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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 -
Proceedings of the National Academy of... Mar 2017Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can...
Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity. Based on lipid structural specificity and kinetic mechanisms, specific interactions of both PS and PC/PE have been inferred. Nevertheless, specific binding sites have not been identified definitively. We address this question with native mass spectrometry (MS) and site-directed mutagenesis. Native MS shows directly that one molecule each of 18:0/18:1 PS and 18:0/20:4 PC can bind specifically to purified human Na,K-ATPase (αβ). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic αL8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), αTM2 and αTM4, abolish the stimulation of activity by 18:0/20:4 PC but do not affect stability. When these data are linked to crystal structures, the underlying mechanism of PS and PC/PE effects emerges. PS (and cholesterol) bind between αTM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the α subunit (site A). PC/PE binds between αTM2, 4, 6, and 9 and accelerates the rate-limiting EP-EP conformational transition (site B). We discuss the potential physiological implications.
Topics: Binding Sites; Enzyme Activation; Humans; Mass Spectrometry; Models, Molecular; Molecular Conformation; Phospholipids; Protein Binding; Protein Stability; Sodium-Potassium-Exchanging ATPase
PubMed: 28242691
DOI: 10.1073/pnas.1620799114 -
Biochimica Et Biophysica Acta.... Oct 2023Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as... (Review)
Review
Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.
Topics: Humans; Viral Envelope; Viral Envelope Proteins; Flavivirus; Zika Virus; Zika Virus Infection; Peptides; Dengue; Antiviral Agents; Phospholipids
PubMed: 37437754
DOI: 10.1016/j.bbamem.2023.184198 -
Journal of Lipid Research Feb 2024Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of...
Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of phospholipid oxidation play a key role in atherosclerosis. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) is a phospholipid oxidation product present in atherosclerotic lesions. It remains unclear whether PGPC causes atherosclerosis by inducing endothelial cell ferroptosis. In this study, human umbilical vein endothelial cells (HUVECs) were treated with PGPC. Intracellular levels of ferrous iron, lipid peroxidation, superoxide anions (O), and glutathione were detected, and expression of fatty acid binding protein-3 (FABP3), glutathione peroxidase 4 (GPX4), and CD36 were measured. Additionally, the mitochondrial membrane potential (MMP) was determined. Aortas from C57BL6 mice were isolated for vasodilation testing. Results showed that PGPC increased ferrous iron levels, the production of lipid peroxidation and O, and FABP3 expression. However, PGPC inhibited the expression of GPX4 and glutathione production and destroyed normal MMP. These effects were also blocked by ferrostatin-1, an inhibitor of ferroptosis. FABP3 silencing significantly reversed the effect of PGPC. Furthermore, PGPC stimulated CD36 expression. Conversely, CD36 silencing reversed the effects of PGPC, including PGPC-induced FABP3 expression. Importantly, E06, a direct inhibitor of the oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine IgM natural antibody, inhibited the effects of PGPC. Finally, PGPC impaired endothelium-dependent vasodilation, ferrostatin-1 or FABP3 inhibitors inhibited this impairment. Our data demonstrate that PGPC impairs endothelial function by inducing endothelial cell ferroptosis through the CD36 receptor to increase FABP3 expression. Our findings provide new insights into the mechanisms of atherosclerosis and a therapeutic target for atherosclerosis.
Topics: Animals; Mice; Humans; Phospholipids; Phosphorylcholine; Phospholipid Ethers; Ferroptosis; Mice, Inbred C57BL; Atherosclerosis; Human Umbilical Vein Endothelial Cells; Endothelium; Glutathione; Iron; Fatty Acid Binding Protein 3; Cyclohexylamines; Phenylenediamines
PubMed: 38218337
DOI: 10.1016/j.jlr.2024.100499 -
The Journal of Biological Chemistry May 2023N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and...
