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1.
Soft Matter ; 20(29): 5745-5753, 2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-38982765

RESUMO

Pulmonary surfactant forms a thin film on the liquid that lines the alveolar air-sacks. When compressed by the decreasing alveolar surface area during exhalation, the films avoid collapse from the air/water interface and reduce surface tension to exceptionally low levels. To define better the structure of compressed films that determines their susceptibility to collapse, we measured how cholesterol affects the structure and collapse of dipalmitoyl phosphatidylcholine (DPPC) monolayers at physiological temperatures. Grazing incidence X-ray diffraction (GIXD) and grazing incidence X-ray off-specular scattering (GIXOS) established the lateral and transverse structures of films on a Langmuir trough at a surface pressure of 45 mN m-1, just below the equilibrium spreading pressure at which collapse begins. Experiments with captive bubbles at a surface pressure of 51 mN m-1 measured how the steroid affects isobaric collapse. Mol fractions of the steroid (Xchol) at 0.05 removed the tilt by the acyl chains of DPPC, shifted the unit cell from centered rectangular to hexagonal, and dramatically decreased the long-range order. Higher Xchol produced no further change in diffraction, suggesting that cholesterol partitions into a coexisting disordered phase. Cholesterol had minimal effect on rates of collapse until Xchol reached 0.20. Our results demonstrate that the decreased coherence length, indicating conversion of positional order to short-range, is insufficient to make a condensed monolayer susceptible to collapse. Our findings suggest a two-step process by which cholesterol induces disorder. The steroid would first convert the film with crystalline chains to a hexatic phase before generating a fully disordered structure that is susceptible to collapse. These results lead to far-reaching consequences for formulation of animal-derived therapeutic surfactants. Our results suggest that removal of cholesterol from these preparations should be unnecessary below Xchol = 0.20.


Assuntos
1,2-Dipalmitoilfosfatidilcolina , Colesterol , Surfactantes Pulmonares , Temperatura , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química
2.
Langmuir ; 39(36): 12541-12549, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37647566

RESUMO

Aberrant levels of glycolipids expressed on cellular surfaces are characteristic of different types of cancers. The oligomer of acylated lysine (OAK) mimicking antimicrobial peptides displays in vitro activity against human and murine melanoma cell lines with upregulated GD3 and GM3 gangliosides. Herein, we demonstrate the capability of OAK to intercalate into the sialo-oligosaccharides of DPPC/GD3 and DPPC/GM3 lipid monolayers using X-ray scattering. The lack of insertion into monolayers containing phosphatidylserine suggests that the mechanism of action by OAKs against glycosylated lipid membranes is not merely driven by charge effects. The fluorescence microscopy data demonstrates the membrane-lytic activity of OAK. Understanding the molecular basis for selectivity toward GD3 and GM3 gangliosides by antimicrobial lipopeptides will contribute to the development of novel therapies to cure melanoma and other malignancies.


Assuntos
Gangliosídeos , Melanoma , Humanos , Animais , Camundongos , Gangliosídeos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Glicolipídeos , Transporte Biológico
3.
Biophys J ; 121(18): 3533-3541, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-35841141

RESUMO

Cholesterol induces faster collapse by compressed films of pulmonary surfactant. Because collapse prevents films from reaching the high surface pressures achieved in the alveolus, most therapeutic surfactants remove or omit cholesterol. The studies here determined the structural changes by which cholesterol causes faster collapse by films of dipalmitoyl phosphatidylcholine, used as a simple model for the functional alveolar film. Measurements of isobaric collapse, with surface pressure held constant at 52 mN/m, showed that cholesterol had little effect until the mol fraction of cholesterol, Xchol, exceeded 0.20. Structural measurements of grazing incidence X-ray diffraction at ambient laboratory temperatures and a surface pressure of 44 mN/m, just below the onset of collapse, showed that the major structural change in an ordered phase occurred at lower Xchol. A centered rectangular unit cell with tilted chains converted to an untilted hexagonal structure over the range of Xchol = 0.0-0.1. For Xchol = 0.1-0.4, the ordered structure was nearly invariant; the hexagonal unit cell persisted, and the spacing of the chains was essentially unchanged. That invariance strongly suggests that above Xchol = 0.1, cholesterol partitions into a disordered phase, which coexists with the ordered domains. The phase rule requires that for a binary film with coexisting phases, the stoichiometries of the ordered and disordered regions must remain constant. Added cholesterol must increase the area of the disordered phase at the expense of the ordered regions. X-ray scattering from dipalmitoyl phosphatidylcholine/cholesterol fit with that prediction. The data also show a progressive decrease in the size of crystalline domains. Our results suggest that cholesterol promotes adsorption not by altering the unit cell of the ordered phase but by decreasing both its total area and the size of individual crystallites.


