Extension of the Scope of Anionic Phospholipid-Based Nanoformulation to Kaempferol and Indometacin

In this work, resveratrol was dispersed with anionic phospholipids of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, and 1,2-distearoyl-sn-glycero-3-phosphoglycerol. Moreover, small-sized nanoparticles of kaempferol and indometacin were successfully prepared by using DPPG as a dispersion agent.

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Phosphatidic acid stimulates cardiac KATPchannels like phosphatidylinositols, but with novel gating kinetics

AJP Cell Physiology ◽

10.1152/ajpcell.00255.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. C94-C102 ◽

Cited By ~ 12

Author(s):

Zheng Fan ◽

Lizhi Gao ◽

Wenxia Wang

Keyword(s):

Phosphatidic Acid ◽

Electrostatic Interaction ◽

Common Mechanism ◽

Lower Sensitivity ◽

Anionic Phospholipid ◽

Gating Kinetics ◽

Anionic Phospholipids ◽

Specific Effects ◽

Open Time ◽

Atp Inhibition

Membrane-bound anionic phospholipids such as phosphatidylinositols have the capacity to modulate ATP-sensitive potassium (KATP) channels through a mechanism involving long-range electrostatic interaction between the lipid headgroup and channel. However, it has not yet been determined whether the multiple effects of phosphatidylinositols reported in the literature all result from this general electrostatic interaction or require a specific headgroup structure. The present study investigated whether phosphatidic acid (PA), an anionic phospholipid substantially different in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native KATP channels of rat heart and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2–1 mg/ml applied to the cytoplasmic side of the membrane) exhibited higher activity, lower sensitivity to ATP inhibition, less Mg2+-dependent nucleotide stimulation, and poor sulfonylurea inhibition. These effects match the spectrum of phosphatidylinositols' effects, but, in addition, PA also induced a novel pattern in gating kinetics, represented by a decreased mean open time (from 12.2 ± 2.0 to 3.3 ± 0.7 ms). This impact on gating kinetics clearly distinguishes PA's effects from those of phosphatidylinositols. Results indicate that multiple effects of anionic phospholipids on KATP channels are related phenomena and can likely be attributed to a common mechanism, but additional specific effects due to other mechanisms may also coincide.

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Size-Tunable Paclitaxel Nanoparticles Stabilized by Anionic Phospholipids for Transdermal Delivery Applications

Natural Product Communications ◽

10.1177/1934578x19900684 ◽

2020 ◽

Vol 15 (3) ◽

pp. 1934578X1990068

Author(s):

Noriyuki Uchida ◽

Masayoshi Yanagi ◽

Hiroki Hamada

Keyword(s):

Stratum Corneum ◽

Transdermal Delivery ◽

Skin Tissue ◽

Composite Nanoparticles ◽

Rat Skin ◽

Cooling Process ◽

Anionic Phospholipid ◽

Anionic Phospholipids ◽

Subsequent Heating

Composite nanoparticles composed of an anionic phospholipid of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG) and paclitaxel (PTX) were successfully prepared by mixing them in water followed by a subsequent heating/cooling process. The size of DPPG-PTX nanoparticle could be easily tuned by ultrasonic fragmentation. Upon addition of small-sized fluorescently labeled paclitaxel (FLPTX) nanoparticles with DPPG (DPPG-FLPTX) to rat skin tissue, part of the FLPTX molecules permeated to the stratum corneum.

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Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

The Journal of General Physiology ◽

10.1085/jgp.201611616 ◽

2016 ◽

Vol 148 (3) ◽

pp. 227-237 ◽

Cited By ~ 31

Author(s):

Sun-Joo Lee ◽

Feifei Ren ◽

Eva-Maria Zangerl-Plessl ◽

Sarah Heyman ◽

Anna Stary-Weinzinger ◽

...

Keyword(s):

Transmembrane Domain ◽

Membrane Lipids ◽

Inward Rectifier ◽

Primary Site ◽

Structural Basis ◽

Channel Activity ◽

Anionic Phospholipid ◽

X Ray ◽

Anionic Phospholipids ◽

Kir Channel

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL−) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL−. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL− binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL− binding and PIP2 sensitivity.

