Improved embryonic cryosurvival observed after in vitro supplementation with conjugated linoleic acid is related to changes in the membrane lipid profile

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudos computacionais de esfingomielinases D: docking, dinâmica molecular e métodos híbridos QM/MM

Pós-graduação em Biofísica Molecular - IBILCE

Estudos computacionais de esfingomielinases D: docking, dinâmica molecular e métodos híbridos QM/MM

Pós-graduação em Biofísica Molecular - IBILCE

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Tip indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.

Proteomic characterisation of toxins isolated from nematocysts of the South Atlantic jellyfish Olindias sambaquiensis

Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including conesnails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological activities of these potential toxins include cytolysins, neurotoxins, phospholipases and toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals sequence orthologs to many representatives of the most important superfamilies of peptide venoms suggesting that their origins in higher organisms arise from deep eumetazoan innovations. Accordingly, cnidarian venoms may possess unique biological properties that might generate new leads in the discovery of novel pharmacologically active drugs.

Systematic studies of the interaction between amyloid beta-protein and lipids

The amyloid peptide (Aß), a normal constituent of neuronal and non-neuronal cells, has been shown to be a major component of the extracellular plaque of Alzheimer’s disease (AD). The interaction of Aß peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of AD. In this study, we have developed peptide-tethered artificial lipid membranes by the Langmuir-Blodgett and Langmuir-Schaefer methods. Anti-Aß40-mAb labeled with a fluorophore was used to probe the Aß40 binding to the model membrane system. Systematic studies on the antibody or Aß-membrane interactions were carried out in our model systems by Surface Plasmon Field-Enhanced Fluorescence Spectroscopy (SPFS). Aß adsorption is critically determined by the lipid composition of the membranes. Aß specifically binds with membranes of sphingomyelin, and this preferential adsorption was markedly amplified by the addition of sterols (cholesterol or 25-OH-Chol). Fluorescence microscopy indicated that 25-OH-Chol could also form micro-domains with sphingomyelin as cholesterol does at the conditions used for the built-up of the model membranes. Our findings suggest that micro-domains composed of sphingomyelin and the sterols could be the binding sites of Aß and the role of sphingomyelin in AD should receive much more attention. The artificial membranes provide a novel platform for the study on AD, and SPFS is a potential tool for detecting Aß-membrane interaction. Numerous investigations indicate that the ability of Aß to form fibrils is considerably dependent upon the levels of ß-sheet structure adopted by Aß. Membrane-mediated conformational transition of Aß has been demonstrated. In this study, we focus on the interaction of Aß and the membranes composed of POPC/SM/25-OH-Chol (2:1:1). The artificial membrane system was established by the methods as described above. Immunoassy based on a pair of monoclonal antibodies (mAbs) against different epitopes was employed to detect the orientation of the Aß at the model membranes. Kinetics of antibody-Aß binding was determined by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The attempt has also been made to probe the change in the conformation of Aß using SPFS combined with immunoassay. Melatonin was employed to induce the conformational change of Aß. The orientation and the conformational change of Aß are evaluated by analysing kinetic/affinity parameters. This work provides novel insight into the investigation on the structure of Aß at the membrane surface.

Untersuchung multimerer Proteinkomplexe mit oberflächenanalytischen und röntgenspektroskopischen Methoden

