Glycerophospholipid polyunsaturation modulates resveratrol action on biomimetic membranes.

The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5 mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.

Optimized 13C-TrEnDi Enhances the Sensitivity of Plasmenyl Ether Glycerophospholipids and Demonstrates Compatibility with Other Derivatization Strategies.

The identification and quantitation of plasmalogen glycerophospholipids is challenging due to their isobaric overlap with plasmanyl ether-linked glycerophospholipids, susceptibility to acid degradation, and their typically low abundance in biological samples. Trimethylation enhancement using diazomethane (TrEnDi) can be used to significantly enhance the signal of glycerophospholipids through the creation of quaternary ammonium groups producing fixed positive charges using 13C-diazomethane in complex lipid extracts. Although TrEnDi requires a strong acid for complete methylation, we report an optimized protocol using 10 mM HBF4 with the subsequent addition of a buffer solution that prevents acidic hydrolysis of plasmalogen species and enables the benefits of TrEnDi to be realized for this class of lipids. These optimized conditions were applied to aliquots of bovine liver extract (BLE) to achieve permethylation of plasmalogen lipids within a complex mixture. Treating aliquots of unmodified and TrEnDi-derivatized BLE samples with 80% formic acid and comparing their liquid chromatography mass spectrometry (LCMS) results to analogous samples not treated with formic acid, enabled the identification of 29 plasmalogen species. On average, methylated plasmalogen species from BLE demonstrated 2.81-fold and 28.1-fold sensitivity gains over unmodified counterparts for phosphatidylcholine and phosphatidylethanolamine plasmalogen species, respectively. Furthermore, the compatibility of employing 13C-TrEnDi and a previously reported iodoacetalization strategy was demonstrated to effectively identify plasmenyl-ether lipids in complex biological extracts at greater levels of sensitivity. Overall, we detail an optimized 13C-TrEnDi derivatization strategy that enables the analysis of plasmalogen glycerophospholipids with no undesired cleavage of radyl groups, boosting their sensitivity in LCMS and LCMS/MS analyses.

LORA, Lipid Over-Representation Analysis Based on Structural Information.

With the increasing number of lipidomic studies, there is a need for an efficient and automated analysis of lipidomic data. One of the challenges faced by most existing approaches to lipidomic data analysis is lipid nomenclature. The systematic nomenclature of lipids contains all available information about the molecule, including its hierarchical representation, which can be used for statistical evaluation. The Lipid Over-Representation Analysis (LORA) web application (https://lora.metabolomics.fgu.cas.cz) analyzes this information using the Java-based Goslin framework, which translates lipid names into a standardized nomenclature. Goslin provides the level of lipid hierarchy, including information on headgroups, acyl chains, and their modifications, up to the "complete structure" level. LORA allows the user to upload the experimental query and reference data sets, select a grammar for lipid name normalization, and then process the data. The user can then interactively explore the results and perform lipid over-representation analysis based on selected criteria. The results are graphically visualized according to the lipidome hierarchy. The lipids present in the most over-represented terms (lipids with the highest number of enriched shared structural features) are defined as Very Important Lipids (VILs). For example, the main result of a demo data set is the information that the query is significantly enriched with "glycerophospholipids" containing "acyl 20:4" at the "sn-2 position". These terms define a set of VILs (e.g., PC 18:2/20:4;O and PE 16:0/20:4(5,8,10,14);OH). All results, graphs, and visualizations are summarized in a report. LORA is a tool focused on the smart mining of epilipidomics data sets to facilitate their interpretation at the molecular level.

Visible-Light Paternò-Büchi Reaction for Lipidomic Profiling at Detailed Structure Levels.

