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Microfluidic diffusional sizing (MDS) is a recent and powerful method for determining the hydrodynamic sizes and interactions of biomolecules and nanoparticles. A major benefit of MDS is that it can report the size of a fluorescently labeled target even in mixtures with complex, unpurified samples. However, a limitation of MDS is that the target itself has to be purified and covalently labeled with a fluorescent dye. Such covalent labeling is not suitable for crude extracts such as native nanodiscs directly obtained from cellular membranes. In this study, we introduce fluorescent universal lipid labeling for MDS (FULL-MDS) as a sparse, noncovalent labeling method for determining particle size. We first demonstrate that the inexpensive and well-characterized fluorophore, Nile blue, spontaneously partitions into lipid nanoparticles without disrupting their structure. We then highlight the key advantage of FULL-MDS by showing that it yields robust size information on lipid nanoparticles in crude cell extracts that are not amenable to other sizing methods. Furthermore, even for synthetic nanodiscs, FULL-MDS is faster, cheaper, and simpler than existing labeling schemes.

The study investigates the antitumor effect of two cationic peptides, R-DIM-P-LF11-215 (RDP215) and the D-amino acid variant 9D-R-DIM-P-LF11-215 (9D-RDP215), targeting the negatively charged lipid phosphatidylserine (PS) exposed by cancer cells, such as of melanoma and glioblastoma. Model studies mimicking cancer and non-cancer membranes revealed the specificity for the cancer-mimic PS by both peptides with a slightly stronger impact by the D-peptide. Accordingly, membrane effects studied by DSC, leakage and quenching experiments were solely induced by the peptides when the cancer mimic PS was present. Circular dichroism revealed a sole increase in β-sheet conformation in the presence of the cancer mimic for both peptides, only 9D-RDP215 showed increased structure already in the buffer. Ex vitro stability studies by SDS-PAGE as well as in vitro with melanoma A375 revealed a stabilizing effect of D-amino acids in the presence of serum, which was also confirmed in 2D and 3D in vitro experiments on glioblastoma LN-229. 9D-RDP215 was additionally able to pass a BBB model, whereupon it induced significant levels of cell death in LN-229 spheroids. Summarized, the study encourages the introduction of D-amino acids in the design of antitumor peptides for the improvement of their stable antitumor activity.

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options—especially during pregnancy.

The aim of this study was to develop effective and specific anti-cancer drugs based on membrane active peptides. In previous studies we showed that human lactoferricin (hLFcin) derived peptides facilitate specific killing of cancer cells. These antitumor peptides were found by conventional melanoma two-dimensional (2D) cell cultures to induce apoptosis of cancer cells and to specifically target lipid phosphatidylserine located on the outside of cancer cell membranes. In order to have a more relevant in vitro model able to mimic the natural microenvironments of tumor tissues we established three-dimensional (3D) multicellular tumor spheroids (MCTS). We used a set of (retro) di-peptides derived from LF11, an 11 amino acid long fragment of hLFcin, which differed in peptide length, positive net charge and hydrophobicity and determined antitumor activity and non-specific toxicity on non-neoplastic cells using 2D and 3D model systems. 2D studies unveiled a correlation between length, positive net charge and hydrophobicity of peptides and their specific antitumor activity. (Retro) di-peptides as R-DIM-P-LF11-215 and DIM-LF11-322 with a net charge of +9 and moderate hydrophobicity exhibited the highest specific antitumor activity. Further evaluation of the peptides anticancer activity by 3D in vitro studies confirmed their higher activity and cancer specificity compared to their parent R-DIM-P-LF11, with the exception of DIM-LF11-339. This highly hydrophobic peptide caused cell death mainly at the border of tumor spheroids indicating that too high hydrophobicity may prevent peptides from reaching the center of the spheroids.

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

Biological membranes can be targets for compounds that either disrupt their barrier function or affect protein function via membrane-mediated processes. Biophysical studies on membrane-mimetic systems composed of membrane lipids have contributed substantially to our knowledge on the pertaining membrane physics and aid the development of membrane-specific drugs. Moreover, lipid membranes and, in particular, liposomes are convenient systems for drug delivery. We review some of our recent work that demonstrates the applicability of X-ray scattering to understanding the molecular mechanisms of drug-membrane interactions. In particular we focus on effects of anesthetics, sphingomyelinase, and antimicrobial peptides. We further discuss X-ray scattering as a quality-control tool for liposomal drug-delivery systems.

