Phospholipid Acyl Chains Research Articles

The Enteric Bacterium Enterococcus faecalis Elongates and Incorporates Exogenous Short and Medium Chain Fatty Acids Into Membrane Lipids.

Enterococcus faecalis incorporates and elongates exogeneous short- and medium-chain fatty acids to chains sufficiently long to enter membrane phospholipid synthesis. The acids are activated by the E. faecalis fatty acid kinase (FakAB) system and converted to acyl-ACP species that can enter the fatty acid synthesis cycle to become elongated. Following elongation the acyl chains are incorporated into phospholipid by the PlsY and PlsC acyltranferases. This process has little effect on de novo fatty acid synthesis in the case of short-chain acids, but a greater effect with medium-chain acids. Incorporation of exogenous short-chain fatty acids in E. faecalis was greatly increased by overexpression of either AcpA, the acyl carrier protein of fatty acid synthesis, or the phosphate acyl transferase PlsX. The PlsX of Lactococcus lactis was markedly superior to the E. faecalis PlsX in incorporation of short-chain but not long-chain acids. These manipulations also allowed unsaturated fatty acids of lengths too short for direct transfer to the phospholipid synthesis pathway to be elongated and support growth of E. faecalis unsaturated fatty acid auxotrophic strains. Short- and medium-chain fatty acids can be abundant in the human gastrointestinal tract and their elongation by E. faecalis would conserve energy and carbon by relieving the requirement for total de novo synthesis of phospholipid acyl chains.

How Membrane Phospholipids Containing Long-Chain Polyunsaturated Fatty Acids and Their Oxidation Products Orchestrate Lipid Raft Dynamics to Control Inflammation

BackgroundLong-chain PUFA (LC-PUFA) influence varying aspects of inflammation. One mechanism by which they regulate inflammation is by controlling the size and molecular composition of lipid rafts. Lipid rafts are sphingolipid/cholesterol-enriched plasma membrane microdomains that compartmentalize signaling proteins and thereby control downstream inflammatory gene expression and cytokine production. ObjectivesThis review summarizes developments in our understanding of how LC-PUFA acyl chains of phospholipids, in addition to oxidized derivatives of LC-PUFAs such as oxidized 1-palmitoyl-2-arachidonyl-phosphatidylcholine (oxPAPC), manipulate formation of lipid rafts and thereby inflammation. MethodsWe reviewed the literature, largely from the past 2 decades, on the impact of LC-PUFA acyl chains and oxidized products of LC-PUFAs on lipid raft biophysical organization of myeloid and lymphoid cells. The majority of the studies are based on rodent or cellular experiments with supporting mechanistic studies using biomimetic membranes and molecular dynamic simulations. These studies have focused largely on the LC-PUFA docosahexaenoic acid, with some studies addressing eicosapentaenoic acid. A few studies have investigated the role of oxidized phospholipids on rafts. ResultsThe biophysical literature suggests a model in which n-3 LC-PUFAs, in addition to oxPAPC, localize predominately to nonraft regions and impart a disordering effect in this environment. Rafts become larger because of the ensuing increase in the difference in order between raft and nonrafts. Biochemical studies suggest that some n-3 LC-PUFAs can be found within rafts. This deviation from homeostasis is a potential trigger for controlling aspects of innate and adaptive immunity. ConclusionOverall, select LC-PUFA acyl chains and oxidized acyl chains of phospholipids control lipid raft dynamics and downstream inflammation. Gaps in knowledge remain, particularly on underlying molecular mechanisms by which plasma membrane receptor organization is controlled in response to oxidized LC-PUFA acyl chains of membrane phospholipids. Validation in humans is also an area for future study.