The Paternò-Büchi (PB) derivatization of carbon-carbon double bond (C═C) has been increasingly employed with tandem mass spectrometry to analyze unsaturated lipids. It enables the discovery of altered or uncanonical lipid desaturation metabolism, which would be otherwise undetected by conventional methods. Although highly useful, the reported PB reactions only provide moderate yield (∼30%). Herein, we aim to determine the key factors that affect the PB reactions and develop a system with improved capabilities for lipidomic analysis. An Ir(III) photocatalyst is chosen as the triplet energy donor for the PB reagent under 405 nm light irradiation, while phenylglyoxalate and its charge-tagging version, pyridylglyoxalate, are developed as the most efficient PB reagents. The above visible-light PB reaction system provides higher PB conversions than all previously reported PB reactions. Around 90% conversion can be achieved at high concentrations (>0.5 mM) for different classes of lipids but drops as the lipid concentration decreases. The visible-light PB reaction has then been integrated with shotgun and liquid chromatography-based workflows. The limits of detection for locating C═C in standard lipids of glycerophospholipids (GPLs) and triacylglycerides (TGs) are in the sub-nM to nM range. More than 600 distinct GPLs and TGs have been profiled at the C═C location level or the sn-position level from the total lipid extract of bovine liver, demonstrating that the developed method is capable of large-scale lipidomic analysis.

Influence of the Nature of the Lipid Building Blocks on the Second-Order Nonlinear Optical Responses of an Embedded Di-8-ANEPPS Probe.

The extensive collection of lipids found in cell membranes is justified by the fact that each lipid contributes to their overall structure, dynamics, and properties and so to the biological processes taking place within them. It also showcases that, in order to deepen our understanding of membranes, we need to have a tool to differentiate lipid bilayers of varying composition. In this work, we investigate a suite of single-component saturated glycerophospholipids varying only in their headgroup structure by analyzing the second harmonic generation (SHG) nonlinear optical (NLO) response of a probe, di-8-ANEPPS, embedded into the membranes. The seven hydrophilic heads chosen (phosphatidylcholine (PC), phosphatidylethanolamine (PE), diaglycerol (GL), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidyc acid (PA)) represent all the major headgroups that are part of mammalian plasma membranes and provide an assortment of neutral, zwiterrionic, and charged species. First, molecular dynamics simulations revealed that the lipidic arrangement is strongly sensitive to the nature of the hydrophilic head and less to the variety in the hydrophobic region. Membranes exhibiting drastically opposite structural properties can be pointed out: 1,2-dihexadecanoyl-rac-glycerol (DPGL) is the thickest and most ordered and aligned system, whereas 1,2-diacyl-sn-glycero-3-phospho-(1'-sn-glycerol) (DPPG) is thinnest and least ordered and aligned system. The structural analyses are then confronted with the molecular NLO responses, ß, computed at the time-dependent density functional theory (TDDFT) level. As the orientation of the chromophore is impacted by the various degrees of order within the lipid bilayers, the diagonal component of the ß tensor parallel to the bilayer normal, ßZZZ, is as well. In the end, this computational approach provides insights into the link between lipid building blocks and the NLO responses of the embedded dye.

Concise Synthesis of Glycerophospholipids.

Glycerophospholipids are major components of cellular membranes and provide important signaling molecules. Besides shaping membrane properties, some bind to specific receptors to activate biological pathways. Untangling the roles of individual glycerophospholipids requires clearly defined molecular species, a challenge that can be best addressed through chemical synthesis. However, glycerophospholipid syntheses are often lengthy due to the contrasting polarities found within these lipids. We now report a general strategy to quickly access glycerophospholipids via opening of a phosphate triester epoxide with carboxylic acids catalyzed by Jacobsen's Co(salen) complex. We show that this method can be applied to a variety of commercially available fatty acids, photoswitchable fatty acids, and other carboxylic acids to provide the corresponding glycerophosphate derivatives.

LipidOA: A Machine-Learning and Prior-Knowledge-Based Tool for Structural Annotation of Glycerophospholipids.

The Paternò-Büchi (PB) reaction is a carbon-carbon double bond (C═C)-specific derivatization reaction that can be used to pinpoint the location(s) of C═C(s) in unsaturated lipids and quantitate the location of isomers when coupled with tandem mass spectrometry (MS/MS). As the data of PB-MS/MS are increasingly generated, the establishment of a corresponding data analysis tool is highly needed. Herein, LipidOA, a machine-learning and prior-knowledge-based data analysis tool, is developed to analyze PB-MS/MS data generated by liquid chromatography-mass spectrometry workflows. LipidOA consists of four key functional modules to realize an annotation of glycerophospholipid (GPL) structures at the fatty acyl-specific C═C location level. These include (1) data preprocessing, (2) picking C═C diagnostic ions, (3) de novo annotation, and (4) result ranking. Importantly, in the result-ranking module, the reliability of structural annotation is sorted via the use of a machine learning classifier and comparison to the total fatty acid database generated from the same sample. LipidOA is trained and validated by four PB-MS/MS data sets acquired using different PB reagents on mass spectrometers of different resolutions and of different biological samples. Overall, LipidOA provides high precision (higher than 0.9) and a wide coverage for structural annotations of GPLs. These results demonstrate that LipidOA can be used as a robust and flexible tool for annotating PB-MS/MS data collected under different experimental conditions using different lipidomic workflows.