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., Highlights ► Deletion of HEM1 causes squalene accumulation in yeast. ► Squalene accumulated in lipid droplets has a disordering effect. ► In biological membranes squalene modulates fluidity/rigidity.

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., Highlights ► Novel marker of glioblastoma, renal cancer, rhabdomyosarcoma ► Novel marker of metastasis ► Marker also present in primary cultures ► Increase of exposure of marker with malignancy ► Demonstration of universality of a novel target for antitumor therapy

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.

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.

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.

In the yeast Saccharomyces cerevisiae, three enzymes of the sterol biosynthetic pathway, namely Erg1p, Erg6p and Erg7p, are located in lipid particles. Whereas Erg1p (squalene epoxidase) is also present in the endoplasmic reticulum (ER) to a significant amount, only traces of Erg6p (sterol C-24 methyltransferase) and Erg7p (lanosterol synthase) are found in the ER. We have chosen these three Erg-proteins as typical representatives of lipid particle proteins to study targeting to their destination. Lipid particle proteins do not contain obvious targeting motifs, but the only common structural feature is the presence of one or two hydrophobic domains near the C-termini. We constructed truncated versions of Erg1p, Erg6p and Erg7p to test the role of these hydrophobic domains in subcellular distribution. Our results demonstrate that lack of the hydrophobic domains prevents at least in part the association of the proteins with lipid particles and causes their retention to the ER. This result strongly supports the view that ER and lipid particles are related organelles.

Cancer with poor prognosis and treatability, like glioblastoma or malignant melanoma demand for new effective treatment and is therefore a focus in our studies. Recently we were able to demonstrate that the negatively charged lipid phosphatidylserine (PS) is specifically exposed by cancer cells and metastases and thus represents a uniform and novel target for cancer therapy (Riedl et al., BBA 2011). This yielded the important basis for the design of cationic antitumor peptides derived from human Lactoferricin (hLFcin).Some of these hLFcin derivatives show highly increased activity towards human malignant melanoma and glioblastoma cells compared to its parent peptide hLFcin. They can discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as PS, specifically exposed on cancer cells. We have improved peptide specificity towards cancer cells by variations of peptide length, net positive charge and hydrophobicity, consequently influencing the secondary structure and the mechanism by which peptides kill the target cell.In our studies we found characteristic features of hLFcin derivatives which facilitate specific killing of cancer cells. Time dependent studies clearly revealed that a highly active but nonselective peptide acts through a direct membranolytic mechanism whereas the selective peptides presumably trigger apoptosis, which is also membrane-mediated by primary interactions with the negatively charged PS exposed by cancer cells and further intracellular targets. The role of cholesterol in triggering a specific peptide-cancer membrane interaction will also be discussed. Studies in mouse melanoma and glioblastoma xenografts confirm successful activity of the derived peptides also in vivo.Acknowledgment: FWF (P24608).

Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH2. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions.

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.

In the yeast Saccharomyces cerevisiae, two acyl-CoA:sterol acyltransferases (ASATs) that catalyze the synthesis of steryl esters have been identified, namely Are2p (Sat1p) and Are1p (Sat2p). Deletion of either ARE1 or ARE2 has no effect on cell viability, and are1are2 double mutants grow in a similar manner to wild-type despite the complete lack of cellular ASAT activity and steryl ester formation [Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T. M., Rothstein, R. & Sturley, S. L. (1996) Science 272, 1353-1356; Yu, C., Kennedy, J., Chang, C. C. Y. & Rothblatt, J. A. (1996) J. Biol. Chem. 271, 24157-24163]. Here we show that both Are2p and Are1p reside in the endoplasmic reticulum as demonstrated by measuring ASAT activity in subcellular fractions of are1 and are2 deletion strains. This localization was confirmed by fluorescence microscopy using hybrid proteins of Are2p and Are1p fused to green fluorescent protein (GFP). Lipid analysis of are1 and are2 deletion strains revealed that Are2p and Are1p utilize sterol substrates in vivo with different efficiency; Are2p has a significant preference for ergosterol as a substrate, whereas Are1p esterifies sterol precursors, mainly lanosterol, as well as ergosterol. The specificity towards fatty acids is similar for both isoenzymes. The lack of steryl esters in are1are2 mutant cells is largely compensated by an increased level of free sterols. Nevertheless, terbinafine, an inhibitor of ergosterol biosynthesis, inhibits growth of are1are2 cells more efficiently than growth of wild-type. In a growth competition experiment are1are2 cells grow more slowly than wild-type after several rounds of cultivation, suggesting that Are1p and Are2p or steryl esters, the product formed by these two enzymes, are more important in the natural environment than under laboratory conditions.