Assuntos
1,2-Dipalmitoilfosfatidilcolina , Surfactantes Pulmonares , 1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Pressão , Surfactantes Pulmonares/química , Tensoativos
4.
Langmuir ; 36(45): 13439-13447, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33080138

RESUMO

When compressed by the shrinking alveolar surface area during exhalation, films of pulmonary surfactant in situ reduce surface tension to levels at which surfactant monolayers collapse from the surface in vitro. Vesicles of pulmonary surfactant added below these monolayers slow collapse. X-ray scattering here determined the structural changes induced by the added vesicles. Grazing incidence X-ray diffraction on monolayers of extracted calf surfactant detected an ordered phase. Mixtures of dipalmitoyl phosphatidylcholine and cholesterol, but not the phospholipid alone, mimic that structure. At concentrations that stabilize the monolayers, vesicles in the subphase had no effect on the unit cell, and X-ray reflection showed that the film remained monomolecular. The added vesicles, however, produced a concentration-dependent increase in the diffracted intensity. These results suggest that the enhanced resistance to collapse results from enlargement by the additional material of the ordered phase.


Assuntos
Surfactantes Pulmonares , 1,2-Dipalmitoilfosfatidilcolina , Fosfolipídeos , Propriedades de Superfície , Tensão Superficial , Tensoativos
5.
ACS Infect Dis ; 5(7): 1214-1222, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31083918

RESUMO

Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ two-component regulatory system that increases resistance to both conventional antibiotics and antimicrobial peptides (AMPs). Acquired resistance to AMPs is contrary to the established narrative that AMPs circumvent bacterial resistance by targeting the general chemical properties of membrane lipids. However, the specific mechanisms underlying AMP resistance remain elusive. Here we report a 2-fold increase in bacteriostatic concentrations of human AMP LL-37 for S. enterica with modified LPSs. LPSs with and without chemical modifications were isolated and investigated by Langmuir films coupled with grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The initial interactions between LL-37 and LPS bilayers were probed using all-atom molecular dynamics simulations. These simulations suggest that initial association is nonspecific to the type of LPS and governed by hydrogen bonding to the LPS outer carbohydrates. GIXD experiments indicate that the interactions of the peptide with monolayers reduce the number of crystalline domains but greatly increase the typical domain size in both LPS isoforms. Electron densities derived from XR experiments corroborate the bacteriostatic values found in vitro and indicate that peptide intercalation is reduced by LPS modification. We hypothesize that defects at the liquid-ordered boundary facilitate LL-37 intercalation into the outer membrane, whereas PhoPQ-mediated LPS modification protects against this process by having innately increased crystallinity. Since induced ordering has been observed with other AMPs and drugs, LPS modification may represent a general mechanism by which Gram-negative bacteria protect against host innate immunity.


Assuntos
Membrana Externa Bacteriana/química , Catelicidinas/farmacologia , Lipopolissacarídeos/química , Salmonella enterica/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos , Membrana Externa Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Humanos , Ligação de Hidrogênio , Lipopolissacarídeos/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Domínios Proteicos , Salmonella enterica/química , Difração de Raios X
6.
Biopolymers ; 110(6): e23274, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30892696

RESUMO

Synthetic polymers mimicking antimicrobial peptides have drawn considerable interest as potential therapeutics. N-substituted glycines, or peptoids, are recognized by their in vivo stability and ease of synthesis. Peptoids are thought to act primarily on the negatively charged lipids that are abundant in bacterial cell membranes. A mechanistic understanding of lipid-peptoid interaction at the molecular level will provide insights for rational design and optimization of peptoids. Here, we highlight recent studies that utilize synchrotron liquid surface X-ray scattering to characterize the underlying peptoid interactions with bacterial and eukaryotic membranes. Cellular membranes are highly complex, and difficult to characterize at the molecular level. Model systems including Langmuir monolayers, are used in these studies to reduce system complexity. The general workflow of these systems and the corresponding data analysis techniques are presented alongside recent findings. These studies investigate the role of peptoid physicochemical characteristics on membrane activity. Specifically, the roles of cationic charge, conformational constraint via macrocyclization, and hydrophobicity are shown to correlate their membrane interactions to biological activities in vitro. These structure-activity relationships have led to new insights into the mechanism of action by peptoid antimicrobials, and suggest optimization strategies for future therapeutics based on peptoids.


