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ACS Infectious Diseases Sep 2021Multidrug-resistant bacteria pose a serious global health threat as antibiotics are increasingly losing their clinical efficacy. A molecular level understanding of the...
Multidrug-resistant bacteria pose a serious global health threat as antibiotics are increasingly losing their clinical efficacy. A molecular level understanding of the mechanism of action of antimicrobials plays a key role in developing new agents to combat the threat of antimicrobial resistance. Daptomycin, the only clinically used calcium-dependent lipopeptide antibiotic, selectively disrupts Gram-positive bacterial membranes to illicit its bactericidal effect. In this study, we use isothermal titration calorimetry to further characterize the structural features of the target bacterial phospholipids that drive daptomycin binding. Our studies reveal that daptomycin shows a clear preference for the phosphoglycerol headgroup. Furthermore, unlike other calcium-dependent lipopeptide antibiotics, calcium binding by daptomycin is strongly dependent on the presence of phosphatidylglycerol. These investigations provide new insights into daptomycin's phospholipid specificity and calcium binding behavior.
Topics: Anti-Bacterial Agents; Calcium; Daptomycin; Gram-Positive Bacteria; Phospholipids
PubMed: 34406007
DOI: 10.1021/acsinfecdis.1c00316 -
Molecules (Basel, Switzerland) Jul 2023The interactions of dsDNA with new targeted drug delivery derivatives of doxorubicin (DOX), such as DOX embedded into phospholipid nanoparticles (NPhs) and DOX with the...
The interactions of dsDNA with new targeted drug delivery derivatives of doxorubicin (DOX), such as DOX embedded into phospholipid nanoparticles (NPhs) and DOX with the NGR targeted peptide-modified NPhs were studied electrochemically by differential pulse voltammetry technique. Screen-printed electrodes (SPEs), modified with stable fine dispersions of carbon nanotubes (CNTs), were used for quantitative electrochemical investigations of direct electrochemical oxidation of guanine, adenine, and thymine heterocyclic bases of dsDNA, and their changes in the presence of DOX nanoderivatives. Analysing the shifts of peak potentials of nucleobases in the presence of drug, we have shown that the doxorubicin with NGR targeted peptide changed the mode of interaction in DNA-drug complexes from intercalative to electrostatic. Binding constants () of DNA-drug complexes were calculated in accordance with adenine, guanine, and thymine oxidation signals. Based on our experiments, we have proven that the surface modification of a drug delivery system with NGR targeted peptide dramatically changed the mechanism of interaction of drug with genetic material. DNA-mediated drug toxicity was calculated based on the concentration-dependent "response" of heterocyclic nucleobases on drug influence. DOX, DOX-loaded phospholipid nanoparticles (NPhs), and DOX with NGR addressed peptide-modified NPhs were moderately toxic in the concentration range of 0.5-290 µM.
Topics: Phospholipids; Nanotubes, Carbon; Thymine; Doxorubicin; Peptides; Drug Delivery Systems; DNA; Nanoparticles; Adenine; Guanine
PubMed: 37513191
DOI: 10.3390/molecules28145317 -
Methods in Molecular Biology (Clifton,... 2016Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs... (Review)
Review
Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS), and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the years, several techniques have emerged in either fixed or live cells to analyze the subcellular localization and dynamics of acidic phospholipids. In this chapter, we review one of them: the use of genetically encoded biosensors that are based on the expression of specific lipid binding domains (LBDs) fused to fluorescent proteins. We discuss how to design such sensors, including the criteria for selecting the lipid binding domains of interest and to validate them. We also emphasize the care that must be taken during data analysis as well as the main limitations and advantages of this approach.
Topics: Biological Transport; Biosensing Techniques; Molecular Imaging; Molecular Probes; Phospholipids; Protein Binding; Protein Interaction Domains and Motifs
PubMed: 26552684
DOI: 10.1007/978-1-4939-3170-5_15 -
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 -
Biochimica Et Biophysica Acta.... Mar 2018Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD),...
Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.