R-DIM-P-LF11-322, derived from the cationic human host defense peptide lactoferricin, exhibits selective antitumor activity against several human cancer cell types. We have shown that cancer cells expose the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane (1), which in healthy cells only comprises neutral lipids as phosphatidylcholine (PC) or cholesterol. Therefore the membrane interaction of R-DIM-P-LF11-322 with membrane mimics composed of mixtures of PS, PC and cholesterol was studied. Lipid analysis of normal melanocytes and melanoma revealed a slight increase of PC and cholesterol in the cancer lipid extracts.Tryptophan fluorescence showed that the peptide penetrated only in the cancer mimics containing ratios of PS. Addition of cholesterol slightly reduced the affinity of the peptide.Calorimetric studies with peptide and mixed liposomes of PS and PC indicated complete perturbation of the PS fraction leaving PC unaffected. Interestingly upon addition of cholesterol the peptide induced separation into PC and PS enriched domains, but seemed to be slightly reduced in its effect.Studies using giant unilamellar vesicles showed that the peptide interacts on cancer model membranes slightly stronger in the absence of cholesterol by formation of extremely deformed and destroyed vesicles. In the presence of cholesterol the peptide however still induced shrinking of the vesicles and formation of many small PS domains.In summary the antitumor peptide strongly interacts only with its target PS on cancer cells. However, cholesterol seems to play a role in the modulation of membrane domain formation and reducing the membrane affinity, which may represent a mechanism for cancer cells to protect themselves against antitumor peptides.Acknowledgment: Austrian Science Foundation FWF (grant no: P24608)(1) Riedl et al. BBA 1808: 2638-2645, 2011.

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.

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Lipopeptides such as polymyxins, octapeptins or daptomycin often show an increased activity against bacteria as compared to their non-acylated analogue. Thus, we have studied N-acylated synthetic peptides derived from a fragment of human lactoferrin (LF-11) to elucidate the interaction of these peptides with Gram-negative and Gram-positive bacteria and membrane mimetic systems using various biophysical and biological methods.Calorimetric studies on liposomes composed of phosphatidylglycerol revealed that the parent peptide induced a phase separation into peptide-enriched and -poor domains, which however consist of a similar domain size as calculated by the cooperative units. In contrast, at the same lipid-to-peptide molar ratio (25:1) the N-acylated derivatives strongly broadened the phase transition range and lowered markedly the main transition temperature. This is indicative for rather small and inhomogeneous domains, which will result in large line defects increasing membrane permeability as observed in intact bacteria. Membrane destabilization of E. coli and S. aureus induced by the peptides was monitored by using the membrane-potential-sensitive dye DiIC1 and the extent of membrane damage caused by the peptides by the cationic dye SYTOX green, which cannot enter intact cells unless its membrane is disrupted by external compounds. In both assays the N-acylated peptides showed a dramatic increase of fluorescence indicating massive membrane damage. This is supported by electron micrographs, which clearly showed a loss of cytoplasmic content and membrane rupture in the presence of the N-acylated peptide. Nevertheless, the extent of cell membrane rupture does not necessarily strongly correlate with the MIC-value of the peptides emphasizing the different mode of interaction of (non)-acylated peptides, which in part may be related to different degree of interaction with cell membrane/wall components such as lipopolysaccharides and lipoteichoic acid.Acknowledgement to EC-projects “ANEPID” and “BIOCONTROL”

Antimicrobial peptides have gained interest as potential anti-cancer agents, e.g. inhibition of tumour growth in human prostate xenografts was shown by host defense like lytic peptides (1). We showed by Annexin V binding that, in prostate tumour cells, negatively charged phosphatidylserine (PS) accumulates in the outer plasma membrane leaflet, which normally resides in the inner leaflet. Thus, surface exposure of PS may make these cells susceptible to killing by these cationic peptides.The aim of this study is to develop short peptide sequences derived from NK-2, which was shown to have anti-tumour activity (2). NKCS (Cys of NK-2 exchanged by Ser) is composed of two α-helices connected with a hinge region. Initially we studied the interaction of the parent peptide and its N- and C-terminal part with membrane mimetic systems. Vesicle leakage experiments revealed that the N-terminal fragment exhibits similar affinity towards PS as NKCS. Thermodynamic experiments indicate that the N-terminal helix resembles the properties of NKCS. Furthermore, calorimetric studies revealed that NKCS and its fragments have no significant effect on the thermotropic behaviour of PC liposomes mimicking healthy mammalian cell membranes. Both circular dichroism and Monte-Carlo simulation using the bilayer parameters derived from our structural characterization of the lipid model systems showed that the selectivity for PS correlated with the alpha-helical content of the peptides. The C-terminal part was less structured showing lower affinity to PS containing membranes. Thus, the shorter N-terminal peptide can be used as a template for further optimization, as in vitro tests on a human prostate carcinoma cell line showed significant cell damage.(1) Papo N. et al., Cancer Res. 66 (2006) 5371-8.(2) Schroder-Borm H. et al., FEBS Lett. 579 (2005) 6128-61.Acknowledgement: EC - Marie-Curie Action: BIOCONTROL (MCRTN - 33439)

The effect of short peptides, derived from lactoferricin, a human host defense peptide exhibiting antibacterial activity and their N-acylated derivatives was studied with biological and membrane mimetic systems. The work carried out during the European RTD-Project “ANEPID” revealed correlation between biological activity against E.coli and interaction with negatively charged lipid model systems, leading to formation of peptide effected lipid domains. Increase of hydrophobicity by addition of hydrophobic amino acids as well as N-acylation improved activity in model and biological systems, but was limited by loss of selectivity for bacterial systems. Addition of peptides to bacterial mimics caused de-mixing into charged peptide effected domains and neutral mainly unaffected domains. Following induction of membrane curvature stress and leakage of cellular contents at defect lines of these induced domains appear to be the major effects of the studied peptides, which could be proven with E.coli mimetic systems. Major perturbance of cytoplasmic membranes of bacteria was also revealed by electron microscopy indicated by peptide induced formation of large membrane blebs and partially by detection of oversized cells that might reflect peptide induced defects in cell-division.

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.

The thermotropic phase behavior and organization of aqueous dispersions of the quadruple-chained, anionic phospholipid tetramyristoyl diphosphatidylglycerol or tetramyristoyl cardiolipin (TMCL) was studied by differential scanning calorimetry, x-ray diffraction, (31)P NMR, and Fourier-transform infrared (FTIR) spectroscopy. At physiological pH and ionic strength, our calorimetric studies indicate that fully equilibrated aqueous dispersions of TMCL exhibit two thermotropic phase transitions upon heating. The lower temperature transition is much less cooperative but of relatively high enthalpy and exhibits marked cooling hysteresis, whereas the higher temperature transition is much more cooperative and also exhibits a relatively high enthalpy but with no appreciable cooling hysteresis. Also, the properties of these two-phase transitions are sensitive to the ionic strength of the dispersing buffer. Our spectroscopic and x-ray diffraction data indicate that the lower temperature transition corresponds to a lamellar subgel (L(c)') to gel (L(beta)) phase transition and the higher temperature endotherm to a L(beta) to lamellar liquid-crystalline (L(alpha)) phase transition. At the L(c)'/L(beta) phase transition, there is a fivefold increase of the thickness of the interlamellar aqueous space from approximately 11 A to approximately 50 A, and this value decreases slightly at the L(beta)/L(alpha) phase transition. The bilayer thickness (i.e., the mean phosphate-phosphate distance across the bilayer) increases from 42.8 A to 43.5 A at the L(c)'/L(beta) phase transition, consistent with the loss of the hydrocarbon chain tilt of approximately 12 degrees , and decreases to 37.8 A at the L(beta)/L(alpha) phase transition. The calculated cross-sectional areas of the TMCL molecules are approximately 79 A(2) and approximately 83 A(2) in the L(c)' and L(beta) phases, respectively, and we estimate a value of approximately 100 A(2) in the L(alpha) phase. The combination of x-ray and FTIR spectroscopic data indicate that in the L(c)' phase, TMCL molecules possess tilted all-trans hydrocarbon chains packed into an orthorhombic subcell in which the zig-zag planes of the chains are parallel, while in the L(beta) phase the untilted, all-trans hydrocarbon chains possess rotational mobility and are packed into a hexagonal subcell, as are the conformationally disordered hydrocarbon chains in the L(alpha) phase. Our FTIR spectroscopic results demonstrate that the four carbonyl groups of the TMCL molecule become progressively more hydrated as one proceeds from the L(c)' to the L(beta) and then to the L(alpha) phase, while the two phosphate moieties of the polar headgroup are comparably well hydrated in all three phases. Our (31)P-NMR results indicate that although the polar headgroup retains some mobility in the L(c)' phase, its motion is much more restricted in the L(beta) and especially in the L(alpha) phase than that of other phospholipids. We can explain most of our experimental results on the basis of the relatively small size of the polar headgroup of TMCL relative to other phospholipids and the covalent attachment of the two phosphate moieties to a single glycerol moiety, which results in a partially immobilized polar headgroup that is more exposed to the solvent than in other glycerophospholipids. Finally, we discuss the biological relevance of the unique properties of TMCL to the structure and function of cardiolipin-containing biological membranes.