3-Acetylpyridine On-Tissue Paternò-Büchi Derivatization Enabling High Coverage Lipid C═C Location-Resolved MS Imaging in Biological Tissues.

Unsaturated lipids containing single or more carbon-carbon double bonds (C═C) within tissues are closely associated with various types of diseases. Mass spectrometry imaging (MSI) has been used to study the spatial distribution of lipid C═C location isomers in tissue sections. However, comprehensive characterization of lipid C═C location isomers using MSI remains challenging. Herein, we established an on-tissue charge-switching Paternò-Büchi (PB) derivatization method using 3-acetylpyridine (3-AP) as a reaction reagent, which can be used to detect and assign C═C location of glycerophospholipids (GPLs) as well as neutral lipids, such as fatty acids (FAs), under the same experimental workflow using matrix-assisted laser desorption/ionization (MALDI)-MSI. High coverage of mono- and poly-unsaturated C═C location isomers among various lipid classes including FA, phosphatidylcholine (PC), and sulfatide (SHexCer) in distinct regions of the mouse brain and kidney was visualized using MALDI-MS/MS imaging. This method has also been applied to map the spatial distribution of lipid C═C location isomers in the Alzheimer's disease (AD) mice model for the first time, which provides a new tool to study the relationships between the distribution of lipid structural diversity and neurodegenerative diseases.

Structural Characterization and Quantitation of Ether-Linked Glycerophospholipids in Peroxisome Biogenesis Disorder Tissue by Ultraviolet Photodissociation Mass Spectrometry.

The biological impact of ether glycerophospholipids (GP) in peroxisomal disorders and other diseases makes them significant targets as biomarkers for diagnostic assays or deciphering pathology of the disorders. Ether lipids include both plasmanyl and plasmenyl lipids, which each contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. This linkage, in contrast to traditional diacyl GPs, precludes their detailed characterization by mass spectrometry via traditional collisional-based MS/MS techniques. Additionally, the isomeric nature of plasmanyl and plasmenyl pairs of ether lipids introduces a further level of complexity that impedes analysis of these species. Here, we utilize 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for detailed characterization of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) plasmenyl and plasmanyl lipids in mouse brain tissue. 213 nm UVPD-MS enables the successful differentiation of these four ether lipid subtypes for the first time. We couple this UVPD-MS methodology to reversed-phase liquid chromatography (RPLC) for characterization and relative quantitation of ether lipids from normal and diseased (Pex7 deficiency modeling the peroxisome biogenesis disorder, RCDP) mouse brain tissue, highlighting the ability to pinpoint specific structural features of ether lipids that are important for monitoring aberrant lipid metabolism in peroxisomal disorders.

Enhancing the Signal-to-Noise of Diagnostic Fragment Ions of Unsaturated Glycerophospholipids via Precursor Exclusion Ultraviolet Photodissociation Mass Spectrometry (PEx-UVPD-MS).

Understanding and elucidating the diverse structures and functions of lipids has motivated the development of many innovative tandem mass spectrometry (MS/MS) strategies. Higher-energy activation methods, such as ultraviolet photodissociation (UVPD), generate unique fragment ions from glycerophospholipids that can be used to perform in-depth structural analysis and facilitate the deconvolution of isomeric lipid structures in complex samples. Although detailed characterization is central to the correlation of lipid structure to biological function, it is often impeded by the lack of sufficient instrument sensitivity for highly bioactive but low-abundance phospholipids. Here, we present precursor exclusion (PEx) UVPD, a simple yet powerful technique to enhance the signal-to-noise (S/N) of informative low-abundance fragment ions produced from UVPD of glycerophospholipids. Through the exclusion of the large population of undissociated precursor ions with an MS3 strategy, the S/N of diagnostic fragment ions from PC 18:0/18:2(9Z, 12Z) increased up to an average of 13x for PEx-UVPD compared to UVPD alone. These enhancements were extended to complex mixtures of lipids from bovine liver extract to confidently identify 35 unique structures using liquid chromatography PEx-UVPD. This methodology has the potential to advance lipidomics research by offering deeper structure elucidation and confident identification of biologically active lipids.

