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PloS One 2018This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this...
This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0-2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 μM, 94.6 ± 47.6 μM, and 0.21 ± 0.07 μM, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10-7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 μM for verapamil IC50, 61.8 ± 34.8 μM for colchicine IC50, and 0.23 ± 0.09 μM for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Biological Transport; Cricetinae; Drug Resistance, Multiple; Proteolipids; Rhodamine 123
PubMed: 29912971
DOI: 10.1371/journal.pone.0199279 -
FEBS Letters Mar 1998The membrane-binding properties and pore-forming potential of the tumor-lysing and antibacterial polypeptide NK-lysin were investigated. Fluorescence quenching...
The membrane-binding properties and pore-forming potential of the tumor-lysing and antibacterial polypeptide NK-lysin were investigated. Fluorescence quenching experiments show a drastic change of accessibility to Trp58 in solution and in association with a lipid membrane. Calcein release from large unilamellar vesicles and fluctuating conductivity observed across a planar lipid bilayer of asolectin show that NK-lysin renders lipid bilayers permeable in a transient fashion, indicating a nonspecific lipid interaction as the mechanism underlying the biological activity. FTIR experiments show the same amount and type of regular secondary structure of NK-lysin in the membrane as in aqueous solution and exclude a structural rearrangement into a set of parallel or antiparallel alpha-helices as the predominant conformation. The molecular mechanism of the membrane-destabilizing effect of NK-lysin is discussed.
Topics: Anti-Infective Agents; Lipid Bilayers; Models, Molecular; Proteolipids; Pulmonary Surfactants
PubMed: 9559676
DOI: 10.1016/s0014-5793(98)00261-0 -
The Biochemical Journal Sep 1987Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective...
Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and 'DM-20' [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.
Topics: Acylation; Acyltransferases; Animals; Brain; Cell-Free System; Chromatography, Gel; Coenzyme A Ligases; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Hydroxylamine; Hydroxylamines; Myelin Proteins; Myelin Proteolipid Protein; Nerve Tissue Proteins; Palmitic Acid; Palmitic Acids; Palmitoyl Coenzyme A; Proteolipids; Rats; Rats, Inbred Strains; Repressor Proteins; Saccharomyces cerevisiae Proteins
PubMed: 2446598
DOI: 10.1042/bj2460611 -
The Journal of Clinical Investigation Mar 2002
Review
Topics: Animals; Carrier Proteins; Collectins; Cytokines; Humans; Immunity, Innate; Lipopolysaccharides; Lung; Models, Immunological; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants
PubMed: 11901176
DOI: 10.1172/JCI15293 -
Proceedings of the National Academy of... Dec 2015NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In...
NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30-35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer's patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cattle; Chromosomes, Mammalian; Escherichia coli; Gene Dosage; Gene Expression Profiling; Gene Order; Microscopy, Electron, Transmission; Molecular Sequence Data; Multigene Family; Organ Specificity; Peptides; Phylogeny; Proteolipids; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid
PubMed: 26668394
DOI: 10.1073/pnas.1519374113 -
Molecules (Basel, Switzerland) Jul 2021Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression...
Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Cell Line; Immunologic Factors; Inflammation; Intestinal Mucosa; Models, Molecular; Pore Forming Cytotoxic Proteins; Proteolipids; Recombinant Proteins; Swine
PubMed: 34299517
DOI: 10.3390/molecules26144242 -
Current Biology : CB Nov 2020Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1, 2]. Although TCJs play...
Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1, 2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained, and remodeled is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembrane-domain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8, 9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10, 11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here, we show that M6 is essential for the assembly of tricellular, but not bicellular, occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a low-mobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices.
Topics: Animals; Animals, Genetically Modified; Cell Membrane; Drosophila Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Epithelial Cells; Female; Fluorescence Recovery After Photobleaching; Intravital Microscopy; Male; Membrane Proteins; Myelin Proteins; Myelin Proteolipid Protein; Nerve Tissue Proteins; Proteolipids; Receptors, Scavenger; Tight Junctions
PubMed: 32857972
DOI: 10.1016/j.cub.2020.08.003 -
Biochimica Et Biophysica Acta Oct 1995
Review
Topics: Detergents; Energy Metabolism; Liposomes; Membrane Proteins; Micelles; Proteolipids; Solubility
PubMed: 7578213
DOI: 10.1016/0005-2728(95)00091-v -
Nature Mar 2016Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose...
Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA-BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate- and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a 'finger helix' that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves 'up' and 'down' in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA's transmembrane channel. This mechanism is validated experimentally by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation.
Topics: Cellulose; Crystallography, X-Ray; Glucose; Glucosyltransferases; Intracellular Membranes; Models, Molecular; Movement; Protein Structure, Secondary; Proteolipids; Rhodobacter sphaeroides; Substrate Specificity
PubMed: 26958837
DOI: 10.1038/nature16966 -
Biophysical Journal Jan 2020The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium...
The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn-Ala and Thr-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.
Topics: Amino Acid Sequence; Humans; Models, Molecular; Muscle Proteins; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Proteolipids; Sarcoplasmic Reticulum Calcium-Transporting ATPases
PubMed: 31858977
DOI: 10.1016/j.bpj.2019.11.3385