Fatty Acyl Moieties Research Articles

Regulation of hepatic lipogenesis by asymmetric arginine methylation

Background and aimsHepatic lipogenesis is elevated in nutrient abundant conditions to convert the excess carbohydrate into triacylglycerol (TAG). Fatty acyl moiety of TAG is eventually transported into adipose tissues by very low density lipoprotein, leading to the accumulation of TAG as a preferred storage form of excess energy. Disruption of the balance between TAG clearance and synthesis leads to the accumulation of lipids in the liver, leading to the progression of non-alcoholic fatty liver disease (NAFLD) including non-alcoholic steatohepatitis. Protein arginine methyltransferase (PRMT) 6 has been linked to the various metabolic processes including hepatic gluconeogenesis, muscle atrophy and lipodystrophy in mouse models. However, the role of PRMT6 in the control of hepatic lipogenesis has not been elucidated to date. MethodsWe assessed the interaction between PRMT6 and LXR alpha by using co-immunoprecipitation assay. The specific arginine residue of LXR alpha that is methylated by PRMT6 was assessed by LC-MS/MS assay and the functional consequences of LXR alpha methylation was explored by mSREBP-1c luciferase assay. The effect of PRMT6 on hepatic lipogenesis was assessed by adenovirus-mediated ectopic expression of PRMT6 or knockdown of PRMT6 via shRNA in hepatocytes. Finally, the role of PRMT6 in hepatic lipid metabolism in vivo was explored by either ectopic expression of LXR alpha mutant that is defective in PRMT6-mediated arginine methylation or knockdown of PRMT6 in liver. ResultsWe found that promoter activity of sterol regulatory element binding protein (SREBP) 1c is robustly activated by PRMT6. Interestingly, we demonstrated that PRMT6 binds to LXR alpha, a transcription factor for SREBP-1c, via its LXXLL motif, leading to the asymmetric dimethylation of an arginine residue and activation of this protein. Indeed, ectopic expression of PRMT6 in hepatocytes led to the enhanced expression of LXR alpha target genes in the lipogenic pathway. Conversely, genetic or pharmacological inhibition of PRMT6 diminished expression of lipogenic genes and the lipid accumulation in primary hepatocytes. Mechanistically, we found that asymmetric dimethylation of LXR alpha led to the dissociation of small heterodimer partner (SHP), a transcriptional co-inhibitor of this factor, resulting in the activation of LXR alpha-mediated transcriptional process. Finally, we showed that disruption of asymmetric dimethylation of LXR alpha in the liver led to the diminished expression of genes in the lipogenesis, resulting in the reduced hepatic lipid accumulation in high fat diet-fed mice in vivo. ConclusionsWe showed that PRMT6 modulates LXR alpha activity by conferring asymmetric dimethylation of arginine 253, thus blocking SHP-mediated inhibition and promoting hepatic lipid accumulation. These results suggest that PRMT6 is critical in the control of lipid homeostasis by regulation of LXR alpha-mediated lipogenesis in the liver.

Characterization of Unique Eukaryotic Sphingolipids with Temperature-Dependent Δ8-Unsaturation from the Picoalga Ostreococcus tauri.

Sphingolipids (SLs) are ubiquitous components of eukaryotic cell membranes and are found in some prokaryotic organisms and viruses. They are composed of a sphingoid backbone that may be acylated and glycosylated. Assembly of various sphingoid base, fatty acyl and glycosyl moieties results in highly diverse structures. The functional significance of variations in SL chemical diversity and abundance is still in the early stages of investigation. Among SL modifications, Δ8-desaturation of the sphingoid base occurs only in plants and fungi. In plants, SL Δ8-unsaturation is involved in cold hardiness. Our knowledge of the structure and functions of SLs in microalgae lags far behind that of animals, plants and fungi. Original SL structures have been reported from microalgae. However, functional studies are still missing. Ostreococcus tauri is a minimal microalga at the base of the green lineage and is therefore a key organism for understanding lipid evolution. In the present work, we achieved the detailed characterization of O. tauri SLs and unveiled unique glycosylceramides as sole complex SLs. The head groups are reminiscent of bacterial SLs, as they contain hexuronic acid residues and can be polyglycosylated. Ceramide backbones show a limited variety, and SL modification is restricted to Δ8-unsaturation. The Δ8-SL desaturase from O. tauri only produced E isomers. Expression of both Δ8-SL desaturase and Δ8-unsaturation of sphingolipids varied with temperature, with lower levels at 24°C than at 14°C. Overexpression of the Δ8-SL desaturase dramatically increases the level of Δ8 unsaturation at 24°C and is paralleled by a failure to increase cell size. Our work provides the first characterization of O. tauri SLs and functional evidence for the involvement of SL Δ8-unsaturation for temperature acclimation in microalgae, suggesting that this function is an ancestral feature in the green lineage.