Varying the position of phospholipid acyl chain unsaturation modulates hopanoid and sterol ordering

The cell membrane must balance mechanical stability with fluidity to function as both a barrier and an organizational platform. Key to this balance is the ordering of hydrocarbon chains and the packing of lipids. Many eukaryotes synthesize sterols, which are uniquely capable of modulating the lipid order to decouple membrane stability from fluidity. Ancient sterol analogs known as hopanoids are found in many bacteria and proposed as ancestral ordering lipids. The juxtaposition of sterols and hopanoids in extant organisms prompts us to ask why both pathways persist, especially in light of their convergent ability to order lipids. In this work, simulations, monolayer experiments, and cellular assays show that hopanoids and sterols order unsaturated phospholipids differently based on the position of double bonds in the phospholipid acyl chain. We find that cholesterol and diplopterol’s methyl group distributions lead to distinct effects on unsaturated lipids. In Mesoplasma florum, diplopterol’s constrained ordering capacity reduces membrane resistance to osmotic stress, unlike cholesterol. These findings suggest that cholesterol’s broader lipid-ordering ability may have facilitated the exploration of a more diverse lipidomic landscape in eukaryotic membranes.

Lysophosphatidylcholine acyltransferase 1 suppresses nanoclustering and function of KRAS.

KRAS is frequently mutated in cancer, contributing to 20% of all human cancer especially pancreatic, colorectal and lung cancer. Signaling of the constitutively active KRAS oncogenic mutants is mostly compartmentalized to proteolipid nanoclusters on the plasma membrane (PM). Signaling nanoclusters of many KRAS mutants selectively enrich phosphatidylserine (PS) lipids with unsaturated sn-2 acyl chains, but not the fully saturated PS species. Thus, remodeling PS acyl chains may suppress KRAS oncogenesis. Lysophosphatidylcholine acyltransferases (LPCATs) remodel sn-2 acyl chains of phospholipids, with LPCAT1 preferentially generating the fully saturated lipids. Here, we show that stable expression of LPCAT1 depletes major PS species with unsaturated sn-2 chains while decreasing minor phosphatidylcholine (PC) species with the corresponding acyl chains. LPCAT1 expression more effectively disrupts the nanoclustering of oncogenic GFP-KRASG12V, which is restored by acute addback of exogenous unsaturated PS. LPCAT1 expression compromises signaling and oncogenic activities of the KRAS-dependent pancreatic tumor lines. LPCAT1 expression sensitizes human pancreatic tumor MiaPaCa-2 cells to KRASG12C specific inhibitor, Sotorasib. Statistical analyses of patient data further reveal that pancreatic cancer patients with KRAS mutations express less LPCAT1. Higher LPCAT1 expression also improves survival probability of pancreatic and lung adenocarcinoma patients with KRAS mutations. Thus, PS acyl chain remodeling selectively suppresses KRAS oncogenesis.

YdbH and YnbE form an intermembrane bridge to maintain lipid homeostasis in the outer membrane of Escherichia coli

The outer membrane (OM) of didermic gram-negative bacteria is essential for growth, maintenance of cellular integrity, and innate resistance to many antimicrobials. Its asymmetric lipid distribution, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet, is required for these functions. Lpt proteins form a transenvelope bridge that transports newly synthesized LPS from the inner membrane (IM) to OM, but how the bulk of phospholipids are transported between these membranes is poorly understood. Recently, three members of the AsmA-like protein family, TamB, YhdP, and YdbH, were shown to be functionally redundant and were proposed to transport phospholipids between IM and OM in Escherichia coli. These proteins belong to the repeating β-groove superfamily, which includes eukaryotic lipid-transfer proteins that mediate phospholipid transport between organelles at contact sites. Here, we show that the IM-anchored YdbH protein interacts with the OM lipoprotein YnbE to form a functional protein bridge between the IM and OM in E. coli. Based on AlphaFold-Multimer predictions, genetic data, and in vivo site-directed cross-linking, we propose that YnbE interacts with YdbH through β-strand augmentation to extend the continuous hydrophobic β-groove of YdbH that is thought to shield acyl chains of phospholipids as they travel through the aqueous intermembrane periplasmic compartment. Our data also suggest that the periplasmic protein YdbL prevents extensive amyloid-like multimerization of YnbE in cells. We, therefore, propose that YdbL has a chaperone-like function that prevents uncontrolled runaway multimerization of YnbE to ensure the proper formation of the YdbH-YnbE intermembrane bridge.

