Carbamyl Analogs of Phosphatidylcholines Synthesis, Interaction With Phospholipases and Permeability Behavior of their Liposomes

A novel class of phospholipase-resisting phosphatidylcholine analogs, in which the C-2 ester group or both C-1 and C-2 ester groups have been replaced by carbamyloxy functions (-NH-C-O-), have been synthesized. These lipids were not degraded by phospholipase A2 while complete hydrolysis occurred with phospholipase C. Ultrasonic irradiation of the aqueous dispersions of the phospholipids in the presence as well as in the absence of cholesterol resulted in the formation of closed bilayer structures as evidenced by negative staining electron microscopy and also by their ability to entrap [14C]glucose. The leakage rates of glucose at 37°C from liposomes of these compounds have also been measured. Liposomes consisting of 1,2-dipentadecanylcarbamyloxy-sn-glycero- 3-phosphorylcholine were found to be more leaky (2.1 %/h) as compared to the liposomes of 1-palmitoyl-2-pentadecanylcarbamyloxy-sn -glycero-3-phosphoryl- choline (O.5%/h). Moreover, inclusion of cholesterol (33 mol%) into the bilayers of the former phospholipid had no effect on the leakage rate (2.4%/h) while it effectively reduced permeability of the latter (O.22%/h). These phosphatidylcholines are useful for studying the possible role of phospholipases in the capture and lysis of liposomes in vivo.

Effect of Altered Polar Headgroup of Phosphatidylethanolamines on Transbilayer Aminophospholipid Distribution in Sonicated Vesicles

The transbilayer aminophospholipid distributions in small unilamellar vesicles comprising of phosphatidylethanolamine or its analogs (bearing modifications in the polar headgroup) and egg hosphatidylcholine were ascertained using trinitrobenzenesulfonic acid as external membrane probe. These vesicles, containing 10-30 mol% phosphatidylethanolamine or its analogs, were formed by sonication and fractionated by centrifugation. Phosphatidylethanolamine at low concentrations (10 mol%) preferentially localized in the outer monolayer. This preference appeared to be reversed at higher phosphatidylethanolamine concentrations (30 mol%). Unlike this finding, phosphatidylethanolamine bearing ethyl, phenyl and benzyl substituents at the carbon atom adjacent to the amino group distributed mainly in the outer surface irrespective of their concentrations. Similar results were obtained when the phosphate and amino groups were separated by three methylene residues. These observations suggest that the effective polar headgroup volume and/or hydrogen-bonding capacity of phospholipids are the important factors that determine their distribution in small unilamellar vesicles.

Characterization of choline uptake in Trypanosoma brucei procyclic and bloodstream forms

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-containing phospholipids, such as phosphatidylcholine (PC). According to published data, Trypanosoma brucei parasites are unable to take up choline from the environment but instead use lyso-phosphatidylcholine as precursor for choline lipid synthesis. We now show that T. brucei procyclic forms in culture readily incorporate [3H]-labeled choline into PC, indicating that trypanosomes express a transporter for choline at the plasma membrane. Characterization of the transport system in T. brucei procyclic and bloodstream forms shows that uptake of choline is independent of sodium and potassium ions and occurs with a Km in the low micromolar range. In addition, we demonstrate that choline uptake can be blocked by the known choline transport inhibitor, hemicholinium-3, and by synthetic choline analogs that have been established as anti-malarials. Together, our results show that T. brucei parasites express an uptake system for choline and that exogenous choline is used for PC synthesis.

Lipid synthesis in protozoan parasites: a comparison between kinetoplastids and apicomplexans.

Lipid metabolism is of crucial importance for pathogens. Lipids serve as cellular building blocks, signalling molecules, energy stores, posttranslational modifiers, and pathogenesis factors. Parasites rely on a complex system of uptake and synthesis mechanisms to satisfy their lipid needs. The parameters of this system change dramatically as the parasite transits through the various stages of its life cycle. Here we discuss the tremendous recent advances that have been made in the understanding of the synthesis and uptake pathways for fatty acids and phospholipids in apicomplexan and kinetoplastid parasites, including Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania. Lipid synthesis differs in significant ways between parasites from both phyla and the human host. Parasites have acquired novel pathways through endosymbiosis, as in the case of the apicoplast, have dramatically reshaped substrate and product profiles, and have evolved specialized lipids to interact with or manipulate the host. These differences potentially provide opportunities for drug development. We outline the lipid pathways for key species in detail as they progress through the developmental cycle and highlight those that are of particular importance to the biology of the pathogens and/or are the most promising targets for parasite-specific treatment.

