KR20090041066A - Synthetic peptide modulating neurotransmitter release - Google Patents

Abstract

A synthetic peptide which is similar to a kernel composite of SNARE(Soluble ethylmaleimide-sensitive factor attachment protein) is provided to control a neutrotransmitter release by hindering the formation of SNARE composite and to ensure the control of the intercellular reaction, improvement of skin wrinkle and pain relieving effects. A peptide fragment controlling a neutrotransmitter release by suppressing the formation of SNARE composite is the whole(SEQ ID NO:2) of peptides configuring N-end part of the core domain of syntaxin 1a protein forming the SNARE composite or a part of peptides; the whole(SEQ ID NO:4) of peptides configuring N-end part of C-helix of SNAP-25 forming the SNARE composite or a part of peptides; and the whole(SEQ ID NO:8) of peptides configuring N-end part of core domain from VAMP-2 protein forming the SNARE composite or a part of peptides.

Description

Synthetic Peptide modulating neurotransmitter release

The present invention relates to synthetic peptides that regulate neurotransmitter release.

 The release of neurotransmitters is caused by the formation of a pathway between the two boundaries after the synaptic vesicle and the presynaptic membrane are fused at the nerve endings containing the neurotransmitter. The vesicle protein VAMP (synaptobrevin) and the plasma membrane binding protein Syntaxin 1a and SNAP-25 are three protein complexes, the SNARE protein, which provides a fundamental force in the fusion of synaptic vesicles and synaptic membranes. Here, the neurotransmitter release pathway is opened by membrane fusion between synaptic vesicles and the synaptic membrane, t-SNARE, a complex of two syntaxin 1a proteins and SNAP-25 protein attached to a target membrane. And the effect of v-SNARE attached to the vesicle. When the membrane is fused, rearrangement of the lipid bilayer occurs. Since the biofilms push strongly against each other, these membranes are not spontaneously fused and have a strong force from outside to overcome the repulsion between the membranes, which is known to provide SNARE protein. As such, the formation of the SNARE complex is a key phenomenon of extracellular excretion, including the release of neurotransmitters.

 Botulinum neurotoxin, which is currently used as an injection for skin wrinkle improvement, is a protease that cleaves SNARE protein, a key protein involved in neurotransmitter release.It cuts SNARE protein to block neurotransmission and consequently invades Botox. Numb the muscle cells. While Botox, which is being used as an injection, blocks nerve transmission by irreversibly cutting the SNARE protein, the so-called "coating Botox" is a kind of "competitive inhibitor" that inhibits neurotransmission by inhibiting the formation of the SNARE complex. A key component of “Bortox” is a hexapeptide called Argireline and has the amino acid sequence of EEMQRR (Blanes-Mira et al., International Journal of Cosmetic Science. 24, 303-310, 2002). This is the site that matches the amino acid sequence of the N-terminal region of SNAP-25. In other words, a small hexapeptide is instead inserted at the site where the intact SNAP-25 should bind, thereby preventing the intact SNAP-25 from binding to other SNARE proteins, syntaxins 1a and VAMP-2. do. However, argireline, the main component of the above-mentioned Botox product, has a disadvantage in that its effect is uncertain.

Accordingly, the present inventors estimated that the N-terminal regions of these specific domains are involved in complex formation in the three proteins forming the SNARE complex, and synthesized peptides constituting these N-terminal ends to confirm neurotransmitter release. . As a result, the present invention was completed by confirming that the peptide constituting the N-terminus of each of the three proteins constituting the SNARE complex effectively regulated the release of neurotransmitters.

It is an object of the present invention to provide synthetic peptides that inhibit the formation of SNARE complexes involved in neurotransmitter release. In addition, an object of the present invention is to provide a neurotransmitter inhibitor composition comprising the synthetic peptide as an active ingredient.