Assuntos
Desenho de Fármacos , Peptoides/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Ciclização , Interações Hidrofóbicas e Hidrofílicas , Peptoides/metabolismo , Fosfatidilgliceróis/química , Termodinâmica , Raios X
7.
Biochim Biophys Acta Biomembr ; 1860(6): 1414-1423, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29621496

RESUMO

Hydrophobic interactions govern specificity for natural antimicrobial peptides. No such relationship has been established for synthetic peptoids that mimic antimicrobial peptides. Peptoid macrocycles synthesized with five different aromatic groups are investigated by minimum inhibitory and hemolytic concentration assays, epifluorescence microscopy, atomic force microscopy, and X-ray reflectivity. Peptoid hydrophobicity is determined using high performance liquid chromatography. Disruption of bacterial but not eukaryotic lipid membranes is demonstrated on the solid supported lipid bilayers and Langmuir monolayers. X-ray reflectivity studies demonstrate that intercalation of peptoids with zwitterionic or negatively charged lipid membranes is found to be regulated by hydrophobicity. Critical levels of peptoid selectivity are demonstrated and found to be modulated by their hydrophobic groups. It is suggested that peptoids may follow different optimization schemes as compared to their natural analogues.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Lipídeos de Membrana/química , Ânions/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cromatografia Líquida de Alta Pressão , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Estrutura Molecular , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato
8.
Langmuir ; 32(48): 12905-12913, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27793068

RESUMO

The peptidomimetic approach has emerged as a powerful tool for overcoming the inherent limitations of natural antimicrobial peptides, where the therapeutic potential can be improved by increasing the selectivity and bioavailability. Restraining the conformational flexibility of a molecule may reduce the entropy loss upon its binding to the membrane. Experimental findings demonstrate that the cyclization of linear antimicrobial peptoids increases their bactericidal activity against Staphylococcus aureus while maintaining high hemolytic concentrations. Surface X-ray scattering shows that macrocyclic peptoids intercalate into Langmuir monolayers of anionic lipids with greater efficacy than for their linear analogues. It is suggested that cyclization may increase peptoid activity by allowing the macrocycle to better penetrate the bacterial cell membrane.


Assuntos
Antibacterianos/farmacologia , Peptoides/farmacologia , Membrana Celular/efeitos dos fármacos , Ciclização , Staphylococcus aureus/efeitos dos fármacos
9.
J Am Chem Soc ; 138(38): 12432-9, 2016 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-27598340

RESUMO

Achieving structurally well-defined catalytic species requires a fundamental understanding of surface chemistry. Detailed structural characterization of the catalyst binding sites in situ, such as single site catalysts on silica supports, is technically challenging or even unattainable. Octadecyltrioxysilane (OTOS) monolayers formed from octadecyltrimethoxysilane (OTMS) at the air-liquid interface after hydrolysis and condensation at low pH were chosen as a model system of surface binding sites in silica-supported Zn(2+) catalysts. We characterize the system by grazing incidence X-ray diffraction, X-ray reflectivity (XR), and X-ray fluorescence spectroscopy (XFS). Previous X-ray and infrared surface studies of OTMS/OTOS films at the air-liquid interface proposed the formation of polymer OTOS structures. According to our analysis, polymer formation is inconsistent with the X-ray observations and structural properties of siloxanes; it is energetically unfavorable and thus highly unlikely. We suggest an alternative mechanism of hydrolysis/condensation in OTMS leading to the formation of structurally allowed cyclic trimers with the six-membered siloxane rings, which explain well both the X-ray and infrared results. XR and XFS consistently demonstrate that tetrahedral [Zn(NH3)4](2+) ions bind to hydroxyl groups of the film at a stoichiometric ratio of OTOS:Zn ∼ 2:1. The high binding affinity of zinc ions to OTOS trimers suggests that the six-membered siloxane rings are binding locations for single site Zn/SiO2 catalysts. Our results show that OTOS monolayers may serve as a platform for studying silica surface chemistry or hydroxyl-mediated reactions.

10.
Ann N Y Acad Sci ; 1374(1): 111-22, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27244102

RESUMO

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1-2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K-dependent and -independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.


Assuntos
Varfarina/efeitos adversos , Varfarina/análise , Animais , Anticoagulantes/efeitos adversos , Anticoagulantes/análise , Anticoagulantes/química , Biomarcadores/análise , Exposição Ambiental/análise , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/patologia , Varfarina/química , Varfarina/intoxicação
11.
Biophys J ; 110(8): 1777-1788, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27119638

RESUMO

Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.