Abstract 7056: The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism

Abstract Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. In PCa the expression of anoctamin 7 (ANO7) has been shown to diminish as the cancer progresses. We have previously linked single-nucleotide polymorphisms in the ANO7 gene to the risk of aggressive prostate cancer and shown that in homozygous carriers of rs77559646, the variant leads to a total loss of ANO7 protein. ANO7 is a prostate-specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. To uncover the cellular functions of ANO7 has proven challenging because ANO7 is not expressed in commercially available cell lines. To study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and gene set enrichment analysis. Interestingly, we found enrichment and downregulation of mitochondrial genes participating in oxidative phosphorylation in ANO7 cells. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the basal and maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells. Furthermore, the glycolysis stress assay showed that glycolytic capacity is increased in ANO7 overexpressing cells. In addition, a targeted metabolite screening revealed that aspartate is decreased in ANO7 cells, which again points towards perturbed mitochondrial function as aspartate is produced from a tricarboxylic acid cycle intermediate. By inhibiting the uptake of extracellular aspartate, we measured slower proliferation of ANO7 expressing cells, while inhibiting aspartate had no effect on control cells. Interestingly, preliminary results also suggest ANO7's involvement in regulation of phospholipid acyl chain length and saturation, offering insights into altered signaling observed in ANO7 cells. Taken together, this study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism, which could explain why it is beneficial for cancer cells to lose ANO7 expression. Citation Format: Christoffer Löf, Nasrin Sultana, Neha Goel, Gudrun Wahlström, Johanna Schleutker. The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7056.

Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration

Synaptotagmin-1 and synaptotagmin-7 are two prominent calcium sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding calcium, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examine the biophysical properties of these two synaptotagmin isoforms and compare their interactions with phospholipid membranes. We discover that synaptotagmin-1—membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a super-penetrator. We exploit these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to calcium.

Abstract 16909: Eicosapentaenoic Acid and Docosahexaenoic Acid Have Competing Effects on Membrane Lipid Dynamics Due to Differences in Structure

Background: Omega-3 fatty acids (n3-FAs) incorporate into vascular cell membranes where they modulate protein function and signal transduction. The n3-FA eicosapentaenoic acid (EPA) delivered in ethyl ester form (icosapent ethyl) reduced a broad range of cardiovascular events in high-risk patients (REDUCE-IT). This was not observed with mixed n3-FA formulations containing docosahexaenoic acid (DHA) in STRENGTH and other trials. These discordant outcomes indicate DHA may counter certain EPA effects due to its additional carbons and double bond. We compared these n3-FA effects on membrane lipid dynamics and width, key determinants of protein function. Methods: Membrane lipid fluidity was measured by fluorescence anisotropy approaches in large unilamellar vesicles (LUVs) of 100 nm diameter containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) and 50 mol% cholesterol with EPA and/or DHA (0-10 mol%). LUVs were treated with the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), at 1 mol% and evaluated using a spectrofluorometer with a polarization accessory. Membrane width ( i.e ., unit cell periodicity) of the samples was determined by small angle x-ray scattering (SAXS). Results: DHA alone increased bulk lipid fluidity as indicated by a dose-dependent reduction in apparent rotational correlation time (ARCT); at 10 mol%, there was a ~20% (p<0.05) reduction from ~19 to ~16 nsec compared to control (untreated membranes). By contrast, EPA had no significant disordering effect except when combined at equimolar levels; the EPA/DHA combination showed a reduction in ARCT to ~18 nsec at 10 mol% that was different from EPA alone (p<0.05). Consistent with these findings, SAXS analysis showed DHA containing membranes had a reduced width compared to EPA by 5% or 3 Å (p<0.05) due to increased trans-gauche isomerizations in the phospholipid acyl chains. Conclusion: EPA maintained membrane fluidity while DHA increased fluidity and thereby reduced membrane width due to its additional carbons and double bond. The EPA/DHA combination increased membrane fluidity compared to control and EPA alone. The disordering effects of DHA may counter EPA stabilizing effects and explain discordant clinical outcomes with different n3-FA formulations.

