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The Journal of Biological Chemistry Feb 2018Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and...
Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with PSD (MBP-His-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λ = 403 nm, λ = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla.
Topics: Carboxy-Lyases; Fluorescence; Maltose-Binding Proteins; Phosphatidylethanolamines; Plasmodium knowlesi; Protozoan Proteins; Recombinant Fusion Proteins
PubMed: 29247006
DOI: 10.1074/jbc.RA117.000525 -
Chemistry and Physics of Lipids Aug 2021Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) are important surface components of plasma lipoproteins, including very-low-density...
Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) are important surface components of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). However, the pathophysiological roles of PC, PE and SM in lipoproteins have not been well characterized owing to the difficulties in quantifying phospholipid classes in lipoproteins. In this study, we assessed the precision and accuracy of the enzymatic fluorometric assays for measuring PC, PE and SM in VLDL, LDL and HDL, which were isolated from human plasma by ultracentrifugation. The within-run coefficients of variation (CV) for the measurements of PC, PE and SM in lipoproteins were 1.5-2.8 %, 1.1-2.4 % and 0.9-2.3 %, respectively, whereas the between-run CVs for the PC, PE and SM assays were 2.7-4.7 %, 2.1-4.5 % and 1.6-3.3 %, respectively. Excellent linearity and almost complete recovery were achieved for all assays measuring PC, PE and SM in VLDL, LDL and HDL. Our preliminary results using these enzymatic fluorometric assays suggested that the phospholipid compositions were different among VLDL, LDL and HDL. In conclusion, we established high-throughput enzymatic fluorometric assays to quantify PC, PE and SM in human plasma VLDL, LDL and HDL, which will be useful for further investigation of pathophysiological roles of phospholipids in lipoproteins.
Topics: Adult; Blood Specimen Collection; Enzyme Assays; Fluorometry; High-Throughput Screening Assays; Humans; Lipoproteins; Male; Middle Aged; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins; Volunteers
PubMed: 34102186
DOI: 10.1016/j.chemphyslip.2021.105102 -
Biochimica Et Biophysica Acta.... Aug 2021Recently, we reported that a ternary lipid bilayer comprising phosphatidylethanolamine (PE), phosphatidylcholine (PC), which were both derived from chicken egg, and...
Recently, we reported that a ternary lipid bilayer comprising phosphatidylethanolamine (PE), phosphatidylcholine (PC), which were both derived from chicken egg, and cholesterol (Chol) generates microdomains that function as specific fusion sites for proteoliposomes. Chol-induced microdomain formation in a completely miscible lipid bilayer is an exceptional phenomenon. Numerous studies have elucidated the formation of domains in liquid ordered (L) and liquid disordered (L) phases of ternary bilayers, which comprise two partially miscible lipids and Chol. Herein, we investigated the composition and mechanism of formation of these unique microdomains in supported lipid bilayers (SLBs) using a fluorescence microscope and an atomic force microscope (AFM). We prepared ternary SLBs using egg-derived PC (eggPC), Chol and three different types of PE: egg-derived PE, 1-palmitoyl-2-oleoyl-PE, and 1,2-didocosahexaenoyl-PE (diDHPE). Fluorescence microscopy observations revealed that fluid and continuous SLBs were formed at PE concentrations (C) of ≥6 mol%. Fluorescence recovery after photobleaching measurement revealed that the microdomain was more fluid than the surrounding region that showed typical diffusion coefficient of the L phase. The microdomains were observed as depressions in the AFM topographies. Their area fraction (θ) increased with C, and diDHPE produced a significantly large θ among the three PEs. The microdomains in the PE+eggPC+Chol-SLBs were rich in polyunsaturated PE and were in the L-like phase. Associating eggPC and Chol caused polyunsaturated PE to segregate, resulting in a microdomain formation by conferring the umbrella effect on Chol, entropic effect of disordered acyl chains, and π-π interactions in the hydrophobic core.
Topics: Cholesterol; Lipid Bilayers; Membrane Microdomains; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids
PubMed: 33901442
DOI: 10.1016/j.bbamem.2021.183626 -
Advances in Nutrition (Bethesda, Md.) Jan 2016Our understanding of the impact of poor hepatic choline/phosphatidylcholine availability in promoting the steatosis characteristic of human nonalcoholic fatty liver... (Review)
Review
Our understanding of the impact of poor hepatic choline/phosphatidylcholine availability in promoting the steatosis characteristic of human nonalcoholic fatty liver disease (NAFLD) has recently advanced and possibly relates to phosphatidylcholine/phosphatidylethanolamine concentrations in various, membranes as well as cholesterol dysregulation. A role for choline/phosphatidylcholine availability in the progression of NAFLD to liver injury and serious hepatic consequences in some individuals requires further elucidation. There are many reasons for poor choline/phosphatidylcholine availability in the liver, including low intake, estrogen status, and genetic polymorphisms affecting, in particular, the pathway for hepatic de novo phosphatidylcholine synthesis. In addition to free choline, phosphatidylcholine has been identified as a substrate for trimethylamine production by certain intestinal bacteria, thereby reducing host choline bioavailability and providing an additional link to the increased risk of cardiovascular disease faced by those with NAFLD. Thus human choline requirements are highly individualized and biomarkers of choline status derived from metabolomics studies are required to predict those at risk of NAFLD induced by choline deficiency and to provide a basis for human intervention trials.
Topics: Bacteria; Choline; Deficiency Diseases; Humans; Liver; Non-alcoholic Fatty Liver Disease; Phosphatidylcholines; Phosphatidylethanolamines; Polymorphism, Genetic
PubMed: 26773011
DOI: 10.3945/an.114.007955 -
Biochimica Et Biophysica Acta.... Oct 2020Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with...
Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with several small molecules, although their identities and structural or functional roles remained unclear. Here, we use solid-state nuclear magnetic resonance (SSNMR) spectroscopy and mass spectrometry to characterize these molecules. Numerous correlations atypical for protein amino acids were found and assigned in the SSNMR spectra. The chemical shift patterns correspond to N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose which are part of the Enterobacterial Common Antigen (ECA). These sugars undergo rapid anisotropic motions and are likely linked flexibly to a rigid anchor that tightly binds ASR. Phosphorus NMR reveals several signals that are characteristic of monophosphates, further suggesting phosphatidylglyceride as the ECA lipid carrier which is anchored to ASR. In addition, NMR signals corresponding to common phospholipid phosphatidylethanolamine (PE) have been detected. The presence of PE tightly interacting with ASR was confirmed using liquid chromatography-mass spectrometry. This article commemorates Professor Michèle Auger and her contributions to membrane biophysics and Nuclear Magnetic Resonance.
Topics: Anabaena; Antigens, Bacterial; Hydrophobic and Hydrophilic Interactions; Mass Spectrometry; Membrane Proteins; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylethanolamines; Protein Binding; Sensory Rhodopsins
PubMed: 32407777
DOI: 10.1016/j.bbamem.2020.183345 -
Biochimica Et Biophysica Acta.... Feb 2018Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the...
Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7M serine and 0.3M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.
Topics: Cell Membrane; Cytoplasmic Vesicles; Lipid Bilayers; Membrane Fusion; Models, Chemical; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipase D; Phospholipids; Spectrometry, Fluorescence
PubMed: 29032301
DOI: 10.1016/j.bbamem.2017.10.011 -
Journal of Lipid Research Jun 2018This article provides a historical account of the discovery, chemistry, and biochemistry of two ubiquitous phosphoglycerolipids, phosphatidylserine (PS) and...
This article provides a historical account of the discovery, chemistry, and biochemistry of two ubiquitous phosphoglycerolipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the ether lipids. In addition, the article describes the biosynthetic pathways for these phospholipids and how these pathways were elucidated. Several unique functions of PS and PE in mammalian cells in addition to their ability to define physical properties of membranes are discussed. For example, the translocation of PS from the inner to the outer leaflet of the plasma membrane of cells occurs during apoptosis and during some other specific physiological processes, and this translocation is responsible for profound life-or-death events. Moreover, mitochondrial function is severely impaired when the PE content of mitochondria is reduced below a threshold level. The discovery and implications of the existence of membrane contact sites between the endoplasmic reticulum and mitochondria and their relevance for PS and PE metabolism, as well as for mitochondrial function, are also discussed. Many of the recent advances in these fields are due to the use of isotope labeling for tracing biochemical pathways. In addition, techniques for disruption of specific genes in mice are now widely used and have provided major breakthroughs in understanding the roles and metabolism of PS and PE in vivo.
Topics: Animals; History, 19th Century; History, 20th Century; History, 21st Century; Humans; Mitochondria; Phosphatidylethanolamines; Phosphatidylserines
PubMed: 29661786
DOI: 10.1194/jlr.R084004 -
MicrobiologyOpen Oct 2021Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that...
Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that certain Gram-negative bacteria, including Vibrio species (cholerae, parahaemolyticus, and vulnificus) are capable of using exogenous polyunsaturated fatty acids (PUFAs) to modify the phospholipids of their membrane. Moreover, exposure to exogenous PUFAs has been shown to affect certain phenotypes that are important factors of virulence. The purpose of this study was to investigate whether V. alginolyticus and V. fischeri are capable of responding to exogenous PUFAs by remodeling their membrane phospholipids and/or altering behaviors associated with virulence. Thin-layer chromatography (TLC) analyses and ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC/ESI-MS) confirmed incorporation of all PUFAs into membrane phosphatidylglycerol and phosphatidylethanolamine. Several growth phenotypes were identified when individual fatty acids were supplied in minimal media and as sole carbon sources. Interestingly, several PUFAs acids inhibited growth of V. fischeri. Significant alterations to membrane permeability were observed depending on fatty acid supplemented. Strikingly, arachidonic acid (20:4) reduced membrane permeability by approximately 35% in both V. alginolyticus and V. fischeri. Biofilm assays indicated that fatty acid influence was dependent on media composition and temperature. All fatty acids caused decreased swimming motility in V. alginolyticus, while only linoleic acid (18:2) significantly increased swimming motility in V. fischeri. In summary, exogenous fatty acids cause a variety of changes in V. alginolyticus and V. fischeri, thus adding these bacteria to a growing list of Gram-negatives that exhibit versatility in fatty acid utilization and highlighting the potential for environmental PUFAs to influence phenotypes associated with planktonic, beneficial, and pathogenic associations.
Topics: Aliivibrio fischeri; Aquatic Organisms; Biofilms; Cell Membrane; Cell Membrane Permeability; Fatty Acids, Unsaturated; Phenotype; Phosphatidylethanolamines; Phosphatidylglycerols; Vibrio Infections; Vibrio alginolyticus; Virulence
PubMed: 34713610
DOI: 10.1002/mbo3.1237 -
Proceedings of the National Academy of... May 2023The sorting of phospholipids between the inner and outer leaflets of the membrane bilayer is a fundamental problem in all organisms. Despite years of investigation, most...
The sorting of phospholipids between the inner and outer leaflets of the membrane bilayer is a fundamental problem in all organisms. Despite years of investigation, most of the enzymes that catalyze phospholipid reorientation in bacteria remain unknown. Studies from almost half a century ago in and revealed that newly synthesized phosphatidylethanolamine (PE) is rapidly translocated to the outer leaflet of the bilayer [Rothman & Kennedy, , 1821-1825 (1977)] but the identity of the putative PE flippase has eluded discovery. Recently, members of the DedA superfamily have been implicated in flipping the bacterial lipid carrier undecaprenyl phosphate and in scrambling eukaryotic phospholipids in vitro. Here, using the antimicrobial peptide duramycin that targets outward-facing PE, we show that cells lacking the DedA paralog PetA (formerly YbfM) have increased resistance to duramycin. Sensitivity to duramycin is restored by expression of PetA or homologs from other bacteria. Analysis of duramycin-mediated killing upon induction of PE synthesis indicates that PetA is required for efficient PE transport. Finally, using fluorescently labeled duramycin we demonstrate that cells lacking PetA have reduced PE in their outer leaflet compared to wildtype. We conclude that PetA is the long-sought PE transporter. These data combined with bioinformatic analysis of other DedA paralogs argue that the primary role of DedA superfamily members is transporting distinct lipids across the membrane bilayer.
Topics: Phosphatidylethanolamines; Phospholipids; Membrane Transport Proteins; Bacteria; Cell Membrane
PubMed: 37155911
DOI: 10.1073/pnas.2301979120 -
Nature Communications May 2018Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology....
Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.
Topics: Amino Acids; Bacterial Toxins; Biological Transport; Biomimetic Materials; Carbocyanines; Gene Expression; Green Fluorescent Proteins; Hemolysin Proteins; Lasers; Lipid Bilayers; Membrane Fusion; Optical Tweezers; Phosphatidylcholines; Phosphatidylethanolamines; RNA, Transfer; Ribonucleotides; Sodium Chloride
PubMed: 29760422
DOI: 10.1038/s41467-018-04282-w