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International Journal of Molecular... Mar 2021The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for...
The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Dimyristoylphosphatidylcholine; Hydrogen Bonding; Lipid Bilayers; Melatonin; Molecular Dynamics Simulation; Serotonin; Solutions; Static Electricity; Thermodynamics; Tryptophan; Water
PubMed: 33799606
DOI: 10.3390/ijms22062842 -
EMBO Reports Oct 2021Phospholipase D (PLD) hydrolyzes membrane lipids to produce phosphatidic acid (PA), a lipid mediator involved in various cellular and physiological processes. Here, we...
Phospholipase D (PLD) hydrolyzes membrane lipids to produce phosphatidic acid (PA), a lipid mediator involved in various cellular and physiological processes. Here, we show that PLDα6 and PA regulate the distribution of GIBBERELLIN (GA)-INSENSITIVE DWARF1 (GID1), a soluble gibberellin receptor in rice. PLDα6-knockout (KO) plants display less sensitivity to GA than WT, and PA restores the mutant to a normal GA response. PA binds to GID1, as documented by liposome binding, fat immunoblotting, and surface plasmon resonance. Arginines 79 and 82 of GID1 are two key amino acid residues required for PA binding and also for GID1's nuclear localization. The loss of PLDα6 impedes GA-induced nuclear localization of GID1. In addition, PLDα6-KO plants attenuated GA-induced degradation of the DELLA protein SLENDER RICE1 (SLR1). These data suggest that PLDα6 and PA positively mediate GA signaling in rice via PA binding to GID1 and promotion of its nuclear translocation.
Topics: Gene Expression Regulation, Plant; Gibberellins; Mutation; Oryza; Phosphatidic Acids; Phospholipases; Plant Growth Regulators; Plant Proteins; Plants, Genetically Modified
PubMed: 34396669
DOI: 10.15252/embr.202051871 -
Pharmacological Research Apr 2023The nicotinic acetylcholine receptor (nAChR) is a transmembrane protein that mediates fast intercellular communication in response to the endogenous neurotransmitter... (Review)
Review
The nicotinic acetylcholine receptor (nAChR) is a transmembrane protein that mediates fast intercellular communication in response to the endogenous neurotransmitter acetylcholine. It is the best characterized and archetypal molecule in the superfamily of pentameric ligand-gated ion channels (pLGICs). As a typical transmembrane macromolecule, it interacts extensively with its vicinal lipid microenvironment. Experimental evidence provides a wealth of information on receptor-lipid crosstalk: the nAChR exerts influence on its immediate membrane environment and conversely, the lipid moiety modulates ligand binding, affinity state transitions and gating of ion translocation functions of the receptor protein. Recent cryogenic electron microscopy (cryo-EM) studies have unveiled the occurrence of sites for phospholipids and cholesterol on the lipid-exposed regions of neuronal and electroplax nAChRs, confirming early spectroscopic and affinity labeling studies demonstrating the close contact of lipid molecules with the receptor transmembrane segments. This new data provides structural support to the postulated "lipid sensor" ability displayed by the outer ring of M4 transmembrane domains and their modulatory role on nAChR function, as we postulated a decade ago. Borrowing from the best characterized nAChR, the electroplax (muscle-type) receptor, and exploiting new structural information on the neuronal nAChR, it is now possible to achieve an improved depiction of these sites. In combination with site-directed mutagenesis, single-channel electrophysiology, and molecular dynamics studies, the new structural information delivers a more comprehensive portrayal of these lipid-sensitive loci, providing mechanistic explanations for their ability to modulate nAChR properties and raising the possibility of targetting them in disease.
Topics: Receptors, Nicotinic; Cell Membrane; Phospholipids; Cholesterol; Synaptic Transmission
PubMed: 36931540
DOI: 10.1016/j.phrs.2023.106729 -
The Journal of Biological Chemistry Aug 2020TMEM16 Ca-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and...
TMEM16 Ca-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.
Topics: Animals; Anoctamins; Catechin; Enzyme Inhibitors; HEK293 Cells; Humans; Mice; Phospholipid Transfer Proteins; Phospholipids; Polyphenols; Tannins
PubMed: 32709749
DOI: 10.1074/jbc.AC120.014872 -
Experimental Eye Research Jan 2021It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane...
