RESUMO
A difficult problem concerning the interaction of DNA with amphiphiles of opposite charge above their critical micelle concentration is the propensity for aggregation of the condensed DNA complexes. In this study, this problem was addressed by attenuating amphiphile charge density within a cholate micelle environment. The amphiphile consisted of a cationic peptide, acetyl-CWKKKPKK-amide, conjugated to dilaurylphos-phatidylethanolamine. In the presence of cholate, multiple equivalents of cationic charge were required to bring about the completion of DNA condensation. At the end point of condensation, stable, soluble DNA-micelle complexes were formed, which by dynamic light scattering exhibited apparent hydro-dynamic diameters between 30 and 60 nm. Aggregation, as measured by static light scattering at 90 degrees and by turbidity, was not observed until further additions of peptide-lipid conjugate were made beyond the end point of DNA condensation. Liposome complexes containing the non-aggregated, compacted DNA were formed by adding dioleoylphosphatidylcholine followed by removing the cholate by dialysis. The resulting complexes were distributed within a narrow density range, the DNA was quantitatively assembled into the liposomes, and liposomes without DNA were not detected. Small particles were formed with a mean hydrodynamic diameter of 77 nm. The liposomal DNA showed complete retention of its supercoiled form and no detectable sensitivity to DNase (25 U/10 microg DNA, 1.5 h, 37 degrees C). The use of an anionic, dialyzable amphiphile to attenuate charge inter-actions between DNA and cationic amphiphiles is a useful technology for the quantitative assembly of compacted DNA into conventional liposomes, with complete protection against nuclease activity.
Assuntos
DNA/química , Lipossomos/química , Micelas , Fosfatidilcolinas , Ânions , Cátions , Fenômenos Químicos , Físico-Química , Colatos/farmacologia , Luz , Peptídeos/química , Fosfatidilcolinas/química , Espalhamento de RadiaçãoRESUMO
We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/microg DNA produced the highest luciferase gene expression, exceeding 1 x 10(9) relative light units/s/mg protein (>3 microg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.
Assuntos
Transfecção/métodos , Sequência de Aminoácidos , Animais , Células COS , Resinas de Troca de Cátion/química , Cloroquina/farmacologia , DNA/química , DNA/genética , Relação Dose-Resposta a Droga , Expressão Gênica , Glicerofosfolipídeos/química , Células HeLa , Humanos , Lipídeos/química , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/química , Fosfatidilcolinas/química , Fosfolipídeos/química , Plasmídeos/química , Plasmídeos/genética , Polietilenoimina/química , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.
Assuntos
Membrana Celular/química , Proteína gp41 do Envelope de HIV/química , HIV-1 , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/química , Dicroísmo Circular , Membrana Eritrocítica/química , Humanos , Lipossomos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Tomato seedlings treated with ethanolamine showed altered phospholipid composition. The changes included altered acyl chain composition as well as changes in the relative amounts of the phospholipid classes. Specifically, there was an increase in phosphatidylethanolamine and phosphatidylserine with a concomitant decrease in phosphatidylcholine and no overall increase in phospholipids. Treatment with ethanolamine increased the relative amount of C18 acyl chains (especially 18 : 2) in phosphatidylethanolamine and phosphatidylcholine at the expense of 16 : 0 and 16 : 1. Acyl composition of other phospholipid classes were unchanged. Labeled ethanolamine was incorporated mostly into phosphatidylethanolamine and phosphatidylcholine. Ethanolamine-stimulated incorporation of labeled oleate was entirely into acyl chains and appeared only as 18 : 1 and 18 : 2. There was greater incorporation, but less conversion of 18 : 1 to 18 : 2 with choline. Stearate was incorporated but desaturated.
Assuntos
Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Membrana Celular/efeitos dos fármacos , Citosol/metabolismo , Etanolaminas/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Ácidos Oleicos/farmacologia , Plantas/metabolismo , Polissorbatos/farmacologia , Sementes/metabolismo , Ácidos Esteáricos/metabolismoRESUMO
We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.