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.

Enhanced Characterization of Cardiolipins via Hybrid 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Cardiolipins (CLs) constitute a structurally complex class of glycerophospholipids with a unique tetraacylated structure accompanied by distinctive functional roles. Aberrations in the composition of this lipid class have been associated with disease states, spurring interest in the development of new approaches to differentiate the structures of diverse CLs in complex mixtures. The structural characterization of these complex lipids using conventional methods, however, suffers from limited resolution and frequently proves unable to discern subtle yet biologically significant features such as unsaturation sites or acyl chain position assignments. Here, we describe the synergistic use of chemical derivatization and hybrid dissociation techniques to characterize CL from complex biological mixtures with both double bond and sn positional isomer resolution in a shotgun mass spectrometry strategy. Utilizing (trimethylsilyl)diazomethane (TMSD), CL phosphate groups were methylated to promote positive-mode ionization by the production of metal-cationized lipids, enabling structural interrogation via hybrid higher-energy collisional activation/ultraviolet photodissociation (HCD/UVPD). This combination of TMSD derivatization and HCD/UVPD fragmentation results in diagnostic product ions that permit distinction and relative quantitation of sn-stereoisomers and the localization of double bonds. Applying this strategy to a total lipid extract from a thyroid carcinoma revealed a previously unreported 18:2/18:1 motif, elucidating a structural feature unique to the lipid class.

Cryogenic infrared spectroscopy provides mechanistic insight into the fragmentation of phospholipid silver adducts.

Tandem mass spectrometry is arguably the most important analytical tool for structure elucidation of lipids and other metabolites. By fragmenting intact lipid ions, valuable structural information such as the lipid class and fatty acyl composition are readily obtainable. The information content of a fragment spectrum can often be increased by the addition of metal cations. In particular, the use of silver ions is deeply rooted in the history of lipidomics due to their propensity to coordinate both electron-rich heteroatoms and C = C bonds in aliphatic chains. Not surprisingly, coordination of silver ions was found to enable the distinction of sn-isomers in glycerolipids by inducing reproducible intensity differences in the fragment spectra, which could, however, not be rationalized. Here, we investigate the fragmentation behaviors of silver-adducted sn- and double bond glycerophospholipid isomers by probing fragment structures using cryogenic gas-phase infrared (IR) spectroscopy. Our results confirm that neutral headgroup loss from silver-adducted glycerophospholipids leads to dioxolane-type fragments generated by intramolecular cyclization. By combining high-resolution IR spectroscopy and computational modelling of silver-adducted fragments, we offer qualitative explanations for different fragmentation behaviors of glycerophospholipid isomers. Overall, the results demonstrate that gas-phase IR spectroscopy of fragment ions can significantly contribute to our understanding of lipid dissociation mechanisms and the influence of coordinating cations.

Lipidomics Analysis of Outer Membrane Vesicles and Elucidation of the Inositol Phosphoceramide Biosynthetic Pathway in Bacteroides thetaiotaomicron.