Steroidogenic Factor-1 form and function: From phospholipids to physiology

Steroidogenic Factor-1 (SF-1, NR5A1) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, consisting of a DNA-binding domain (DBD) connected to a transcriptional regulatory ligand binding domain (LBD) via an unstructured hinge domain. SF-1 is a master regulator of development and adult function along the hypothalamic pituitary adrenal and gonadal axes, with strong pathophysiological association with endometriosis and adrenocortical carcinoma. SF-1 was shown to bind and be regulated by phospholipids, one of the most interesting aspects of SF-1 regulation is the manner in which SF-1 interacts with phospholipids: SF-1 buries the phospholipid acyl chains deep in the hydrophobic core of the SF-1 protein, while the lipid headgroups remain solvent-exposed on the exterior of the SF-1 protein surface. Here, we have reviewed several aspects of SF-1 structure, function and physiology, touching on other transcription factors that help regulate SF-1 target genes, non-canonical functions of SF-1, the DNA-binding properties of SF-1, the use of mass spectrometry to identify lipids that associate with SF-1, how protein phosphorylation regulates SF-1 and the structural biology of the phospholipid-ligand binding domain. Together this review summarizes the form and function of Steroidogenic Factor-1 in physiology and in human disease, with particular emphasis on adrenal cancer.

Growth of Enterococcus faecalis ∆plsX strains is restored by increased saturated fatty acid synthesis.

The Enterococcus faecalis acyl-acyl carrier protein (ACP) phosphate acyltransferase PlsX plays an important role in phospholipid synthesis and exogenous fatty acid incorporation. Loss of plsX almost completely blocks growth by decreasing de novo phospholipid synthesis, which leads to abnormally long-chain acyl chains in the cell membrane phospholipids. The ∆plsX strain failed to grow without supplementation with an appropriate exogenous fatty acid. Introduction of a ∆fabT mutation into the ∆plsX strain to increase fatty acid synthesis allowed very weak growth. The ∆plsX strain accumulated suppressor mutants. One of these encoded a truncated β-ketoacyl-ACP synthase II (FabO) which restored normal growth and restored de novo phospholipid acyl chain synthesis by increasing saturated acyl-ACP synthesis. Saturated acyl-ACPs are cleaved by a thioesterase to provide free fatty acids for conversion to acyl-phosphates by the FakAB system. The acyl-phosphates are incorporated into position sn1 of the phospholipids by PlsY. We report the tesE gene encodes a thioesterase that can provide free fatty acids. However, we were unable to delete the chromosomal tesE gene to confirm that it is the responsible enzyme. TesE readily cleaves unsaturated acyl-ACPs, whereas saturated acyl-ACPs are cleaved much more slowly. Overexpression of an E. faecalis enoyl-ACP reductase either FabK or FabI which results in high levels of saturated fatty acid synthesis also restored the growth of the ∆plsX strain. The ∆plsX strain grew faster in the presence of palmitic acid than in the presence of oleic acid with improvement in phospholipid acyl chain synthesis. Positional analysis of the acyl chain distribution in the phospholipids showed that saturated acyl chains dominate the sn1-position indicating a preference for saturated fatty acids at this position. High-level production of saturated acyl-ACPs is required to offset the marked preference of the TesE thioesterase for unsaturated acyl-ACPs and allow the initiation of phospholipid synthesis.

Azadirachtin exposure inhibit ovary development of Spodoptera litura (Lepidoptera: Noctuidae) by altering lipids metabolism event and inhibiting insulin signaling pathways

