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Proton circuits within biological membranes, the foundation of natural bioenergetic systems, are significantly influenced by the lipid compositions of different biological membranes. In this study, we investigate the influence of mixed lipid membrane composition on the proton transfer (PT) properties on the surface of the membrane. We track the excited-state PT (ESPT) process from a tethered probe to the membrane with timescales and length scales of PT relevant to bioenergetic systems. Two processes can happen during ESPT: the initial PT from the probe to the membrane at short timescales, followed by diffusion of dissociated protons around the probe on the membrane, and the possible geminate recombination with the probe at longer timescales. Here, we use membranes composed of mixtures of phosphatidylcholine (PC) and phosphatidic acid (PA). We show that the changes in the ESPT properties are not monotonous with the concentration of the lipid mixture; at a low concentration of PA in PC, we find that the membrane is a poor proton acceptor. Molecular dynamics simulations indicate that the membrane is more structured at this specific lipid mixture, with the least number of defects. Accordingly, we suggest that the structure of the membrane is an important factor in facilitating PT. We further show that the composition of the membrane affects the geminate proton diffusion around the probe, whereas, on a timescale of tens of nanoseconds, the dissociated proton is mostly lateral restricted to the membrane plane in PA membranes, while in PC, the diffusion is less restricted by the membrane., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration, and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner one yield less stretchable membranes with higher compressibility modulus compared with their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. In addition, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol %), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such domain formation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Lipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column., Results: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H] - ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation., Significance: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode., Competing Interests: Declaration of competing interest There is no conflict of interest from the authors of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCβ (opto-PLCβ). Opto-PLCβ uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCβ triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCβ can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCβ offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Phosphatidic acid and phosphatidylserine are anionic phospholipids with emerging signalling roles in cells. Determination of how phosphatidic acid and phosphatidylserine change location and quantity in cells over time requires selective fluorescent sensors that can distinguish these two anionic phospholipids. However, the design of such synthetic sensors that can selectively bind and respond to a single phospholipid within the complex membrane milieu remains challenging. In this work, we present a simple and robust strategy to control the selectivity of synthetic sensors for phosphatidic acid and phosphatidylserine. By changing the coordination metal of a dipicolylamine (DPA) ligand from Zn(II) to Ni(II) on the same synthetic sensor with a peptide backbone, we achieve a complete switch in selectivity from phosphatidic acid to phosphatidylserine in model lipid membranes. Furthermore, this strategy was largely unaffected by the choice and the position of the fluorophores. We envision that this strategy will provide a platform for the rational design of targeted synthetic phospholipid sensors to probe plasma and intracellular membranes.

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Motor proteins transport diverse membrane-bound vesicles along microtubules inside cells. How specific lipids, particularly rare lipids, on the membrane recruit and activate motors is poorly understood. To address this, we prepare spherical supported lipid bilayers (SSLBs) consisting of a latex bead enclosed within a membrane of desired lipid composition. SSLBs containing phosphatidic acid recruit dynein when incubated with Dictyostelium fractions but kinesin-1 when incubated with rat brain fractions. These SSLBs allow controlled biophysical investigation of membrane-bound motors along with their regulators at the single-cargo level in vitro. Optical trapping of single SSLBs reveals that motor-specific inhibitors can "lock" a motor to a microtubule, explaining the paradoxical arrest of overall cargo transport by such inhibitors. Increasing their size causes SSLBs to reverse direction more frequently, relevant to how large cargoes may navigate inside cells. These studies are relevant to understand how unidirectional or bidirectional motion of vesicles might be generated., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metal pollutants are a growing concern due to increased use in mining and other industrial processes. Moreover, the use of metals in daily life is becoming increasingly prevalent. Metals such as manganese (Mn), cobalt (Co), and nickel (Ni) are toxic in high amounts whereas lead (Pb) and cadmium (Cd) are acutely toxic at low µM concentrations. These metals are associated with system dysfunction in humans including cancer, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, and other cellular process'. One known but lesser studied target of these metals are lipids that are key membrane building blocks or serve signalling functions. It was shown that Mn, Co, Ni, Pb, and Cd cause rigidification of liposomes and increase the phase transition in membranes composed of both saturated or partly unsaturated phosphatidic acid (PA) and phosphatidylserine (PS). The selected metals showed differential effects that were more pronounced on saturated lipids. In addition, more rigidity was induced in the biologically relevant liquid-crystalline phase. Moreover, metal affinity, induced rigidification and liposome size increases also varied with the headgroup architecture, whereby the carboxyl group of PS appeared to play an important role. Thus, it can be inferred that Mn, Co, Ni, Cd, and Pb may have preferred binding coordination with the lipid headgroup, degree of acyl chain unsaturation, and membrane phase., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Phosphatidic acid (PA), the simplest phospholipid, acts as a key metabolic intermediate and second messenger that impacts diverse cellular and physiological processes across species ranging from microbes to plants and mammals. The cellular levels of PA dynamically change in response to stimuli, and multiple enzymatic reactions can mediate its production and degradation. PA acts as a signalling molecule and regulates various cellular processes via its effects on membrane tethering, enzymatic activities of target proteins, and vesicular trafficking. Because of its unique physicochemical properties compared to other phospholipids, PA has emerged as a class of new lipid mediators influencing membrane structure, dynamics, and protein interactions. This review summarizes the biosynthesis, dynamics, and cellular functions and properties of PA., (© 2023 Federation of European Biochemical Societies.)