Approximately one-third of the human colonic microbiome is formed by bacteria from the genus Bacteroides. These bacteria produce a large amount of uniformly sized outer membrane vesicles (OMVs), which are equipped with hydrolytic enzymes that play a role in the degradation of diet- and host-derived glycans. In this work, we characterize the lipid composition of membranes and OMVs from Bacteroides thetaiotaomicron VPI-5482. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that OMVs carry sphingolipids, glycerophospholipids, and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMVs. The most abundant sphingolipids in OMVs are ethanolamine phosphoceramide (EPC) and inositol phosphoceramide (IPC). Bioinformatics analysis allowed the identification of the BT1522-1526 operon putatively involved in IPC synthesis. Mutagenesis studies revealed that BT1522-1526 is essential for the synthesis of phosphatidylinositol (PI) and IPC, confirming the role of this operon in the biosynthesis of IPC. BT1522-1526 mutant strains lacking IPC produced OMVs that were indistinguishable from the wild-type strain, indicating that IPC sphingolipid species are not involved in OMV biogenesis. Given the known role of sphingolipids in immunomodulation, we suggest that OMVs may act as long-distance vehicles for the delivery of sphingolipids in the human gut. IMPORTANCE Sphingolipids are essential membrane lipid components found in eukaryotes that are also involved in cell signaling processes. Although rare in bacteria, sphingolipids are produced by members of the phylum Bacteroidetes, human gut commensals. Here, we determined that OMVs carry sphingolipids and other lipids of known signaling function. Our results demonstrate that the BT1522-1526 operon is required for IPC biosynthesis in B. thetaiotaomicron.

Each phospholipase A2 type exhibits distinct selectivity toward sn-1 ester, alkyl ether, and vinyl ether phospholipids.

Glycerophospholipids are major components of cell membranes and have enormous variation in the composition of fatty acyl chains esterified on the sn-1 and sn-2 position as well as the polar head groups on the sn-3 position of the glycerol backbone. Phospholipase A2 (PLA2) enzymes constitute a superfamily of enzymes which play a critical role in metabolism and signal transduction by hydrolyzing the sn-2 acyl chains of glycerophospholipids. In human cell membranes, in addition to the conventional diester phospholipids, a significant amount is the sn-1 ether-linked phospholipids which play a critical role in numerous biological activities. However, precisely how PLA2s distinguish the sn-1 acyl chain linkage is not understood. In the present study, we expanded the technique of lipidomics to determine the unique in vitro specificity of three major human PLA2s, including Group IVA cytosolic cPLA2, Group VIA calcium-independent iPLA2, and Group V secreted sPLA2 toward the linkage at the sn-1 position. Interestingly, cPLA2 prefers sn-1 vinyl ether phospholipids known as plasmalogens over conventional ester phospholipids and the sn-1 alkyl ether phospholipids. iPLA2 showed similar activity toward vinyl ether and ester phospholipids at the sn-1 position. Surprisingly, sPLA2 preferred ester phospholipids over alkyl and vinyl ether phospholipids. By taking advantage of molecular dynamics simulations, we found that Trp30 in the sPLA2 active site dominates its specificity for diester phospholipids.

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen.

The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout Enterobacterales (e.g., Salmonella, Klebsiella, and Yersinia). There are two OM forms of ECA (phosphoglyceride-linked ECAPG and lipopolysaccharide-linked ECALPS) and one periplasmic form of ECA (cyclic ECACYC). ECAPG, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECAPG is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECAPG biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between elyC and ECAPG biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found ΔelyC was lethal specifically in strains producing ECAPG without other ECA forms, suggesting ECAPG biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECAPG synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECACYC. Our data demonstrate ECACYC can regulate ECAPG synthesis in strains wild type for elyC. Overall, our data demonstrate ElyC and ECACYC act in a novel pathway that regulates the production of ECAPG, supporting a model in which ElyC provides feedback regulation of ECAPG production based on the periplasmic levels of ECACYC. IMPORTANCE Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales, including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECAPG. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECACYC, genetically interact to inhibit the synthesis of ECAPG, potentially through feedback regulation based on ECACYC levels. This is the first insight into the pathway responsible for synthesis of ECAPG and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECAPG levels.

Novel Glycerophospholipid, Lipo- and N-acyl Amino Acids from Bacteroidetes: Isolation, Structure Elucidation and Bioactivity.

