RESUMO
The primary function of lung surfactant is to form monolayers at the alveolar interface capable of lowering the normal surface tension to near zero. To accomplish this process, the surfactant must be capable of maintaining a coherent, tightly packed monolayer that avoids collapse during expiration. The positively charged amino-terminal peptide SP-B1-25 of lung surfactant-specific protein SP-B increases the collapse pressure of an important component of lung surfactant, palmitic acid (PA), to nearly 70 millinewtons per meter. This alteration of the PA isotherms removes the driving force for "squeeze-out" of the fatty acids from the primarily dipalmitoylphosphatidylcholine monolayers of lung surfactant. An uncharged mutant of SP-B1-25 induced little change in the isotherms, suggesting that a specific charge interaction between the cationic peptide and the anionic lipid is responsible for the stabilization. The effect of SP-B1-25 on fatty acid isotherms is remarkably similar to that of simple poly-cations, suggesting that such polymers might be useful as components of replacement surfactants for the treatment of respiratory distress syndrome.
Assuntos
Proteolipídeos/fisiologia , Surfactantes Pulmonares/fisiologia , 1,2-Dipalmitoilfosfatidilcolina/química , Ácidos Eicosanoicos/química , Humanos , Ácido Palmítico , Ácidos Palmíticos/química , Fragmentos de Peptídeos/química , Proteolipídeos/química , Surfactantes Pulmonares/química , Tensão Superficial , TermodinâmicaRESUMO
Both human lung surfactant protein, SP-B, and its amino-terminal peptide, SP-B1-25, inhibit the formation of condensed phases in monolayers of palmitic acid, resulting in a new fluid phase. This fluid phase forms a network, separating condensed-phase domains at coexistence. The network persists to high surface pressures, altering the nucleation, growth, and morphology of monolayer collapse structures, leading to lower surface tensions on compression and more reversible respreading on expansion. The network is stabilized by the low line tension between the fluid phase and the condensed phase as confirmed by the formation of "stripe" phases.
Assuntos
Ácidos Palmíticos/química , Fragmentos de Peptídeos/química , Proteolipídeos/química , Surfactantes Pulmonares/química , Microscopia de Fluorescência , Ácido Palmítico , Pressão , Propriedades de Superfície , TemperaturaRESUMO
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
The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0 degrees C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospho lipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at -25 degrees C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.
Assuntos
Ácidos Graxos Insaturados , Corantes Fluorescentes , Membranas Intracelulares/ultraestrutura , Lipídeos de Membrana/análise , Mitocôndrias Cardíacas/ultraestrutura , Fosfolipídeos/análise , Animais , Bovinos , Congelamento , Espectrometria de FluorescênciaRESUMO
The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Proteolipídeos/antagonistas & inibidores , Surfactantes Pulmonares/antagonistas & inibidores , Adulto , Sequência de Aminoácidos , Cálcio/farmacologia , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/síntese química , Fosfolipídeos/metabolismo , Proteolipídeos/síntese química , Surfactantes Pulmonares/síntese química , Propriedades de SuperfícieRESUMO
Pulmonary surfactant contains at least three unique proteins: SP-A, SP-B and SP-C. SP-B and SP-C from bovine surfactant are markedly hydrophobic and have molecular masses between 3 and 26 kDa. We identify surfactant proteins under nonreducing conditions on polyacrylamide gels with approximate molecular mass of 5, 14, 26 kDa (SP-5, 14, 26) when organic solvent-soluble material is eluted from a Sephadex LH-20 size exclusion column followed by separation on a high-performance reverse-phase chromatography system. These bands correspond to monomeric SP-C, oligomeric SP-C and oligomeric SP-B, respectively. Computer analysis (Eisenberg-hydrophobic moment) of sequences for these proteins suggests that SP-B contains surface-seeking amphiphilic segments. In contrast, SP-C resembles a more hydrophobic transmembrane anchoring peptide. Dispersions containing dipalmitoylphosphatidylcholine, phosphatidylglycerol, palmitic acid and multimeric SP-B and SP-C duplicate the surface activity of natural surfactant when assayed in a pulsating bubble surfactometer. We speculate that oligomers of SP-B and monomers and oligomers of SP-C may act cooperatively in affecting surfactant function. An important function of SP-B and SP-C may be to affect the ordering of surfactant lipids so that rates of transport of surfactant lipids to the hypophase surface in the alveoli are enhanced.