Assuntos
1,2-Dipalmitoilfosfatidilcolina , Produtos do Gene nef/metabolismo , HIV-1/metabolismo , Lipossomos , Fusão de Membrana , Fragmentos de Peptídeos/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Produtos do Gene nef/biossíntese , Produtos do Gene nef/química , Luz , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND: Exogenous surfactant has been accepted worldwide as a therapy of RDS in premature and term infants. Exogenous surfactant is usually derived from lung extracts containing phospholipids and the surfactant proteins SP-B and SP-C. Synthetic peptides of SP-B and SP-C are being tested with the aim to develop a completely synthetic surfactant preparation. Nevertheless, the effects of these peptides on the endogenous surfactant metabolism remain unknown. OBJECTIVES: The effect of synthetic SP-B peptides on uptake of surfactant-like liposomes was investigated in alveolar cells. Native SP-B and seven SP-B peptides were included: monomeric and dimeric SP-B(1-25) (Cys-11 --> Ala-11), SP-B(63-78)and Ala-SP-B(63-78) (Cys-71 --> Ala-71;Cys-77 --> Ala-77)and their serine mutants. METHODS: In vitro, alveolar macrophages (AM) and alveolar type II cells (ATII) were incubated with liposomes containing SP-B or one of its peptides. In vivo, rats received intratracheally various SP-B peptides (SP-B/lipid ratio 1:33 w/w) incorporated in fluorescent surfactant-like liposomes. One hour after instillation, AM and ATII were isolated and cell-associated fluorescence was determined using flow cytometry. Confocal laser microscopy was performed to ensure internalization of the liposomes. RESULTS: In vitro uptake by AM or ATII was not influenced by the SP-B peptides. In vivo, SP-B(1-25) and Ser-SP-B(1-25) increased the uptake by AM whereas dSP-B(1-25) decreased the uptake. Neither SP-B(1-25) nor dSP-B(1-25 )affected total uptake by ATII. The overall uptake by SP-B(63-78) variants was not changed. CONCLUSIONS: Surface-active synthetic SP-B peptides do not interfere with the normal uptake of surfactant by ATII.
Assuntos
Lipossomos/farmacocinética , Fragmentos de Peptídeos/farmacologia , Alvéolos Pulmonares/metabolismo , Proteína B Associada a Surfactante Pulmonar/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Citometria de Fluxo , Humanos , Cinética , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/fisiologia , Fragmentos de Peptídeos/síntese química , Alvéolos Pulmonares/efeitos dos fármacos , Proteína B Associada a Surfactante Pulmonar/química , Proteína B Associada a Surfactante Pulmonar/isolamento & purificação , SuínosRESUMO
Using the spin probe 5-doxylstearic acid, we studied the structural perturbations of rat liver mitochondrial membranes produced by exposure to ethanol in vitro and by chronic ethanol feeding. The addition of ethanol in vitro to mitochondria from control animals appears to "fluidize" the membranes, as evidenced by a pronounced decrease in the order parameter. By contrast, in membranes from rats fed ethanol chronically, there was no effect on the order parameter. This resistance of the mitochondrial membranes from chronically intoxicated animals to the fluidizing effect of ethanol probably results from a change in the composition of the phospholipids, because the same differential response to ethanol was observed upon using vesicles of mitochondrial phospholipids extracted from control and chronically treated rats. In the presence of 0.025--0.1 M ethanol, a range that prevails in the blood of chronic alcoholics, the order parameter of mitochondrial membranes from rats fed ethanol was comparable to that of control membranes without ethanol in vitro. Analysis of extracted mitochondrial phospholipids showed that the cardiolipin from ethanol-fed animals had fatty acyl residues that are more saturated than those of controls. These findings point to the underlying molecular mechanism of our previous observation that mitochondria from chronic alcoholic rats are more resistant to uncoupling by ethanol at physiological temperature [Rottenberg, H., Robertson, D. E. & Rubin, E. (1980) Lab. Invest. 42, 318--326]. We suggest that an adaptive change in the phospholipid composition leads to structural alterations, which result in increased resistance to disruption of mitochondrial membranes by ethanol. These changes in lipid composition and structure may explain many, if not all, of the mitochondrial abnormalities that have been previously reported to result from chronic ethanol intoxication.