Topics: Animals; Carrier Proteins; Cholesterol; Dietary Fats; Fatty Acid-Binding Proteins; Female; Liver; Male; Mice; Mice, Knockout; Non-alcoholic Fatty Liver Disease; Phospholipids
PubMed: 29307784
DOI: 10.1016/j.bbalip.2017.12.013 -
The Journal of Biological Chemistry Aug 2022Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total...
Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology. It has been reported that KcsA activity is regulated by membrane phospholipids, such as phosphatidylglycerol. However, there has been no quantitative analysis of the correlation between direct lipid binding and the functional modification of KcsA, and it is unknown whether PIPns modulate KcsA function. Here, using contact bubble bilayer recording, we observed that the open probability of KcsA increased significantly (from about 10% to 90%) when the membrane inner leaflet contained only a small percentage of PIPns. In addition, we found an increase in the electrophysiological activity of KcsA correlated with a larger number of negative charges on PIPns. We further analyzed the affinity of the direct interaction between PIPns and KcsA using microscale thermophoresis and observed a strong correlation between direct lipid binding and the functional modification of KcsA. In conclusion, our approach was able to reconstruct the direct modification of KcsA by PIPns, and we propose that it can also be applied to elucidate the mechanism of modification of other ion channels by PIPns.
Topics: Bacterial Proteins; Membrane Lipids; Phosphatidylinositols; Phospholipids; Potassium Channels
PubMed: 35839854
DOI: 10.1016/j.jbc.2022.102257 -
The Journal of Biological Chemistry Sep 2019A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process,...
A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the β-adrenergic receptor (βAR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted βAR, the βAR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted βAR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of βAR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that βAR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress βAR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.
Topics: Detergents; Humans; Phospholipids; Receptors, Adrenergic, beta-2
PubMed: 31362983
DOI: 10.1074/jbc.AC119.009848 -
Chinese Journal of Traumatology =... Apr 2020With the deepening of research, proteomics has developed into a science covering the study of all the structural and functional characteristics of proteins and the... (Review)
Review
With the deepening of research, proteomics has developed into a science covering the study of all the structural and functional characteristics of proteins and the dynamic change rules. The essence of various biological activities is revealed from the perspectives of the biological structure, functional activity and corresponding regulatory mechanism of proteins by proteomics. Among them, phospholipid-binding protein is one of the hotspots of proteomics, especially annexin A1, which is widely present in various tissues and cells of the body. It has the capability of binding to phospholipid membranes reversibly in a calcium ion dependent manner. In order to provide possible research ideas for researchers, who are interested in this protein, the biological effects of annexin A1, such as inflammatory regulation, cell signal transduction, cell proliferation, differentiation and apoptosis are described in this paper.
Topics: Annexin A1; Apoptosis; Calcium; Cell Proliferation; Humans; Inflammation; Phospholipids; Protein Binding; Proteomics; Signal Transduction
PubMed: 32201231
DOI: 10.1016/j.cjtee.2020.02.002 -
International Journal of Molecular... Nov 2020Phosphoinositides play a crucial role in regulating many cellular functions, such as actin dynamics, signaling, intracellular trafficking, membrane dynamics, and... (Review)
Review
Phosphoinositides play a crucial role in regulating many cellular functions, such as actin dynamics, signaling, intracellular trafficking, membrane dynamics, and cell-matrix adhesion. Central to this process is phosphatidylinositol bisphosphate (PIP2). The levels of PIP2 in the membrane are rapidly altered by the activity of phosphoinositide-directed kinases and phosphatases, and it binds to dozens of different intracellular proteins. Despite the vast literature dedicated to understanding the regulation of PIP2 in cells over past 30 years, much remains to be learned about its cellular functions. In this review, we focus on past and recent exciting results on different molecular mechanisms that regulate cellular functions by binding of specific proteins to PIP2 or by stabilizing phosphoinositide pools in different cellular compartments. Moreover, this review summarizes recent findings that implicate dysregulation of PIP2 in many diseases.
Topics: Cell Membrane; Disease; Humans; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Signal Transduction
PubMed: 33172190
DOI: 10.3390/ijms21218342 -
Scientific Reports May 2016Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of...
Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.
Topics: Bacteria; Cytoplasmic Granules; Phospholipids; Polyhydroxyalkanoates
PubMed: 27222167
DOI: 10.1038/srep26612