RESUMEN
Ceramide 1-phosphate (C1P) is a lipid mediator that specifically binds and activates cytosolic phospholipase A2α (cPLA2α). To elucidate the structure-activity relationship of the affinity of C1P for cPLA2α in lipid environments, we prepared a series of C1P analogs containing structural modifications in the hydrophilic parts and subjected them to surface plasmon resonance (SPR). The results suggested the presence of a specific binding site for cPLA2α on the amide, 3-OH and phosphate groups in C1P structure. Especially, dihydro-C1P exhibited enhanced affinity for cPLA2α, suggesting the hydrogen bonding ability of 3-hydroxy group is important for interactions with cPLA2α. This study helps to understand the influence of specific structural moieties of C1P on the interaction with cPLA2α at the atomistic level and may lead to the design of drugs that regulate cPLA2α activation.
Asunto(s)
Ceramidas , Diseño de Fármacos , Resonancia por Plasmón de Superficie , Ceramidas/química , Ceramidas/síntesis química , Ceramidas/metabolismo , Relación Estructura-Actividad , Fosfolipasas A2 Grupo IV/metabolismo , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Humanos , Estructura Molecular , Sitios de UniónRESUMEN
We examined the volumetric behavior of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol binary bilayer system with high accuracy and more cholesterol concentrations to reveal the detailed molecular states in the liquid-disordered (Ld) phase, the liquid-ordered (Lo) phase and the gel phase. We measured the average specific volume of the binary bilayer at several temperatures by the neutral flotation method and calculated the average volume per molecule to estimate the partial molecular volumes of DPPC and cholesterol in each phase. As a result, we found that the region with intermediate cholesterol concentrations showed a more complicated behavior than expected from simple coexistence of Ld and Lo domains. We also measured fluorescence decay of trans-parinaric acid (tPA) added into the binary bilayer with more cholesterol concentrations to get further insight into the cholesterol-induced formation of the Lo phase. On the basis of these results we discuss the molecular interaction between DPPC and cholesterol molecule in the Lo phase and the manner of Ld/Lo phase coexistence.
RESUMEN
Sphingomyelins are important phospholipids in plasma membranes of most cells. Because of their dominantly saturated nature, they affect the lateral structure of membranes, and contribute to the regulation of cholesterol distribution within membranes, and in cells. However, the abundance of molecular species present in cells also implies that sphingomyelins have other, more specific functions. Many of these functions are currently unknown, but are under extensive study. Mostly model membrane studies have shown that sphingomyelins (and other sphingolipids), in contrast to glycerophospholipids, have important hydrogen bonding properties which in several important ways confer specific functional properties to this abundant class of membrane phospholipids. The often very asymmetric nature of sphingomyelins, arising from mismatch in length between the long chain base and N-acyl chains, also impose specific properties (e.g., interdigitation) to sphingomyelins not seen with glycerophospholipids. In this review, the latest sphingomyelin literature will be scrutinized, and an effort will be made to correlate the molecular structure of sphingomyelin with functional properties. In particular, the effects of head group properties, interfacial hydrogen bonding, long chain base hydroxylation, N-acyl chain hydroxylation, and N-acyl chain methyl-branching will be discussed.
Asunto(s)
Esfingomielinas/química , Esfingomielinas/metabolismo , Animales , Ceramidas/metabolismo , Humanos , Enlace de Hidrógeno , Relación Estructura-ActividadRESUMEN
Long-chain saturated ceramides possess the ability to form gel domains in fluid bilayer membranes. Such domains may also contain sphingomyelin, but generally exclude cholesterol. We studied the effect of N-acyl chain methylations on the ability of ceramide to form ceramide- and sphingomyelin-containing gel domains that displace sterol. Fluorescence quenching of probes displaying different lateral partitioning in heterogeneous lipid bilayers showed that the methyl branches induced position-dependent changes in the lateral distribution of the ceramides. Distally monomethylated ceramides interacted with sphingomyelin and displaced sterol, whereas proximally monomethylated and polymethylated ceramides appeared to be located outside of sterol/sphingomyelin-enriched domains. The branched ceramides also markedly reduced the bilayer affinity for sterol as determined from the equilibrium partitioning of sterol between lipid vesicles and cyclodextrin. Altogether, alterations in intermolecular interactions induced by the methyl branches markedly affected the molecular properties of ceramide in artificial bilayers.
Asunto(s)
Membrana Celular/metabolismo , Ceramidas/química , Ceramidas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Membrana Celular/química , Ceramidas/síntesis química , Colorantes Fluorescentes/química , Membrana Dobles de Lípidos/química , Metilación , Esfingomielinas/metabolismo , Esteroles/metabolismo , Relación Estructura-ActividadRESUMEN
In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.