Assuntos
Proteolipídeos , Surfactantes Pulmonares , Sequência de Aminoácidos , Animais , Líquido da Lavagem Broncoalveolar , Bovinos , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância de Spin Eletrônica , Processamento Eletrônico de Dados , Eletroforese em Gel de Poliacrilamida , Lipídeos/isolamento & purificação , Dados de Sequência Molecular , Oxirredução , Relação Estrutura-AtividadeRESUMO
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
The effect of administration of D-Trp6-Luteinizing Hormone-Releasing Hormone (LH-RH) on synthesis and degradation of cyclic nucleotides was studied in the rat. There were no significant changes in the rate of synthesis and degradation of cyclic AMP in the ovary, testis and pituitary gland of D-Trp6-LH-RH-treated rats as compared to controls. On the other hand, the levels of cyclic GMP and activity of guanylate cyclase were significantly higher in the ovary and testis as well as in the pituitary gland of animals which received the analog. The rate of hydrolysis of cyclic GMP was unchanged by the administration of D-Trp6-LH-RH. Interestingly, the cyclic CMP phosphodiesterase seemed to be activated in animals treated with D-Trp6-LH-RH.
Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Hormônio Liberador de Gonadotropina/análogos & derivados , Ovário/metabolismo , Hipófise/metabolismo , Testículo/metabolismo , Animais , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Guanilato Ciclase/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Prolactina/metabolismo , Ratos , Testosterona/metabolismo , Pamoato de TriptorrelinaRESUMO
The failure of some infants with respiratory distress syndrome to respond to therapy with surfactant may be explained in part by inactivation of surfactant caused by leakage of plasma constituents into air spaces. Surfactant-associated proteins (SP-A, SP-B and SP-C) reduce the susceptibility of surfactants to inactivation in vitro. To study this phenomenon further, we used full length synthetic proteins, SP-B [1-78] and SP-C [1-31], mixed with surfactant lipids in different ratios and different concentrations. Equilibrium and minimum surface tensions of these mixtures, with or without serum and calcium, were measured using a pulsating surfactometer. Mixtures containing both SP-B and SP-C had optimal minimum and equilibrium surface tensions of < 5 and < 28 mN/m, respectively. Mixtures with SP-B had optimal minimum surface tensions, but equilibrium surface tensions averaged 35 mN/m. Mixtures with SP-C had high minimal (19 mN/m) and high equilibrium surface tensions (35 mN/m). When serum was added to these mixtures, the least inactivation was found with mixtures containing 3% protein at 1:1 ratio of SP-B/SP-C with 2 mM calcium chloride. These data indicate that SP-B and SP-C, particularly in the presence of calcium, reduce surfactant inactivation that may be caused by plasma constituents. The results lead to the hypothesis that charge interactions among ions, lipids, surfactant proteins, and serum inactivators are a major element in pathophysiological surfactant inactivation.
Assuntos
Proteolipídeos/sangue , Surfactantes Pulmonares/sangue , Adulto , Sequência de Aminoácidos , Animais , Cloreto de Cálcio/farmacologia , Cães , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Dados de Sequência Molecular , Proteolipídeos/química , Proteolipídeos/fisiologia , Surfactantes Pulmonares/química , Surfactantes Pulmonares/fisiologia , Tensão Superficial/efeitos dos fármacosRESUMO
Functional studies assessed the cytolytic activity of the amino terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37 degrees C, with 40% lysis at 32 microM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526-531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519-524 and residues 526-536) nor a N-terminal flanking peptide (e.g., residues 512-523) induced red cell hemolysis, the entire 23-residue (519-541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4(+)-bearing Hut-78 cells, with 40% lysis at approx. 150 microM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327-328).
Assuntos
Linfócitos T CD4-Positivos/citologia , Proteína gp41 do Envelope de HIV/fisiologia , Hemólise , Fragmentos de Peptídeos/fisiologia , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Morte Celular , Humanos , Meliteno/farmacologia , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis.
