Acidic Lipids Research Articles

Anatomical and histochemical aspects of the peach palm (Bactris gasipaes Kunth) seed

Bactris gasipaes Kunth (peach palm) is a palm tree (Arecaceae) widely used by Amazon people, whose seeds have dormancy of unknown cause, which result in difficulties on producing uniform seedlings. This study aimed at identifying anatomical and histochemical aspects of peach palm seeds that may have influence on germination. Histological sections were performed with seed material embedded into hystoresin, which were then stained with toluidine blue and assessed under optical microscope to verify structural characteristics. Histological sections were manually performed to identify primary and secondary metabolites; and histochemical test were performed for fatty acids, alkaloids, starch, phenolic compounds, lignin, neutral and acidic lipids, pectins, polysaccharides, proteins, tannins, and terpenoids. Tegument is thin and contains two layers. The external layer is thicker and has vascular bundles; and the internal layer is thinner containing irregular cells replete of phenolic compounds. Endosperm is whitish in color, with cells with shapes that vary from oblong or ellipsoidal until oval; containing lipids, proteins, pectins, and polysaccharides. Embryo is relatively small as related to seed size, conical and vascularized at distal portion. On longitudinal sections, plumule appears in proximal region as three foliar primordia. At seedling protrusion region the cells contain acidic lipids, proteins, and neutral polysaccharides.

3P069 ヒトカルシトニンの酸性膜存在下でのアミロイド線維形成機構の解明(01C.蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

3P069 ヒトカルシトニンの酸性膜存在下でのアミロイド線維形成機構の解明(01C.蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

1P053 QCMによるグルカゴンと酸性膜との相互作用解析(01C. 蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

1P053 QCMによるグルカゴンと酸性膜との相互作用解析(01C. 蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

МЕХАНОХИМИЧЕСКИЕ ПРЕВРАЩЕНИЯ КИСЛОРОДСОДЕРЖАЩИХ СОЕДИНЕНИЙ ЛИПИДОВ ТОРФА

The effect of mechanical activation of peat on the changes in lipid yield and in composition of resin fraction is shown. Mechanochemical transformations of carboxylic acids in acidic lipid fractions are followed by a slight decrease in their number and increase in the fraction of compounds with lower molecular weight. A significant decrease in saturated monohydric alcohols is observed after the mechanical activation of peat.

Dual Regulation of Diacylglycerol Kinase (DGK)-θ: POLYBASIC PROTEINS PROMOTE ACTIVATION BY PHOSPHOLIPIDS AND INCREASE SUBSTRATE AFFINITY

Diacylglycerol kinases are important mediators of lipid signaling cascades, and insight into their regulation is of increasing interest. Using purified DGK-θ, we show that this isoform is subject to dual regulation and that the previously characterized stimulation by acidic phospholipids is dependent on the presence of a positively charged protein or peptide. Polybasic cofactors lowered the K(m) for diacylglycerol at the membrane surface (K(m)((surf))), and worked synergistically with acidic phospholipids to increase activity 10- to 30-fold, suggesting that the purified enzyme is autoinhibited. Vesicle pulldown studies showed that acidic phospholipids recruit polybasic cofactors to the vesicle surface but have little effect on the membrane association of DGK-θ, suggesting that a triad of enzyme, acidic lipid and basic protein are necessary for interfacial activity. Importantly, these data demonstrate that the interfacial association and catalytic activity of DGK-θ are independently regulated. Finally, we show that DGK-θ directly interacts with, and is activated by, basic proteins such as histone H1 and Tau with nm affinity, consistent with a potential role for a polybasic protein or protein domain in the activation of this enzyme.