Lipids are main energy source for insects reproduction, which are becoming emerging target for pest management. Azadirachtin (AZA) is a multi-targeted and promising botanical insecticide, but its reproduction toxicity mechanism related to lipids metabolism is poorly understood. Here, we applied lipidomic and transcriptomic to provide a comprehensive resource for describing the effect of AZA on lipids remodeling in ovary of Spodoptera litura. The results showed that AZA exposure obviously altered the contents of 130 lipids subclasses (76 upregulated and 54 downregulated). In detail, AZA exposure changed the length and saturation degrees of fatty acyl chain of most glycerolipid, phospholipid and sphingolipid as well as the expression of genes related to biosynthesis of unsaturated fatty acids and fatty acids elongation. Besides, following the abnormal lipids metabolism, western blot analysis suggested that AZA induce insulin resistance-like phenotypes by inhibiting insulin receptor substrates (IRS) /PI3K/AKT pathway, which might be responsible for the ovary abnormalities of S. litura. Collectively, our study provided insights into the lipids metabolism event in S. litura underlying AZA exposure, these key metabolites and genes identified in this study would also provide important reference for pest control in future.

The effect of rhamnolipids on fungal membrane models as described by their interactions with phospholipids and sterols: An in silico study.

Introduction: Rhamnolipids (RLs) are secondary metabolites naturally produced by bacteria of the genera Pseudomonas and Burkholderia with biosurfactant properties. A specific interest raised from their potential as biocontrol agents for crop culture protection in regard to direct antifungal and elicitor activities. As for other amphiphilic compounds, a direct interaction with membrane lipids has been suggested as the key feature for the perception and subsequent activity of RLs. Methods: Molecular Dynamics (MD) simulations are used in this work to provide an atomistic description of their interactions with different membranous lipids and focusing on their antifungal properties. Results and discussion: Our results suggest the insertion of RLs into the modelled bilayers just below the plane drawn by lipid phosphate groups, a placement that is effective in promoting significant membrane fluidification of the hydrophobic core. This localization is promoted by the formation of ionic bonds between the carboxylate group of RLs and the amino group of the phosphatidylethanolamine (PE) or phosphatidylserine (PS) headgroups. Moreover, RL acyl chains adhere to the ergosterol structure, forming a significantly higher number of van der Waals contact with respect to what is observed for phospholipid acyl chains. All these interactions might be essential for the membranotropic-driven biological actions of RLs.

Divergent unsaturated fatty acid synthesis in two highly related model pseudomonads.

The genomes of the best-studied pseudomonads, Pseudomonas aeruginosa and Pseudomonas putida, which share 85% of the predicted coding regions, contain a fabA fabB operon (demonstrated in P. aeruginosa, putative in P. putida). The enzymes encoded by the fabA and fabB genes catalyze the introduction of a double bond into a 10-carbon precursor which is elongated to the 16:1Δ9 and 18:1Δ11 unsaturated fatty acyl chains required for functional membrane phospholipids. A detailed analysis of transcription of the P. putida fabA fabB gene cluster showed that fabA and fabB constitute an operon and disclosed an unexpected and essential fabB promoter located within the fabA coding sequence. Inactivation of the fabA fabB operon fails to halt the growth of P. aeruginosa PAO1 but blocks growth of P. putida F1 unless an exogenous unsaturated fatty acid is provided. We report that the asymmetry between these two species is due to the P. aeruginosa PAO1 desA gene which encodes a fatty acid desaturase that introduces double bonds into the 16-carbon acyl chains of membrane phospholipids. Although P. putida F1 encodes a putative DesA homolog that is 84% identical to the P. aeruginosa PAO1, the protein fails to provide sufficient unsaturated fatty acid synthesis for growth when the FabA FabB pathway is inactivated. We report that the P. putida F1 DesA homolog can functionally replace the P. aeruginosa DesA. Hence, the defect in P. putida F1 desaturation is not due to a defective P. putida F1 DesA protein but probably to a weakly active component of the electron transfer process.

Multimodal high-resolution nano-DESI MSI and immunofluorescence imaging reveal molecular signatures of skeletal muscle fiber types.

