Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “PHOSPHOLIPIDS”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Copine1 regulates neural stem cell functions during brain development

Highlights: • Copine1 is highly expressed in neural lineage cells at early developmental stages. • Copine1 regulates the proliferation of embryonic neural stem cells (NSCs). • Copine1 regulates the differentiation of NSCs into neuronal and glial lineages. • mTOR signaling pathway is involved in Copine1-meidated neural development. Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Osh6p, a homologue of the oxysterol-binding protein, is involved in production of functional cytochrome P450 belonging to CYP52 family in n-alkane-assimilating yeast Yarrowia lipolytica

Highlights: • Yarrowia lipolytica has four OSBP homolog genes, OSH1, OSH3, OSH4, and OSH6. • OSH3 and OSH6 are required for growth on n-alkanes. • OSH6 is involved in the production of functional P450 of CYP52 family. • Osh6p might provide an ideal ER membrane environment for CYP52 P450 in Y. lipolytica. In this study, we investigated the role of OSH6, which encodes a homolog of the oxysterol-binding protein, in the assimilation of n-alkanes in the yeast Yarrowia lipolytica. The deletion mutant of OSH6 showed growth defects on n-alkanes of 10–16 carbons. In the deletion mutant, production of the functional cytochrome P450 was not observed. However, transcription of ALK1, encoding a major P450 belonging to the CYP52 family that plays a critical role in n-alkane hydroxylation, and further translation of its transcript were noted in the deletion mutant as well as in the wild-type strain. The phospholipid composition was altered and, the ratio of phosphatidylserine (PS) was reduced by the deletion of OSH6. Residues involved in the transport of PS and phosphatidylinositol-4-phosphate in Osh6 of Saccharomyces cerevisiae are conserved in Y. lipolytica Osh6p and substitutions of these residues resulted in a defect in the n-alkane assimilation by Y. lipolytica. From these results, we propose a hypothesis that Osh6p provides an ideal endoplasmic reticulum membrane environment for Alk proteins to have a functional conformation via lipid transport activity in Y. lipolytica.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Detecting phospholipase activity with the amphipathic lipid packing sensor motif of ArfGAP1

Highlights: • The amphipathic lipid packing sensor (ALPS) motif of ArfGAP1 binds membranes of high curvature. • The activity of phospholipase A2, C and D on membranes generate lipids with high spontaneous curvature. • This in-situ phospholipase activity induces ALPS binding to membranes with small curvature. • We propose ALPS not only as a curvature sensor but also as a tool to monitor phospholipase activity. The amphipathic lipid packing sensor (ALPS) motif of ArfGAP1 brings this GTPase activating protein to membranes of high curvature. Phospholipases are phospholipid-hydrolyzing enzymes that generate different lipid products that alter the lateral organization of membranes. Here, we evaluate by fluorescence microscopy how in-situ changes of membrane lipid composition driven by the activity of different phospholipases promotes the binding of ALPS. We show that the activity of phospholipase A2, phospholipase C and phospholipase D drastically enhances the binding of ALPS to the weakly-curved membrane of giant liposomes. Our results suggest that the enzymatic activity of phospholipases can modulate the ArfGAP1-mediated intracellular traffic and that amphiphilic peptides such as the ALPS motif can be used to study lipolytic activities at lipid membranes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Domain architecture and catalysis of the Staphylococcus aureus fatty acid kinase

Fatty acid kinase (Fak) is a two-component enzyme that generates acyl-phosphate for phospholipid synthesis. Fak consists of a kinase domain protein (FakA) that phosphorylates a fatty acid enveloped by a fatty acid binding protein (FakB). The structural basis for FakB function has been established, but little is known about FakA. Here, we used limited proteolysis to define three separate FakA domains: the amino terminal FakA_N, the central FakA_L, and the carboxy terminal FakA_C. The isolated domains lack kinase activity, but activity is restored when FakA_N and FakA_L are present individually or connected as FakA_NL. The X-ray structure of the monomeric FakA_N captures the product complex with ADP and two Mg 2+ ions bound at the nucleotide site. The FakA_L domain encodes the dimerization interface along with conserved catalytic residues Cys240, His282, and His284. AlphaFold analysis of FakA_L predicts the catalytic residues are spatially clustered and pointing away from the dimerization surface. Furthermore, the X-ray structure of FakA_C shows that it consists of two subdomains that are structurally related to FakB. Analytical ultracentrifugation demonstrates that FakA_C binds FakB, and site-directed mutagenesis confirms that a positively charged wedge on FakB meshes with a negatively charged groove on FakA_C. Finally, small angle X-ray scattering analysis is consistent with freely rotating FakA_N and FakA_C domains tethered by flexible linkers to FakA_L. These data reveal specific roles for the three independently folded FakA protein domains in substrate binding and catalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Pregnancy is accompanied by larger high density lipoprotein particles and compositionally distinct subspecies

Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Plant and microbial biomarkers suggest mechanisms of soil organic carbon accumulation in a Mojave Desert ecosystem under elevated CO 2

We ireport on how properties of soil organic matter (SOM) were altered after 10 years exposure to elevated atmospheric CO 2 concentration ([CO 2 ]) in a Mojave Desert ecosystem, using plant and microbial biomarkers. We focused on roles of Larrea tridentata, the dominant evergreen shrub which form islands of fertility, and biological soil crusts which have extensive cover in plant interspace. Soils to 5 cm in depth were collected under L. tridentata and plant interspace, and biological soil crusts to 0.5 cm in depth under three cover types, Pleuraphis rigida , a C 4 grass, shrubs and plant interspace. Soil organic carbon contents were not significantly different between elevated and ambient [CO 2 ]. Yet, significantly higher abundance of n-alkanes, a major constituent of foliage wax material, occurred in the elevated compared to ambient [CO 2 ] plots in the soils collected under L. tridentata , but no such difference was found in the soils at plant interspace or the biological soil crusts. There was no critical difference in abundance of microbial phospholipid fatty acids between the CO 2 treatments in the soils of either cover types. However, neutral lipid fatty acid abundance was significantly higher under elevated than ambient [CO 2 ] in the soils under L. tridentata , whereas no such significant difference was observed at plant interspace. These results emphasize important roles of the dominant shrubs in SOM formation under elevated [CO 2 ] in arid ecosystems. Elevated [CO 2 ] stimulated growth of L. tridentata in wet years, and aboveground litter deposition via senescence contributed to SOM formation in islands of fertility. In addition, elevated [CO 2 ] stimulated soil microbial turnover rates in rhizosphere of L. tridentata , which left more soil microbial necromass, a major SOM source. We concluded that responses of dominant shrubs to elevated [CO 2 ] can stimulate SOM formation in arid ecosystems, but biological soil crusts may have limited capacity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Comparison of TCDD-elicited genome-wide hepatic gene expression in Sprague–Dawley rats and C57BL/6 mice

Although the structure and function of the AhR are conserved, emerging evidence suggests that downstream effects are species-specific. In this study, rat hepatic gene expression data from the DrugMatrix database (National Toxicology Program) were compared to mouse hepatic whole-genome gene expression data following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For the DrugMatrix study, male Sprague–Dawley rats were gavaged daily with 20 μg/kg TCDD for 1, 3 and 5 days, while female C57BL/6 ovariectomized mice were examined 1, 3 and 7 days after a single oral gavage of 30 μg/kg TCDD. A total of 649 rat and 1386 mouse genes (|fold change| ≥ 1.5, P1(t) ≥ 0.99) were differentially expressed following treatment. HomoloGene identified 11,708 orthologs represented across the rat Affymetrix 230 2.0 GeneChip (12,310 total orthologs), and the mouse 4 × 44K v.1 Agilent oligonucleotide array (17,578 total orthologs). Comparative analysis found 563 and 922 orthologs differentially expressed in response to TCDD in the rat and mouse, respectively, with 70 responses associated with immune function and lipid metabolism in common to both. Moreover, QRTPCR analysis of Ceacam1, showed divergent expression (induced in rat; repressed in mouse) functionally consistent with TCDD-elicited hepatic steatosis in the mouse but not the rat. Functional analysis identified orthologs involved in nucleotide binding and acetyltransferase activity in rat, while mouse-specific responses were associated with steroid, phospholipid, fatty acid, and carbohydrate metabolism. These results provide further evidence that TCDD elicits species-specific regulation of distinct gene networks, and outlines considerations for future comparisons of publicly available microarray datasets. - Highlights: ► We performed a whole-genome comparison of TCDD-regulated genes in mice and rats. ► Previous species comparisons were extended using data from the DrugMatrix database. ► Less than 15% of TCDD-regulated orthologs were common to mice and rats. ► Considerations for the comparison of publicly available datasets are described.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Acute chlorine gas exposure produces transient inflammation and a progressive alteration in surfactant composition with accompanying mechanical dysfunction