Studies of antibacterial activity (in vitro and in vivo) and mode of action for des-acyl tridecaptins (DATs)

Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.

Biomolecular condensates create phospholipid-enriched microenvironments.

Proteins and RNA can phase separate from the aqueous cellular environment to form subcellular compartments called condensates. This process results in a protein-RNA mixture that is chemically different from the surrounding aqueous phase. Here, we use mass spectrometry to characterize the metabolomes of condensates. To test this, we prepared mixtures of phase-separated proteins and extracts of cellular metabolites and identified metabolites enriched in the condensate phase. Among the most condensate-enriched metabolites were phospholipids, due primarily to the hydrophobicity of their fatty acyl moieties. We found that phospholipids can alter the number and size of phase-separated condensates and in some cases alter their morphology. Finally, we found that phospholipids partition into a diverse set of endogenous condensates as well as artificial condensates expressed in cells. Overall, these data show that many condensates are protein-RNA-lipid mixtures with chemical microenvironments that are ideally suited to facilitate phospholipid biology and signaling.

Comprehensive Screening for EPA/DHA-Structured Phospholipids in Aquatic Products by a Specific Precursor Ion Scanning-Based HILIC-MS/MS Method

Comprehensive screening for functional substances from natural resources is always a hot research topic. Eicosapentaenoic acid- (EPA) and docosahexaenoic acid (DHA)-structured phospholipids (PLEPA/DHA) have versatile cardiovascular benefits as well as superior bioavailability. Herein, the abundance of PLEPA/DHA in 16 aquatic products was specifically and selectively screened using a recently developed precursor ion scan-driven hydrophilic interaction chromatography-mass spectrometry (PreIS-HILIC/MS) method with the fatty acyl moieties of EPA (m/z 301.6) and DHA (m/z 327.6) locked. The aim focused on the characteristics and differences in the varieties and contents of EPA/DHA-structured phosphatidylcholine (PCEPA/DHA) and EPA/DHA-structured phosphatidylethanolamine (PEEPA/DHA) molecular species. A total of 80 PLEPA/DHA molecules were identified in these natural sources, including 47 PCEPA/DHA and 33 PEEPA/DHA. After analysis, PC 16:0/20:5 and PC 16:0/22:6 are present in all aquatic products and at high levels. Antarctic krill was found to be the best resource of PLEPA/DHA in total (2574.69 μg·g-1), followed by mackerel (2330.11 μg·g-1), salmon (2109.91 μg·g-1), and Farrer's scallop (1883.59 μg·g-1), while abalone contained the lowest level of PLEPA/DHA (310.44 μg·g-1). Besides, sea cucumber and sea bass contained the highest contents of EPA-structured and DHA-structured ether phospholipids, respectively, which could be highly recommended as dietary sources of special functional phospholipids. Finally, the multiple discrepancies between the 16 aquatic products were revealed by multivariate statistical analysis. These findings improve the awareness of the composition and content of PLEPA/DHA contained in aquatic products, providing a reference for their integrated development.

Stearoyl-CoA desaturase 1 deficiency exacerbates palmitate-induced lipotoxicity by the formation of small lipid droplets in pancreatic β-cells