In order to achieve the above object, the present inventors synthesized any peptide from three proteins involved in SNARE complex formation, ie, syntaxin 1a, SNAP-25 and VAMP-2. Syntaxin 1a and VAMP-2 provide one core helix each, and SNAP-25 constitutes a SNARE complex by providing two core helixes, the N-terminal helix and the C-terminal helix. In the syntaxin 1a protein, a SynN peptide corresponding to the N-terminus of the core domain, a SynM peptide corresponding to the middle region of the core domain and a SynC peptide corresponding to the C-terminus of the core domain of the syntaxin 1a protein were synthesized. From the SNAP-25 protein, including the SNN corresponding to the N-terminus of the N-helix, the SNM peptide corresponding to the middle region of the core domain of the SNAP-25 N-helix and the SNC peptide corresponding to the C-terminus of the core domain Was synthesized. In addition, from the SNAP-25 protein, an SCM peptide in the middle portion and an SCC peptide in the C-terminal portion were synthesized, including the SCN corresponding to the N-terminal portion of the C-helix. VpM peptides and VpC peptides were synthesized from the VAMP-2 protein, including VpN corresponding to the N-terminus of the core domain.

The synthetic peptide was used to investigate whether membrane fusion was inhibited, and whether the neurotransmitter was inhibited in the PC12 cell line and SNARE complex formation was inhibited. In addition, argireline peptides and other peptides Lib. (SAAEAFAKLYAEAFAKG, Blanes-Mira C, Pastor MT, Valera E, Fernandez-Ballester G, Merino JM, Gutierrez LM, Perez-Paya E & Ferrer-Montiel A) have been reported in the literature. (2003) Identification of SNARE complex modulators that inhibit exocytosis from an alpha-helix-constrained combinatorial library.Biochem J 375 , 159-166.

The synthetic peptide composition according to the present invention is useful as an inhibitor of SNARE complex formation, a modulator of intracellular response involving SNARE by regulating neurotransmitter release, and for skin wrinkle improvement and pain relief. In addition, since the synthetic peptide is chemically synthesized, the quality can be controlled, and as a result, the reproducibility of the test results is ensured.

As three proteins involved in SNARE complex formation, the amino acid sequences of syntaxin 1a, SNAP-25 and VAPMP-2 proteins are shown in FIGS. 1, 2 and 3, respectively.

In FIG. 1, syntaxin 1a consists of 288 amino acids and consists of a Habc domain, a H3 domain (core domain), and a transmembrane domain. SynN peptides consist of an amino acid sequence representing the N-terminal end (SEQ ID NO: 2) of the core domain of the syntaxin 1a protein. (See FIG. 5)

In FIG. 2, SNAP-25 has two core helixes connected in a long loop, and SCN peptide is an amino acid representing the N-terminal (SEQ ID NO: 5) region of SNAP-25 protein C-helix. Consists of sequences. (See FIG. 5)

In FIG. 3, the VAMP-2 protein consists of 116 amino acids and consists of a non-structural region, a core helix and a transmembrane domain, and the VpN peptide is an amino acid sequence representing the N-terminal (SEQ ID NO: 8) region of the core domain. It consists of. With respect to the syntaxin 1a protein, three peptides (SynN, SynM, and SynC) were synthesized. Six peptides (SNN, SNM, SNC and SCN, SCM, SCC) were synthesized with respect to SNAP-25. Three peptides (VpN, VpM and VpC) were synthesized from VAMP-2. (See FIG. 5)

M로 가하고 형광강도를 측정하였다. (도 7a 내지 7c 참조) Using the synthetic peptide of the present invention, in order to confirm the membrane fusion inhibitory effect, a fluorescent material labeled liposomes were prepared. A solution containing a mixture of SNAP-25, syntaxin 1a, and liposomes, and a liposome prepared by adding rhodamine as a VAMP-2 and a fluorescent substance was obtained at 9: 1. Next, the peptide 200 synthesized in the present invention

M was added and the fluorescence intensity was measured. (See Figures 7A-7C)

Inhibition of neurotransmitter release in PC12 cells was investigated. After incorporating the noradrenaline assay buffer and the synthetic peptide of the present invention into a PC12 cell line, it was confirmed whether noradrenaline was released. (See FIG. 8)

In the present invention, three peptides that inhibit the formation of SNARE complex among the synthetic peptides to be tested for efficacy and are most effective in regulating neurotransmitter release were selected as follows.

