-
Advances in Biological Regulation Jan 2018The phospholipase A superfamily of enzymes plays a significant role in the development and progression of numerous inflammatory diseases. Through their catalytic action... (Review)
Review
The phospholipase A superfamily of enzymes plays a significant role in the development and progression of numerous inflammatory diseases. Through their catalytic action on membrane phospholipids, phospholipases are the upstream regulators of the eicosanoid pathway releasing free fatty acids for cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes which produce various well-known inflammatory mediators including leukotrienes, thromboxanes and prostaglandins. Elucidating the association of phospholipases A with the membrane, the extraction and binding of phospholipid substrates, and their interactions with small-molecule inhibitors is crucial for the development of new anti-inflammatory therapeutics. Studying phospholipases has been challenging because they act on the surface of cellular membranes and micelles. Multidisciplinary approaches including hydrogen/deuterium exchange mass spectrometry, molecular dynamics simulations, and other computer-aided drug design techniques have been successfully employed by our laboratory to study interactions of phospholipases with membranes, phospholipid substrates and inhibitors. This review summarizes the application of these techniques to study four human recombinant phospholipases A.
Topics: Cell Membrane; Deuterium Exchange Measurement; Humans; Mass Spectrometry; Molecular Dynamics Simulation; Phospholipases A2; Phospholipids
PubMed: 29248300
DOI: 10.1016/j.jbior.2017.10.009 -
Langmuir : the ACS Journal of Surfaces... Aug 2022Liposomes represent important drug carrier vehicles in biological systems. A fusogenic liposomal system composed of equimolar mixtures of the cationic lipid DOTAP and...
Liposomes represent important drug carrier vehicles in biological systems. A fusogenic liposomal system composed of equimolar mixtures of the cationic lipid DOTAP and the phospholipid DOPE showed high fusion and delivery efficiencies with cells and lipid vesicles. However, aspects of the thermodynamics involving the interaction of these fusogenic liposomes and biomimetic systems remain unclear. Here, we investigate the fusion of this system with large unilamellar vesicles (LUVs) composed of the zwitterionic lipid POPC and increasing fractions of the anionic lipid POPG and up to 30 mol % cholesterol. The focus here is to concomitantly follow changes in size, zeta-potential, and enthalpy binding upon membrane interaction and fusion. Isothermal titration calorimetry (ITC) data showed that membrane fusion in our system is an exothermic process in the absence of cholesterol, suggesting that electrostatic attraction is the driving force for fusion. An endothermic component appeared and eventually dominated the titration at 30 mol % cholesterol, which we propose is caused by membrane fluidification when cholesterol is diluted upon fusion. The inflection points of the ITC data occurred around 0.5-0.7 POPG/DOTAP for all systems, the same stoichiometry for which zeta-potential and dynamic light scattering measurements showed an increase in size coupled with charge neutralization of the system, which is consistent with the fact that fusion in our system is charge-mediated. Microscopy observations of the final mixtures revealed the presence of giant vesicles, which is a clear indication of fusion, coexisting with intermediate-sized objects that could be the result of both fusion and/or aggregation. The results show that the fusion efficiency of the DOTAP:DOPE fusogenic system is modulated by the charge and membrane packing of the acceptor membrane and explain why the system fuses very efficiently with cells.
Topics: Calorimetry; Cholesterol; Liposomes; Membrane Fusion; Phospholipids; Unilamellar Liposomes
PubMed: 35977420
DOI: 10.1021/acs.langmuir.2c01169 -
The Journal of Biological Chemistry Mar 2019Toll-like receptors (TLRs) coupled to intracellular signaling cascades function as central elements of innate immunity that control transcription of numerous... (Review)
Review
Toll-like receptors (TLRs) coupled to intracellular signaling cascades function as central elements of innate immunity that control transcription of numerous pro-inflammatory genes. Two minor anionic phospholipids present in the pulmonary surfactant complex, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), antagonize the cognate ligand activation of TLRs 2 and 4. The lipids block recognition of activating ligands by the TLRs, either directly or via the TLR4 coreceptors CD14 and MD2. Antagonism of TLR activation results in inhibition of the initiating step of the pro-inflammatory signaling pathways. Evidence for this mechanism of action comes from direct binding studies between CD14 and MD2 with POPG and PI. Additional evidence for this mechanism of antagonism also comes from monitoring the reduction of protein phosphorylation events that characterize the intracellular signaling by activated TLRs. The pathogenesis of respiratory syncytial virus (RSV) and influenza A virus (IAV) have been linked to TLR4 activation, and we examined the action of POPG and PI as potential antagonists of the pathology of these viruses. Surprisingly, POPG and PI dramatically curtail infection, in addition to inhibiting inflammatory sequelae associated with RSV and IAV infections. The mechanism of action by the lipids is disruption of virus particle binding to host cell plasma membrane receptors, required for viral uptake. The antagonism of activation of TLRs and virus binding to the alveolar epithelium by resident constituents of the pulmonary surfactant system suggests that POPG and PI function in homeostasis, to prevent inflammatory processes that result in reductions in gas exchange within the alveolar compartment.
