Background Phenylalanine (Phe) is involved in physiological and pathological processes in cell membranes in which expanded and condensed states coexist. In this direction, it was reported that surface hydration is important for the binding affinity of the amino acid which significantly perturbs 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer structure and morphology. A deeper insight showed that Phe inserts in DPPC monolayer defects as a monomer at pH 5 and forms aggregates that adsorb to the membrane surface generating a reconfiguration of the lipid arrangement in areas of higher packing. This new arrangement in the monolayer causes the reorientation of dipoles of lipid and water molecules which is congruent with the dehydration and surface tension changes reported above. With this background, this article studies the affinity of Phe in liquid-expanded 1,2-dimyristoyl-sn-glycero-3 phosphocholine (LE DMPC) and liquid-condensed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (LC DPPC) monolayers and their effects on membrane properties. Results The adsorption of Phe can be described by a cooperative process in non-independent sites suggesting that Phe/lipid systems reorganize to form new structures at a high degree of coverage. Compressibility modulus and Brewster angle microscopy (BAM) images allow to propose that Phe causes a new phase in 1,2-dimyristoyl-sn-glycero-3 phosphocholine (DMPC) and DPPC. Conclusions Phe imposes new arrangements in the lipid phase to form new structures with different compressibility behavior than lipid binary mixtures of DMPC and DPPC. Phe interaction with the LC and LE phases gives place to a process in which a synergistic effect between non-independent sites can be produced. These features of Phe/lipid interaction would be of great importance to understand the multiple effects of Phe on cell membranes.
In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.
Hepatic stellate cells (HSCs) are professional lipid-storing cells and are unique in their property to store most of the retinol (vitamin A) as retinyl esters in large-sized lipid droplets. Hepatic stellate cell activation is a critical step in the development of chronic liver disease, as activated HSCs cause fibrosis. During activation, HSCs lose their lipid droplets containing triacylglycerols, cholesteryl esters, and retinyl esters. Lipidomic analysis revealed that the dynamics of disappearance of these different classes of neutral lipids are, however, very different from each other. Although retinyl esters steadily decrease during HSC activation, triacylglycerols have multiple pools one of which becomes transiently enriched in polyunsaturated fatty acids before disappearing. These observations are consistent with the existence of preexisting "original" lipid droplets with relatively slow turnover and rapidly recycling lipid droplets that transiently appear during activation of HSCs. Elucidation of the molecular machinery involved in the regulation of these distinct lipid droplet pools may open new avenues for the treatment of liver fibrosis.
Lipid droplets (LDs) are unique intracellular organelles that are mainly constituted by neutral lipids (triglycerides, sterol esters). As such they serve as the main site of energy storage in the cell and they are akin to oil emulsions in water. To prevent the direct exposure of the hydrophobic neutral lipids to the aqueous environment of the cytosol, LDs are surrounded by a monolayer of phospholipids that thus behave as a natural surfactant. This interfacial structure is rather unique inside the cell, but a molecular understanding of how the LD structure modulates its functions is still lacking, mainly due to technical challenges in both experimental and computational approaches to investigate oil-in-water emulsions. Recently, we have investigated the structure of LDs using a combination of existing and newly developed computational approaches that are optimized to study oil-water interfaces.1 Our simulations provide a comprehensive molecular characterization of the unique surface properties of LDs, suggesting structure-function relationship in several LD-related metabolic processes.
Membrane lipid homeostasis is maintained by de novo synthesis, intracellular transport, remodeling, and degradation of lipid molecules. Glycerophospholipids, the most abundant structural component of eukaryotic membranes, are subject to acyl chain remodeling, which is defined as the post-synthetic process in which one or both acyl chains are exchanged. Here, we review studies addressing acyl chain remodeling of membrane glycerophospholipids in Saccharomyces cerevisiae, a model organism that has been successfully used to investigate lipid synthesis and its regulation. Experimental evidence for the occurrence of phospholipid acyl chain exchange in cardiolipin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine is summarized, including methods and tools that have been used for detecting remodeling. Progress in the identification of the enzymes involved is reported, and putative functions of acyl chain remodeling in yeast are discussed.
Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM-ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM-ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions.
Castor oil, produced from castor beans, has long been considered to be of important commercial value primarily for the manufacturing of soaps, lubricants, and coatings, among others. Global castor oil production is concentrated primarily in a small geographic region of Gujarat in Western India. This region is favorable due to its labor-intensive cultivation method and subtropical climate conditions. Entrepreneurs and castor processors in the United States and South America also cultivate castor beans but are faced with the challenge of achieving high castor oil production efficiency, as well as obtaining the desired oil quality. In this manuscript, we provide a detailed analysis of novel processing methods involved in castor oil production. We discuss novel processing methods by explaining specific processing parameters involved in castor oil production.