The ins and outs of phosphatidylethanolamine synthesis in Trypanosoma brucei.

Phospholipids are not only major building blocks of biological membranes but fulfill a wide range of critical functions that are often widely unrecognized. In this review, we focus on phosphatidylethanolamine, a major glycerophospholipid class in eukaryotes and bacteria, which is involved in many unexpected biological processes. We describe (i) the ins, i.e. the substrate sources and biochemical reactions involved in phosphatidylethanolamine synthesis, and (ii) the outs, i.e. the different roles of phosphatidylethanolamine and its involvement in various cellular events. We discuss how the protozoan parasite, Trypanosoma brucei, has contributed and may contribute in the future as eukaryotic model organism to our understanding of phosphatidylethanolamine homeostasis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Placental ABCA1 and ABCG1 expression in gestational disease: pre-eclampsia affects ABCA1 levels in syncytiotrophoblasts

INTRODUCTION Transplacental feto-maternal lipid exchange through the ATP-binding cassette transporters ABCA1 and ABCG1 is important for normal fetal development. However, only scarce and conflicting data exist on the involvement of these transporters in gestational disease. METHODS Placenta samples (n = 72) derived from common gestational diseases, including pre-eclampsia (PE), HELLP, intrauterine growth restriction (IUGR), intrahepatic cholestasis of pregnancy and gestational diabetes, were assessed for their ABCA1 and ABCG1 expression levels and compared to age-matched control placentas with qRT-PCR and immunohistochemistry. ABCA1 expression was additionally investigated with immunoblot in placental membrane vesicles. Furthermore, placental cholesterol and phospholipid contents were assessed. RESULTS ABCA1 mRNA levels differed significantly between preterm and term control placentas (p = 0.0013). They were down-regulated in isolated PE and PE with IUGR (p = 0.0006 and p = 0.0012, respectively), but unchanged in isolated IUGR, isolated HELLP and other gestational diseases compared to gestational age-matched controls. Correspondingly, in PE, ABCA1 protein expression was significantly reduced in the apical membrane of the villous syncytiotrophoblast (p = 0.011) and in villous fetal endothelial cells (p = 0.036). Furthermore, in PE there was a significant increase in the placental content of total and individual classes of phospholipids which were partially correlated with diminished ABCA1 expression. Conversely, ABCG1 mRNA and protein levels were stable in the investigated conditions. CONCLUSIONS In gestational disease, there is a specific down-regulation of placental ABCA1 expression at sites of feto-maternal lipid exchange in PE. At a functional level, the increase in placental lipid concentrations provides indirect evidence of an impaired transport capacity of ABCA1 in this disease.

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Molecular genetic approaches to defining lipid function.

Lipids fulfill multiple and diverse functions in cells. Establishing the molecular basis for these functions has been challenging due to the lack of catalytic activity of lipids and the pleiotropic effects of mutations that affect lipid composition. By combining molecular genetic manipulation of membrane lipid composition with biochemical characterization of the resulting phenotypes, the molecular details of novel lipid functions have been established. This review summarizes the results of such a combined approach to defining lipid function in bacteria.

AUTOIMMUNE RESPONSES TO ATHEROSCLEROTIC

Atherosclerosis is a chronic, complex arterial disease characterized by intimal lipid accumulation and inflammation. A unique lipid-binding molecule, namely cluster of differentiation 1d (CD1d), may impact atherosclerosis. Structurally, CD1d acts as a nonpolymorphic cell-surface receptor, resembling the major histocompatibility complex-I (MHC-I). While MHC-I restricts peptide antigen presentation to T cells, CD1d presents lipid antigens to T cells named CD1d-restrictedd T cells. Although increased expression of CD1d has been found in human plaques, the exact nature of CD1d-recognized lipids in atherosclerosis remains to be determined. Three groups of lipids may undergo oxidation in atherosclerosis producing atherogenic lipids: phospholipids, fatty acids, and cholesterol. The central hypothesis is that CD1d recognizes and present oxidative lipids to activate CD1d-restricted T cells, and trigger proinflammatory signal transduction In the first part of this study, oxidative phospholipids were identified and characterized as potential autoantigen for CD1d-restricted T cells. Derived from phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine by oxidization, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) is commonly found in atherosclerotic plaques. Upon stimulation with PGPC, spleen-derived CD1d-restricted T cells produced higher levels of cytokines and proliferated at higher rates than those without PGPC stimulation. CD1d deficiency compromised the PGPC-triggered T cell activation, suggesting that PGPC may function as a potentially novel autoantigen for T cells in atherosclerosis. In the second part of this study, CD1d-mediated proinflammatory signaling was evaluated in murine models. Enhanced CD1 expression occurred in spleens of db/db mice with hyperlipidemia. Tumor necrosis factor-alpha (TNF-α) was increased in db/db spleen, while TNF-α receptor expression augmented in the db/db murine heart, in comparison with those in normal mice. The nuclear factor-κ B (NF-κB) expression was enhanced in the db/db heart, whereas CD1d-null mice showed lower NF-κB, implying the involvement of CD1d in inflammation of the spleen and heart tissues in the mice with hyperlipidemia. The current study has identified PGPC as a novel lipid antigen recognized by CD1d-restricted T cells in atherosclerosis. The animal study has also provided evidence that CD1d regulates NF-κB-mediated proinflammatory signaling. Hence, CD1d-restricted T cell responses to autolipid antigen and mediated inflammatory signal may represent a new molecular pathway that triggers cardiovascular tissue injury in atherosclerosis and hyperlipidemia.

Calcification of a collagen-liposome complex

The purpose of the study was to evaluate in vitro calcification potential among liposomes composed of phospholipids with variations in fatty acid chains and polar head groups. The liposome was also modified by utilizing mixed phospholipids, incorporation of different types of protein to the liposome, or complexing with various collagen preparations. The samples were then incubated in a metastable calcium phosphate solution for the proposed time period. Calcium and phosphate uptake were measured. Resulting precipitates were processed for x-ray diffraction and electron microscopy. Acidic phospholipid, Dioleoylphosphatidic acid and mixed phospholipids, Dioleoylphosphatidic acid/Dipalmitoylphosphatidylethanolamine liposomes calcified at a faster rate and to a greater degree than other phospholipids tested. The incorporation of polylysine, fibronectin, bone protein, or the complexing with collagen decreased the rate and amount of calcification. Electron microscopy demonstrated the similarity of the calcified collagen-liposome complex to the natural calcification matrix. These preparations may be used as a model to study the role of membrane lipids and collagen-phospholipid during the process of calcification.^ The in vivo study was designed to determine whether the potential existed for the promotion of bone healing by the synthetic liposome-collagen complex. The implant materials were modified to provide decreased antigenicity, biocompatability while maintaining their bone conduction properties. The samples were placed subcutaneously and/or subperiosteally and/or in 8 mm calvarium defects of adult rats. Histological and immunological studies demonstrated that the implant itself retained minimal antigenicity and did not inhibit bone formation. However, modification of the implant may contain the bone induction property and be utilized to stimulate bony healing. ^

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

Characterization of choline uptake in Trypanosoma brucei and involvement of a mitochondrial carrier in resistance to choline analogs

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-­containing phospholipids, such as phosphatidylcholine (PC). Our experiments showed – for the first time – that Trypanosoma brucei, the causative agent of human African sleeping sickness, is able to take up choline from the culture medium to use for PC synthesis, indicating that trypanosomes express a transporter for choline at the plasma membrane. Further characterization in procyclic and bloodstream forms revealed that choline uptake is saturable and can be inhibited by HC-3, a known inhibitor of choline uptake in mammalian cells. To obtain additional insights on choline uptake and metabolism, we investigated the effects of choline-analogs that were previously shown to be toxic for T. brucei parasites in culture. Interestingly, we found that all analogs tested effectively inhibited choline uptake into both bloodstream and procyclic form parasites. Subsequently, selected compounds were used to search for possible candidate genes encoding choline transporters in T. brucei, using an RNAi library in bloodstream forms. We identified a protein belonging to the mitochondrial carrier family, previously annotated as TbMCP14, as prime candidate. Down‐regulation of TbMCP14 by RNAi prevented drug-­induced loss of mitochondrial membrane potential and conferred 8­‐fold resistance of T. brucei bloodstream forms to choline analogs. Conversely, over‐expression of the carrier increased parasite susceptibility more than 13-­fold. However, subsequent experiments demonstrated that TbMCP14 was not involved in metabolism of choline. Instead, growth curves in glucose‐depleted medium using RNAi or knock‐out parasites suggested that TbMCP14 is involved in metabolism of amino acids for energy production. Together, our data demonstrate that the identified member of the mitochondrial carrier family is involved in drug uptake into the mitochondrion and has a vital function in energy production in T. brucei.