Topics: Animals; Humans; Immunity, Innate; Influenza A virus; Phospholipids; Pulmonary Surfactants; Respiratory Syncytial Viruses; Respiratory Tract Infections
PubMed: 30733339
DOI: 10.1074/jbc.AW118.003229 -
The Journal of Physical Chemistry. B May 2023In this work, the influence of membrane curvature on the Ca binding to phospholipid bilayers is investigated by means of molecular dynamics simulations. In particular,...
In this work, the influence of membrane curvature on the Ca binding to phospholipid bilayers is investigated by means of molecular dynamics simulations. In particular, we compared Ca binding to flat, elastically buckled, or uniformly bent zwitterionic and anionic phospholipid bilayers. We demonstrate that Ca ions bind preferably to the concave membrane surfaces in both types of bilayers. We also show that the membrane curvature leads to pronounced changes in Ca binding including differences in free ion concentrations, lipid coordination distributions, and the patterns of ion binding to different chemical groups of lipids. Moreover, these effects differ substantially for the concave and convex membrane monolayers. Comparison between force fields with either full or scaled charges indicates that charge scaling results in reduction of the Ca binding to curved phosphatidylserine bilayers, while for phosphatidylcholine membranes, calcium binds only weakly for both force fields.
Topics: Phospholipids; Lipid Bilayers; Calcium; Molecular Dynamics Simulation; Phosphatidylcholines; Ions
PubMed: 37191140
DOI: 10.1021/acs.jpcb.3c01962 -
Cells May 2021Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on... (Review)
Review
Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.
Topics: Animals; Binding Sites; Humans; Phospholipids; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Signal Transduction; Structure-Activity Relationship; src Homology Domains; src-Family Kinases
PubMed: 34068055
DOI: 10.3390/cells10051191 -
Journal of Integrative Plant Biology Jan 2021The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in... (Review)
Review
The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associated proteins, microtubule-associated proteins (MAPs) and actin-binding proteins (ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.
Topics: Cytoskeleton; Microfilament Proteins; Microtubule-Associated Proteins; Phospholipids; Protein Processing, Post-Translational; Signal Transduction
PubMed: 33274838
DOI: 10.1111/jipb.13046 -
Journal of Pharmaceutical and... Oct 2016HPLC methods that use chromatographic retention times for gaining information about the properties of compounds for the purpose of designing drug molecules are reviewed.... (Review)
Review
HPLC methods that use chromatographic retention times for gaining information about the properties of compounds for the purpose of designing drug molecules are reviewed. Properties, such as lipophilicity, protein binding, phospholipid binding, and acid/base character can be incorporated in the design of molecules with the right biological distribution and pharmacokinetic profile to become an effective drug. Standardization of various methodologies is suggested in order to obtain data suitable for inter-laboratory comparison. The published HPLC methods for lipophilicity, acid/base character, protein and phospholipid binding are critically reviewed and compared with each other using the solvation equation approach. One of the most important discussion points is how these data can be used in models and how they can influence the drug discovery process. Therefore, the published models for volume of distribution, unbound volume of distribution and drug efficiency are also discussed. The general relationships between the chemical structure and biomimetic HPLC properties are described in view of ranking and selecting putative drug molecules.
Topics: Biomimetics; Chromatography, High Pressure Liquid; Drug Discovery; Lipids; Phospholipids
PubMed: 27084527
DOI: 10.1016/j.jpba.2016.04.009 -
Molecules (Basel, Switzerland) Apr 2020Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance,...
Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance, insulin stability as well as adipose tissue inflammation, parameters seriously affected by obesity. Some of these effects require inhibition of target proteases such as kallikrein 7(KLK7) and many studies have demonstrated vaspin-mediated activation of intracellular signaling cascades in various cells and tissues. So far, little is known about the exact mechanism how vaspin may trigger these intracellular signaling events. In this study, we investigated and characterized the interaction of vaspin with membrane lipids and polyphosphates as well as their potential regulatory effects on serpin activity using recombinant vaspin and KLK7 proteins and functional protein variants thereof. Here, we show for the first time that vaspin binds to phospholipids and polyphosphates with varying effects on KLK7 inhibition. Vaspin binds strongly to monophosphorylated phosphatidylinositol phosphates (PtdInsP) with no effect on vaspin activation. Microscale thermophoresis (MST) measurements revealed high-affinity binding to polyphosphate 45 (K: 466 ± 75 nM) and activation of vaspin in a heparin-like manner. Furthermore, we identified additional residues in the heparin binding site in β-sheet A by mutating five basic residues resulting in complete loss of high-affinity heparin binding. Finally, using lipid overlay assays, we show that these residues are additionally involved in PtdInsP binding. Phospholipids play a major role in membrane trafficking and signaling whereas polyphosphates are procoagulant and proinflammatory agents. The identification of phospholipids and polyphosphates as binding partners of vaspin will contribute to the understanding of vaspins involvement in membrane trafficking, signaling and beneficial effects associated with obesity.
Topics: Binding Sites; Heparin; Humans; Kinetics; Membrane Lipids; Models, Molecular; Multiprotein Complexes; Phospholipids; Polyphosphates; Protein Binding; Serpins; Structure-Activity Relationship
PubMed: 32344508
DOI: 10.3390/molecules25081992 -
Proceedings of the National Academy of... Jan 2022Lipoprotein-associated phospholipase A (Lp-PLA) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized...
Lipoprotein-associated phospholipase A (Lp-PLA) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA's specificity toward oxidized phospholipids.
Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Allosteric Regulation; Binding Sites; Catalysis; Catalytic Domain; Fatty Acids; Humans; Hydrolysis; Lipoproteins, HDL; Membranes; Molecular Dynamics Simulation; Phospholipids; Substrate Specificity
PubMed: 34996868
DOI: 10.1073/pnas.2102953118 -
The Journal of Biological Chemistry May 2022Lipids play critical roles in several major chronic diseases of our times, including those that involve inflammatory sequelae such as metabolic syndrome including... (Review)
Review
Lipids play critical roles in several major chronic diseases of our times, including those that involve inflammatory sequelae such as metabolic syndrome including obesity, insulin sensitivity, and cardiovascular diseases. However, defining the substrate specificity of enzymes of lipid metabolism is a challenging task. For example, phospholipase A (PLA) enzymes constitute a superfamily of degradative, biosynthetic, and signaling enzymes that all act stereospecifically to hydrolyze and release the fatty acids of membrane phospholipids. This review focuses on how membranes interact allosterically with enzymes to regulate cell signaling and metabolic pathways leading to inflammation and other diseases. Our group has developed "substrate lipidomics" to quantify the substrate phospholipid specificity of each PLA and coupled this with molecular dynamics simulations to reveal that enzyme specificity is linked to specific hydrophobic binding subsites for membrane phospholipid substrates. We have also defined unexpected headgroup and acyl chain specificity for each of the major human PLA enzymes, which explains the observed specificity at a structural level. Finally, we discovered that a unique hydrophobic binding site-and not each enzyme's catalytic residues or polar headgroup binding site-predominantly determines enzyme specificity. We also discuss how PLAs release specific fatty acids after allosteric enzyme association with membranes and extraction of the phospholipid substrate, which can be blocked by stereospecific inhibitors. After decades of work, we can now correlate PLA specificity and inhibition potency with molecular structure and physiological function.
Topics: Allosteric Regulation; Fatty Acids; Humans; Phospholipases A2; Phospholipids; Substrate Specificity
PubMed: 35358512
DOI: 10.1016/j.jbc.2022.101873