Phosphatidic acid (PA) is a key signaling lipid that plays a crucial role in regulating various cellular processes. Studies have shown that azobenzene-containing PA analogues can be used as an all-chemical strategy for light-mediated control of PA signaling. These photoswitchable lipids offer a solution to the limitations of traditional bulk dosing methods by allowing for light- and shape-dependent interactions with protein effectors and lipid-metabolizing enzymes. This chapter describes how to synthesize AzoPA and dAzoPA., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Phospholipase D (PLD) is an enzyme with many functions, one of which is the synthesis of phosphatidic acid (PA), a molecule with a myriad of effects on various organ systems and processes. These numerous roles make it hard to understand the true action of PA in cellular and bodily processes. Imaging PLD activity is one way to better understand the synthesis of PA and start to elucidate its function. However, many of the current imaging techniques for PLD come with limitations. This chapter presents a thorough methodology of a new imaging technique for PLD activity with clickable alcohols via transphosphatidylation (IMPACT) and Real-Time IMPACT (RT-IMPACT) that takes advantage of clickable chemistry to overcome current limitations. Using strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA), and the synthesis of various organic compounds, this chapter will explain a step-by-step procedure of how to perform the IMPACT and RT-IMPACT method(s)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells.In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT.IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid-protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling.PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling.Collectively, the strategies described here summarize our progress to date in tackling the challenge of assigning precise functions to defined pools of phospholipids in cells. They also point to new challenges and directions for future study, including extension of imaging and membrane editing tools to other classes of lipids. We envision that continued application of bioorthogonal chemistry, optogenetics, and directed evolution will yield new tools and discoveries to interrogate the phospholipidome and reveal new mechanisms regulating phospholipid homeostasis and roles for phospholipids in cell signaling.

Diacylglycerol kinases (DGKs) are important enzymes in molecular membrane biology, as they can lower the concentration of diacylglycerol through phosphorylation while at the same time producing phosphatidic acid. Dysfunction of DGK is linked with multiple diseases including cancer and autoimmune disorders. Currently, the high-resolution structures have not been determined for any of the 10 human DGK paralogs, which has made it difficult to gain a more complete understanding of the enzyme's mechanism of action and regulation. In the present study, we have taken advantage of the significant developments in protein structural prediction technology by artificial intelligence (i.e., Alphafold 2.0), to conduct a comprehensive investigation on the properties of all 10 human DGK paralogs. Structural alignment of the predictions reveals that the C1, catalytic, and accessory domains are conserved in their spatial arrangement relative to each other, across all paralogs. This suggests a critical role played by this domain architecture in DGK function. Moreover, docking studies corroborate the existence of a conserved ATP-binding site between the catalytic and accessory domains. Interestingly, the ATP bound to the interdomain cleft was also found to be in proximity of the conserved glycine-rich motif, which in protein kinases has been suggested to function in ATP binding. Lastly, the spatial arrangement of DGK, with respect to the membrane, reveals that most paralogs possess a more energetically favorable interaction with curved membranes. In conclusion, AlphaFold predictions of human DGKs provide novel insights into the enzyme's structural and functional properties while also paving the way for future experimentation.

Metal ion-membrane interactions have gained appreciable attention over the years resulting in increasing investigations into the mode of action of toxic and essential metals. More work has focused on essential ions like Ca or Mg and toxic metals like Cd and Pb, whereas this study investigates the effects of the abundant essential trace metal manganese with model lipid systems by screening zwitterionic and anionic glycerophospholipids. Despite its essentiality, deleterious impact towards cell survival is known under Mn stress. The fluorescent dyes Laurdan and diphenylhexatriene were used to assess changes in membrane fluidity both in the head group and hydrophobic core region of the membrane, respectively. Mn-rigidified membranes composed of the anionic phospholipids, phosphatidic acid, phosphatidylglycerol, cardiolipin, and phosphatidylserine. Strong binding resulted in large shifts of the phase transition temperature. The increase was in the order phosphatidylserine > phosphatidylglycerol > cardiolipin, and in all cases, saturated analogues > mono-unsaturated forms. Dynamic light scattering measurements revealed that Mn caused extensive aggregation of liposomes composed of saturated analogues of phosphatidic acid and phosphatidylserine, whilst the mono-unsaturated analogue had significant membrane swelling. Increased membrane rigidity may interfere with permeability of ions and small molecules, possibly disrupting cellular homeostasis. Moreover, liposome size changes could indicate fusion, which could also be detrimental to cellular transport. Overall, this study provided further understanding into the effects of Mn with biomembranes, whereby the altered membrane properties are consequential to the proper structural and signalling functions of membrane lipids., (© 2022. European Biophysical Societies' Association.)

The clathrin coat assembly protein AP180 drives endocytosis, which is crucial for numerous physiological events, such as the internalization and recycling of receptors, uptake of neurotransmitters and entry of viruses, including SARS-CoV-2, by interacting with clathrin. Moreover, dysfunction of AP180 underlies the pathogenesis of Alzheimer's disease. Therefore, it is important to understand the mechanisms of assembly and, especially, disassembly of AP180/clathrin-containing cages. Here, we identified AP180 as a novel phosphatidic acid (PA)-binding protein from the mouse brain. Intriguingly, liposome binding assays using various phospholipids and PA species revealed that AP180 most strongly bound to 1-stearoyl-2-docosahexaenoyl-PA (18:0/22:6-PA) to a comparable extent as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), which is known to associate with AP180. An AP180 N-terminal homology domain (1-289 aa) interacted with 18:0/22:6-PA, and a lysine-rich motif (K38-K39-K40) was essential for binding. The 18:0/22:6-PA in liposomes in 100 nm diameter showed strong AP180-binding activity at neutral pH. Notably, 18:0/22:6-PA significantly attenuated the interaction of AP180 with clathrin. However, PI(4,5)P 2 did not show such an effect. Taken together, these results indicate the novel mechanism by which 18:0/22:6-PA selectively regulates the disassembly of AP180/clathrin-containing cages., Competing Interests: Declaration of competing interest All authors have declared no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Complement receptor 3 (CD11b/CD18) is an important receptor that mediates adhesion, phagocytosis and chemotaxis in various immunocytes. The conidia of the medically-important pathogenic fungus, Aspergillus fumigatus can be internalized into alveolar epithelial cells to disseminate its infection in immunocompromised host; however, the role of CR3 in this process is poorly understood. In the present study, we investigated the potential role of CR3 on A. fumigatus internalization into type II alveolar epithelial cells and its effect on host intracellular PA content induced by A. fumigatus . We found that CR3 is expressed in alveolar epithelial cells and that human serum and bronchoalveolar lavage fluid (BALF) could improve A. fumigatus conidial internalization into A549 type II alveolar epithelial cell line and mouse primary alveolar epithelial cells, which were significantly inhibited by the complement C3 quencher and CD11b-blocking antibody. Serum-opsonization of swollen conidia, but not resting conidia led to the increase of cellular phosphatidic acid (PA) in A549 cells during infection. Moreover, both conidial internalization and induced PA production were interfered by CD11b-blocking antibody and dependent on FAK activity, but not Syk in alveolar epithelial cells. Overall, our results revealed that CR3 is a critical modulator of Aspergillus fumigatus internalization into alveolar epithelial cells.

Phosphatidic acid (PA) is one of the simplest membrane phospholipids, yet it plays a crucial role in various biologically relevant processes that take place in cells. Since PA generation may be triggered by a variety of factors, very often of antagonistic character, the specific nature of physiological responses driven by PA is not clear. In order to shed more light on these issues, we carried out a systematic characterization of membranes containing one of the three biologically significant PA molecular species. The effect of these molecules on the properties of membranes composed of phosphatidylcholine and/or cholesterol was assessed in a multidisciplinary approach, including molecular dynamic simulations, flicker noise spectroscopy, and Langmuir monolayer isotherms. The first enables the determination of various macroscopic and microscopic parameters such as lateral diffusion, membrane thickness, and defect analysis. The obtained data revealed a strong interaction between unsaturated PA species and phosphatidylcholine. On the other hand, the behavior of saturated PA was greatly influenced by cholesterol. Additionally, a strong effect on mechanical properties was observed in the case of three-component systems, which could not be explained by the simple extrapolation of parameters of the corresponding two-component systems. Our data show that various PA species are not equivalent in terms of their influence on lipid mono- and bilayers and that membrane composition/properties, particularly those related to the presence of cholesterol, may strongly modulate PA behavior.

Although there are many phosphatidic acid (PA) molecular species based on its fatty acyl compositions, their interacting partners have been poorly investigated. Here, we identified synaptojanin-1 (SYNJ1), Parkinson's disease-related protein that is essential for regulating clathrin-mediated synaptic vesicle endocytosis via dually dephosphorylating D5 and D4 position phosphates from phosphatidylinositol (PI) (4,5)-bisphosphate, as a 1-stearoyl-2-docosahexaenoyl (18:0/22:6)-PA-binding protein. SYNJ1 failed to substantially associate with other acidic phospholipids. Although SYNJ1 interacted with 18:0/20:4-PA in addition to 18:0/22:6-PA, the association of the enzyme with 16:0/16:0-, 16:0/18:1-, 18:0/18:0-, or 18:1/18:1-PA was not considerable. 18:0/20:4- and 18:0/22:6-PAs bound to SYNJ1 via its SAC1 domain, which preferentially hydrolyses D4 position phosphate. Moreover, 18:0/20:4- and 18:0/22:6-PA selectively enhanced the D4-phosphatase activity, but not the D5-phosphatase activity, of SYNJ1., (© 2021 Federation of European Biochemical Societies.)

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production., (© 2021. The Author(s).)

The C2 domain of novel protein kinases C (nPKC) binds to membranes in a Ca 2+ -independent way contributing to the activation of these enzymes. We have studied the C2 domain of one of these nPKCs, namely PKCε, and confirmed that it establishes a strong interaction with POPA, which is clearly visible through changes in chemical shifts detected through 31 P-MAS-NMR and the protection that it exerts on the domain against thermal denaturation seen through DSC and FT-IR. In this study, using two-dimensional correlation analysis (2D-COS) applied to infrared spectra, we determined the sequence of events that occur during the thermal unfolding of the domain and highlighted some differences when phosphatidic acid or cardiolipin are present. Finally, by means of FRET and DLS experiments, we wanted to determine the effect of membrane curvature on the domain/membrane interaction by using lysophosphatidylcholine to introduce positive curvature as a control and we observed that the effect of these phospholipids on the protein binding is not exerted through the change of membrane curvature., (Copyright © 2021. Published by Elsevier Inc.)

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca 2+ ) dynamics in cultured human cerebral microvascular endothelial cells (hCMEC/D3), as an in vitro human blood-brain barrier (BBB) model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment actively increased both the duration and the area under the curve (A.U.C) of the ATP-evoked Ca 2+ waves in cultured hCMEC/D3 cells as well as in cultured astrocytes. mApoE-PA-LIP increased the ATP-evoked intracellular Ca 2+ waves even under 0 [Ca 2+ ] e conditions, thus indicating that the increased intracellular Ca 2+ response to ATP is mainly due to endogenous Ca 2+ release. Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca 2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca 2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca 2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca 2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer's disease (AD).

Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). In humans, the alpha isoform (DGKα) has emerged as a potential target in the treatment of cancer due to its anti-tumor and pro-immune responses. However, its mechanism of action at a molecular level is not fully understood. In this work, a systematic investigation of the role played by the membrane in the regulation of the enzymatic properties of human DGKα is presented. By using a cell-free system with purified DGKα and model membranes of variable physical and chemical properties, it is shown that membrane physical properties determine human DGKα substrate acyl chain specificity. In model membranes with a flat morphology; DGKα presents high enzymatic activity, but it is not able to differentiate DAG molecular species. Furthermore, DGKα enzymatic properties are insensitive to membrane intrinsic curvature. However, in the presence of model membranes with altered morphology, specifically the presence of physically curved membrane structures, DGKα bears substrate acyl chain specificity for palmitic acid-containing DAG. The present results identify changes in membrane morphology as one possible mechanism for the depletion of specific pools of DAG as well as the production of specific pools of PA by DGKα, adding an extra layer of regulation on the interconversion of these two potent lipid-signaling molecules. It is proposed that the interplay between membrane physical (shape) and chemical (lipid composition) properties guarantee a fine-tuned signal transduction system dependent on the levels and molecular species of DAG and PA., (© 2021 Federation of American Societies for Experimental Biology.)

In this study, we developed a method to analyze liposomal binding to a cell membrane receptor using fluorescence-labeled liposomes and demonstrated that scavenger class B type 1 (SR-B1) plays a crucial role in binding of liposomes containing phosphatidylcholine (PC) to HEK293T cell membrane and phosphatidic acid (PA) can modulate it. Site-directed mutagenesis of SR-B1 revealed that S112F and T175A mutations in its ectodomain abrogated binding and endocytosis of PC liposomes in HEK293T cells. K151A and K156A mutations attenuated their binding and endocytosis too. Although the effects of mutations on binding and endocytosis were similar between PC liposomes and PC/PA and PA liposomes, SR-B1 dependency appeared to be PC > PC/PA > PA liposomes. Our data indicate that (i) nanoparticles including high-density lipoprotein (HDL), silica, and liposomes bind to a common or close site of SR-B1, and (ii) PC/PA and PA liposomes bind not only to SR-B1 but also other receptor(s) in HEK293T cells. In addition, PC/PA liposomes induced lipid droplet (LD) formation in HEK293T cells more than PC liposomes. Treatment of HEK293T cells with SR-B1 siRNA suppressed PC/PA liposome-induced LD formation. Taken together, our results demonstrate that SR-B1 plays an essential role in binding PC-containing liposomes and the subsequent induction of cellular responses, while PA can modulate them., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomyces antibioticus termed TNYR SaPLD was developed capable of synthesising 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild-type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism., (© 2021 The Author(s).)

Radiodynamic therapy (RDT), an emerging therapeutic approach for cancer treatment by employing ionizing irradiation to induce localized photodynamic therapy (PDT) can overcome the drawbacks of the limited penetration depth for traditional PDT and the unconcentrated energy in the tumor for traditional radiotherapy (RT). Taking advantage of aggregation-induced emission (AIE) photosensitizers with bright fluorescence and efficient singlet oxygen production in the aggregate state, Hf-AIE coordination polymer nanoparticles (CPNs), which show both strong RT and RDT effect under X-ray irradiation, are developed. Furthermore, to enhance the tumor accumulation and prolong the tumor retention of the CPNs, bioorthogonal click chemistry is applied in the system through coupling between dibenzocyclooctyne (DBCO)-modified CPNs (Hf-AIE-PEG-DBCO) (PEG: poly(ethylene glycol)) and azide groups on the cell membrane formed by metabolic glycoengineering. Thanks to the high penetration of X-ray irradiation, the bioorthogonal-assisted RT and RDT combination therapy realizes significant killing of cancer cells without showing noticeable biotoxicity after intravenous administration of CPNs., (© 2021 Wiley-VCH GmbH.)

While previous studies have characterized the fatty acids and global lipid families of the chicken egg yolk, there have been no publications characterizing the individual lipids in these lipid families. Such an in-depth characterization of egg yolk lipids is essential to define the potential benefits of egg yolk consumption for the supply of structural and anti-inflammatory lipids. Historically, the major focus has been on the cholesterol content of eggs and the potential negative health benefits of this lipid, while ignoring the essential roles of cholesterol in membranes and as a precursor to other essential sterols. A detailed analysis of egg yolk lipids, using high-resolution mass spectrometric analyses and tandem mass spectrometry to characterize the fatty acid substituents of complex structural lipids, was used to generate the first in-depth characterization of individual lipids within lipid families. Egg yolks were isolated from commercial eggs (Full Circle Market) and lipids extracted with methyl-t-butylether before analyses via high-resolution mass spectrometry. This analytical platform demonstrates that chicken egg yolks provide a rich nutritional source of complex structural lipids required for lipid homeostasis. These include dominant glycerophosphocholines (GPC) (34:2 and 36:2), plasmalogen GPC (34:1, 36:1), glycerophosphoethanolamines (GPE) 38:4 and 36:2), plasmalogen GPE (36:2 and 34:1), glycerophosphoserines (36:2 and 38:4), glycerophosphoinositols (38:4), glycerophosphoglycerols (36:2), N-acylphosphatidylethanolamines (NAPE) (56:6), plasmalogen NAPE (54:4 and 56:6), sphingomyelins (16:0), ceramides (22:0 and 24:0), cyclic phosphatidic acids (16:0 and 18:0), monoacylglycerols (18:1 and 18:2), diacylglycerols (36:3 and 36:2), and triacylglycerols (52:3). Our data indicate that the egg yolk is a rich source of structural and energy-rich lipids. In addition, the structural lipids possess ω-3 and ω-6 fatty acids that are essential precursors of endogenous anti-inflammatory lipid mediators. These data indicate that eggs are a valuable nutritional addition to the diets of individuals that do not have cholesterol issues., (Copyright © 2020. Published by Elsevier Inc.)

Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [ 3 H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation., Competing Interests: The authors declare no conflict of interest.

Lipids have emerged as important actors in an ever-growing number of key functions in cell biology over the last few years. Among them, glycerophospholipids are major constituents of cellular membranes. Because of their amphiphilic nature, phospholipids form lipid bilayers that are particularly useful to isolate cellular content from the extracellular medium, but also to define intracellular compartments. Interestingly, phospholipids come in different flavors based on their fatty acyl chain composition. Indeed, lipidomic analyses have revealed the presence in cellular membranes of up to 50 different species of an individual class of phospholipid, opening the possibility of multiple functions for a single class of phospholipid. In this review we will focus on phosphatidic acid (PA), the simplest phospholipid, that plays both structural and signaling functions. Among the numerous roles that have been attributed to PA, a key regulatory role in secretion has been proposed in different cell models. We review here the evidences that support the idea that mono- and poly-unsaturated PA control distinct steps in hormone secretion from neuroendocrine cells., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

TRAAK is an ion channel from the two-pore domain potassium (K 2P ) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K 2P 4.1 toward lipids remains poorly understood. Here we show the two isoforms of K 2P 4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN 1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K 2P 4.1. Liposome potassium flux assays with K 2P 4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K 2P 4.1.

Phosphatidic acid (PA) consists of various molecular species that have different fatty acyl chains at the sn-1 and sn-2 positions; and consequently, mammalian cells contain at least 50 structurally distinct PA molecular species. However, the different roles of each PA species are poorly understood. In the present study, we attempted to identify dipalmitoyl (16:0/16:0)-PA-binding proteins from mouse skeletal muscle using liposome precipitation and tandem mass spectrometry analysis. We identified L-lactate dehydrogenase (LDH) A, which catalyzes conversion of pyruvate to lactate and is a key checkpoint of anaerobic glycolysis critical for tumor growth, as a 16:0/16:0-PA-binding protein. LDHA did not substantially associate with other phospholipids, such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphoinositides and cardiolipin at physiological pH (7.4), indicating that LDHA specifically bound to PA. Interestingly, 18:0/18:0-, 18:0/20:4- and 18:0/22:6-PA also interacted with LDHA, and their binding activities were stronger than 16:0/16:0-PA at pH 7.4. Moreover, circular dichroism spectrometry showed that 18:0/20:4- and 18:0/22:6-PA, but not 16:0/16:0- or 18:0/18:0-PA, significantly reduced the α-helical structure of LDHA. Furthermore, 18:0/20:4- and 18:0/22:6-PA attenuated LDH activity. Taken together, we demonstrated for the first time that LDHA is a PA-binding protein and is a unique PA-binding protein that is structurally and functionally controlled by associating with 18:0/20:4- and 18:0/22:6-PA., Competing Interests: Declaration of competing interest All authors have declared no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PA). The η isozyme of DGK is abundantly expressed in C2C12 myoblasts. However, the role of DGKη in skeletal muscle cells remains unknown. In the present study, we showed that DGKη was downregulated at an early stage of myogenic differentiation. The knockdown of DGKη by siRNAs significantly inhibited C2C12 myoblast proliferation but did not inhibit differentiation. Moreover, the suppression of DGKη expression decreased the expression levels of mammalian target of rapamycin (mTOR), which is a key regulator of cell proliferation, and fatty acid synthase (FASN), which catalyzes the de novo synthesis of fatty acids for cell proliferation and is transcriptionally regulated via mTOR signaling. Furthermore, the knockdown of mTOR or raptor, which is a component of mTOR complex 1 (mTORC1), decreased the amount of FASN. These results indicate that DGKη regulates myoblast proliferation through the mTOR (mTORC1)-FASN pathway. Interestingly, the knockdown of mTOR reduced the expression levels of DGKη, implying mutual regulation between DGKη and mTOR. In DGKη-knockdown myoblasts, C30-C36-PA species, mTOR activators, were decreased, suggesting that the modulation of mTOR activity through these PA species also plays an important role in myoblast proliferation., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Lipids function not only as the major structural components of cell membranes, but also as molecular messengers that transduce signals to trigger downstream signaling events in the cell. Phosphatidic acid (PA), the simplest and a minor class of glycerophospholipids, is a key intermediate for the synthesis of membrane and storage lipids, and also plays important roles in mediating diverse cellular and physiological processes in eukaryotes ranging from microbes to mammals and higher plants. PA comprises different molecular species that can act differently, and is found in virtually all organisms, tissues, and organellar membranes, with variations in total content and molecular species composition. The cellular levels of PA are highly dynamic in response to stimuli and multiple enzymatic reactions can mediate its production and degradation. Moreover, its unique physicochemical properties compared with other glycerophospholipids allow PA to influence membrane structure and dynamics, and interact with various proteins. PA has emerged as a class of new lipid mediators modulating various signaling and cellular processes via its versatile effects, such as membrane tethering, conformational changes, and enzymatic activities of target proteins, and vesicular trafficking., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Anti-tumour lipids are synthetic analogues of lysophosphatidylcholine. These drugs are both cytotoxic and cytostatic, and more interestingly, exert these effects preferentially in tumour cells. While the exact mechanism of action isn't fully elucidated, these drugs appear to preferentially partition into rigid lipid domains in cell membranes. Upon insertion, the compounds alter membrane domain organization, disrupt normal signal transduction, and cause cell death. Recently, it has been reported that these drugs induce accumulation of diacylglycerol in yeast cells which in turn sensitizes cells to the drugs. Conversely, phosphatidic acid accumulation appears to protect cells against the drugs. In the current work, the aim was to compare the biophysical effects of the drugs edelfosine, miltefosine and perifosine on monolayers of dimyristoyl phosphatidic acid, dimyristoyl glycerol and an equimolar mixture, to understand how these lipids modulate the mode of action. Surface pressure - area isotherms, compression moduli and Brewster angle microscopy were used to compare drug effects on lipid packing, monolayer compressibility and lateral domain organization of these films. Results suggest that edelfosine and miltefosine have stabilizing effects on all of the monolayers, while perifosine destabilizes dimyristoyl glycerol and the equimolar mixture. Additionally, all three drugs change the morphology of the domains observed. Based on these results the stabilization of diacylgylcerol by edelfosine and miltefosine may contribute to the mode of action as diacylglycerol is a known disruptor of bilayers. Perifosine however does not stabilize diacylglycerol, and therefore cell death may occur through a more direct inhibition of specific signal transduction. These results suggest that perifosine may illicit cytotoxicity through a different mechanism compared to the other antitumor lipid drugs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Large-scale compound screening in adult flies is hampered by the lack of continuous drug delivery systems and poor solubility of numerous compounds. Here we found that gum Arabic (Acacia/Senegal gum), a widely used stabilizer, can also emulsify lipophilic compounds and profoundly increase their accessibility to target tissues in Drosophila and mice. We further developed a gum Arabic-based drug delivery system, wherein the drug was ground into gum Arabic and emulsified in liquid food fed to flies by siphoning through a U-shape glass capillary. This system did not affect food intake nor cell viability. Since drugs were continuously delivered by siphoning, minimal compound waste and less frequent food changes make this system ideal for large-scale long-term screenings. In our pilot screening for antitumor drugs in the NCI DTP library, we used a Drosophila model of colorectal cancer and identified two drugs that are especially hydrophobic and were not identified in previous screenings. Our data demonstrated that gum Arabic facilitates drug delivery in animal models and the system is suitable for long-term high-throughput drug screening in Drosophila This system would accelerate drug discovery for chronic and cognitive conditions., Competing Interests: Competing interestsA provisional patent application related to this work has been filed by Tongji University., (© 2020. Published by The Company of Biologists Ltd.)

Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that α-synuclein (α-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of α-Syn (α-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 μm. α-Syn-N colocalized with PA-producing enzymes, diacylglycerol kinase (DGK) β at the plasma membrane (PM), myristoylated DGKζ at the Golgi apparatus, phorbol ester-stimulated DGKγ at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, α-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that α-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor., (© 2019 Federation of European Biochemical Societies.)

Serotonin transporter (SERT) is involved in serotonergic system regulation and in the pathophysiology/therapeutics of serotonin-/SERT-related diseases such as obsessive-compulsive disorder, depression, autism, and schizophrenia. We recently revealed that diacylglycerol (DG) kinase (DGK) δ induces ubiquitination/degradation of SERT in a DGK activity-dependent manner through Praja-1 E3 ubiquitin-protein ligase. However, it is still unclear how Praja-1 activity is regulated by DGKδ. Here, we reveal that 1-stearoyl-2-docosahexaenoyl (18:0/22:6)-phosphatidic acid (PA) and 18:0/22:6-DG are simultaneously decreased and accumulated, respectively, in the DGKδ-knockout mouse brain, indicating that DGKδ selectively phosphorylates 18:0/22:6-DG to generate 18:0/22:6-PA. Moreover, we find that 18:0/22:6-PA selectively binds to Praja-1 and enhances its activity. These results strongly suggest that 18:0/22:6-PA generated by DGKδ activates Praja-1 to degrade SERT in the brain., (© 2020 Federation of European Biochemical Societies.)

Diacylglycerol kinase (DGK) α enhances the proliferation of melanoma and hepatocellular carcinoma cells whereas, in contrast, DGKα induces a nonproliferative state in T cells. We previously found that DGKα produces palmitic acid (16:0)-containing PA species, such as 16:0/16:0- and 16:0/18:0-PA, in melanoma cells under serum-starved (nonproliferative) conditions. In the present study, we identified the PA species generated by DGKα in T cells under serum-starved (nonproliferative) conditions. We found that serum starvation markedly increased the levels of many PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:1/18:2-, 16:1/18:1-, 16:0/18:1-, 16:0/18:0-, 18:1/18:2-, 18:1/18:1- and 18:0/18:1-PA, in Jurkat T cells. In lysates from serum-starved Jurkat T cells, DGKα activity, which was Ca 2+ -dependent and sensitive to a DGKα-specific inhibitor (CU-3), was substantially increased, indicating its activation. Moreover, CU-3 (1-10 μM) significantly reduced the amounts of palmitic acid- and/or palmitoleic acid (16:1)-containing PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:0/18:1- and 16:0/18:0-PA, which were increased by serum starvation. These results indicate that DGKα generates different PA species in starved melanoma cells (palmitic acid-containing PA species) and T cells (palmitic acid- and/or palmitoleic acid (16:1)-containing PA species). Therefore, the differences in the PA molecular species may account for the opposing functions of DGKα in melanoma and T cells., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with the contents of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The signaling lipids phosphatidic acid (PA) and diacylglycerol pyrophosphate (DGPP) are involved in regulating the stress response in plants. PA and DGPP are anionic lipids consisting of a negatively charged phosphomonoester or pyrophosphate group attached to diacylglycerol, respectively. Changes in the pH modulate the protonation of their head groups modifying the interaction with other effectors. Here, we examine in a controlled system how the presence of Ca 2+ modulates the surface organization of dioleyl diacylglycerol pyrophosphate (DGPP) and its interaction with dioleoyl phosphatidic acid (DOPA) at different pHs. Both lipids formed expanded monolayers at pH 5 and 8. At acid and basic pHs, monolayers formed by DOPA or DGPP became denser when Ca 2+ was added to the subphase. At pH 5, Ca 2+ also induced an increase of surface potential of both lipids. Conversely, at pH 8 the effects induced by the presence of Ca 2+ on the surface potential were reversed. Mixed monolayers of DOPA and DGPP showed a non-ideal behavior. The addition of even tiny amounts of DGPP to DOPA films caused a reduction of the mean molecular area. This effect was more evident at pH 8 compared to pH 5. Our finding suggests that low amounts of DGPP in an film enriched in DOPA could lead to a local increase in film packing with a concomitant change in the local polarization, further regulated by local pH. This fact may have implications for the assigned role of PA as a pH-sensing phospholipid or during its interaction with proteins., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The effects of lipid charge and head group size on liposome partitioning by detergents is an important consideration for applications such as liposomal drug delivery or proteoliposome formation. Yet, the solubilization of mixed-lipid liposomes, those containing multiple types of lipids, by detergents has received insufficient attention. This study examines the incorporation into and subsequent dissolution of mixed-lipid liposomes comprised of both egg phosphatidylcholine (ePC) and egg phosphatidic acid (ePA) by the detergent Triton-X100 (TX). Liposomes were prepared with mixtures of the two lipids, ePC and ePA, at molar ratios from 0 to 1, then step-wise solubilized with TX. Changes in turbidity, size distribution, and molar heat power at constant temperature throughout the solubilization process were assessed. The data suggest that the difference in lipid shapes (shape factors = 0.74 and 1.4 [1,2]) affects packing in membranes, and hence influences how much TX can be incorporated before disruption. As such, liposomes containing the observed ratios of ePA incorporated higher concentrations of TX before initiating dissolution into detergent and lipid mixed-micelles. The cause was concluded to be increased mismatching in the bilayer from the conical shape of ePA compared to the cylindrical shape of ePC. Additionally, the degree to which ePA is approximated as conical versus cylindrical was modulated with pH. It was confirmed that less conical ePA behaved more similarly to ePC than more conical ePA. The understanding gained here on lipid shape in liposome incorporation of TX enables research to use in vitro liposomes that more closely mimic native membranes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A major challenge in liposomal research is to minimize the leakage of encapsulated cargo from either uncontrolled passive permeability across the liposomal membrane or upon fusion with other membranes. We previously showed that liposomes made from pure Archaea-inspired bipolar tetraether lipids exhibit exceptionally low permeability of encapsulated small molecules due to their capability to form more tightly packed membranes compared to typical monopolar lipids. Here, we demonstrate that liposomes made of synthetic bipolar tetraether lipids can also undergo membrane fusion, which is commonly accompanied by content leakage of liposomes when using typical bilayer-forming lipids. Importantly, we demonstrate calcium-mediated fusion events between liposome made of glycerolmonoalkyl glycerol tetraether lipids with phosphatidic acid headgroups (GMGTPA) occur without liposome content release, which contrasts with liposomes made of bilayer-forming EggPA lipids that displayed ~80% of content release under the same fusogenic conditions. NMR spectroscopy studies of a deuterated analog of GMGTPA lipids reveal the presence of multiple rigid and dynamic conformations, which provide evidence for the possibility of these lipids to form intermediate states typically associated with membrane fusion events. The results support that biomimetic GMGT lipids possess several attractive properties (e.g., low permeability and non-leaky fusion capability) for further development in liposome-based technologies.

The literature is replete with information describing the composition of the root lipidome in several plant species grown under various environmental conditions. However, it is unknown to what extent the root membrane lipidome vary between silage-corn genotypes, and how such variation could influence agronomic performances during field cultivation in cool climate. To address this issue, the root membrane lipidome and agronomic performance were assessed for five silage-corn genotypes (Fusion-RR, Yukon-R, A4177G3-RIB, DKC23-17RIB, DKC26-28RIB) cultivated under cool climatic conditions. Leaf area, plant height and biomass production were used as agronomic performance indicators. Varieties DKC26-28RIB and Yukon-R expressed significantly higher leaf area, plant height and biomass production compared to the other genotypes. A strong positive Spearman rank-order correlation (P = 0.001) was observed between biomass production and root phosphatidic acid (PA). The high correlation observed between PA and agronomic performance indicates PA could potentially be used as biomarker to assist in the selection of silage-corn genotypes with superior agronomic performance ideally suited for field cultivations in cool climatic conditions., (© 2018 Scandinavian Plant Physiology Society.)

Cardiolipins (CL) are anionic dimeric phospholipids bearing four fatty acids, found in inner mitochondrial membrane as structural components and are involved in several processes as oxidative phosphorylation or apoptotic signalling. As other phospholipids, CL can be modified by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can modulate various cellular functions. Modifications of CL by RNS remain largely unstudied although other nitrated lipids are emerging as bioactive molecules. In this work, we developed a C30-LC-HRMS/MS methodology to identify the nitrated and nitroxidized tetralinoleoyl-cardiolipin (TLCL), using a biomimetic model of nitration, and to disclose specific fragmentation pathways under HCD MS/MS. Using this lipidomics approach, we were able to separate and identify nitro, nitroso, nitronitroso, and nitroxidized TLCL derivatives, comprising 11 different nitrated compounds. These products were identified using accurate mass measurements and the fragmentation pattern acquired in higher-energy collision dissociation (HCD)-tandem MS/MS experiments. These spectra showed classifying fragmentation pathways, yielding phosphatidic acid (PA - ), lysophosphatidic acid (LPA - ), and carboxylate fragment ions with the modifying moiety. Remarkably, the typical neutral losses associated with the added moieties were not observed. In conclusion, this work has developed a new method for the identification of nitroso, nitrated and nitroxidized cardiolipin products by using a C30LC-MS platform method, potentially allowing their detection in biological samples., (Copyright © 2019. Published by Elsevier Inc.)

