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Lysophosphatidylcholine inhibits membrane-associated SNARE complex disassembly.

Shin L, Wang S, Lee JS, Flack A, Mao G, Jena BP - J. Cell. Mol. Med. (2012)

Bottom Line: Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes.In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly.Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA.

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Schematic drawing depicting cholesterol- and LPC-associated PC:PS vesicles reconstituted with t-SNAREs and v-SNARE, interact to form t-/v-SNARE ring complexes. In this study, the interaction between the opposing t- and v-SNARE vesicles containing either cholesterol or LPC was investigated, both in presence and absence on NSF–ATP.
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fig01: Schematic drawing depicting cholesterol- and LPC-associated PC:PS vesicles reconstituted with t-SNAREs and v-SNARE, interact to form t-/v-SNARE ring complexes. In this study, the interaction between the opposing t- and v-SNARE vesicles containing either cholesterol or LPC was investigated, both in presence and absence on NSF–ATP.

Mentions: All lipids were obtained from Avanti Polar Lipids (Alabaster, AL, USA). For the control group, a 5 mM lipid stock solution was prepared by mixing lipid solution in chloroform-DOPC (1,2-dioleoyl phosphatidylcholine): DOPS (1,2-dioleoyl phosphatidylserine) in 70:30 mol/mol ratios in glass test tubes. For the cholesterol group, a 5 mM lipid stock solution was prepared by mixing lipid solution in chloroform-DOPC (1,2-dioleoyl phosphatidylcholine): DOPS (1,2-dioleoyl phosphatidylserine): cholesterol in 63:27:10 mol/mol/mol ratios in glass test tubes. For the LPC group, 5 mM lipid stock solution was prepared by mixing lipid solution in chloroform-DOPC: DOPS: LPC at the molar ratio of 63:27:10 in glass test tubes. The lipid mixture was dried under gentle stream of nitrogen and resuspended in 5 mM sodium phosphate buffer, pH 7.5, by vortexing for 5 min. at room temperature. Unilamellar vesicles were formed following sonication (50 times at 10 sec./sonication), followed by extrusion using 50 nm pore-size membrane. Typically, vesicles ranging in size from 45 to 50 nm in diameter were obtained as assessed by AFM and photon correlation spectroscopy. Two sets of proteoliposomes were prepared by gently mixing either t-SNARE complex (Syntaxin-1A-His6/SNAP-25-His6; final concentration 25 μM) or VAMP2-His6 (final concentration 25 μM) with liposomes [1, 13], followed by three freeze/thaw cycles to enhance protein reconstitution at the vesicles membrane (Fig. 1).


Lysophosphatidylcholine inhibits membrane-associated SNARE complex disassembly.

Shin L, Wang S, Lee JS, Flack A, Mao G, Jena BP - J. Cell. Mol. Med. (2012)

Schematic drawing depicting cholesterol- and LPC-associated PC:PS vesicles reconstituted with t-SNAREs and v-SNARE, interact to form t-/v-SNARE ring complexes. In this study, the interaction between the opposing t- and v-SNARE vesicles containing either cholesterol or LPC was investigated, both in presence and absence on NSF–ATP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822683&req=5

fig01: Schematic drawing depicting cholesterol- and LPC-associated PC:PS vesicles reconstituted with t-SNAREs and v-SNARE, interact to form t-/v-SNARE ring complexes. In this study, the interaction between the opposing t- and v-SNARE vesicles containing either cholesterol or LPC was investigated, both in presence and absence on NSF–ATP.
Mentions: All lipids were obtained from Avanti Polar Lipids (Alabaster, AL, USA). For the control group, a 5 mM lipid stock solution was prepared by mixing lipid solution in chloroform-DOPC (1,2-dioleoyl phosphatidylcholine): DOPS (1,2-dioleoyl phosphatidylserine) in 70:30 mol/mol ratios in glass test tubes. For the cholesterol group, a 5 mM lipid stock solution was prepared by mixing lipid solution in chloroform-DOPC (1,2-dioleoyl phosphatidylcholine): DOPS (1,2-dioleoyl phosphatidylserine): cholesterol in 63:27:10 mol/mol/mol ratios in glass test tubes. For the LPC group, 5 mM lipid stock solution was prepared by mixing lipid solution in chloroform-DOPC: DOPS: LPC at the molar ratio of 63:27:10 in glass test tubes. The lipid mixture was dried under gentle stream of nitrogen and resuspended in 5 mM sodium phosphate buffer, pH 7.5, by vortexing for 5 min. at room temperature. Unilamellar vesicles were formed following sonication (50 times at 10 sec./sonication), followed by extrusion using 50 nm pore-size membrane. Typically, vesicles ranging in size from 45 to 50 nm in diameter were obtained as assessed by AFM and photon correlation spectroscopy. Two sets of proteoliposomes were prepared by gently mixing either t-SNARE complex (Syntaxin-1A-His6/SNAP-25-His6; final concentration 25 μM) or VAMP2-His6 (final concentration 25 μM) with liposomes [1, 13], followed by three freeze/thaw cycles to enhance protein reconstitution at the vesicles membrane (Fig. 1).

Bottom Line: Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes.In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly.Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA.

Show MeSH
Related in: MedlinePlus