SynN (LSEIETRHSEIIKLENS, SEQ ID NO: 3), SCN (DARENEMDENLEQVSGI, SEQ ID NO: 6) and VpN (RRLQQTQAQVDEVVDIM, SEQ ID NO: 9) in the N-terminal region were effective in inhibiting neurotransmitter release. Herein, SCN, SynN and VpN according to the present invention are not limited thereto because they are simply representative amino acid sequences.

 The synthetic peptide composition of the present invention contains ingredients commonly used in cosmetics and pharmaceuticals in addition to the synthetic peptides described herein as active ingredients. Such ingredients include, but are not limited to, conventional auxiliaries and carriers such as, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and flavorings.

Synthetic peptide compositions of the present invention can be prepared in any formulation conventionally prepared in the art, such as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, It may be formulated as a powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto.

When the formulations of the present invention are pastes, creams and gels, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc and zinc oxide may be used as carrier components. It is possible, but not limited to.

When the formulations of the present invention are powders and sprays, lactose, talc, silica, aluminum hydroxide, calcium silicate and polyamide powders can be used as carrier components, especially in the case of sprays additionally chlorofluorohydrocarbons, propane Propellants such as butane and dimethyl ether may be included, but are not limited thereto.

When the formulations of the present invention are solutions and emulsions, solvents, solubilizers and emulsions are used as carrier components, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic esters, polyethylene glycols and sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol and propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals Castle cellulose, aluminum metahydroxy, bentonite, agar and tracant and the like can be used, but are not limited thereto.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidoventins, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives and ethoxylated glycerol fatty acid esters and the like can be used, but are not limited thereto.

Hereinafter, the present invention will be described in more detail based on the following examples. The following examples are intended to illustrate the invention, not to limit the invention. All documents disclosed in the present invention are incorporated by reference.

Experimental material

1-palmitoyl-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), 1,2- Dioleoyl-sn-glycero-3-phosphoseline-N- (7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PS) and 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N- (rissamine rhodamine B sulfenyl) (rhodamine-PE) was purchased from Avanti Polar Lipid (Alabama, USA).

RPMI 1640, 10% fetal calf serum, 5% fetal bovine serum was purchased from GIBCO-BRL (Grand Island, NY, USA). Argirerin (6-mer peptide, EEMQRR) and LIB (17-mer peptide, SAAEAFAKLYAEAFAKG) were used as controls. Synthetic peptide having a pattern of the SNARE model of the present invention was commissioned to peptron (Daejeon, Korea) as a 17-mer peptide designed directly through a rational design. The synthetic peptide has a purity of 95% or more and dissolved in distilled water or DMSO.

Example 1: Expression of SNARE Protein

The pGEX-2T vector (GE Healthcare, Inc.) with amino-terminal glutathione S-transferase (GST) -tag cleaved with thrombin was used for the protein of the invention, ie full-length syntaxin 1a (FIG. 1). , SEQ ID NO: 1), SNAP-25 (FIG. 2, SEQ ID NO: 4), full length VAMP-2 (FIG. 3, SEQ ID NO: 7), a soluble SNARE motif of syntaxin 1a (SynH3, FIG. 1, SEQ ID NO: 10) and soluble SNARE motif (VpS, FIG. 3, SEQ ID NO: 11) of VAMP. All recombinant proteins were expressed in E. coli (DE3, Novagen). All N-terminal GST tagged proteins were purified by affinity chromatography using glutathione agarose beads. Proteins were cleaved with thrombin in digestion buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) and 12.5% n-octyl-D-glucopyranoside (OG) was added to syntaxin 1a and VAMP-2. . Purified protein was electrophoresed on Sodhit Dodecyl Sulfate-Polyacrylamide (SDS-PAGE) to confirm that all proteins were 90% pure.