Assuntos
4-Hidroxicumarinas/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Ratos
12.
Biophys J ; 109(12): 2537-2545, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26682812

RESUMO

Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted as monolayers at the air-water interface, and their properties, as well as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region, but was prevented from this penetration into the modified lipopolysaccharides. Results correlate with behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Novobiocina/farmacologia , Salmonella/citologia , Salmonella/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeo A/metabolismo , Novobiocina/química , Novobiocina/metabolismo , Permeabilidade
13.
Biochim Biophys Acta ; 1838(10): 2492-2502, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24878450

RESUMO

Antimicrobial peptides or their synthetic mimics are a promising class of potential new antibiotics. Herein we assess the effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial α-peptide-ß-peptoid chimeras. Langmuir monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) were used to model cytoplasmic membranes of both Gram-positive and Gram-negative bacteria, while lipopolysaccharide Kdo2-lipid A monolayers were mimicking the outer membrane of Gram-negative species. We report the results of the measurements using an array of techniques, including high-resolution synchrotron surface X-ray scattering, epifluorescence microscopy, and in vitro antimicrobial activity to study the molecular mechanisms of peptidomimetic interaction with bacterial membranes. We found guanidino group-containing chimeras to exhibit greater disruptive activity on DPPG monolayers than the amino group-containing analogues. However, this effect was not observed for lipopolysaccharide monolayers where the difference was negligible. Furthermore, the addition of the nitrobenzoxadiazole fluorophore did not reduce the insertion activity of these antimicrobials into both model membrane systems examined, which may be useful for future cellular localization studies.


Assuntos
Anti-Infecciosos/química , Bactérias/química , Membrana Celular/química , Guanidina/química , Peptidomiméticos/química , Anti-Infecciosos/farmacologia , Bactérias/metabolismo , Membrana Celular/metabolismo , Guanidina/farmacologia , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Peptidomiméticos/farmacologia , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo
14.
Phys Rev Lett ; 108(23): 238103, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-23003994

RESUMO

Biomembranes undergo extensive shape changes as they perform vital cellular functions. The mechanisms by which lipids and proteins control membrane curvature remain unclear. We use x-ray reflectivity, grazing incidence x-ray diffraction, and epifluorescence microscopy to study binding of HIV-1 glycoprotein gp41's membrane-bending domain to DPPC/cholesterol monolayers of various compositions at the air-liquid interface. The results offer a new insight into how membrane curvature could be regulated by cholesterol during fusion of the viral lipid envelope and the host cell membranes.


Assuntos
Membrana Celular/química , Colesterol/química , Fusão de Membrana , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Colesterol/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Difração de Raios X
15.
Biochim Biophys Acta ; 1818(11): 2521-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22692008

RESUMO

Membrane-activity of the glycoprotein 41 membrane-proximal external region (MPER) is required for HIV-1 membrane fusion. Consequently, its inhibition results in viral neutralization by the antibody 4E10. Previous studies suggested that MPER might act during fusion by locally perturbing the viral membrane, i.e., following a mechanism similar to that proposed for certain antimicrobial peptides. Here, we explore the molecular mechanism of how MPER permeates lipid monolayers containing cholesterol, a main component of the viral envelope, using grazing incidence X-ray diffraction and X-ray reflectivity. Our studies reveal that helical MPER forms lytic pores under conditions not affecting the lateral packing order of lipids. Moreover, we observe an increment of the surface area occupied by MPER helices in cholesterol-enriched membranes, which correlates with an enhancement of the 4E10 epitope accessibility in lipid vesicles. Thus, our data support the view that curvature generation by MPER hydrophobic insertion into the viral membrane is functionally more relevant than lipid packing disruption.


Assuntos
Colesterol/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Dicroísmo Circular , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/química , HIV-1/imunologia , HIV-1/fisiologia , Fusão de Membrana/fisiologia , Dados de Sequência Molecular , Difração de Raios X
16.
Biophys J ; 101(10): 2426-35, 2011 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-22098741