Phosphoinositides and cytokinesis: The “PIP” of the iceberg

Phosphoinositides [Phosphatidylinositol (PtdIns), phosphatidylinositol 3-monophosphate (PtdIns3P), phosphatidylinositol 4-monophosphate (PtdIns4P), phosphatidylinositol 5-monophosphate (PtdIns5P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2) ), phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2) ), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2) ), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3) )] are lowly abundant acidic lipids found at the cytosolic leaflet of the plasma membrane and intracellular membranes. Initially discovered as precursors of second messengers in signal transduction, phosphoinositides are now known to directly or indirectly control key cellular functions, such as cell polarity, cell migration, cell survival, cytoskeletal dynamics, and vesicular traffic. Phosphoinositides actually play a central role at the interface between membranes and cytoskeletons and contribute to the identity of the cellular compartments by recruiting specific proteins. Increasing evidence indicates that several phosphoinositides, particularly PtdIns(4,5)P(2) , are essential for cytokinesis, notably after furrow ingression. The present knowledge about the specific phosphoinositides and phosphoinositide modifying-enzymes involved in cytokinesis will be first presented. The review of the current data will then show that furrow stability and cytokinesis abscission require that both phosphoinositide production and hydrolysis are regulated in space and time. Finally, I will further discuss recent mechanistic insights on how phosphoinositides regulate membrane trafficking and cytoskeletal remodeling for successful furrow ingression and intercellular bridge abscission. This will highlight unanticipated connections between cytokinesis and enzymes implicated in human diseases, such as the Lowe syndrome.

Roles of Acidic Phospholipids and Nucleotides in Regulating Membrane Binding and Activity of a Calcium-independent Phospholipase A2 Isoform

Phospholipase A(2) activity plays key roles in generating lipid second messengers and regulates membrane topology through the generation of asymmetric lysophospholipids. In particular, the Group VIA phospholipase A(2) (GVIA-iPLA(2)) subfamily of enzymes functions independently of calcium within the cytoplasm of cells and has been implicated in numerous cellular processes, including proliferation, apoptosis, and membrane transport steps. However, mechanisms underlying the spatial and temporal regulation of these enzymes have remained mostly unexplored. Here, we examine the subset of Caenorhabditis elegans lipases that harbor a consensus motif common to members of the GVIA-iPLA(2) subfamily. Based on sequence homology, we identify IPLA-1 as the closest C. elegans homolog of human GVIA-iPLA(2) enzymes and use a combination of liposome interaction studies to demonstrate a role for acidic phospholipids in regulating GVIA-iPLA(2) function. Our studies indicate that IPLA-1 binds directly to multiple acidic phospholipids, including phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, and phosphorylated derivatives of phosphatidylinositol. Moreover, the presence of these acidic lipids dramatically elevates the specific activity of IPLA-1 in vitro. We also found that the addition of ATP and ADP promote oligomerization of IPLA-1, which probably underlies the stimulatory effect of nucleotides on its activity. We propose that membrane composition and the presence of nucleotides play key roles in recruiting and modulating GVIA-iPLA(2) activity in cells.

Virus assembly and plasma membrane domains: Which came first?

Viral assembly is a key step in the virus life cycle. In this review, we focus mainly on the ability of retroviruses, especially HIV-1, to assemble at the plasma membrane of their host cells. The assembly process of RNA enveloped viruses necessitates a fine orchestration between the different viral components and specific interactions between viral proteins and lipids of the host cell membrane. Searching for a comparison with another RNA enveloped virus, we refer to influenza virus to show how it could share (or not) some common features with HIV-1 assembly since both viruses are believed to assemble mainly in raft microdomains. We also discuss the role of RNA and the cellular actin cytoskeleton in enhancing these viral assembly processes. Finally, based on the literature and on new results we have obtained by molecular docking, we propose another mechanism for HIV-1 assembly in membrane domains. This mechanism involves the trapping of acidic lipids by the viral Gag protein by means of ionic protein–lipid interactions, inducing thereby formation of acidic lipid-enriched microdomains (ALEM).