Example 2: Membrane Fusion Inhibitory Effect

In order to search for membrane fusion inhibitors, the effects of membrane fusion inhibition on the synthetic peptides of the present invention were tested.

M 농도로 가하고, 형광강도(모델명: SpectraMax M2, 제조사: Molecular Device)를 측정했다. 결과는 도 7a, 도 7b 및 도 7c에 도시하였다. 도 7a, 도 7b 및 도 7c에서 형광강도는 SNARE 단백질간의 막 융합 현상을 의미하므로 보다 더 낮은 형광강도는 막융합 저해 효과가 우수함을 나타낸다.To prepare liposomes, which are microencapsulated particles labeled with phosphors, POPC (62 mol%), DOPS (35 mol%), NBD-PS (1.5 mol%) and phosphor Rhodamine (PE, 1.5 mol%) ) Was mixed to prepare a 10 mM liposome (v-vesicle). Next, in order to prepare liposomes that are not labeled with fluorescent material, 50 mM liposomes were prepared by mixing DOPS and POPC in a molar concentration of 35:65. In order to prepare a complex of purified SNAP-25 and syntaxin 1a, they were mixed so that their molarity was 1: 1 and reacted at room temperature for 1 hour, so that the molar ratio of liposomes with no fluorescent substance labeled with 100: 1 would be obtained. After mixing, VAMP-2 was bound to a molar ratio of 50: 1 with liposomes containing fluorescent material. Subsequently, dialysis of the two liposomes followed by mixing at a ratio of 3: 7 (v-vesicle: t-vesicle), followed by synthesis of the present invention into the above mixture comprising SNARE-25, syntaxin 1a and VAMP-2 Peptide 200

M concentration was added, and fluorescence intensity (model name: SpectraMax M2, manufacturer: Molecular Device) was measured. The results are shown in Figures 7a, 7b and 7c. 7A, 7B, and 7C, the fluorescence intensity means a membrane fusion phenomenon between SNARE proteins, and thus, lower fluorescence intensity shows an excellent membrane fusion inhibitory effect.

In FIG. 7A and FIG. 7B, the solid red line represents the path through which the control reaction proceeds. FIG. 7C is a bar graph of the results of FIGS. 7A and 7B calculated based on the solid red line, and Con. Is methanol in FIGS. 7A and 7B. About 40 to 60% of the response of the membrane control was expressed as a reference, and statistical treatment was performed by ANOVA (Analysis of variance). Significant differences between the three measured data averages were represented by Duncan's mutiple range test and statistical system (version 8.2, SAS, USA), with SCN, SynN and VpN deteriorating fusion. The effect of letting is recognized. In contrast, Lib. Peptides had a very slight effect.

