RESUMEN

Molecular dynamics simulations amounting to ≈8 µs demonstrate that the glucose transporter GLUT1 undergoes structural fluctuations mediated by the fluidity of the lipid bilayer and the proximity to glucose. The fluctuations of GLUT1 increase as the glucose concentration is raised. These fluctuations are more pronounced when the lipid bilayer is in the fluid compared to the gel phase. Glucose interactions are confined to the extra-membranous residues when the lipid is in the gel phase but diffuses into the transmembrane regions in the fluid phase. Proximity of glucose to GLUT1 causes asynchronous expansions of key bottlenecks at the internal and external openings of the central pore. This is accomplished only by small conformational changes at the single residue level that lower the resistance to glucose movements, thereby permitting unsteered glucose and water movements along the entire length of the pore. When glucose is near salt bridges located at the external and internal openings of the central pore, the distance separating the polar amino acid residues guarding these apertures tends to increase in both fluid and gel phases. It is evident that the multiplicity of glucose interactions, obtained with high concentrations, amplifies the structural fluctuations in GLUT1. The findings that most of the salt bridges and the bottlenecks appear to be operated by glucose proximity suggest that the main triggers to activation of transport are located within the solvent accessible linker regions in the extramembranous zones.

Asunto(s)

RESUMEN

The structure of fully hydrated bilayers composed of equimolar proportions of palmitoylsphingomyelin (PSM) and cholesterol has been examined by synchrotron X-ray powder diffraction and atomistic molecular dynamics (MD) simulations. Two coexisting bilayer structures, which are distinguished by the transbilayer phosphate-phosphate distance of coupled PSM molecules, are observed by diffraction at 37 °C. The MD simulations reveal that PSM molecules in the thicker membrane are characterized by more ordered, more extended, and less interdigitated hydrocarbon tails compared to those in the thinner membrane. Intermolecular hydrogen bonds further distinguish the two bilayer structures, and we observe the disruption of a sphingomyelin intermolecular hydrogen bond network induced by the proximity of cholesterol. Through an unsupervised clustering of interatomic distances, we show for the first time that the asymmetry of phospholipids is important in driving their interactions with cholesterol. We identify four distinct modes of interaction, two of which lead to the dehydration of cholesterol. These two modes of interaction provide the first description of precise physical mechanisms underlying the umbrella model, which itself explains how phospholipids may shield cholesterol from water. The most dehydrating mode of interaction is particular to the N-acylated fatty acid moiety of PSM and thus may explain the long-held observation that cholesterol preferentially mixes with sphingomyelins over glycerophospholipids.

Asunto(s)

RESUMEN

L-Valine is one of the three branched-chain amino acids (valine, leucine, and isoleucine) essential for animal health and important in metabolism; therefore, it is widely added in the products of food, medicine, and feed. L-Valine is predominantly produced through microbial fermentation, and the production efficiency largely depends on the quality of microorganisms. In recent years, continuing efforts have been made in revealing the mechanisms and regulation of L-valine biosynthesis in Corynebacterium glutamicum, the most utilitarian bacterium for amino acid production. Metabolic engineering based on the metabolic biosynthesis and regulation of L-valine provides an effective alternative to the traditional breeding for strain development. Industrially competitive L-valine-producing C. glutamicum strains have been constructed by genetically defined metabolic engineering. This article reviews the global metabolic and regulatory networks responsible for L-valine biosynthesis, the molecular mechanisms of regulation, and the strategies employed in C. glutamicum strain engineering.

