciumchannel blocker, did not have an additional inhibitory effect on noradrenaline release Also, we were able to confirm that SNARE complex formation was dramatically reduced by N-terminal peptides, whereas other peptides were almost neutral with regard to SNARE complex formation in PC12 cells (Fig 4D) In conclusion, N-terminal peptides inhibit newly forming SNARE complex in PC12 cells and block the regeneration of neuroexocytosis in synaptic vesicles We speculate that the neurotransmitter release and SNARE complex band shown in Fig were the sum of effects derived from partially zipped SNARE complex and newly forming complex By eliminating the effect derived from already-zipped complex, the effect of N-terminal peptides on SNARE complex formation and neuroexocytosis could be more distinctive Discussion Depolarization of PC12 cells induces the influx of Ca2+, leading to the fusion of synaptic vesicles which are docked at the active zone of neurotransmitter A X B D 3058 X C release [43] When the PC12 cells were depolarized with a short-pulse high concentration of K+, the synthetic peptides derived from the N-terminal regions of the SNARE motifs SCN, SynN and VpN inhibited [3H]-noradrenaline release in detergent-permeabilized PC12 cells (Fig 3) These results correlate strongly with the reduction in SNARE complex formation in the PC12 cells (Fig 3B,C), suggesting that the peptides inhibit [3H]-noradrenaline release by competing with parental SNAREs for complex formation Recently, it has been shown that N-terminal nucleation of the SNARE complex can promote rapid membrane fusion [37] Conversely, one could predict that the disruption of N-terminal nucleation would affect the rate of membrane fusion significantly (route A in Fig 5) A variety of experiments including the lipidmixing assay, the neurotransmitter release assay in PC12 cells and western blot analysis consistently confirmed that SNARE-mediated fusion is profoundly affected by N-terminal patterned peptides Therefore, it appears that the N-terminal regions of SNARE motifs are the potential targets for developing potent blockers of SNARE-mediated membrane fusion The relative inefficiency of other peptides in inhibiting fusion can be rationalized in the context of the zipper model for SNARE complex formation [31,44– 47] The zipper model predicts that SNARE assembly starts in the N-terminal region and proceeds towards the C-terminal region One may envisage that a Fig Schematic presentation showing the effect of SNARE motif-patterned peptides on the inhibition of membrane fusion (A) N-Terminal docking, which is believed to be the rate-limiting step, is inhibited in the presence of N-terminal-mimicking peptide (e.g VpN in the figure), resulting in profoundly reduced SNARE-driven membrane fusion (B) In the absence of inhibitory peptide, SNARE complex drives membrane fusion (C) Middle- or C-terminal-mimicking peptides (e.g VpC in the figure) did not inhibit membrane fusion indicating that the peptide might be easily removed from t-SNARE complex [37] (D) In the presence of SynN, t-SNARE complex cannot accept VAMP2 for binding as in (A) Yellow, membranes; red, SNARE motif of Syntaxin or Syntaxin-patterned peptides; green, two SNARE motifs of SNAP-25; blue, SNARE motif of VAMP2 or VAMP2-patterned peptides FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS C H Jung et al peptide bound to the middle or C-terminal region of t-SNARE complex (route C in Fig 5) is peeled off easily as zippering progresses [37], resulting in no or only moderate effects on membrane fusion (Figs 2–4) A current model for SNARE assembly predicts that VAMP2 in synaptic vesicles associates with the binary complex of t-SNARE Syntaxin 1a and SNAP-25 in presynaptic membranes Thus, it is likely that VAMP2-patterned peptides bind to the t-SNARE complex and compete with VAMP2 for binding Because Syntaxin 1a and SNAP-25 can form a complex with : stoichiometry, it is likely that syntaxin-patterned peptides also have their binding sites within the t-SNARE complex When SynN is bound to the : t-SNARE