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.


The SDS-resistant ability of the truncated SNARE complex.Four groups of different molecular ratio of one time full-length syntaxin 1A mixes with two times wild-type or truncated SNAP-25 and various soluble VAMP2 and runs the SDS-PAGE without boiling. The markers, from top to bottom, are 66, 45, 29 and 14.2 kDa. (A) Wild-type SNAP-25 with syntaxin 1A and soluble VAMP2, (B) BoNT/A truncated SNAP-25A (Δ9), (C) BoNT/E truncated SNAP-25E (Δ26). The red arrows indicate the position of the SNARE complex; the red brackets indicate the higher order of SNARE complexes due to SNAP-25 swapping.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-4: The SDS-resistant ability of the truncated SNARE complex.Four groups of different molecular ratio of one time full-length syntaxin 1A mixes with two times wild-type or truncated SNAP-25 and various soluble VAMP2 and runs the SDS-PAGE without boiling. The markers, from top to bottom, are 66, 45, 29 and 14.2 kDa. (A) Wild-type SNAP-25 with syntaxin 1A and soluble VAMP2, (B) BoNT/A truncated SNAP-25A (Δ9), (C) BoNT/E truncated SNAP-25E (Δ26). The red arrows indicate the position of the SNARE complex; the red brackets indicate the higher order of SNARE complexes due to SNAP-25 swapping.

Mentions: Former studies show that SNARE proteins can form a very stable complex that is resistant to SDS denaturation and high temperatures (>90 °C) (Hayashi et al., 1995; Wei et al., 2000). Therefore, we compared the SDS resistant property of different SNARE complexes, composed of truncated SNAP-25, syntaxin 1A and VAMP2 (Fig. 4). We mixed syntaxin 1A and wild-type SNAP-25, or truncated SNAP-25A (Δ9) or SNAP-25E (Δ26) at the molar ratio of 1:2, and with four different amounts of soluble VAMP2 (1-, 2-, 4-, 6-fold of syntaxin 1A) at room temperature for one hour, and ran the SDS-PAGE gel without boiling. As expected, the wild-type SNAP-25 formed the SDS-resistant complex with the other two SNARE proteins (Fig. 4A, red arrow). We also found that there were many high molecular weight complexes besides the monomeric SNARE complex, which may be due to the aggregation of SNARE complexes by SNAP-25 swapping (Fig. 4A, red bracket). SNAP-25A (Δ9) formed a SDS-resistant mono-complex and small amounts of high molecular weight complexes (Fig. 4B), while SNAP-25E (Δ26) did not form any SDS-resistant complex (Fig. 4C). Therefore, these results suggest that the SNARE complexes composed of the truncated SNAP-25 display a low stability to the SDS detergent.


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

Lu B - PeerJ (2015)

The SDS-resistant ability of the truncated SNARE complex.Four groups of different molecular ratio of one time full-length syntaxin 1A mixes with two times wild-type or truncated SNAP-25 and various soluble VAMP2 and runs the SDS-PAGE without boiling. The markers, from top to bottom, are 66, 45, 29 and 14.2 kDa. (A) Wild-type SNAP-25 with syntaxin 1A and soluble VAMP2, (B) BoNT/A truncated SNAP-25A (Δ9), (C) BoNT/E truncated SNAP-25E (Δ26). The red arrows indicate the position of the SNARE complex; the red brackets indicate the higher order of SNARE complexes due to SNAP-25 swapping.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-4: The SDS-resistant ability of the truncated SNARE complex.Four groups of different molecular ratio of one time full-length syntaxin 1A mixes with two times wild-type or truncated SNAP-25 and various soluble VAMP2 and runs the SDS-PAGE without boiling. The markers, from top to bottom, are 66, 45, 29 and 14.2 kDa. (A) Wild-type SNAP-25 with syntaxin 1A and soluble VAMP2, (B) BoNT/A truncated SNAP-25A (Δ9), (C) BoNT/E truncated SNAP-25E (Δ26). The red arrows indicate the position of the SNARE complex; the red brackets indicate the higher order of SNARE complexes due to SNAP-25 swapping.
Mentions: Former studies show that SNARE proteins can form a very stable complex that is resistant to SDS denaturation and high temperatures (>90 °C) (Hayashi et al., 1995; Wei et al., 2000). Therefore, we compared the SDS resistant property of different SNARE complexes, composed of truncated SNAP-25, syntaxin 1A and VAMP2 (Fig. 4). We mixed syntaxin 1A and wild-type SNAP-25, or truncated SNAP-25A (Δ9) or SNAP-25E (Δ26) at the molar ratio of 1:2, and with four different amounts of soluble VAMP2 (1-, 2-, 4-, 6-fold of syntaxin 1A) at room temperature for one hour, and ran the SDS-PAGE gel without boiling. As expected, the wild-type SNAP-25 formed the SDS-resistant complex with the other two SNARE proteins (Fig. 4A, red arrow). We also found that there were many high molecular weight complexes besides the monomeric SNARE complex, which may be due to the aggregation of SNARE complexes by SNAP-25 swapping (Fig. 4A, red bracket). SNAP-25A (Δ9) formed a SDS-resistant mono-complex and small amounts of high molecular weight complexes (Fig. 4B), while SNAP-25E (Δ26) did not form any SDS-resistant complex (Fig. 4C). Therefore, these results suggest that the SNARE complexes composed of the truncated SNAP-25 display a low stability to the SDS detergent.

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.