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MSphere Jan 2021Melinda A. Brindley works in the field of virology with specific interests in understanding how viruses enter cells. In this mSphere of Influence article, she reflects...
Melinda A. Brindley works in the field of virology with specific interests in understanding how viruses enter cells. In this mSphere of Influence article, she reflects on how the paper "Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells" by J. Mercer and A. Helenius (Science 320:531-535, 2008, https://doi.org/10.1126/science.1155164) made an impact on her by expanding our understanding of virus-host interactions and virus-cell binding.
Topics: Apoptosis; Host Microbial Interactions; Phosphatidylserines; Vaccinia virus; Virus Internalization
PubMed: 33504657
DOI: 10.1128/mSphere.00034-21 -
Langmuir : the ACS Journal of Surfaces... Dec 2023Phosphatidylserine (PS) exposure on the plasma membrane is crucial for many cellular processes including apoptotic cell recognition, blood clotting regulation, cellular...
Phosphatidylserine (PS) exposure on the plasma membrane is crucial for many cellular processes including apoptotic cell recognition, blood clotting regulation, cellular signaling, and intercellular interactions. In this study, we investigated the arrangement of PS headgroups in mixed PS/phosphatidylcholine (PC) bilayers, serving as a simplified model of the outer leaflets of mammalian cell plasma membranes. Combining atomistic-scale molecular dynamics (MD) simulations with Langmuir monolayer experiments, we unraveled the mutual miscibility of POPC and POPS lipids and the intricate intermolecular interactions inherent to these membranes as well as the disparities in position and orientation of PC and PS headgroups. Our experiments revealed micrometer-scale miscibility at all mole fractions of POPC and POPS, marked by modest deviations from ideal mixing with no apparent microscale phase separation. The MD simulations, meanwhile, demonstrated that these deviations were due to strong electrostatic interactions between like-lipid pairs (POPC-POPC and POPS-POPS), culminating in lateral segregation and nanoscale clustering. Notably, PS headgroups profoundly affect the ordering of the lipid acyl chains, leading to lipid elongation and subtle PS protrusion above the zwitterionic membrane. In addition, PC headgroups are more tilted with respect to the membrane normal, while PS headgroups align at a smaller angle, making them more exposed to the surface of the mixed PC/PS membranes. These findings provide a detailed molecular-level account of the organization of mixed PC/PS membranes, corroborated by experimental data. The insights gained here extend our comprehension of the physiological role of PSs.
Topics: Lipid Bilayers; Phosphatidylcholines; Phosphatidylserines; Membranes, Artificial; Cell Membrane
PubMed: 38096496
DOI: 10.1021/acs.langmuir.3c03061 -
The Biochemical Journal Apr 2014Human coagulation FXa (Factor Xa) plays a key role in blood coagulation by activating prothrombin to thrombin on 'stimulated' platelet membranes in the presence of its...
Human coagulation FXa (Factor Xa) plays a key role in blood coagulation by activating prothrombin to thrombin on 'stimulated' platelet membranes in the presence of its cofactor FVa (Factor Va). PS (phosphatidylserine) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used FRET to monitor changes in FXa length in response to (i) soluble short-chain PS [C6PS (dicaproylphosphatidylserine)], (ii) PS membranes, and (iii) FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa Fluor® 555) at the FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites: a regulatory site at the N-terminus [identified previously as involving the Gla (γ-carboxyglutamic acid) and EGFN (N-terminus of epidermal growth factor) domains] and a presumptive protein-recognition site in the catalytic domain. Binding of C6PS to the regulatory site increased the interprobe distance by ~3 Å (1 Å=0.1 nm), whereas saturation of both sites increased the distance by a further ~6.4 Å. FXa binding to a membrane produced a smaller increase in length (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane from when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS- or PS-containing membranes were bound to FXa, the overall change in length was comparable (~5.6-5.8 Å), indicating that C6PS- and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analysing structure-function relationships in FXa and in the FXa·FVa2 complex.
Topics: Allosteric Regulation; Animals; Cell Line; Cricetinae; Factor Va; Factor Xa; Humans; Phosphatidylserines; Protein Binding; Quantitative Structure-Activity Relationship
PubMed: 24467409
DOI: 10.1042/BJ20131099 -
Journal of Molecular Medicine (Berlin,... Nov 2023Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular...
Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EV) with an average size of 7.5 μm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EV also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EV were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EV. Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EV was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B-positive cells (P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 10 Mreg/ml) led to a reduction of T-cell activation (number of granzyme B-positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EV (P < 0.05 for 3.2 × 10 L-EV/ml). A differential analysis of the effects of Mreg and L-EV on CD4 and CD8 T-cells showed an inhibition of CD4 T-cells by Mreg (P < 0.01) and L-EV (P < 0.05 for 1.6 × 10 L-EV/ml; P < 0.01 for 3.2 × 10 L-EV/ml). A moderate inhibition of CD8 T-cells was observed by Mreg (P < 0.05) and by L-EV (P < 0.01 for 1.6 × 10 L-EV/ml and 3.2 × 10 L-EV/ml). PS was restricted to confined regions of the Mreg surface, while L-EV showed strong signals for PS in the exoplasmic leaflet. L-EV attenuate CD3/CD28-mediated activation of CD4 and CD8 T-cells. L-EV may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity. KEY MESSAGES: Mreg release large extracellular vesicles (L-EV) with an average size of 7.5 µm L-EV exhibit phosphatidylserine positivity L-EV suppress CD4 and CD8 T-cells L-EV hold clinical potential in T-cell-related diseases.
Topics: Humans; CD28 Antigens; CD8-Positive T-Lymphocytes; Granzymes; Phosphatidylserines; Macrophages; Lymphocyte Activation; CD4-Positive T-Lymphocytes
PubMed: 37725101
DOI: 10.1007/s00109-023-02374-9 -
Plant Signaling & Behavior Feb 2017A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant...
A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells.
Topics: Anions; Biosensing Techniques; Cell Membrane; Eukaryota; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Static Electricity
PubMed: 28102755
DOI: 10.1080/15592324.2017.1282022 -
Journal of Lipid Research Jun 2018This article provides a historical account of the discovery, chemistry, and biochemistry of two ubiquitous phosphoglycerolipids, phosphatidylserine (PS) and...
This article provides a historical account of the discovery, chemistry, and biochemistry of two ubiquitous phosphoglycerolipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the ether lipids. In addition, the article describes the biosynthetic pathways for these phospholipids and how these pathways were elucidated. Several unique functions of PS and PE in mammalian cells in addition to their ability to define physical properties of membranes are discussed. For example, the translocation of PS from the inner to the outer leaflet of the plasma membrane of cells occurs during apoptosis and during some other specific physiological processes, and this translocation is responsible for profound life-or-death events. Moreover, mitochondrial function is severely impaired when the PE content of mitochondria is reduced below a threshold level. The discovery and implications of the existence of membrane contact sites between the endoplasmic reticulum and mitochondria and their relevance for PS and PE metabolism, as well as for mitochondrial function, are also discussed. Many of the recent advances in these fields are due to the use of isotope labeling for tracing biochemical pathways. In addition, techniques for disruption of specific genes in mice are now widely used and have provided major breakthroughs in understanding the roles and metabolism of PS and PE in vivo.
Topics: Animals; History, 19th Century; History, 20th Century; History, 21st Century; Humans; Mitochondria; Phosphatidylethanolamines; Phosphatidylserines
PubMed: 29661786
DOI: 10.1194/jlr.R084004 -
Cancer Communications (London, England) Mar 2022Understanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo...
BACKGROUND
Understanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo spontaneous apoptotic cell death in tumor microenvironment, these apoptotic cells are histologically co-localized with immunosuppressive macrophages. However, the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood. In this study, we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved.
METHODS
Apoptotic cells and macrophages in tumors were detected by immunohistochemical staining. Morphological analysis was performed with Giemsa staining. Lipids generated from apoptotic cells were detected by liquid chromatography-mass spectrometry. Phosphatidylserine-containing liposomes were prepared to mimic apoptotic cells. The expression of protein was determined by real-time PCR, immunohistochemistry enzyme-linked immunosorbent assay and Western blotting. Mouse malignant ascites and subcutaneous tumor models were designed for in vivo analysis. Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies.