N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and phosphatidylethanolamine, plays a crucial role in the visual cycle and visual pigment photoregeneration. However, N-Ret-PE can react with another molecule of retinal to form toxic di-retinoids if not removed from photoreceptors through its transport across photoreceptor membranes by the ATP-binding-cassette transporter ABCA4. Loss-of-function mutations in ABCA4 are known to cause Stargardt disease (STGD1), an inherited retinal degenerative disease associated with the accumulation of fluorescent di-retinoids and severe loss in vision. A larger assessment of retinal-phospholipid Schiff-base conjugates in photoreceptors is needed, along with further investigation of ABCA4 residues important for N-Ret-PE binding. In this study we show that N-Ret-PE formation is dependent on pH and phospholipid content. When retinal is added to liposomes or photoreceptor membranes, 40 to 60% is converted to N-Ret-PE at physiological pH. Phosphatidylserine and taurine also react with retinal to form N-retinylidene-phosphatidylserine and N-retinylidene-taurine, respectively, but at significantly lower levels. N-retinylidene-phosphatidylserine is not a substrate for ABCA4 and reacts poorly with retinal to form di-retinoids. Additionally, amino acid residues within the binding pocket of ABCA4 that contribute to its interaction with N-Ret-PE were identified and characterized using site-directed mutagenesis together with functional and binding assays. Substitution of arginine residues and hydrophobic residues with alanine or residues implicated in STGD1 significantly reduced or eliminated substrate-activated ATPase activity and substrate binding. Collectively, this study provides important insight into conditions which affect retinal-phospholipid Schiff-base formation and mechanisms underlying the pathogenesis of STGD1.
Topics: Humans; ATP-Binding Cassette Transporters; Phosphatidylserines; Phospholipids; Retinoids; Stargardt Disease
PubMed: 36931393
DOI: 10.1016/j.jbc.2023.104614 -
Nature Communications Aug 2023Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels...
Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. Arabidopsis thaliana CLCa (AtCLCa) transporter localizes to the tonoplast which imports NO and to a less extent Cl from cytoplasm. The activity of AtCLCa and many other CLCs is regulated by nucleotides and phospholipids, however, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with NO and Cl, respectively. Both structures are captured in ATP and PI(4,5)P bound conformation. Structural and electrophysiological analyses reveal a previously unidentified N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ- phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance. Our data further show that PI(4,5)P or PI(3,5)P binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios and provides new insights for future study of CLCs.
Topics: Arabidopsis; Nucleotides; Protons; Nitrates; Phospholipids; Arabidopsis Proteins; Anions; Adenosine Triphosphate; Chloride Channels
PubMed: 37573431
DOI: 10.1038/s41467-023-40624-z -
Biochimica Et Biophysica Acta.... Oct 2020The plasma membrane phospholipid distribution of animal cells is markedly asymmetric. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are concentrated in the... (Review)
Review
The plasma membrane phospholipid distribution of animal cells is markedly asymmetric. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are concentrated in the inner leaflet, whereas phosphatidylcholine (PC) and sphingomyelin (SM) are concentrated in the outer leaflet. This non-equilibrium situation is maintained by lipid pumps (flippases or floppases), which utilize energy in the form of ATP to translocate lipids from one leaflet to the other. Scramblases, which are activated when physiologically required, transport lipids in both directions across the membrane and can abolish lipid asymmetry. Lipid asymmetry also causes imbalances in the areas occupied by lipid in the two membrane leaflets, contributing to membrane curvature. The asymmetry of PS across the plasma membrane plays a crucial signalling role in numerous physiological processes. Exposure of PS on the external surface of blood platelets stimulates blood coagulation. PS exposure by other cells during apoptosis provides an "eat me" signal to surrounding macrophages. Many peripheral and integral membrane proteins have polybasic PS-binding domains on their cytoplasmic surfaces which either provide a membrane anchor or affect activity. These domains can also determine trafficking within the cell and control regulation via an electrostatic switch mechanism, as well as potentially acting as "death sensors" when cytoplasmic PS is transferred to the extracellular leaflet during apoptosis. Apart from these physiological roles, external PS exposure by microorganisms, viruses and cancer cells allows them to mimic the immunosuppressive anti-inflammatory action of apoptotic cells and proliferate, thus providing a strong medical motivation for future research in the field of lipid asymmetry in membranes.
Topics: Animals; Cell Membrane; Humans; Phospholipids; Signal Transduction
PubMed: 32511979
DOI: 10.1016/j.bbamem.2020.183382 -
Cell Research Dec 2020In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the...
In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the unique dual-membrane barrier to exclude the entry of most antibiotics. Understanding the mechanisms of phospholipid translocation between the inner and outer membrane represents one of the major challenges surrounding bacterial phospholipid homeostasis. The conserved MlaFEDB complex in the inner membrane functions as an ABC transporter to drive the translocation of phospholipids between the inner membrane and the periplasmic protein MlaC. However, the mechanism of phospholipid translocation remains elusive. Here we determined three cryo-EM structures of MlaFEDB from Escherichia coli in its nucleotide-free and ATP-bound conformations, and performed extensive functional studies to verify and extend our findings from structural analyses. Our work reveals unique structural features of the entire MlaFEDB complex, six well-resolved phospholipids in three distinct cavities, and large-scale conformational changes upon ATP binding. Together, these findings define the cycle of structural rearrangement of MlaFEDB in action, and suggest that MlaFEDB uses an extrusion mechanism to extract and release phospholipids through the central translocation cavity.