Example 3: Inhibition of Neurotransmitter Release in PC12 Cells

Ci/ml), 합성 펩타이드와 같이 첨가한 후 탄산가스 배양기에서 90분간 도입하였다. 반응 후 배지를 제거한 후 고농도의 K⁺ 완충액(115 mM NaCl, 30 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 11mM 글루코스, 15 mM HEPES-tris, pH 7.4)을 넣은 후 15분간 탄산가스 배양기에서 배양하고, 상등액을 회수하여 [³H]-아드레날린의 방출을 저해하는지를 신틸레이션 계수관으로 측정하였다. 상기의 방법으로 합성 펩타이드가 PC12 세포내에서 도파민 유도체인 노르아드레날린의 방출을 저해하는지를 확인하고, 그 결과를 도 8에 제시하였다. 도 8에서 Con.은 메탄올이고, CPM은 Count Per minute을 의미한다. 그 결과는 도 7a, 도 7b 및 도 7c의 실험결과와 일치하였으며, SCN, SynN 및 VpN이 PC12 세포내에서 도파민 유도체인 노르아드레날린의 방출을 저해하는 효과가 상대적으로 높게 측정되었다. 이에 반하여 아르기렐린 및 Lib 펩타이드는 매우 미미한 효과를 나타내었다.PBS (Phosphate) after aspirating a medium containing 10% fetal calf serum, 5% fetal bovine serum and antibiotics to collagen-coated plates (60 mm dish) of PC12 cells (Korea Cell Line Bank) Buffered 2 ml of Saline) was added and pipetted to separate the cells from the dish wall, and the cells were collected by centrifugation at 1,000 × g for 5 minutes. Cell pellets were then dispersed by pipetting with fresh RPMI 1640 medium, and then placed in fresh culture plates and subcultured in an incubator fed with 37 ° C. and 5% CO ₂ gas. [ ³ H] -noradrenaline used in the experiment was purchased from Amersham. After inhaling the medium from the plate of PC12 cells, PBS was added, the cells were removed from the plate wall, and the number of cells was measured using a hematocytometer and dispersed in fresh medium at a concentration of 2 × 10 ⁵ cells / ml and inoculated. Digitonin (C ₅₆ H ₉₂ O ₂₉ ) was added to noradrenaline assay buffer ([ ³ H] -NA, 1.5 to increase cell permeability).

Ci / ml), and then added together with the synthetic peptide and introduced in a carbon dioxide gas incubator for 90 minutes. After the reaction, the medium was removed and a high concentration of K ⁺ buffer (115 mM NaCl, 30 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, 15 mM HEPES-tris, pH 7.4) was added and carbonated for 15 minutes. Incubation was carried out in a gas incubator, and the supernatant was recovered to determine whether it inhibited the release of [ ³ H] -adrenaline using a scintillation counter. It was confirmed whether the synthetic peptides inhibit the release of the dopamine derivative noradrenaline in PC12 cells by the above method, and the results are shown in FIG. 8. In FIG. 8, Con. Is methanol and CPM means Count Per minute. The results were in agreement with the experimental results of FIGS. 7A, 7B and 7C, and the effects of SCN, SynN, and VpN inhibiting the release of the dopamine derivative noradrenaline in PC12 cells were relatively high. In contrast, argireline and Lib peptides showed very slight effects.

Example 4: Inhibition of SNARE Complex Formation in PC12 Cells

PC12 cells were cultured as in Example 3. After inhaling the medium from the plate of PC12 cells, PBS was added, the cells were removed from the plate wall, and the number of cells was measured by a hematocytometer, and the cells were inoculated by dispersing in a fresh medium at a concentration of 2 ㅧ 10 ⁶ cell / 100π. After 24 hours, dikitonin and synthetic peptide were added to increase cell permeability and reacted for 2 hours. After removing the medium, the high concentration of the K ⁺ buffer, and then incubated in a carbon dioxide gas incubator for 15 minutes, the supernatant was removed and the cell membrane was separated using a lysis buffer. The separated cell membrane proteins were subjected to electrophoresis using 5 ~ 21% SDS-PAGE gel. The electrotransfer was performed as follows. The NC membrane (Nitrocellulose membrane) was immersed in the transfer buffer for 5 minutes and placed on a cassette, and the gel to be transferred was slightly wetted with transfer buffer and placed on the membrane. Prepare a transfer by preparing a gel sandwich (gel sandwich), transfer for 1 hour at 100 V, then disassemble the device, dye the transferred gel in Ponceau S solution (Sigma-Altrik) for 5 minutes and stained with distilled water Washed to see the transferred band. The dyed Ponceau S was completely decolorized with PBST and blocked by blocking for 1 hour (room temperature) in blocking buffer. After washing with PBST, the primary antibody (SNAP-25) was added, stirred at 4 ° C. overnight, washed twice with PBST for 5 minutes, and then the secondary antibody (Goth anti-mouse IgG and peroxidase was combined and affinity was isolated. Antibody, No. A4416, Lot. 125K6059, Sigma) was diluted with a blocking solution according to the recommended dilution ratio (1: 2000) and then reacted with stirring for 1 hour at room temperature. After the reaction, washed three times with PBST and then mixed in an enhanced chemiluminescense (ECL) kit to react evenly to the membrane to detect.