The skeletal muscle is a highly heterogeneous tissue comprised of different fiber types with varying contractile and metabolic properties. The complexity in the analysis of skeletal muscle fibers associated with their small size (30-50 μm) and mosaic-like distribution across the tissue tnecessitates the use of high-resolution imaging to differentiate between fiber types. Herein, we use a multimodal approach to characterize the chemical composition of skeletal fibers in a limb muscle, the gastrocnemius. Specifically, we combine high-resolution nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) with immunofluorescence (IF)-based fiber type identification. Computational image registration and segmentation approaches are used to integrate the information obtained with both techniques. Our results indicate that the transition between oxidative and glycolytic fibers is associated with shallow chemical gradients (<2.5 fold change in signals). Interestingly, we did not find any fiber type-specific molecule. We hypothesize that these findings might be linked to muscle plasticity thereby facilitating a switch in the metabolic properties of fibers in response to different conditions such as exercise and diet, among others. Despite the shallow chemical gradients, cardiolipins (CLs), acylcarnitines (CAR), monoglycerides (MGs), fatty acids, highly polyunsaturated phospholipids, and oxidized phospholipids, were identified as molecular signatures of oxidative metabolism. In contrast, histidine-related compounds were found as molecular signatures of glycolytic fibers. Additionally, the presence of highly polyunsaturated acyl chains in phospholipids was found in oxidative fibers whereas more saturated acyl chains in phospholipids were found in glycolytic fibers which suggests an effect of the membrane fluidity on the metabolic properties of skeletal myofibers.

Abstract PR015: Excess dietary oleic acid primes the pancreas for cancer

Abstract Human epidemiologic studies support a strong link between increased pancreatic adenocarcinoma (PDAC) risk and high fat diet (HFD) consumption, obesity, and overall energy imbalance. Given the rapid rise in worldwide obesity rates and the prevalence of western diets rich in fat, deciphering these mechanisms is not only a societal imperative but also represents a key untapped target to develop novel strategies for prevention and therapy. The translational relevance of diet research, however, has been limited by inconsistencies in fat source and consumption across human populations and mouse studies. Therefore, whether and how specific dietary fatty acids drive cancer development is poorly understood. To identify commonly consumed dietary fats capable of promoting pancreatic tumorigenesis, we fed a novel panel of 12 isocaloric HFDs – differing solely in fat source and representing the diversity of modern human fat consumption (as per statistics from the USDA) – to an oncogenic Kras-driven mouse model (KC: Pdx1-Cre; KrasLSL-G12D/+) that closely mimics the genetic and histologic features of human PDAC progression. Surprisingly, we found that diets rich in oleic acid (OA), a monounsaturated fat typically associated with good health, were strongly correlated with enhanced precancerous pancreatic intraepithelial neoplasia (PanIN) formation, arguing that OA enhances Kras-induced cellular transformation and early progression. High-oleic diets (HODs) and OA treatment of primary acinar cells induced loss of acinar cell identity and acquisition of ductal markers, consistent with a direct role for OA in acinar-to-ductal metaplasia (ADM), a prerequisite step in early PDAC development. Lipidomic analyses of plasma, liver, and muscle of mice fed HODs revealed greater circulating OA levels and increased tissue incorporation of OA into the acyl chains of phospholipids and sphingolipids, which make up the plasma membrane of cells and mediate intracellular and extracellular signaling. Furthermore, plasma and tissue OA levels more strongly correlated with tumor development, suggesting that direct OA pancreatic tissue incorporation could drive tumorigenesis. Indeed, molecular and biochemical analyses confirmed upregulation in the expression of de novo lipogenesis genes, alterations in lipid metabolism, and enhanced mTOR signaling in pancreata of mice fed HODs. Overall, these results directly link dietary OA to pancreatic lipid metabolism and transformation during PDAC development and highlight the complex pleiotropic effects of dietary fatty acids on health and disease: OA, while beneficial for heart health, may promote the development of certain cancers, such as PDAC. Uncovering the links between specific dietary fats and tumorigenesis are critical to enable precision nutritional guidance for the prevention and treatment of PDAC and potentially other obesity-associated cancers. Citation Format: Christian F. Ruiz, Rylee McDonnell, Jennifer Kaplan, Jasper de Jong, Christine Shugrue, Michael C. Rudolph, Fred S. Gorelick, John Wysolmerski, Matthew Rodeheffer, Mandar D. Muzumdar. Excess dietary oleic acid primes the pancreas for cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR015.