Trichomes micromorphology and essential oil variation at different developmental stages of cultivated and wild growing Mentha pulegium L. populations from Portugal

The indumentum of Mentha pulegium L., studied by light and scanning electron microscopy, was characterized by non-glandular and glandular trichomes, which corresponded to the common arrangement described for the Lamiaceae family. Histochemistry revealed the presence of pectins, total lipids, acidic lipids and essential oils in the glandular trichomes secretions. The essential oil yield ranged from 0.3% (w/d.w.) in the vegetative phase to 1.6% at full flowering. Gas chromatography and gas chromatography–mass spectrometry essential oils composition analysis at full flowering revealed mostly quantitative rather than qualitative variations, with pulegone as the major compound (52–82%), followed by isomenthone (2–36%), menthone (0.1–17%), and piperitenone (1–15%). Comparative evaluation of cultivated and wild growing populations showed differences in the relative amounts of the main components of the essential oils isolated from plants harvested at different developmental stages.

Cis - and trans -membrane interactions of synaptotagmin-1

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca(2+) triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca(2+)-dependent and -independent cis-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca(2+)-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca(2+)-dependent cis-binding prevents trans-interactions if the cis-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis- and trans-binding, which may play an important modulatory role in synaptic transmission.

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.

The importance of an asymmetric distribution of acidic lipids for synaptotagmin 1 function as a Ca2+ sensor

Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3-10-fold without Ca2+. However, with Ca2+ there was an additional 2-5-fold enhancement. This Ca2+-dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+-dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+-dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane.

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase

The SecA ATPase associates with the SecY complex to push preproteins across the bacterial membrane. Because a single SecY is sufficient to create the conducting channel, the function of SecY oligomerization remains unclear. Here, we have analyzed the translocation reaction using nanodiscs. We show that one SecY copy is sufficient to bind SecA and the preprotein, but only the SecY dimer together with acidic lipids supports the activation of the SecA translocation ATPase. In discs, the dimer is predominantly arranged in a back-to-back manner and remains active even if a constituent SecY copy is defective for SecA binding. In membrane vesicles and in intact cells, the coproduction of two inactive SecYs, one for channel gating and the other for SecA binding, recreates a functional translocation unit. These results indisputably argue that the SecY dimer is crucial for the activation of SecA, which is necessary for preprotein transport.

Fusion Activity of HIV gp41 Fusion Domain Is Related to Its Secondary Structure and Depth of Membrane Insertion in a Cholesterol-Dependent Fashion

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30 mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30 mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.

Structural basis for membrane targeting by the MVB12-associated β-prism domain of the human ESCRT-I MVB12 subunit

MVB12-associated β-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-Å atomic resolution crystal structure of the MVB12B MABP domain reveals a β-prism fold, a hydrophobic membrane-anchoring loop, and an electropositive phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.

Acidic Lipid, NBD-PG has Crucial Effect on the Voltage-Dependence of Virus Fusion

Previously we have shown that cell-cell fusion mediated by Class II and Class III proteins is voltage-dependent, whereas fusion of cells expressing Class I fusion protein is not affected by voltage. Moreover, by testing chimeras consisting of the ectodomain of a Class I protein and the transmembrane domain (TMD) of a Class III protein, we found that TMD was the candidate for the voltage sensor in fusion mediated by Class II and Class III viral proteins.We hypothesized that, since flip-flop of acidic lipids inherently potential-dependent, incorporation of these lipids into a target membrane should facilitate the fusion at trans-positive membrane potential through affecting the TMD.To test this hypothesis we incorporated a fluorescent acidic lipid (NBD-PG) into target cells membrane and allowed fusion with VSV-G effector cells maintained at negative (−40mV) or positive potential (+40mV) created by the ionophore, SQI-Pr.We determined that when NPD-PG was present in the target (or effector) cell membranes, the extent of fusion was the same at negative and positive potentials, whereas fusion with untreated cells was inhibited in the presence of ionophore.Our result show that accumulation of acidic lipids in membranes during fusion directly affects to potential-dependence of fusion.

Regulation of plasma membrane Ca2 +-ATPase activity by acetylated tubulin: Influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca2+-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca2+ transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.

