Phospholipid composition of the rat lens is independent of diet

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

Differences in membrane acyl phospholipid composition between an endothermic mammal and an ectothermic reptile are not limited to any phospholipid class

This study examined questions concerning differences in the acyl composition of membrane phospholipids that have been linked to the faster rates of metabolic processes in endotherms versus ectotherms. In liver, kidney, heart and brain of the ectothermic reptile, Trachydosaurus rugosus, and the endothermic mammal, Rattus norvegicus, previous findings of fewer unsaturates but a greater unsaturation index (UI) in membranes of the mammal versus those of the reptile were confirmed. Moreover, the study showed that the distribution of phospholipid head-group classes was similar in the same tissues of the reptile and mammal and that the differences in acyl composition were present in all phospholipid classes analysed, suggesting a role for the physical over the chemical properties of membranes in determining the faster rates of metabolic processes in endotherms. The most common phosphatidylcholine (PC) molecules present in all tissues (except brain) of the reptile were 16:0/18:1, 16:0/18:2, 18:0/18:2, 18:1/18:1 and 18:1/18:2, whereas arachidonic acid (20:4), containing PCs 16:0/ 20: 4, 18: 0/ 20: 4, were the common molecules in the mammal. The most abundant phosphatidylethanolamines ( PE) used in the tissue of the reptile were 18:0/18:2, 18:0/20:4, 18:1/18:1, 18:1/18:2 and 18:1/20:4, compared to 16: 0/ 18: 2, 16: 0/ 20: 4, 16: 0/ 22: 6, 18: 0/ 20: 4, 18: 0/ 22: 6 and 18:1/20: 4 in the mammal. UI differences were primarily due to arachidonic acid found in both PC and PEs, whereas docosahexaenoic acid (22:6) was a lesser contributor mainly within PEs and essentially absent in the kidney. The phospholipid composition of brain was more similar in the reptile and mammal compared to those of other tissues.

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the Structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance (G(H)(+)) was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to Saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes. (C) 2009 Elsevier Inc. All rights reserved.

Heterogeneous Distribution of the Unusual Phospholipid Semilysobisphosphatidic Acid through the Golgi Complex

To investigate the distribution of lipids through the Golgi complex, we analyzed the envelopes of several viruses that assemble in different subcompartments of the Golgi, as well as subcellular fractions. Our results indicate that each Golgi subcompartment has a distinct phospholipid composition due mainly to differences in the relative amounts of semilysobisphosphatidic acid (SLBPA), sphingomyelin, phosphatidylserine, and phosphatidylinositol. Interestingly, SLBPA is enriched in the adjacent Golgi networks compared with the Golgi stack, and this enrichment varies with cell type. The heterogeneous distribution of SLBPA through the Golgi complex suggests it may play an important role in the structure and/or function of this organelle.

Instability of the cellular lipidome with age

The human lens nucleus is formed in utero, and from birth onwards, there appears to be no significant turnover of intracellular proteins or membrane components. Since, in adults, this region also lacks active enzymes, it offers the opportunity to examine the intrinsic stability of macromolecules under physiological conditions. Fifty seven human lenses, ranging in age from 12 to 82 years, were dissected into nucleus and cortex, and the nuclear lipids analyzed by electrospray ionization tandem mass spectrometry. In the first four decades of life, glycerophospholipids (with the exception of lysophosphatidylethanolamines) declined rapidly, such that by age 40, their content became negligible. In contrast the level of ceramides and dihydroceramides, which were undetectable prior to age 30, increased approximately 100-fold. The concentration of sphingomyelins and dihydrosphingomyelins remained unchanged over the whole life span. As a consequence of this marked alteration in composition, the properties of fiber cell membranes in the centre of young lenses are likely to be very different from those in older lenses. Interestingly, the identification of age 40 years as a time of transition in the lipid composition of the nucleus coincides with previously reported macroscopic changes in lens properties (e.g., a massive age-related increase in lens stiffness) and related pathologies such as presbyopia. The underlying reasons for the dramatic change in the lipid profile of the human lens with age are not known, but are most likely linked to the stability of some membrane lipids in a physiological environment.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Metabolism and Translocation of Aminophospholipids in Mammalian Cells

In this study we investigated the metabolism, i.e. remodeling and translocation, of the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). A new method for introduction of exogenous PS and PE molecular species to cultured cells was developed, and combined with mass spectrometry it enabled more detailed follow-up of the metabolism of single molecular species than previously. We found that I) exogenous PS and PE molecular species can be efficiently introduced to cultured cells without compromising cell integrity, II) PS and PE molecular species are remodeled by several phospholipases displaying selectivity based on phopholipid head group and acyl chain composition, III) PS decarboxylase (PSD) and Kennedy pathways provide a different PE molecular species composition to the cellular PE pool. In addition, PE species produced by these pathways are translocated from the site of synthesis to other cell compartments depending on their acyl chain composition. The data obtained in the present study helps to understand cellular phospholipid metabolism in more depth. The data show that effective labeling of cultured cells by exogenous phospholipids does not compromise cell viability and may be used to disturb cellular phospholipid composition to study lipid homeostasis. Remodeling and translocation of PS and PE molecular species is highly selective. The developed method and mass- spectrometric techniques may be used in future studies to understand disturbances in lipid homeostasis for example in diabetes mellitus, thus opening doors to optional scientific approaches to study mechanisms behind pathologies related to lipid disturbances.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Perfil lipídico da membrana citoplasmática de espermatozoides in natura e criopreservados de jumentos e cavalos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Photodynamic Efficiency of Cationic meso-Porphyrins at Lipid Bilayers: Insights from Molecular Dynamics Simulations

Porphyrin derivatives have applications as photoactive drugs in photodynamic therapy. However, little is known about their interactions with phospholipid membranes at the molecular level. We employed molecular dynamics simulations to model the binding between a series of cationic meso-(N-methyl-4-pyridinium)phenylporphyrins and anionic phosphatidylglycerol lipid bilayers. This was done in the presence of molecular oxygen within the membrane. The ability of various porphyrins to cause photodamage was quantified in terms of their immersion depth and degree of exposition to a higher oxygen concentration inside the membrane. Simulations showed that the photodynamic efficiency could be improved as the number of hydrophobic phenyl substituents attached to the porphyrinic ring increased. In the specific case of porphyrins containing two hydrophobic and two charged substituents, the cis isomer was significantly more efficient than the trans. These results correlate well with previous experimental observations. They highlight the importance of both the total charge and amphiphilicity of the photosensitizer for its performance in photodynamic therapy.

Flagellar membranes are rich in raft-forming phospholipids.

The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

Role of phosphatidylethanolamine in the function and assembly of phenylalanine-specific permease of Escherichia coli

Transmembrane segments of polytopic membrane proteins once inserted are generally considered stably oriented due to the large free energy barrier for topological reorientation of adjacent extra-membrane domains. However, proper topology and function of the polytopic membrane protein lactose permease (LacY) of Escherichia coli is dependent on the membrane phospholipid composition revealing topological dynamics of transmembrane domains (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107–2116). The high affinity phenylalanine permease PheP shares many topological similarities with LacY. In this study, mutant E. coli cells lacking phosphatidylethanolamine (PE) as a membrane component were used to evaluate the role of PE in the function and assembly of PheP. Active transport of phenylalanine by cells lacking PE was severely inhibited (both Vmax and Km were altered), whereas the PheP protein level in membranes was unaffected. Cysteine residues were introduced into predicted periplasmic or cytoplasmic segments of cysteine-less PheP, and the topology of the protein was explored using a membrane-impermeable thiol-specific biotinylated probe. Based on the biotinylation patterns of PheP in whole cells, the N-terminus and adjoining transmembrane hairpin of PheP adopted an inverted topological orientation in PE-lacking cells. Introduction of PE following the assembly of PheP triggered a reorientation of the N-terminus and adjacent hairpin to their native orientation associated with regain of wild type transport function. These results coupled with the results for LacY support a specific role for membrane lipid composition in determining topological organization and function of membrane proteins. Several other secondary symporters are compromised for activity in PE-lacking cells suggesting that lipid-assisted topogenesis is a general property of such transporters. The reversible orientation of these secondary transport proteins in response to a change of phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly. ^

Differences in surfactant lipids collected from pleural and pulmonary lining fluids

Purpose. The type and relative importance of saturated and unsaturated phospholipid components of surfactant within the epithelial lining fluid (ELF) of the inner and outer surfaces of the lung is not known. Methods. Seven healthy dogs were anesthetized and a bronchoalveolar lavage (BAL) was performed, immediately followed by a pleural lavage (PL). Lipid was extracted from lavage fluid and then analyzed for saturated, primarily dipalmitoylphosphatidylcholine (DPPC), and unsaturated phosphatidylcholine (PC) species using high-performance liquid chromatography (HPLC) with combined fluorescence and ultraviolet detection. Dilution of ELF in lavage fluids was corrected for using the urea method. Results. DPPC (494.7 +/- 213.9 mu g/mL) was the predominant PC present in ELF collected from the alveolar surface. In contrast, significantly higher (p = 0.028) proportions of unsaturated PC species were measured in PL fluid (similar to 105 mu g/mL), particularly stearoyl-linoleoyl-phosphatidylcholine (SLPC), which could not be measured in fluid collected from the alveoli, compared to DPPC (2.6 +/- 2.0 mu g/mL). Conclusions. This study indicates that unsaturated PC species seem to be more important than saturated species, particularly DPPC, in the pleural cavity, which has implications for surfactant replenishment following pleural disease or thoracic surgery.

Comparison of surfactant lipids between pleural and pulmonary lining fluids

Saturated phospholipids (PCs), particularly dipalmitoylphosphatidylcholine (DPPC), predominate in surfactant lining the alveoli, although little is known about the relationship between saturated and unsaturated PCs on the outer surface of the lung, the pleura. Seven healthy cats were anesthetized and a bronchoalveolar lavage (BAL) was performed, immediately followed by a pleural lavage (PL). Lipid was extracted from lavage fluid and then analyzed for saturated, primarily dipalmitoylphosphatidylcholine (DPPC), and unsaturated PC species using high-performance liquid chromatography (HPLC) with combined fluorescence and ultraviolet detection. Dilution of epithelial lining fluid (ELF) in lavage fluids was corrected for using the urea method. The concentration of DPPC in BAL fluid (85.3 +/- 15.7 mu g/mL) was significantly higher (P=0.021) than unsaturated PCs (similar to 40 mu g/mL). However, unsaturated PCs (similar to 34 mu g/mL), particularly stearoyl-linoleoyl-phosphatidylcholine (SLPC; 17.4 +/- 6.8), were significantly higher (P = 0.021) than DPPC (4.3 +/- 1.8 mu g/mL) in PL fluid. These results show that unsaturated PCs appear functionally more important in the pleural cavity, which may have implications for surfactant replenishment following pleural disease or thoracic surgery. (c) 2005 Published by Elsevier Ltd.