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Virus Research Nov 2014The MA domain mediates plasma membrane (PM) targeting of HIV-1 Gag, leading to particle assembly at the PM. The interaction between MA and acidic phospholipids, in... (Review)
Review
The MA domain mediates plasma membrane (PM) targeting of HIV-1 Gag, leading to particle assembly at the PM. The interaction between MA and acidic phospholipids, in addition to N-terminal myristoyl moiety, promotes Gag binding to lipid membranes. Among acidic phospholipids, PI(4,5)P2, a PM-specific phosphoinositide, is essential for proper HIV-1 Gag localization to the PM and efficient virus particle production. Recent studies further revealed that MA-bound RNA negatively regulates HIV-1 Gag membrane binding and that PI(4,5)P2 is necessary to overcome this RNA-imposed block. In this review, we will summarize the current understanding of Gag-membrane interactions and discuss potential roles played by acidic phospholipids.
Topics: Cell Membrane; HIV Antigens; HIV-1; Humans; Lipid Metabolism; Lipids; Membrane Microdomains; Phosphatidylinositols; Phospholipids; Protein Binding; Protein Interaction Domains and Motifs; RNA, Viral; gag Gene Products, Human Immunodeficiency Virus
PubMed: 24998886
DOI: 10.1016/j.virusres.2014.06.015 -
Journal of Thrombosis and Haemostasis :... Mar 2022Cellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the...
BACKGROUND
Cellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the plasma membrane, thereby allowing binding of many blood clotting proteins. We previously proposed the Anything But Choline (ABC) hypothesis to explain how PS and PE synergize to support binding of clotting proteins with gamma-carboxyglutamate (Gla)-rich domains, which posited that each Gla domain binds to a limited number of PS molecules and multiple PE molecules. However, the minimal number of PS molecules required to stably bind a Gla-domain-containing blood clotting protein in the presence of excess PE was unknown.
OBJECTIVE
To test the ABC hypothesis for factor X by determining the threshold binding requirement of PS molecules under conditions of PS-PE synergy.
METHODS
We used surface plasmon resonance to investigate the stoichiometry of factor X binding to nanoscale membrane bilayers (Nanodiscs) of varying phospholipid composition.
RESULTS AND CONCLUSIONS
We quantified 1.05 ± 0.2 PS molecules per bound factor X molecule in Nanodiscs containing a mixture of 10% PS, 60% PE, and 30% phosphatidylcholine. Hence, there appears to be one truly PS-specific binding site per Gla domain, while the remaining membrane binding interactions can be satisfied by PE.
Topics: Binding Sites; Cell Membrane; Factor X; Humans; Phosphatidylserines; Phospholipids; Protein Binding
PubMed: 34894064
DOI: 10.1111/jth.15620 -
Cellular Physiology and Biochemistry :... 2018The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at...
BACKGROUND/AIMS
The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at least partially effective by triggering suicidal tumor cell death. Side effects of afatinib treatment include anemia. At least in theory, afatinib induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, and increase of ceramide abundance. The present study explored, whether afatinib induces eryptosis and, if so, whether its effect involves Ca2+ entry, oxidative stress, and/or ceramide.
METHODS
Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies.
RESULTS
A 48 hours exposure of human erythrocytes to afatinib (≥ 4 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Afatinib significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance. The effect of afatinib on annexin-V-binding and forward scatter was significantly blunted by removal of extracellular Ca2+.
CONCLUSIONS
Afatinib triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, and ceramide.
Topics: Afatinib; Anemia; Calcium Signaling; Eryptosis; Erythrocyte Membrane; Humans; Phospholipids; Quinazolines
PubMed: 29913444
DOI: 10.1159/000490221 -
Scientific Reports Mar 2017Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of...
Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of polymerizable bis-imidazolium and urea hosts were evaluated as cationic and neutral hosts for phosphates and phosphonates, the latter used as mimics of the phospholipid head groups. The bis-imidazolium hosts interacted with the guests in a cooperative manner leading to the presence of tight and well defined 1:2 ternary complexes. Optimized monomer combinations were subsequently used for imprinting of phosphatidic acid as an endotoxin dummy template. Presence of the aforementioned ternary complexes during polymerization resulted in imprinting of lipid dimers - the latter believed to crudely mimic the endotoxin Lipid A motif. The polymers were characterized with respect to template rebinding, binding affinity, capacity and common structural properties, leading to the identification of polymers which were thereafter subjected to an industrially validated endotoxin removal test. Two of the polymers were capable of removing endotoxin down to levels well below the accepted threshold (0.005 EU/mg API) in pharmaceutical production.
Topics: Cross-Linking Reagents; Drug Contamination; Endotoxins; Imidazoles; Methacrylates; Molecular Imprinting; Molecular Mimicry; Organophosphonates; Pharmaceutical Preparations; Phosphates; Phospholipids; Polymerization; Urea
PubMed: 28303896
DOI: 10.1038/srep44299 -
Methods in Molecular Biology (Clifton,... 2017Releasing sterols to the extracellular milieu is an important part of sterol homeostasis in cells and in the body. ATP-binding cassette transporter A1 (ABCA1) plays an...
