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The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion.

Lu B - PeerJ (2015)

Bottom Line: The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25.CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants.We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Membrane Biology, University of Virginia , Charlottesville, VA , USA.

ABSTRACT
Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

No MeSH data available.


Related in: MedlinePlus

Lipid mixing assay for the nitroxide spin-labeled of SNAP-25 mutants.The curves represent the lipid mixing when the wild-type or nitroxide spin-labeled SNAP-25 at positions of 181 and 188 were used in the proteoliposome fusion assay (protein/lipid ratio 1:200). The data were normalized against the maximum fluorescence intensity (MFI) obtained by adding 0.1% reduced Triton-X-100. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without SNAP-25 (grey curve). The inset in the left corner is the electron micrograph of the SNAREs-reconstituted vesicles used in assays. The vesicles were stained with 1% phosphotungstic acid on the carbon grids. The size of the vesicles was 90 ± 15 nm.
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fig-3: Lipid mixing assay for the nitroxide spin-labeled of SNAP-25 mutants.The curves represent the lipid mixing when the wild-type or nitroxide spin-labeled SNAP-25 at positions of 181 and 188 were used in the proteoliposome fusion assay (protein/lipid ratio 1:200). The data were normalized against the maximum fluorescence intensity (MFI) obtained by adding 0.1% reduced Triton-X-100. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without SNAP-25 (grey curve). The inset in the left corner is the electron micrograph of the SNAREs-reconstituted vesicles used in assays. The vesicles were stained with 1% phosphotungstic acid on the carbon grids. The size of the vesicles was 90 ± 15 nm.

Mentions: The EPR lineshape analysis revealed that the C-terminal of SNAP-25 SN2 in the binary complex is partially free, and that the sharp components can be detected even in the ternary complex. One might then wonder whether the formation of the completely assembled SNARE complex is required for membrane fusion. To address this question, we examined the fusion activity of the nitroxide spin-labeled SNAP-25 using the fluorescence lipid mixing assay. The SNAREs reconstituted vesicles that were employed in the study are shown in Fig. 3. The nitroxide side chain was relatively bulky, similar in size to that of tryptophan. Therefore, if the formation of the completed coiled coil is necessary for membrane fusion, the alterations at the internal positions (‘a’ or ‘d’) (Poirier et al., 1998) might cause some serious perturbations. As Fig. 3 shows, we detected the distinct lipid mixing activity decreasing with the introduction of the mutations, as compared to the wild-type SNAP-25. Moreover, when the spin-labeled position was closer to the central of SNAP-25 SNARE motif, the perturbation was stronger. SNAP-25 C188 showed a 78% fusion activity of wild-type SNAP-25, while SNAP-25 C181 was just left of 40%. Therefore, our data confirm that SN2 of SNAP-25 is required for activating membrane fusion.


The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion.

Lu B - PeerJ (2015)

Lipid mixing assay for the nitroxide spin-labeled of SNAP-25 mutants.The curves represent the lipid mixing when the wild-type or nitroxide spin-labeled SNAP-25 at positions of 181 and 188 were used in the proteoliposome fusion assay (protein/lipid ratio 1:200). The data were normalized against the maximum fluorescence intensity (MFI) obtained by adding 0.1% reduced Triton-X-100. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without SNAP-25 (grey curve). The inset in the left corner is the electron micrograph of the SNAREs-reconstituted vesicles used in assays. The vesicles were stained with 1% phosphotungstic acid on the carbon grids. The size of the vesicles was 90 ± 15 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493708&req=5

fig-3: Lipid mixing assay for the nitroxide spin-labeled of SNAP-25 mutants.The curves represent the lipid mixing when the wild-type or nitroxide spin-labeled SNAP-25 at positions of 181 and 188 were used in the proteoliposome fusion assay (protein/lipid ratio 1:200). The data were normalized against the maximum fluorescence intensity (MFI) obtained by adding 0.1% reduced Triton-X-100. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without SNAP-25 (grey curve). The inset in the left corner is the electron micrograph of the SNAREs-reconstituted vesicles used in assays. The vesicles were stained with 1% phosphotungstic acid on the carbon grids. The size of the vesicles was 90 ± 15 nm.
Mentions: The EPR lineshape analysis revealed that the C-terminal of SNAP-25 SN2 in the binary complex is partially free, and that the sharp components can be detected even in the ternary complex. One might then wonder whether the formation of the completely assembled SNARE complex is required for membrane fusion. To address this question, we examined the fusion activity of the nitroxide spin-labeled SNAP-25 using the fluorescence lipid mixing assay. The SNAREs reconstituted vesicles that were employed in the study are shown in Fig. 3. The nitroxide side chain was relatively bulky, similar in size to that of tryptophan. Therefore, if the formation of the completed coiled coil is necessary for membrane fusion, the alterations at the internal positions (‘a’ or ‘d’) (Poirier et al., 1998) might cause some serious perturbations. As Fig. 3 shows, we detected the distinct lipid mixing activity decreasing with the introduction of the mutations, as compared to the wild-type SNAP-25. Moreover, when the spin-labeled position was closer to the central of SNAP-25 SNARE motif, the perturbation was stronger. SNAP-25 C188 showed a 78% fusion activity of wild-type SNAP-25, while SNAP-25 C181 was just left of 40%. Therefore, our data confirm that SN2 of SNAP-25 is required for activating membrane fusion.

Bottom Line: The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25.CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants.We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Membrane Biology, University of Virginia , Charlottesville, VA , USA.

ABSTRACT
Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

No MeSH data available.


Related in: MedlinePlus