Assuntos
Membrana Eritrocítica/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Hemólise , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Dicroísmo Circular , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Análise de Fourier , Proteína gp41 do Envelope de HIV/química , Humanos , Meliteno/farmacologia , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Espectrofotometria Infravermelho , Marcadores de SpinRESUMO
The ability of synthetic peptides based on the amino-terminus of HIV-1 glycoprotein 41,000 (gp41) to fuse human erythrocytes was investigated. Previous site-directed mutagenesis studies have shown an important role for the N-terminal gp41 domain in HIV-fusion, in which replacement of hydrophobic amino acids with polar residues inhibits viral infection and syncytia formation. Here, a synthetic peptide (FP; 23 amino acid residues 519-541) corresponding to the N-terminus of HIV-1 gp41, and also a FP analog (FP526L/R) with Arg replacing Leu-526, were prepared with solid phase techniques. The lipid mixing and leakage of resealed ghosts triggered by these peptides were examined with fluorescence quenching techniques. Peptide-induced aggregation of human erythrocytes was studied using Coulter counter sizing and scanning electron microscopy (SEM). Using resealed erythrocyte ghosts at physiologic pH, FP induces rapid lipid mixing between red cell membranes at doses previously shown to hemolyze intact cells. FP also causes leakage from resealed ghosts, and promotes the formation of multicelled aggregates with whole erythrocytes. Contrarily, similar FP526L/R concentrations did not induce red cell lysis, lipid mixing, leakage or aggregation. Since the fusogenic potency of FP and FP526L/R parallels earlier gp41 mutagenesis studies showing that substitution of Arg for Leu-526 blocks fusion activity, these data suggest that the N-terminal gp41 domain in intact HIV participates in fusion.
Assuntos
Eritrócitos/efeitos dos fármacos , Proteína gp41 do Envelope de HIV/farmacologia , Peptídeos/farmacologia , Agregação Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/ultraestrutura , Proteína gp41 do Envelope de HIV/química , Humanos , Peptídeos/síntese químicaRESUMO
We purified two new minidefensins (RTD-2 and RTD-3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD-1, a circular (theta) defensin previously described by Tang et al. (Science, 286, 498-502, 1999). However, whereas the 18 residues of RTD-1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD-2 and -3 comprise tandem nonapeptide repeats derived from only one of the RTD-1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.
Assuntos
Defensinas/genética , Defensinas/metabolismo , Macaca mulatta/metabolismo , Sequência de Aminoácidos , Animais , Medula Óssea/metabolismo , Clonagem Molecular , Defensinas/química , Dissulfetos/química , Variação Genética , Humanos , Macaca mulatta/genética , Dados de Sequência Molecular , Oxirredução , Peptídeos Cíclicos/síntese química , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids.
Assuntos
Proteínas de Transporte/química , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Proteolipídeos/química , Surfactantes Pulmonares/química , Amidas/química , Sequência de Aminoácidos , Dicroísmo Circular , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oxalatos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Marcadores de Spin , Propriedades de Superfície , Triptofano/químicaRESUMO
Synthetic peptides based on the native human sequence of surfactant protein B have been used to generate polyclonal monospecific antibodies against specific segments of the native SP-B protein. Circular dichroism analysis of the synthetic peptides shows they have a dominant helical content in structure promoting environments and tensiometric measurements indicate these peptides lower surface tension at air-water interfaces implying that they contain amphipathic alpha helical motifs. Antibodies directed against the C-terminal segment of SP-B react with the native protein in the oxidized and reduced state. Antibodies directed against the N-terminal sequence of SP-B react with the native protein only in the reduced state suggesting that this domain has a conformation dependent on disulfide bond formation.
Assuntos
Proteolipídeos/imunologia , Surfactantes Pulmonares/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Técnicas In Vitro , Dados de Sequência Molecular , Peso Molecular , Oxirredução , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Surfactantes Pulmonares/ultraestrutura , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide 'calcitermin' since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl(2). Analysis revealed a putative zinc-binding consensus sequence as well as an alpha-helical conformation in structure-promoting solvents.
