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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

EPR spectra of the spin-labeled C-terminal of SNAP-25 SN2 in binary and ternary SNARE complexes.(A) EPR spectra for three mutants at the C-terminal of SNAP-25 SN2 (177C, 184C and 198C) in solution. (B) EPR spectra for the same spin-labeled positions in membrane-bound syntaxin 1A and SNAP-25 binary complex. (C) Syntaxin 1A-SNAP-25-souble VAMP2 (1-94) ternary complex. All the binary and ternary complexes are prepared by His-tag syntaxin 1A pulling down spin-labeled GST-tag SNAP-25 mutants in the absence or presence of GST-tag soluble VAMP2.
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fig-2: EPR spectra of the spin-labeled C-terminal of SNAP-25 SN2 in binary and ternary SNARE complexes.(A) EPR spectra for three mutants at the C-terminal of SNAP-25 SN2 (177C, 184C and 198C) in solution. (B) EPR spectra for the same spin-labeled positions in membrane-bound syntaxin 1A and SNAP-25 binary complex. (C) Syntaxin 1A-SNAP-25-souble VAMP2 (1-94) ternary complex. All the binary and ternary complexes are prepared by His-tag syntaxin 1A pulling down spin-labeled GST-tag SNAP-25 mutants in the absence or presence of GST-tag soluble VAMP2.

Mentions: We further investigated the C-terminal conformation of SNAP-25 SN2 in the ternary SNARE complex. His6-tagged syntaxin 1A was incubated with two-fold excess of spin-labeled GST-tag SNAP-25 and four-fold excess of GST-tag soluble VAMP2. At three C-terminal positions (C177, 184 and 198), when adding soluble VAMP2, all the EPR spectra became broader, indicating that the C-terminal of SNAP-25 SN2 interacted with VAMP2 and formed the trans-SNARE ternary complex (Fig. 2). However, we still detected some sharp components at these positions, which may be because the trans-SNARE complex easily splays out at the end of the C-terminus. This also implies that the complete SNARE assembly may require other regulatory proteins (such as synaptotagmin and complexin) at the late zippering step to promote the tight SNARE bundle formation, which is crucial for fusion pore opening.


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 C-terminal of SNAP-25 SN2 in binary and ternary SNARE complexes.(A) EPR spectra for three mutants at the C-terminal of SNAP-25 SN2 (177C, 184C and 198C) in solution. (B) EPR spectra for the same spin-labeled positions in membrane-bound syntaxin 1A and SNAP-25 binary complex. (C) Syntaxin 1A-SNAP-25-souble VAMP2 (1-94) ternary complex. All the binary and ternary complexes are prepared by His-tag syntaxin 1A pulling down spin-labeled GST-tag SNAP-25 mutants in the absence or presence of GST-tag soluble VAMP2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-2: EPR spectra of the spin-labeled C-terminal of SNAP-25 SN2 in binary and ternary SNARE complexes.(A) EPR spectra for three mutants at the C-terminal of SNAP-25 SN2 (177C, 184C and 198C) in solution. (B) EPR spectra for the same spin-labeled positions in membrane-bound syntaxin 1A and SNAP-25 binary complex. (C) Syntaxin 1A-SNAP-25-souble VAMP2 (1-94) ternary complex. All the binary and ternary complexes are prepared by His-tag syntaxin 1A pulling down spin-labeled GST-tag SNAP-25 mutants in the absence or presence of GST-tag soluble VAMP2.
Mentions: We further investigated the C-terminal conformation of SNAP-25 SN2 in the ternary SNARE complex. His6-tagged syntaxin 1A was incubated with two-fold excess of spin-labeled GST-tag SNAP-25 and four-fold excess of GST-tag soluble VAMP2. At three C-terminal positions (C177, 184 and 198), when adding soluble VAMP2, all the EPR spectra became broader, indicating that the C-terminal of SNAP-25 SN2 interacted with VAMP2 and formed the trans-SNARE ternary complex (Fig. 2). However, we still detected some sharp components at these positions, which may be because the trans-SNARE complex easily splays out at the end of the C-terminus. This also implies that the complete SNARE assembly may require other regulatory proteins (such as synaptotagmin and complexin) at the late zippering step to promote the tight SNARE bundle formation, which is crucial for fusion pore opening.

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