complex, however, the structure of the t-SNARE complex might change to that of the : complex, which is known to be a dead-end product (route D in Fig 5) [48] Such a conformational change would definitely inhibit the association with VAMP2, and thus membrane fusion By contrast, N- and C-terminal-mimicking peptides from SNAP-25 (SNN and SCN, respectively) had rather intricate effects on neuroexocytosis (Figs 2–4) SNN did not inhibit SNARE complex formation at all SNN might not bind to any of the SNARE complexes involved in the SNARE assembly pathway However, SCN was as effective as SynN and VpN SCN may bind to the partially assembled t-SNARE complex in which the C-terminal SNARE motifs of SNAP-25 are not yet engaged [49], competing with the binding of the C-terminal motif of SNAP-25 The ineffectiveness of several well-known peptides that are known to regulate neuronal exocytosis is interesting For example, a peptide mimicking the C-terminal domain of SNAP-25 (corresponding to SCC in this study) reportedly blocked Ca2+-dependent exocytosis in chromaffin cells with an IC50 of 20 lm [20] However, we did not observe such an inhibitory effect of SCC on SNARE assembly and neuroexocytosis in PC12 cells (Figs 2–4) In addition, LIB, which was selected from an a-helical peptide library of 137 180 sequences, was also not effective in the lipid-mixing assay or the noradrenaline release and SNARE complex formation assays in PC12 cells (Figs and 3) Previously, LIB was screened by measuring the SDSresistant complex formation with the purified SNARE proteins We speculate that the discrepancy between our results and those found previously on the efficacies of SCC and LIB arises from differences in the measurement scale of the assays used In a screening process, determining the selection criteria depends on the level of the background and experimental conditions As such, a potential inhibitory peptide screened Inhibition of neurotransmitter release from an experiment may not necessarily be reproduced in other experiments using different assays or differently prepared cells Therefore, a fair comparison of the effectiveness of different SNARE-patterned peptides can only be made under identical experimental conditions In this study, the 13 representative peptides were tested under the same experimental conditions and their relative inhibition efficacies were ranked Our results showed that SCN, SynN and VpN are much more effective than any other SNARE-patterned peptides It is also noteworthy that we were able to reproduce part of the previous results with LIB When LIB was tested for its inhibitory effect on SDS-resistant complex formation of purified cytoplasmic domains of SNARE proteins in vitro it reduced SNARE complex formation by  80% at 200 lm (data not shown) This is comparable with previous results in which LIB inhibited SNARE complex formation by  95% at 0.5 mgỈmL)1 ( 250 lm) [24] However, LIB did not show any significant effects on membrane fusion and neurotransmitter release in our study (Figs and 3) Interestingly, the inhibition of SNARE complex formation by SCN, SynN and VpN was less than that by LIB when purified SNAREs were used (data not shown), although these peptides were much more potent in inhibiting membrane fusion and release in PC12 cells These results suggest that the peptides screened by SDS-resistant complex formation using purified SNARE proteins might not correlate well with the inhibition of neuroexocytosis There is evidence that the membrane has crucial roles in SNARE complex formation and membrane fusion [14,50] Membranes may also restrict the geometry in which SNAREs assemble Thus, inhibition of SDS-resistant complex formation of purified soluble proteins does not necessarily mirror the inhibition of membraneanchored SNARE complex formation Because neuronal exocytosis is triggered on a millisecond time scale, synaptic vesicles are believed to be present at the active zone in a ready-to-fire state However, we not know in which state SNAREs are arrested before formation of the fusion pore: at the