It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane of the eye lens. The fiber cell plasma membrane of the eye lens consists of four major phospholipids (PLs), i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). Despite several attempts to study the interaction of α-crystallin with PLs of the eye lens membrane, the role of individual PL for the binding with α-crystallin is still unclear. We recently developed the electron paramagnetic resonance (EPR) spin-labeling method to study the binding of α-crystallin to the PC membrane (Mainali et al., 2020a). Here, we use the recently developed EPR method to explicitly measure the binding affinity (K) of α-crystallin to the individual (PE*, PS, and SM) and two-component mixtures (SM/PE, SM/PS, and SM/PC in 70:30 and 50:50 mol%) of PL membranes as well as the physical properties (mobility parameter and maximum splitting) of these membranes upon binding with α-crystallin. One of the key findings of this study was that the K of α-crystallin binding to individual PL membranes followed the trends: K(PC) > K(SM) > K(PS) > K(PE*), indicating PE* inhibits binding the most whereas PC inhibits binding the least. Also, the K of α-crystallin binding to two-component mixtures of PL membranes followed the trends: K(SM/PE) > K(SM/PS) > K(SM/PC), indicating SM/PC inhibits binding the most whereas SM/PE inhibits binding the least. Except for the PE* membrane, for which there was no binding of α-crystallin, the mobility parameter for all other membranes decreased with an increase in α-crystallin concentration. It represents that the membranes become more immobilized near the headgroup regions of the PLs when more and more α-crystallin binds to them. The maximum splitting increased only for the SM and the SM/PE (70:30 mol%) membranes, with an increase in the binding of α-crystallin. It represents that the PL headgroup regions of these membranes become more ordered after binding of α-crystallin to these membranes. Our results showed that α-crystallin binds to PL membranes in a saturable manner. Also, our data suggest that the binding of α-crystallin to PL membranes likely occurs through hydrophobic interaction between α-crystallin and the hydrophobic fatty acid core of the membranes, and such interaction is modulated by the PL headgroup's size and charge, hydrogen bonding between headgroups, and PL curvature. Thus, this study provides an in-depth understanding of α-crystallin interaction with the PL membranes made of individual and two-component mixtures of the four major PLs of the eye lens membranes.
Topics: Cell Membrane; Electron Spin Resonance Spectroscopy; Humans; Lens, Crystalline; Phospholipids; Protein Binding; alpha-Crystallins
PubMed: 33127344
DOI: 10.1016/j.exer.2020.108337 -
PloS One 2021The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of...
The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of the cell membrane, but PS can be flipped to the outer surface in a process called PS exposure. PS exposure is important in many cell functions, yet the mechanisms that control PS exposure have not been extensively studied. Copines (Cpn), found in most eukaryotic organisms, make up a family of calcium-dependent phospholipid binding proteins. In Dictyostelium, which has six copine genes, CpnA strongly binds to PS and translocates from the cytosol to the plasma membrane in response to a rise in calcium. Cells lacking the cpnA gene (cpnA-) have defects in adhesion, chemotaxis, membrane trafficking, and cytokinesis. In this study we used both flow cytometry and fluorescent microscopy to show that cpnA- cells have increased adhesion to beads and bacteria and that the increased adhesion was not due to changes in the actin cytoskeleton or cell surface proteins. We found that cpnA- cells bound higher amounts of Annexin V, a PS binding protein, than parental cells and showed that unlabeled Annexin V reduced the increased cell adhesion property of cpnA- cells. We also found that cpnA- cells were more sensitive to Polybia-MP1, which binds to external PS and induces cell lysis. Overall, this suggests that cpnA- cells have increased PS exposure and this property contributes to the increased cell adhesion of cpnA- cells. We conclude that CpnA has a role in the regulation of plasma membrane lipid composition and may act as a negative regulator of PS exposure.
Topics: Cell Adhesion; Cell Membrane; Dictyostelium; Mutation; Phosphatidylserines; Protozoan Proteins
PubMed: 34043641
DOI: 10.1371/journal.pone.0250710 -
Biochimica Et Biophysica Acta.... Jun 2020Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to...
Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways.