Assuntos
Alcoolismo/patologia , Etanol/farmacologia , Lipídeos de Membrana/fisiologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosfolipídeos/fisiologia , Animais , Óxidos N-Cíclicos , Modelos Animais de Doenças , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Membranas Intracelulares/efeitos dos fármacos , Lipossomos , Masculino , Ratos , Marcadores de SpinRESUMO
Heavy sarcoplasmic reticulum (SR) was prepared from skeletal muscle of chronic alcoholic rats and control rats. Compared with control rat SR, the SR prepared from alcoholic rats is leakier to Ca2+ and has a lower level of maximum storable Ca2+. With in vitro exposure to ethanol, the Ca2+ release of alcoholic rat SR was not enhanced as much as that of SR prepared from control rats. By using the spin-probes 5-, 7-, 12-, and 16-doxylstearic acid, the disordering effect of ethanol on the SR membrane was studied. The results suggest that SR membrane of chronic alcoholic rats is less ordered than that of control rats even in the absence of in vitro ethanol. However, chronic SR membrane shows a greater resistance to the disordering effect of in vitro addition of ethanol than control rat SR.
Assuntos
Alcoolismo/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Músculos/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Animais , Cálcio/metabolismo , Etanol/administração & dosagem , Humanos , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Lipossomos , Fluidez de Membrana/efeitos dos fármacos , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos , Retículo Sarcoplasmático/metabolismoRESUMO
Here we report the characterization and optimization of a peptide/non-cationic lipid gene delivery system that successfully produces high levels of gene expression when delivered by microinjection into chicken embryos in vivo. In addition to plasmid DNA, the delivery complex consisted of four components: 1) a "condensing" peptide with both hydrophobic and cationic amino acid segments; 2) a "fusogenic" peptide with both membrane insertion and amphipathic helical segments; 3) a relatively short-chain phosphatidylcholine (14:1 cis-9); and 4) polyethyleneglycol conjugated to dioleoylphosphatidylethanolamine through a disulfide linkage. Optimum amounts of each component were determined by measuring expression of a luciferase reporter gene following a 24-hour incubation with chick embryo fibroblast (CEF) cells in culture. When relatively low amounts of condensing peptide, fusogenic peptide, or lipid were assembled into the complexes, relatively large concentrations of complex were required to reach maximum gene expression. When the amounts of peptide or lipid were increased, less complex was required to achieve maximum expression, but expression fell substantially with higher amounts of added complex. The polyethyleneglycol component significantly increased gene expression. With some preparations, luciferase activities in the CEF cells reached 1x10(10) relative light units per second per mg protein within 24 hours. Following the optimization experiments with the CEF cells, formulations containing low levels, intermediate levels, and high levels of the delivery system components were assembled with green fluorescent protein plasmid DNA, then microinjected into somite regions of chicken embryos in vivo. It was found that intermediate levels of the components gave the most reliable formulations for inducing localized gene expression in the somitic cells.
Assuntos
Técnicas de Transferência de Genes , Lipídeos , Microinjeções , Peptídeos , Animais , Células Cultivadas , Embrião de Galinha , Dissulfetos , Fibroblastos , Expressão Gênica , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Fosfatidilcolinas/administração & dosagem , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/administração & dosagem , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Plasmídeos/administração & dosagem , Plasmídeos/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Modelos Químicos , Fosfolipídeos/química , Sítios de Ligação , Difusão , Espectroscopia de Ressonância Magnética , Membranas Artificiais , Rotação , Propriedades de Superfície , Termodinâmica , Água/químicaRESUMO
We cloned the precursor of a novel peptide from a cDNA library prepared from pharyngeal tissues of the tunicate, Styela clava. Its sequence predicted a histidine-rich, amidated 23-residue peptide (FLRF(IG)SVIHGIGHLVHHIGVAL-NH2) that we named clavaspirin. A synthetic clavaspirin was prepared and it was found that it killed Gram-positive and Gram-negative bacteria, permeabilized the outer and inner membranes of Escherichia coli, lysed phosphatidylglycerol (POPG) liposomes, and was potently haemolytic towards human and bovine erythrocytes. Each of these activities was performed more effectively at an acidic pH. Circular dichroism measurements of synthetic clavaspirin revealed a largely alpha-helical structure and polarized and residue-specific FTIR spectrometry showed that its association with phospholipid membranes was influenced by pH. Peptides such as clavaspirin may equip tunicate haemocytes to mediate cytotoxicity and participate in antimicrobial defence.