free protein stage, when N-terminal tips are already zipped or when the full complex is formed but the fusion pore is not yet made In Fig 4, we show that SCN, SynN and VpN inhibited N-terminal zippering and that the already-zipped complex was not inhibited by those peptides (Fig 4A,B) Together with the fact that N-terminal peptides target newly forming SNARE complex (Fig 4C,D), we might rule out the possibility that SNARE proteins are arrested before fusion at the state of free proteins In conclusion, the inhibitory effects of the N-terminal-mimicking peptides on neuro- FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3059 Inhibition of neurotransmitter release C H Jung et al transmitter release are very promising in the context of their applications in pharmaceuticals and cosmetics Experimental procedures Materials POPC, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphoserineN-(7-nitro-2-1,3-benzoxadiazol-4-yl) and 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) were obtained from Avanti Polar Lipids Inc (Alabaster, AL, USA) RPMI-1640, penicillin– streptomycin, horse serum and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY, USA) Triton X-100, 2-mercaptoethanol and all other chemicals were purchased from Sigma-Aldrich Co (St Louis, MO, USA) All peptides used (Fig 1A) were synthesized by Peptron, Inc (Dajeon, Korea), and were ‡ 95% pure as judged by MS Synthetic peptides were dissolved in distilled water or dimethylsulfoxide depending on their solubility Argireline [26] and LIB [23] were also synthesized as controls Expression and purification of recombinant protein preparation Protein expression and purification procedures for neuronal SNARE proteins have been described previously [14] In brief, the pGEX-2T vector encoding a thrombin-cleavable N-terminal glutathione S-transferase tag was used for the expression of the following constructs: full-length syntaxin 1a (amino acids 1–288), full-length VAMP2 (amino acids 1)116) and SNAP-25 (amino acids 1)206) All recombinant proteins were expressed in an Escherichia coli CodonPlusRIL (DE3) (Novagen, Darmstadt, Germany) All N-terminal glutathione S-transferase-tagged fusion proteins were purified by affinity chromatography using glutathione–agarose beads The protein was cleaved by thrombin in cleavage buffer (50 mm Tris ⁄ HCl, 150 mm NaCl, pH 8.0) We added 1% n-octyl-d-glucopyranoside for syntaxin 1a and VAMP2 Purified proteins were examined by 12.5% SDS ⁄ PAGE, and the purity was at least 90% for all proteins (Fig 1B) Reconstitution of SNARE proteins into membranes Large unilamellar vesicles with a diameter of 100 nm were prepared as described previously [28] In brief, a mixture of POPC and DOPS (molar ratio of 65 : 35) in chloroform was dried in a vacuum and resuspended in buffer (50 mm Tris ⁄ HCl, 150 mm NaCl, pH 8.0) for a total lipid concentration of 50 mm Protein-free large unilamellar vesicles ( 100 nm dia.) were prepared by extrusion through poly- 3060 carbonate filters (Avanti Polar Lipids Inc., Alabaster, AL, USA) Syntaxin 1a and SNAP-25 were mixed at room temperature for  60 to allow the formation of t-SNARE complex The t-SNARE complex was then mixed with the prepared large unilamellar vesicles at a 50 : lipid ⁄ protein molar ratio For the v-SNARE vesicles, 10 mm fluorescent liposomes, containing POPC, DOPS, 1,2-dioleoyl-snglycero-3-phosphoserine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and rhodamine-PE at a molar ratio of 62 : 35 : 1.5 : 1.5, were mixed with VAMP2 at a 50 : lipid ⁄ protein ratio The liposome ⁄ protein mixture was diluted twice, which brought the concentration of n-octyl-d-glucopyranoside below the critical micelle concentration After dialyzing against dialysis buffer (25 mm Hepes, 100 mm KCl, 5% w ⁄ v glycerin, pH 7.4) at °C overnight to remove detergent, the sample was treated with Bio-Beads SM2 (Bio-Rad, Hercules, CA, USA) to eliminate any remaining detergent The solution was then centrifuged at 10 000 g for 30 to remove protein and lipid aggregates The final t-SNARE liposome solution contained  2.5 