The 'core' metabolome of the Bacteroidetes genus Chitinophaga was recently discovered to consist of only seven metabolites. A structural relationship in terms of shared lipid moieties among four of them was postulated. Here, structure elucidation and characterization via ultra-high resolution mass spectrometry (UHR-MS) and nuclear magnetic resonance (NMR) spectroscopy of those four lipids (two lipoamino acids (LAAs), two lysophosphatidylethanolamines (LPEs)), as well as several other undescribed LAAs and N-acyl amino acids (NAAAs), identified during isolation were carried out. The LAAs represent closely related analogs of the literature-known LAAs, such as the glycine-serine dipeptide lipids 430 (2) and 654. Most of the here characterized LAAs (1, 5-11) are members of a so far undescribed glycine-serine-ornithine tripeptide lipid family. Moreover, this study reports three novel NAAAs (N-(5-methyl)hexanoyl tyrosine (14) and N-(7-methyl)octanoyl tyrosine (15) or phenylalanine (16)) from Olivibacter sp. FHG000416, another Bacteroidetes strain initially selected as best in-house producer for isolation of lipid 430. Antimicrobial profiling revealed most isolated LAAs (1-3) and the two LPE 'core' metabolites (12, 13) active against the Gram-negative pathogen M. catarrhalis ATCC 25238 and the Gram-positive bacterium M. luteus DSM 20030. For LAA 1, additional growth inhibition activity against B. subtilis DSM 10 was observed.

Molecular-dynamics-simulation-guided membrane engineering allows the increase of membrane fatty acid chain length in Saccharomyces cerevisiae.

The use of lignocellulosic-based fermentation media will be a necessary part of the transition to a circular bio-economy. These media contain many inhibitors to microbial growth, including acetic acid. Under industrially relevant conditions, acetic acid enters the cell predominantly through passive diffusion across the plasma membrane. The lipid composition of the membrane determines the rate of uptake of acetic acid, and thicker, more rigid membranes impede passive diffusion. We hypothesized that the elongation of glycerophospholipid fatty acids would lead to thicker and more rigid membranes, reducing the influx of acetic acid. Molecular dynamics simulations were used to predict the changes in membrane properties. Heterologous expression of Arabidopsis thaliana genes fatty acid elongase 1 (FAE1) and glycerol-3-phosphate acyltransferase 5 (GPAT5) increased the average fatty acid chain length. However, this did not lead to a reduction in the net uptake rate of acetic acid. Despite successful strain engineering, the net uptake rate of acetic acid did not decrease. We suggest that changes in the relative abundance of certain membrane lipid headgroups could mitigate the effect of longer fatty acid chains, resulting in a higher net uptake rate of acetic acid.

Membrane lipid replacement with nano-micelles in human sperm cryopreservation improves post-thaw function and acrosome protein integrity.

RESEARCH QUESTION: Membrane lipid replacement (MLR) of oxidized membrane lipids can restore sperm cellular membrane functionality and help improve surface protein stability during cryopreservation. What are the effects of MLR with nano-micelles made from a glycerophospholipid (GPL) mixture and cholesterol-loaded cyclodextrin (CLC), on the cryosurvival and expression of acrosome-related proteins in thawed human spermatozoa? DESIGN: Twenty samples were used to determine the optimum level of nano-micelles by incubation of semen with different concentrations of GPL (0.1 and 1%) and CLC (1 and 2 mg/ml) (including GPL-0.1, GPL-1, CLC-1, CLC-2, CLC-1/GPL-0.1, CLC-2/GPL-0.1, CLC-1/GPL-1 and CLC-2/GPL-1) before cryopreservation. Then, 30 semen samples were collected, and each sample was divided into the following three aliquots: fresh, frozen control and frozen incubated with optimum level of nano-micelles (0.1% GPL and 1 mg/ml CLC). RESULTS: CLC-1/GPL-0.1 and GPL-0.1 significantly increased motility parameters. CLC-1, GPL-0.1 and CLC-1/GPL-0.1 significantly improved viability rate compared with frozen control group. Significantly higher mitochondrial activity and acrosome integrity, and a lower rate of apoptosis, were observed in the CLC-1/GPL-0.1 compared with the frozen control group. The expression ratios of arylsulfatase A (ARSA), serine protease 37 (PRSS37), serine protease inhibitor Kazal-type 2 (SPINK2) and equatorin (EQTN) significantly increased compared with the frozen control group. CONCLUSIONS: Modification of membrane cholesterol and GPL mixtures in spermatozoa enhances their acrosome protein integrity by inhibiting early apoptotic changes and spontaneous acrosome reactions.

Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization.

Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity.