RESUMEN
NK-2 is an antimicrobial peptide derived from helices 3 and 4 of the pore-forming protein of natural killer cells, NK-lysin. It has potent activities against Gram-negative and Gram-positive bacteria, fungi and protozoan parasites without being toxic to healthy human cells. In biophysical assays its membrane activities were found to require phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), lipids which dominate the composition of bacterial membranes. Here the structure and activities of NK-2 in binary mixtures of different PE/PG composition were investigated. CD spectroscopy reveals that a threshold concentration of 50 % PG is needed for efficient membrane association of NK-2 concomitant with a random coil - helix transition. Association with PE occurs but is qualitatively different when compared to PG membranes. Oriented solid-state NMR spectroscopy of NK-2 specifically labelled with 15N indicates that the NK-2 helices are oriented parallel to the PG bilayer surface. Upon reduction of the PG content to 20 mol% interactions are weaker and/or an in average more tilted orientation is observed. Fluorescence spectroscopy of differently labelled lipids is in agreement of an interfacial localisation of both helices where the C-terminal end is in a less hydrophobic environment. By inserting into the membrane interface and interacting differently with PE and PG the peptides probably induce high curvature strain which result in membrane openings and rupture.
Asunto(s)
Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Membrana Dobles de Lípidos , Fosfatidiletanolaminas , Proteolípidos , Humanos , Membrana Dobles de Lípidos/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Péptidos/químicaRESUMEN
Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.
Asunto(s)
Catelicidinas/farmacología , Membrana Celular/metabolismo , Interacciones Huésped-Patógeno , Lipopolisacáridos/farmacología , Pruebas de Neutralización , Polimixina B/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , Femenino , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer's disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.
Asunto(s)
Proteína ADAM10/metabolismo , Membrana Celular/metabolismo , Activación Enzimática , Proteína ADAM10/química , Secuencia de Aminoácidos , Animales , Anoctaminas/metabolismo , Biomarcadores , Células COS , Línea Celular , Chlorocebus aethiops , Eritrocitos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Fosfoserina/metabolismo , Conejos , Relación Estructura-ActividadRESUMEN
ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates including TGF-alpha, Amphiregulin (AREG) and TNF-Receptor 1 (TNFR1). We recently presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. Anoctamin-6 (ANO6) has Ca2+-dependent phospholipid scramblase activity and it followed that the functions of ANO6 and ADAM17 might be linked. We report that overexpression of ANO6 in HEK293T cells led to increased Ca2+-mediated PS-exposure that was indeed accompanied by enhanced release of AREG and TGF-alpha. The effect was not observed when cells were treated with the PKC-dependent ADAM17 activator PMA. Transformation of cells with a constitutively active ANO6 mutant led to spontaneous PS-exposure and to the release of ADAM17-substrates in the absence of any stimuli. Inhibitor experiments indicated that ANO6-mediated enhancement of substrate cleavage simultaneously broadened the spectrum of participating metalloproteinases. In complementary experiments, siRNA-mediated downregulation of ANO6 was shown to decrease ionophore-mediated release of TNFR1 in human umbilical vein endothelial cells (HUVECs). We conclude that ANO6, by virtue of its scramblase activity, may play a role as an important regulator of the ADAM-network in the plasma membrane.
Asunto(s)
Proteínas ADAM/metabolismo , Anoctaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Proteína ADAM17/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ionomicina/farmacología , Modelos Biológicos , Mutación , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi-)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti-cancer therapeutics. Peptide NK-2, derived from porcine NK-lysin, was originally discovered due to its broad-spectrum antimicrobial activities. Today, also potent anti-cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non-abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK-2 and structurally improved anti-cancer variants thereof against two patient-derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle-based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface-exposed phosphatidylserine is of crucial importance for the activity of peptide NK-2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Antineoplásicos/farmacología , Colon/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Péptidos/farmacología , Fosfatidilserinas/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Pollos , Colon/metabolismo , Colon/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Liposomas/química , Especificidad de Órganos , Péptidos/química , Péptidos/metabolismo , Fosfatidilserinas/química , Electricidad Estática , Propiedades de Superficie , PorcinosRESUMEN
ADAM17, a prominent member of the 'Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca(2+) elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function.