Assuntos
Antibacterianos/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Bactérias/efeitos dos fármacos , Sequência de Bases , Proteínas Sanguíneas/química , Northern Blotting , Permeabilidade da Membrana Celular , Dicroísmo Circular , Clonagem Molecular , Hemólise , Humanos , Lipossomos/metabolismo , Testes de Sensibilidade Microbiana/métodos , Dados de Sequência Molecular , Peptídeos/química , Homologia de Sequência de Aminoácidos , Espectroscopia de Infravermelho com Transformada de Fourier , UrocordadosRESUMO
Langmuir isotherms, fluorescence microscopy, and atomic force microscopy were used to study lung surfactant specific proteins SP-B and SP-C in monolayers of dipalmitoylphosphatidylglycerol (DPPG) and palmitoyloleoylphosphatidylglycerol (POPG), which are representative of the anionic lipids in native and replacement lung surfactants. Both SP-B and SP-C eliminate squeeze-out of POPG from mixed DPPG/POPG monolayers by inducing a two- to three-dimensional transformation of the fluid-phase fraction of the monolayer. SP-B induces a reversible folding transition at monolayer collapse, allowing all components of surfactant to remain at the interface during respreading. The folds remain attached to the monolayer, are identical in composition and morphology to the unfolded monolayer, and are reincorporated reversibly into the monolayer upon expansion. In the absence of SP-B or SP-C, the unsaturated lipids are irreversibly lost at high surface pressures. These morphological transitions are identical to those in other lipid mixtures and hence appear to be independent of the detailed lipid composition of the monolayer. Instead they depend on the more general phenomena of coexistence between a liquid-expanded and liquid-condensed phase. These three-dimensional monolayer transitions reconcile how lung surfactant can achieve both low surface tensions upon compression and rapid respreading upon expansion and may have important implications toward the optimal design of replacement surfactants. The overlap of function between SP-B and SP-C helps explain why replacement surfactants lacking in one or the other proteins often have beneficial effects.
Assuntos
Pulmão , Membranas Artificiais , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Sequência de Aminoácidos , Ânions/metabolismo , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Ligação Proteica , Proteolipídeos/química , Proteolipídeos/genética , Surfactantes Pulmonares/química , Surfactantes Pulmonares/genética , Tensão Superficial , TemperaturaRESUMO
Protegrins are 2-kDa antimicrobial peptides that contain 16-18 amino acid residues and two intramolecular disulfide bonds. We studied the contribution of these disulfide bonds to the bactericidal activity of protegrins in physiological concentrations of NaCl by comparing protegrin PG-1 with variants that lacked one or both cysteine disulfides. Whereas the bactericidal and liposome-lytic properties of protegrin PG-1 were enhanced by adding 100 mM NaCl to the phosphate-buffered medium, NaCl addition strongly inhibited the effects of its linearized, disulfide-free variant, [A6, A8, A13, A15]protegrin-1. Whereas protegrin PG-1 manifested beta-sheet structure by CD (circular dichroism) and ATR-FTIR (attenuated-total-reflectance-Fourier-transform-infrared) spectroscopy in buffer or membrane-mimetic environments, [A6, A8, A13, A15]protegrin-1 manifested disordered structure in phosphate buffer and alpha-helical characteristics in membrane-mimetic environments. Both single-disulfide protegrin variants, [A8, A13]protegrin-1 and [A6, A15]protegrin-1, assumed beta-sheet conformations with liposomes that simulated bacterial membranes, and both retained substantial bactericidal activity when 100 mM NaCl was present. These findings demonstrate that the intramolecular disulfide bonds of protegrins are required for their antiparallel beta-sheet conformation in membrane-mimetic environments and for their potent antimicrobial activity in media containing NaCl concentrations comparable to those found in serum and extracellular fluids.