Acute Cl{sub 2} exposure following industrial accidents or military/terrorist activity causes pulmonary injury and severe acute respiratory distress. Prior studies suggest that antioxidant depletion is important in producing dysfunction, however a pathophysiologic mechanism has not been elucidated. We propose that acute Cl{sub 2} inhalation leads to oxidative modification of lung lining fluid, producing surfactant inactivation, inflammation and mechanical respiratory dysfunction at the organ level. C57BL/6J mice underwent whole-body exposure to an effective 60 ppm-hour Cl{sub 2} dose, and were euthanized 3, 24 and 48 h later. Whereas pulmonary architecture and endothelial barrier function were preserved, transient neutrophilia, peaking at 24 h, was noted. Increased expression of ARG1, CCL2, RETLNA, IL-1b, and PTGS2 genes was observed in bronchoalveolar lavage (BAL) cells with peak change in all genes at 24 h. Cl{sub 2} exposure had no effect on NOS2 mRNA or iNOS protein expression, nor on BAL NO{sub 3}{sup −} or NO{sub 2}{sup −}. Expression of the alternative macrophage activation markers, Relm-α and mannose receptor was increased in alveolar macrophages and pulmonary epithelium. Capillary surfactometry demonstrated impaired surfactant function, and altered BAL phospholipid and surfactant protein content following exposure. Organ level respiratory function was assessed by forced oscillation technique at 5 end expiratory pressures. Cl{sub 2} exposure had no significant effect on either airway or tissue resistance. Pulmonary elastance was elevated with time following exposure and demonstrated PEEP refractory derecruitment at 48 h, despite waning inflammation. These data support a role for surfactant inactivation as a physiologic mechanism underlying respiratory dysfunction following Cl{sub 2} inhalation. - Highlights: • Effect of 60 ppm*hr Cl{sub 2} gas on lung inflammation and mechanical function examined. • Pulmonary inflammation is transient and minor. • Alterations in surfactant homeostasis and pulmonary mechanics are noted. • No increase in the caliber of larger airways was suggested. • Small airways stability appears impaired based on PEEP response of mechanics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug

A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this drug. - Highlights: • Identified susceptible and resistant mouse strains to liver injury induced by a CAD • Liver injury characterized by single cell necrosis, and phospholipidosis • Decreased gene expression associated with protein ubiquitination in sensitive mice • Altered protein ubiquitination may cause oxidized protein accumulation in the liver.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Assessment of serum biomarkers in rats after exposure to pesticides of different chemical classes

There is increasing emphasis on the use of biomarkers of adverse outcomes in safety assessment and translational research. We evaluated serum biomarkers and targeted metabolite profiles after exposure to pesticides (permethrin, deltamethrin, imidacloprid, carbaryl, triadimefon, fipronil) with different neurotoxic actions. Adult male Long–Evans rats were evaluated after single exposure to vehicle or one of two doses of each pesticide at the time of peak effect. The doses were selected to produce similar magnitude of behavioral effects across chemicals. Serum or plasma was analyzed using commercial cytokine/protein panels and targeted metabolomics. Additional studies of fipronil used lower doses (lacking behavioral effects), singly or for 14 days, and included additional markers of exposure and biological activity. Biomarker profiles varied in the number of altered analytes and patterns of change across pesticide classes, and discriminant analysis could separate treatment groups from control. Low doses of fipronil produced greater effects when given for 14 days compared to a single dose. Changes in thyroid hormones and relative amounts of fipronil and its sulfone metabolite also differed between the dosing regimens. Most cytokine changes reflected alterations in inflammatory responses, hormone levels, and products of phospholipid, fatty acid, and amino acid metabolism. These findings demonstrate distinct blood-based analyte profiles across pesticide classes, dose levels, and exposure duration. These results show promise for detailed analyses of these biomarkers and their linkages to biological pathways. - Highlights: • Pesticides typical of different classes produced distinct patterns of change in biomarker panels. • Based on the panels used, alterations suggest impacts on immune, metabolism, and homeostasis functions. • Some changes may reflect actions on neurotransmitter systems involved in immune modulation. • Fipronil effects on thyroid and kinetics differed with acute and repeated administration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Increased serum bile acid concentration following low-dose chronic administration of thioacetamide in rats, as evidenced by metabolomic analysis

A liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS)-based metabolomics approach was employed to identify endogenous metabolites as potential biomarkers for thioacetamide (TAA)-induced liver injury. TAA (10 and 30 mg/kg), a well-known hepatotoxic agent, was administered daily to male Sprague–Dawley (SD) rats for 28 days. We then conducted untargeted analyses of endogenous serum and liver metabolites. Partial least squares discriminant analysis (PLS-DA) was performed on serum and liver samples to evaluate metabolites associated with TAA-induced perturbation. TAA administration resulted in altered levels of bile acids, acyl carnitines, and phospholipids in serum and in the liver. We subsequently demonstrated and confirmed the occurrence of compromised bile acid homeostasis. TAA treatment significantly increased serum levels of conjugated bile acids in a dose-dependent manner, which correlated well with toxicity. However, hepatic levels of these metabolites were not substantially changed. Gene expression profiling showed that the hepatic mRNA levels of Ntcp, Bsep, and Oatp1b2 were significantly suppressed, whereas those of basolateral Mrp3 and Mrp4 were increased. Decreased levels of Ntcp, Oatp1b2, and Ostα proteins in the liver were confirmed by western blot analysis. These results suggest that serum bile acids might be increased due to the inhibition of bile acid enterohepatic circulation rather than increased endogenous bile acid synthesis. Moreover, serum bile acids are a good indicator of TAA-induced hepatotoxicity. - Highlights: • Endogenous metabolic profiles were assessed in rat after treatment of thioacetamide. • It significantly increased the levels of bile acids in serum but not in the liver. • Expression of the genes related to bile acid secretion and reuptake was decreased. • Increased serum bile acids result from block of enterohepatic circulation of bile acids.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Mechanistic review of drug-induced steatohepatitis

Drug-induced steatohepatitis is a rare form of liver injury known to be caused by only a handful of compounds. These compounds stimulate the development of steatohepatitis through their toxicity to hepatocyte mitochondria; inhibition of beta-oxidation, mitochondrial respiration, and/or oxidative phosphorylation. Other mechanisms discussed include the disruption of phospholipid metabolism in lysosomes, prevention of lipid egress from hepatocytes, targeting mitochondrial DNA and topoisomerase, decreasing intestinal barrier function, activation of the adenosine pathway, increasing fatty acid synthesis, and sequestration of coenzyme A. It has been found that the majority of compounds that induce steatohepatitis have cationic amphiphilic structures; a lipophilic ring structure with a side chain containing a cationic secondary or tertiary amine. Within the last decade, the ability of many chemotherapeutics to cause steatohepatitis has become more evident coining the term chemotherapy-associated steatohepatitis (CASH). The mechanisms behind drug-induced steatohepatitis are discussed with a focus on cationic amphiphilic drugs and chemotherapeutic agents. - Highlights: • Reviewed the mechanisms underlying drug-induced steatohepatitis for many compounds • Mitochondrial dysfunction is critical in the development of drug-induced steatohepatitis. • Majority of drugs that induce steatohepatitis are cationic amphiphilic drugs. • Chemotherapeutics that induce CASH are cationic amphiphilic drugs. • Majority of drugs that induce steatohepatitis are carnitine palmitoyltransferase-I inhibitors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

On the evolution of the pulmonary alveolar lipofibroblast

The pulmonary alveolar lipofibroblast was first reported in 1970. Since then its development, structure, function and molecular characteristics have been determined. Its capacity to actively absorb, store and ‘traffic’ neutral lipid for protection of the alveolus against oxidant injury, and for the active supply of substrate for lung surfactant phospholipid production have offered the opportunity to identify a number of specialized functions of these strategically placed cells. Namely, Parathyroid Hormone-related Protein (PTHrP) signaling, expression of Adipocyte Differentiation Related Protein, leptin, peroxisome proliferator activator receptor gamma, and the prostaglandin E2 receptor EP2- which are all stretch-regulated, explaining how and why surfactant production is ‘on-demand’ in service to ventilation–perfusion matching. Because of the central role of the lipofibroblast in vertebrate lung physiologic evolution, it is a Rosetta Stone for understanding how and why the lung evolved in adaptation to terrestrial life, beginning with the duplication of the PTHrP Receptor some 300 mya. Moreover, such detailed knowledge of the workings of the lipofibroblast have provided insight to the etiology and effective treatment of Bronchopulmonary Dysplasia based on physiologic principles rather than on pharmacology. - Highlights: • development and function of the pulmonary alveolar lipofibroblast phenotype. • Regulation of lipofibroblast neutral lipid metabolism. • Exploiting the deep homologies regulating lipofibroblast structure and function. • Utilizing such in-depth knowledge to understand the etiology and effective treatment of Bronchopulmonary Dysplasia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Direct Probes of 4 nm Diameter Gold Nanoparticles Interacting with Supported Lipid Bylayers