The accelerating accumulation of surplus lipids in the pancreas triggers structural and functional changes in type 2 diabetes-affected islets. Pancreatic β-cells exhibit a restricted capacity to store fat reservoirs in lipid droplets (LDs), which act as transient buffers to prevent lipotoxic stress. With the increasing incidence of obesity, growing interest has been seen in the intracellular regulation of LD metabolism for β-cell function. Stearoyl-CoA desaturase 1 (SCD1) is critical for producing unsaturated fatty acyl moieties for fluent storage into and out of LDs, likely affecting the overall rate of β-cell survival. We explored LD-associated composition and remodeling in SCD1-deprived INS-1E cells and in pancreatic islets in wildtype and SCD1−/− mice in the lipotoxic milieu. Deficiency in the enzymatic activity of SCD1 led to decrease in the size and number of LDs and the lower accumulation of neutral lipids. This occurred in parallel with a higher compactness and lipid order inside LDs, followed by changes in the saturation status and composition of fatty acids within core lipids and the phospholipid coat. The lipidome of LDs was enriched in 18:2n−6 and 20:4n−6 in β-cells and pancreatic islets. These rearrangements markedly contributed to differences in protein association with the LD surface. Our findings highlight an unexpected molecular mechanism by which SCD1 activity affects the morphology, composition and metabolism of LDs. We demonstrate that SCD1-dependent disturbances in LD enrichment can impact pancreatic β-cells and islet susceptibility to palmitate, which may have considerable diagnostic and methodological value for the characterization of LDs in human β-cells in type 2 diabetes patients.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism

Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.

Molecular Mechanism for Hepatic Glycerolipid Partitioning of n-6/n-3 Fatty Acid Ratio in an Obese Animal Biomodels.

The n-6/n-3 metabolic pathway associated with hepatic glycerolipid portioning plays a key role in preventing obesity. In this nutrition metabolism study, we used in vivo monitoring techniques with 40 obese male Sprague-Dawley strain rats attached with jugular-vein cannula after obesity was induced by a high-fat diet to determine the molecular mechanism associated with hepatic glycerolipid partitioning involving the n-6/n-3 metabolic pathway. Rats were randomly assigned to four groups (10 animals per group), including one control group (CON, n-6/n-3 of 71:1) and three treatment groups (n-6/n-3 of 4:1, 15:1 and 30:1). They were fed with experimental diets for 60 days. Incorporation rates of [14C]-labeling lipid into glycerolipid in the liver were 28.87-37.03% in treatment groups fed with diets containing an n-6/n-3 ratio of 4:1, 15:1 and 30:1, which were significantly (p < 0.05) lower than that in the CON (40.01%). However, 14CO2 emission % of absorbed dose showed the opposite trend. It was significantly (p < 0.05) higher in a treatment groups (n-6/n-3 of 4:1, 15:1 and 30:1, 30.35-45.08%) than in CON (27.71%). Regarding the metabolic distribution of glycerolipid to blood from livers, phospholipid/total glycerolipid (%) was significantly (p < 0.05) lower in CON at 11.04% than in treatment groups at 18.15% to 25.15%. Moreover, 14CO2/[14C]-total glycerolipid (%) was significantly (p < 0.05) higher in treatment groups at 44.16-78.50% than in CON at 39.50%. Metabolic distribution of fatty acyl moieties flux for oxidation and glycerolipid synthesis in the liver were significantly (p < 0.05) better in order of 4:1 > 15:1 > 30:1 than in the CON. Our data demonstrate that n-6/n-3 of 4:1 could help prevent obesity by controlling the mechanism of hepatic partitioning through oxidation and esterification of glycerolipid in an obese animal biomodel.

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg2+-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.

Crystal Structure of the Bifunctional Wax Synthase 1 from Acinetobacter baylyi Suggests a Conformational Change upon Substrate Binding and Formation of Additional Substrate Binding Sites

Wax esters (WE) are neutral lipids that are formed by the transesterification of an activated fatty acyl moiety to a fatty alcohol. Due to their diverse physicochemical properties, WE are used as industrial lubricants, in cosmetics, or for coating. There is substantial interest in producing WE in bacteria and plants by genetic engineering to improve their sustainability and to reduce production costs. However, we lack a detailed understanding of the catalytic mechanism and structural determinants that influence substrate specificities of WE-synthesizing enzymes, which is essential for tailored WE production. One class of well-studied WE-producing enzymes are the bifunctional bacterial wax synthases/acyl-CoA:diacylglycerol O-acyltransferases (WSD). Here, we report the 1.95 Å crystal structure of Acinetobacter baylyi WSD1 (AbWSD1) with a fatty acid molecule bound in the active site. The location of a cocrystallized myristic acid confirms a previously proposed acyl-CoA binding site. A comparison of this AbWSD1 structure to a published Marinobacter aquaeolei WSD1 (MaWSD1) structure of the apoenzyme revealed a major structural difference in the C-terminal part of AbWSD1. This leads us to propose a conformational change in AbWSD1 induced by substrate binding. This conformational change forms then a potential coenzyme A (CoA) binding site. Furthermore, we have identified an additional cavity in AbWSD1 and could show through mutational studies that two amino acids lining the cavity are crucial for the acyl-CoA:diacylglycerol O-acyltransferase (DGAT) activity of the enzyme. Our findings provide a foundation for designing WSD variants that lack DGAT activity.