Asunto(s)
Proteína ADAM17/metabolismo , Fosfatidilserinas/metabolismo , Proteína ADAM17/química , Proteína ADAM17/deficiencia , Proteína ADAM17/genética , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Trastornos de la Coagulación Sanguínea/sangre , Trastornos de la Coagulación Sanguínea/genética , Línea Celular , Activación Enzimática , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Meliteno/farmacología , Ratones , Ratones Noqueados , Modelos Biológicos , Dominios Proteicos , Especificidad por SustratoRESUMEN
Host defense peptides (HDP) constitute effector molecules of the innate immune system. Besides acting against microbia and fungi, they exhibit broad and selective oncolytic activity. The underlying mechanism is at least partially attributable to elevated surface-exposed levels of phosphatidylserine (PS) on tumor targets. In this study, comprehensive analysis of NK-2-based derivatives (C7A, C7A-D21K, and C7A-Δ) was done on patient-derived ultra-low passage colorectal carcinoma (CRC) cell lines. Peptides were designed to improve antitumoral potential. Mellitin was used as positive control and a non-toxic peptide (NK11) served as negative control. Subsequently, effectiveness of local HDP application was determined in xenopatients. Generally, CRC lines displayed a heterogeneous pattern of surface-exposed PS, which was usually below standard CRC cells. Of note, five out of seven cell lines were susceptible towards HDP-mediated lysis (lytic activity of peptides: C7A-D21K > C7A-Δ= C7A). Oncolytic activity correlated mostly with surface-exposed PS levels. Apoptosis as well as necrosis were involved in killing. In an in vivo experiment, substantial growth inhibition of HROC24 xenografts was observed after HDP therapy and, surprisingly, also after NK11 treatment. These promising data underline the high potential of HDPs for oncolytic therapies and may provide a rationale for optimizing preclinical treatment schedules based on NK-2.
Asunto(s)
Neoplasias Colorrectales/terapia , Péptidos/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Péptidos/inmunologíaRESUMEN
Antimicrobial peptides (AMPs) are important effector molecules of the innate immune system of all species. AMPs are highly selective and can be used as lead structures for the development of new drugs complementing standard antibiotic therapies. Understanding the crucial parameters of peptide-membrane interactions is necessary for elucidation of the molecular mechanisms of action. Phospholipid monolayers, as simple 2D models of the membrane surface, can be effectively used for studies of peptide-membrane interactions. The present study is focused on the recently discovered peptide arenicin-1 (Ar-1), which possesses antibacterial and antifungal activities. A linear derivative with serine residues instead of cysteines (C/S-Ar-1) was additionally used to investigate the influence of the AMP on the phase behavior of lipid monolayers at the air/liquid interface. Using the Langmuir balance technique and IRRAS allows us to conclude that both original and modified arenicins reveal a strong influence on the phase transition of anionic phospholipids (fluidization of the lipid hydrocarbon chains), whereas the thermodynamic properties of the zwitterionic phospholipid layers are not affected. A strong effect of the modified peptide on the ordering of negatively charged phospholipids at the air-water interface compared to zwitterionic phospholipids has been observed using GIXD measurements, supported by IRRAS simulations for the spectral range corresponding to the lipid hydrocarbon chains. At lateral pressures above 30 mN/m, both peptides are squeezed out from zwitterionic lipid monolayers, but remains attached to and partly incorporated in anionic lipid monolayers. This study points at the importance of the interplay between hydrophobic and electrostatic interactions for the membrane disruption by AMPs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteínas del Helminto/química , Fosfolípidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Transición de Fase , Electricidad Estática , Propiedades de SuperficieRESUMEN
Equine sarcoid is a topically accessible model for the evaluation of anticancer peptides acting by physical membrane disruption avoiding the complexity of a systemic application. We aim at evaluating and improving natural peptides for host defence as lead structures, where we focus on the cationic and amphipathic peptide NK-2. Cytotoxicity tests, fluorescence microscopy and a chip-based biosensor, which enabled real-time monitoring of cell metabolism, were applied. Cancer cell killing was dynamic with an initial phase of increased cellular respiration, followed by membrane destruction. NK-2 was substantially improved and shortened. Novel peptides exhibited a fivefold improved activity against sarcoid cells, while haemolysis remained almost unaltered. Similar Zeta potential and similar amount of surface phosphatidylserine of sarcoid and normal skin cells are responsible for a lack of selectivity between these two cell types.
Asunto(s)
Caballos/virología , Microscopía Fluorescente , Péptidos/química , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Técnicas Biosensibles , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Hemólisis , Humanos , Péptidos/síntesis química , Péptidos/farmacología , Fosfatidilserinas/química , Neoplasias Cutáneas/metabolismoRESUMEN
Most chemotherapeutics harm normal cells causing severe side effects and induce the development of resistance in cancer cells. Antimicrobial peptides (AMPs), recognized as anti-cancer agents, may overcome these limitations. The most studied mechanism underlying multi-drug resistance (MDR) is the over-expression of cell membrane transporter P-glycoprotein (P-gp), which extrudes a variety of hydrophobic drugs. Additionally, P-gp contributes to cell membrane composition and increases the net negative charge on cell surface. We postulated that NK-lysin derived cationic peptide NK-2 might discriminate and preferentially eliminate P-gp over-expressing cancer cells. To test this hypothesis, we employed MDR non-small cell lung carcinoma (NCI-H460/R) and colorectal carcinoma (DLD1-TxR) cell lines with high P-gp expression. MDR cancer cells that survived NK-2 treatment had decreased P-gp expression and were more susceptible to doxorubicin. We found that NK-2 more readily eliminated P-gp high-expressing cells. Acting in 'carpet-like' manner NK-2 co-localized with P-gp on the MDR cancer cell membrane. The inhibition of P-gp reduced the NK-2 effect in MDR cancer cells and, vice versa, NK-2 decreased P-gp transport activity. In conclusion, NK-2 could modulate MDR in unique way, eliminating the P-gp high-expressing cells from heterogeneous cancers and making them more vulnerable to classical drug treatment.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Péptidos/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Proteolípidos/metabolismoRESUMEN
Some antimicrobial peptides have emerged as potential anticancer agents. In contrast to chemotherapeutics, they act primarily by physical disruption of the cancer cell membrane. Selective targeting of these cationic peptides still remains elusive. We focus on the interaction of α-helical peptides NK-2, cathelicidin LL32, and melittin with PC-3 prostate cancer cells, and we provide strong evidence that, amongst the anionic glycans covering the cell surface, sulphated carbohydrates rather than sialic acids are the preferred interaction sites of the peptides. To test the significance of cell surface carbohydrates, a glycan microarray screen with fluorescently labelled peptides has been performed. Amongst 465 mammalian glycan structures on the chip, more than 20 different sulphated glycans were detected as the preferred binding partners of the peptide NK-2. The amount of peptide bound to sialic acid containing oligosaccharides was close to background level. These findings were consistent with microcalorimetric experiments revealing high and low binding enthalpies of peptides to sulphated carbohydrates and to sialic acid, respectively. Enzymatic desialylation of PC-3 cells did not affect peptide-mediated changes in cell metabolism, cell membrane permeabilisation, killing rate, and kinetics. Finally, the cytotoxicity of all peptides could be drastically impaired through the competitive inhibition by chondroitin sulphate, but not by sialic acid and sialylated fetuin.
Asunto(s)
Antineoplásicos/farmacología , Membrana Celular/efectos de los fármacos , Péptidos/farmacología , Polisacáridos/química , Polisacáridos/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/farmacología , Secuencia de Carbohidratos , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Masculino , Meliteno/farmacología , Datos de Secuencia Molecular , Neoplasias de la Próstata/química , Neoplasias de la Próstata/metabolismo , Ácidos Siálicos/metabolismo , Sulfatos/química , Sulfatos/metabolismo , CatelicidinasRESUMEN
The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Pared Celular/efectos de los fármacos , Bacterias/inmunología , Bacterias/metabolismo , Pared Celular/inmunología , Pared Celular/metabolismo , HumanosRESUMEN
The first barrier that an antimicrobial agent must overcome when interacting with its target is the microbial cell wall. In the case of Gram-negative bacteria, additional to the cytoplasmic membrane and the peptidoglycan layer, an outer membrane (OM) is the outermost barrier. The OM has an asymmetric distribution of the lipids with phospholipids and lipopolysaccharide (LPS) located in the inner and outer leaflets, respectively. In contrast, Gram-positive bacteria lack OM and possess a much thicker peptidoglycan layer compared to their Gram-negative counterparts. An additional class of amphiphiles exists in Gram-positives, the lipoteichoic acids (LTA), which may represent important structural components. These long molecules cross-bridge the entire cell envelope with their lipid component inserting into the outer leaflet of the cytoplasmic membrane and the teichoic acid portion penetrating into the peptidoglycan layer. Furthermore, both classes of bacteria have other important amphiphiles, such as lipoproteins, whose importance has become evident only recently. It is not known yet whether any of these amphiphilic components are able to stimulate the immune system under physiological conditions as constituents of intact bacteria. However, all of them have a very high pro-inflammatory activity when released from the cell. Such a release may take place through the interaction with the immune system, or with antibiotics (particularly with those targeting cell wall components), or simply by the bacterial division. Therefore, a given antimicrobial agent must ideally have a double character, namely, it must overcome the bacterial cell wall barrier, without inducing the liberation of the pro-inflammatory amphiphiles. Here, new data are presented which describe the development and use of membrane-active antimicrobial agents, in particular antimicrobial peptides (AMPs) and lipopolyamines. In this way, essential progress was achieved, in particular with respect to the inhibition of deleterious consequences of bacterial infections such as severe sepsis and septic shock.