Influence of Culture Medium on Fungal Biomass Composition and Biosorption Effectiveness

Zygomycetes such as Cunninghamella elegans seem to be promising biosorbents for pollutants removal from wastewaters because of their particular cell wall characteristics. In this article the effect of ten culture media on C. elegans biomass composition was investigated by means of Fourier transform infra red spectroscopy (FTIR). Biomasses grown on starches from potatoes and cereals were characterised by high amount of chitin and polysaccharides, the glucose gave rise to a biomass rich in acidic polysaccharides and lipids. By contrast, biomasses grown on corn steep liquor were poor in acidic polysaccharides and, when N sources and micronutrients were added, rich in proteins. The lipid content of the biomass generally increased by halving nutrients. Biosorption yields of these biomasses towards four wastewater models were assessed in terms of colour, salts and toxicity reduction. The biomasses rich in proteins and acid polysaccharides were less effective in removing reactive and direct dyes, whereas those rich in cationic polysaccharides showed a higher affinity for these dyes. Both chromatography and FTIR analyses showed that biomasses cultured in halved C and N had the highest affinity for salts. The wastewaters detoxification was quite always achieved, with values often lower that the Italian legal threshold limit.

SB202190-Induced Cell Type-Specific Vacuole Formation and Defective Autophagy Do Not Depend on p38 MAP Kinase Inhibition

SB202190, a widely used inhibitor of p38 MAPKα and β, was recently described to induce autophagic vacuoles and cell death in colon and ovarian cancer cells lines and, therefore, this effect was supposed to be specific for transformed cells and to open therapeutic options. Here, we demonstrate that SB202190 and the structurally related inhibitor SB203580 induce pro-autophagic gene expression and vacuole formation in various cancer and non-cancer cell lines of human, rat, mouse and hamster origin. This effect seems to induce defective autophagy leading to the accumulation of acidic vacuoles, p62 protein and lipid conjugated LC3. Using further p38 inhibitors we show that p38 MAPK inhibition is not sufficient for the autophagic response. In line with these results, expression of a SB202190-resistant mutant of p38α, which significantly increases activity of the p38 pathway under inhibitory conditions, does not block SB202190-dependent vacuole formation, indicating that lack of p38α activity is not necessary for this effect. Obviously, the induction of autophagic vacuole formation by SB203580 and SB202190 is due to off-target effects of these inhibitors on post-translational protein modifications, such as phosphorylation of the MAPKs ERK1/2 and JNK1/2, ribosomal protein S6, and PKB/Akt. Interestingly, the PI3K-inhibitor wortmannin induces transient vacuole formation indicating that the PI3K-PKB/Akt-mTOR pathway is essential for preventing autophagy and that cross-inhibition of this pathway by SB202190 could be the reason for the early part of the effect observed.

Rous Sarcoma Virus Gag Has No Specific Requirement for Phosphatidylinositol-(4,5)-Bisphosphate for Plasma Membrane Association In Vivo or for Liposome Interaction In Vitro

The MA domain of the retroviral Gag protein mediates interactions with the plasma membrane, which is the site of productive virus release. HIV-1 MA has a phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] binding pocket; depletion of this phospholipid from the plasma membrane compromises Gag membrane association and virus budding. We used multiple methods to examine the possible role of PI(4,5)P₂ in Gag-membrane interaction of the alpharetrovirus Rous sarcoma virus (RSV). In contrast to HIV-1, which was tested in parallel, neither membrane localization of RSV Gag-GFP nor release of virus-like particles was affected by phosphatase-mediated depletion of PI(4,5)P₂ in transfected avian cells. In liposome flotation experiments, RSV Gag required acidic lipids for binding but showed no specificity for PI(4,5)P₂. Mono-, di-, and triphosphorylated phosphatidylinositol phosphate (PIP) species as well as high concentrations of phosphatidylserine (PS) supported similar levels of flotation. A mutation that increases the overall charge of RSV MA also enhanced Gag membrane binding. Contrary to previous reports, we found that high concentrations of PS, in the absence of PIPs, also strongly promoted HIV-1 Gag flotation. Taken together, we interpret these results to mean that RSV Gag membrane association is driven by electrostatic interactions and not by any specific association with PI(4,5)P₂.