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The Electrostatic Basis of Diacylglycerol Pyrophosphate—Protein Interaction

Cells ◽

10.3390/cells11020290 ◽

2022 ◽

Vol 11 (2) ◽

pp. 290

Author(s):

Zachary Graber ◽

Desmond Owusu Kwarteng ◽

Shannon M. Lange ◽

Yannis Koukanas ◽

Hady Khalifa ◽

...

Keyword(s):

Phosphatidic Acid ◽

Protein Interaction ◽

Anionic Phospholipid ◽

Tryptophan Residues ◽

Switch Mechanism ◽

31P Mas Nmr ◽

Calcium And Magnesium ◽

Ionic Binding ◽

Novel Model ◽

Positively Charged

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP’s overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP’s charge. Our results lead to a novel model for DGPP—protein interaction.

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Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

Biochemical Journal ◽

10.1042/bcj20160792 ◽

2017 ◽

Vol 474 (3) ◽

pp. 385-398 ◽

Cited By ~ 14

Author(s):

Ryan P. Emptage ◽

Mark A. Lemmon ◽

Kathryn M. Ferguson

Keyword(s):

Membrane Surface ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Anionic Phospholipid ◽

Anionic Phospholipids ◽

Disease States ◽

C Terminus ◽

Regulatory Module ◽

Correct Location ◽

Mechanistic Basis

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

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Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

Journal of Biological Chemistry ◽

10.1074/jbc.m809600200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16099-16107 ◽

Cited By ~ 22

Author(s):

Mingming Pu ◽

Xiaomin Fang ◽

Alfred G. Redfield ◽

Anne Gershenson ◽

Mary F. Roberts

Keyword(s):

Enzyme Activity ◽

Phospholipase C ◽

Lateral Diffusion ◽

Lipid Vesicles ◽

Membrane Lipid ◽

Correlation Spectroscopy ◽

Anionic Phospholipid ◽

Anionic Phospholipids ◽

Substrate Analogue ◽

Nmr Methods

The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (τc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased τc. PI-PLC activity and phospholipid τc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC τc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.

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Ligand-regulated secretion of recombinant annexin V from cultured thyroid epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00553.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. C1313-C1321 ◽

Cited By ~ 7

Author(s):

Xiuqiong Wang ◽

Marcia A. Kaetzel ◽

Sung E. Yoo ◽

Paul S. Kim ◽

John R. Dedman

Keyword(s):

Mammalian Cells ◽

Secretory Pathway ◽

External Surface ◽

Annexin V ◽

Therapeutic Agents ◽

Anionic Phospholipid ◽

Thyroid Cells ◽

Tissue Ischemia ◽

Anionic Phospholipids ◽

Activated Platelets

The exposure of anionic phospholipids on the external surface of injured endothelial cells and activated platelets is a primary biological signal to initiate blood coagulation. Disease conditions that promote the formation of ectopic thrombi result in tissue ischemia. Annexins, Ca2+-dependent anionic phospholipid binding proteins, are potential therapeutic agents for the inhibition of coagulation. We have designed a transgene that targets secretion of annexin V from cultured thyroid cells under the control of doxycycline. Our results indicate that annexin V in the endoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis, processing, and secretion of thyroglobulin. ER luminal Ca2+was moderately increased and can be released by inositol 1,4,5-trisphosphate. Our study demonstrates that targeting and secretion of annexin V through the secretory pathway of mammalian cells does not adversely affect cellular function. Regulated synthesis and release of annexin V may exert anticoagulatory and anti-inflammatory effects systemically and may prove useful in further developing therapeutic strategies for conditions including antiphospholipid syndrome.

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Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells

Biochemical Journal ◽

10.1042/bj3141027 ◽

1996 ◽

Vol 314 (3) ◽

pp. 1027-1033 ◽

Cited By ~ 30

Author(s):

Karine AUPEIX ◽

Florence TOTI ◽

Nathalie SATTA ◽

Pierre BISCHOFF ◽

Jean-Marie FREYSSINET

Keyword(s):

Tissue Factor ◽

Annexin V ◽

Antigen Expression ◽

Resting Cells ◽

Dependent Manner ◽

Anionic Phospholipid ◽

Monocytic Cells ◽

Anionic Phospholipids ◽

Phosphatidylserine Exposure ◽

And Control

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7β-hydroxycholesterol (7β-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7β-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may result from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.

