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 C-terminal of SNAP-25 SN1 and SN2 in t-SNAREs binary complex under room temperature.(A) The diagram of the spin-labeled SNAP-25 SN1 and SN2 with syntaxin 1A SNARE motif in t-SNAREs binary complex. The corresponding labeled positions of SNAP-25 SN1 are in red and that of SN2 are in blue. (B) EPR spectra of the spin-labeled C-terminal of SNAP-25 SN1 and SN2 in the full-length His-tag syntaxin 1A and spin-labeled GST-tag SNAP-25 binary complex. EPR spectra of the binary complex in the detergent and in the membrane are shown. The immobile and mobile 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-1: EPR spectra of the C-terminal of SNAP-25 SN1 and SN2 in t-SNAREs binary complex under room temperature.(A) The diagram of the spin-labeled SNAP-25 SN1 and SN2 with syntaxin 1A SNARE motif in t-SNAREs binary complex. The corresponding labeled positions of SNAP-25 SN1 are in red and that of SN2 are in blue. (B) EPR spectra of the spin-labeled C-terminal of SNAP-25 SN1 and SN2 in the full-length His-tag syntaxin 1A and spin-labeled GST-tag SNAP-25 binary complex. EPR spectra of the binary complex in the detergent and in the membrane are shown. The immobile and mobile components are indicated in red and blue arrows, respectively.

Mentions: To compare the SN1 and SN2 conformations in the t-SNAREs binary complex, we employed site-directed spin labeling (SDSL) and continuous wave (CW) EPR spectroscopy and prepared three cysteine mutants located in the C-terminus of SN1 and SN2, respectively (Fig. 1A): C63, 70 and 77 of SNAP-25 SN1, and C184, 191 and 198 of SNAP-25 SN2. The selected residues were all at the predicted “g” position in the heptad repeats of SNAP-25 SNARE motifs (Poirier et al., 1998). In order to avoid the oligomerization of syntaxin 1A, we used His6-tagged full-length syntaxin 1A to pull down the spin-labeled GST-tag SNAP-25. Two-fold excess of purified SNAP-25 were incubated with syntaxin 1A, and then washed extensively to get rid of the free SNAP-25.


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

Lu B - PeerJ (2015)

EPR spectra of the C-terminal of SNAP-25 SN1 and SN2 in t-SNAREs binary complex under room temperature.(A) The diagram of the spin-labeled SNAP-25 SN1 and SN2 with syntaxin 1A SNARE motif in t-SNAREs binary complex. The corresponding labeled positions of SNAP-25 SN1 are in red and that of SN2 are in blue. (B) EPR spectra of the spin-labeled C-terminal of SNAP-25 SN1 and SN2 in the full-length His-tag syntaxin 1A and spin-labeled GST-tag SNAP-25 binary complex. EPR spectra of the binary complex in the detergent and in the membrane are shown. The immobile and mobile 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-1: EPR spectra of the C-terminal of SNAP-25 SN1 and SN2 in t-SNAREs binary complex under room temperature.(A) The diagram of the spin-labeled SNAP-25 SN1 and SN2 with syntaxin 1A SNARE motif in t-SNAREs binary complex. The corresponding labeled positions of SNAP-25 SN1 are in red and that of SN2 are in blue. (B) EPR spectra of the spin-labeled C-terminal of SNAP-25 SN1 and SN2 in the full-length His-tag syntaxin 1A and spin-labeled GST-tag SNAP-25 binary complex. EPR spectra of the binary complex in the detergent and in the membrane are shown. The immobile and mobile components are indicated in red and blue arrows, respectively.
Mentions: To compare the SN1 and SN2 conformations in the t-SNAREs binary complex, we employed site-directed spin labeling (SDSL) and continuous wave (CW) EPR spectroscopy and prepared three cysteine mutants located in the C-terminus of SN1 and SN2, respectively (Fig. 1A): C63, 70 and 77 of SNAP-25 SN1, and C184, 191 and 198 of SNAP-25 SN2. The selected residues were all at the predicted “g” position in the heptad repeats of SNAP-25 SNARE motifs (Poirier et al., 1998). In order to avoid the oligomerization of syntaxin 1A, we used His6-tagged full-length syntaxin 1A to pull down the spin-labeled GST-tag SNAP-25. Two-fold excess of purified SNAP-25 were incubated with syntaxin 1A, and then washed extensively to get rid of the free SNAP-25.

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.