A calorimetric study of the thermotropic behavior of aqueous dispersions of natural and synthetic sphingomyelins

Barenholz, Y.; Suurkuusk, J.; Mountcastle, Donald B.; Thompson, T. E.; Biltonen, Rodney L.

doi:10.1021/bi00656a030

Cited by 190 publications

(110 citation statements)

References 20 publications

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“…5 shows normalized emission spectra of laurdan in BBSM-containing SUV. According to differential scanning calorimetry, BBSM undergoes a broad chain melting transition around 37°C (32). The emission maxima reflect not only the ordered and fluid states of the bilayer (23 and 45°C, respectively) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Binding of α-Synuclein Affects the Lipid Packing in Bilayers of Small Vesicles

Kamp

Beyer

2006

Journal of Biological Chemistry

113

114

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The intracellular deposition of fibrillar aggregates of ␣-synuclein is a characteristic feature of Parkinson disease. Alternatively, as a result of its unusual conformational plasticity, ␣-synuclein may fold into an amphipathic helix upon contact with a lipid-water interface. Using spin label ESR and fluorescence spectroscopy, we show here that ␣-synuclein affects the lipid packing in small unilamellar vesicles. The ESR hyperfine splittings of spin-labeled phospholipid probes revealed that ␣-synuclein induces chain ordering at carbon 14 of the acyl chains below the chain melting phase transition temperature but not in the liquid crystalline state of electroneutral vesicle membranes. Binding of ␣-synuclein leads to an increase in the temperature and cooperativity of the phase transition according to the fluorescence anisotropy of the hydrophobic polyene 1,6-diphenylhexatriene and of the fluorescence emission maxima of the amphiphilic probe 6-dodecanoyl-2-dimethylaminonaphthalene. Binding parameters were obtained from the fluorescence anisotropy measurements in combination with our previous determinations by titration calorimetry (Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J. Biol. Chem. 279, 21966 -21975). We also show that ␣-synuclein interacts with vesicle membranes containing sphingomyelin and cholesterol. We propose that the protein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion. ␣-Synuclein (␣S)2 is a small cytosolic protein with unknown function that is abundant in nerve terminals of the dopaminergic system. This protein is associated with Parkinson disease and other neurodegenerative disorders (1, 2). The hallmark feature in these conditions is the deposition of ␣S in insoluble intracellular aggregates (Lewy bodies) (3). Three single site ␣S mutations are known to be associated with rare, early onset variants of the disease (4 -6), whereas the overwhelming number of Parkinson cases are of the sporadic type. Recombinant ␣S remains monomeric and almost entirely unfolded unless its aqueous solution reaches a concentration threshold at which it may form different aggregates such as spherical or doughnut-shaped oligomers and amyloid-like fibrils (7,8). The formation of large ␣S fibrils may be preceded by assembly into protofibrillar aggregation intermediates as shown by chromatography and by an investigation of the aggregation kinetics (9).The interaction of ␣S with small unilamellar vesicles (SUV) obtained by sonication of a phospholipid suspension reveals particularly intriguing features. Upon binding to the lipid-water interface, the "natively unfolded" protein assumes a conformation characterized by two helical domains interrupted by a short non-helical region (10 -12). The vesicles are neither disrupted nor permeabilized by the monomeric protein, whereas influx of metal ions or efflux of dopamine has been observed upon addition of ␣S protofibrils (13). Protofibrillar intermediates have b...