It was confirmed whether the synthetic peptides inhibit SNARE complex formation in PC12 cells by the above method, and the results are shown in FIGS. 9A and 9B. In FIG. 9A and FIG. 9B, SNARE complex 1 is a SNARE complex consisting of one each of syntaxin 1a, SNAP-25, and VAMP-2, and SNARE complex 2 is a complex in which the SNARE complex is large through domain swapping. Accordingly, the results were consistent with the experimental results of Examples 2 and 3 of the present invention, and the peptide SCN, SynN and VpN inhibitory effects of inhibiting SNARE complex formation in PC12 cells were relatively high.

Although the above has been described with reference to a preferred embodiment of the present invention, those skilled in the art will be able to variously modify or change the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated.

While the effects of argireline, the main ingredient of current Botox products, are uncertain and have disadvantages of causing skin side effects, the synthetic peptides according to the present invention are chemically synthesized, so that the quality can be controlled, and as a result The reproducibility of the test results is guaranteed, and the industrial use effect is large.

1 depicts the amino acid sequence of syntaxin 1a protein among the three proteins involved in SNARE complex formation.

Figure 2 shows the amino acid sequence of the SNAP-25 protein among the three proteins involved in SNARE complex formation.

Figure 3 shows the amino acid sequence of the VAMP-2 protein among the three proteins involved in SNARE complex formation.

4 illustrates complex formation and membrane fusion of SNARE proteins.

Figure 5 shows the amino acid sequence of the 17-mer synthetic peptide according to the present invention.

Figure 6a shows the domain structure of recombinant SNARE protein used in the neurotransmitter inhibition experiment of the present invention. 6B shows the result of SDS-PAGE analysis of recombinant SNARE protein.

Figures 7a, 7b and 7c are measured by the fluorescence intensity of the results of membrane fusion phenomenon to search for inhibitors of SNARE complex formation of the synthetic peptide of the present invention.

Figure 8 is a synthetic peptide of the present invention was measured by the scintillation counting the results of the inhibition of noradrenaline release as a neurotransmitter in PC12 cells.

Figure 9a shows the results of Western blot using the antibody SNAP-25 the synthetic peptide of the present invention results in inhibition of SNARE complex formation in PC12 cells.