The best understood consequence of the collapse of lipid asymmetry is exposure of phosphatidylserine (PS) in the external leaflet of the plasma membrane bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. Lipid asymmetry is collapsed by activation of phospholipid scramblase(s) that catalyze bidirectional transbilayer movement of the major classes of phospholipid. The protein corresponding to this activity is not yet known. Observations on cells from patients with Scott syndrome, a rare hereditary bleeding disorder resulting from impaired lipid scrambling, have shown that there are multiple activation pathways that converge on scramblase activity.
In most reviews addressing intracellular lipid trafficking, spontaneous diffusion of lipid monomers between the cellular organelles is considered biologically irrelevant because it is thought to be far too slow to significantly contribute to organelle biogenesis. This view is based on intervesicle transfer experiments carried out in vitro with few lipids as well as on the view that lipids are highly hydrophobic and thus cannot undergo spontaneous intermembrane diffusion at a significant rate. However, besides that single-chain lipids can translocate between vesicles in seconds, it has been demonstrated that the rate of spontaneous transfer of two-chain polar lipids can vary even 1000-fold, depending on the number of carbons and double bonds in the acyl chains. In addition, the rate of spontaneous lipid transfer can strongly depend on the experimental conditions such as vesicle composition and concentration. This review examines the studies suggesting that spontaneous lipid transfer is probably more relevant to intracellular trafficking of amphipathic lipids than commonly thought.
Negative feedback regulation of cholesterol metabolism in mammalian cells ensures a proper balance of cholesterol with other membrane lipids, principal among these being the major phospholipid phosphatidylcholine (PC). Processes such as cholesterol biosynthesis and efflux, cholesteryl ester storage in lipid droplets, and uptake of plasma lipoproteins are tuned to the cholesterol/PC ratio. Cholesterol-loaded macrophages in atherosclerotic lesions display increased PC biosynthesis that buffers against elevated cholesterol levels and may also facilitate cholesterol trafficking to enhance cholesterol sensing and efflux. These same mechanisms could play a generic role in homeostatic responses to acute changes in membrane free cholesterol levels. Here, I discuss the established and emerging roles of PC metabolism in promoting intracellular cholesterol trafficking and membrane lipid homeostasis.
Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed.
Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane. The glycans are first assembled as precursor glycolipids, on the cytosolic surface of the ER, which are tethered to the membrane by attachment to a long-chain polyisoprenyl phosphate (dolichol) containing a reduced α-isoprene. The lipid-anchored building blocks then migrate transversely (flip) across the ER membrane to the lumenal surface, where final assembly of the glycan is completed. This strategy allows the cell to export high-energy biosynthetic intermediates as lipid-bound glycans, while constraining the glycosyl donors to the site of assembly on the membrane surface. This review focuses on the flippases that participate in protein glycosylation in the ER.
Diverse acyl-CoA species and acyltransferase isoenzymes are components of a complex system that synthesizes glycerophospholipids and triacylglycerols. Saccharomyces cerevisiae has four main acyl-CoA species, two main glycerol-3-phosphate 1-O-acyltransferases (Gat1p, Gat2p), and two main 1-acylglycerol-3-phosphate O-acyltransferases (Lpt1p, Slc1p). The in vivo contribution of these isoenzymes to phospholipid heterogeneity was determined using haploids with compound mutations: gat1Δlpt1Δ, gat2Δlpt1Δ, gat1Δslc1Δ, and gat2Δslc1Δ. All mutations mildly reduced [3H]palmitic acid incorporation into phospholipids relative to triacylglycerol. Electrospray ionization tandem mass spectrometry identified few differences from wild type in gat1Δlpt1Δ, dramatic differences in gat2Δslc1Δ, and intermediate changes in gat2Δlpt1Δ and gat1Δslc1Δ. Yeast expressing Gat1p and Lpt1p had phospholipids enriched with acyl chains that were unsaturated, 18 carbons long, and paired for length. These alterations prevented growth at 18.5°C and in 10% ethanol. Therefore, Gat2p and Slc1p dictate phospholipid acyl chain composition in rich media at 30°C. Slc1p selectively pairs acyl chains of different lengths.
Statins, a class of cholesterol-lowering medications that inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, are commonly administered to treat atherosclerotic cardiovascular disease. Statin use may expand considerably given its potential for treating an array of cholesterol-independent diseases. However, the lack of conclusive evidence supporting these emerging therapeutic uses of statins brings to the fore a number of unanswered questions including uncertainties regarding patient-to-patient variability in response to statins, the most appropriate statin to be used for the desired effect, and the efficacy of statins in treating cholesterol-independent diseases. In this review, the adverse effects, costs, and drug–drug and drug–food interactions associated with statin use are presented. Furthermore, we discuss the pleiotropic effects associated with statins with regard to the onset and progression of autoimmune and inflammatory diseases, cancer, neurodegenerative disorders, strokes, bacterial infections, and human immunodeficiency virus. Understanding these issues will improve the prognosis of patients who are administered statins and potentially expand our ability to treat a wide variety of diseases.