Releasing sterols to the extracellular milieu is an important part of sterol homeostasis in cells and in the body. ATP-binding cassette transporter A1 (ABCA1) plays an essential role in cellular phospholipid and sterol release to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), the major apolipoprotein in high-density lipoprotein (HDL), and constitutes the first step in the formation of nascent HDL. Loss-of-function mutations in the ABCA1 gene lead to a rare disease known as Tangier disease that causes severe deficiency in plasma HDL level. Mammalian cells receive exogenous cholesterol mainly from low-density lipoprotein. In addition, they synthesize cholesterol endogenously, as well as multiple precursor sterols that are sterol intermediates en route to be converted to cholesterol. HDL contains phospholipids, cholesterol, and precursor sterols, and ABCA1 has an ability to release phospholipids and various sterol molecules. Recent studies using model cell lines showed that ABCA1 prefers to use sterols newly synthesized endogenously as its preferred substrate, rather than cholesterol derived from LDL or cholesterol being recycled within the cells. Here, we describe several methods at the cell culture level to monitor ABCA1-dependent release of sterol molecules to apoA-I present at the cell exterior. Sterol release can be assessed by using a simple colorimetric enzymatic assay, and/or by monitoring the radioactivities of radiolabeled cholesterol incorporated into the cells, and/or of sterols biosynthesized from radioactive acetate, and/or by using gas chromatography-mass spectrometry analysis of various sterols present in medium and in cells. We also discuss the pros and cons of these methods. Together, these methods allow researchers to detect the release not only of cholesterol but also of other sterols present in minor quantities.
Topics: ATP Binding Cassette Transporter 1; Animals; Apolipoprotein A-I; Cell Line; Cholesterol; Humans; Phospholipids; Tangier Disease
PubMed: 28205180
DOI: 10.1007/978-1-4939-6875-6_19 -
Cellular Physiology and Biochemistry :... 2017The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal...
BACKGROUND/AIMS
The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca2+ entry and/or oxidative stress.
METHODS
Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence.
RESULTS
A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Gefitinib further significantly increased DCFDA fluorescence.
CONCLUSIONS
Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca2+ and paralleled by oxidative stress.
Topics: Calcium; Calcium Signaling; Cell Death; Erythrocyte Membrane; Female; Gefitinib; Humans; Male; Oxidative Stress; Phospholipids; Quinazolines; Reactive Oxygen Species
PubMed: 28359067
DOI: 10.1159/000471823 -
Biochimica Et Biophysica Acta.... Mar 2018Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKVPPTKVKVKVK), an engineered AMP and...
Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKVPPTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a "flip and dip" mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.
Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Circular Dichroism; Lipid Bilayers; Liposomes; Membrane Lipids; Membrane Proteins; Models, Molecular; Molecular Dynamics Simulation; Phospholipids; Protein Folding; Protein Structure, Secondary; Spectrometry, Fluorescence; Static Electricity
PubMed: 29291379
DOI: 10.1016/j.bbamem.2017.12.019 -
The Journal of Biological Chemistry Mar 2020Eukaryotic cells are compartmentalized to form organelles, whose functions rely on proper phospholipid and protein transport. Here we determined the crystal structure of...
Eukaryotic cells are compartmentalized to form organelles, whose functions rely on proper phospholipid and protein transport. Here we determined the crystal structure of human VAT-1, a cytosolic soluble protein that was suggested to transfer phosphatidylserine, at 2.2 Å resolution. We found that VAT-1 transferred not only phosphatidylserine but also other acidic phospholipids between membranes Structure-based mutational analyses showed the presence of a possible lipid-binding cavity at the interface between the two subdomains, and two tyrosine residues in the flexible loops facilitated phospholipid transfer, likely by functioning as a gate to this lipid-binding cavity. We also found that a basic and hydrophobic loop with two tryptophan residues protruded from the molecule and facilitated binding to the acidic-lipid membranes, thereby achieving efficient phospholipid transfer.
Topics: Binding Sites; Biological Transport; Crystallography, X-Ray; Humans; Liposomes; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Phosphatidylserines; Phospholipids; Protein Domains; Protein Structure, Tertiary; Tryptophan; Vesicular Transport Proteins
PubMed: 32005660
DOI: 10.1074/jbc.RA119.011019 -
Molecular Pharmaceutics Jun 2018Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by...
Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F. The induction of NL did not further increase drug binding but led to altered F due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.
Topics: 3T3 Cells; Adipocytes; Animals; Biological Availability; Cytoplasm; Hydrogen-Ion Concentration; Lysosomes; Mice; Pharmacokinetics; Phospholipids
PubMed: 29709195
DOI: 10.1021/acs.molpharmaceut.8b00064 -
EMBO Reports Jun 2021In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca ion and...
In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.
Topics: Calcium; Endoplasmic Reticulum; Humans; Mitochondria; Mitochondrial Proteins; Phospholipids; Protein Tyrosine Phosphatases
PubMed: 33938112
DOI: 10.15252/embr.202051323