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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 truncated SNARE complex.Fluorescence changes normalized against the maximum fluorescence intensity (MFI) are shown for the lipid: protein molar ratio of 100:1 (A) and 200:1 (B). Wild-type SNAP-25 and the truncated SNAP-25A (Δ9) and SNAP-25E (Δ26) are used in the proteoliposome fusion assay. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without the SNAP-25 (black curve).
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fig-5: Lipid mixing assay for the truncated SNARE complex.Fluorescence changes normalized against the maximum fluorescence intensity (MFI) are shown for the lipid: protein molar ratio of 100:1 (A) and 200:1 (B). Wild-type SNAP-25 and the truncated SNAP-25A (Δ9) and SNAP-25E (Δ26) are used in the proteoliposome fusion assay. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without the SNAP-25 (black curve).

Mentions: The less stable truncated SNARE complexes, due to the truncated SNAP-25, may decrease their fusion activity. To test this idea, we used the fluorescence lipid mixing assay to detect the function of the truncated SNAP-25. As Figs. 5A and 5B show, the truncated SNAP-25E (Δ26) formed SNARE complex totally abolished the function to induce membrane fusion, just as in the case of negative control, which has no SNAP-25. The SNAP-25A (Δ9) mutant had only about 6% of maximum fluorescence intensity (MFI), while the wild-type SNAP-25 had about 20% of MFI. So, it is clear that the truncated SNAP-25A lost most of its function, although it could still form the SDS-resistant SNARE complex (Fig. 4B). Our lipid mixing results were also in agreement with the in vivo studies, which showed the same tendency of different membrane fusion activities in neurotoxins treated spinal cord cell cultures (Bergey, Bigalke & Nelson, 1987; Williamson, Fitzgerald & Neale, 1992).


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 truncated SNARE complex.Fluorescence changes normalized against the maximum fluorescence intensity (MFI) are shown for the lipid: protein molar ratio of 100:1 (A) and 200:1 (B). Wild-type SNAP-25 and the truncated SNAP-25A (Δ9) and SNAP-25E (Δ26) are used in the proteoliposome fusion assay. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without the SNAP-25 (black curve).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-5: Lipid mixing assay for the truncated SNARE complex.Fluorescence changes normalized against the maximum fluorescence intensity (MFI) are shown for the lipid: protein molar ratio of 100:1 (A) and 200:1 (B). Wild-type SNAP-25 and the truncated SNAP-25A (Δ9) and SNAP-25E (Δ26) are used in the proteoliposome fusion assay. The control runs with the v-SNARE vesicles and the t-SNARE vesicles without the SNAP-25 (black curve).
Mentions: The less stable truncated SNARE complexes, due to the truncated SNAP-25, may decrease their fusion activity. To test this idea, we used the fluorescence lipid mixing assay to detect the function of the truncated SNAP-25. As Figs. 5A and 5B show, the truncated SNAP-25E (Δ26) formed SNARE complex totally abolished the function to induce membrane fusion, just as in the case of negative control, which has no SNAP-25. The SNAP-25A (Δ9) mutant had only about 6% of maximum fluorescence intensity (MFI), while the wild-type SNAP-25 had about 20% of MFI. So, it is clear that the truncated SNAP-25A lost most of its function, although it could still form the SDS-resistant SNARE complex (Fig. 4B). Our lipid mixing results were also in agreement with the in vivo studies, which showed the same tendency of different membrane fusion activities in neurotoxins treated spinal cord cell cultures (Bergey, Bigalke & Nelson, 1987; Williamson, Fitzgerald & Neale, 1992).

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