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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 -
Cell Cycle (Georgetown, Tex.) Nov 2016
Topics: ADAM17 Protein; Cell Membrane; Enzyme Activation; Phosphatidylserines
PubMed: 27463373
DOI: 10.1080/15384101.2016.1211449 -
Nature Communications Feb 2018Molecular logic gates are expected to play an important role on the way to information processing therapeutic agents, especially considering the wide variety of physical...
Molecular logic gates are expected to play an important role on the way to information processing therapeutic agents, especially considering the wide variety of physical and chemical responses that they can elicit in response to the inputs applied. Here, we show that a 1:2 demultiplexer based on a Zn-terpyridine-Bodipy conjugate with a quenched fluorescent emission, is efficient in photosensitized singlet oxygen generation as inferred from trap compound experiments and cell culture data. However, once the singlet oxygen generated by photosensitization triggers apoptotic response, the Zn complex then interacts with the exposed phosphatidylserine lipids in the external leaflet of the membrane bilayer, autonomously switching off singlet oxygen generation, and simultaneously switching on a bright emission response. This is the confirmatory signal of the cancer cell death by the action of molecular automaton and the confinement of unintended damage by excessive singlet oxygen production.
Topics: Apoptosis; Cell Line; Cell Membrane; Cells; Flow Cytometry; Humans; Phosphatidylserines; Photochemistry; Photosensitizing Agents; Singlet Oxygen; Zinc
PubMed: 29476048
DOI: 10.1038/s41467-018-03259-z -
Cell Reports Aug 2018Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine...
Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an "eat-me" signal allowing recognition of degenerating neurites by phagocytes. Here we show that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. Our findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo.
Topics: Animals; Drosophila; Drosophila Proteins; Nerve Degeneration; Neurites; Phosphatidylserines
PubMed: 30157423
DOI: 10.1016/j.celrep.2018.07.095 -
Cellular and Molecular Life Sciences :... Jun 1997Annexin V belongs to a family of phospholipid binding proteins, the Annexins. It binds in the presence of Ca(2+)-ions with high affinity to negatively charged... (Review)
Review
Annexin V belongs to a family of phospholipid binding proteins, the Annexins. It binds in the presence of Ca(2+)-ions with high affinity to negatively charged phospholipids like phosphatidylserine (PS). On the basis of its protein structure and biological activity Annexin V is considered as a protein exhibiting its hitherto unknown function within the intracellular environment. One argument comes from the understanding that PS is predominantly located in membrane leaflets, which face the cytosol. However, recent findings show that each cell type has the molecular machinery to expose PS at its cell surface. This machinery is activated during the execution of apoptosis. Once PS is exposed at the cell surface it exhibits procoagulant and proinflammatory activities. Annexin V will bind to the PS-exposing apoptotic cell and can inhibit thereby the procoagulant and pro-inflammatory activities of the dying cell. These findings together with the presence of Annexin V in the extracellular space depict a novel (patho)Physiological significance for Annexin V in vivo.
Topics: Animals; Annexin A5; Apoptosis; Blood Coagulation; Humans; Inflammation; Membranes, Artificial; Phosphatidylserines
PubMed: 9230931
DOI: 10.1007/s000180050067 -
PloS One 2016Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of...
Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 μM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process.
Topics: Blood Proteins; Humans; Phosphatidylethanolamines; Phosphatidylserines; Protein Binding
PubMed: 27584039
DOI: 10.1371/journal.pone.0161896 -
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 -
Biochimica Et Biophysica Acta.... Feb 2018Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the...
Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7M serine and 0.3M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.
Topics: Cell Membrane; Cytoplasmic Vesicles; Lipid Bilayers; Membrane Fusion; Models, Chemical; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipase D; Phospholipids; Spectrometry, Fluorescence
PubMed: 29032301
DOI: 10.1016/j.bbamem.2017.10.011 -
Cell Communication and Signaling : CCS Jan 2020The establishment of parasitic infection is dependent on the development of efficient strategies to evade the host defense mechanisms. Phosphatidylserine (PS) molecules... (Review)
Review
The establishment of parasitic infection is dependent on the development of efficient strategies to evade the host defense mechanisms. Phosphatidylserine (PS) molecules are pivotal for apoptotic cell recognition and clearance by professional phagocytes. Moreover, PS receptors are able to trigger anti-inflammatory and immunosuppressive responses by phagocytes, either by coupled enzymes or through the induction of regulatory cytokine secretion. These PS-dependent events are exploited by parasites in a mechanism called apoptotic mimicry. Generally, apoptotic mimicry refers to the effects of PS recognition for the initiation and maintenance of pathogenic infections. However, in this context, PS molecules can be recognized on the surface of the infectious agent or in the surface of apoptotic host debris, leading to the respective denomination of classical and non-classical apoptotic mimicry. In this review, we discuss the role of PS in the pathogenesis of several human infections caused by protozoan parasites. Video Abstract.
Topics: Animals; Apoptosis; Host-Parasite Interactions; Humans; Parasites; Parasitic Diseases; Phosphatidylserines
PubMed: 31941500
DOI: 10.1186/s12964-019-0482-8 -
Aging Cell Aug 2004It is important for the resolution of inflammation that the number and activity of immune cells are reduced. Clearance of immune cells may be achieved by apoptosis and... (Review)
Review
It is important for the resolution of inflammation that the number and activity of immune cells are reduced. Clearance of immune cells may be achieved by apoptosis and phagocytosis of cell fragments by macrophages. However, signalling shutdown by immune cells committed to apoptosis occurs early in the progression of these cells towards fragmentation and, it could be argued, is a key feature of apoptosis. There is surprisingly little known about the mechanisms that underlie this signalling shutdown, in particular the shutdown of Ca(2+) influx. The consequences and the potential mechanisms by which Ca(2+) influx shutdown is achieved are discussed. In addition, the potential consequences for cell signalling of cytochrome c release from mitochondria and of phosphatidyl-serine externalization are discussed. The aim of the review is therefore to highlight the evidence for various signalling shutdown strategies in immune cells that may limit their activity during progression towards apoptosis.
Topics: Apoptosis; Calcium Signaling; Cell Death; Cell Membrane; Cytochromes c; Humans; Immune System; Lymphocytes; Mitochondria; Neutrophils; Phosphatidylserines; Signal Transduction
PubMed: 15268747
DOI: 10.1111/j.1474-9728.2004.00100.x