9b shows the Western blot results in a bar graph.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Synthetic Peptide modulating neurotransmitter release <130> IPM-34420 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Syntaxin 1a <400> 1 Met Lys Asp Arg Thr Gln Glu Leu Arg Thr Ala Lys Asp Ser Asp Asp   1 5 10 15 Asp Asp Asp Val Thr Val Thr Val Asp Arg Asp Arg Phe Met Asp Glu              20 25 30 Phe Phe Glu Gln Val Glu Glu Ile Arg Gly Phe Ile Asp Lys Ile Ala          35 40 45 Glu Asn Val Glu Glu Val Lys Arg Lys His Ser Ala Ile Leu Ala Ser      50 55 60 Pro Asn Pro Asp Glu Lys Thr Lys Glu Glu Leu Glu Glu Leu Met Ser  65 70 75 80 Asp Ile Lys Lys Thr Ala Asn Lys Val Arg Ser Lys Leu Lys Ser Ile                  85 90 95 Glu Gln Ser Ile Glu Gln Glu Glu Gly Leu Asn Arg Ser Ser Ala Asp             100 105 110 Leu Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg Lys Phe Val         115 120 125 Glu Val Met Ser Glu Tyr Asn Ala Thr Gln Ser Asp Tyr Arg Glu Arg     130 135 140 Cys Lys Gly Arg Ile Gln Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr 145 150 155 160 Thr Ser Glu Glu Leu Glu Asp Met Leu Glu Ser Gly Asn Pro Ala Ile                 165 170 175 Phe Ala Ser Gly Ile Ile Met Asp Ser Ser Ile Ser Lys Gln Ala Leu             180 185 190 Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Asn Ser         195 200 205 Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu Val Glu     210 215 220 Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu His Ala 225 230 235 240 Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala Val Lys                 245 250 255 Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile Met Ile Ile Is Cys Cys             260 265 270 Val Ile Leu Gly Ile Ile Ile Ala Ser Thr Ile Gly Gly Ile Phe Gly         275 280 285 <210> 2 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> N-terminal Peptide of Syntaxin 1a <400> 2 Leu Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Asn   1 5 10 15 Ser Ile Arg Glu Leu His Asp Met Phe Met              20 25 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> SynN <400> 3 Leu Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Asn   1 5 10 15 Ser     <210> 4 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> SNAP-25 <400> 4 Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg   1 5 10 15 Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met              20 25 30 Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val          35 40 45 Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met      50 55 60 Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp  65 70 75 80 Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys                  85 90 95 Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val             100 105 110 Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala         115 120 125 Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn     130 135 140 Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Gly Asn Leu 145 150 155 160 Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg                 165 170 175 Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile             180 185 190 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly         195 200 205 <210> 5 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> N-terminal Peptide of SNAP-25 <400> 5 Asp Ala Arg Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly   1 5 10 15 Ile Ile Gly Asn Leu Arg His Met Ala Leu              20 25 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> SCN <400> 6 Asp Ala Arg Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly   1 5 10 15 Ile     <210> 7 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VAMP-2 <400> 7 Met Ser Ala Thr Ala Ala Thr Val Pro Pro Ala Ala Pro Ala Gly Glu   1 5 10 15 Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Asn Arg Arg Leu              20 25 30 Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg Val          35 40 45 Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Asp      50 55 60 Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser  65 70 75 80 Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met                  85 90 95 Ile Ile Leu Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile Val             100 105 110 Tyr Phe Ser Thr         115 <210> 8 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> N-terminal Peptide of VAMP-2 <400> 8 Asn Leu Thr Ser Asn Arg Arg Leu Gln Gln Thr Gln Ala Gln Val Asp   1 5 10 15 Glu Val Val Asp Ile Met Arg              20 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VpN <400> 9 Arg Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile   1 5 10 15 Met     <210> 10 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> SyNH3 <400> 10 Ala Leu Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu   1 5 10 15 Asn Ser Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu              20 25 30 Val Glu Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu          35 40 45 His Ala Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala      50 55 60 Val Lys Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile  65 70 75 <210> 11 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> VpS <400> 11 Met Ser Ala Thr Ala Ala Thr Val Pro Pro Ala Ala Pro Ala Gly Glu   1 5 10 15 Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Asn Arg Arg Leu              20 25 30 Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg Val          35 40 45 Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Asp      50 55 60 Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser  65 70 75 80 Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met                  85 90 95  

Claims (6)

The entire peptide (SEQ ID NO: 2) or part thereof constituting the N-terminus of the core domain of the syntaxin 1a protein forming a SNARE complex; The entire peptide (SEQ ID NO: 4) or a portion thereof constituting the N-terminus of the C-helix of the SNAP-25 protein forming a SNARE complex; Or a peptide fragment that regulates neurotransmitter release by inhibiting SNARE complex formation, which is part or all of the peptide corresponding to the N-terminal end of the core domain from the VAMP-2 protein forming the SNARE complex. The peptide fragment according to claim 1, which has an amino acid sequence of SEQ ID NO: 3, an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence of SEQ ID NO: 9. A composition comprising the peptide fragment of claim 1 or 2 as an active ingredient to inhibit neurotransmitter release by inhibiting the formation of the SNARE complex. Pain relief composition containing the peptide fragment of claim 1 or 2 as an active ingredient. A composition for improving skin wrinkles comprising the peptide fragment of claim 1 or 2 as an active ingredient. Composition for the regulation of intracellular responses containing the peptide fragment of claim 1 or 2 as an active ingredient.

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