Topics: Adenosine Triphosphate; Cryoelectron Microscopy; Escherichia coli; Escherichia coli Proteins; Models, Biological; Models, Molecular; Phospholipids; Protein Binding; Protein Structure, Secondary; Protein Subunits
PubMed: 32884137
DOI: 10.1038/s41422-020-00404-6 -
Proceedings of the National Academy of... Feb 2022A high extracellular adenosine triphosphate (ATP) concentration rapidly and reversibly exposes phosphatidylserine (PtdSer) in T cells by binding to the P2X7 receptor,...
A high extracellular adenosine triphosphate (ATP) concentration rapidly and reversibly exposes phosphatidylserine (PtdSer) in T cells by binding to the P2X7 receptor, which ultimately leads to necrosis. Using mouse T cell transformants expressing P2X7, we herein performed CRISPR/Cas9 screening for the molecules responsible for P2X7-mediated PtdSer exposure. In addition to Eros, which is required for the localization of P2X7 to the plasma membrane, this screening identified Xk and Vps13a as essential components for this process. Xk is present at the plasma membrane, and its paralogue, Xkr8, functions as a phospholipid scramblase. Vps13a is a lipid transporter in the cytoplasm. Blue-native polyacrylamide gel electrophoresis indicated that Xk and Vps13a interacted at the membrane. A null mutation in or blocked P2X7-mediated PtdSer exposure, the internalization of phosphatidylcholine, and cytolysis. Xk and Vps13a formed a complex in mouse splenic T cells, and Xk was crucial for ATP-induced PtdSer exposure and cytolysis in CD25CD4 T cells. and are responsible for McLeod syndrome and chorea-acanthocytosis, both characterized by a progressive movement disorder and cognitive and behavior changes. Our results suggest that the phospholipid scrambling activity mediated by XK and VPS13A is essential for maintaining homeostasis in the immune and nerve systems.
Topics: Adenosine Triphosphate; Amino Acid Transport Systems, Neutral; Animals; CRISPR-Cas Systems; Cell Death; Cell Line; Gene Deletion; Gene Expression Regulation; Genome-Wide Association Study; HEK293 Cells; Humans; Mice; Mice, Transgenic; Mutation; Phosphatidylserines; Phospholipids; Receptors, Purinergic P2X7; T-Lymphocytes; Vesicular Transport Proteins
PubMed: 35140185
DOI: 10.1073/pnas.2119286119 -
Biochimica Et Biophysica Acta.... Oct 2022Over the past decades an extensive effort has been made to provide a more comprehensive understanding of Wnt signaling, yet many regulatory and structural aspects remain...
Over the past decades an extensive effort has been made to provide a more comprehensive understanding of Wnt signaling, yet many regulatory and structural aspects remain elusive. Among these, the ability of Dishevelled (DVL) protein to relocalize at the plasma membrane is a crucial step in the activation of all Wnt pathways. The membrane binding of DVL was suggested to be mediated by the preferential interaction of its C-terminal DEP domain with phosphatidic acid (PA). However, due to the scarcity and fast turnover of PA, we investigated the role on the membrane association of other more abundant phospholipids. The combined results from computational simulations and experimental measurements with various model phospholipid membranes, demonstrate that the membrane binding of DEP/DVL constructs is governed by the concerted action of generic electrostatics and finely-tuned intermolecular interactions with individual lipid species. In particular, while we confirmed the strong preference for PA lipid, we also observed a weak but non-negligible affinity for phosphatidylserine, the most abundant anionic phospholipid in the plasma membrane, and phosphatidylinositol 4,5-bisphosphate. The obtained molecular insight into DEP-membrane interaction helps to elucidate the relation between changes in the local membrane composition and the spatiotemporal localization of DVL and, possibly, other DEP-containing proteins.
Topics: Cell Membrane; Dishevelled Proteins; Phosphatidic Acids; Proteins; Static Electricity
PubMed: 35750206
DOI: 10.1016/j.bbamem.2022.183983