Assuntos
Antibacterianos/farmacologia , Dissulfetos/química , Proteínas/química , Proteínas/farmacologia , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos , Bactérias/efeitos dos fármacos , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Fluoresceínas/metabolismo , Lipossomos/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas/genética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Rhesus monkey bone marrow expresses a cathelicidin whose C-terminal domain comprises a 37-residue alpha-helical peptide (RL-37) that resembles human LL-37. Like its human counterpart, RL-37 rapidly permeabilized the membranes of Escherichia coli ML-35p and lysed liposomes that simulated bacterial membranes. When tested in media whose NaCl concentrations approximated those of extracellular fluids, RL-37 was considerably more active than LL-37 against staphylococci. Whereas human LL-37 contains five acidic residues and has a net charge of +6, rhesus RL-37 has only two acidic residues and a net charge of +8. Speculating that the multiple acidic residues of human LL-37 reduced its efficacy against staphylococci, we made a peptide (LL-37 pentamide) in which each aspartic acid of LL-37 was replaced by an asparagine and each glutamic acid was replaced by a glutamine. LL-37 pentamide's antistaphylococcal activity was substantially greater than that of LL-37. Thus, although the precursor of LL-37 is induced in human skin keratinocytes by injury or inflammation, its insufficiently cationic antimicrobial domain may contribute to the success of staphylococci in colonizing and infecting human skin.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Sequência de Bases , Catelicidinas , Dicroísmo Circular , DNA Complementar/análise , Escherichia coli/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Lipossomos/metabolismo , Macaca mulatta , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Homologia de Sequência de Aminoácidos , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.
Assuntos
Pulmão/metabolismo , Ácido Palmítico/farmacologia , Proteolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Tensoativos/química , Adsorção , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Biofísica , Bovinos , Cromatografia Líquida de Alta Pressão , Fenômenos Eletromagnéticos , Humanos , Membranas Artificiais , Microscopia de Força Atômica , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptídeos/química , Dobramento de Proteína , TemperaturaRESUMO
This work reports the first x-ray scattering measurements to determine the effects of SP-B(1-25), the N-terminus peptide of lung surfactant-specific protein SP-B, on the structure of palmitic acid (PA) monolayers. In-plane diffraction shows that the peptide fluidizes a portion of the monolayer but does not affect the packing of the residual ordered phase. This implies that the peptide resides in the disordered phase, and that the ordered phase is essentially pure lipid, in agreement with fluorescence microscopy studies. X-ray reflectivity shows that the peptide is oriented in the lipid monolayer at an angle of approximately 56 degrees relative to the interface normal, with one end protruding past the hydrophilic region into the fluid subphase and the other end embedded in the hydrophobic region of the monolayer. The quantitative insights afforded by this study lead to a better understanding of the lipid/protein interactions found in lung surfactant systems.
Assuntos
Metabolismo dos Lipídeos , Membranas Artificiais , Proteolipídeos/química , Proteolipídeos/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , Difração de Raios X/instrumentação , Difração de Raios X/métodos , Sequência de Aminoácidos , Conformação Molecular , Dados de Sequência Molecular , Ácido Palmítico/metabolismo , Estrutura Terciária de Proteína , Cloreto de Sódio/metabolismo , Síncrotrons , Temperatura , Água/metabolismoRESUMO
Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.
Assuntos
Fosfatidilgliceróis , Proteolipídeos/química , Surfactantes Pulmonares/química , Radioisótopos de Carbono , Dicroísmo Circular , Humanos , Lipossomos , Modelos Moleculares , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Conformação Proteica , Análise de Sequência de Proteína , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-B's function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo.