Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids.

Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP).

Lipid Conversion by Cell-Free Synthesized Phospholipid Methyltransferase Opi3 in Defined Nanodisc Membranes Supports an in Trans Mechanism.

Biomembranes composed of lipids and proteins play central roles in physiological processes, and the precise balance between different lipid species is crucial for maintaining membrane function. One pathway for the biosynthesis of the abundant lipid phosphatidylcholine in eukaryotes involves a membrane-integrated phospholipid methyltransferase named Opi3 in yeast. A still unanswered question is whether Opi3 can catalyze phosphatidylcholine synthesis in trans, at membrane contact sites.

Tranexamic Acid-Encapsulating Thermosensitive Liposomes for Site-Specific Pharmaco-Laser Therapy of Port Wine Stains.

Site-specific pharmaco-laser therapy (SSPLT) is a developmental stage treatment modality designed to non-invasively remove superficial vascular pathologies such as port wine stains (PWS) by combining conventional laser therapy with the prior administration of a prothrombotic and/or antifibrinolytic pharmaceutical-containing drug delivery system. For the antifibrinolytic SSPLT component, six different PEGylated thermosensitive liposomal formulations encapsulating tranexamic acid (TA), a potent antifibrinolytic lysine analogue, were characterized for drug:lipid ratio, encapsulation efficiency, size, endovesicular TA concentration (C TA), phase transition temperature (T m), and assayed for heat-induced TA release. Assays were developed for the quantification of liposomal TA and heat-induced TA release from two candidate formulations.

Lipid Acyl Chain Remodeling in Yeast.

Membrane lipid homeostasis is maintained by de novo synthesis, intracellular transport, remodeling, and degradation of lipid molecules. Glycerophospholipids, the most abundant structural component of eukaryotic membranes, are subject to acyl chain remodeling, which is defined as the post-synthetic process in which one or both acyl chains are exchanged. Here, we review studies addressing acyl chain remodeling of membrane glycerophospholipids in Saccharomyces cerevisiae, a model organism that has been successfully used to investigate lipid synthesis and its regulation.

Phosphatidylcholine's functions beyond that of a membrane brick.

Since its discovery in the 19th century, phosphatidylcholine (PC) has been regarded primarily as a structural lipid. However, more recent evidence, much of it in the last five years, strongly suggests that PC has other roles. Here, we explore some of that new evidence and consider the possibility that the ultimate role of phosphatidylcholine may not be predictable.

E. coli MG1655 modulates its phospholipid composition through the cell cycle.

This paper describes a study of the phospholipid profile of Escherichia coli MG1655 cultures at the B and D periods of the cell cycle. The results indicate that the phosphatidyl glycerol fraction grows relatively rapidly and that the size of the cardiolipin (CL) fraction does not grow at all during cell elongation. This is consistent with observations that CL is located preferentially at the poles of E.

Ligation strategies for targeting liposomal nanocarriers.

Liposomes have been exploited for pharmaceutical purposes, including diagnostic imaging and drug and gene delivery. The versatility of liposomes as drug carriers has been demonstrated by a variety of clinically approved formulations. Since liposomes were first reported, research of liposomal formulations has progressed to produce improved delivery systems.

Checks and balances in membrane phospholipid class and acyl chain homeostasis, the yeast perspective.

Glycerophospholipids are the most abundant membrane lipid constituents in most eukaryotic cells. As a consequence, phospholipid class and acyl chain homeostasis are crucial for maintaining optimal physical properties of membranes that in turn are crucial for membrane function. The topic of this review is our current understanding of membrane phospholipid homeostasis in the reference eukaryote Saccharomyces cerevisiae.

Plasma membrane--endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis.

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)--ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N-methyltransferase enzyme, Opi3.

Yeast cells accumulate excess endogenous palmitate in phosphatidylcholine by acyl chain remodeling involving the phospholipase B Plb1p.

In the yeast Saccharomyces cerevisiae, the molecular species profile of the major membrane glycerophospholipid phosphatidylcholine (PC) is determined by the molecular species-selectivity of the biosynthesis routes and by acyl chain remodeling. Overexpression of the glycerol-3-phosphate acyltransferase Sct1p was recently shown to induce a strong increase in the cellular content of palmitate (C16:0). Using stable isotope labeling and mass spectrometry, the present study shows that wild type yeast overexpressing Sct1p incorporates excess C16:0 into PC via the methylation of PE, the CDP-choline route, and post-synthetic acyl chain remodeling.

The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p.

The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA.

Quantitative profiling of PE, MMPE, DMPE, and PC lipid species by multiple precursor ion scanning: a tool for monitoring PE metabolism.

We report a method for the simultaneous identification and quantification of phosphatidylethanolamine (PE), monomethyl-phosphatidylethanolamine (MMPE), dimethyl-phosphatidylethanolamine (DMPE), and phosphatidylcholine (PC) species in lipid extracts. The method employs a specific "mass-tag" strategy where DMPE, MMPE, and PE species are chemically methylated with deuterated methyliodide (CD(3)I) to produce PC molecules having class-specific mass offsets of 3, 6 and 9Da, respectively. The derivatized aminoglycerophospholipids release characteristic phosphorylcholine-like fragment ions having specific mass offsets that powers sensitive and quantitative analysis by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer.

Proteome-wide detection of phospholipid-protein interactions in mitochondria by photocrosslinking and click chemistry.

Photoactivatable lipid analogues are uniquely suited for the detection of lipid-protein interactions in biological membranes. Based on photocrosslinking, new methodology has been developed for the proteome-wide detection of lipid-protein interactions. Bifunctional lipid analogues containing a tag for click chemistry in addition to the photoactivatable moiety enable the enrichment of the crosslinked proteins that is required for subsequent identification by mass spectrometry.

The influence of the acyl chain composition of cardiolipin on the stability of mitochondrial complexes; an unexpected effect of cardiolipin in alpha-ketoglutarate dehydrogenase and prohibitin complexes.

The role of cardiolipin acyl chain composition in assembly/stabilization of mitochondrial complexes was investigated using three yeast deletion mutants (acb1Delta strain; taz1Delta strain; and acb1Deltataz1Delta strain). Deletion of the TAZ1 gene, involved in cardiolipin acyl chain remodeling, is known to increase the content of monolyso-cardiolipin (MLCL) at the expense of CL, and to decrease the unsaturation of the remaining CL. Deletion of the ACB1 gene encoding the acyl-CoA-binding protein, involved in fatty acid elongation, decreases the average length of the CL acyl chains.

Nanocapsules of platinum anticancer drugs: development towards therapeutic use.

Platinum-based anticancer agents have been widely used in the clinic to successfully treat many different types of cancer. However, the therapeutic efficacy of platinum drugs is limited by serious side effects and the occurrence of inherent or acquired resistance of tumor cells. Nanoparticulate drug-delivery systems have the potential to reduce side effects and circumvent drug resistance.

Cardiolipin molecular species with shorter acyl chains accumulate in Saccharomyces cerevisiae mutants lacking the acyl coenzyme A-binding protein Acb1p: new insights into acyl chain remodeling of cardiolipin.

The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p.

On the interaction of fluorophore-encapsulating PEGylated lecithin liposomes with hamster and human platelets.

Polyethylene glycol (PEG)-grafted phosphatidylcholine liposomes are used as drug carriers due to their low immunogenicity and prolonged circulation time. The interaction between sterically stabilized lecithin liposomes and platelets has not been investigated before, and deserves to be subjected to scrutiny inasmuch as the uptake of liposomes by platelets could be detrimental for drug delivery and primary hemostasis. Consequently, the interaction between resting and convulxin-activated hamster and human platelets and calcein- or 5,6-carboxyfluorescein-encapsulating PEGylated liposomes composed of distearoyl- and dipalmitoyl phosphatidylcholine and PEG-derivatized distearoyl phosphatidylethanolamine was investigated by flow cytometry, confocal microscopy, and a glass capillary thrombosis model.

High cytotoxicity of cisplatin nanocapsules in ovarian carcinoma cells depends on uptake by caveolae-mediated endocytosis.

Purpose: Cisplatin nanocapsules, nanoprecipitates of cisplatin encapsulated in phospholipid bilayers, exhibit increased in vitro toxicity compared with the free drug toward a panel of human ovarian carcinoma cell lines. To elucidate the mechanism of cell killing by nanocapsules and to understand the cell line dependence of nanocapsule efficacy, the route of uptake and the intracellular fate of the nanocapsules were investigated.

Experimental Design: Intracellular platinum accumulation and cisplatin-DNA-adduct formation were measured in cell lines that differ in sensitivity to cisplatin nanocapsules.

Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.

New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively.

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al.

Antitumor activity and biodistribution of cisplatin nanocapsules in nude mice bearing human ovarian carcinoma xenografts.

Cisplatin nanocapsules represent a novel lipid formulation of the anticancer drug cis-diamminedichloridoplatinum(II) (cisplatin), characterized by an unprecedented cisplatin-to-lipid molar ratio, and exhibiting strongly increased in-vitro cytotoxicity compared with the free drug. In this study, antitumor efficacy and biodistribution of PEGylated cisplatin nanocapsules were compared with those of the free drug in a mouse tumor model. Nude mice bearing human ovarian carcinoma OVCAR-3 xenografts were treated twice with a 1-week interval by intravenous administration of cisplatin nanocapsules or cisplatin in solution, and the growth inhibitory effects were determined by measurement of tumor volumes.

Nanocapsules: a novel lipid formulation platform for platinum-based anti-cancer drugs.

Platinum-based anti-cancer agents have been used for many years to treat many different types of cancer. However, the efficacy of these drugs is limited by serious side effects. One of the strategies to reduce the side effects is encapsulation of the drug in a lipid formulation.

Phosphatidylcholine is essential for efficient functioning of the mitochondrial glycerol-3-phosphate dehydrogenase Gut2 in Saccharomyces cerevisiae.

Gut2, the mitochondrial glycerol-3-phosphate dehydrogenase, was previously shown to become preferentially labelled with photoactivatable phosphatidylcholine (PC), pointing to a functional relation between these molecules. In the present study we analyzed whether Gut2 functioning depends on the PC content of yeast cells, using PC biosynthetic mutants in which the PC content was lowered. PC depletion was found to reduce growth on glycerol and to increase glycerol excretion, both indicating that PC is needed for optimal Gut2 functioning in vivo.