Introduction The increasing occurrence of pathogenic bacteria that are resistant to commercially available antibiotics has led to a growing interest in the development of peptides as a novel class of antibacterial drugs. Since bacteria have evolved to present multiple resistances to a large number of existing antibiotics, a new class of compounds is more likely to minimize the rapid emergence of bacterial resistance. Nature has taught us that effector molecules of mammalian innate immunity can provide a first line of defense against a substantial array of pathogenic microorganisms. In particular, host-defense peptides are considered to be multifunctional effector molecules and represent novel sources for the development of therapeutic agents with which to overcome antimicrobial resistance. Today, several hundred host defense peptides have been isolated from natural sources and their functions characterized [reviewed in 1-3]. These peptides are important components of innate defenses, since they not only have the ability to kill bacteria, but are also able to modulate inflammatory responses. While many conventional antibiotics damage or kill bacteria over a period of days, most antimicrobial peptides affect the bacterial membrane and kill almost instantaneously, i.e., within minutes. Therefore, a permeabilizing peptide may be able to counteract mechanisms of antibiotic resistance relying on an efficient membrane permeability barrier and be of great value in a combination therapy against drug-resistant bacterial strains. In the aim to test whether antimicrobial peptides may be used as permeabilizing agents that render bacteria more vulnerable to conventional antibiotics, we compared the antimicrobial and permeabilizing activities of a panel of 70 peptides (7-11 amino acid) rich in positively charged residues derived from a human lactoferrin fragment (LF11, FQWQRNIRKVR-NH2 [4]).

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.

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidyl-serine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.

Antimicrobial peptides have gained interest as potential anti-cancer agents. Phosphatidylserine (PS), which normally resides in the inner leaflet of the plasma membrane, can move to the outer leaflet and act as a surface marker of cancer cells. The surface exposure of negatively charged PS on prostate tumour cells makes these cells susceptible to killing by cationic membranolytic peptides such as NK-2 [1]. Shai et al. [2] have also shown the inhibition of tumor growth in human prostate xenografts by host defense like lytic peptides.The aim of this study is to develop short peptide sequences acting selectively towards PS exposed on cancer cells. As a prerequisite it is necessary to analyze the lipid composition of prostate cancer cell - and non cancer cell plasma membranes. Further as a basis for peptide activity studies the biophysical characteristics of cancer cell membranes and healthy counterparts with respect to lipid composition were determined by DSC and X-ray studies with liposomal mimics. Fluorescence spectroscopy was applied to test the release of marker molecules from liposomes, which revealed that some NK-2 derived peptides have a high affinity towards PS.Results of the in-vitro testing of optimized peptides on prostate cancer cell lines together with biophysical data help us to shed light on the future evaluation of their usage as anticancer therapeutics.[1] Schroder-Borm H. et al., FEBS Lett. 579 (2005) 6128-61.[2] Shai Y. et al., Cancer Research 2006; 66 (10) May 15.Acknowledgement: Marie Curie Actions “Biocontrol”.

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.

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., Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means. Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.

Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.