RESULTS
The location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas. Phosphatidylserine, a lipid molecule generated in apoptotic cells, induced polarization and accumulation of M2-like macrophages in vivo and in vitro. Moreover, sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcutaneous tumor models. Further analyses suggested that phosphoserine induced a M2-like phenotype in macrophages, which was related to the activation of phosphoserine receptors including T-cell immunoglobin mucin 4 (TIM4) and the FAK-SRC-STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain-containing protein 3 (JMJD3). Administration of specific inhibitors of these pathways could reduce tumor progression.
CONCLUSIONS
This study suggest that apoptotic cell-generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors, and the related pathways might be potential therapeutic targets for cancer therapy.
Topics: Animals; Apoptosis; Ascites; Jumonji Domain-Containing Histone Demethylases; Macrophages; Mice; Neoplasms; Phosphatidylserines; Phosphoserine; STAT3 Transcription Factor; Tumor Microenvironment
PubMed: 35191227
DOI: 10.1002/cac2.12272 -
Biochemistry Dec 2018The plasma membrane of the cell is a complex, tightly regulated, heterogeneous environment shaped by proteins, lipids, and small molecules. Ca ions are important...
The plasma membrane of the cell is a complex, tightly regulated, heterogeneous environment shaped by proteins, lipids, and small molecules. Ca ions are important cellular messengers, spatially separated from anionic lipids. After cell injury, disease, or apoptotic events, anionic lipids are externalized to the outer leaflet of the plasma membrane and encounter Ca, resulting in dramatic changes in the plasma membrane structure and initiation of signaling cascades. Despite the high chemical and biological significance, the structures of lipid-Ca nanoclusters are still not known. Previously, we demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy that upon binding to Ca, individual phosphatidylserine lipids populate two distinct yet-to-be-characterized structural environments. Here, we concurrently employ extensive all-atom molecular dynamics (MD) simulations with our accelerated membrane mimetic and detailed NMR measurements to identify lipid-Ca nanocluster conformations. We find that major structural characteristics of these nanoclusters, including interlipid pair distances and chemical shifts, agree with observable NMR parameters. Simulations reveal that lipid-ion nanoclusters are shaped by two characteristic, long-lived lipid structures induced by divalent Ca. Using ab initio quantum mechanical calculations of chemical shifts on MD-captured lipid-ion complexes, we show that computationally observed conformations are validated by experimental NMR data. Both NMR measurements of diluted specifically labeled lipids and MD simulations reveal that the basic structural unit that reshapes the membrane is a Ca-coordinated phosphatidylserine tetramer. Our combined computational and experimental approach presented here can be applied to other complex systems in which charged membrane-active molecular agents leave structural signatures on lipids.
Topics: Biomimetic Materials; Calcium; Cell Membrane; Ions; Magnetic Resonance Spectroscopy; Membrane Lipids; Molecular Conformation; Molecular Dynamics Simulation; Nanostructures; Phosphatidylserines
PubMed: 30456950
DOI: 10.1021/acs.biochem.8b01069 -
Cellular and Molecular Life Sciences :... Nov 2007The surge in apoptosis research and the discovery of the phosphatidylserine binding properties of annexin A5 have propelled a tremendous interest in cell death detection... (Review)
Review
The surge in apoptosis research and the discovery of the phosphatidylserine binding properties of annexin A5 have propelled a tremendous interest in cell death detection technologies. In the past years, annexin A5 has evolved from an efficient assay for detection of apoptotic cells in vitro to an in vivo molecular imaging technology with potential clinical use. A second key discovery, the specific internalization properties of annexin A5, has opened the opportunity to use annexin A5 for therapeutic applications. Annexin A5-mediated internalization creates a novel therapeutic platform for targeted drug delivery and cell entry to treat various diseases, including cancer and cardiovascular disease.
Topics: Animals; Annexin A5; Apoptosis; Cardiovascular Diseases; Drug Delivery Systems; Humans; Neoplasms; Phosphatidylserines
PubMed: 17876516
DOI: 10.1007/s00018-007-7297-2 -
Biochimica Et Biophysica Acta.... Feb 2022Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to...
Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, H). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.
Topics: Annexin A2; Biophysical Phenomena; Calcium; Carrier Proteins; Cell Communication; Cell Membrane; Cytoskeleton; Humans; Membrane Lipids; Phosphatidylserines; Phospholipids
PubMed: 34699769
DOI: 10.1016/j.bbamem.2021.183810