Elucidating the binding mechanism of LPA species and analogs in an LPA4 receptor homology model

Lysophosphatidic acid receptor 4 (LPA4) has emerged as a potential therapeutic target for the treatment of a variety of diseases, including cancer and obesity-induced diabetes, but its structure remains to be revealed. In the present work, a homology model of LPA4 was built for studying the binding mechanism of LPA species and analogs. Then five selected LPA species and analogs with structural variations in their phosphate groups, substitutions on the glycerol backbone, and fatty acyl chains were docked into the LPA4 model, followed by molecular dynamics simulations and energy analyses. The computational results revealed that the aliphatic residues located at the vertical cleft of LPA4 may form a hydrophobic environment for the fatty acyl moiety of LPA species and their analogs. Meanwhile, the positively charged residues in the central cavity of LPA4 may form ionic interactions with the negatively charged hydrophilic head group of LPA species and their analogs. In addition, it was noted that a different binding mode of the hydrophilic head group in each species with the central cavity of the LPA4 might lead to a special rearrangement of the fatty acyl moiety. Taken together, these results may facilitate understanding of the activation mechanism of LPA4 and help design selective ligands to modulate its function for therapeutic purposes.

Manufacturing specialized wax esters in plants

Biologically produced wax esters can fulfil different industrial purposes. These functionalities almost drove the sperm whale to extinction from hunting. After the ban on hunting, there is a niche in the global market for biolubricants with properties similar to spermaceti. Wax esters can also serve as a mechanism for producing insect sex pheromone fatty alcohols. Pheromone-based mating disruption strategies are in high demand to replace the toxic pesticides in agriculture and manage insect plagues threatening our food and fiber reserves. In this study we set out to investigate the possibilities of in planta assembly of wax esters, for specific applications, through transient expression of various mix-and-match combinations of genes in Nicotiana benthamiana leaves. Our synthetic biology designs were outlined in order to pivot plant lipid metabolism into producing wax esters with targeted fatty acyl and fatty alcohols moieties. Through this approach we managed to obtain industrially important spermaceti-like wax esters enriched in medium-chain fatty acyl and/or fatty alcohol moieties of wax esters. Via employment of plant codon-optimized moth acyl-CoA desaturases we also managed to capture unusual, unsaturated fatty alcohol and fatty acyl moieties, structurally similar to moth pheromone compounds, in plant-accumulated wax esters. Comparison between outcomes of different experimental designs identified targets for stable transformation to accumulate specialized wax esters and helped us to recognize possible bottlenecks of such accumulation.

Looking in Depth at Oxidized Cholesteryl Esters by LC-MS/MS: Reporting Specific Fragmentation Fingerprints and Isomer Discrimination.

Cholesteryl esters (CE) are prone to oxidation under increased oxidative stress conditions, but little is known about oxidized CE species (oxCE). To date, only a few oxCE have been identified, however, mainly based on the detection of molecular ions by mass spectrometry (MS) or target approaches for specific oxCE. The study of oxCE occurring from radical oxidation is still scarcely addressed. In this work, we made a comprehensive assessment of oxCE derivatives and their specific fragmentation patterns to identify detailed structural features and isomer differentiation using high-resolution C18 HPLC-MS- and MS/MS-based lipidomic approaches. The LC-MS/MS analysis allowed us to pinpoint oxCE structural isomers of long-chain and short-chain species, eluting at different retention times (tR). Data analysis revealed that oxCE can be modified either in the fatty acyl moiety or in the cholesterol ring. The location of the hydroxy/hydroperoxy group originates characteristic fragment ions, namely the unmodified cholestenyl cation (m/z 369) for the isomer with oxidation in the fatty acyl chain or ions at m/z 367 and m/z 385 (369 + 16) when oxygenation occurs in the cholesterol ring. Additionally, we identified CE 18:2 and 20:4 aldehydic and carboxylic short-chain products that showed a clear fragmentation pattern that confirmed the modification in the fatty acyl chain. Specific fragmentation fingerprinting allowed discrimination of the isobaric short-chain species, namely carboxylic short-chain products, from hydroxy aldehyde short-chain products, with a hydroxycholesterol moiety. This new information is important to identify different oxCE in biological samples and will contribute to unraveling their role in biological conditions and diseases such as cardiovascular disease.