Salinity and hydraulic retention time induce membrane phospholipid acyl chain remodeling in Halanaerobium congolense WG10 and mixed cultures from hydraulically fractured shale wells.

Bacteria remodel their plasma membrane lipidome to maintain key biophysical attributes in response to ecological disturbances. For Halanaerobium and other anaerobic halotolerant taxa that persist in hydraulically fractured deep subsurface shale reservoirs, salinity, and hydraulic retention time (HRT) are important perturbants of cell membrane structure, yet their effects remain poorly understood. Membrane-linked activities underlie in situ microbial growth kinetics and physiologies which drive biogeochemical reactions in engineered subsurface systems. Hence, we used gas chromatography-mass spectrometry (GC-MS) to investigate the effects of salinity and HRT on the phospholipid fatty acid composition of H. congolense WG10 and mixed enrichment cultures from hydraulically fractured shale wells. We also coupled acyl chain remodeling to membrane mechanics by measuring bilayer elasticity using atomic force microscopy (AFM). For these experiments, cultures were grown in a chemostat vessel operated in continuous flow mode under strict anoxia and constant stirring. Our findings show that salinity and HRT induce significant changes in membrane fatty acid chemistry of H. congolense WG10 in distinct and complementary ways. Notably, under nonoptimal salt concentrations (7% and 20% NaCl), H. congolense WG10 elevates the portion of polyunsaturated fatty acids (PUFAs) in its membrane, and this results in an apparent increase in fluidity (homeoviscous adaptation principle) and thickness. Double bond index (DBI) and mean chain length (MCL) were used as proxies for membrane fluidity and thickness, respectively. These results provide new insight into our understanding of how environmental and engineered factors might disrupt the physical and biogeochemical equilibria of fractured shale by inducing physiologically relevant changes in the membrane fatty acid chemistry of persistent microbial taxa. GRAPHICAL ABSTRACTSalinity significantly alters membrane bilayer fluidity and thickness in Halanaerobium congolense WG10.

Membrane Association Allosterically Regulates Phospholipase A2 Enzymes and Their Specificity.

Water-soluble proteins as well as membrane-bound proteins associate with membrane surfaces and bind specific lipid molecules in specific sites on the protein. Membrane surfaces include the traditional bilayer membranes of cells and subcellular organelles formed by phospholipids. Monolayer membranes include the outer monolayer phospholipid surface of intracellular lipid droplets of triglycerides and various lipoproteins including HDL, LDL, VLDL, and chylomicrons. These lipoproteins circulate in our blood and lymph systems and contain triglycerides, cholesterol, cholesterol esters, and proteins in their interior, and these are sometimes interspersed on their surfaces. Similar lipid-water interfaces also occur in mixed micelles of phospholipids and bile acids in our digestive system, which may also include internalized triglycerides and cholesterol esters. Diacyl phospholipids constitute the defining molecules of biological membranes. Phospholipase A1 (PLA1) hydrolyzes phospholipid acyl chains at the sn-1 position of membrane phospholipids, phospholipase A2 (PLA2) hydrolyzes acyl chains at the sn-2 position, phospholipase C (PLC) hydrolyzes the glycerol-phosphodiester bond, and phospholipase D (PLD) hydrolyzes the polar group-phosphodiester bond. Of the phospholipases, the PLA2s have been the most well studied at the mechanistic level. The PLA2 superfamily consists of 16 groups and numerous subgroups, and each is generally described as one of 6 types. The most well studied of the PLA2s include extensive genetic and mutational studies, complete lipidomics specificity characterization, and crystallographic structures. This Account will focus principally on results from deuterium exchange mass spectrometric (DXMS) studies of PLA2 interactions with membranes and extensive molecular dynamics (MD) simulations of their interactions with membranes and specific phospholipids bound in their catalytic and allosteric sites. These enzymes either are membrane-bound or are water-soluble and associate with membranes before extracting their phospholipid substrate molecule into their active site to carry out their enzymatic hydrolytic reaction. We present evidence that when a PLA2 associates with a membrane, the membrane association can result in a conformational change in the enzyme whereby the membrane association with an allosteric site on the enzyme stabilizes the enzyme in an active conformation on the membrane. We sometimes refer to this transition from a "closed" conformation in aqueous solution to an "open" conformation when associated with a membrane. The enzyme can then extract a single phospholipid substrate into its active site, and catalysis occurs. We have also employed DXMS and MD simulations to characterize how PLA2s interact with specific inhibitors that could lead to potential therapeutics. The PLA2s constitute a paradigm for how membranes interact allosterically with proteins, causing conformational changes and activation of the proteins to enable them to extract and bind a specific phospholipid from a membrane for catalysis, which is probably generalizable to intracellular and extracellular transport and phospholipid exchange processes as well as other specific biological functions. We will focus on the four main types of PLA2, namely, the secreted (sPLA2), cytosolic (cPLA2), calcium-independent (iPLA2), and lipoprotein-associated PLA2 (Lp-PLA2) also known as platelet-activating factor acetyl hydrolase (PAF-AH). Studies on a well-studied specific example of each of the four major types of the PLA2 superfamily demonstrate clearly that protein subsites can show precise specificity for one of the phospholipid hydrophobic acyl chains, often the one at the sn-2 position, including exquisite sensitivity to the number and position of double bonds.

