Dagmar Zweytick

Intracellular lipid particles of eukaryotic cells

In this review article we describe characterization of intracellular lipid particles of three different eukaryotic species, namely mammalian cells, plants and yeast. Lipid particles of all types of cells share a general structure. A hydrophobic core of neutral lipids is surrounded by a membrane monolayer of phospholipids which contains a minor amount of proteins. Whereas lipid particles from mammalian cells and plants harbor specific classes of polypeptides, mainly perilipins and oleosins, respectively, yeast lipid particles contain a more complex set of enzymes which are involved in lipid biosynthesis. Function of lipid particles as storage compartment and metabolic organelle, and their interaction with other subcellular fractions are discussed. Furthermore, models for the biogenesis of lipid particles are presented and compared among the different species.

Membrane-active host defense peptides--challenges and perspectives for the development of novel anticancer drugs.

Highlights ► Outlined the need of novel strategies for cancer therapies that can counteract problems arising particularly in chemotherapy due to resistance to current drugs and their low specificity. ► Elaborated the differences in membrane composition and properties between cancer and non-cancer cells, the basis for the use of anticancer peptides derived from host defense peptides as new weapons against cancer. ► Described the current knowledge on the mode of action of these peptides and the status of in vivo studies. ► Summarized the challenges and perspectives for the development of host defense peptides as novel anticancer agents.

In search of a novel target - phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy.

This study was performed in the aim to identify potential targets for the development of novel therapy to treat cancer with poor outcome or treatment efficacy. We show that the negatively charged phospholipid phosphatidylserine (PS) is exposed in the outer leaflet of their plasma membrane not only in tumor cell lines, but also in metastases and primary cultures thereof, which contrasts with a lack of PS exposure by differentiated non-tumorigenic counterparts. Studied tumor cell lines were derived from non-tumorigenic and malignant melanomas, prostate- and renal cancer, glioblastoma and a rhabdomyosarcoma. Importantly, also metastases of melanoma expose PS and there is a correlation between malignancy of melanoma cell lines from different stages of tumor progression and PS exposure. The PS exposure we found was neither of apoptotic nor of experimental artificial origin. Finally potentially malignant and non-malignant cells could be differentiated by sorting of a primary cell culture derived from a glioblastoma based on PS exposure, which has so far not been possible within one culture due to lack of a specific marker. Our data provide clear evidence that PS could serve as uniform marker of tumor cells and metastases as well as a target for novel therapeutic approaches based on e.g. PS-specific host defense derived peptides.

Structural and Biochemical Properties of Lipid Particles from the Yeast Saccharomyces cerevisiae

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.

Biochemical characterization and subcellular localization of the sterol C-24(28) reductase, Erg4p, from the yeast Saccharomyces cerevisiae

The yeast ERG4 gene encodes sterol C‐24(28) reductase which catalyzes the final step in the biosynthesis of ergosterol. Deletion of ERG4 resulted in a complete lack of ergosterol and accumulation of the precursor ergosta‐5,7,22,24(28)‐tetraen‐3β‐ol. An erg4 mutant strain exhibited pleiotropic defects such as hypersensitivity to divalent cations and a number of drugs such as cycloheximide, miconazole, 4‐nitroquinoline, fluconazole, and sodium dodecyl sulfate. Similar to erg6 mutants, erg4 mutants are sensitive to the Golgi‐destabilizing drug brefeldin A. Enzyme activity measurements with isolated subcellular fractions revealed that Erg4p is localized to the endoplasmic reticulum. This view was confirmed in vivo by fluorescence microscopy of a strain expressing a functional fusion of Erg4p to enhanced green fluorescent protein. We conclude that ergosterol biosynthesis is completed in the endoplasmic reticulum, and the final product is supplied from there to its membranous destinations.

Membrane curvature stress and antibacterial activity of lactoferricin derivatives.

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127–6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1∆ deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1∆ mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.

Killing of melanoma cells and their metastases by human lactoferricin derivatives requires interaction with the cancer marker phosphatidylserine

Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl derivative also slightly affects PC. Tryptophan fluorescence of selective peptide R-DIM-P-LF11-322 revealed specific peptide penetration into the PS membrane interface and circular dichroism showed change of its secondary structure by increase of proportion of β-sheets just in the presence of the cancer mimic. Data correlated with in vitro studies with cell lines of human melanomas, their metastases and melanocytes, revealing R-DIM-P-LF11-322 to exhibit strongly increased specificity for cancer cells. This indicates the need of high affinity to the target PS, a minimum length and net positive charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity.

N-acylated Peptides Derived from Human Lactoferricin Perturb Organization of Cardiolipin and Phosphatidylethanolamine in Cell Membranes and Induce Defects in Escherichia coli Cell Division

Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill bacteria by interacting with bacterial membrane lipids but only N-acylated peptides interact with both charged cardiolipin and zwitterionic phosphatidylethanolamine resulting in remodeling of the natural phospholipid domains in the E. coli membrane that leads to defects in cell division.

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.