The homeostasis of most organelles requires membrane fusion mediated by s oluble N -ethylmaleimide-sensitive factor (NSF) a ttachment protein re ceptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis -SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N -ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N- ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics., (© 2019 Sparks et al.)

Morusin is a prenylated flavonoid found in mulberry that shows antimicrobial activity against foodborne pathogens. The MIC values of morusin toward S. aureus ATCC 6538 and S. aureus ATCC 25923 were both 14.9 μmol L-1. This study further investigated the antimicrobial mechanism of morusin in inhibiting the growth of Staphylococcus aureus ATCC 6538. Scanning electron microscopy and transmission electron microscopy revealed that morusin disrupted the integrity of the bacterial cell membrane. Morusin may also affect the phospholipid-repair system of bacteria, which repairs membrane structures. To test this hypothesis, quantitative real-time PCR was used to examine the effect of morusin treatment of S. aureus on the regulation of genes associated with the cell phosphatidic acid biosynthesis pathway. Gas chromatography-mass spectrometry was used to investigate the fatty acid components, which are used to synthesize bacterial phosphatidic acids. In summary, the results revealed that morusin showed a potent antibacterial effect by disrupting the cell membrane architecture and inhibiting the phosphatidic acid biosynthesis pathway of S. aureus.

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r 12AS , fluorescence anisotropy of 12-AS). Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA. When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r 12AS ). In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake., (Copyright © 2019 Elsevier B.V. All rights reserved.)

High stability and extended circulation time in vivo are quite favorable for practical biomedical applications of nanomaterials, because they greatly facilitate the preferential tumor accumulation of nanomaterials, resulting in enhanced signal fidelity for imaging and improved therapeutic effect for treatment. Although many surface modification approaches have been employed to improve the stability and circulating behavior of nanomaterials, it still remains challenging in acquiring stable and long-lasting nanomaterials for in vivo bioimaging and therapy, especially for nanoscale metal-organic frameworks (NMOFs) due to their intrinsic instability in physiological conditions. Herein, a facile, one-step strategy is reported to encapsulate the zirconium (Zr)-based NMOF UiO-66 within 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) lipid bilayer (DOPA-LB). Contrary to UiO-66 NMOFs functionalized with polyethylene glycol, the obtained UiO-66@DOPA-LB presents significantly enhanced stability and impressive blood circulation time, allowing a higher accumulation of UiO-66@DOPA-LB in the tumor tissue. Benefited from these meritorious features, UiO-66@DOPA-LB labeled with near-infrared dye, IRDye 800CW, can not only achieve highly sensitive imaging of breast cancer tumor (5 mm), but also exhibits superior capability for early tumor (1-2 mm) detection. This study enriches the surface modification approach of NMOFs, and is of great importance for practical application of NMOFs in biomedical areas., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ultrasound (US) is a widely used diagnostic imaging tool because it is inexpensive, safe, portable, and broadly accessible. Ultrasound contrast agents (UCAs) are employed to enhance backscatter echo and improve imaging contrast. The most frequently utilized UCAs are echogenic bubbles made with a phospholipid or protein-stabilized hydrophobic gas core. While clinically utilized, applications of UCAs are often limited by rapid signal decay (<5 min) in vivo under typical ultrasound imaging protocols. Here, we report on a formulation of lipid shell-stabilized perfluoropropane (C 3 F 8 ) microbubbles and nanobubbles with a significantly prolonged in vivo stability. Microbubbles (875 ± 280 nm) of the target size were prepared by utilizing a multiple-step centrifugation cycle, while nanobubbles (299 ± 189 nm) were isolated from the activated vial using a single centrifugation step. To provide in-depth acoustic characterization of the new construct we evaluated the effect of size and concentration on their in vitro and in vivo performance. In vitro and in vivo characterization were carried out for a range of bubble concentrations normalized by total gas volume quantified via headspace gas chromatography/mass spectrometry (GC/MS). In vitro characterization revealed that nanobubbles at different concentrations are more consistently stable over time with the highest and lowest dilutions (50-fold decrease) only differing in US signal after 8 min exposure by 10.34%, while for microbubbles the difference was 86.46%. As expected, due to the difference in hydrodynamic diameter and scattering cross section difference, nanobubbles showed lower overall initial signal intensity. In vivo experiments showed that both microbubbles and nanobubbles with similar initial peak signal intensity are comparably stable over time with 66.8% and 60.6% remaining signal after 30 min, respectively. This study demonstrates that bubble concentration has significant effects on the persistence of both microbubbles and nanobubbles in vitro and in vivo, but the effects are more pronounced in larger bubbles. These effects should be taken into account when selecting the appropriate bubble parameters for future imaging applications.

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process.

Using Arabidopsis thaliana wild type (WT) plants and diacylglycerol kinase knockouts (single mutants - dgk3, dgk1, dgk6; double mutants - dgk3dgk7, dgk5dgk6, dgk1dgk2) we observed that the inhibitor of brassinosteroid (BR) biosynthesis, brassinazole (BRZ), drastically decreased germination of dgk mutants under salt stress, while BRZ co-administration with 24-epibrassinolide (EBL) partially improved germination rates. We also observed a statistically significant decrease in alternative and cytochrome respiratory pathways in response to BRZ treatment under salinity conditions. We showed that production of the lipid second messenger phosphatidic acid (PA) is impaired in dgk mutants in response to EBL treatment and inhibitor of diacylglycerol kinase (DGK) - R59022. This study demonstrates that dgk mutants possess lower germination rates, lower total respiration rates, an alternative respiratory pathway and PA content under optimal and high salinity conditions in response to EBL treatment comparing to WT plants., (Copyright © 2019. Published by Elsevier Inc.)