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A Complex Interplay of Anionic Phospholipid Binding Regulates 3′-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation

Scientific Reports ◽

10.1038/s41598-019-50742-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Gloria de las Heras-Martínez ◽

Véronique Calleja ◽

Remy Bailly ◽

Jean Dessolin ◽

Banafshé Larijani ◽

...

Keyword(s):

Conformational Dynamics ◽

Pi3k Pathway ◽

Correlation Spectroscopy ◽

Fluorescence Lifetime Imaging ◽

Anionic Phospholipid ◽

Direct Role ◽

Time Resolved ◽

Anionic Phospholipids ◽

Spatio Temporal ◽

Phosphoinositide Dependent Kinase

Abstract 3′-Phosphoinositide-dependent-Kinase-1 (PDK1) is a master regulator whereby its PI3-kinase-dependent dysregulation in human pathologies is well documented. Understanding the direct role for PtdIns(3,4,5)P3 and other anionic phospholipids in the regulation of PDK1 conformational dynamics and its downstream activation remains incomplete. Using advanced quantitative-time-resolved imaging (Fluorescence Lifetime Imaging and Fluorescence Correlation Spectroscopy) and molecular modelling, we show an interplay of antagonistic binding effects of PtdIns(3,4,5)P3 and other anionic phospholipids, regulating activated PDK1 homodimers. We demonstrate that phosphatidylserine maintains PDK1 in an inactive conformation. The dysregulation of the PI3K pathway affects the spatio-temporal and conformational dynamics of PDK1 and the activation of its downstream substrates. We have established a new anionic-phospholipid-dependent model for PDK1 regulation, depicting the conformational dynamics of multiple homodimer states. We show that the dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 homodimer conformations. Our findings provide a role for the PtdSer binding site and its previously unrewarding role in PDK1 downregulation, suggesting a possible therapeutic strategy where the constitutively active dimer conformer of PDK1 may be rendered inactive by small molecules that drive it to its PtdSer-bound conformer.

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Antiphospholipid Antibody Syndrome: The Flow Cytometric Annexin A5 Competition Assay as a Diagnostic Tool.

Blood ◽

10.1182/blood.v110.11.3981.3981 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3981-3981

Author(s):

Aaron Tomer ◽

Shira Bar-Lev ◽

Stela Fleisher ◽

Boris Shenkman ◽

Michael Friger ◽

...

Keyword(s):

Lupus Erythematosus ◽

Vascular Complications ◽

Relative Sensitivity ◽

Annexin A5 ◽

Antiphospholipid Antibody ◽

Antiphospholipid Antibody Syndrome ◽

Flow Cytometric Assay ◽

Anionic Phospholipid ◽

Anionic Phospholipids ◽

Flow Cytometric

Abstract The mechanism underlying hypercoagulability in antiphospholipid antibody syndrome (APS) is uncertain. Here, we present a flow-cytometric assay (FCA) based on the hypothesis that anti-platelet-anionic-phospholipid autoantibodies (aPL) interfere with the activity of the natural anticoagulant protein annexin A5, thereby accelerating platelet procoagulant activity. This study assessed the clinical utility of the feasible FCA, which demonstrates the competition of the patient’s aPL with the binding of annexin A5 to the platelet-anionic-phospholipids, in the diagnosis of APS. Sixty-two (94%) of 66 APS patients, 20 (51%) of 39 patients with systemic lupus erythematosus and two (4%) of 49 healthy individuals were positive by FCA. Compared with the anticardiolipin (aCL) assay, the relative sensitivity was 82% and the specificity 73∴3%. However, 19 (25%) aCL-negative patients were positive by FCA; 12 were positive for lupus-anticoagulant (LA). Compared with LA assay, the relative sensitivity was 85% and the specificity 72∴2%. However, 21 (26%) LA-negative patients were FCA-positive, 12 were positive for aCL. The FCA was particularly sensitive for APS patients with arterial (97∴0%) and gestational vascular complications (100%) with overall sensitivity of 95% and specificity of 97%. Our findings suggest that the FCA is practical, sensitive and specific for the detection of clinically relevant aPL in the diagnosis of APS.

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