Ribosomally derived lipopeptides containing distinct fatty acyl moieties

Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non-gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.

Lipidome Atlas of the Developing Heart Uncovers Dynamic Membrane Lipid Attributes Underlying Cardiac Structural and Metabolic Maturation.

Precise metabolic rewiring during heart organogenesis underlies normal cardiac development. Herein, we utilized high-coverage, quantitative lipidomic approaches to construct lipidomic atlases of whole hearts (861 lipids; 31 classes) and mitochondria (587 lipids; 27 classes) across prenatal and postnatal developmental stages in mice. We uncovered the progressive formation of docosahexaenoyl-phospholipids and enhanced remodeling of C18:2, C20:3, and C20:4 fatty acyl moieties into cardiolipins as cardiac development progresses. A preferential flow of ceramides toward sphingomyelin biosynthesis over complex glycosphingolipid formation was also noted. Using maSigPro and GPclust algorithms, we identified a repertoire of 448 developmentally dynamic lipids and mapped their expression patterns to a library of 550 biologically relevant developmentally dynamic genes. Our combinatorial transcriptomics and lipidomics approaches identified Hadha, Lclat1, and Lpcat3 as candidate molecular drivers governing the dynamic remodeling of cardiolipins and phospholipids, respectively, in heart development. Our analyses revealed that postnatal cardiolipin remodeling in the heart constitutes a biphasic process, which first accumulates polyunsaturated C78-cardiolipins prior to tetralinoleoyl cardiolipin forming the predominant species. Multiomics analyses supplemented with transmission electron microscopy imaging uncovered enhanced mitochondria-lipid droplet contacts mediated by perilipin-5. Our combinatorial analyses of multiomics data uncovered an association between mitochondrial-resident, docosahexaenoic acid-phospholipids and messenger RNA levels of proton-transporting adenosine triphosphate synthases on inner mitochondrial membranes, which adds credence to the membrane pacemaker theory of metabolism. The current findings offer lipid-centric biological insights potentially important to understanding the molecular basis of cardiac metabolic flexibility and disease pathology.

Lipids in Pathophysiology and Development of the Membrane Lipid Therapy: New Bioactive Lipids

Membranes are mainly composed of a lipid bilayer and proteins, constituting a checkpoint for the entry and passage of signals and other molecules. Their composition can be modulated by diet, pathophysiological processes, and nutritional/pharmaceutical interventions. In addition to their use as an energy source, lipids have important structural and functional roles, e.g., fatty acyl moieties in phospholipids have distinct impacts on human health depending on their saturation, carbon length, and isometry. These and other membrane lipids have quite specific effects on the lipid bilayer structure, which regulates the interaction with signaling proteins. Alterations to lipids have been associated with important diseases, and, consequently, normalization of these alterations or regulatory interventions that control membrane lipid composition have therapeutic potential. This approach, termed membrane lipid therapy or membrane lipid replacement, has emerged as a novel technology platform for nutraceutical interventions and drug discovery. Several clinical trials and therapeutic products have validated this technology based on the understanding of membrane structure and function. The present review analyzes the molecular basis of this innovative approach, describing how membrane lipid composition and structure affects protein-lipid interactions, cell signaling, disease, and therapy (e.g., fatigue and cardiovascular, neurodegenerative, tumor, infectious diseases).

Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)–based fluorescence quencher

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted “super-substrates” with catalytic constants of up to 15,000,000 M−1s−1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD+ has IC50 values below 200 pM.

Fast and Specific Screening of EPA/DHA-Enriched Phospholipids in Fish Oil Extracted from Different Species by HILIC-MS.