The biosynthesis of cellular polysaccharides and glycoconjugates often involves lipid-linked intermediates that need to be translocated across membranes. Essential pathways such as N-glycosylation in eukaryotes and biogenesis of the peptidoglycan (PG) cell wall in bacteria share a common strategy where nucleotide-sugars are used to build a membrane-bound oligosaccharide precursor that is linked to a phosphorylated isoprenoid lipid. Once made, these lipid-linked intermediates must be translocated across a membrane so that they can serve as substrates in a different cellular compartment. How translocation occurs is poorly understood, although it clearly requires a transporter or flippase. Identification of these transporters is notoriously difficult, and, in particular, the identity of the flippase of lipid II, an intermediate required for PG biogenesis, has been the subject of much debate. Here, I will review the body of work that has recently fueled this controversy, centered on proposed flippase candidates FtsW, MurJ, and AmJ.
Increasing evidence suggests that oxysterol-binding protein-related proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-binding protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling.
We examined whether serum from obese, compared to non-obese, PCa (prostate cancer) patients creates a growth-enhancing tumor micro-environment in vitro. Serum from 80 subjects was divided into four groups: normal weight men with and without PCa and overweight/obese men with and without PCa. Cell proliferation, migration, and invasion were measured in LNCaP, and PC3 cells treated with patient serum were obtained from the above groups. The results reveal that proliferation of LNCaP cells was significantly ( P = 0.05) greater with serum from non-obese (mean = 1.26 ± 0.20) compared to that from obese patients (mean = 1.16 ± 0.19). Serum from obese PCa patients compared to non-obese PCa patients induced significantly greater amounts of cell migration ( P < 0.01) in PC3 cells. Serum from obese patients induced significantly ( P < 0.01) lower amounts of cell invasion (mean = 8.2 ± 4.5) compared to non-obese patients (mean = 18.1 ± 5.0) when treated on PC3 cells. Serum TNF-α (tumor necrosis factor alpha) levels correlated with LNCaP cell proliferation in vitro in non-obese PCa ( P < 0.01) and non-obese control groups ( P = 0.05). All statistical calculations controlled for age, since the PCa patient groups were significantly older than the control groups ( P < 0.01). In conclusion, serum from obese PCa patients induced greater PCa cell migration and lower cell proliferation and invasion in vitro.
The action of hypochlorous acid (HOCl) and γ-radiation on aqueous lysosphingolipid dispersions was found to produce 2-hexadecenal (Hex). This process includes the stages of formation of nitrogen-centered radicals from the starting molecules and the subsequent fragmentation of these radicals via the rupture of C–C and O–H bonds. These findings prove the existence of a nonenzymatic pathway of sphingolipid destruction leading to the formation of Hex, which possesses a wide spectrum of biological activity. Analysis of the effect of HOCl on transplantable rat glioma C6 cells and human embryonic kidney 293 cells points to the formation of Hex. This suggests that the described mechanism of free-radical destruction of sphingolipids may be replicated on cell culture under the stress of active chlorine forms.
Cholesterol transport between cellular organelles comprised vesicular trafficking and nonvesicular exchange; these processes are often studied by quantitative fluorescence microscopy. A major challenge for using this approach is producing analogs of cholesterol with suitable brightness and structural and chemical properties comparable with those of cholesterol. This review surveys currently used fluorescent sterols with respect to their behavior in model membranes, their photophysical properties, as well as their transport and metabolism in cells. In the first part, several intrinsically fluorescent sterols, such as dehydroergosterol or cholestatrienol, are discussed. These polyene sterols (P-sterols) contain three conjugated double bonds in the steroid ring system, giving them slight fluorescence in ultraviolet light. We discuss the properties of P-sterols relative to cholesterol, outline their chemical synthesis, and explain how to image them in living cells and organisms. In particular, we show that P-sterol esters inserted into low-density lipoprotein can be tracked in the fibroblasts of Niemann-Pick disease using high-resolution deconvolution microscopy. We also describe fluorophore-tagged cholesterol probes, such as BODIPY-, NBD-, Dansyl-, or Pyrene-tagged cholesterol, and eventual esters of these analogs. Finally, we survey the latest developments in the synthesis and use of alkyne cholesterol analogs to be labeled with fluorophores by click chemistry and discuss the potential of all approaches for future applications.
Ceramide and sphingosine and their phosphorylated counterparts are recognized as "bioactive sphingolipids" and modulate membrane integrity, the activity of enzymes, or act as ligands of G protein-coupled receptors. The subcellular distribution of the bioactive sphingolipids is central to their function as the same lipid can mediate diametrically opposite effects depending on its location. To ensure that these lipids are present in the right amount and in the appropriate organelles, cells employ selective lipid transport and compartmentalize sphingolipid-metabolizing enzymes to characteristic subcellular sites. Our knowledge of key mechanisms involved in sphingolipid signaling and trafficking has increased substantially in the past decades-thanks to advances in biochemical and cell biological methods. In this review, we focus on the bioactive sphingolipids and discuss how the combination of studies in cells and in model membranes have contributed to our understanding of how they behave and function in living organisms.