Interfacial charge densities and potentials are determined for silica-supported phospholipid bilayers formed from lipids having zwitterionic, negatively charged, and positively charged headgroups. Quartz crystal microbalance with dissipation (QCM-D), fluorescence recovery after photobleaching (FRAP), and atomic force microscopy demonstrate the presence of well-formed supported lipid bilayers, which, as probed by vibrational sum frequency generation (SFG), undergo negligible structural changes along their alkyl chains when NaCl concentration is raised from 0.001 to 0.1 M. From second harmonic generation (SHG) measurements we estimate that each zwitterionic headgroup of the bilayer formed from pure DOPC is associated with an apparent charge of -0.028(+0.008/-0.007)×10-¹⁹C, corresponding to 1.8 ± 0.5 % of an elementary negative charge. Moreover, we show that a supported lipid bilayer carrying an apparent negative interfacial potential may interact with not just positively charged 4-nm diameter gold nanoparticles but also negatively charged gold nanoparticles. In this latter case, charge-charge repulsion does not appear to inhibit particle-bilayer interactions and is likely overcome by multivalent interactions that are estimated to involve 3-5 hydrogen-bond equivalents. FRAP, QCM-D, and SFG measurements indicate that the bilayers remain intact under the conditions of the experiments. SHG charge screening experiments are consistent with an apparent zero net charge density associated with the positively charged gold nanoparticles when they are attached to a supported lipid bilayer carrying an apparent negative potential. The results presented here serve to benchmark experimental and computational studies of the nano-bio interface.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2013

Structural basis of substrate discrimination and integrin binding by autotaxin

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure of a eukaryotic cholinephosphotransferase-1 reveals mechanisms of substrate recognition and catalysis

Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic cell membranes. In eukaryotes, two highly homologous enzymes, cholinephosphotransferase-1 (CHPT1) and choline/ethanolamine phosphotransferase-1 (CEPT1) catalyze the final step of de novo PC synthesis. CHPT1/CEPT1 joins two substrates, cytidine diphosphate-choline (CDP-choline) and diacylglycerol (DAG), to produce PC, and Mg 2+ is required for the reaction. However, mechanisms of substrate recognition and catalysis remain unresolved. Here we report structures of a CHPT1 from Xenopus laevis (xlCHPT1) determined by cryo-electron microscopy to an overall resolution of ~3.2 Å. xlCHPT1 forms a homodimer, and each protomer has 10 transmembrane helices (TMs). The first 6 TMs carve out a cone-shaped enclosure in the membrane in which the catalysis occurs. The enclosure opens to the cytosolic side, where a CDP-choline and two Mg 2+ are coordinated. The structures identify a catalytic site unique to eukaryotic CHPT1/CEPT1 and suggest an entryway for DAG. The structures also reveal an internal pseudo two-fold symmetry between TM3-6 and TM7-10, and suggest that CHPT1/CEPT1 may have evolved from their distant prokaryotic ancestors through gene duplication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Diffusion and spectroscopy of water and lipids in fully hydrated dimyristoylphosphatidylcholine bilayer membranes

Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in the membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10{sup −5} cm{sup 2}/s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10{sup −8} cm{sup 2}/s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2–8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a result of its interaction with lipids. We have thoroughly analyzed the physical meaning of all spectral features from lipid atomic sites and correlated them with experimental data. Our findings include a “wagging of the tails” frequency around 30 cm{sup −1}, which essentially corresponds to motions of the tail-group along the instantaneous plane formed by the two lipid tails, i.e., in-plane oscillations are clearly of bigger importance than those along the normal-to-the plane direction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Modeling kinetics and equilibrium of membranes with fields: Milestoning analysis and implication to permeation

Coarse graining of membrane simulations by translating atomistic dynamics to densities and fields with Milestoning is discussed. The space of the membrane system is divided into cells and the different cells are characterized by order parameters presenting the number densities. The dynamics of the order parameters are probed with Milestoning. The methodology is illustrated here for a phospholipid membrane system (a hydrated bilayer of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) lipid molecules). Significant inhomogeneity in membrane internal number density leads to complex free energy landscape and local maps of transition times. Dynamics and distributions of cavities within the membrane assist the permeation of nonpolar solutes such as xenon atoms. It is illustrated that quantitative and detailed dynamics of water transport through DOPC membrane can be analyzed using Milestoning with fields. The reaction space for water transport includes at least two slow variables: the normal to the membrane plane, and the water density.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