mm lipids and 1.9 mgỈmL)1 of protein, and the v-SNARE liposome solution contained  mm lipids and 0.25 mgỈmL)1 of protein The reconstitution efficiency was determined using SDS ⁄ PAGE The amount of protein in the liposomes was estimated by comparing the band in the gel with that of a known concentration of the same protein SNARE-driven membrane fusion assay The total lipid-mixing fluorescence assay has been described elsewhere [29] To measure total lipid mixing, v-SNARE liposomes were mixed with t-SNARE liposomes at a ratio of : The final solution contained mm lipids, in a total volume of 100 lL, in the presence of synthetic peptide Fluorescence was measured at excitation and emission wavelengths of 465 and 530 nm, respectively Changes in fluorescence were recorded with a Spectra Max M2 (Molecular Devices Inc., Palo Alto, CA, USA) fluorescence spectrophotometer The maximum fluorescence intensity was obtained by adding Triton X-100 All lipid mixing experiments were carried out at 37 °C PC12 cell culture PC12 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) The cells were plated onto poly-(d-lysine)coated culture dishes and were kept in RPMI-1640 containing 100 lgỈmL)1 streptomycin, 100 mL)1 penicillin, mm l-glutamine, 10% heat-inactivated horse serum and 5% fetal bovine serum at 37 °C in a 5% CO2 incubator Cell cultures were split once a week, and the medium was refreshed three times a week PC12 cells were treated with NGF (7S, 50 ngỈmL)1; Invitrogen, Carlsbad, CA, USA) for days prior to [3H]-noradrenaline uptake and release FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS C H Jung et al Determination of [3H]-noradrenaline release from detergent-permeabilized PC12 cells The amount of secreted [3H]-noradrenaline was determined in digitonin-permeabilized cells as described previously [20,27] In brief, PC12 cells were grown in 12-well plates at a density of · 105 cellsỈdish)1 After the cells adhered to the plates (20–24 h), they were preincubated with peptides and [3H]-noradrenaline (1 lCiỈmL)1) at 37 °C for 90 in Krebs ⁄ Hepes solution (140 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 2.5 mm CaCl2, 1.2 mm MgSO4, 11 mm glucose and 15 mm Hepes ⁄ Tris, pH 7.4) containing 10 lm digitonin to permeabilize the cell Cells were washed four times to remove the unincorporated radiolabeled compound Cells were then depolarized with a high-K+ solution (115 mm NaCl, 50 mm KCl, 1.2 mm KH2PO4, 2.5 mm CaCl2, 1.2 mm MgSO4, 11 mm glucose and 15 mm Hepes ⁄ Tris, pH 7.4) for 15 to assess the stimulated release Extracellular media were transferred to scintillation vials and then measured by liquid scintillation counting The amount of [3H]-noradrenaline release was calculated according to the following equation: amount of [3H]-noradrenaline release = (cpm of high K+-stimulated sample – cpm of basal level release) per mg of protein Immunoblots Cells were washed twice with ice-cold NaCl ⁄ Pi after treatment, and then lysed with RIPA buffer (Cell Signaling #9806) directly in the plate wells after the removal of media Cell lysates were obtained by centrifugation at 13 000 g for 15 at °C Protein concentrations were determined by the Bio-Rad protein assay kit using BSA as a standard Equal amounts of protein (50 lg) from the cell lysates were dissolved in Laemmli’s sample buffer (not boiled), electrophoresed (SDS ⁄ PAGE) and transferred to a nitrocellulose membrane The membranes were then blocked with NaCl ⁄ Pi ⁄ 0.1% Tween 20 (NaCl ⁄ Pi-T buffer) containing 1% skim milk and 1% BSA for h at room temperature Thereafter, the membranes were incubated overnight at °C with a : 1000 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SNARE assembly predicts that VAMP2 in synaptic vesicles associates with the binary complex of t-SNARE Syntaxin 1a and SNAP-25 in presynaptic membranes Thus, it is likely that VAMP2-patterned peptides. .. SNARE-independent membrane fusion was shown as ‘% of maximum fluorescence intensity’ (gray bar) (C) Western blot analysis of SNARE complex formation was performed after the membrane fusion assay