Asunto(s)

RESUMEN

Experimental evidence has shown a close correlation between the composition and physical state of the membrane bilayer and glucose transport activity via the glucose transporter GLUT1. Cooling alters the membrane lipids from the fluid to gel phase, and also causes a large decrease in the net glucose transport rate. The goal of this study is to investigate how the physical phase of the membrane alters glucose transporter structural dynamics using molecular-dynamics simulations. Simulations from an initial fluid to gel phase reduce the size of the cavities and tunnels traversing the protein and connecting the external regions of the transporter and the central binding site. These effects can be ascribed solely to membrane structural changes since in silico cooling of the membrane alone, while maintaining the higher protein temperature, shows protein structural and dynamic changes very similar to those observed with uniform cooling. These results demonstrate that the protein structure is sensitive to the membrane phase, and have implications for how transmembrane protein structures respond to their physical environment.

Asunto(s)

RESUMEN

3-deoxy-d-manno-octulosonic acid-lipid A (Kdo2 -lipid A) is the essential component of lipopolysaccharide in most Gram-negative bacteria and the minimal structural component to sustain bacterial viability. It serves as the active component of lipopolysaccharide to stimulate potent host immune responses through the complex of Toll-like-receptor 4 (TLR4) and myeloid differentiation protein 2. The entire biosynthetic pathway of Escherichia coli Kdo2 -lipid A has been elucidated and the nine enzymes of the pathway are shared by most Gram-negative bacteria, indicating conserved Kdo2 -lipid A structure across different species. Yet many bacteria can modify the structure of their Kdo2 -lipid A which serves as a strategy to modulate bacterial virulence and adapt to different growth environments as well as to avoid recognition by the mammalian innate immune systems. Key enzymes and receptors involved in Kdo2 -lipid A biosynthesis, structural modification and its interaction with the TLR4 pathway represent a clear opportunity for immunopharmacological exploitation. These include the development of novel antibiotics targeting key biosynthetic enzymes and utilization of structurally modified Kdo2 -lipid A or correspondingly engineered live bacteria as vaccines and adjuvants. Kdo2 -lipid A/TLR4 antagonists can also be applied in anti-inflammatory interventions. This review summarizes recent knowledge on both the fundamental processes of Kdo2 -lipid A biosynthesis, structural modification and immune stimulation, and applied research on pharmacological exploitations of these processes for therapeutic development.

Asunto(s)

RESUMEN

INTRODUCTION: Automated lipidomic methods based on mass spectrometry (MS) are now proposed to screen a large variety of candidate drugs available that inhibit de novo lipid synthesis and replace tedious methods based on radiotracer incorporation. A major new interest in inhibitors of de novo lipogenesis is their proapoptotic effect observed in cancerous cells. AREAS COVERED: In this review, the authors focus on the screening methods of antilipogenic inhibitors targeting the synthesis of malonylCoA (carbonic anhydrase, acetylCoA carboxylase), palmitylCoA (fatty acid synthase condensing and thioesterase subunits) and monounsaturated fatty acids (Δ9-desaturase). The consequences of inhibition depend on how the pathway deviates above the blockade: accelerated mitochondrial fatty acid oxidation following the decreased malonylCoA level, accumulation of ketone bodies and increased cholesterol synthesis following the increased acetylCoA level. Side effects such as anorexia and skin defects may critically decrease therapeutic indices in the long term. The authors emphasize the need for assessment of toxicity in short-term treatments inducing proapoptotic effects observed in aggressive hormone-dependent malignancies. EXPERT OPINION: The activity of lipogenesis inhibitors can be recognised in lipid profiles established by a combination of MS-based measurements and multivariate analysis processing hundreds of lipid molecular species. Because the method can be automated, it is suitable for screening large chemical libraries, with particular focus on anticancer activities.

Asunto(s)

RESUMEN

The calcium-mediated interaction of DNA with monolayers of the non-toxic, zwitterionic phospholipid, 1,2-distearoyl-sn-glycero-3-phosphocholine when mixed with 50 mol% of a second lipid, either the zwitteronic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or neutral cholesterol was investigated using a combination of surface pressure-area isotherms, Brewster angle microscopy, external reflectance Fourier transform infrared spectroscopy and specular neutron reflectivity in combination with contrast variation. When calcium and DNA were both present in the aqueous subphase, changes were observed in the compression isotherms as well as the surface morphologies of the mixed lipid monolayers. In the presence of calcium and DNA, specular neutron reflectivity showed that directly underneath the head groups of the lipids comprising the monolayers, DNA occupied a layer comprising approximately 13 and 18% v/v DNA for the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesterol-containing monolayers, respectively. The volume of the corresponding layer for 1,2-distearoyl-sn-glycero-3-phosphocholine only containing monolayers was ∼15% v/v DNA. Furthermore regardless of the presence and nature of the second lipid and the surface pressure of the monolayer, the specular neutron reflectivity experiments showed that the DNA-containing layer was 20-27 Šthick, suggesting the presence of a well-hydrated layer of double-stranded DNA. External reflectance Fourier transform infrared studies confirmed the presence of double stranded DNA, and indicated that the strands are in the B-form conformation. The results shed light on the interaction between lipids and nucleic acid cargo as well as the role of a second lipid in lipid-based carriers for drug delivery.

Asunto(s)

RESUMEN

Lipid domain formation in membranes underlies the concept of rafts but their structure is controversial because the key role of cholesterol has been challenged. The configuration of glycosphingolipid receptors for agonists, bacterial toxins and enveloped viruses in plasma membrane rafts appears to be an important factor governing ligand binding and infectivity but the details are as yet unresolved. I have used X-ray diffraction methods to examine how cholesterol affects the distribution of glycosphingolipid in aqueous dispersions of an equimolar mixture of cholesterol and egg-sphingomyelin containing different proportions of glucosylceramide from human extracts. Three coexisting liquid-ordered bilayer structures are observed at 37°C in mixtures containing up to 20mol% glycosphingolipid. All the cholesterol was sequestered in one bilayer with the minimum amount of sphingomyelin (33mol%) to prevent formation of cholesterol crystals. The other two bilayers consisted of sphingomyelin and glucosylceramide. Asymmetric molecular species of glucosylceramide with N-acyl chains longer than 20 carbons form an equimolar complex with sphingomyelin in which the glycosidic residues are arranged in hexagonal array. Symmetric molecular species mix with sphingomyelin in proportions less than equimolar to form quasicrystalline bilayers. When the glycosphingolipid exceeds equimolar proportions with sphingomyelin cholesterol is incorporated into the structure and formation of a gel phase of glucosylceramide is prevented. The demonstration of particular structural features of ceramide molecular species combined with the diversity of sugar residues of glycosphingolipid classes paves the way for a rational approach to understanding the functional specificity of lipid rafts and how they are coupled across cell membranes.

Asunto(s)

RESUMEN

Phospholipid vesicles (liposomes) formed in pharmaceutically acceptable nonaqueous polar solvents such as propylene glycol are of interest in drug delivery because of their ability to improve the bioavailability of drugs with poor aqueous solubility. We have demonstrated a stabilizing effect of cholesterol on lamellar phases formed by dispersion of distearoylphosphatidylcholine (DSPC) in water/propylene glycol (PG) solutions with glycol concentrations ranging from 0 to 100%. The stability of the dispersions was assessed by determining the effect of propylene glycol concentration on structural parameters of the lamellar phases using a complementary combination of X-ray and neutron scattering techniques at 25 °C and in the case of X-ray scattering at 65 °C. Significantly, although stable lamellar phases (and liposomes) were formed in all PG solutions at 25 °C, the association of the glycol with the liposomes' lamellar structures led to the formation of interdigitated phases, which were not thermostable at 65 °C. With the addition of equimolar quantities of cholesterol to the dispersions of DSPC, stable lamellar dispersions (and indeed liposomes) were formed in all propylene glycol solutions at 25 °C, with the significant lateral phase separation of the bilayer components only detectable in propylene glycol concentrations above 60% (w/w). We propose that the stability of lamellar phases of the cholesterol-containing liposomes formed in propylene glycol concentrations of up to 60% (w/w) represent potentially very valuable drug delivery vehicles for a variety of routes of administration.

Asunto(s)

RESUMEN

Sphingomyelin and cholesterol are of interest to biologists because they interact to form condensed structures said to be responsible for a variety of functions that membranes perform. Synchrotron X-ray diffraction methods have been used to investigate the structure of bilayers of D-erythro palmitoyl-sphingomyelin and complexes formed by palmitoyl- and egg-sphingomyelin with cholesterol in aqueous multibilayer dispersions. D-erythro palmitoyl sphingomyelin bilayers exist in two conformers that are distinguished by their lamellar repeat spacing, bilayer thickness, and polar group hydration. The distinction is attributed to hydrogen bonding to water or to intermolecular hydrogen bonds that are disrupted by the formation of ripple structure. The coexisting bilayer structures of pure palmitoyl sphingomyelin are observed in the presence of cholesterol-rich bilayers that are characterized by different bilayer parameters. The presence of cholesterol preferentially affects the conformer of D-erythro sphingomyelin with thicker, more hydrated bilayers. Coexisting bilayers of sphingomyelin and complexes with cholesterol are in register and remain coupled at temperatures at least up to 50 °C. Cholesterol forms a complex of 1.8 mols of sphingomyelin per cholesterol at 37 °C that coexists with bilayers of pure sphingomyelin up to 50 °C. Redistribution of the two lipids takes place on cooling below the fluid- to gel-phase transition temperature, resulting in the withdrawal of sphingomyelin into gel phase and the formation of coexisting bilayers of equimolar proportions of the two lipids. Cholesterol-rich bilayers fit a stripe model at temperatures less than 37 °C characterized by alternating rows of sphingomyelin and cholesterol molecules. A quasicrystalline array models the arrangement at higher temperatures in which each cholesterol molecule is surrounded by seven hydrocarbon chains, each of which is in contact with two cholesterol molecules. The thickness of bilayer complexes of sphingomyelin and cholesterol is less than that of coexisting bilayers of pure sphingomyelin. The implications for protein sorting theories based on bilayer thickness are discussed.

Asunto(s)

RESUMEN

Cells grown in culture are frequently employed to model lipid metabolism in vivo. There are reasons of convenience for this but examination of the lipidome of cultured cells and their metabolic responses to lipid supplementation give cause to indicate disparity with their counterparts in living animals. The reason is mainly that homeostatic regulation is exercised in animals supplied with an adequate diet in which the adipose tissue and liver represent plentiful sources of lipid integrated via inter-organ collaboration and able to buffer transient fluctuations in dietary lipid and essential fatty acids (EFAs). Moreover, conventional culture media are generally deficient in total lipids as well as essential EFAs. Cultured cells exposed to high glucose concentrations and lipid deficit typically manifest accelerated rates of lipogenesis evidenced by high rates of de novo FA biosynthesis. A more realistic model may be obtained by increasing supplements of lipid especially enriched in essential EFAs in the growth medium. Increasing concentrations of ω3 FAs, in particular, attenuate the rate of de novo lipogenesis. The improvement of cell culture models for pharmacological screening of drug-candidates targeting lipid or glucose metabolism is highlighted.

Asunto(s)

RESUMEN

L-threonine, one of the three major amino acids produced throughout the world, has a wide application in industry, as an additive or as a precursor for the biosynthesis of other chemicals. It is predominantly produced through microbial fermentation the efficiency of which largely depends on the quality of strains. Metabolic engineering based on a cogent understanding of the metabolic pathways of L-threonine biosynthesis and regulation provides an effective alternative to the traditional breeding for strain development. Continuing efforts have been made in revealing the mechanisms and regulation of L-threonine producing strains, as well as in metabolic engineering of suitable organisms whereby genetically-defined, industrially competitive L-threonine producing strains have been successfully constructed. This review focuses on the global metabolic and regulatory networks responsible for L-threonine biosynthesis, the molecular mechanisms of regulation, and the strategies employed in strain engineering.

