Limits...
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.


EPR spectra of the spin-labeled full-length syntaxin 1A mutants involved in the wild-type or truncated SNAP-25 and soluble VAMP2 ternary complex.(A) and (B) EPR spectra for spin-labeled positions at 199, 206, 248 and 249 of syntaxin 1A in the ternary SNARE complex. These four positions correspond to the upstream of SNAP-25 SN2 neurotoxins cleavage sites. (A) is the ternary complex in the detergent, (B) is in the membrane. (C) and (D) EPR spectra for positions at 256, 257, 258 and 259 of syntaxin 1A in the ternary SNARE complex. These four positions are the last four amino acids of the syntaxin 1A SNARE motif. (C) is the ternary complex in the detergent, (D) is in the membrane. The mobile and immobile components are indicated in red and blue arrows, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493708&req=5

fig-6: EPR spectra of the spin-labeled full-length syntaxin 1A mutants involved in the wild-type or truncated SNAP-25 and soluble VAMP2 ternary complex.(A) and (B) EPR spectra for spin-labeled positions at 199, 206, 248 and 249 of syntaxin 1A in the ternary SNARE complex. These four positions correspond to the upstream of SNAP-25 SN2 neurotoxins cleavage sites. (A) is the ternary complex in the detergent, (B) is in the membrane. (C) and (D) EPR spectra for positions at 256, 257, 258 and 259 of syntaxin 1A in the ternary SNARE complex. These four positions are the last four amino acids of the syntaxin 1A SNARE motif. (C) is the ternary complex in the detergent, (D) is in the membrane. The mobile and immobile components are indicated in red and blue arrows, respectively.

Mentions: We are very interested in exploring why there was such a big difference between the wild-type and truncated SNARE complexes. Therefore, we employed CW-EPR spectroscopy to investigate the local conformational changes of the truncated ternary SNARE complex. The spin-labeled syntaxin 1A proteins were incubated with soluble VAMP2 and the truncated SNAP-25 mutants, and then formed ternary complexes. We selected four positions in syntaxin 1A, corresponding to the upstream positions of SNAP-25 neurotoxins cleavage sites (N-terminus). As Figs. 6A and 6B show, in the N-terminal SNARE motif of syntaxin 1A, the spectra were all very broad, both for the wild-type and the truncated SNARE complexes. This suggests that SNARE proteins form the complete coiled coil regardless of the kind of truncated SNAP-25 involved. Since the BoNT/A and BoNT/E cleavage sites were both at a distance from these spin-labeled positions, the truncated SNAP-25 did not disrupt the SNARE core formation.


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

Lu B - PeerJ (2015)

EPR spectra of the spin-labeled full-length syntaxin 1A mutants involved in the wild-type or truncated SNAP-25 and soluble VAMP2 ternary complex.(A) and (B) EPR spectra for spin-labeled positions at 199, 206, 248 and 249 of syntaxin 1A in the ternary SNARE complex. These four positions correspond to the upstream of SNAP-25 SN2 neurotoxins cleavage sites. (A) is the ternary complex in the detergent, (B) is in the membrane. (C) and (D) EPR spectra for positions at 256, 257, 258 and 259 of syntaxin 1A in the ternary SNARE complex. These four positions are the last four amino acids of the syntaxin 1A SNARE motif. (C) is the ternary complex in the detergent, (D) is in the membrane. The mobile and immobile components are indicated in red and blue arrows, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-6: EPR spectra of the spin-labeled full-length syntaxin 1A mutants involved in the wild-type or truncated SNAP-25 and soluble VAMP2 ternary complex.(A) and (B) EPR spectra for spin-labeled positions at 199, 206, 248 and 249 of syntaxin 1A in the ternary SNARE complex. These four positions correspond to the upstream of SNAP-25 SN2 neurotoxins cleavage sites. (A) is the ternary complex in the detergent, (B) is in the membrane. (C) and (D) EPR spectra for positions at 256, 257, 258 and 259 of syntaxin 1A in the ternary SNARE complex. These four positions are the last four amino acids of the syntaxin 1A SNARE motif. (C) is the ternary complex in the detergent, (D) is in the membrane. The mobile and immobile components are indicated in red and blue arrows, respectively.
Mentions: We are very interested in exploring why there was such a big difference between the wild-type and truncated SNARE complexes. Therefore, we employed CW-EPR spectroscopy to investigate the local conformational changes of the truncated ternary SNARE complex. The spin-labeled syntaxin 1A proteins were incubated with soluble VAMP2 and the truncated SNAP-25 mutants, and then formed ternary complexes. We selected four positions in syntaxin 1A, corresponding to the upstream positions of SNAP-25 neurotoxins cleavage sites (N-terminus). As Figs. 6A and 6B show, in the N-terminal SNARE motif of syntaxin 1A, the spectra were all very broad, both for the wild-type and the truncated SNARE complexes. This suggests that SNARE proteins form the complete coiled coil regardless of the kind of truncated SNAP-25 involved. Since the BoNT/A and BoNT/E cleavage sites were both at a distance from these spin-labeled positions, the truncated SNAP-25 did not disrupt the SNARE core formation.

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.