Eicosapentaenoic acid- and docosahexaenoic acid-enriched phospholipids (PLEPA/DHA) have versatile health-beneficial functions and can be well absorbed in the intestine. Herein, a precursor ion scan-driven hydrophilic interaction chromatography mass spectrometry (PreIS-HILIC-MS) method with the fatty acyl moieties of m/z 301.6 and 327.6 locked was established to specifically and selectively screen PLEPA/DHA in different fish oil samples, including saury, grass carp, hairtail, and yellow croaker. Taking saury oil as an example, a total of 24 PLEPA/DHA were successfully identified and quantified, including 20 PCEPA/DHA and 4 PEEPA/DHA. Finally, this method was validated in terms of sensitivity (limit of detection ≤ 4.15 μg·mL-1), linearity (≥0.9979), precision (RSDintraday ≤ 4.65%), and recovery (≥78.6%). The performance of the PreIS-HILIC-MS method was also compared with that of the traditional full-scan mode, and the former demonstrated its unique superiority in targeted screening of PLEPA/DHA in fish oils.

High resolution mass spectrometric analysis reveals that Giardia cysts are metabolically active and generates membrane lipids with long chain fatty acyl moieties

Giardia lamblia, the causative agent of “giardiasis” affects people throughout the world. Metronidazole (Mtz, Flagyl®), a nitroimidazole group of compounds is the commonly prescribed treatment for giardiasis. However, Mtz is a genotoxic agent and a potential carcinogen. Therefore, it is imperative to identify new and specific targets in Giardia that could be exploited to develop non-Mtz classes of drugs to treat giardiasis. Because of its limited lipid synthetic ability, Giardia was proposed to scavenge phospholipids, sterols and free fatty acids from the hosts or its environment for membrane and organelle biogenesis. In the current study, we utilized a quantitative lipidomic and proteomic approach to further elucidate the lipid metabolic pathways in Giardia. Vegetative and encysting trophozoites as well as osmotically resistant cysts were cultured in the laboratory, collected, and subjected to lipid extraction or tryptic digestion followed by the high-resolution mass spectrometric analysis (Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray (HESI-II) or a Nanospray Flex Ion source, Thermo Fisher). Lipid identification and confirmation was performed on open-source software MS-DIAL and LipidMatch flow. Proteomic analysis was performed on a licensed version of Proteome Discoverer. Lipidomic analysis revealed the dynamic changes of overall lipid and fatty acid (mostly C16, C18 moieties) profiles throughout the life stages of Giardia. Lipid classes that were found in cysts included LPC, P- and O-LPC, LPE, DG, TG, CE, PC, P-PC (Plasmalogen), and ceramides. Lipids, such as SM, PG, BMP were present mostly in encysting trophozoites rather than cysts. Ether linked PC species revealed to be most abundant in trophozoites. Proteomic analysis identified increased levels of several enzymes from lipid elongation pathway i.e., acyl-CoA ligases, long-chain fatty acid elongase, oxidoreductase and enoyl-CoA reductase in cysts.These studies suggest that although limited, Giardia maintains an active and robust lipid metabolism in all stages including the water-resistant non-motile cysts. It is possible that active lipid metabolism in cysts is required for the successful excystation to produce infective membranous trophozoites.

Effect of pasteurisation, homogenisation and freeze‐drying on bovine and buffalo milk fat triacylglycerols profile

Milk fat triacylglycerols (TAGs) analysis is considered to be a challenging task; considerable numbers of fatty acyl moieties form one of the most complicated natural fats, with potentially thousands of molecular species of TAGs. Herein, TAGs fingerprinting of raw and processed bovine and buffalo milk was carried out using ultra‐performance liquid chromatography‐mass spectrometry. Firstly, milk samples were subjected to pasteurisation, homogenisation and freeze‐drying, and the TAGs profile was then analysed. Palmitic, oleic, myristic and stearic were the predominant fatty acids detected. The results revealed that the TAG profile was not significantly influenced by the applied technological treatments. Bovine milk TAGs exhibited 28–54 total acyl carbons, with 0–5 double bonds at 80 different retention times. However, buffalo milk TAGs possessed 26–54 total acyl carbon number containing 0–4 double bonds at 84 different retention times. The findings of this study could offer valuable knowledge to the field of dairy technology.