Regina Leber

Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomycescerevisiae.

Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Δ mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.

Application of inductively coupled plasma mass spectrometry to phospholipid analysis

Phospholipids are the main constituents of membranes in all types of prokaryotic and eukariotic cells. Due to their complexity and heterogeneity in biological samples, qualitative and quantitative analyses of membrane phospholipids in cellular extracts represent major analytical challenges, mainly due to suitable and sensitive detection methods. The inductively coupled plasma mass spectrometer (ICP-MS) is a suitable detector for selective determination of phospholipids as they all contain phosphorus. Phospholipids are extractable with organic solvents, therefore liquid chromatography with an organic mobile phase was used for separation of different lipid species. Solvent load to the plasma was reduced by splitting the mobile phase prior to reaching the nebulizer, by chilling the spray chamber to −5 °C and by optimisation of carrier gas flow for maximum condensation of organic vapours. Despite desolvation, oxygen was added to prevent carbon deposition on interface cones. To reduce polyatomic interferences at m/z ratio 31 (e.g.31CH3O+) and to improve detection limits, helium was used as a collision gas. The achieved absolute detection limits were between 0.21 and 1.2 ng of phosphorus and were superior to those obtained by an evaporative light scattering detector, which provides an alternative detection system for lipid analysis. The usefulness of the developed method was demonstrated by analysis of lipid extracts from the yeast Saccharomyces cerevisiae.

Phospholipid-driven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems.

OP-145, a synthetic antimicrobial peptide developed from a screen of the human cathelicidin LL-37, displays strong antibacterial activities and is--at considerably higher concentrations--lytic to human cells. To obtain more insight into its actions, we investigated the interactions between OP-145 and liposomes composed of phosphatidylglycerol (PG) and phosphatidylcholine (PC), resembling bacterial and mammalian membranes, respectively. Circular dichroism analyses of OP-145 demonstrated a predominant α-helical conformation in the presence of both membrane mimics, indicating that the different membrane-perturbation mechanisms are not due to different secondary structures. Membrane thinning and formation of quasi-interdigitated lipid-peptide structures was observed in PG bilayers, while OP-145 led to disintegration of PC liposomes into disk-like micelles and bilayer sheets. Although OP-145 was capable of binding lipoteichoic acid and peptidoglycan, the presence of these bacterial cell wall components did not retain OP-145 and hence did not interfere with the activity of the peptide toward PG membranes. Furthermore, physiological Ca++ concentrations did neither influence the membrane activity of OP-145 in model systems nor the killing of Staphylococcus aureus. However, addition of OP-145 at physiological Ca++-concentrations to PG membranes, but not PC membranes, resulted in the formation of elongated enrolled structures similar to cochleate-like structures. In summary, phospholipid-driven differences in incorporation of OP-145 into the lipid bilayers govern the membrane activity of the peptide on bacterial and mammalian membrane mimics.

liposomal nanoparticles encapsulating iloprost exhibit enhanced vasodilation in pulmonary arteries

Prostacyclin analogues are standard therapeutic options for vasoconstrictive diseases, including pulmonary hypertension and Raynaud’s phenomenon. Although effective, these treatment strategies are expensive and have several side effects. To improve drug efficiency, we tested liposomal nanoparticles as carrier systems. In this study, we synthesized liposomal nanoparticles tailored for the prostacyclin analogue iloprost and evaluated their pharmacologic efficacy on mouse intrapulmonary arteries, using a wire myograph. The use of cationic lipids, stearylamine, or 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) in liposomes promoted iloprost encapsulation to at least 50%. The addition of cholesterol modestly reduced iloprost encapsulation. The liposomal nanoparticle formulations were tested for toxicity and pharmacologic efficacy in vivo and ex vivo, respectively. The liposomes did not affect the viability of human pulmonary artery smooth muscle cells. Compared with an equivalent concentration of free iloprost, four out of the six polymer-coated liposomal formulations exhibited significantly enhanced vasodilation of mouse pulmonary arteries. Iloprost that was encapsulated in liposomes containing the polymer polyethylene glycol exhibited concentration-dependent relaxation of arteries. Strikingly, half the concentration of iloprost in liposomes elicited similar pharmacologic efficacy as nonencapsulated iloprost. Cationic liposomes can encapsulate iloprost with high efficacy and can serve as potential iloprost carriers to improve its therapeutic efficacy.

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine ☆

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity.

Synergism of Antimicrobial Frog Peptides Couples to Membrane Intrinsic Curvature Strain

Mixtures of the frog peptides magainin 2 and PGLa are well-known for their pronounced synergistic killing of Gram-negative bacteria. We aimed to gain insight into the underlying biophysical mechanism by interrogating the permeabilizing efficacies of the peptides as a function of stored membrane curvature strain. For Gram-negative bacterial-inner-membrane mimics, synergism was only observed when the anionic bilayers exhibited significant negative intrinsic curvatures imposed by monounsaturated phosphatidylethanolamine. In contrast, the peptides and their mixtures did not exhibit significant activities in charge-neutral mammalian mimics, including those with negative curvature, which is consistent with the requirement of charge-mediated peptide binding to the membrane. Our experimental findings are supported by computer simulations showing a significant decrease of the peptide-insertion free energy in membranes upon shifting intrinsic curvatures toward more positive values. The physiological relevance of our model studies is corroborated by a remarkable agreement with the peptide’s synergistic activity in Escherichia coli. We propose that synergism is related to a lowering of a membrane-curvature-strain-mediated free-energy barrier by PGLa that assists membrane insertion of magainin 2, and not by strict pairwise interactions of the two peptides as suggested previously.

Antifungal Host Defense Peptides

Fungi infect billions of people every year, yet their contribution to the global burden of disease is largely unrecognized and the repertoire of antifungal agents is rather limited. Thus, treatment of life-threatening invasive fungal infections is still based on drugs discovered several decades ago. In addition, recent data on resistance emergence of fungi emphasize the urgent need for novel antifungal treatments. One alternative strategy is based on host defense peptides. Among the large number of antimicrobial peptides, a group of peptides show primarily antifungal activity by interfering with enzymes of cell wall biosynthesis or specific membrane lipids such as ergosterol. Both are promising targets for antifungal peptides, as they are absent in mammalian cells and hence low toxicity of peptides can be expected. However, most of the antimicrobial peptides exhibit a broad spectrum activity including antifungal activity. These peptides act on the cell membrane level and although their structures vary largely, they share a positive net charge, which facilitates electrostatic interactions with negatively charged lipids of the target cell, and an amphipathic structure, which facilitates incorporation into the cell membrane and in turn membrane disruption. Thereby, membrane lipids differing between mammals and fungi play a central role concerning specificity and efficacy of these peptides. Hence, understanding their molecular mechanism(s) of action will aid in the design of novel antifungal agents. Finally, some of these peptides were shown to act synergistically with conventional drugs, which would further widen the armory to treat especially life-threatening invasive fungal infections.