Phospholipid headgroups govern area per lipid and emergent elastic properties of bilayers

Phospholipid bilayers are liquid-crystalline materials whose intermolecular interactions at mesoscopic length scales have key roles in the emergence of membrane physical properties. Here we investigated the combined effects of phospholipid polar headgroups and acyl chains on biophysical functions of membranes with solid-state 2H NMR spectroscopy. We compared the structural and dynamic properties of phosphatidylethanolamine and phosphatidylcholine with perdeuterated acyl chains in the solid-ordered (so) and liquid-disordered (ld) phases. Our analysis of spectral lineshapes of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the so (gel) phase indicated an all-trans rotating chain structure for both lipids. Greater segmental order parameters (SCD) were observed in the ld (liquid-crystalline) phase for DPPE-d62 than for DPPC-d62 membranes, while their mixtures had intermediate values irrespective of the deuterated lipid type. Our results suggest the SCD profiles of the acyl chains are governed by methylation of the headgroups and are averaged over the entire system. Variations in the acyl chain molecular dynamics were further investigated by spin-lattice (R1Z) and quadrupolar-order relaxation (R1Q) measurements. The two acyl-perdeuterated lipids showed distinct differences in relaxation behavior as a function of the order parameter. The R1Z rates had a square-law dependence on SCD, implying collective mesoscopic dynamics, with a higher bending rigidity for DPPE-d62 than for DPPC-d62 lipids. Remodeling of lipid average and dynamic properties by methylation of the headgroups thus provides a mechanism to control the actions of peptides and proteins in biomembranes.

Chain-locked precursor ion scanning based HPLC–MS/MS for in-depth molecular analysis of lipase-catalyzed transesterification of structured phospholipids containing ω-3 fatty acyl chains

Lipase-catalyzed transesterification of structured phospholipids (sPLs) is a hot topic, but the structural variation of the fatty acyl chains in intact phospholipids at the molecular level remains unclear to date. The present study explored the detailed characteristics of synthesized phospholipids through high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in precursor ion scan mode. The optimal conditions were in-depth inspected and determined for the reaction system, including phospholipase A1 as catalyst, 15% lipase loading, and 1% water content. The sPLs enriched with EPA/DHA were structurally and quantitatively characterized by focusing on the fragments of m/z 301.6 (eicosapentaenoic acid, EPA) and m/z 327.6 (docosahexaenoic acid, DHA), and the results were statistically analyzed using partial least squares discriminant analysis and clustered heatmap hierarchical clustering analysis. PC 38:6 (18:1/20:5), PC 38:7 (18:2/20:5), PC o-40:6 (o-18:0/22:6), and PE 40:8 (18:2/22:6) etc. were revealed as the main variables that were active in the reaction.