RESUMO

Electron microscopy structural determinations suggest that the membrane-proximal external region (MPER) of glycoprotein 41 (gp41) may associate with the HIV-1 membrane interface. It is further proposed that MPER-induced disruption and/or deformation of the lipid bilayer ensue during viral fusion. However, it is predicted that the cholesterol content of this membrane (∼45 mol %) will act against MPER binding and restructuring activity, in agreement with alternative structural models proposing that the MPER constitutes a gp41 ectodomain component that does not insert into the viral membrane. Here, using MPER-based peptides, we test the hypothesis that cholesterol impedes the membrane association and destabilizing activities of this gp41 domain. To that end, partitioning and leakage assays carried out in lipid vesicles were combined with x-ray reflectivity and grazing-incidence diffraction studies of monolayers. CpreTM, a peptide combining the carboxyterminal MPER sequence with aminoterminal residues of the transmembrane domain, bound and destabilized effectively cholesterol-enriched membranes. Accordingly, virion incubation with this peptide inhibited cell infection potently but nonspecifically. Thus, CpreTM seems to mimic the envelope-perturbing function of the MPER domain and displays antiviral activity. As such, we infer that CpreTM bound to cholesterol-enriched membranes would represent a relevant target for anti-HIV-1 immunogen and inhibitor development.


Assuntos
Capsídeo/metabolismo , Membrana Celular/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Colesterol/química , HIV-1/patogenicidade , Membranas Artificiais , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosfatidilcolinas/química , Relação Estrutura-Atividade , Difração de Raios X
18.
Phys Rev Lett ; 104(10): 108101, 2010 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-20366454

RESUMO

We report a structural study of cholesterol-DPPC (1,2-dipalmitoyl-sn-glycero-3-phophocholine) monolayers using x-ray reflectivity and grazing incidence x-ray diffraction. Reflectivity reveals that the vertical position of cholesterol relative to phospholipids strongly depends on its mole fraction (chi(CHOL)). Moreover, we find that at a broad range of chi(CHOL) cholesterol and DPPC form alloylike mixed domains of short-range order and the same stoichiometry as that of the film. Based on the data presented, we propose a new model of cholesterol-phospholipid organization in mixed monolayers.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Modelos Químicos , Difração de Raios X
19.
FASEB J ; 24(6): 1904-13, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20124435

RESUMO

Oligomers of acylated lysines (OAKs) are synthetic mimics of host defense peptides (HDPs) with promising antimicrobial properties. Here we challenged the OAK concept for its ability to generate both systemically efficient and economically viable lead compounds for fighting multidrug-resistant bacteria. We describe the design and characterization of a miniature OAK composed of only 3 lysyls and 2 acyls (designated C(12(omega7))K-beta(12)) that preferentially targets gram-positive species by a bacteriostatic mode of action. To gain insight into the mechanism of action, we examined the interaction of OAK with various potential targets, including phospholipid bilayers, using surface plasmon resonance, and Langmuir monolayers, using insertion assays, epifluorescence microscopy, and grazing incidence X-ray diffraction, in a complementary manner. Collectively, the data support the notion that C(12(omega7))K-beta(12) damages the plasma-membrane architecture similarly to HDPs, that is, following a near-classic 2-step interaction including high-affinity electrostatic adhesion and a subsequent shallow insertion that was limited to the phospholipid head group region. Notably, preliminary acute toxicity and efficacy studies performed with mouse models of infection have consolidated the potential of OAK for treating bacterial infections, including systemic treatments of methicillin-resistant Staphylococcus aureus. Such simple yet robust chemicals might be useful for various antibacterial applications while circumventing potential adverse effects associated with cytolytic compounds.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Biomimética , Animais , Fibroblastos/metabolismo , Lisina/química , Masculino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Ressonância de Plasmônio de Superfície , Difração de Raios X
20.
Biochim Biophys Acta ; 1798(5): 851-60, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19800862

RESUMO

This work investigates the discrimination of lipid monolayers by the ovine antimicrobial peptide SMAP-29 and compares it to that of the human LL-37 peptide. Fluid phospholipid monolayers were formed in a Langmuir trough and subsequently studied with the X-ray scattering techniques of X-ray reflectivity and grazing incidence X-ray diffraction. Any changes in the phospholipid structure after injection of peptide under the monolayer were considered to be due to interactions between the peptides and lipids. The data show that SMAP-29 discriminates against negatively charged phospholipids in a similar way to LL-37. However, it is even more interesting to note that despite a higher concentration of SMAP-29 near the monolayer, ensured by its greater charge as compared to LL-37, the amount of SMAP-29 needed to observe monolayer disruption was around three and a half times the number of molecules of LL-37 used to see similar changes with the same system. This result suggests that the structure, amino acid sequence or size of the peptide may well be as important as electrical charge and therefore gives many implications for the further study of antimicrobial peptides with regards to novel drug design and development.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas Sanguíneas/química , Lipídeos/química , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Catelicidinas , Desenho de Fármacos , Humanos , Ovinos , Água/química , Difração de Raios X
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