Asunto(s)
Antiinfecciosos/farmacología , Bacterias/química , Pared Celular/química , Poliaminas/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Datos de Secuencia MolecularRESUMEN
Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.
Asunto(s)
Proteínas ADAM/metabolismo , Queratinocitos/efectos de los fármacos , Meliteno/farmacología , Receptores Purinérgicos P2X7/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Adenosina Trifosfato/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Western Blotting , Cadherinas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/citología , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Fosforilación/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein with a conserved cationic effector domain, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS and MARCKS-related protein specifically bind to LPS and that the addition of the MARCKS effector peptide inhibited LPS-induced production of TNF-α in mononuclear cells. The LPS binding site within the effector domain of MARCKS was narrowed down to a heptapeptide that binds to LPS in an extended conformation as determined by nuclear magnetic resonance spectroscopy. After LPS stimulation, MARCKS moved from the plasma membrane to FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. TLR4 signaling was enhanced by the ablation of MARCKS, which had no effect on stimulation by TLR2, TLR3, and TLR5 agonists. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/inmunología , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Proteínas de la Membrana/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Endosomas/inmunología , Fibroblastos/inmunología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inmunidad Innata , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Péptidos/química , Péptidos/farmacología , Unión Proteica , Transporte de Proteínas/inmunología , ARN Interferente Pequeño/genética , Transducción de Señal , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The interaction of selected endotoxin preparations (lipid A from Erwinia carotovora and LPS Re and Ra from Salmonella enterica sv. Minnesota strains R595 and R60, respectively) with selected bile acids was investigated biophysically. Endotoxin aggregates were analyzed for their gel-to-liquid crystalline phase behavior, the type of their aggregates, the conformation of particular functional groups, and their Zeta potential in the absence and presence of the bile acids by applying Fourier-transform infrared spectroscopy, differential scanning calorimetry, measurements of the electrophoretic mobility, and synchrotron radiation X-ray scattering. In addition, the ability of the endotoxins to induce cytokines in human mononuclear cells was tested in the absence and presence of varying concentrations of bile acids. The data show that the endotoxin:bile acid interaction is not governed by Coulomb forces, rather a hydrophobic interaction takes place. This leads to an enhanced formation of the inherent cubic aggregate structures of the endotoxins, concomitant with a slight disaggregation, as evidenced by freeze-fracture electron microscopy. Parallel to this, the addition of bile acids increased the bioactivity of lipid A and, to a lower degree, also that of the tested rough mutant LPS at lower concentrations of the endotoxin preparation, a finding similar as reported for the interaction of other agents such as hemoglobin. These data imply that there are general mechanisms that govern the expression of biological activities of endotoxins.
Asunto(s)
Ácidos y Sales Biliares/química , Endotoxinas/química , Biofisica , Rastreo Diferencial de Calorimetría , Ácido Quenodesoxicólico/química , Citocinas/biosíntesis , Ácido Deshidrocólico/química , Ácido Desoxicólico/química , Electroquímica , Técnica de Fractura por Congelación , Humanos , Técnicas In Vitro , Lípido A/farmacología , Ácido Litocólico/química , Monocitos/metabolismo , Pectobacterium carotovorum/química , Salmonella enterica/química , Colato de Sodio/química , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
Antimicrobial peptides are promising anti-cancer agents with a unique mode of action. We established the usage of a chip-based sensor to monitor the dynamic interplay between cells on the chip and peptides and compared it with endpoint tests. Human neuroblastoma cancer cells and spontaneously immortalized non-cancer keratinocytes were perfused with representative peptides (NK-2, NK11, and melittin). The sensor system enabled continuous recording of cell layer impedance (adhesion/confluence), oxygen consumption (respiration) and extracellular acidification (glycolysis) and provided insights in cell damage, stress response and recovery. Cells responded differentially to peptide treatment. During perfusion, peptides accumulated on the cell surface until they reached a critical concentration. Preceding to cell death, melittin triggered glycolysis, suggesting stress response. NK-2 induced no change in energy metabolism, but led to an increase in impedance, i.e. a temporarily altered morphology, which appeared to be an excellent parameter to detect subtle structural changes of cell layers.
Asunto(s)
Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Péptidos/farmacología , Línea Celular , Línea Celular Tumoral , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Queratinocitos/citología , Meliteno/farmacología , Neuroblastoma/patología , Péptidos/síntesis químicaRESUMEN
To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.
Asunto(s)
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Lactoferrina/química , Acilación , Péptidos Catiónicos Antimicrobianos/química , Biofisica/métodos , Rastreo Diferencial de Calorimetría/métodos , Hemólisis , Humanos , Leucocitos Mononucleares/metabolismo , Liposomas/química , Microscopía Electrónica de Transmisión/métodos , Péptidos/química , Fosfolípidos/química , Estructura Terciaria de ProteínaRESUMEN
Modification of the membrane lipid phosphatidylglycerol (PG) of Staphylococcus aureus by enzymatic transfer of a l-lysine residue leading to lysyl-PG converts the net charge of PG from -1 to +1 and is thought to confer resistance to cationic antimicrobial peptides (AMPs). Lysyl-PG synthesis and translocation to the outer leaflet of the bacterial membrane are achieved by the membrane protein MprF. Consequently, mutants lacking a functional mprF gene are in particular vulnerable to the action of AMPs. Hence, we aim at elucidating whether and to which extent lysyl-PG modulates membrane binding, insertion, and permeabilization by various AMPs. Lysyl-PG was incorporated into artificial lipid bilayers, mimicking the cytoplasmic membrane of S. aureus. Moreover, we determined the activity of the peptides against a clinical isolate of S. aureus strain SA113 and two mutants lacking a functional mprF gene and visualized peptide-induced ultrastructural changes of bacteria by transmission electron microscopy. The studied peptides were: (i) NK-2, an α-helical fragment of mammalian NK-lysin, (ii) arenicin-1, a lugworm ß-sheet peptide, and (iii) bee venom melittin. Biophysical data obtained by FRET spectroscopy, Fourier transform infrared spectroscopy, and electrical measurements with planar lipid bilayers were correlated with the biological activities of the peptides. They strongly support the hypothesis that peptide-membrane interactions are a prerequisite for eradication of S. aureus. However, degree and mode of modulation of membrane properties such as fluidity, capacitance, and conductivity were unique for each of the peptides. Altogether, our data support and underline the significance of lysyl-PG for S. aureus resistance to AMPs.
Asunto(s)
Aminoaciltransferasas/metabolismo , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Farmacorresistencia Bacteriana/fisiología , Membrana Dobles de Lípidos/metabolismo , Lisina/metabolismo , Fosfatidilgliceroles/metabolismo , Staphylococcus aureus/metabolismo , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/ultraestructura , Membrana Dobles de Lípidos/química , Lisina/química , Lisina/genética , Mutación , Fosfatidilgliceroles/química , Fosfatidilgliceroles/genética , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/ultraestructuraRESUMEN
Sepsis (blood poisoning) is a severe infectious disease with high mortality, and no effective therapy is actually known. In the case of Gram-negative bacteria, endotoxins (lipopolysaccharides) are known to be responsible for the strong inflammation reaction leading to the systemic infection. Peptides based on endotoxin-binding domains of human or animal proteins represent a promising approach in sepsis research. Although so far no medicament is available, the progress in recent years might lead to a breakthrough in this field. In this review, recent investigations are summarised, which may lead to an understanding of the mechanisms of action of peptides to suppress the inflammation reaction in vitro and in vivo (animal models) and thus may allow the development of effective anti-septic drugs.