Assuntos
Antibacterianos/isolamento & purificação , Mucosa Nasal/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Proteínas S100/isolamento & purificação , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Dicroísmo Circular , Escherichia coli/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos , Conformação Proteica , Proteínas S100/química , Proteínas S100/farmacologia , Homologia de Sequência de Aminoácidos , Espectrofotometria Atômica , Zinco/farmacologiaRESUMO
Structural and functional studies were made to assess interactions between human serum albumin (HSA) and the amino-terminal peptide (FP-I; 23-residue peptide 519-541) of glycoprotein 41,000 (gp41) of human immunodeficiency virus type-1 (HIV-1). Circular dichroism (CD) spectroscopy indicated that the peptide binds to albumin with dominant alpha-helical character. Peptide binding to albumin was also examined using FP-I spin labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). Electron spin resonance (ESR) spectra of FP-II bound to HSA at 38 degrees C indicated that the spin label at the amino-terminal residue (Ala-519) was motionally restricted. The ESR spectrum of 12-nitroxide stearate (12-NS)-labeled HSA was identical to that obtained with FP-II, indicating that the reporter groups for the 12-NS and FP-II probes are similarly bound to albumin. Contrarily, ESR spectra of HSA labeled with FP-III indicated high mobility for the reporter group (Met-537) at the aqueous-protein interface. This suggests that the N-terminal gp41 peptide binds as an alpha helix (residues 519-536) to fatty acid sites on HSA, such that Ala-519 of the peptide residues in the interior of the protein while Met-537 lies outside the protein in aqueous solution. It is also of interest that addition of HSA to human red blood cells dramatically reduced the ability of FP-I to induce hemolysis, presumably through peptide-albumin binding that inhibited FP-I interactions with red cell membranes. The significance of these results focuses on the following three points. The first is that high serum levels of albumin may limit the efficacy of anti-HIV therapies using peptides based on the N-terminal gp41 domain. The second is that the elucidation of FP-I and HSA interactions with physical techniques may provide clues on the molecular features underlying viral FP-I combination with receptors on the target cell surface. Last, the affinity of albumin for the N-terminal gp41 peptide may play a subordinate role in the blocking of HIV infectivity in vitro that has been reported for chemically modified albumins.
Assuntos
Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Albumina Sérica/metabolismo , Sequência de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação/genética , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Hemólise , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Peptídeos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Albumina Sérica/química , Marcadores de SpinRESUMO
Structural and functional studies were performed to assess the membrane actions of peptides based on HIV-1 glycoprotein 41,000 (gp41). Previous site-directed mutagenesis of gp41 has shown that amino acid changes in either the N-terminal fusion or N-leucine zipper region depressed viral infection and syncytium formation, while modifications in the C-leucine zipper domain both increased and decreased HIV fusion. Here, synthetic peptides were prepared corresponding to the N-terminal fusion region (FP-I; gp41 residues 519-541), the nearby N-leucine zipper domain (DP-107; gp41 residues 560-597), and the C-leucine zipper domain (DP-178; gp41 residues 645-680). With erythrocytes, FP-I or DP-107 induced dose-dependent hemolysis and promoted cell aggregation; FP-I was more hemolytic than DP-107, but each was equally effective in aggregating cells. DP-178 produced neither hemolysis nor aggregation, but blocked either FP-I- or DP-107-induced hemolysis and aggregation. Combined with previous nuclear magnetic resonance and Fourier transform infrared spectroscopic results, circular dichroism (CD) spectroscopy showed that the alpha-helicity for these peptides in solution decreased in the order: DP-107 >> DP-178 > FP-I. CD analysis also indicated binding of DP-178 to either DP-107 or FP-I. Consequently, DP-178 may inhibit the membrane actions mediated by either FP-I or DP-107 through direct peptide interactions in solution. These peptide results suggest that the corresponding N-terminal fusion and N-leucine zipper regions participate in HIV infection, by promoting membrane perturbations underlying the merging of the viral envelope with the cell surface. Further, the C-leucine zipper domain in "prefusion" HIV may inhibit these membrane activities by interacting with the N-terminal fusion and N-leucine zipper domains in unactivated gp41. Last, exogenous DP-178 may bind to the N-terminal and N-leucine zipper domains of gp41 that become exposed on HIV stimulation, thereby preventing the fusogenic actions of these gp41 regions leading to infection.