Báo cáo khoa học: A search for synthetic peptides that inhibit soluble N-ethylmaleimide sensitive-factor attachment receptor-mediated membrane fusion pot

A search for synthetic peptides that inhibit soluble
N-ethylmaleimide sensitive-factor attachment
receptor-mediated membrane fusion
Chang H. Jung
1,
*, Yoo-Soo Yang
1,
*, Jun-Seob Kim
1
, Jae-Il Shin
1
, Yong-Su Jin
1
, Jae Y. Shin
1
, Jong
H. Lee
2
, Koo M. Chung
2
, Jae S. Hwang
3
, Jung M. Oh
1
, Yeon-Kyun Shin
4
and Dae-Hyuk Kweon
1
1 School of Biotechnology and Bioengineering, Sungkyunkwan University, Gyeonggi-do, Korea
2 School of Bioresource Sciences, Andong National University, Gyeongsangbuk-do, Korea
3 Amorepacific R&D Center, Yongin, Korea

4 Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA
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 2 (v)-SNARE synaptobrevin (VAMP2) associ-
ates 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 mem-
brane fusion, while other accessory proteins are
involved in docking, tethering, Ca
2+
-sensing and
Keywords
exocytosis; membrane fusion;
neurotransmitter release; SNARE; SNARE-
patterned 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:
*These authors contributed equally to this
work

(Received 28 December 2007, revised 9
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 SNARE-
driven membrane fusion. Therefore, the results suggest that the N-terminal
regions of SNARE motifs are excellent targets for the development of
drugs to block SNARE-mediated membrane fusion and neurotransmitter
release.
Abbreviations
DOPS, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine; POPC, 1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphatidylcholine; SNAP-25, synaptosome-
associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide sensitive factor attachment receptor; VAMP2, vesicle-associated membrane
protein 2.
FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3051
recycling. A fusion pore is created as a result of mem-

brane fusion and neurotransmitters are released
through this pore [7–12].
The SNARE complex is a parallel four-helical bun-
dle [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 trans-
membrane 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 forma-
tion can be faithfully transferred to the membranes
[13–15].
Impaired SNARE function is known to block neuro-
nal exocytosis. For example, synaptic SNARE proteins
are the specific substrates of eight clostridial neuro-
toxins (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 para-
lytic syndromes of botulism and tetanus. Low doses of
BoNT ⁄ A are now widely used to alleviate the symp-

toms of various disorders, including paralytic strabis-
mus, blephoraspasm, cervical dystonia and severe
hyperhydrosis [18,19]. The neurotoxin is also widely
used for cosmetic purposes. Clostridial toxins have pro-
ven 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 inter-
actions 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 forma-
tion to differing degrees, careful assessment of the
effect of SNARE-patterned peptides on SNARE
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 digitonin-
permeabilized PC12 cells and SNARE complex forma-

tion 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 forma-
tion and neurotransmitter release, we synthesized thir-
teen 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 1.
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 pep-
tides, a fluorescence lipid-mixing assay was performed

using SNARE-reconstituted liposomes (see Experi-
mental 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 vesi-
cles (100 nm diameter) composed of 1-palmitoyl-2-
dioleoyl-sn-glycero-3-phosphatidylcholine (POPC) and
1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) in
Inhibition of neurotransmitter release C. H. Jung et al.
3052 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 flu-
orescence 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 solu-
bility in water, whereas others had good solubility in
buffer. Therefore, dimethylsulfoxide was used to dis-
solve peptides with poor water solubility and peptide ⁄
dimethylsulfoxide solutions were injected directly into
the fusion reaction. The added dimethylsulfoxide influ-
enced 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 concentra-
tion 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 con-
trol (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 pep-
tides 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 cyto-
plasmic domain of VAMP2 (VpS, amino acids 1–96)
has been used frequently as an inhibitor for SNARE-
driven membrane fusion [3]. VpS suppressed mem-
brane 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 syn-
thetic peptides.
37
kDa
25
15
10
SNAP-25
Syntaxin 1a
VAMP-2

20
A
B
Fig. 1. 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 synthe-
sized patterned after VpN, VpM and VpC sequences. The amino
acid sequences of synthetic peptides are shown in Table 1. (B)
SDS ⁄ PAGE analysis of purified recombinant SNARE proteins used
in this study.
Table 1. Amino acid sequences of synthetic peptides. Glutamine and arginine residues in the zero layer are shown in bold italics.
SNN (7–23)
MRNELEEMQRRADQLAD
SNM (45–61)
RTLVMLDEQGEQLERIE
SNC (65–71)
DQINKDMKEAEKNLTDL
SCN (140–156)
DARENEMDENLEQVSGI
SCM (166–182)
DMGNEIDTQNRQIDRIM
SCC (190–206)
TRIDEANQRATKMLGSG
SynN (192–208)
LSEIETRHSEIIKLENS
SynM (218–224)
DMAMLVESQGEMIDRIE
SynC (245–261)
ERAVSDTKKAVKYQSKA

VpN (30–46)
RRLQQTQAQVDEVVDIM
VpM (48–64)
VNVDKVLERDQKLSELD
VpC (76–92)
QFETSAAKLKRKYWWKN
LIB
SAAEAFAKLYAEAFAKG
Argireline
EEMQRR
VpN1
RRLQQT
VpN2
QAQVDE
VpN3
EVVDIM
VpM1
VNVDKV
VpM2
LERDQK
VpM3
KLSELD
VpC1
QFETSA
VpC2
KLKRKY
VpC3
KYWWKN
C. H. Jung et al. Inhibition of neurotransmitter release
FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3053

A
BC
Fig. 2. Inhibitory effects of the synthetic peptides on SNARE-driven membrane fusion. Peptides were added at 200 lM. (A) Percent of maxi-
mum 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 dis-
tilled water (DW). VpS, soluble domain of VAMP2 (amino acids 1–96). (B) The inhibitory effect of synthetic peptides was converted to ‘% of con-
trol’ (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 ver-
sus 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 inhib-
ited SNARE complex formation. Numbers below the figure are relative band intensities. Asterisks denote peptides dissolved in dimethylsulfox-
ide and should be compared with dimethylsulfoxide as the control; others were dissolved in water and should be compared with distilled water.
Inhibition of neurotransmitter release C. H. Jung et al.
3054 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 previ-
ously been shown that a synthetic peptide from the
C-terminal region of VAMP2 promoted SNARE-medi-
ated 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.
Inhibition of neurotransmitter release from PC12
cells by the synthetic peptides
In order to measure the effects of synthetic peptides on
neuronal exocytosis, we prepared permeabilized PC12
cells loaded with [
3
H]-noradrenaline. Depolarization of
these cells by high K
+
concentrations (50 mm KCl)
results in the release of neurotransmitters such as nor-
adrenaline, acetylcholine and arachidonic acid [38,39].
Inhibition of this release by synthetic peptides could be
assessed by measuring [
3
H]-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 [
3

H]-
noradrenaline release, compared with the basal levels.
We tested the effects of the synthetic peptides on the
high K
+
concentration-stimulated release of [
3
H]-nor-
adrenaline. Addition of SCN, SynN and VpN effi-
ciently 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
[
3
H]-noradrenaline release by the N-terminal-mimick-
ing peptides SCN, SynN and VpN was as much as
20–30%, when compared with controls (Fig. 3A).
Verapamil, an L-type calcium-channel blocker, inhib-
ited neurotransmitter release by  70% at the same
concentration (10 lm) [40,41]. Thus, N-terminal-
mimicking peptides serve as good SNARE-targeting
neuroexocytosis inhibitors. We were not able to test
A
B
C
Fig. 3. Inhibitory effects of the synthetic peptide on [
3
H]-noradrena-
line release in high K

+
-stimulated PC12 cells. (A) The high K
+
-stimu-
lated [
3
H]-noradrenaline release over 15 min was measured in the
presence or absence of synthetic peptides. The amount of [
3
H]-nor-
adrenaline released measured in the presence of an inhibitory pep-
tide was divided by that measured in the absence of the peptide,
where the amount of [
3
H]-noradrenaline release is (cpm of high
K
+
-stimulated sample – cpm of basal level release) per mg of pro-
tein. 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 2 h prior to stimulation with K
+

(50 mM KCl), and then further
incubated for 15 min.
C. H. Jung et al. Inhibition of neurotransmitter release
FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3055
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). Consis-
tent with other results, SCN, SynN and VpN inhibited
SNARE complex formation significantly (Fig. 3B,C).
VpN inhibited SNARE complex formation with a sim-
ilar 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 notice-
able inhibitory effects on SNARE complex formation.
Therefore, the results show that the reduction in neu-
roexocytosis by SCN, VpN and SynN was a direct
consequence of the inhibition of SNARE complex
formation.
Smaller peptides might be beneficial in terms of syn-
thesis costs, as well as membrane penetration. There-
fore, 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-ter-
minal-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 prein-
cubation of t- and v-SNARE vesicles at 4 °C overnight
[3]. SNN and SCC were selected as negative controls
because they did not inhibit membrane fusion and
neurotransmitter release (Figs 2 and 3).
Preincubation of t- and v-SNARE vesicle mixture at
4 °C overnight accelerated the membrane fusion reac-
tion 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-ter-
minal zippering. When membrane fusion was complete,
the amount of SNARE complex was measured by wes-
tern 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 for-
mation very efficiently when added before preincuba-
tion. 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 do not inhibit membrane fusion if the N-ter-
minals 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
+
, inhibi-
tory 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. 4. 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 4 °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.
Inhibition of neurotransmitter release C. H. Jung et al.
3056 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS
A
B
D
C
C. H. Jung et al. Inhibition of neurotransmitter release
FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3057
reduced by N-terminal-mimicking peptides after pre-
treatment 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 calcium-
channel blocker, did not have an additional inhibitory
effect on noradrenaline release. Also, we were able to
confirm that SNARE complex formation was dramati-
cally reduced by N-terminal peptides, whereas other
peptides were almost neutral with regard to SNARE
complex formation in PC12 cells (Fig. 4D). In conclu-
sion, N-terminal peptides inhibit newly forming
SNARE complex in PC12 cells and block the regenera-
tion of neuroexocytosis in synaptic vesicles. We specu-
late that the neurotransmitter release and SNARE
complex band shown in Fig. 3 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
Ca
2+
, leading to the fusion of synaptic vesicles which
are docked at the active zone of neurotransmitter
release [43]. When the PC12 cells were depolarized
with a short-pulse high concentration of K
+
, the syn-
thetic peptides derived from the N-terminal regions of

the SNARE motifs SCN, SynN and VpN inhibited
[
3
H]-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 [
3
H]-noradrenaline release by competing with
parental SNAREs for complex formation.
Recently, it has been shown that N-terminal nucle-
ation of the SNARE complex can promote rapid mem-
brane 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 lipid-
mixing assay, the neurotransmitter release assay in
PC12 cells and western blot analysis consistently con-
firmed 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 inhi-
biting 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

B
C
D
A
X
X
Fig. 5. 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 pro-
foundly reduced SNARE-driven membrane
fusion. (B) In the absence of inhibitory pep-
tide, SNARE complex drives membrane
fusion. (C) Middle- or C-terminal-mimicking
peptides (e.g. VpC in the figure) did not inhi-
bit membrane fusion indicating that the pep-
tide 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-pat-
terned peptides; green, two SNARE motifs
of SNAP-25; blue, SNARE motif of VAMP2
or VAMP2-patterned peptides.
Inhibition of neurotransmitter release C. H. Jung et al.
3058 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS
peptide bound to the middle or C-terminal region of

t-SNARE complex (route C in Fig. 5) is peeled off eas-
ily 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 com-
plex and compete with VAMP2 for binding. Because
Syntaxin 1a and SNAP-25 can form a complex with
2 : 1 stoichiometry, it is likely that syntaxin-patterned
peptides also have their binding sites within
the t-SNARE complex. When SynN is bound to the
1 : 1 t-SNARE complex, however, the structure of
the t-SNARE complex might change to that of the
2 : 1 complex, which is known to be a dead-end prod-
uct (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 com-
plexes 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 inter-
esting. For example, a peptide mimicking the C-termi-
nal domain of SNAP-25 (corresponding to SCC in this
study) reportedly blocked Ca
2+
-dependent exocytosis
in chromaffin cells with an IC
50
of 20 lm [20]. How-
ever, 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 com-
plex formation assays in PC12 cells (Figs 2 and 3).
Previously, LIB was screened by measuring the SDS-
resistant 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 condi-
tions. As such, a potential inhibitory peptide screened
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 condi-
tions. 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 com-
plex 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 mgÆmL
)1
( 250 lm) [24]. However, LIB did not
show any significant effects on membrane fusion and
neurotransmitter release in our study (Figs 2 and 3).
Interestingly, the inhibition of SNARE complex for-
mation 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 com-
plex formation and membrane fusion [14,50]. Mem-

branes 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 membrane-
anchored SNARE complex formation.
Because neuronal exocytosis is triggered on a milli-
second time scale, synaptic vesicles are believed to be
present at the active zone in a ready-to-fire state. How-
ever, we do 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-
C. H. Jung et al. Inhibition of neurotransmitter release
FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS 3059
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-phosphoserine-
N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and 1,2-dioleoyl-sn-
glycero-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 syn-
taxin 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 pro-
teins were purified by affinity chromatography using gluta-
thione–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 syn-
taxin 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 concen-
tration of 50 mm. Protein-free large unilamellar vesicles
( 100 nm dia.) were prepared by extrusion through poly-
carbonate filters (Avanti Polar Lipids Inc., Alabaster, AL,
USA). Syntaxin 1a and SNAP-25 were mixed at room tem-
perature for  60 min to allow the formation of t-SNARE
complex. The t-SNARE complex was then mixed with the
prepared large unilamellar vesicles at a 50 : 1 lipid ⁄ protein
molar ratio. For the v-SNARE vesicles, 10 mm fluorescent
liposomes, containing POPC, DOPS, 1,2-dioleoyl-sn-
glycero-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 : 1 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 4 °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 min to remove protein and lipid aggregates. The
final t-SNARE liposome solution contained  2.5 mm

lipids and 1.9 mgÆmL
)1
of protein, and the v-SNARE lipo-
some solution contained  1mm lipids and 0.25 mgÆmL
)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 1 : 9. The final solution contained 1 mm lipids, in a total
volume of 100 lL, in the presence of synthetic peptide. Flu-
orescence was measured at excitation and emission wave-
lengths of 465 and 530 nm, respectively. Changes in
fluorescence were recorded with a Spectra Max M2 (Molec-
ular Devices Inc., Palo Alto, CA, USA) fluorescence spec-
trophotometer. The maximum fluorescence intensity was
obtained by adding Triton X-100. All lipid mixing experi-
ments 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 contain-
ing 100 lgÆmL
)1
streptomycin, 100 UÆmL
)1

penicillin, 2 mm
l-glutamine, 10% heat-inactivated horse serum and 5% fetal
bovine serum at 37 °C in a 5% CO
2
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 ngÆmL
)1
; Invitrogen, Carlsbad, CA, USA) for 5 days
prior to [
3
H]-noradrenaline uptake and release.
Inhibition of neurotransmitter release C. H. Jung et al.
3060 FEBS Journal 275 (2008) 3051–3063 ª 2008 The Authors Journal compilation ª 2008 FEBS
Determination of [
3
H]-noradrenaline release from
detergent-permeabilized PC12 cells
The amount of secreted [
3
H]-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 4 · 10
5
cellsÆdish
)1
. After the cells adhered to
the plates (20–24 h), they were preincubated with peptides

and [
3
H]-noradrenaline (1 lCiÆmL
)1
)at37°C for 90 min in
Krebs ⁄ Hepes solution (140 mm NaCl, 4.7 mm KCl, 1.2 mm
KH
2
PO
4
, 2.5 mm CaCl
2
, 1.2 mm MgSO
4
,11mm 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 KH
2
PO
4
, 2.5 m m CaCl
2
,
1.2 mm MgSO
4

,11mm glucose and 15 mm Hepes ⁄ Tris,
pH 7.4) for 15 min to assess the stimulated release. Extra-
cellular media were transferred to scintillation vials and
then measured by liquid scintillation counting. The amount
of [
3
H]-noradrenaline release was calculated according to
the following equation: amount of [
3
H]-noradrenaline relea-
se = (cpm of high K
+
-stimulated sample – cpm of basal
level release) per mg of protein.
Immunoblots
Cells were washed twice with ice-cold NaCl ⁄ P
i
after treat-
ment, 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 min at 4 °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 trans-
ferred to a nitrocellulose membrane. The membranes were
then blocked with NaCl ⁄ P
i
⁄ 0.1% Tween 20 (NaCl ⁄ P

i
-T
buffer) containing 1% skim milk and 1% BSA for 1 h at
room temperature. Thereafter, the membranes were incu-
bated overnight at 4 °C with a 1 : 1000 dilution of anti-
(SNAP-25) mAb. Membranes were washed three times
with NaCl ⁄ P
i
-T and further incubated with a 1 : 1000
dilution of horseradish peroxidase-conjugated secondary
antibodies for 1 h at room temperature. Membranes were
then washed extensively three more times with NaCl ⁄ P
i
-T
and developed using an enhanced chemiluminescence
solution.
Statistical analysis
All experimental data were examined by analysis of vari-
ance (anova) procedures, and significant differences among
the means from triplicate determinations were assessed
using Duncan’s multiple range tests and the Statistical
Analysis System, version 8.2 (SAS Institute Inc., Cary, NC,
USA).
Acknowledgement
This study was supported by a Korea Research Foun-
dation Grant (KRF-2004-C00197).
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