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1.
Anal Biochem ; 687: 115447, 2024 04.
Artículo en Inglés | MEDLINE | ID: mdl-38141800

RESUMEN

Membrane proteins (MPs) are affected by binding of specific lipids. We previously developed a methodology for systematically analyzing MP-lipid interactions leveraging surface plasmon resonance (SPR). In this method, the gold sensor chip surface was modified with a self-assembled monolayer (SAM), which allowed for a larger amount of MP-immobilization. However, the laborious lipid purification step remained a bottleneck. To address this issue, a new strategy has been developed utilizing gold nanoparticles (AuNPs) instead of the gold sensor chip. AuNPs were coated with SAM, on which MP was covalently anchored. The MP-immobilized AuNPs were mixed with a lipid mixture, and the recovered lipids were quantified by LC-MS. Bacteriorhodopsin (bR) was used as an MP to demonstrate this concept. We optimized immobilization conditions and confirmed the efficient immobilization of bR by dynamic light scattering and electron micrographs. Washing conditions for pulldown experiments were optimized to efficiently remove non-specific lipids. A new binding index was introduced to qualitatively reproduce the known affinity of lipids for bR. Consequently, the low-abundant and least-studied lipid S-TeGD was identified as a candidate for bR-specific lipids. This technique can skip the laborious lipid purification process, accelerating the screening of MP-specific lipids from complex lipid mixtures.


Asunto(s)
Lípidos de la Membrana , Nanopartículas del Metal , Oro , Proteínas de la Membrana , Resonancia por Plasmón de Superficie/métodos
2.
Analyst ; 149(14): 3747-3755, 2024 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-38829210

RESUMEN

In biological membranes, lipids often interact with membrane proteins (MPs), regulating the localization and activity of MPs in cells. Although elucidating lipid-MP interactions is critical to comprehend the physiological roles of lipids, a systematic and comprehensive identification of lipid-binding proteins has not been adequately established. Therefore, we report the development of lipid-immobilized beads where lipid molecules were covalently immobilized. Owing to the detergent tolerance, these beads enable screening of water-soluble proteins and MPs, the latter of which typically necessitate surfactants for solubilization. Herein, two sphingolipid species-ceramide and sphingomyelin-which are major constituents of lipid rafts, were immobilized on the beads. We first showed that the density of immobilized lipid molecules on the beads was as high as that of biological lipid membranes. Subsequently, we confirmed that these beads enabled the selective pulldown of known sphingomyelin- or ceramide-binding proteins (lysenin, p24, and CERT) from protein mixtures, including cell lysates. In contrast, commercial sphingomyelin beads, on which lipid molecules are sparsely immobilized through biotin-streptavidin linkage, failed to capture lysenin, a well-known protein that recognizes clustered sphingomyelin molecules. This clearly demonstrates the applicability of our beads for obtaining proteins that recognize not only a single lipid molecule but also lipid clusters or lipid membranes. Finally, we demonstrated the screening of lipid-binding proteins from Neuro2a cell lysates using these beads. This method is expected to significantly contribute to the understanding of interactions between lipids and proteins and to unravel the complexities of lipid diversity.


Asunto(s)
Esfingomielinas , Esfingomielinas/química , Animales , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Ceramidas/química , Toxinas Biológicas
3.
J Dairy Res ; 90(4): 367-375, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-38226400

RESUMEN

The milk fat globule membrane (MFGM) is formed by complex cell biological processes in the lactating mammary epithelial cell which result in the release of the milk fat globule (MFG) into the secretory alveolus. The MFG is bounded by a continuous unit membrane (UM), separated from the MFG lipid by a thin layer of cytoplasm. This unique apocrine secretion process has been shown in all of the mammary species so far investigated. Once the MFG is released into the alveolus there is a considerable transformation of the UM with its attached cytoplasm. This is the MFGM. The transformation is stable and expressed milk shows the same transformed MFGM structure. Again, this transformation of structure is common to all mammalian species so far investigated. However, the explanation of the transformation very much depends on the method of investigation. Transmission electron microscope (TEM) studies suggest a literal breakdown to a discontinuous UM plus cytoplasm in patches and strands, whereas more recent confocal laser scanning light microscopy (CLSM) studies indicate a separation, in a continuous UM, of two phases, one liquid ordered and the other liquid disordered. This review is designed to show that the TEM and CLSM results show different views of the same structures once certain deficiencies in techniques are factored in.


Asunto(s)
Lactancia , Gotas Lipídicas , Proteínas de la Leche , Femenino , Animales , Proteínas de la Leche/química , Glucolípidos/química , Glicoproteínas/química , Mamíferos/metabolismo
4.
Biochim Biophys Acta Biomembr ; 1866(7): 184366, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38960300

RESUMEN

Ginsenoside Rh2 (Rh2) is a ginseng saponin comprising a triterpene core and one unit of glucose and has attracted much attention due to its diverse biological activities. In the present study, we used small-angle X-ray diffraction, solid-state NMR, fluorescence microscopy, and MD simulations to investigate the molecular interaction of Rh2 with membrane lipids in the liquid-disordered (Ld) phase mainly composed of palmitoyloleoylphosphatidylcholine compared with those in liquid-ordered (Lo) phase mainly composed of sphingomyelin and cholesterol. The electron density profiles determined by X-ray diffraction patterns indicated that Rh2 tends to be present in the shallow interior of the bilayer in the Ld phase, while Rh2 accumulation was significantly smaller in the Lo phase. Order parameters at intermediate depths in the bilayer leaflet obtained from 2H NMR spectra and MD simulations indicated that Rh2 reduces the order of the acyl chains of lipids in the Ld phase. The dihydroxy group and glucose moiety at both ends of the hydrophobic triterpene core of Rh2 cause tilting of the molecular axis relative to the membrane normal, which may enhance membrane permeability by loosening the packing of lipid acyl chains. These features of Rh2 are distinct from steroidal saponins such as digitonin and dioscin, which exert strong membrane-disrupting activity.

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