Asunto(s)

RESUMEN

S-Adenosyl-L-methionine is an important bioactive sulfur-containing amino acid. Large scale preparation of the amino acid is of great significance. S-Adenosyl-L-methionine can be synthesized from L-methionine and adenosine triphosphate in a reaction catalyzed by methionine adenosyltransferase. In order to enhance S-adenosyl-L-methionine biosynthesis by industrial microbial strains, various strategies have been employed to optimize the process. Genetic manipulation has largely focused on enhancement of expression and activity of methionine adenosyltransferase. This has included its overexpression in Pichia pastoris, Saccharomyces cerevisiae and Escherichia coli, molecular evolution, and fine-tuning of expression by promoter engineering. Furthermore, knocking in of Vitreoscilla hemoglobin and knocking out of cystathionine-ß-synthase have also been effective strategies. Besides genetic modification, novel bioprocess strategies have also been conducted to improve S-adenosyl-L-methionine synthesis and inhibit its conversion. This has involved the optimization of feeding modes of methanol, glycerol and L-methionine substrates. Taken together considerable improvements have been achieved in S-adenosyl-L-methionine accumulation at both flask and fermenter scales. This review provides a contemporary account of these developments and identifies potential methods for further improvements in the efficiency of S-adenosyl-L-methionine biosynthesis.

Asunto(s)

RESUMEN

There is considerable interest in lateral domain structure in biological membranes not least because a variety of physiological processes are believed to require assembly and mutual organization of particular membrane components for their execution. Domain structure is known to be created by differences in physicochemical properties between membrane lipids such as phase transition temperature, intermolecular hydrogen bonding and ionic functional groups. Domains are also created by specific interactions between different membrane lipids to form stoichiometric complexes. Such complexes often form ordered structures referred to as membrane rafts. The present challenge is to define the balance of line tension between lateral membrane domains in individual leaflets of the bilayer and coupling forces operating at the midplane of the bilayer responsible for maintaining lipids in opposing domains on either side of the structure in register. A review of the current evidence relating to these questions is presented.

Asunto(s)

RESUMEN

There are numerous phospholipid formulations that incorporate α-tocopherol as a stabilizing agent but there are few studies of the effect of α-tocopherol on phospholipid structure and bilayer permeability. This study uses synchrotron X-ray powder diffraction methods to investigate how α-tocopherol changes the structure of distearoylphosphatidylcholines bilayers. Increasing proportions of α-tocopherol up to 20 mol% induces ripple structures in the bilayers. Two types of ripple structure are produced which are seen in electron micrographs of freeze-fracture replicas with periodicities of 16 and 12 nm, respectively. The stoichiometry of phospholipid: α-tocopherol in the ripple structures at 37 °C is 8:1. The presence of α-tocopherol tends to reduce the angle of tilt of the hydrocarbon chains of the phospholipid in the gel phase from about 34° to the bilayer normal at 20 °C into a more vertical orientation. Increasing proportions of α-tocopherol progressively decrease the temperature of the gel to liquid-crystal phase transition of the phospholipid. The presence of up to 20 mol% α-tocopherol in 1-palmitoyl-2-oleoyl-phosphocholine inhibits leakage of phenol red dye from liposomes. The effect of 7 mol% α-tocopherol on leakage was compared with phospholipid liposomes containing 50 mol% cholesterol. The cholesterol-containing liposomes inhibited leakage to a greater extent than the vesicles incorporating α-tocopherol but the effect of α-tocopherol at equivalent molar proportions was comparable to cholesterol.