Zusammenfassung rnrnIn dieser Arbeit wurden Untersuchungen an zwei verschiedenen multimeren Proteinkomplexen durchgeführt: Zum einen am Hämocyanin aus Homarus americanus mittels Röntgen-L-Kantenspektroskopie und zum anderen am α-Toxin aus Staphylococcus aureus, hinsichtlich der Interaktion an speziellen Raft-artigen Membranabschnitten, mittels AFM.rnFür das Hämocyanin aus Homarus americanus konnte ein neuer Aspekt bezüglich der Bindung von Sauerstoff aufgezeigt werden. Ein zuvor nicht in Betracht gezogener und diskutierter Einfluss von Wassermolekülen auf diesen Vorgang konnte mittels der Methode der Röntgen-L-Kantenspektroskopie dargestellt werden. Erstmals war es möglich die beiden verschiedenen Beladungszustände (Oxy-, Deoxy-Zustand) des Hämocyanin mittels dieser Methode in physiologisch ähnlicher Umgebung zu untersuchen. Vergleiche der erhaltenen L-Kanten-Spektren mit denen anorganischer Vergleichslösungen ließen auf eine Interaktion von Wassermolekülen mit den beiden Kupferatomen des aktiven Zentrums schließen. Dadurch wurde erstmals ein möglicher Einfluss des Wassers auf den Oxygenierungsprozess des Hämocyanins auf elektronischer Ebene aufgezeigt. Vergleichende Betrachtungen von Röntgenkristallstrukturen verschiedener Typ-3-Kupferproteine bestätigten, dass auch hier ein Einfluss von Wassermolekülen auf die aktiven Zentren möglich ist. Vorgeschlagen wird dabei, dass an Stelle der Überlappung der 3d-Orbitale des Kupfers mit den 2p-Orbitalen des Sauerstoffs, wie sie im sauerstoffbeladenen Zustand auftritt, im sauerstoffunbeladenen Zustand eine Wechselwirkung der 3d-Orbitale des Kupfers mit den LUMOS der Wassermoleküle möglich wird, und ein Elektronen- bzw. Ladungstransfer von den Kupfern auf die Wassermoleküle erfolgen kann. rnAFM-Untersuchungen hinsichtlich der Interaktion des α-Toxins aus Staphylococcus aureus mit oberflächenunterstützten Modellmembranen wiesen darauf hin, dass eine bevorzugte Anbindung und zumindest teilweise Integration der α-Toxine in Raft-artige Membranbereiche stattfindet. Für verschiedene ternäre Lipidsysteme konnten phasenseparierte Modellmembranen abgebildet und die unterschiedlichen Domänenformen zugeordnet werden. Der Anbindungsprozess der Toxine an diese oberflächenunterstützte Modellmembranen erfolgte dann wahrscheinlich vornehmlich an den speziellen Raft-artigen Domänen, wohingegen die Insertion der Poren vorrangig an den Grenzbereichen zwischen den Domänen auftrat. Mögliche Ursache dafür sind die räumlichen Besonderheiten dieser Grenzflächen. Membranen weisen an den Schnittstellen zwischen zwei Domänenformen eine erhöhte Unordnung auf, was sich u.a. in einer geringeren Packungsdichte der Phospholipide und dem erhöhten Freiheitsgrad ihrer Kopfgruppen bemerkbar macht. Außerdem kommt es auf Grund der Interaktion der beteiligten Membranbestandteile Sphingomyelin und Cholesterol untereinander zu einer speziellen Ausrichtung der Phosphocholin-Kopfgruppen und innerhalb der Raft-artigen Domänen zu einer erhöhten Packungsdichte der Phospholipide. Die in dieser Arbeit präsentierten Ergebnisse unterstützten demnach die in der Literatur postulierte Vermutung der bevorzugten Interaktion und Integration der Toxin-Moleküle mit Raft-artigen Membrandomänen. Die Insertion der Pore erfolgt aber wahrscheinlich bevorzugt an den Grenzbereichen zwischen den auftretenden Domänen.rn

Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis

Although the protozoan parasite, Trypanosoma brucei, can acquire lipids from its environment, recent reports have shown that it is also capable of de novo synthesis of all major phospholipids. Here we provide an overview of the biosynthetic pathways involved in phospholipid formation in T. brucei and highlight differences to corresponding pathways in other eukaryotes, with the aim of promoting trypanosomes as an attractive model organism to study lipid biosynthesis. We show that de novo synthesis of phosphatidylethanolamine involving CDP-activated intermediates is essential in T. brucei and that a reduction in its cellular content affects mitochondrial morphology and ultrastructure. In addition, we highlight that reduced levels of phosphatidylcholine inhibit nuclear division, suggesting a role for phosphatidylcholine formation in the control of cell division. Furthermore, we discuss possible routes leading to phosphatidylserine and cardiolipin formation in T. brucei and review the biosynthesis of phosphatidylinositol, which seems to take place in two separate compartments. Finally, we emphasize that T. brucei represents the only eukaryote so far that synthesizes all three sphingophospholipid classes, sphingomyelin, inositolphosphorylceramide and ethanolaminephosphorylceramide, and that their production is developmentally regulated.

Biochemical characterization of detergent-resistant membranes: a systematic approach

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms. Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [(14)CO(2)]-liberation. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides (p<0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity (p<0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc (p<0.05) and ODC (p<0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARgamma-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.

Rapid effects of 1alpha,25-dihydroxyvitamin D3 on osteoblasts: Studies of membrane transducers that affect calcium signals

1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] exerts pleiotropic effects on osteoblasts via both long-term nuclear receptor-mediated and rapid membrane-initiated pathways during bone remodeling and mineral homeostasis. This study explored the membrane transducers that mediate rapid effects of 1,25(OH)2D3 on osteoblasts, including sphingomyelinase (SMase) and L-type voltage sensitive calcium channels (VSCCs). ^ It was previously demonstrated that 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through VSCCs in ROS 17/2.8 osteoblasts, however the molecular identity of 1,25(OH)2D 3-regulated VSCC has not been known. In this study, on the basis of in vitro tests of three unique ribozymes specifically cleaving a1C mRNA, I transfected ROS 17/2.8 cells with vectors coding recombinant ribozyme modified with U1 snRNA structure, and successfully selected stable clonal cells in which the expression of a1C was strikingly reduced. Ca2+ influx studies in these cells compared to control transfectants showed selective attenuation of depolarization- and 1,25(OH)2D3-regulated Ca2+ responses. These results allow us to conclude that the cardiac ( a1C ) subtype of the L-type VSCC is the major membrane transducer of Ca 2+ influx in osteoblasts. ^ I also demonstrated that 1,25(OH)2D3 induces a rapid hydrolysis of membrane sphingomyelin (SM) in ROS 17/2.8 cells, with the concomitant generation of ceramide, detectable at 15 minute, and maximal at 1 hour after addition. Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC), downstream products of SM hydrolysis, but not ceramide, elicit Ca 2+ release from intracellular stores. Considering ceramide, sphingosine, and SPP as second messengers modulating intracellular kinases or phosphatases, these findings implicate sphingolipid-signaling pathways in transducing rapid effects of 1,25(OH)2D3 on osteoblasts. In structure/function analyses of sphingolipid signaling, it was observed that psychosine elicits Ca2+ release from intracellular stores. This challenges the dogma that sphingosine phosphorylation permits mobilization of Ca2+ , because psychosine is a sphingosine analog galactosylated at 1-carbon, preventing phosphorylation at that site. Psychosine is the pathological metabolite found in patients with Krabbe's disease, suggesting that psychosine disrupts the physiological sphingolipid signaling by chronic release of Ca2+ from intracellular stores. ^ Slower SM turnover than Ca2+ influx through VSCCs in response to 1,25(OH)2D3 demonstrates ceramide does not mediate the 1,25(OH)2D3-induced Ca2+ signaling, a conclusion endorsed further by the failure of ceramide to induce Ca 2+ signaling. ^

Ceramide in Plasma Membrane Repair

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca2+ and an efflux of cytoplasmic proteins. In order to ensure cellular survival, lesions have to be identified, plugged and removed from the membrane. The Ca2+-driven fusion of lysosomes with the plasma membrane leads to hydrolysis of sphingomyelin by acid sphingomyelinase and a formation of ceramide platforms in the outer leaflet of the lipid bilayer. We propose that the negative curvature, promoted by tighter packing of lipids in the outer layer, leads to an inward vesiculation of the damaged area for its endocytotic uptake and internal degradation. In contrast, the activation of neutral sphingomyelinase triggers the production of ceramide within the inner leaflet of the lipid bilayer, thereby promoting an outward curvature, which enables the cell to shed the membrane-containing toxin pore into the extracellular space. In this process, ceramide is supported by members of the annexin protein family which act as Ca2+ sensors and as membrane fusion agents.

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.

Decreased phosphatidylcholine plasmalogens - A putative novel lipid signature in patients with stable coronary artery disease and acute myocardial infarction.

OBJECTIVE Glycerophospholipids and sphingolipids are structurally heterogeneous due to differences in the O- and N-linked fatty acids and head groups. Sphingolipids also show a heterogeneity in their sphingoid base composition which up to now has been little appreciated. The aim of this study was to investigate the association of certain glycerophospholipid and sphingolipid species with stable coronary artery disease (CAD) and acute myocardial infarction (AMI). METHODS The lipid profile in plasma from patients with stable CAD (n = 18) or AMI (n = 17) was compared to healthy subjects (n = 14). Sixty five glycerophospholipid and sphingolipid species were quantified by LC-MS. The relative distribution of these lipids into lipoprotein fractions was analyzed. RESULTS In the CAD cohort, 45 glycerophospholipid and sphingolipid species were significantly lower compared to healthy controls. In the AMI group, 42 glycerophospholipid and sphingolipid species were reduced. Four PC plasmalogens (PC33:1, PC33:2, PC33:3 and PC35:3) showed the most significant difference. Out of eleven analyzed sphingoid bases, four were lower in the CAD and six in the AMI group. Sphingosine-1-phosphate (S1P) levels were reduced in the AMI group whereas an atypical C16:1 S1P was lower in both groups. Phosphatidylcholine and sphingomyelin species were exclusively present in lipoprotein particles, whereas lysophosphatidylcholines were mainly found in the lipoprotein-free fraction. The observed differences were not explained by the use of statins as confirmed in a second, independent cohort. CONCLUSIONS Reduced levels of four PC plasmalogens (PC33:1, PC33:2, PC33:3 and PC35:3) were identified as a putatively novel lipid signature for CAD and AMI.

Fas-induced apoptosis of T cells occurs independently of ceramide generation

The Fas receptor is one of a number of important physiological inducers of programmed cell death (apoptosis). Current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases. Induced changes in cellular levels of such sphingosine-based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate by treatment of cellular lipid extracts with bacterial diacylglycerol kinase (DAGK). To allow direct study of cellular sphingosine- and sphinganine-based ceramide levels, we developed a mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations. Contrary to current models, we detected no changes in cellular ceramide levels up to 2 hr poststimulation of Jurkat T cells with an anti-Fas IgM, although this treatment did induce apoptosis. We also determined in the same system that, when utilizing the DAGK assay, increased phosphorylation of substrates that comigrated with ceramide standards was apparent but that this effect was due to an enhancement of DAGK activity rather than increases in levels of cellular ceramides as substrates per se. Thus, the first direct measurement of ceramides present in cells undergoing apoptosis indicates that, insofar as it can be measured, the induction of apoptosis does not involve the generation of sphingosine-based ceramides, contrary to many published accounts.