Topics: Immunoglobulin E; Lipid Metabolism; Models, Molecular; Molecular Structure; Olea; Oleic Acid; Phospholipids; Plant Proteins; Protein Binding
PubMed: 32142819
DOI: 10.1016/j.bbamem.2020.183258 -
The Journal of Physical Chemistry. B Jul 2019In this study, we provide a quantitative description of the adsorption of water-soluble -substituted glycine oligomers (peptoids) to supported lipid bilayers that mimic...
In this study, we provide a quantitative description of the adsorption of water-soluble -substituted glycine oligomers (peptoids) to supported lipid bilayers that mimic mammalian plasma membranes. We prepared a small array of systematically varied peptoid sequences ranging in length from 3 to 15 residues. Using the nonlinear optical method second harmonic generation (SHG), we directly monitored adsorption of aqueous solutions of 3- and 15-residue peptoids to phospholipid membranes of varying physical phase, cholesterol content, and head group charge in physiologically relevant pH buffer conditions without the use of extrinsic labels. Equilibrium binding constants and relative surface coverages of adsorbed peptoids were determined from fits to the Langmuir model. Three- and 15-residue peptoids did not interact with cholesterol-containing lipids or charged lipids in the same manner, suggesting that a peptoid's adsorption mechanism changes with sequence length. In a comparison of four three-residue peptoids, we observed a correlation between equilibrium binding constants and calculated log values. Cationic charge modulated surface coverage. Principles governing how peptoid sequence and membrane composition alter peptoid-lipid interactions may be extended to predict physiological effects of peptoids used as therapeutics or as coatings in medical devices.
Topics: Binding Sites; Hydrogen-Ion Concentration; Molecular Structure; Peptoids; Phospholipids; Solubility; Water
PubMed: 31251622
DOI: 10.1021/acs.jpcb.9b04641 -
Langmuir : the ACS Journal of Surfaces... May 2022Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and...
Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine's influence on the cell membrane, including permeability, binding, and rearrangement at the molecular level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid composition on the membrane interactions of quinacrine. Using electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Our EIS data reveal that the drug is permeable through ternary DOPC:SM:Chol and DOPC-only bilayer compositions. In contrast, the binary cholesterol/DOPC membrane arrested permeation, yet the drug binds or intercalates at this membrane as reflected by an increase in membrane impedance. SERS supported the EIS data, which was utilized to gain structural insights into the drug-membrane interaction. Our SERS data also provides a simple but powerful label-free assessment of drug permeation because a significant SERS enhancement of the drug's Raman signature was observed only if the drug accessed the plasmonic interior of the pore cavity passing through the membrane. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophysical insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug-membrane interactions, making it a powerful tool for drug screening.
Topics: Cell Membrane; Cholesterol; Dielectric Spectroscopy; Lipid Bilayers; Phosphatidylcholines; Quinacrine
PubMed: 35561255
DOI: 10.1021/acs.langmuir.2c00524 -
Biochemical Society Transactions Oct 2019The phosphoinositide (PIPn) family of signalling phospholipids are central regulators in membrane cell biology. Their varied functions are based on the phosphorylation... (Review)
Review
The phosphoinositide (PIPn) family of signalling phospholipids are central regulators in membrane cell biology. Their varied functions are based on the phosphorylation pattern of their inositol ring, which can be recognized by selective binding domains in their effector proteins and be modified by a series of specific PIPn kinases and phosphatases, which control their interconversion in a spatial and temporal manner. Yet, a unique feature of PIPns remains largely unexplored: their unusually uniform acyl chain composition. Indeed, while most phospholipids present a range of molecular species comprising acyl chains of diverse length and saturation, PIPns in several organisms and tissues show the predominance of a single hydrophobic backbone, which in mammals is composed of arachidonoyl and stearoyl chains. Despite evolution having favoured this specific PIPn configuration, little is known regarding the mechanisms and functions behind it. In this review, we explore the metabolic pathways that could control the acyl chain composition of PIPns as well as the potential roles of this selective enrichment. While our understanding of this phenomenon has been constrained largely by the technical limitations in the methods traditionally employed in the PIPn field, we believe that the latest developments in PIPn analysis should shed light onto this old question.
Topics: 1-Phosphatidylinositol 4-Kinase; Animals; Binding Sites; Phosphatidylinositols; Signal Transduction
PubMed: 31657437
DOI: 10.1042/BST20190205