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Section: Resultsmentioning

confidence: 99%

Binding of α-Synuclein Affects the Lipid Packing in Bilayers of Small Vesicles

Kamp

Beyer

2006

Journal of Biological Chemistry

113

114

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“…2 show that the protein is also insoluble when it is in liposomes containing lipids in the gel phase. Lipids with high T m , such as sphingolipids, can form a gel phase at or near physiological temperatures (30). Alternatively, they can form an l o phase in the presence of cholesterol (13).…”

Section: Cholesterol Is Not Required For Detergent Resistance Of Plapmentioning

confidence: 99%

Cholesterol and Sphingolipid Enhance the Triton X-100 Insolubility of Glycosylphosphatidylinositol-anchored Proteins by Promoting the Formation of Detergent-insoluble Ordered Membrane Domains

Schroeder¹,

Ahmed²,

Zhu³

et al. 1998

Journal of Biological Chemistry

397

315

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Glycosylphosphatidylinositol (GPI)-anchored proteins can be isolated from both cells and sphingolipid and cholesterol-rich liposomes (SCRLs) in association with detergent-insoluble membranes. We found previously that detergent insolubility of lipids was characteristic of phases in which lipid acyl chains are ordered. We presented evidence that GPI-anchored proteins are insoluble because they associate with cholesterol and sphingolipid-rich lipid domains with properties similar to the liquid-ordered phase. Here, this model was tested by a variety of approaches. First, we demonstrated that saponin, which removes cholesterol from cell membranes and allows solubilization of GPI-anchored proteins by Triton X-100, had the same effect on the GPIanchored protein alkaline phosphatase (PLAP) in SCRLs of appropriate lipid composition. The similarity of saponin action in cells and simple liposomes suggests that the compound disrupts protein-lipid interactions. However, direct interactions between PLAP and cholesterol were not needed for insolubility, because the protein was also insoluble in cholesterol-free liposomes containing lipid in an ordered phase. Instead, cholesterol acted by greatly enhancing the formation of a detergent-insoluble phase by sphingolipids, which have a tendency to form ordered phases. We propose that saponin solubilizes GPI-anchored proteins because the lipid composition of cell membranes (and the SCRLs used above) supports ordered phase formation in the presence but not the absence of cholesterol. Supporting this model, saponin did not promote Triton X-100 solubilization of PLAP in SCRLs with sphingolipid levels high enough to allow ordered phase formation in the absence of cholesterol. We also showed that two additional GPI-anchored proteins are detergent-insoluble in SCRLs and that detergent does not artifactually create ordered domains or cause components of solubilized membranes to associate with detergent-resistant membranes present in separate bilayers in the same lysate. We conclude that the ordered domain model explains the behavior of detergent-resistant membranes in liposomes and cells.

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“…This effect was attributed to the more heterogeneous mixture of acyl chains found with natural lipid systems and would likely lessen the order of l o domains when compared with l o domains formed from synthetic lipids (Jin et al, 2006). Similarly, a calorimetric study by Barenholz et al (1976) examined the thermotropic behavior of lipid vesicles composed of natural and synthetic sphingomyelin lipid species. It was reported that membrane organization of sphingomyelin is a complex function of its specific composition and that the transition temperature of natural sphingomyelin was much broader than the synthetic forms.…”

Section: Resultsmentioning

confidence: 99%

Relationship between CYP1A2 Localization and Lipid Microdomain Formation as a Function of Lipid Composition

et al. 2013

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Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (l o ) domains. The goal of this study was to determine if l o domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in "ER-like" lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form l o domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/ acceptor FRET pair in both ER-like and model l o -forming systems demonstrated the coexistence of l o -and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the l o regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into l o membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing l o domains.

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A calorimetric study of the thermotropic behavior of aqueous dispersions of natural and synthetic sphingomyelins

Cited by 190 publications

References 20 publications

Binding of α-Synuclein Affects the Lipid Packing in Bilayers of Small Vesicles

Binding of α-Synuclein Affects the Lipid Packing in Bilayers of Small Vesicles

Cholesterol and Sphingolipid Enhance the Triton X-100 Insolubility of Glycosylphosphatidylinositol-anchored Proteins by Promoting the Formation of Detergent-insoluble Ordered Membrane Domains

Relationship between CYP1A2 Localization and Lipid Microdomain Formation as a Function of Lipid Composition

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