Asunto(s)

RESUMEN

The lipidome of the liver and the secreted circulating lipoproteins can now be interrogated conveniently by automated mass spectrometric methods. Multivariate analysis of the liver and serum lipid composition in various animal modes or in human patients has pointed to specific molecular species markers. The perturbations of lipid metabolism can be categorized on the basis of three basic pathological mechanisms: (1) an accelerated rate of de novo lipogenesis; (2) perturbation of the peroxisome pathway of ether-lipid and very-long-chain fatty acid biosynthesis; (3) a change in the rate of interconversion of essential omega-3 and -6 polyunsaturated fatty acids. This review provides examples to illustrate the practicalities of lipidomic studies in biomedicine.

RESUMEN

The rising incidence of cardiovascular and metabolic diseases in industrialized countries has led the pharmaceutical industry to make them key areas of drug development. These diseases imply a clustering of metabolic factors where lipid metabolites play a relevant role. Measurement of pharmacodynamic endpoints of drugs on lipid metabolism pathways and downstream biological processes appear crucial for a rational drug discovery/development. Fortunately, recent mass spectrometers with an enhanced sensitivity and resolution in combination with multivariate statistical analysis provide the practical possibility to analyze and measure wide portions of the lipidome. The final goal is to identify lipid signatures which fit with specific pharmacologic responses to therapeutic intervention. Focusing on applications of lipidomics for drug development this review outlines the methodological steps, from analytical measurements to data processing and to graphical representation, for an efficient implementation of informative lipid signatures.

Asunto(s)

RESUMEN

Interaction between membrane lipids creates lateral domains within which essential membrane processes like trans-membrane signaling, differentiation etc. take place. Attention has focused on liquid-ordered phases formed by sphingomyelin and cholesterol but formation of ordered domains on the cytoplasmic membrane surfaces has largely been neglected. Synchrotron X-ray powder diffraction methods were used to investigate the interaction between two components of the cytoplasmic leaflet of the plasma membrane, phosphatidylethanolamine and glucosylceramide. Multilamellar dispersions of binary mixtures of different molecular species of phosphatidylethanolamine and glucosylceramide were examined. Stoichiometric complexes are formed when the phosphatidylethanolamine has at least one unsaturated fatty acid. The stoichiometry of the complexes was 2.0 fluid phospholipids per glucosylceramide with C22/24 N-acyl chains and 1.8 with C-12 chains. Saturated molecular species of phosphatidylethanolamines were immiscible with glucosylceramide. The complexes formed with unsaturated phosphatidylethanolamines and glucosylceramide are stable above physiological temperatures. A putative role of these matrices in membrane rafts is considered.

Asunto(s)

RESUMEN

Specific lipid-lipid interactions are believed to be responsible for lateral domain formation in the lipid bilayer matrix of cell membranes. The miscibility of glucocerebroside and sphingomyelin extracted from biological tissues has been examined by synchrotron X-ray powder diffraction methods. Fully hydrated binary mixtures of egg-sphingomyelin codispersed with glucosylceramide rich in saturated C22 and C24 N-acyl fatty acids were subjected to heating scans between 20 and 90 °C at 2 °C·min(-1). X-ray scattering intensity profiles were recorded at 1 °C intervals simultaneously in both small-angle and wide-angle scattering regions. A gel phase characterized by a single symmetric peak in the wide-angle scattering region was transformed in all mixtures examined to a fluid phase at about 40 °C, similar to dispersions of pure egg-sphingomyelin. A coexisting lamellar structure was identified at temperatures up to about 75 °C which was characterized by a broad Bragg reflection. The scattering intensity of this structure increased relative to the structure assigned as bilayers of pure sphingomyelin with increasing proportions of glucosylceramide in the mixture. The relationship between the scattering intensities of the two peaks and the relative mass fractions of the two lipids showed that the bilayers assigned to a glucosylceramide-rich structure were composed of sphingomyelin and glucosylceramide in molar ratios of 1 : 1 and 2 : 1, respectively, at temperatures below and above the order-disorder phase transition temperature of the sphingomyelin (40 °C).

Asunto(s)

RESUMEN

L-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize L-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of L-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including L-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined L-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered.

Asunto(s)